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The Survival of Different Fungal Spores During Tabletting

Raghad A. Al-Shikli , Alaa A.Abdul-Rasool and Mustafa M. Al-Hiti

Design and Synthesis of New Non-Steroidal Anti-inflammatory Agents with

Expected Selectivity toward Cyclooxygenase-2 Inhibition

Nadeem A. Abdul Razzak , Samera.F.Hussan and Ahlam J. Qaseer

Bioequivalence of Two Formulations of Amoxicillin in Human Healthy

Volunteers on (HPLC) Technique

Alaa K. Jabbar Alhamd

Formulation of Metoprolol Bilayer Tablets as an Oral Modified Release

Dosage Form

Mohammed S. Jabbar and Yehia I. Khalil

In Vitro Effect of Cholesterol and Different Sugars on Digitonin Production

in Multiplied Shoots of Digitalis purpurea L. Plant

Zainab J.Awad

Synthesis, Characterization and Antibacterial Activities of Ligand Type N2O4

Schiff base and its Novel Complexes with Co(II), Ni(II), Cu(II) and Zn(II) ions.

Ahmed T. Numan , Manhel R. Aziz and Sinaa M. Shaker

Phytochemical Study of some Flavonoids Present in the Fruits of Two Ammi L.

Species wildly Grown in Iraq

Thukaa Z. Abdul-Jalil , Kawkab Saour and Abdul- MutalibA. Nasser

The Effect of Long Term use of Glibenclamide on Serum and Urinary Sodium

and Potassium Level in Type 2 DM Patients Ali A. Ali

Detection of Carbohydrate Antigen CA19-9 Levels in Sera and Tissues'

Homogenate of Breast and Thyroid Benign Cases

Gheid H. Alubaidi , Zeyan A. Ali and Abdulrahman R. Mahmood

The Effect of Chronic Renal Failure on Thyroid Hormones

Layla K. Ali

Effect of Chronic Exposure of Cadmium Chloride in Drinking Water on

Structural and Functional Aspects of Thyroid Gland in Mature Male Rabbits

Samir H. Cheyad , Kalisa K. Khudier and Khtan A. AL-Mzain

Study the Prevalence of Helicobacter pylori Infection by Different Diagnostic

Methods

Ibtihal N. Saeed and Aseel I. Ibrahim

Enhancement of Atorvastatin Tablet Dissolution Using Acid Medium

Kahtan j. Hasson

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Iraqi J Pharm Sci, Vol.19(1) 2010 Survival of fungal spores during tabletting

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The Survival of Different Fungal Spores During Tabletting Raghad A.Al-Shikli*,1 , Alaa A.Abdul-Rasool ** and Mustafa M. Al-Hiti *

* Department of Clinical Laboratory Science,College of Pharmacy, University of Baghdad, Baghdad, Iraq. **Department of Pharmaceutics, College of Pharmacy, University of Baghdad, Baghdad, Iraq.

Abstract The survival of dried spores of A.flavus, Penicillia Spp., and Cladosporia Spp.inoculated into multivitamins and folic acid tablets were examined at different compression pressures.Survival of fungal spores decreased with increasing compression pressure. The level of survival at particular pressures was shown to depend on the size of the contaminating fungal spores.The lethal effect of tabletting was attributed to shearing forces upon the contaminating spores generated by interparticulate movement. This hypothesis was supported by the dependence of survival upon spore size. Key words: Fungal spores, microbial contamination.

الخالصة

واع مـن ة اـن وتم إدخـال ثالـث ة وهي حامض الفولك ومجموعة الفيتامينات ن من األقراص الدوائية الصيدالني لقد تم تصنيع نوعيوبتركيزين الفطريات وهي بنيسيلية ة سبارجلس كالدوسبوري ة الكبس المباشر تحت ضغوط غرام/سبور ٤۱۰ ,۲۱۰ ا وتم كبسها بطريق

وغ م الـب ى حـج ت ضـغط معـين يعتمـد عـل ـح اء ت ن معدل البـق وا عدد السبورات ن زيادة الضغط تؤدي الى نقصان في وقد تبين إ ة مختلفةِ tablettingالتأثير القاتل ل.الملوث ولّـَد بحرـك ْت . interparticulate ُنِسبَ إلى َقّص القواِت على بويغاِت الَتلويث ِة َكاـن ذه الفرضـي ـه

ويغِة على حجِم الب .مدعومة من قبل إعتماِد البقاءِ Introduction Microbiologically contaminated tablets may cause disease and under humid storage conditions lead to visible deterioration of the tablet.Various workers have examined the effect of compaction process upon natural contaminants present in the granulate.In all cases, compression of granules to significant reduction in the levels of microbial contamination. (1- 3)Tabletting machines exert some antimicrobial effect . The shearing forces and localized heat involved in pressing tablets is sufficient to destroy many mould spores and vegetative organisms, although Bacillus spores appear to survive. Reduction (67-93)% in total viable counts of dry blended product has been reported to be produced by tabletting (1)Increasing compression pressures and tabletting speed enhance the anti microbial effect(4-8.) .Fassihi and Parker(9)granulated lactose powder using a granulating fluid containing A. niger spores .They demonstrated a linear relationship between the log survival and the applied pressure (28-271 MN/m2). Such linearity was not observed by Yanagita et al. (10) for Rhodotorula glutinins, E.coil and bacillus subtilis contaminated crystalline cellulose.plumpton(11) found that the levels of survival within four formulations were similar for a given organism, yet patterns emerged according to the mechanism of compaction of the material.Survival following compaction in

lactose and emdex , which compact by a process of fracture, was inversely related to pressure over the entire range tested , whilst for sta-Rx and potassium chloride such relationship was only exhibited up to compaction pressure of (194 MN/m2). At low compaction pressure (0-39 MN/m2)there was a little inactivation of the microorganisms in any of the four formulations. Also Plumpton(11)

found that levels of survival were inversely related to the size of the organisms for any given pressure, survival being greatest for B.megaterium spores (cell dameter approximately 1.5µm), intermediary for A.niger spores (4µdiameter) and least for S.cerevisiae (mean cell diameter approximately 8 µm). Similar trends in survival according to cell size were suggested from the work of Yanagiitu et al. (10). These workers examined the survival during compaction of vegetative cells of E.coli and R.glutinis and spores of B.subtillis within an Avicel/skin milk formulation. B.subitillis spores were found to be highly resistant to tabletting. Where as vegetative cellsof R.glutinis were much more sensitive to tabletting than those of E.coil. Blair(7) found that extent of kill on tablet compression was great for the larger organism, E.cloacase, on studying separate batches of lactose monohydrate , maize starch and cellulose

1 Corresponding author E- mail : [email protected] Received : 28 / 12 / 2008 Accepted : 31 / 5 / 2009


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microcrystalline contaminated with Staphylococus aureus or Enterbacter cloacae and tabletted at various compression force,indicating that size is important and cell rupture by shear,rather than heat,is the mechanism of kill.In this study,we examined the effect of tabletting on survival of dried spores of A.flavus,Penicillia Spp. and Cladosporia Spp. inoculated into the multi vitamines and folic acid tabletsduring compression with two levels (102 and 104)spore/gram at three compression pressure and one conpression pressure (102and104) spore/gram respectively. Materials, instruments and methods Chemicals Acetonitrile ,Acetone, Ammonium Hydroxide ,Anhydrous Sodium Sultate , Benzene, Chloroform, Glacial Acetic Acid ,Hydrous Disodium Hydrogen Phosphate (Na2Hpo4.12H2o), , Methanol, Potassium Hydroxide, Potassium Chloride, Sulfuric Acid, Sodium Hydroxide, Sodum 1-Hexane Sulfonate, Sodium Perchlorate, Sodium Chloride, and Tween 80 (Polysorbate 80) Suppied by BDY England. Monobasic Potassium Phosphate From Fluka-Swizerland. Heexan supplied by Merch-W. Germany. Microcrystalline cellulose (Avicel pH 101, Avicel pH 301), Folic Acid, Maize Starch, Vitamin B1 (Thiamin mononitrite), Vitamin B2 (Riboflavin),Vitamin B6 (pyridoxine Hcl), Methionine, Talc and Magnesium Stearate supplied by FMC coproation and Kindly supplied from (Sammara Drug Industires SDI. Iraq). Microorganisms Aspergillus flavus , penicillia Spp. and Cladospria Cladosporoids were obtained from College of Agriculture ,University of Baghdad. Culatures were stored on Sabouared Ager slants following incubation at 25°C for 5 days. Fresh cultures were prepared every 4 weeks . Culture media Sabouraud Dextrose Agar. RoseBengal Agar MacConkey Broth Solution used for dilutions and preparation of spore suspension Relative Humidity of the prepared solutions . Extraction Solvent of Aflatoxin. Relative Humidity Containers . Tablet formulations Prepation of dried spore powder 0.1 ml aliquots of 7days cultures of A.flavus, Cladosporia and Penicillia were inoculated onto the surfaces of predied

sabourad dextrose agar plates, incubated at 25°C for 5days. After this time spores were clearly visible on all the plates. Three ml of sterile water containing 0.1%tween 80 (as a dispersin agent) Were add to each plate . the spores were then dislodged by using glass spreader. The spore suspensions obtained were then stirred by using a vortex mixer for one minute. The spore suspension were then filtered through a sterile cotton wool in order to get rid of the hypha. The filtrates were then harvested by centrifugation at (10,000xy for10 min ). The supernatant liquids were then decanted and the residues were resuspended in 20 ml of sterile distilled water and washing were repeated three times. The number of spores of the resultant sopre suspensions was determined by viable count technique, spore suspensions were adjusted to obtain 2.16x106 spore/ml for A.flavus, 1.68x106 spore/ml for Penicillia Spp. and 1.98x106 spore/ml for C. cladsporoids. Multivitamin tablets and folic acid tablets were prepared using the formulas listed in tables (1) and (2), respectively . The following excipient were used Avicel pH 301 as a direct compression excipient, starch (5% w/w)as a disintegrant, magnesium stearate (0.5%)and stearic acid (2% w/w) as lubricants, were mixed with the active ingredients and compressed directly using a single punch tabletting machine with 7-mm flat –faced punches. Table 1 : The formula of the prepared folic acid tablet

Ingredie nt Amount / tab

Folic acid 1mg

Avicel pH 301 118.6 mg

Maize starch 6.5 mg

Mg.stearte 2.6 mg

Talc 1.3 mg

Total 130 mg

Table 2 : The formula of the prepared multivitamin table t

Ingredient Amount/tab Thiamin Mononitrite 1.5 mg

Riboflavin U.S.P 20 mg Pyridoxine Hcl U.S.P 2.0 mg Methionine 2.0 mg Avicel pH 301 105 mg Maize Starch 6.5 mg Talc U.S.P 6 mg Stearic acid ( powdered ) 3.0 mg

Mg.Stearte ( powdered ) 2.0 mg Total 130 mg


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Preparation of contaminated tablets For preparation of 500 contaminated tablets a(0.3 ml, 30µl) of A.flavus .(0.4 ml 40µl) of penicillia Spp and ( 0.3 ml, 30 µl) of C. cladosporiods were transferred to a sterile mortars and placed in the incubator until completely evaporation of water .Dried spores were scraped off and were included in direct compression formulation by dry mixing to 102 get spore / gram and 104 spore / gram for each of A.flavus, penicillia Spp. and C. cladosporids respectively. Ingredients including dried microorganisms spores weighed and lightly mixed in a glass morter by the method of geometric dilution technique for 20 min. Preliminary experiments had established that this method gives a uniform distribution of the microorganisms within the formulation , screen in the lubricant (magnesium stearte or stearic acid) and mixed for an additional 5 minutes. Quantities each of 130mg were accurately weighed and poured into 7-mm diameter die. Determination of viable number of spores in the prepared tablets Viable number of spores in prepared tablets was determined immediately after their production at different compression forces and after storage up to 8 weeks. Eight tablets (total wt.=1gm) were disintegrated in trypic soy broth (9ml) according to B.P 98 using a flask and suitable serial dilution in tryptic soy broth were prepared. One- ml sample of each dilution was poured in a sterile petridish and then 15 ml of molten dextrose agar was added to the plate. The sample and molten sabouraud dextrose agar were mixed together in forward and backward movement and swirled movement. The plates were allowed to solidify on a leveled surface. The plates were incubated at 35°C for 2-5 days. Survivals as colony forming units were estimated as the mean of triplicate determinations and expressed as a percentage relative to an uncompressed control sample of the contaminated formulations. Physical properties of tablets The result of physical properties of tablets are shown in table (3). Tablet weight and thickness, friability, hardness and disintegration time were measured.

Table 3 : Physical ,Chemical and Microbiological ( Control ) evaluation of folic acid and multivitamin tablet Tablet evaluation Multivitamin Folic acid

We ight ( 7min ) 130 mg 130 mg

C.pressure 148.3MN/m3

Wt.Unifo rmity 0 .9% 0.9%

Hardness (Kp) 6.6 0.4 6.5 0.6

Thickness ( mm ) 2 .85 2.65

Friability % 0.2 0.2

Disintegration time 2 .3 min 2 min

Assay

B6 Pyridoxine % 133.76%

B1 Thiamin % 148.2%

Folic acid % 905

Microbiological Quality

One day after preparatio n Less than 10 *C FU/gm

After storage for 8 week at 35 C 75 % RH Less than 10 *C FU/gm After storage for 8 week at 35 C 85 % RH Less than 10 *CFU/gm

A fter storage for 8 week at 35 C 95 % RH Less than 10 *CFU/gm

Interrelation of compression pressure and survival of A. flavus, penicillia and Cladosporia spores in multivitamin and folic acid tablets Multivitamin formulation were contaminated with A.flavus spores or cladosporium spores or penicillia spores using 102 and 104 spore/gm. Tablets were prepared at a variety of compression pressure (137.9, 144.8, and 148.3) MN/m2 and the number of survival was determined immediately upon ejection of tablets from the die. The number of survival was plotted as logarithmic function of compression pressure.

Results and Discussion Tablet evaluation Folic acid and multivitamin tablets were prepared as previously mentioned (Tables 1 and 2, respectively). The prepared folic acid and multivitamin tablets were evaluated physically, chemically and microbiologically. The results areshown in table 3. Interrelation of compaction pressure and survival of A. flavus, penicillia and cladosporia spores in multivitamin and folic acid tablets The results of the effect of compression force upon percent survival are shown in figure(1) and table (4a)for a contamination level of 102 spore/gm using three compression pressures of (137.9, 144.8 and 148.3 MN/m2) to get tablets with optimum physical properties. The results (fig. 1) and (table 4a) in which a 102 was used indicate that a loss of


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50.0, 49.0, and 94.0 percent in viability of the spores was obtained at pressure of 148.3 MN/m2 for A.flavus spores , Penicillia Spp. Spores, and C.cladosporoids spores, respectively. The data also show that the survival at a contamination level of 102 spore/gm was inversely proportional to the compression pressure i.e, increasing pressure from (137.9 to 148.3)MN/m2 caused a decrease in survival from 60 to 50.0 percent for A.flavus spores, from 50.0 to 41.0 percent for Penicillia Spp . spores and from 10.0 to 6.0 percent for C.cladospooids. That reduction is statistically significant (p=0.05). In general a higher reduction in viability was observed for Cladosporia than of Penicillia and A.flavus, size of the spores were in order of(3-6)µm in diameter, (4-6)µm in diameter, 3-7(-11)x2-4(5) µm in diameter )for A.flavus, Penicillia, and C.cladosporoids, respectively. On the other hand, Figure (2) and table (4b) show the effect of compression pressure upon percent of survival using 104 spore/gm contamination level and (148.3MN/m2) compression pressure to to obtain optimum physical properties of the tablet. The results indicate that the pressure applied caused a significant reduction in percent of survival (p=0.05)as 50.0 ,37.0 and 16.0 percent for A.flavus, penicillia, and C.cladosporoids ,as shown in figure (2). (4b).These results are well support the hypothesis that an inverse relationship is existed between spore size and compression pressure. Chesworth et al.(1) attributed the lethal effects of compression to a combination of two events, generation of heat and shearing between particles causing mechanical damage to the cells.Fassihi et al. (12) regarded localized heating within the formulation as being the critical parameter associated with inactivation of A.niger spores during compression of lactose granules .They proposed that during compression process, the pressures applied between the upper and lower punches were exerted only upon those particles directly in contact with the punch surface and that stresses on the remaining particles were through inter particulate contact . Such a phenomenon would result in very high pressures being exerted over small areas of inter particulate contact .This might cause the existence of localized (hot spots) within the tablets and bring about death of the microorganisms by heat alone. Such high temperatures have been shown to result in the melting of some materials during compression (13-18). If such a mechanism were applicable in our systems one would expect the levels of survival for different organisms to reflect their heat sensitivity rather than cell size. This was not the case, results suggested

therefore that shearing forces(size) are contributory to the observed lethal effects of compression rather than heat per sec.If pressure alone was responsible for the observed inactivation of microbial spores then once again no differences would have been expected between organism types. Conversely, larger sized microorganisms would be more likely to be subjected to shearing forces within a compact than smaller sized ones, supporting the hypothesis that inactivation is brought about by a direct physical trauma.

Figure 1 : e ffect of compression pressure upon the survival of ( 102 spore /gram ) diffe re nt fungal spores in Multivita min table t L.S.D = 3.7

Figure 2 : survival of 104 ASP flavus penicillia and cladosporia spores at constant pressure in multivita min table t


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Table 4: e ffect of pressure on survival of different fungal spores expressed as a % in multivitamin and folic acid tablets using 102 spore /gm and using 102 spore/gm contamination leve ls

4- a

4- b

Conclusion The results emphasize on the existence of relationship between survival of fungal spores and the pressure used during tableting. Survival of fungal spores decreased with increasing compression pressure and level of survival at particular pressure depends upon the size of the spore. The lethal effect of tabletting was attributed to shearing forces upon the contaminating spores. References 1. chesworth, K.A.c.; Sincletr, A.; atertten ,

R.J; and Hayer,W.p. Micro bios lettus 1977,4:41.

2. Morris,S.J- proceedings of thye Guild of Hospital oharmacists, hondon, ,1981 10:63.

3. Ayorinde, J.o. odeku, o.A. and Tida, o.A. the survival of B.subtilis spore in dicacium phosphate, hactose and starch and ther binary mixtures during tableting. Pharmaceutical technology, 2005, 29 (12):56-67-

4. plumpton E.J.;gilbert,p.; and fell, J.T the survival of microgranisms during tableting Int.J. pharm. 1986a ,30:241-246;

5. Plumpton E.J.;Jilbert,P.;and Fell J.T.Effect of special distribution on contaminant microorganisms with in tablet formulations on subsequent in in activation through compaction Int.J.Pharm.1986b,30:237-240;

6. Fassihi,A.R;and Parker,M.S.Inimicable effects of compaction speed on

microorganisms in powder systems with dis-similar compaction mechanisms J.pharm.sci,1987, 76:466-470.

7. Blair T.C. ; Buckton ,G. ;and Bloomfield ,S.F; Baird, R;Leak R.E .;and Leech , R (ods). Microbial quality assurance in pharmaceuticals , cosmetics and Toiletries .Ellis.Horwood,Chichest :1991,pp:104-161.

8. Odeku,O.A,Awe,O.O.,Popoola,B.Odenigi,M.A.and Itida,O.A.Compreccssion and me mechanical properties of tablet formulations containing corn,sweet potato and cocoyam starches.Phar.Tech ,2005 29(4):82-90.

9. Fassihi,A.R.;and parker,M.S.Journal of applied acteriology,43meetings xvii;1977a

10. Yanagtta,T.;Miki,T.;Sakat,T.;and Itorkoshi ,chemical and pharmaceutical bulletin, 1987 ,26:185-190.

11. PlumptonE.J.Studies upon the survival of various microorganisms in solid dosage forms,ph.D.thesis, university of manchester;1982

12. Fassihi,A.R.;and parker, M.S.the influence of water activity and oxygen tension upon the survival of aspergillus and pencillia species and tablets .Int .Biodeterior . Bull , 1977,13 :75-80.

13. Bowden,F.P.and tabor , D . (Friction and lubrication of solids) , vol. 2 , Clarendon press,oxford;1964 .

14. Jayasinghe ,S.S;pllpei,N.and Harwood ,C.F.Materals science and Engineering ,5 ,287 ;1969,1970

15. Odeka,O.Aand Itiolao A.Evaluation of Khaya gum as abinder in paracetol tablet formulation on pharm.pharmacol commum, 1998, 4:183 -188.

16. Espinasse.V,Mperria cornet.J, Amartceat, Gervais. P.High pressure in activation of dried microorganisms. Drug developed and industrial pharmacy, 2002,81ssae: 329-337.

17. IF Eynwa F.Obuekwe and Florence Eichie , Actapolonae pharmaceutica-drug research , ;2006 ,6 (2) :121-125.

18. Obuekwae,I.F.,Ogbimi A . O. , Pakistan J. sc. Ind. Res. 2002,45:341.

Pressure (MN/m2)

% Mean No. o f spore / gm fo r 102 spore/gm contamination level

A.flavus Penicillin Spp.

C. cladosporoids

137.9 60.0 50.0 10.0

144.8 56.0 45.7 7.7

148.3 50.0 41.0 6.0

Pressure (MN/m2)

% Mean No. o f spore / gm fo r 102 spore/gm contamination level

A.flavus Penicillin Spp.

C. cladosporoids

148.3 50.0 37.0 16.0

Iraqi J Pharm Sci, Vol.19(1) 2010 Bioequivalence of two formulations of amoxicillin

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Bioequivalence of Two Formulations of Amoxicillin in Human Healthy Volunteers on (HPLC) Technique

Alaa K. Jabbar Alhamd*, 1

* Department of Medicinal Sciences, College of Pharmacy, Al-Mustansirya University, Baghdad , Iraq Abstract Amoxicillin is commercially available in the form of capsules and tablets containing 250mg or 500mg for oral administration. It is also available in the form of suspension containing "25mg/ml” . Amoxicillin is presently used as the most common antibiotics .Ten healthy Human volunteers were characterized respected to their pharmacokinetic and bioavailability of two formulations of Amoxicillin from two sources of industrial companies after a single dose administration was given orally. A procedure is described for determination the concentration levels of Amoxicillin in human plasma of healthy volunteers using high performance liquid chromatography (HPLC) with reversed-phase isocratic column at low wave length of UV-visible detection "230nm". An efficient drug extraction procedure was used for the separation of Amoxicillin after simple extraction with cold methanol using ODS-C18-DB column. The pharmacokinetic 500mg of Amoxicillin capsule orally administrated treatment through 10 hours has been examined. The Amoxicillin was eluted for "10.0 minutes" at flow Rate "1.5ml/min." and Temperature equal to 298 K .The retention time of Amoxicillin was observed at 7.0 minutes. The mean absolute recovery of Amoxicillin in blood plasma of all healthy volunteers were 94.1% at 1.0ppm, 102% at 5.0ppm, 103% at 10.0ppm 102% at 20ppm, 99.3% at 40ppm and 104% at 50ppm respectively. The assay showed excellent relationships between area under the curve ratios and drug concentration levels (P>0.002) .Oral Amoxicillin administration in ten healthy volunteers gave maximum concentration peak plasma at two hours and decline through ten hours. Treatment with Iraqi formulation Amoxicillin produced higher area under the curve “AUC” and maximum concentration “C (max)” of Amoxicillin than Indian formulation. Key word: Amoxicillin, Bioe quivale nce, ODS -DB column.

الخالصةحتوي االولى على على شكل كبسوالت و حبوب ت ى 250يتوفر االموكسيلين تجاريا عل ة والثاني ملي غرام كما 500ملي غرام

الجرعة عن طريق الفم ويعتبر االموكسلين المضاد لتر حيث توخذملي / ملي غرام 25ويتوفر على شكل محلول عالق بتركيز وليفتين من كبسوالت االموكسلين لشركتين مختلفتين طبقت . الحيوي االكثر أستخداما على ت والتوافر الحيوي درست حركية الدواء

ن بجرعة م 500على عشرة من الناس االصحاء المتطوعي غرام اخذت عن طريق الف ويات توضح الطريقة الت. ملي ي حسبت فيها مستتراكيز االموكسيلين في بالزما الدم لعدد من الناس االصحاء والمتطوعين باستخدام جهاز كروموتوغرفيا السائل ذات االداء العالي

)HPLC ( ميثانول المبرد على عمود من نوع ن بال ستخلص منها االموكسيلي .)ODS-C18-DB (على نماذج بالزما الدم اعلى جهاز كروموتوغرافيا السائل ذات االداء العالي بزمن قدره عشر دقائق وبسرعة طور ن عينت مستويات تراكيز االموكسيلي

دقائق 7درجة كلفن و وقت االحتجاز لالموكسيلين 298دقيقة وبمحيط حراري / ملي ليتر 1.5متحرك حسبت النسبة المئوية ,, 99.3, 102, 103, 102, 94.1( على التوالي )بي بي أم 50, 40, 20, 10, 5, 1(زلالسترجاع في بالزما الدم للتراكي

ة % ) 104 على تركيز في بالزما الدم تكون خالل ساعتين من وقت الجرعة المأخوذة ) P>0.002 (بمستوى ثق اشارت النتائج بان ان سلوك التكافوء الحيوي عن طريق الفم ثم يبدأ التركيز بالنزول حتى الساعة العاشرة من وقت الجرعة كما وان النتائج اشارت با

ى توافر حيوي عل ة ا وليفة العراقي والتركيز االعلى للتوليفتيين الن الت غير مشابه واالختالف في المساحة ن وليفتي وليفة الهندية للت .من الت Introduction

Amoxicillin capsules and Amoxicillin suspensions were analyzed for their drug content as described in the united state pharmacopeia (1). Amoxicillin is the most commonly prescribed antibiotic for the treatment of phyaryngitis in the US.(2) Pharmacokinetic and bioavailability may be vital to ensure successful protocol in the clinic as well as in researches. Often the clinical evaluation of drugs is carried out on the basis of some secondary response because of the non existence of directly measurable parameter

which is related to the treatment of disease by the drugs (3) .Many times no response is measured at all, and the clinician attempts to make objective and subjective assessment of the patients general welfare. A dosage regimen for a new drug may in fact be based on such an evaluation and may not include a comparison with a standard drug or analog, a dosage regimen based upon such studies can be only a rough approximation at best. this point is well illustrated if one compares drug dosage regimens (e.g. sulfonamides) calculated

1Corresponding author E- mail : [email protected] [email protected] Received : 11 /3 /2009 Accepted : 28/12/2009

mailto:[email protected]


15

from pharmacokinetic data to those commonly used in the clinic. The same importance from this point of view does not concern only medicines but all other compounds including pharmaceuticals which are introduced to the organism for the diagnostic purposes. There is a through pharmacokinetic examination of a diagnostic agent which is extremely important from the point of view of decreasing of the possible risk (4).Pharmacokinetics are the studies of the movement of drugs in the body through the time course; it must not be hidden in mathematics itself. It is extremely important for everybody who is interested in pharmacotherapeutics in drug researches and in drugs productions to understand what the pharmacokinetics really means (5). Pharmacokinetic study of children that assessed the single-dose administration of an investigational oral Amoxicillin sprinkle designed to sequentially deliver an immediate-release and multiple delayed-release pulses of Amoxicillin to provide prolonged plasma concentrations of Amoxicillin (6). A new pharmacokinetically enhanced formulation of Amoxicillin has recently become available (7). The new formulation can maintain the main Amoxicillin serum concentration about 49% of the dosing interval in contrast with only 34% for three times daily regimen (7,8). The pharmacodynamic and pharmacokinetic properties of the new formulation predict high rates of success against respiratory tract pathogens (8). Amoxicillin (AMO) is oral semi-synthetic penicillin structurally related to Ampicillin as shown below:

The present of benzyl ring in the side chain extends the antibacterial activity to gram-negative bacteria (9). Amoxicillin presents as a highly absorption after oral administration and is not altered by the concomitant ingestion with food (10). Amoxicillin exhibits low binding with plasma proteins and is quickly distributed through the body. It has an elimination half life of one hour (11). Amoxicillin pharmacokinetics was obtained across pregnancy states. Oral clearance and renal clearance were higher while the half life was shorter during pregnancy, these changes suggest that the Amoxicillin exposure will be less while pregnancy that maintenance of trough concentrations will be difficult (12). Anew per oral Amoxicillin \ Clavulanate therapeutic system was developed and evaluated by in vivo bioavailability study (13,14,15). Amoxicillin was effective in reducing oral micro organism level up to 12 hour post- dose (16). The treatment with Amoxicillin for 3 to 7 days had similar clinical efficiency and also similar selection of oral streptococci with reduced susceptibility to Amoxicillin (17). Amoxicillin is commercially available in the form of capsules and tablets (250 or 500 mg) for oral administration and also available in the form of suspensions containing "25 or 50 mg / ml". Amoxicillin is one as the most commonly used as antibiotic. To understand the bioavailability and pharmacokinetic behavior of this drug in human which is needed reliable qualitative and quantitative methods. There are several high performance liquid chromatography (HPLC) methods were used for separation and determination of Amoxicillin in body fluids .Some of these methods were developed to use direct UV-visible detection at low wave length (225-229nm) (18—22). The bioavailability of two brands of melixican (7.5mg and 15mg) tablets and to obtained pharmacokinetic parameters of this molecules on Mexican population using modified and validated high performance liquid chromatography “HPLC” technique pharmacokinetic parameters AUC , C(max) , and T(max) were determined from plasma concentration levels of both formulations that the results indicated in a C(max) 120% larger and T(max) 65% faster than those reported(23,24). Others were used fluorimetric detection (25—28). Some others special techniques have been used to enhance the sensitivity and selectivity such as ion-pairing reagents and post column derivatization (21, 28, 29). There are several different methods used to preparation the sample that have been applied prior to chromatographic analysis mostly based on


16

liquid- liquid extraction (28) , deproteinization by precipitation (19,25) and solid phase extraction (18,30,31). Plasmatic Amoxicillin concentration levels were determined by combined reversed-phase liquid chromatography and mass spectrometry with positive ion electro spray ionization using the selective ion monitoring technique (32, 33). The paper presents the favored approach of clinical studies involved in qualitative and quantitative assay of pharmacokinetics of two Amoxicillin formulations firstly, Iraqi formulation of capsule containing 500mg Amoxicillin compared with Indian formulation capsule containing 500mg Amoxicillin. After evaluation of the various condition of the (HPLC) assay, a suitable and simple assay for the determination of Amoxicillin in human plasma of healthy volunteers was developed using reversed-phase isocratic (HPLC) at direct low wave length of UV-visible detection (230nm) and temperature equal to 298 K for subsequent study of pharmacokinetics and bioavailability. Experimental Materials and methods Chemicals and drugs All chemicals used in this study were the highest analytical grade purchased from commercial sources and used without any further purification. The deionized distilled-water was used for all preparation. Methanol (Absolute MeOH) and acetonitrile (Absolute ACN) “HPLC grade” were purchased from (FLUKA). Amoxicillin capsules from two sources one from Iraq (S D I, Iraq ) and the other from India(MICRO LABS LIMITED ,Bangalore ,India) Potassium dihydrogen phosphate (KH2PO4), Dipotassium hydrogen phosphate (K2HPO4), Phosphoric acid and 1-octane sulphonic acid sodium salt were purchased from (BDH, England). Sepelco-ODS-C18-DB column (250 X 4.6mm I.D.) was purchased from (sepelco, United Kingdom). Standard solutions Amoxicillin (1.0 mg) was dissolved in 100ml of freshly prepared mixture of water: methanol (95:5) “10000 ppm”. The standard solution was filtered, degassed and stored at 253 K for further use. The standards were prepared freshly every month. Stock solution of Amoxicillin (1mg/ml) was prepared in a mixture of (water: methanol) (95: 5). The applied standard solutions were prepared from stock solution by sequential dilution with the same mixture to produce final concentrations (1, 5, 10, 20, 40, 50ppm). The

stock and applied solutions were protected from light and stored at 253 K. Calibration standard curve were performed to achieve the concentration of (0.1, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0ppm) Figure-1.

Figure 1: Linearity of diffe re nt concentration levels of Amoxicillin using HPLC technique on ODS-DB column Extraction of Amoxicillin Blood sample (3--5ml) were drawn from vein by syrings in to hyparinized blood tubes, then transferred immediately into polyproplene tubes and centrifuged within 5 min. at 500G for 15 min. One milliliter of sodium metabisulphate, (pH equal to 8.0) was added for each one milliliter of plasma. Amoxicillin was extracted from human plasma samples by deproteinization using precipitation process. A 500µl aliquot form each plasma sample was transferred to a 5.0ml polypropylene tube. One milliliter of cold methanol was added. After slightly vortex mixing, the tubes were centrifuged for 15min. at 500G. A 100µl aliquot of the supernatant was transferred to the injection vials and 10µl were injected into chromatographic system. All samples from volunteers were analyzed on the same day in order to avoid inter-assay variation. Plasma solutions were protected from the light and stored in a deep freezer at (203 K). HPLC Instrumentation This study was performed on Shimadzu instruments model LC-6A HPLC system. The unit was operated in the isocratic model using solvent reservoirs fitted with 0.22 µm stainless steel filter at the end of polytriflouroethylene (PTFE) tubes, transferring the mobile phase from reservoirs to the pump, the system also involved an injector with 50µL sample loop


17

model ( Reseadyre 7125 ) , Column in type of ODS-C18-DB "250 X 4.6mm I.D.", Thermostatic oven model CTO-6A Shimadzu, UV-visible detector model "SPD" and chromatopac unit model R4-6A Shimadzu. HPLC Operation Condition For routine HPLC analysis of Amoxicillin use the following estimation condition. The mobile-phase was phosphate buffer conc. 10mM that containing 0.1mM 1-octane Sulphonic acid sodium salt: methanol (95:5) (v/v), pH buffer equal to six, column temperature 298 K, flow rate equal to (1.5ml/min.) and UV-visible detection at 230nm. The typical chromatograms of standard solution and blood plasma samples of Amoxicillin are shown in fig-2 and fig-3 respectively.

Figure 2: Typical chromatogram of HPLC analysis of Amoxicillin on ODS-DB column Figure 3: Typical chromatogram of HPLC analysis of plasma Amoxicillin on ODS-DB column

Pharmacokinetics and statistical analysis The observation of maximum plasma concentration levels (C(max)) and time consuming to reach it (T(max)) were obtained fro m drug concentration versus time curves. The area under the curve ”AUC” of the Amoxicillin concentration levels versus time fro m 5.0 minute to ten hours were estimated fro m "figure-4".

Figure 4: Pharmacokinetics of Iraqi formulation and Indian formulation Amoxicillin in blood plasma of healthy volunteers Results The isocratic reverse-phase HPLC technique described and used here for estimation of drug provides the appropriated sensitivity, specificity and high sample accuracy for bioavailability and pharmacokinetic studies. Fig.1 shows the retention time of Amoxicillin standard solution that under described chromatographic condition. The retention time of Amoxicillin was 7.0 minutes. The optimal chromatogram of analysis was given an ideal shape, symmetrical, and good resolution of peak. Fig.-2 shows the typical chromatogram of Amoxicillin in blood plasma samples of healthy volunteers which was appeared no endogenous Interfering peaks at the retention time of interest compound. The mean absolute recovery of Amoxicillin in blood plasma was 94.1% at 1.0ppm, 102% at 5.0ppm, 103% at 10.0ppm 102% at 20ppm, 99.3% at 40ppm and 104% at 50ppm respectively. The calibration curve was linear with regression coefficient R2 =0.989 (Table-1). The analytical precision and accuracy values was obtained from assays of six quality control (1, 5.0, 10.0, 20.0, 40.0, and 50.0 ppm) are shown in table-1 .The accuracy were 94.1% , 102% , 103% ,

Re tention Time (minute )

Re te ntion Time (minute )


18

102%, 99.3% and 100% respectively and there is not significant degradation of Amoxicillin was observed during the period of storage. Table 1: the linearity, precision and accuracy of blood plasma Amoxicillin samples.

Spiked concentration

(ppm) 1.0 5.0 10.0 20.0 40.0 50.0

Recovered average con.

(ppm) 0.94 5.1 10.3 20.4 39.7 50.2

Slope R2 P.V

1134 0.989 0.001

Discussion The HPLC technique presented in this study decreases the lower limit of quantitation of Amoxicillin to about 0.1ppm. It was appeared that is more sensitive than many other assays. The low limit of the estimation of the plasma concentration of Amoxicillin was sufficient to perform the pharmacokinetics study of drug. Amoxicillin plasma concentration levels were measured by several methods in combination with UV-visible detection. The lowest plasma concentration levels of Amoxicillin was obtained by UV-visible detection which was 0.05ppm but the process was consumed long time that was 30min. (10,14) .Charles et al were described a procedure for determination of Amoxicillin in urine(8). Other complicated procedures for extraction and estimation of plasma concentration levels of Amoxicillin by using Solid-phase extraction has been also reported (7, 9, 18, &19). Nevertheless, Solid-phase extraction (SPE) procedures are laborious and require SPE cartridges, increasing the cost of analysis. In order to improve the sensitivity, a column ion-pair HPLC with post column derivatization has been used(16). Where the low limit of quatitation was 0.01ppm, this procedure was more complicated due to the more step of post column derivatization and their retention time that will be longer than 10min.which is compared with our procedure that the retention time of Amoxicillin peak was 7min. and the full time of process not greater than 10min. . Also these procedures cannot be used in pharmacokinetics studies in human where a large number of samples were analyzed.The pharmacokinetics study was done in "10 hours" and the results indicate that the Iraqi formulation has higher bioavailability compared to the Indian formulation depending

on the area under the curve AUC and C(max) .Our technique was evaluated and produced the best results in terms of selectivity and sensitivity consideration the fact that the present technique involves a shorter running time and a simple sample preparation process. Conclusion Our HPLC technique was employed here proved to be fast, simple, precise, selective and sensitive enough to be used in clinical pharmacokinetic and bioavailability study for Amoxicillin in plasma human. The AUC and C(max) of Iraqi formulation are higher than Indian formulation of Amoxicillin and the T(max) of both two formulations are similar which is shown in table-2 and the relative bioavailability of Indian to Iraqi formulation was estimated equal to 64.11% . Table 2 : The pharmacokinetic parameters “ Cmax , Tmax and AUC” for the Iraqi and Indian formulations of Amoxicillin .

Pharmacokinetic parameter

Indian fo rmulation

”A”

Iraqi formulation

“B”

C(max) “ppm” 8.9 11.5

T(max) “hour” 2 2

AUC 30.25 47.18

Acknowledgment I wish to thank Miss. Kafa Q. Al-Obydee for carrying out the experiments that associated with preparation of samples and Kolood I. Mohamad for their technical assistance. References 1. US pharmacopeial ,(2005), USP 28\NF 23

.The united states pharmacopeial convention, Inc, ROCKVILLE, MD , USA.

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6. M. E. Pichichero ,J. R. Casey, S. L. Block et al , Pharmacodynamic analysis and clinical trials of Amoxicillin sprinkle


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administrated one daily for 7 days compared to pencillin V potassium administrated four times daily for 10 days in the treatment of tonsillopharyngitis to streptococous pyrogenes in children . J. Antimicrobial Agents Chemother. ( 2008) 52(7):2512---2520.

7. Kaye CM ,Allen A, Perry S. et al , The clinical pharmacokinetics of a new pharmacokinetically enhance formulation of Amoxicillin \ Clavulanate , Clin. Ther., (2001) 23: 578---584.

8. White AR , Kaye CM, Poupard J. et al , Augmentin ( Amoxicillin| Clavulanate ) in the treatment of community – acquired respiratory tract infection : a review of the continuing development of an antimicrobial agent , J. Antimicrob. Chemother. (2004) 53: 13---20 .

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13. Kerc J., Opara J. , A new Amoxicillin \ Clavulanate therapeutic system : preparation , in vitro and pharmacokinetic evaluation . Int. J. Pharm. , (2007) 335(2): 106---113.

14. Wirongnong Chierakul, Jinda Wangboonskul, Thida Singtoroj et al , Pharmacokinetic and pharmacodynamic assessment of co-amoxiclav in treatment of meliodosis, J. Antimicrobial Chemother. , (2006) 58(6): 1215---1220.

15. Guillterme Suarez-kurtz , Frederico Mota Ribero, Flaviol Vicente and Claudio J. Struchiner , Development and validation of limited – sampling strategies for predicating Amoxicillin pharmacokinetic and pharmacodynamic preparation ,Antimicrobial Agent and Chemother. , (2001) 45(11): 3029---3036.

16. Baglie S. , Del Ruenis AP. , Motta RH, et al , Plasma and salivary Amoxicillin concentration and effect against oral micro

organism , Int. J. Clin. Pharmacol.Ther., (2007): 45(10) 556---562.

17. Chardin H., Yasukawu K. , Nonucer N. , et al , Reduced susceptibility to Amoxicillin of oral streptococci following Amoxicillin exposure . J. Med. Microbio. , (2009) 58(8): 1092-1097.

18. Krauwinkel W. J. ,Volkers-kamermans N. J. , Van Zijtveld J. , Determination of Amoxicillin on human plasma by hjgh performance liquid chromatography and solid phase extraction , J. Chromatogr., (1993) 617: 334-338.

19. Charles B. , Chulavatnatol S. , Simple analysis of Amoxicillin in plasma byhigh performance liquid chromatography with internal standardization and ultra- violet detection . , Biomed. Chromatogr. (1993) 7: 204-207 .

20. Yaun Z. , Russlie H. Q. , Canafax D. M. , Sensitive assay for measuring Amoxicillin in human plasma and middle ear fluid using solid phase extraction and reversed phase high performance liquid chromatography ., J. Chromatogr. B, (1995) 674: 93-99 .

21. Muth P. , Metz R. , Beck H. ,Bolten W. W. , Verdin H. , Improved high performance liquid chromatographic determination of Amoxicillin in human plasma by means of column switching . J (1996) 259-266 .

22. Sourgens H. , Stinbrede H. , Verschoor J. S. , Bertola M. A. , Rayer B. , Bioequivalence study of a novel solutab tablet formulation of Amoxicillin / Clavulanic acid versus the originator film-coated tablet. Int. J. Clin.pharmacol. ther. (2001) 39: 75-82 .

23. G. Marcelin – Jimenez , Jose A. Hernandexz , Alionka P. Angeles , et al , Bioquivalance evaluation of two brands of meloxican tablet ( promotion and mobicox): Pharmacokinetics in healthy female Mexican population , Biopharm. Drug Dispos., (2005) 26 : 167---171.

24. Foroutan S. M. , Zarghi A. , Shafaai A. , Khaddam A. , et al , Simultaneous determination of Amoxicillin and Clavulaic acid in human plasma by isocratic reversed-phase HPLC using UV detection , J. Pharm. Biomed. Analysis(UK) , (2007) 45:370---374.

25. Hoizy G. , lamiable D. , Frances C. , Trenque T. , Kaltenbach M., Denis J. , Millart H. , Simultaneous determination of Amoxicillin and Clavulanic acid in human plasma by HPLC with UV


20

detection . J. Pharm. Biomed. Anal.. (2002) 30: 661-666.

26. Miyazaki K. , Ohtani K. , Sunada K. , Arita T., . Dtermination of Ampicillin,Amoxicillin , Cephalexin and Cephradine in plasma by high performanceliquid chromatography using fluorimetric detection . J. Chromatogr. , (1986) 276: 478-482 .

27. Marscher H. , Kikuta C. , Determination of Amoxicillin in plasma by high performance liquid chromatography with fluorescence after online oxidation, J.Chromatogr. A , (1990) 506: 417-421 .

28. Carlqvist J. , Westerlund D. , Automated determination of Amoxicillin in biological fluids by column switching in ion-pair reversed phase liquid chromatographic system with post column derivation , J. Chromatogr. (1985) 344:285-296 .

29. Henion J. , Brewer E. , Rule G. , Simple preparation for LC/MS/MS analyzing biological and environmental samples , Anal. Chem. (1998) 70: 650-656 .

30. Maurer H. H., Liquid chromatography & mass spectrometry in forensic and clinical toxicology , J. Chromatogr. , B , (1998) 713: 3-25 .

31. Oliveira C. H. , Abib E. Vannuchi Y. B. , Sucupira M. , Llha J. , De Nucci G. , Comparative bioavailability of 4 Amoxicillin Formulations in healthy human volunteers after asingl dose administration .Int. L. Clin. Pharm. And Ther. (39), 4 : (2001) 167-172 .

32. Baglie S. , Rosalen PL. , Franco LM , et al , Comparative bioavailability of 875 mg Amoxicillin tablets in healthy human volunteers , Int. J. Clin. Pharmacol. Ther., (2005) 43(7) : 350---354.

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Iraqi J Pharm Sci, Vol.19 (1) 2010 Metoprolol bilayer tablets

21

Formulation of Metoprolol Bilayer Tablets as an Oral Modified Release Dosage Form

Mohammed S. Jabbar* ,1 and Yehia I. Khalil** *Department of Pharmaceutics, College of Pharmacy, University of Basrah, Basrah ,Iraq. **Department o f Pharmaceutics, College of Pharmacy, University of Baghdad, Baghdad, Iraq.

Abstract Metoprolol is a β1 adrenergic blocker used in treatment of heart diseases. Metoprolol (100mg) tablets was formulated as a modified release oral system utilizing the concept of bilayer system, first layer contained (30mg) as immediate release and the other (70mg) in the sustained release matrix. The immediate release layer consisted of lactose or microcrystalline cellulose as diluents with sodium starch glycolate or sodium croscarmellose as disintegrants. The result showed that the layer contains microcrystalline cellulose and 2% sodium starch glycolate gave disintegration time similar to that of conventional metoprolol tartrate tablet. This result was subjected in the subsequent preparation of the bilayer tablet. The sustained release layer was prepared using three polymers: ethylcellulose (EC), Hydroxypropyl methylcellulose (HPMC) and hydroxyl ethylcellulose (HEC) as retardant materials. It was found that the combination of EC with HPMC in ratio of 2:1 in F11 was best formula because of it’s release profile and the tablet integrity and dimensions were conserved for the period of the test, but according to similarity factor (f 2), F15 (which contained EC:HPMC in ratio 2:1 with polyvinyl pyrrolidone (PVP) as a binder) was the best formula showed higher (f2) among all other formulas and equals to 72.3 comparing to reference product. Key words: Me toprolol, Bilayer table t, Imme diate re lease, Sustained re lease.

الخالصة ب أدريناليني في عالج أمراض القلب 1الميتوبرولول هو حاصر يستخدم تصييغ. تم كنظام )مغ100(حبوب الميتوبرولول

األولى ة الطبق ة٬ ثنائي الطبق نظام مبدأ خدام ست بإ التحرر متغير التحرر وو )مغ30(تحوي دوائي فموي ) مغ70(ال تكون مباشرةوا .قالب بطيئ التحرر في االخرى ة مباشرة التحرر تتكون من كل من الالكتوز أ سليلوزالطبق وم ل ور كمخففات مع صودي مجهري التبل

ة التي تحوي على ا. نشأالكاليكولت أو صوديوم الكروسكارملوس كمفككات سليلوزتبين النتائج بإن الطبق ور مع ل من % 2مجهري التبل للحبوب ثنائية صوديوم نشأالكاليكولت تع الميتوبرولول التقليدية ولذلك ٌاستخدمت في التحضيرات الالحقة لحبة طي وقت تفكك مشابه

و الهايدروكسي . الطبقة وليمرات هي االثيل سليلوز ٬الهايدروكسي بروبيل مثيل سليلوز ة ب ة التحرر بإستخدام ثالث ُحضرت الطبقة بطيئسليلوز كمثبطات تحرر سليلوز بنسبة وجد إن خليط .اثيل الهايدروكسي بروبيل مثيل هو 11في الصيغة 1:2من االثيل سليلوز و

ة الحبة وأبعادها للتحرر وهيكلي بسبب الشكل الجيد ممحافظ عليه كان أفضل صيغة عامل " إعتمادا خالل فترة اإلختبار٬ لكن على الصيغة ه٬ شاب الهايدروكسي بروب( 15الت و ة التي تحوي االثيل سليلوز بنسب بايروليدون كمادة 1:2يل مثيل سليلوز البولي فنيل مع

على عامل تشابه وهو ) رابطة ة بالمنتج المرجعي 72.3كانت أفضل صيغة وأظهرت ا . مقارنIntroduction Tablets may be defined as solid pharmaceutical dosage forms containing drug substances with or without suitable diluents and have traditionally prepared by either compression, or molding methods. Recently, punching of laminated sheets, electronic deposition methods, and three-dimensional printing methods have been used to formulate tablets (1). Modified-release tablets are coated or uncoated tablets that contain special excipients or they are prepared by special procedures, or both, designed to modify the rate, place or time of release of the active substance(s) (2). Layered tablets, type of modified release, are prepared by compressing several different granulations fed into a die in succession, one on top of another, in layers. Each layer comes from a separate feed frame with individual weight control to form two- or

three- layered tablets, depending on the number of separate fills. Each layer may contain a different medicinal agent, separated for reasons of chemical or physical incompatibility, staged drug release, or simply for unique appearance of the layered tablet (3). Some layers may be formulated to exert certain functions. Rahmanz Z. et al, designed a bilayer floating tablet of captopril in which one layer responsible for floating of the tablet for prolongation the gastric residence time with a controlled release mechanism(4). The bioavailability of propranolol was enhanced by formulating a bilayer and multilayer buccal mucoadhesive tablet in which one layer adhere to the buccal mucosa so localized the drug in the oral cavity and avoid hepatic first pass metabolism (5).

1Corresponding author E- mail : [email protected]@yahoo.com Received : 1 /8 /2009 Accepted : 28/12/2009



22

The bioavailabilty of rosiglitazone was improved by developing a bilayer and floating-bioadhesive drug delivery system exhibiting a unique combination of floatation and bioadhesion to prolong residence of rosiglitazone in the stomach (It is highly soluble in 0.1 mol/l HCl) so improve its bioavailability (6).Bilayer tablet is suitable for sequential release of two drugs in combination, with one layer of drug for immediate release while the second layer designed to release drug in an extended release manner. Bhavesh S. et al developed bilayer tablet for treatment of migraine consisting from Ibuprofen in the sustained release layer and metoclopramide (to enhance the absorption of non-steroidal anti-inflammatory drug) in the immediate release layer (7).The most important application of the bilayer is a quick/slow release system provides an initial burst of drug release followed by a constant rate (ideally) of release over a defined period of time. This type of system is used primarily when a maximum response needs to be achieved quickly, and it is followed by a sustained release phase to avoid repeated administration. When a single constant rate for drug release does not entirely satisfy the therapeutic objective, the quick/slow delivery system may be an interesting alternative. This biphasic release system can be achieved by the application of an immediate release layer to the conventional layered matrix tablet (8).Suitable candidate drugs for this type of administration include nonsteroidal anti-inflammatory drugs (NSAIDs) and antihypertensive, antihistaminic, and anti-allergic agents(9). Metoprolol (MT) is a cardioselective β1blocker exert equilibrium-competitive antagonism of the actions of catecholamines and other adrenomimetics at β-receptors, used in treatment of hypertension, angina pectoris, myocardial infarction, cardiac arrhythmias and heart failure (10). Peak plasma concentrations are achieved within 1.5 to 2 hours after a single oral dose, but because of the drug’s short half-life (3 to 4 hours), the therapeutic plasma concentration can be maintained only if the metoprolol is administered frequently (11). These characteristics make metoprolol a suitable candidate for administration by a quick/slow delivery system. The purpose of this study is to design a quick/slow delivery dosage form as biphasic bilayer tablet in which one layer containing superdisintegrant like sodium starch glycolate or sodium croscarmellose responsible for immediate release of the drug and the other is matrix layer of retardant polymer like Ethylcellulose (EC), Hydroxypropyl methylcellulose (HPMC) and

hydroxyethylcellulose (HEC) responsible for sustained action.

Materials and Methods Materials Metoprolol tartrate, hydroxypropyl methylcellulose and colloidal silicon dioxide (CabOSil) from Sigma, Germany. Metoprolol tartrate 50mg tablet: UK limited, England. Metoprolol tartrate 100mg sustained release: Ratiopharm, Germany. E thylcellulose from Acros organics, United States. Hydroxyethylcellulose from Merck, Germany. Lactose from Riedel-DeHaen, Germany. Microcrystalline cellulose (Avicel PH 102) and croscarmellose from Hekma Drug Industry, Jordan. Starch and talc from AFCO, India. Polyvinylpyrrolidone, sodium starch glycolate (Explotab) and magnesium stearate from BDH laboratory, England. Methods Preparation of Immediate Release (IR) Granules Different formulas (table 1) were prepared using wet granulation method to achieve most acceptable pharmacopial requirements and consider as comparable test with the reference one (Metoprolol tartrate UK limited 50 mg) tablet.After weighing the drug and the excipients enough to prepare 40 tablets in dried form, and then blend the ingredients using mortar and pestle, the powders were mixed with binding solution (10% alcoholic starch paste) gradually until proper ball test consistency was resulted.The wet mass was screened through sieve (10 mesh) and dried in pre warmed oven at 60 º C for one hour. The dried granules then reduced in size by screening them through 12 mesh size sieve. A known weight of granules equivalent to the stated dose was mixed with calculated amount of magnesium stearate and talc powder for five minutes. Table 1: Different formulas of metoprolol granules as immediate release (IR) laye r

IInnggrreedd iieenntt ((mmgg )) IIRR11 IIRR22 IIRR33 IIRR44 IIRR55 IIRR66

MMeettoopprroo lloo ll ttaarrttrraa ttee 30 30 30 30 30 30

MMiicc rroo ccrryyssttaalllliinn ee cceelllluulloossee ((MMCCCC))

103 100 100

LLaacc ttoossee 103 100 100

SSoodd iiuumm ssttaarrcc hh ggllyycc oollaattee

3 3

CCrroo ssccaarrmmee lllloossee 3 3

1100%% aa llcc oohhoolliicc ssttaarrcchh ppaassttee

15 15 15 15 15 15

MMgg sstteeaa rraattee 1 1 1 1 1 1

TTaallcc 1 1 1 1 1 1

TToottaall wwtt 150 150 150 150 150 150


23

Preparation of Sustained Release (SR) granules Different formulas (table 2) were prepared using wet granulation method like immediate release granule to achieve most acceptable pharmacopial requirements. After weighing the drug, polymer(s) and diluent enough to prepare 40 tablets in dried form, and then blend the ingredients using mortar and pestle, the powders were mixed with binding

solution gradually until proper ball test consistency was resulted. The wet mass was screened through sieve (10 mesh) and dried in pre warmed oven at 60 º C for one hour. The dried granules then reduced in size by screening them through 12 mesh size sieve. A known weight of granules equivalent to the stated dose was mixed with calculated amount of magnesium stearate, CabOSil and talc powder for five minutes.

Table 2 : Diffe re nt formulas of metoprolol granules as sustained re lease layer

VVaa rr ii aa bb ll ee

ff oo rr mmuu

ll aa ss

MMee tt oo pp

rr oo ll oo ll tt aa rr tt rr aa

tt ee

EECC

HHPP

MMCC

HHEE

CC

LLaa

cc tt oo ss ee

mmii cc rr oo cc rr yy ss tt aa

ll ll iinn

ee cc ee ll ll uull oo ss ee

11 00%%

aall cc oo hh

ooll ii cc

ssttaarrcchh

ppaassttee

PPoo

ll yyvv ii nn

yy ll pp

yy rr rr oo ll ii ddoo nn ee

MMgg SS

tt ee aarr aa tt ee

CCaa

bbOO

SSii ll

TTaa

llcc

TToo

tt aa ll LLaa yy

ee rr WW

tt mmgg

EEffffeecc tt ooff EECC

FF11 70 70 172 35 1 1 1 350

FF22 70 140 102 35 1 1 1 350

FF33 70 210 32 35 1 1 1 350

EEffffeecc tt ooff HHPPMMCC

FF44 70 70 172 35 1 1 1 350

FF55 70 140 102 35 1 1 1 350

FF66 70 210 32 35 1 1 1 350

EEffffeecc tt ooff HHEECC

FF77 70 70 172 35 1 1 1 350

FF88 70 140 102 35 1 1 1 350

FF99 70 210 32 35 1 1 1 350

EEffffeecc tt ooff EECC-- HHPPMMCC

FF 1100 70 70 70 102 35 1 1 1 350

FF 1111 70 140 70 32 35 1 1 1 350

EEffffeecc tt ooff EECC-- HHEECC

FF 1122 70 70 70 102 35 1 1 1 350

FF 1133 70 140 70 32 35 1 1 1 350 DDiilluuee nntt

ttyyppee FF 1144 70 140 70 32 35 1 1 1 350

BBiinnddee rr ttyyppee FF 1155 70 140 70 32 35 1 1 1 350

Preparation of Metoprolol Bilayer Tablet The quantities of material required to produce the tablets were individually weighed. The SR layer was manually poured into the 10 mm die and was gently precompressed so that a flat surface was created. Then the other IR layer was poured into the die to create the second layer. The layered granules were then compressed to a specified maximum compression.

Evaluation of Bilayer Tablet Disintegration Test The different formulas of IR layer were compressed alone at the same compression forced and their disintegration times were compared with the reference conventional tablet (Metoprolol 50 mg).The disintegration time was determined in 0.1N HCl (pH 1.2) (2).


24

Friability Test This test was done for 20 tablets, starting by weighing them and then operating the friabilator at 25 rpm for 4 minutes, re-weighing the tablets to determine the loss in their weight (12). Tensile Strength The tensile strength of five tablets of each prepared formula was determined from diametrical compression tests, which were performed using a universal hardness tester to measure the maximal diametrical crushing force (F) accurately. Together with the measured diameter (d) and thickness (t) of the tablets (by micrometer), the tensile strength (σ t) is then calculated according to the following equation (13):

dtF

t

2

Uniformity of Dosage Units The content uniformity was

performed for the prepared metoprolol bilayer tablet by taking five tablets and assayed individually. The requirement for this test is met if the amount of ingredient in each of the five tablets lies within the range of 90-110% of the label claim (14). Dissolution behavior A suitable dissolution tests were carried out to demonstrate the appropriate release of metoprolol, the dissolution studies were carried out using the USP basket method at 37 º C ± 0.5 º C and rotation speed 100 rpm as follows (14):- One tablet of each prepared formula was running out in a dissolution jar containing 900ml of 0.1 N HCl (pH 1.2) for two hours, then in 500ml of phosphate buffer (pH 6.8) for the rest of the experiment. Samples of 5ml were withdrawn at specific time intervals (first at 5, 10 and 15 min then at 1,2,3,4,5,6,7,8,9,10,11 and 12 hr), the volume of samples were replaced with the same buffer solution. Samples were filtered and diluted, analyzed using UV spectrophotometer at 223 nm, the procedure was triplicated for each run test. Some variables that affect the dissolution behavior had been studied like retardant polymer type, polymer concentration, polymer – polymer ratio, type of diluent, type of binder and pH of dissolution media (1.2, 4.6 and 6.8). Swelling and Erosion Study The swelling and erosion studies were performed for optimized formulation. Briefly, weighed tablets with watch glass [H1] and placed in dissolution vessel, containing 0.1 M hydrochloric acid for first 2 hr followed by 6.8 pH phosphate buffer for 4 hr using paddle method at stirring speed of 100 rpm. At selected time interval (1 to 6 hr) the tablets

were withdrawn. The watch glass and tablets were blotted dry to remove water and weighed [H3]. The axial and radial swelling was measured using micrometer. The wetted tablets were dried in an oven at 110 °C for 24 hr, cooled and dried in a dessicator and finally weighed as dry weight [H2] (15): The percent absorption was calculated as

A%=100[H3-H2]/H2 The percent erosion was calculated as E%=100[H1-H2]/H1 Fourier Transform Infrared Studies (FT-IR) The FT-IR spectra of metoprolol tartrate, physical mixtures and granules of immediate release and sustained release layers were obtained after making potassium bromide discs in the range of 4000 cm -1 to 500 cm -1 to detect drug–excipients interaction, if any (16). Statistical Analysis The results of the experiments are given as a mean of triplicate samples ± standard deviation and were analyzed according to the one way analysis of variance (ANOVA) at the level of (P < 0.05).

Results and Discussion Evaluation of Bilayer Tablet Disintegration Test All the disintegration times of the prepared formulas are shown in table (3) are within the pharmacopial requirements but the IR3 is the closest one to the standard metoprolol tartrate 50 mg conventional tablet so it was used for the further bilayer formulation. Table (3) the disintegration time of the pre pared immediate release (IR) laye r

Friability Test and Tensile Strength As shown in table (4), all the prepared formula had acceptable weight loss (not more than 1%) and high tensile strength this is mainly due to the composition of each layer. Microcrystalline cellulose used as diluent in the immediate release layer, is well known to

FFoorr mmuullaass DDiissiinnttee gg rraattiioonn ttiimmee

((mmiinn)) IIRR11 11 66±±22 IIRR22 55±±22 IIRR33 ((1111±± 11..55)) IIRR44 33±±11 IIRR55 33..55±± 00..55 IIRR66 22..55±± 00..55

SSttaannddaa rrdd ttaabb llee tt ((MMeettoopp rroolloo ll ttaa rrttrraattee 5500mmgg

ttaabblleett))

((11 00±±11 ))


25

deform in a predominantly plastic manner which is a direct result of the presence of slip planes or dislocations and is thought to be an important factor affecting the compressibility of microcrystalline cellulose (17) and result in the consolidation of granules and increased intraparticulate bonding during compaction in addition to the homogenous distribution of bonds in the compact (18). On the other hand, all the sustained release layers containing a considerable amount of the retarding polymer (20% to 60% of tablet weight) which improve the compressibility of the matrix and this elevated with increase the polymer concentration (19). The formulas (F7, F8 and F9) have lower strength than that of other formulas this may be attributed to the better capability of the granulating solvent to dissolve the polymeric support of EC (and not to HEC), thus improving the strength properties of tablets (20). Table (4) Physical prope rties of the prepared bilayered tablet

Uniformity of Dosage Units Table (4) shows that, all the prepared formulas complied with the United State pharmacopial specification which is 90-110% of metporolol content in each individual tablet(14).

Variables Affecting the Dissolution Profile of Metoprolol Bilayer Tablets The Effect of Retarding Polymer Type The release of metoprolol from formulas F2, F5 and F8 which contain (1:2) drug: polymer ratio for EC, HPMC and HEC respectively is shown in figure (1). A significant difference (P < 0.05) was found among the cumulative amount of metoprolol released (after the first hour) with time depending on the nature of each polymer. EC is hydrophobic polymer and the drug release occurs by dissolution of the active ingredient through capillaries composed of interconnecting drug particle clusters and the pore network as drug release continues, the interconnecting clusters increase the pore network through which interior drug clusters can diffuse causing eventually rapture of the matrix system and rapid release (21). HPMC and HEC are hydrophilic polymers on first contact of theirs matrix system with aqueous fluid rapid uptake of water occurs, chains gradually uncoil and extend but never form into linear coils, as the water content increases, the polymer becomes hydrated and the layer takes on the full characteristics of a gel. This is followed by retardation of water uptake by the rest of the tablet due to the formation of this gel layer. The major difference in the release profile of these two polymers due to the difference in the rate of hydration resulted from type of substitution on the cellulosic backbone (22).

Figure 1: The effect of polymer type at (1:2) drug: polyme r ratio on the cumulative release of MT at 37 ºC. The Effect of Retarding Polymer Concentration The effect of polymer concentration on metoprolol release from the bilayer tablets was studied for each type of polymers and the result illustrated in the figure (2). Formulas F1, F2 and F3 were prepared utilizing EC at ratio

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

T ime (h r )

% M

T R

elea

sed

F 2 ( E C )

F 5 ( H P M C )

F 8 ( H E C )

p H 1 .2 p H 6 .8

FFoo rrmmuullaass FFrriiaa bbiilliittyy

((%%))

TTeenn ssiillee

SSttrree nngg tthh

kkgg //ccmm 22

MMee ttoo pprroo lloo ll

ccoonn tteenntt

((%%))

FF11 00 ..4455 1133..11 9999±±44 ..55

FF22 00 ..3388 1133..33 9933±±22 ..99

FF33 00 ..2255 1155..66 9977±±55 ..99

FF44 00 ..5511 1100..44 9999±±44 ..88

FF55 00 ..4444 1122..99 9988±±44 ..22

FF66 00 ..3355 1144..33 9955±±22 ..11

FF77 00 ..9900 55 ..99 9988±±44 ..99

FF88 00 ..7799 66 ..99 9955±±44 ..44

FF99 00 ..5511 77 ..66 9933±±22 ..22

FF 1100 00 ..3377 1166..00 9955±±11 ..99

FF 1111 00 ..2255 1166..44 9988±±44 ..22

FF 1122 00 ..5577 1122..88 9922±±11 ..22

FF 1133 00 ..5500 1133..55 9966±±55

FF 1144 00 ..2200 1155..00 9933±±44 ..99

FF 1155 00 ..1155 1177..44 9922±±33 ..55


26

of 1, 2 and 3 (in respect to the metoprolol) respectively. The same ratios were used for HPMC in formulas F4, F5 and F6, and for HEC in formulas F7, F8 and F9 respectively. It appears that there is a significant difference (P < 0.05) in the release of metoprolol from matrices of EC (F 1-3), HPMC (F 4-6) and HEC (F 7-9) when the polymer concentration was increased. In case of EC the results can be attributed to the decrease in the porosity with a concomitant increase in the tortuosity of matrix (19). The most important factor affecting the rate of release from hydrophilic matrices is the drug:polymer ratio. The increase in polymer concentration causes an increase in the viscosity of the gel and formation of a gel layer with a longer diffusional path leading to a decrease in the drug release (23).

Figure 2: The effect of varying drug:plymer ratio on the cumulative release of MT at 37ºC. The Effect of Polymer-Polymer Ratio Four formulas (F10, F11, F12 and F13) were prepared by mixing hydrophobic EC with HPMC and HEC (both hydrophilic polymer) in different ratios 1:1 and 2:1 and the result shown in figure (3).As shown in the figure (2) EC alone was not able to sustained the release of metoprolol beyond 6hr even with 60% of the polymer used (as in F3) in spite of excellent granular and tablet properties this is mainly due to high solubility of the metoprolol that lead to rapid ingress of water and loss the integrity of the matrix (24). The use of hydrophilic matrix alone for extending drug release for highly water soluble drugs is restricted due to rapid diffusion of the dissolved drug through the hydrophilic gel network. For such drugs it becomes essential to include hydrophobic polymers in the matrix system (25). So the mixing of hydrophobic with hydrophilic polymer to improve the retardant ability resulting in good release profile and the tablet integrity and dimensions were conserved for the period of the test.

Figure 3: The effect of EC: Hydrophilic polyme r ratio on the on the cumulative release of MT at 37 ºC.

E C po lym e r

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8

T ime (h r)

% M

T R

ele

ase

d

F1 (1 :1 )

F2 (1 :2 )

F3 (1 :3 )

pH 1 .2 pH 6 .8

H P M C p o lym e r

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2T im e (h r)

% M

T R

ele

ase

d

F 4 ( 1: 1)

F 5 ( 1: 2)

F 6 ( 1: 3)

pH 1 .2 p H 6 .8

H E C p o lym e r

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

0 1 2 3 4 5 6 7 8 9 1 0 1 1

T im e (h r)

% M

T R

ele

as

e

F 7 (1 :1 )

F 8 (1 :2 )

F 9 (1 :3 )

p H 1.2 p H 6.8

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10 11 12

T im e (h r)

% M

T R

ele

ase

F1 0: EC :HP M C (1:1 )

F1 1: EC :HP M C (2:1 )

p H 6.8pH 1.2

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10 11 12

T im e (h r)

% M

T R

ele

ase

F 12: E C :H EC (1:1)

F 13: E C :H EC (2:1)

pH 6 .8pH 1.2


27

The Effect of Diluent Type To study the effect of diluent, F11 which contain lactose as a diluent compared with the F14 which contain microcrystalline cellulose as in figure (4). Although both lactose and microcrystalline cellulose consider as hydrophilic filler, there was retardation in the release when changing from lactose to microcrystalline cellulose (from 84% to 75% at 8hr). This may be due to the high solubility of lactose so that it will act as channeling agent, permitting a rapid ingress of dissolution medium into the matrix tablets, thus facilitating drug release (26). While the presence of a swelling, insoluble filler like microcrystalline cellulose changes the release profile due to a change in the rate of swelling at the tablet surface (27).

Figure 4: The effect of diluent type on the cumulative release of MT from formulas F11 (Lactose) and F14 (MCC) at 37 ºC. The Effect of Binder Type To study the effect of binder, F11 which contain starch as a binder compared with the F15 which contain polyvinylpyrrolidone (PVP) as in figure (5). It was seen that slight retardation in the release of metoprolol from matrix when the starch was replaced by PVP (from 84% to 77% at 8hr). It was hypothesized that this may have been resulted from a change in the properties of the ‘pseudo-gel layer’ surrounding the tablet, which controls the drug release rate through intimate mixing during hydration of the dosage form and through the formation of strong hydrogen bonds between PVP and HPMC (28). The Effect of the pH of the Dissolution Media Three tablets of F11 were running out separately in dissolution jars containing 900ml of pH 1.2, 4.6 and 6.8 the procedure was triplicated for each run test and the result of the drug released compared with that obtained normally (2 hr at pH 1.2 then at 6.8) as in

figure (6). There was no significance differences (P> 0.05) among the release of metoprolol at different pH media this mainly due to two reasons. First, the metoprolol is highly soluble drug and its solubility dose not affected by the pH (29). Second, the mechanism of the drug release is by swelling the polymer to form barrier gel that resist the diffusion and the HPMC swelling property not affected by the pH of the media (22).

Figure 5: The effect of binder type on the cumulative re le ase of MT from formulas F11 (Starch) and F15 (PVP) at 37 ºC.

Figure 6: The effect of pH variation on the total amount of metoprolol re lease at 37 ºC. Selection of the Best Formula To select the most promised formula in this study, reference product equivalent to 100 mg of metoprolol as a sustained release tablet (Metoprolol Ratiopharm® 100 sustained release) was utilized as a standard one, this was carried out using similarity factor (f 2) introduced by Moore and Flanner as shown in equation (3). A difference not exceeding 10% at any sampling time point between reference and test products may be acceptable and f 2 value of 50-100 indicates similarity in the dissolution profiles (30):

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10 11 12

Time (hr)

% M

T r

ele

ase

d

F11 (Lactose)

F14 (MCC)

pH 1.2 pH 6.8 0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10 11 12

Tim e (hr)%

MT

Rel

ase

d

F1 1 (S ta rch )

F1 5 (PVP)

pH 1.2 pH 6.8

0

10

20

30

40

50

60

70

80

90

0 1 2 3 4 5 6

T ime (hr)

% R

elea

sed

F 11 (2hr at pH 1.2 then 6.8)

F 11 (at pH 1.2)

F 11 (at pH 4.6)

F 11 (at pH 6.8)


28

1000.5n

1t

2t

Tt

Rn

11log50

2f

…… (3)

Where n is the number of dissolution time points, Rt and Tt are the reference and test dissolution values at time t. F15 (which contain EC:HPMC in ratio of 2:1 and PVP as a binder) showed higher (f 2) among all other formulas and equals to 72.3 as shown in figure(7).

Figure 7: The dissolution profile of se lected formula F15 versus reference standard (ST) with (f ¬2¬) =72.3 at 37 ºC. Swelling and Erosion Swelling and erosion had been studied to the best formula F15 and the result shown in figure (8) revealed that the tablets were swollen and retained their physical integrity till the end. Although there is an increase in both percentage water absorbed and percentage erosion of the tablet, but the percentage water absorbed was more dominant than erosion indicating that the release was biphasic mechanism mainly by diffusion than erosion(31).

Figure 8: Swelling-eroding behavior of optimize d se lected formula F15. FT-IR Studies The FT-IR spectra of metoprolol tartrate, physical mixtures and granules of immediate release and sustained release layers are shown

in figures (9) and (10). The O-H stretching vibration peak of metoprolol is seen in 3458 cm -1 and the C-N peak is seen at 2872 cm -1, N-H bending vibration at 1590 cm -1, at 1112 cm -1 the C-O stretching bond, at 1458 cm -1 the C=C, and at 1244 cm -1 the C-O stretching bond are obvious (32). Similar peaks are observed in the corresponding physical mixture and granules. Furthermore, the broadening of O-H stretching vibration peak mainly due to hydrogen bonding of the drug with the polymers and other excipients, also the absence of significant shifts in the wave numbers of the IR peaks of the granules compared with physical mixture indicates lack the possibility of interaction between metoprolol tartrate and excipients used in this bilayer tablets (16).

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9

Time (hr)

% M

T R

ele

ase

d

F15 ST

pH 1.2 pH 6.8

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

1 2 3 4 5 6

Ti me hr

% S

wel

lin

g

0.00

0.50

1.00

1.50

2.00

2.50

% E

rosi

on

Swell ing

Erosion

pH 1.2 pH 6.8


29

Figure 9: FT-IR spectra of Metoprolol, physical mixture and granules of the IR laye r. Figure 10: FT-IR spectra of Metoprolol, physical mixture and granules of the SR laye r. References 1. Remington: The science and practice of

pharmacy, 21st ed., Lippincot, Williams, & Wilkins; U.S.A, (2006), 889.

2. British Pharmacopoeia BP (CD). The stationary office, Crown copyright, London, (2009).

3. Ansel, H., Allen, L., Popovich, N., Pharmaceutical Dosage Forms and Drug Delivery Systems .8 th ed. Lippincot, Williams, & Wilkins. 2005. p. 227-260.

4. Ziyaur Rahman, Mushir Ali and Rk Khar. Design and evaluation of bilayer floating

tablets of captopril, Acta Pharm. 2006; 56: 49–57.

5. Vishnu M., Bhupendra G. and Madhabhai M., Formulation, Evaluation, and Comparison of Bilayered and Multilayered Mucoadhesive Buccal Devices of Propranolol Hydrochloride, AAPS PharmSciTech. 2007; 8 (1) Article 22: E1-E8.

6. Girish S., Devendra K. and Dhananjay M. Preparation and in vitro evaluation of bilayer and floating-bioadhesive tablets of rosiglitazone maleate, Asian Journal of Pharmaceutical Sciences. 2007; 2 (4): 161-169.

7. Bhavesh S., Surendra G. and Sanjay S., Formulation and Evaluation of Bilayer Tablet of Metoclopramide Hydrochloride and Ibuprofen, AAPS PharmSciTech. 2008; 9(3): 818-827.

8. Carla M., José M. and Joã o F. , Compressed Matrix Core Tablet as a Quick/Slow Dual-Component Delivery System Containing Ibuprofen, AAPS PharmSciTech. 2007; 8 (3) Article 76: E1-E8.

9. Lauretta M., Evelyn O. and Maria L., Formulation of biphasic release tablets containing slightly soluble drugs. Eur J Pharm Biopharm.1999; 48:37-42.

10. Betram G. Katzung, Anthony J. Trevor, Pharmacology Examination and Board Review. 8 th edition. McGraw-Hill Education. 2008. p. 85-87.

11. Martindale, The complete Drug Reference. 35 thed. .The pharmaceutical press, 2007.

12. Qureshi S., Tablet Testing. Encyclopedia of Pharmaceutical Technology. 2007; 3: 3707-3716.

13. ChuanY., Jonathan P., A comparative study of compaction properties of binary and bilayer tablets, Powder Technology. 2009; 189:285-294.

14. USP, XXX. The United States Pharmacopoeia, 2007.

15. Reynolds T., Gehrke S., Polymer erosion and drug release characterization of Hydroxypropyl methylcellulose matrices. J. Pharm. Sci. 1998; 87 (9):1115-1123.

16. Silversteine R., Webster F. and Kiemle D., Infrared Spectrometry in Spectrometric Identification of Organic Compounds. 7th edition. John Wily and Sons, Inc. 2005. p. 72-126.

17. Inman S., Briscoe B., The non-uniformity of microcrystalline cellulose bilayer tablets, Powder Technology. 2009; 188: 283–294.


30

18. Kyriakos K., Malamataris S., Compact size and mechanical strength of pharmaceutical diluents. Eur. J. Pharm. Sci. 2005; 24: 169-177.

19. Pruthvipathy R., Katikaneni P. and Upadrashta S., Ethylcellulose matrix controlled release tablets of a water-soluble drug. Int. J. pharm. 1995; 123: 119-125.

20. Holgado M., Caraballo I. and Alvarez-Fuentes J., Influence of diluents and manufacturing method on carteolol hydrochloride dissolution from ma trix tablets. Int. J. pharm. 1995; 118: 151-160.

21. Michael M., Britta S., Physicochemical properties and mechanism of drug release from ethyl cellulose matrix tablets prepared by direct compression and hot-melt extrusion. Int. J. pharm. 2004; 269: 509–522.

22. Chi L., Luigi G. Martini, T he use of hypromellose in oral drug delivery. JPP 2005, 57: 533–546.

23. Victoria M., James L., Influence of drug:Hydroxypropyl methylcellulose ratio, drug and polymer particle size and compression force on the release of diclofenac sodium fro m HPMC tablets, Journal of Controlled Release. 1999; 57: 75–85.

24. Hadi M. and Seyed A., The Release Behavior and Kinetic Evaluation of Diltiazem HCl from Various Hydrophilic and Plastic Based Matrices. Iranian Journal of Pharmaceutical Research. 2005; 3: 137-146.

25. Atul K., Ashok K., Formulation and In Vitro, In Vivo Evaluation of Extended release Matrix Tablet of Zidovudine:

Influence of Combination of Hydrophilic and Hydrophobic Matrix Formers. AAPS PharmSciTech 2006; 7 (1) Article 1 E1-E9.

26. Furlanetto S., Cirri M. and Maestrelli F., Study of formulation variables influencing the drug release rate from matrix tablets by experimental design. Eur. J. Pharm. Biopharm. 2006; 62: 77-84.

27. Ranjani V., Gurvinder S., Development of metoprolol tartrate extended-release matrix tablet formulations for regulatory policy consideration. Journal of Controlled Release.1998; 50: 247–256.

28. Ian J., Anne W., Modulation of drug release kinetics from hydroxypropyl methyl cellulose matrix tablets using polyvinyl pyrrolidone. Int. J. pharm. 2007; 337: 246–253.

29. Sandra K. and Jennifer B., Comparison of Drug Release From Metoprolol Modified Release Dosage Forms in Single Buffer versus a pH-Gradient Dissolution Test. Dissolution technologies. 2006; 13(1): 6-12.

30. Moore J., Flanner H., Mathematical comparison of curves with an emphasis on in vitro dissolution profiles. Pharm. Tech. 1996; 20(6): 64-74.

31. Jayabalan N., Srinivasan S., Bilayer Tablets of Atorvastatin Calcium and Nicotinic Acid: Formulation and Evaluation, Chem. Pharm. Bull 2008; 56(10) 1455-1458.

32. Jaleh V., Hossein F., Preparation and Characterization of Metoprolol Controlled Release Solid Dispersions. Drug Delivery. 2006; 13: 295-302.

Iraqi J Pharm Sci, Vol.19(1) 2010 In Vitro Digitonin production in Digitalis plant

31

In Vitro Effect of Cholesterol and Different Sugars on Digitonin Production in Multiplied Shoots of Digitalis purpurea L. Plant

Zainab J.Awad*,1

* Department of Pharmacognosy ,College of Pharmacy, University of Baghdad ,Baghdad, Iraq. Abstract Production of the steroidal saponin digitonin in multiplied shoots of Digitalis purpurea , (var. Excelsior Mixed) has been achieved in vitro by two experiments. In the experiment 1, shoot tips ( 1cm length ) explants from the sterilized seedlings were excised and cultured on MS medium ( Murashige and Skoog medium) supplemented with 0.5 mg/L TDZ (thidiazuron) and cholesterol at the concentrations 0.0, 0.1, 0.3, 0.5, 1.0, 1.5, 2.0 or 4.0 mg/L. After 45 day, results showed that the treatment with 0.5 mg/L TDZ and 2.0mg/L cholesterol had a positive effected on increasing the dry weight of multiplied shoots and their production of digitonin when compared with other treatments, where this treatment gave 2.98 g dry weight of multiplied shoots and digitonin at amount of 64.42 g/g dry weight, while the other studied characteristics of multiplied shoots (content the total chlorophyll, soluble sugars and starch.) also this treatment had appositive effected and was gave the following values:- (3.51 mg/g fresh weight, 5.06% ,6.09% ) respectively. After that experiment 2 was carried out . The objective of this experiment was to increase the degree of digitonin production in multiplied shoots compared with the experiment 1.Therfore in the experiment 2, we were selected supplements the best treatment of experiment 1(0.5mg /L TDZ and 2.0mg/ L cholesterol ) and supplemented to the MS medium with the sugars glucose, fructose, sucrose or maltose at the concentrations 30,50,70 or 90 g/L for each sugar. After 45 days results showed that the treatment with 0.5 mg/L TDZ, 2.0 mg/L cholesterol and 50g/L maltose was the best a compared with other treatments, where this treatment gave 4.52 g dry weight of multiplied shoots and digitonin at amount of 191.87 g/g dry weight . The content of multiplied shoots from the total chlorophyll, soluble sugars and starch also this treatment gave highest values and they are 4.97 g/g fresh weight , 5.91% and 8.30% respectively . Key words: Digitalis purpurea, Cholesterol, Sugars, Digitonin .

الخالصةن وني الديجيت الصابوني السيتروئيد لنبات الديجيتالس (Digitonin)انتاج Digitalis purpuseaفي االفرع المتضاعفة

سم 1ول تم انجازه خارج الجسم الحي بواسطة تجربتين في التجربة األولى٬ ُفصلت اطراف االفرع بط) Excelsior Mixedضرب (ُمعقمة وزرعت على وسط البادرات ال شيك وسكوك( MSمن اليه ) مرا TDZمضافًا بالتراكيز /ملغم 0.5بتركيز والكولسترول لتر

ة من 45لتر٬ بعد /ملغم 4.0او ٬2.0 ٬1.5 ٬1.0 ٬0.5 ٬0.3 ٬0.1 0.0 متالف أظهرت النتائج بان المعاملة ال TDZلتر /ملغم 0.5يومًاةً مع لتر /ملغم 2.0مع ونين مقارن واها من الديجيت ة ومحت كولسترول كانت ذات تأثير ايجابي على زيادة الوزن الجاف لألفرع المتضاعف

ة ى الديجيتونين بمقدار 2.98المعامالت االخرى٬ حيث بلغ الوزن الجاف لألفرع المتضاعف عل ة وي غم وزن /مايكروغم 64.42غم محتما الصفات االخرى التي ُدرست لال. جاف والنشاء٬ فقد كانت ا ة واها من الكلوروفيل الكلي٬ السكريات الذائب فرع المتضاعفة وهي محت

الصفات أيضاهذه المعاملة على هذه وهي حيث أعطت ذات تأثير إيجابي القيم غم وزن طري /ملغم 3.51( :أفضل ,5.06% ٬ن في األفرع . وعلى التوالي%) 6.09 ة الثانية والهدف منها هو إمكانية إحداث زيادة أكثر في إنتاج الديجيتوني بعد ذلك أجريت التجرب

لذلك اضيف الى وسط االولى٬ األولى وهي MSالمتضاعفة مقارنة مع التجربة في التجربة 0.5اضافات أفضل معاملة لتر /ملغمTDZ او مالتوز وبالتراكيز /ملغم 2.0و كلوكوز٬ فركتوز السكريات سكروز٬ و 30 ٬50 ٬70لتر كولسترول مع لكل /غم 90ا لتر

لتر /غم 50لتر كولسترول مع /ملغم ٬2.0 TDZلتر /ملغم 0.5يومًا٬ اظهرت النتائج بان المعاملة المتالفة من 45بعد . ُسكر على حده هي كانت مالتوز ة المتضاعف لألفرع جاف ال الوزن بلغ حيث 4.52األفضل٬ بمقدار جيتونين الدي مادة على محتوية 191.87غم

ة ايضًا /مايكروغم سكريات الذائبة والنشاء فقد أعطت هذه المعامل غم وزن جاف٬ أما محتوى األفرع المتضاعفة من مادة الكلورفيل٬ الى التوالي%) ٬8.30 %5.91غم وزن طري٬ /ملغم4.97: (أفضل القيم وهي .عل

Introduction Digitalis purpurea ( Fam: Scrophularaceae ) is a biennial herb plant (1). In the medicinal and pharmacy fields this plant has a great benefit because it contains the cardiac glycosides like" Digitoxin" and steroidal saponin glycosides like" Digitonin"

both of these groups representing compounds of considerable importance to the pharmaceutical industry(2). As yet, these compounds can not be synthesized economically by chemical or microbiological methods (3) .

1Corresponding author E- mail : merciful2003@ yahoo.com Received : 24/3/2009 Accepted : 9/1/2010


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In the field of pharmacy and medicine, the steroidal saponin digitonin had a great importance because it was used to determine the ratio of cholesterol in the blood, plasma and bile acids but the great importance and interest of this compound and some steroidal saponins like the Diosgenin owing to their relation ship with some important substances as progesteron hormone, cortisone and vitamin D where the structures of these steroidal saponins have the same nucleus of these substances , then these steroidal saponins can be used as a precursors for the production of these substances . Originally these substances were isolated from expensive and limit sources like adrenal cortex and bile acids of cattle (4 ,5)

For this important many studies using modern technology like tissue culture technique ( in vitro) to increasing the production of steroidal saponins in some plants . Where via the usage of this technology different explants could be cultured and inducted to different stage of growths as a source for these important medical compounds and in a high concentrations without being restricted to the environmental conditions as well as cleans of the pharmaco which could be obtained (6 ).T herefore the objective of our study using this technique to inducted the multiplication shoots of Digitalis purpurea (var exlecior mixed) and increasing their production from the digitonin compound . Material and method First :- Tissue culture of the plant Digitalis purpurea; this work went through the following steps:- 1. Preparation of sterilized seedlings : That

was, initiation a specific culture of seedlings free from any contamination which it will be a source for shoot tips and that was performed by:- A. Medium preparation : Basic

medium composition was that of MS(7) with 30g/L sucrose. The pH of this medium was adjusted to 5.5 then gelled with 8g/L Difco Bacto agar . Ten milliliters of this medium was dispensed into 25x125 ml tubes. After capped with clear autoclavable lids, tubes were autoclaved at 1210C for 20 min .

B. Seeds germination : Seeds of the plant Digitalis purpurea ( var exlecior mixed) were surface sterilized in 70% ethyl alcohol for 30 second. This was followed with three sterilize distilled rinses( 8), after that the seeds were cultured on MS

medium , and allowed to germinate by incubated in growth room at 25 2 C with artificial illumination given by fluorescent light of about 1600 lux at 16 hour / day( 9). Incubation was for30 days and then the seedlings are ready to take the explants shoot tips which excised and cultured in step 2.

2. Shoots multiplication and the production of steroidal saponin "Digitonin": These processes were inducted by two experiments:-

Experiment 1 This experiment was carried out to inducted multiplication the cultured shoot tips and their digitonin production and by the following steps:- 1. Preparation of the nutrient medium:-

Basic medium MS was used supplemented with 30g/L sucrose plus 0.5 mg/L TDZ (10) (for multiply the cultured shoot tip) and Cholesterol at a concentrations of 0.0, 0.1, 0.3 , 0.5, 1.0, 1.5, 2.0 or 4.0mg/L ( for induct the production of digitonin ). The pH of the media were adjusted to 5.5 and gelled 8g/L Difco Bacto agar. Twenty five milliliter medium was dispensed into 25x125 ml tubes. After capped with clear autoclavable, tubes were autoclaved at 121 0C for 25 min .

2. Cultured the shoot tip:- Excised the shoot tips ( 1cm length ) from the sterilized seedlings and one shoot tip in every tube was cultured, then allowed to multiplication by incubated in a growth room at the same condition of seeds germination ( 11) .

Experiment 2 This experiment was carried out after experiment 1 .The objective of this experiment was to increasing the degree of digitonin production in multiplied shoots compared with the experiment 1. Therefore we were selected supplements the best treatment of experiment 1 (0.5 mg/L TDZ and 2.0 mg/L cholesterol) and supplemented to MS medium with the sugars glucose, fructose, sucrose or maltose at a concentrations of 30,50,70 or 90 g/L for each sugar , the pH of these media and their gelled , distribution, autoclaving , cultured the shoot tip and condition the incubated in a growth room as in the experiment 1. Second :- Analysis of the multiplied shoots . After 45 days in cultures, multiplied shoots are ready for studying the following characteristics:-


33

1. Total chlorophyll content:- This characteristic was estimated according to goodwing method(12 ).

2. Dry weight estimation:- The multiplied shoots were taken out of the tubs and washed in water to remove agar from them, after wiped with clean cloth ,the shoots were separated and differentiated on filter paper to be dried in an oven at 40C until they reached constant weight (13 ), later, shoots were grinded to hard powder and kept in paper sacks in dry condition and dark place .

3. Soluble sugars and starch content:- These characteristics were estimated according to joslyn method (14 ) .

4. Extraction of the steroidal saponin "digitonin" according to the procedure of Brain et al(15 )

5. HPLC analysis of digitonin:-This analysis was carried out by using ( 16) C 18 – column and the mobile phase used was acetonitril: water (25:75). The flow rate was 1.5 ml/min .The measurements were carried out , using uv detector at 205 nm. Statistical analyses in our study were made by using complete randomized

design. The mean differences were tested utilizing LSD test. ( 17)

Results Multiplication of cultured shoot tip and yielded the highest number of shoots in the experiment 1 and 2 could be inducted by supplemented 0.5 mg/L TDZ to MS medium . In experiment 1, supplemented different concentrations of cholesterol to MS medium with o.5 mg/L TDZ , showed positive effect on digitonin production in multiplied shoots and other studied characteristics for these shoots ( total chlorophyll content, soluble sugar, starch and dry weight ) as show in table (1). But a significant e ffect was observed with the concentration 2.0 mg/L cholesterol and 0.5 mg/L TDZ. Therefore we were selected this treatment and was used in the experiment 2 with different sugars . In this experiment , all concentrations of maltose sugar had signification effect on digitonin production, but the best concentration was 50 g/L which had more effect on the production of this compound and other studied characteristics of multiplied shoots as compared with other treatments of this experiment (Table 2 ,3 ,4 and 5 ).

Table 1 : Effect of diffe rent concentrations of Cholesterol. with the presence of 0.5 mg/L TDZ on the conte nt of multiplie d shoots of Digitalis purpurea from the total chlorophyll , soluble sugars , starch , digitonin and on their dry weight.

Concentration mg/L

Total Chlorophyll mg/g fresh

weight

Soluble Sugars (%)

Starch(%) Digitonin g/g

dry weight

Dry we ight

(g)

0.0 3.31 4.36 5.11 33.13 2.11 0.1 3.39 4.44 5.14 33.60 2.09 0.3 3.33 4.42 5.31 35.71 2.13 0.5 3.47 4.54 5.24 37.56 2.36 1.0 3.49 4.61 5.66 43.49 2.41 1.5 3.47 4.88 5.84 49.46 2.71 2.0 3.51 5.06 6.09 64.42 2.98 4.0 3.48 4.41 5.86 44.40 2.31

L.S.D.5% 0.05 0.21 0.17 6.19 0.23

Table 2 : Effect of different conce ntrations of sugars with the prese nce of 0.5mg/L TDZ and 2mg/L Cholesterol on total chlorophyll content of multiplied shoots of Digitalis purpurea .

Total Chlorophyll mg/g fresh we ight. Concentration

g/L Sugars

Maltose Fructose Glucose Sucrose 4.69 3.18 3.41 3.51 30 4.97 3.85 3.51 3.90 50 4.31 3.84 3.56 3.96 70 3.60 3.61 3.15 3.42 90

0.23 L.S.D 5%


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Table 3 :- Effect of diffe re nt concentration of sugars with the presence of 0.5 mg/L TDZ and 2mg/L Chole sterol on soluble sugars and starch content of multiplie d shoots of Digitalis purpurea .

Table 4 :- Effect of diffe rent concentrations of sugars with the presence of 0.5 mg/L TDZ and 2mg/L Cholesterol on digitonin content of multiplied shoots of Digitalis purpurea .

Table 5 :- Effect of differe nt concentrations of the sugars with the prese nce of 0.5 mg/L TDZ and 2mg/L Cholesterol on the dry weight of multiplied shoots of Digitalis purpurea .

Dry weight (g) Concentration g/L Sugars


0.25 L.S.D 5% Discussion In the field of tissue culture, shoots cultures (multiplied shoots) of Digitalis plant was considered one of the main sources to obtain some medicinal compounds like the cardiac and saponin glycosides (18)

because the site of biosynthesis of these medicinal compounds is the shoots of this plant(19) for this reason , in the present study we were inducted multiplication the cultured shoot tip

and formation the shoots cultures by supplemented the growth regulator TDZ ( cytokinin – like ) to MS medium at a concentration 0.5 mg/L( 10), and because the strong effects of this growth regulator(20) , shoot cultures in our study were appeared as leaf masses consisted from a high number of small and dwarf shoots as shown in figure (1).

Soluble Sugars (%) Concentration g/L Sugars


0.30 L.S.D 5%

Starch(%) Concentration g/L Sugars


0.41 L.S.D 5%

Digitonin g/g dry weight. Concentration g/L Sugars


17.35 L.S.D 5%


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Figure 1 : Multiplication shoot tip of Digitalis purpurea (Var. excelsior Mixe d) in the treatment 0.5 mg/L TDZ and 2.0 mg/L cholesterol afte r 45 days. So it was difficult to distinguish the stem tips of these shoots then it was not possible to count their number and arrange the statistical analysis for them.Total chlorophyll content, soluble sugars and starch of the shoot cultures was studied as an indicator to degree of the changes in quantity of digitonin in these shoots, where if the content of chlorophyll was high this mean the green plastids have high degree of differentiation and that reflected positively on the ability of green plastids to provide some necessary enzymes to produce these compounds (21, 22) . As for the soluble sugars and starch , these compounds are the main source for energy and carbon element which are necessary to formation the great carbon structure of steroidal compounds (23). Thus the rich places of chlorophyll , sugars and starch will be also rich in these medicinal compounds.In experiment 1, Presence of the cholesterol with 0.5 mg/ TDZ in MS medium had effected on increasing the internal level of digitonin inside the tissue of multiplied shoots especially in the concentration 2.0 mg/L , because this material was considered as a precursor to form the digitogenin ( aglycon part of digitonin ) (24) as shown in figure (2). Also presence the cholesterol in MS medium was led to increasing the content of multiplied shoots from the total chlorophyll, soluble sugars and starch. This increasing may be due to analysis this precursor and supply of some elements for these shoots like the carbon element which is necessary to formation the chlorophyll , soluble sugars and starch (25) and reflected that positively on shoots production from the digitonin. All of these increases

reflected positively on increasing the dry weight of these shoots ecpically in the concentration 2.0 mg/L . Therefore we were selected this concentration with 0.5 mg/ L TDZ and used in the experiment 2 with different sugars. In the experiment 2, the treatment 50 g /L maltose with 2.0 mg/ L cholesterol and 0.5 mg/L TDZ significantly increased the digitonin , total chlorophyll, soluble sugars and starch in multiplied shoots and their dry weight as compared with other treatments of this experiment . The reason may be due, the maltose sugar is disaccharide and consist of two molecule of glucose . Therefore presence of this sugar in MS medium was led to increasing the number of glucose molecules inside the tissue of these shoots and that reflected positively on different bioprocesses of these shoots like: 1- increasing the supply of sugar part (glucose) which is necessary to building the digitonin where this compound consist fro m 2 molecules of glucose, 2 molecules of galctose and 1 molecule of xylose (25 ). 2 – increasing the supply of energy which is necessary to growth and development of the shoots ( 23), 3- increasing the supply of carbon element which is necessary to building the great carbon structure of chlorophyll, different sugars, starch and digitonin (5,25 ).


36

digitogenin Figure 2 : The biosynthesis of steroidal sapogenin digitoge nin(24)


37

References 1. Tyler ,V.E, Brod , L.R . and Robbers ,

J.E.1988. Pharmacognosy.9th edition .K.M. Varghese , com , Bombay.

2. Evans , F. J . and cowleg , P.S . 1972 .variation in cardenolides and sapogenins in Digitalis Purpurea during germination phytochemsitry 11.2729 – 2733 .

3. James , A.G.1999. Physiology of plants and their cells : Phytochemistry (saponins ).Pergamon press INC .New York .

4. Trease , G.E. and Evans,W.C . 1983. Pharmacognosy ( Saponins , cardio – active drug and other steroids ) . 12 th edition . Published by Bailliere Tindall a division of cassell LTd .

5. Aua Zeid , S.N.1986 . Medicinal Plants and Herbales . Dar Al Bihar . Bayrute .

6. Stafford, A. 1986. Secondary Metabolism in plant cell culture .(Eds.).Cambridge University Press . Cambridge , London , New Rochelle , melbourme , Sydney .

7. Murashige, T. and Skoog, F. 1962 .Arevised medium for rapid growth and bioassay with tobacco tissue culture . Physiol . Plant .15:473 – 497 .

8. Kubalakova , M., Spitzova , I . and Novak , F.J.1987 stability of lanatoside C content in the in vitro propagated Digitalis lanata clones . Biologia plant arum .29(1):7 – 9 .

9. Hay, F.R. , Robert , R.J. and coomber , S.A . 1997 . Development o f desiccation tolerance and longevity in seed detached capsules of foxgloves ( Digitalis purpurea ) Annals .Botany . 79 : 419 – 427 .

10. Zeinab , J.A. 2002 . The production of cardiac glycosides from the plant of zahrat al kishtiban ( Digitalis purpurea L .) by using tissue culture technique .Ph.D thesis Agric.College .Baghdad University .Iraq .

11. Coner, S.and Reinhard , E.1986 . Longterm cultivation of Digitalis lanata clones propagated in vitro : cardenolide glycosides tent of the regenerated plants . Plant Medica . 7:478 – 481 .

12. Goodwing , T.W .1976 . Chemistry and biochemistry of plant pigments , 2nded Academic press ,London , New York , sanfrencisco

13. -Brugidou ,C. ,Jacques ,M., Cosson ,L., Jarreau F.X. and ogerau , T.1988 . Growth and digoxim content in Digitalis lanata in controlled condition and natural environment .Plant medical 262 – 265 .

14. Joslyn , M.A. 1970 . Method in food analysis , physical , chemical and instrumental methods of

analysis ,2nd .Academic press , New York and London .

15. Brain , G.L. , Brain , K.R. 1976.Influence of hormonal supplementation on steroid level during callus induction from seeds of Trigonella foenum - graecum . Phytochemistry , 15: 1655 – 1660 .

16. Barnes , J. , Anderson , L.A . and Phillipson , J.D. 2002 . Herbal medicines , 2nd ed , Pharmaceutical Press ,UK .

17. Alrawi, K.M. and khalaf – Allah, A.M. 1980 . Design and analysis of agricultural experiments , Mosul University . Agric and Forest College Iraq .

18. Abdul – Mutalib , S.M . and Mubashar , S.O. 1990 . The basic conception in plant cell , Tissue and Organ Culture. University of Baghdad , Ministry of Higher Education and Scientific Research . Iraq .

19. Stuhlemmer , U., Kreis, w., Eisenbesis , M. and Reinhard , E. 1993 . Cardiac glyrosides in party submerged shoots of Digitalis lanata .Planta Media ,59 : 539 – 545 .

20. Carl , A.H. and Jhon ,E.P.1993 . Thidiazuron a potent cytokinin for woody plant culture .palnt cell tissue and organ culture .33:105 – 119 .

21. Hagimori , M ., Matsumoto , T. and Obi , Y.1982 .Studies on the production of Digitalis cardenolides by plant tissue culture .II.Effect of light and plant growth substances on digitoxin formation by undifferentiated cells and shoots – forming cultures of Digitalis Purpurea grown in light media .Plant physiol .69 : 635 – 656 .

22. Morales , C., Cusido , R., Palzon , J., Bonfill , M.and Pinol L.1999 . Digitalis Plants and tissue culture , improved conditions for cardinolide production .Plant Biology , 1:35.

23. Alrawi, A.M. and Sulieman ,R.R.1988 .Metabolitei bioefficiencies .Ministry of high education and scientific research ,Baghdad university ,Iraq .

24. Margaret, L.V. and Vickery , B.1981 .Secondary Plant metabolism . The macmillan press LTD. London and Basingstoke .

25. Mohamed, A.A.K.and Younis , M.A.1991 .Principles of Plant Physiology. V2 . ministry of higher education and scientific research .Baghdad University . . College of Agriculture .Iraq .

Iraqi J Pharm Sci, Vol.19(1) 2010 Schiff base of aminobenzoic acid

38

Synthesis, Characterization and Antibacterial Activities of Ligand Type N2O4 Schiff base and its Novel Complexes with

Co(II), Ni(II), Cu(II) and Zn(II) ions. Ahmed T. Numan* , Manhel R. Aziz*,1 and Sinaa M. Shaker**

*Department of Chemistry,College of Education, Ibn-Al-Haitham,University of Baghdad,Baghdad, Iraq. ** Microbiology, Medical City, Teaching Laboratories, Baghdad, Iraq.

Abstract The reaction of 2-amino benzoic acid with 1,2-dichloroethane under reflux in methanol and KOH as a base to gave the precursor [H4L]. The precursor under reflux and drops of CH3COOH which reacted with (2mole) from salicycaldehyde in methanol to gave a new type N2O4 ligand [H2L], this ligand was reacted with (MCl2) Where [M= Co (II), Ni(II), Cu(II) and Zn(II)] in (1:1) ratio at reflux in methanol using KOH as a base, to give complexes of the general formula [M(L)]. All compounds have been characterized by spectroscopic methods [1H NMR ( just to the ligand), FTIR, uv-vis, atomic absorption], melting point, conductivity, chloride content, as well as magnetic susceptibility measurements. From the above data, the proposed molecular structure of [Co(L)], [Ni(L)], [Cu(L)] and[Zn(L)] complexes adopting an octahedral about this metal ions. The synthesized ligand, along with their metal complexes were screened for their in vitro antibacterial activity against ten local strains of E. coli as gram-negative bacteria in addition to ten strains of Salmonella typhi and to ten strains of Acinetobacter baumannii and Ten gram- positive bacteria utilizing for locally strains of Staphylococcus aureus, were tested also using the agar diffusion technique. Keywords: Schiff base , Comple xes .

الخالصةر الليكاند ة [H2L] تضمن البحث تحضي عل تحت التصعيد داي كلورو أيثان 2,1مع أمينو حامض البنزويك 2وذلك من مفا

وم كقاعدة اذ اعطى التفاعل المـادة الوسـطية االرجاعي في الميثانول وبوج ادة [H4L] ود هيدروكسيد الصودي ـم عـل ال ومـن خـالل تفاة الثلجي ٬ اذ اعطى CH3COOHتحت التصعيد االرجاعي في الميثانول وقطرات من Salicylaldehydeمول من 2مع الوسطي

ة باستخدام الميثانول وسطاً للتفاعـل وبنسـبة (N2O2)نوع [H2L]التفاعل الليكاند زي ) 1:1(٬ ثم مفاعلة الليكاند مع بعض العناصر الفلوتاسيومو عدة بوجود هيدروكسيد الب حيث , [M(L)]حيث تكونت معقدات جديدة ذوات الصيغة العامة , وتحت التصعيد األرجاعي كقا

M= Co(II), Ni(II), Cu(II), and Zn(II) . ة التالية االشعة تحت الحمراء واالشعة فوق (شخصت جميع المركبات بالطرق الطيفية رنين النووي المغناطيسي و المرئية –البنفسجي دقيق للعناصـر مـع التوصـيلية و) )فقط لليكاند( 1HNMRوطيف ال حليل الكمي اـل الت

رح غـي المقـت ائج البحـث كـان الشـكل الفرا ن نـت الموالرية الكهربائية ومحتوى الكلور ودرجة االنصهار والحساسية المغناطيسـية ٬ ـم[Co(L)], [Ni(L)], [Cu(L)], [Zn(L)] ة لليكان.ثماني السطوح حي ولوجية خارج الخلية ال ه ضددرست الفعالية الباي عشر د ومعقدات

رام E. coliمن ال عزالت مـن وعشـر عـزالت Salmonella typhiأخـرى مـن ال و عشـر عـزالت كبكتريا سالبة لصـبغة كـAcinetobacter baumannii من ال عشر عزالتوStaphylococcus aureus ع ك ث جمـي ة لصـبغة كـرام حـي بكتريا موجـب

. األصناف أختبرت بأستخدام تقنية الحيودIntroduction Schiff bases and their coordination compounds have played a great importance in medicine, industry and biochemistry. Schiff bases are characterized by the (-N=CH-) (imine) group which imports in elucidating the mechanism of transamination rasemination reaction in biological (1,2). During the past two decades, considerable attention has been paid to the chemistry of metal complexes containing nitrogen and other donor (3). This may be attributed to their stability, biological activity (4)and potential application in many fields such as oxidation catalysis (5) and electrochemistry (6). We have already drawn attention (7-11) to the strong relationship

between metals or their complexes, and antibacterial (12-18), and anticancer (19,20) activities. In 2009 A. Thabit and co-workers reported the synthesis of a Novel ligand type N2O2 and its complexes with Co(II), Cu(II), Zn(II), and Cd(II), which have been characterized by spectroscopy and elemental analysis (21). This paper reports the synthesis and characterization of new derived from the reaction of α-amino carboxylic acid with 1,2-dichloroethane in methanol, resulted (precursor)[H4L], which reacted with salicylaldehyde and its Co(II), Ni(II), Cu(II) and Zn(II) complexes.

1Corresponding author E- mail : [email protected]@yahoo.com Received : 13/10/2009 Accepted : 24/1/2010



39

Experimental Chemicals used were analytical grades; metals were used as chloride salts. The complexes were determined by absorption technique, using Shimadza (A.A) 680 G atomic absorption spectrophotometer. I.R data were recorded as (KBr) disc using a Schimadza 4800 s FTIR spectrophotometer in the range (4000-400) cm-1 which measured at the laboratories of Ibn-Sinaa Company. 1H-NMR spectra were recorded in DMSO-d6 using Burcker 300 MHz spectrometer1 which measured at Amman - Jordan. (UV-Vis.) spectra were obtained in (MeOH) on a CECIL, CE 2700 spectrophotometer in the range (200-900) nm using quartz cell which measured at the college of Ibn- Al-Haithem. Magnetic measurements were carried out on solid compounds using 6 Bruker B.M. Melting points were recorded on an electro thermal Stuart apparatus and are not corrected. Electrical conductivity measurements of the complexes were recorded at 25C for 10 -3M solutions in (MOH) as a solvent using a Wissenchaftilich technique werksttaten, D1820 bweilheimI.F 42 conductivity meter which measured at the college of Ibn- Al-Haithem. The chloride contents for complexes was determined by potentiometric titration method on (686-titro processor-665), Dosinat-metrom Swiss, which measured at the laboratories of Ibn-Sinaa Company. Antibacterial screening was done at laboratories of medical city, Baghdad using agar diffusion technique (22,23). The ligand along with their metal complexes were screened for their in vitro antibacterial activity against gram negative bacteria (E. coli), gram-positive bacteria (Staphylococcus aureus), Salmonella typhi and Acinetobacter baumannii bacterial strains. The ligand and their complexes have shown varied antibacterial activities against one or more

bacterial strains and this activity enhanced on coordination / chelating.

Preparation of the ligand [H2L] The ligand was prepared by two steps: Step (1): A solution of α-amino carboxylic acid (0.35 g, 2.25 mmole) in methanol (10ml) was added to it dichloroethane (0.2g, 2.23mmole) , (0.2 ml) with KOH (0.18ml) as a base, the mixture was refluxed for 3 hours with stirring. Then the mixture was allowed to cool at room temperature. The resulting a gray solid (precursor) [H4L] was obtained which filtered off and then washed with ethanol. Yield (37%), (0.25g), mp (245-250 C˚ dec.). Step (2): A solution of salicalyaldehyde (0.2 g, 1.66mmole), (0.18 ml) in methanol (5 ml) was added to the precursor solution [H4L] (0.25 g, 0.83 mmole), then (8) drops of CH3COOH was added slowly to the reaction mixture. The mixture was refluxed for (5 hours) with stirring. The resulting was orange solid of [H2L] as product was filtered of and then washed with ethanol. Yield (42%), (0.18g), mp (220-226 C˚ dec.). Preparation of the ligand [H2L] with metal ions (0.15 g, 2.94 mmole) of ligand solution in methanol (10ml), with KOH as a base was added to a solution of (0.07 g, 2.94 mmole) CoCl2.6H2O in methanol (10ml), the mixture was refluxed with stirring for (4 hours). The resulting was dark brown solid as product which was filtered of and then washed by water and re-crystallized with ethanol. The complexes [Ni(L)], [Cu(L)] and[Zn(L)], were obtained in a similar method to that mentioned in the preparation of [Co(H2L)2] complex described above, (Table1) stated the quantity of starting materials and some physical properties of the prepared complexes.

Table 1 : The microanalysis results and some physical properties for the prepared ligand H2L and its complexes

Co mpounds Formula Co lour M.p(C˚) Yield% Chloride Metal M .wt

H4L C16H16N2O4 Gray 245-250 dec. 37 Nil ----- 300.31

H2L C30H24N2O6 Orange 220-226 dec. 42 Nil ----- 508.52

[Co(L)](1) C30H22N2CoO6 Dark brown 230 dec. 60 Nil 10.42 (9.18)

565.44

[Ni(L)](2) C30H22N2NiO6 Yellowish green 292 dec. 78 Nil 10.38 (11.18)

565.20

[Cu(L)](3) C30H22N2CuO6 Dark green 320 dec. 52 Nil 11.15 (11.37)

570.05

[Zn(L)](4) C30H22N2ZnO6 Light green 230 dec. 53 Nil 11.43 (12.44)

571.90


40

N H 2

C O O H

+ C l C H 2 C H 2 C l2 K O H

M e t h a n o l

R e f l u x3 h o u r s

2 - a m in o b e n z o i c a c i d d i c h lo r o e th a n e

H 2 N

C

O O

C H 2H 2 C

OC

O

N H 2

+2

C H

O H

O

2 - h y d r o x y b e n z a l d e h y d e

e t h a n e - 1 , 2 -d i y l b i s (2 - a m i n o b e n z o a t e )

N

C

O O

C H 2H 2 C

OC

O

N

C H H C

H OO H

( Z ) - e th a n e - 1 , 2 - d i y l b i s ( 2 - ( ( Z ) - 2 - h y d r o x y b e n z y li d e n e a m in o ) b e n z o a t e )


M e th a n o l

C H 3 C O O H8 d r o p s

+ M C l 2

N

C

O O

C H 2H 2 C

OC

O

N

C H H C

OO

M

K O H

M e t h a n o l


Results and Discussion Synthesis of the ligand The ligand [H2L] was prepared according to the general method shown in Scheme (1). The I.R spectral of the precursor [H4L] of the ligand [H2L], is shown in Fig (1), the results were summarized in Table (2) The figure exhibited band at (3329,3379) cm-1 which attributed to the stretching vibration of asymmetric and symmetric (N-H) for NH2 group, also the spectrum was showed bands at (1612)cm-1, (1249) cm-1 and (1192) cm-1 attributed to υ (C=O) of ester group, υ (C-O) ester group and υ (C-O) phenolic respectively (21,24-27) , By comparing with the I.R spectrum of the ligand [H2L], Fig (2), Table (2) exhibited bands (3421) cm-1, which can be attributed to υ (O-H) and (disappeared NH2 groups), also the spectrum showed bands at (1612)cm-1, (1608) cm-1, (1273) cm-1 and (1239) cm-1 attributed to υ (C=O) ester group, υ (C=N) imine group, υ (C-O) ester group and υ (C-O) phenolic respectively [21,24-27]. The (U.V-Vis) spectra for precursor [H4L], Fig (3), the results were summarized in Table (3), the figure exhibits two high intense absorption peaks at (248) nm εmax (2532) molar-1cm-1, and (319) nm εmax (2484) molar-1cm-1, which

assigned to (π→π*), and (n→π*) transition respectively [28,29], While The (U.V-Vis) spectrum of the ligand [H2L] Fig (4), (Table3) exhibits three high intense absorption peaks at (250) nm εmax (2631) molar-1cm-1, (293) nm εmax (2660) molar-1cm-1, and (360) nm εmax (2581) molar-1cm-1, which assigned to (π→π*), (π→π*) and (n→π*) transition respectively (28,29). The 1H NMR spectrum of the ligand (H2L), in DMSO-d6, Fig (5) shows proton of (O–H) group (ph–OH) which appears as a singlet peak signal at (10.2) ppm. The proton of (C–H) imine group appears as a singlet peak at (8.7) ppm. The multiples signals peaks at the range between (7-8)ppm, are due to aromatic hydrogen of carbon for the benzene ring which bonded with (C=O) carbonyl group, while the multiples signals peaks at the range between (6-7)ppm, are due to aromatic hydrogen of carbon for the benzene ring which bonded with (C=N) imine group, also the spectrum of the ligand appears a triplet peak at (4.7)ppm, which assigned to (-CH2-) methelene group, as soon as a singlet high peak at (2.5)pmm for the DMSO-d6 solvent [24,25,30,31].

Where M= Co(II),Ni(II),Cu(II),and Zn(II) Scheme 1 : The preparation of the ligand [H2L] and its complexes


41

Table 2 : Infrared spectral data(wave number ύ) cm-1 for the ligand H2L and its complexes

Figure 1: Infrare d spectrum of the precursor (H4L)

Figure 2 : Infrare d spectrum of the ligand (H2L)

Figure 3 : Electronic spectrum of the precursor (H4L)

Compound υ(C=N) υ(C=O) ester

υ(C-O) ester

υ(C -O) phenolic

υ(O-H) υ(M-O) phenolic

υ(M-O) esteric

υ(M-N)

H4L

----- 1612 1249 1192 ----- ---- ----- -----

H2L 1608 1612 1273 1239 3421 ----- ----- ----- [Co(L)](1) 1593 1612 1327 1300 ----- 505 461 416 [Ni (L)](2) 1593 1612 1327 1300 ------ 536 440 430 [Cu (L)](3) 1585 1609 1319 1288 ------ 540 468 428 [Zn (L)](4) 1593 1611 1327 1300 ----- 516 459 445


42

Table 3 : Electronic spectral data for the ligand H2L and its comple xes

C.T= Charge transfer

Figure 4: Electronic spectrum of the ligand (H2L)

Figure 5: 1H NMR spectrum of the ligand (H2L)

Compound

Λ nm

εmax molar-1cm-1

Assignment

Ratio

Mo lar conductivity S.cm2.mo l-1

Magnetic susceptibility

B.M

coordination

H4L

248.9 319.7

2532 2484

π→π* n→π*

-----

------

------

-------

H2L 250.0 293.6 360.8

2631 2660 2581

π→π* π→π* n→π*

-----

-------

------

------

[Co(L)](1) 248.5 295.6 414.0 629.8

2642 2531 1264 96

Ligand field Ligand field

C.T 4T1g(F)→4T2g(F)

neutral

18.1

3.87

(4.15)

Octahedral

[Ni(L)](2) 245.0 305.3 400.1 580.0

2597 1617 1317 62


C.T 3A2(g)→3T 1g (p)

neutral

14.9

2.83 (3.2)

Octahedral

[Cu(L)](3) 243.5 300.0 406.1 698.9

2608 2551 2636 52


C.T 2E →2T 2

neutral

12.46

1.7 (1.9)

Octahedral

[Zn(L)](4) 220.3 289.0 388.5

1483 584 371


C.T

neutral 13.65

------ Octahedral


43

Synthesis of the complexes The reaction the of ligand [H2L] with Co(II), Ni(II), Cu(II) and Zn(II) was carried out in MeOH. These complexes are stable in solution. The analytical and physical data, Table (1) and spectral data, Table (2) and Table (3) are compatible with the suggested structure Scheme (1). The I.R spectra of the complexes [Co(L)](1), [Ni(L)](2), [Cu(L)](3), and [Zn(L)](4). Fig (6) and Fig (7), respectively, the results were summarized in Table (2), the figure exhibited at (1608) cm-1 in the free ligand spectrum which assigned to υ (C=N) imine group Shifted to lower frequency and appeared at (1593) cm-1, (1593) cm-1, (1585) cm-1 and (1593) cm-1 for the complexes (1),(2),(3), and (4) respectively (25-28). These bands were assigned the υ (C=N) stretches of reduced bond order, this can be attributed to the delocalization of metal-electron density into the ligand π-system (HOMO→LUMO) [32,33]. (HOMO=Highest occupied molecular orbital, LUMO= Lowest unoccupied molecular orbital). The phenolic (C-O) stretching vibration appeared at (1239) cm-1 in the free ligand was Shifted to higher frequency and appeared at (1300) cm-1, (1300) cm-1, (1288) cm-1, and (1300) cm-1 for the complexes (1), (2), (3) and (4) respectively, as well as ester group (C-O) stretching vibration appeared at (1273) cm-1 in the free ligand was shifted to higher frequency too, and appeared at (1327) cm-1, (1327) cm-1, (1319) cm-1 and (1327) cm-1 for the complexes (1), (2), (3) and (4) respectively, all that indicated a linkage between oxygen of phenolic group and oxygen of ester group and the metal (24,32,34). The spectra showed the appearance of bands at (416) cm-1, (430) cm-1, (428) cm-1 and (445) cm-1 refer to υ (M-N) for complexes (1), (2), (3) and (4) respectively, These bands confirm the coordination of the nitrogen atom to the metal center, while the bands at [(505),(461)] cm-1, [(536),(440)] cm-1 and [(540),(468)] cm-1 [(516),(459)] cm-1assigned to υ (M-O) of complexes (1),(2),(3), and (4) respectively,

Theses bands indicating the phenolic and esteric oxygen in the ligand is involved the coordination with metal ions in complexes [34-37]. The (U.V-Vis) spectra for the complexes are shown in Fig (8) and Fig (9), Table(3), Complex [Co(L)]: showed two high intense peak at (248) nm εmax (2642) molar-1cm-1 and (295) nm εmax (2531) molar-1cm-1 were assigned to the ligand field, while a medium intense peak at (414) nm εmax (1264) molar-

1cm-1 was assigned to the charge transfer (C.T), a weak broad peak at (629) nm εmax (96) molar-1cm-1 was assigned to(d-d)electronic transition (4T1g(F)→4T2g(F)) suggesting octrahedral geometry(29). Comple x[Ni(L)]: showed two high intense absorption peaks at (245) nm εmax (2597) molar-1cm-1 and (305) nm εmax (1617) molar-1cm-1 are due to the ligand field, another high intense peak at (400) nm εmax (1317) molar-1cm-1 was assigned to (C.T), while a weak broad peak at (580)nm εmax (62) molar-1cm-1 was assigned to (d-d) electronic transition (3A2(g)→3T1g(p)) suggesting octahedral geometry (29). Complex[Cu(L)]: showed two high intense absorption peaks at (243) nm εmax (2608) molar-1cm-1 and (300) nm εmax (2551) molar-1cm-1 are due to the ligand field. a high intense absorption peak at (406) nm εmax (2636) molar-1cm-1 was assigned to (C.T), while a weak broad peak at (698) nm εmax (52) molar-1cm-1 was assigned to (d-d) electronic transition ( 2E →2T2 ) suggesting octahedral geometry(29). Complex[Zn(L)]: showed two peaks at (220) nm εmax (1483) molar-1cm-1 and (289) nm εmax (584) molar-1cm-1 are due to the ligand field. while a weak broad peak at (388) nm εmax (371) molar-1cm-1 was assigned to (C.T), the d10 configuration of ZnII ion along with the data obtained confirms a octahedral structure around the ion (29). The molar conductance of the complexes in methanol lie in the range(12.46-18.1 Ohm-1cm-2mol-1), Table(3), indicting their non-electrolyte having molar ratio of metal:ligand as 1:1 (38). The magnetic moments for the complexes are shown in Table (3) (39).


44

Figure 6: Infrared spectrum of the complex [Co(L)](1)

Figure 7: Infrare d spectrum of the complex [Ni(L)](2)

Figure 8: Electronic spectrum of the ligand [Co(L)](1)

Figure 9: Electronic spectrum of the ligand [Ni(L)](2) Biological activity The antibacterial activity of the synthesized ligand [H2L] and its complexes [Co(L)](1), [Ni(L)](2) , [Cu(L)](3) , and [Zn(L)](4) Table (4, 5, 6 and7), Fig(10 and 11) were tested utilizing the agar diffusion

technique (40). The organisms tested were Staphylococcus aureus , E. collie , Salmonella typhi , and Acinetobacter baumannii. The agar media (Muller-Hinton agar) were inoculated with test organisms and a solution of the tested compound (100μg/ml) (41), was placed


45

separately in cups (6 mm diameter) in the agar medium. The inhibition zones were measured after 24 hours incubation at 35 C .̊ Separate studies were carried out with the solution alone of DMSO and the showed no activity against any bacterial strains (41). The results of these studies revealed that metals complexes showed an effective in the inhibition of Acinetobacter baumannii, Table (4). The ligand and (Co+2, Ni+2, Cu+2,Zn+2) complexes were showed an inhibition in some strains in each of Staphylococcus , E. coli, and Salmonella typhi, as shown in Table (5, 6, and 7) . Biological activity of the previous compounds in inhibition of bacterial growth could be attributed to one of the following mechanisms, the first mechanism is by the inhibition of the bacterial cell wall synthesis by bounding to the precursor of the cell wall (42), second

mechanism revealed that some antibodies have similar stereo structure to substrate (D-alanyl D-alanine). So it will act competitive inhibitions for the enzymes (transpeptidase and /or carboxpeptidase) which are the main enzymes catalyzed the end step in the biosynthesis of peptidoglycans of the bacterial cell wall (43). Other mechanisms could contributed to the results found in the study which include the inhibition of biosynthesis of bacterial proteins by linking to the ribosoms by doing so, the ribosomes will not be in contact with tRNA, so the bacteria will not survive (44). An other mechanisms were postulated that some antibodies inhibit the denovo synthesis of bacterial DNA by splitting DNA in DNA-enzyme complexes by inhibition DNA ligase (45).

Table 4 : Biological activity of Acinetobacter baumannii bacteria of the ligand [H2L] and its complexes

A= Acinetobacter baumannii bacteria

Table 5 : Biological activity of Staphylococcus aureus bacteria of the ligand [H2L] and its complexes S= Staphylococcus aureus bacteria Table 6 : Biological activity of E. coli bacteria of the ligand [H2L] and its complexes

E= E. coli bacteria Table 7 : Biological activity of Salmonella typhi of the ligand [H2L] and its complexes

Compound

Bacteria (zone of inhibition (diameter mm))

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10

H2L 14 11 Nil 14 16 14 15 12 13 16

[Co(L)](1) 15 10 Nil 15 14 15 12 Nil 16 15

[Ni(L)](2) 14 10 12 14 13 16 13 12 15 15 [Cu(L)](3) 14 10 11 12 13 12 13 13 12 13 [Zn(L)](4) 11 11 Nil 14 13 14 14 12 15 16

Compound


S1 S2 S3 S4 S5 S6 S7 S8 S9 S10

H2L Nil Nil 16 Nil Nil Nil Nil Nil Nil Nil

[Co(L)](1) Nil Nil 16 Nil Nil Nil Nil Nil Nil Nil

[Ni(L)](2) Nil Nil 16 Nil Nil Nil Nil Nil Nil Nil [Cu(L)](3) 9 8 17 9 10 9 8 9 8 11 [Zn(L)](4) Nil Nil 18 12 Nil Nil 12 Nil Nil Nil

Compound


E1 E2 E3 E4 E5 E6 E7 E8 E9 E10

H2L Nil Nil Nil 12 12 10 Nil 11 Nil 10

[Co(L)](1) 9 Nil Nil 10 7 10 Nil Nil Nil 10

[Ni(L)](2) 10 Nil Nil 11 8 12 Nil 10 Nil 8 [Cu(L)](3) Nil Nil Nil 12 8 12 Nil 10 Nil 10 [Zn(L)](4) Nil Nil Nil 8 8 8 Nil 10 Nil Nil

Compound


Sal1 Sal 2 Sal 3 Sal 4 Sal 5 Sal 6 Sal 7 Sal 8 Sal 9 Sal10


46

Sal= Salmonella typhi bacteria

Figure 10: Inhibition zones for Acinetobacter baumannii utilizing agar diffus ion technique

Figure 11: Inhibition zones for Staphylococcus aureus utilizing agar diffus ion technique

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[Ni(L)](2) Nil Nil 10 Nil Nil Nil Nil Nil Nil Nil [Cu(L)](3) Nil Nil Nil Nil Nil Nil 12 9 Nil Nil [Zn(L)](4) 10 Nil Nil Nil Nil Nil Nil Nil Nil Nil


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Iraqi J Pharm Sci, Vol.19(1) 2010 Detection of flavonoids in Ammi L. species

48

Phytochemical Study of some Flavonoids Present in the Fruits of Two Ammi L. Species Wildly Grown in Iraq

Thukaa Z. Abdul-Jalil * , Kawkab Saour **,1 and Abdul- MutalibA. Nasser***

*Department of Pharmacognosy , College of Pharmacy , University of Baghdad , Baghdad , Iraq . ** Department of Pharmaceutical Chemistry, College of Pharmacy,University of Baghdad , Baghdad, Iraq . *** Department of Pharmacognosy , Baghdad College of Pharmacy , Baghdad , Iraq.

Abstract Ammi species belong to the family Umbellifereae that provide a host of bioactive compounds (mainly coumarins and flavonoids) of important biological activities, like prevention and treatment of heart and vascular disease and some types of cancer. Literature survey revealed that there was no study concerning Ammi flavonoids in Iraq. Ammi majus and Ammi visnaga , which are wildly grown in Iraq, were chosen for this study. This study concerned with extraction, identification, isolation, and purification of some biologically important flavonols quercetin and kaempferol from the fruits of Ammi majus and Ammi visnaga. Extraction of these flavonols was carried out using 85% methanol and 90% ethanol. Identification of these flavonols quercetin and kaempferol was done using thin Layer chromatography (TLC) where different solvent systems had been tried. Ultra violet (UV) Light and iodine vapor where used for detection. This identification was further augmented by using high performance Liquid chromatography (HPLC) and then these flavonols were isolated and purified. The most suitable extraction, isolation and purification procedures of flavonols were fully described in this study. The identification of isolated flavonols (quercetin & kaempferol) was carried out using melting point (M.P.), Thin Layer chromatography (TLC), and infrared spectroscopy (IR).This study confirms the presence of quercetin and kaempferol in Ammi majus & an Ammi visnaga fruit, the percentage of quercetin was higher in Ammi visnaga than Ammi majus , while the percentage of kaempferol was higher in Ammi majus than Ammi visnaga . Key words : Ammi majus , Ammi visnaga , que rcetin , kaempferol .

الخــالصـةة المظلية عائل ة من ال حتوي على الكثير من المركبات ) Umbellifereae(جنس الخل ذات ) والفالفينويداتمنها الكومارين (و هو ي

واع السـرطانات والشرايين وبعض ان وقاية وعالج امراض القلب ة االحيائية التي تستخدم لل حـول . الفعالي رآ لعـدم وجـود دراسـات ـظ ونق خلة في العرا ويدات في نبات ال ة الشيطاني ,الفالفين خل ة ومنها ال ) .Ammi majus L(لذلك اصبح من االهمية دراسة بعض اصناف الخل

ي العـراق ) .Ammi visnaga Lam(الخلة البلدي و آ ـف ة بعـض .التي تنمو برـي سـتخالص وكشـف وفصـل وتنقـي م ا ذه الدراسـة ـت ي ـه ـفرالنوع ة وهما الكورستين والكامبفيرول من ثما ويدات المهمة من الناحية االحيائي ) Ammi visnaga(والنوع )Ammi majus(الفالفين

ة % ۸٥العضوي ميثانول بنسبة حيث تم االستخالص باستخدام المذيب م الكشـف عـن %. ۹۰ومن ثم المذيب العضوي ايثانول بنسـب ـتويدات والكامبفيرول(الفالفين ن ة كوسيط ناقل والكشف ) الكورستي ة الرقيقة ٬ باستخدام مذيبات مختلف ة كروماتوغرافيا الطبق باستخدام تقني

ة وبخار االيودين٬وكذ ة الفصـل عنها باستخدام االشعة فوق البنفسجي وبعـدها تمـت عملـي ة ي السـائل ا االداء العـال لك تقنية كروماتوغرافـية ة . والتنقي ة متكامـل ة وشرحها بطريق ة لالستخالص والفصل والتنقي سة اختيار الطريقة المناسب وكـذلك اسـتخدمت . كما تم في هذه الدرا

حقيق من نوعية المركبات المفصولة و الك(مجموعة من التقنيات للت ن درجة االنصهار : ودرجة نقاوتها والتي شملت ) امبفيرولكورستيراء ت الحمـ ـح رقيقة وكذلك مطياف االشـعة ت ة وكروماتوغرافيا الطبقة ال دات .للمركبات المفصول ة وجـود الفالفينوـي ت هـذه الدراسـ اثبـت

ي العـراق ) الكورستين والكامبفيرول ( و الخلة البلدي التي تنمو بريا ـف ة الشيطاني ظهـرت هـ . في ثمار الخل ة ذكمـا أ ن كمـي سـة أ ه الدراة البلدي خل ة الشيطاني من ال ما الكامبفيرول فأن كميته أكثر في الخل ة الشيطاني أ خل .الكورستين كانت أكثر في الخلة البلدي من ال

Introduction Ammi is a genus of 3-6 species of flowering plants in the Apiaceae Family; They are native in southern Europe, northen Africa and south west Asia.(1,2) Ammi majus and Ammi visnaga are one of the most important medicinal plant species in the world ( figure 1 , figure 2 ).(3) In Iraq Ammi majus

usually found in fields and gardens and by the side of channels, often as weed of cultivation. It's collected from Kut, Baghdad, Hawija and many other areas. While Ammi visnaga are widely distributed in Erbil, Mousl, Baghdad, Sulaimania and Kirkuk in north of Iraq.(4)

1Corresponding author E- mail : [email protected]@yahoo.com Received : 13/10/2009 Accepted : 13/2 / 2010



49

Figure (1) photography of Ammi majus

figure (2) photography of Ammi visnaga Epidemiological data, clinical investigations, and animal studies provide strong evidence that the main active constituents of this genus are: Coumarin and their derivatives, flavonoids, volatile oil and fixed oil.(5) All the flavonoids described in Ammi species can be classified into flavonols (quercetin , kaempferol , isorhamnetine ) and flavones (apigenin , luteolin , chrysoeriol ) these types of flavonoid have been show to be powerful antioxidant and free radical scavengers. (6-

8)Among these flavonoids : quercetin which is a flavonol type of flavonoids that has been shown to help prevent the development of a variety of condition related to inflammation and free radical damage, including arthritis, allergies, macular degeneration, heart disease, gout, and various forms of cancer.(9,10)The other flavonol found in these plants is the kaempferol, which is a strong antioxidant and help to prevent oxidative damage of our cells, lipids and Deoxyribonucleic acid (DNA). It seems to prevent arteriosclerosis by inhibiting the oxidation of low density lipoprotein and the formation of platelets in the blood. Studies have also confirmed that Kaempferol acts a chemopreventive agent which means that it inhibits the formation of cancer cell (9,11)

Materials and methods A. Plant materials The plant materials (dried ripe fruits) of Ammi majus L. (Apiaceae) was collected during the months of March and April from local fields about 2 Km south of Kut. While the fruits of Ammi visnaga (Apiaceae) were collected from the botany garden in the College of Pharmacy, Uuniversity of Baghdad. (Pharmacognosy department). Both of them were identified by the department of pharmacognosy, College of Pharmacy, University of Baghdad and authenticated by National Iraqi Herbarium, Botany Directorate at Abu – Ghraib. A 50 gm of powdered fruits of Ammi majus and Ammi visnaga were packed in a thimble of soxhlet extractors. 500 ml of petroleum ether (b.p 40 – 60 c0) was used in a soxhlet extractor for three hours to get rid of lipids and fat.(9) The defatted powdered fruits after drying over night were extracted with 500 ml of 85% methanol for 12 hours by use of soxhlet apparatus for flavonoids extraction as free and glycosides.(6,7)The methanolic extract was then filtered and then a portion had been taken and kept aside for further work. The remaining portion of filtrate was evaporated under reduced pressure at a temperature not exceeding 40 °C (F1).The methanolic extract (F1) residue was weighted and subjected for identification and purification procedures. To continue the extraction process, the previously extracted plant materials (fruits) of both Ammi majus and Ammi visnaga were dried at room temperature, and then were extracted again with 500 ml of 90 % ethanol for 12 hours by use of reflux apparatus for extracting the remaining flavonoids.(6,7). The contents of the flask were filtered while hot and the ethanolic extract was allowed to cool at room temperature. A portion of ethanolic extract had been taken and kept aside for further work. The remaining portion of ethanolic extract was concentrated under reduced pressure to dryness (F2) and the dried extract (F2) was weighted and subjected for identification and purification procedures.Later on, equal volumes of methanolic extract and ethanolic extract of both Ammi majus and Ammi visnaga were mixed together to give methanolic ethanolic extract which also concentrated under reduced pressure to dryness (F3). The dry extract (F3) was weighted and subjected for identification and purification procedures.Figure (3 and 4) show Schematic procedure for the extraction method.


50

50 gm of powdered fruits of Ammi majus Soxhelt with petroleum ether (b.p 40 – 60 c 0) for 3 hours Filtrate Marc defatted fruits No flavonoid appear after test Soxhelt with 85 % methanol for 12 hours Marc (fruits) Filtrate

Reflux with 90 % ethanol for 12 hours Evaporation to dryness (F1) Filter hot Filtrate Marc Evaporation to dryness (F2) Figure 3 : Sche matic procedure for the extraction method of flavonoids from Ammi majus fruits. 50 gm of powdered fruits of Ammi visnaga Soxhelt with petroleum ether (b.p 40 -60 c 0) for 3 hours Filtrate Marc defatted fruits No flavonoid appear after test Soxhelt with 85% methanol for 12 hours Marc (fruits) Filtrate Reflux with 90 % ethanol For 12 hours Evaporation to Dryness (F1) Filter hot Filtrate Marc Evaporation to dryness (F2) Figure 4 : Sche matic procedure for the extraction method of flavonoids from Ammi visnaga

fruits.


51

B. Identification of flavonoid Identification of F1,F2,F3 were carried by thin Layer chromatography (TLC) using a ready made aluminum plates of silica gel GF254, two different detection methods, first by using UV light wave length 254 nm and 366 nm, second by using iodine vapor in the jar,in comparison with three different solvent systems S1,S2,S3. Standard flavonoids: Quercetin (FLUKA-Austia) Kaempferol (Sigma-Aldrich,USA) Different developing solvent systems that were:(12-15) S1= chloroform:Aceton:Formic acid(75: 16.5 :

8.5 ) S2 = chloroform: methanol (90:10) S3 = toluene: chloroform: Aceton (40: 25: 35) C. Isolation and purification of quercetin and

kaempferol After locating of quercetin and kaempferol of the extract in comparison with standards, preparative thin layer chromatograghy was done to isolate and purify them. The portion of mixture methanolic-ethanolic extract (F3) was used to obtain the final product by applying it as a concentrated

solution in arrow of spots using capillary tube and the standard sample was applied in one side of the plate. the mobile phase used was S1 = chloroform : Aceton : formic acid ( 75 : 16.5 : 8.5 ) the separated compound appear as a band identified using u.v light detection method.The band corresponding to the standard was scrapped out and collected in a beaker and eluted with gentle heating and filtered. Then the filtrate was evaporated to dryness under reduced pressure to give yellow precipitate. The precipitate then recrystallized using hot ethanol and maintained for TLC and measuring melting point and Infra red spectrum. D. Qualitative and quantitative estimation of

flavonoid using HPLC technique Qualitative and quantitative estimations of quercetin and kaempferol were done by using Knauer/Germany High Performance Liquid Chromatography (HPLC) in which identifications were made by comparism of retention time obtained at identical chromatographic conditions of analyzed samples and authentic standards.The HPLC conditions are listed in the following table (1):

Table 1 : HPLC Conditions (16)

Sample Mobile phase Column Flow rate Detection

Quercetin

Acetonitrile: methanol : glacial acetic acid

(70:30: 0.1)

C18 5 mm x 150 mm

0.5 ml / min

UV. Detector at λ 306 nm

Kaempferol

methanol : water ( 7.5 : 92.5 )

C18 ODS 1.5

ml / min UV. detector at

λ 308 nm

Results and Discussion Three extraction portions were obtained from the experimental work in which methanolic extract (F1), ethanolic extract (F2) and the third extract portion, which is a mixture of methanolic – ethanolic extract (F3).

Results showed that the third extract portion was the best, because the amount of both extract and flavonoid were higher than the two other extract portions. As shown in (table 2).

Table 2 : Percentages of extract and flovonoids (que rcetin, and kae mpferol) in the fruits of Ammi majus and Ammi visnaga.

Plant Ammi majus Ammi visnaga Extraction portions F1 F2 F3 F1 F2 F3

Percentage of extract 8.5 4.8 10.0 9.2 6.0 11.2

Percentage of quercetin 0.019 Traces 0.036 0.020 Traces 0.042

Percentage of kaempferol 0.025 Traces 0.045 0.018 Traces 0.035


52

Identification of Flavonoids by TLC TLC of the extracts (F1,F2,F3) obtained from dried ripe fruits of Ammi majus and Ammi visnaga , confirms the presence of

quercetin and kaempferol in all extraction portions in comparison with standards. As represented in table (3) and figure (5) .

Table 3 : showed the Rf values of flavonoid (quercetin and kaempfe rol) and their standards in different developing solvent systems in TLC.

Solvent system S1 S2 S3

Rf value of standard Quercetin 0.4 0.45 0.78 Rf value of quercetin in Ammi majus 0.38 0.43 0.76 Rf value of quercetin in Ammi visnaga 0.39 0.44 0.77 Rf value of standard kaempferol 0.52 0.6 0.85 Rf value of kaempferol in Ammi majus 0.51 0.61 0.84 Rf value of kaempferol in Ammi visnaga 0.49 0.59 0.86

MEe Q K MEe MEe Q K MEe A.V. A.M. A.V. A.M.

( 1 ) ( 2 ) MEe Q K MEe A.V. A.M. ( 3 ) Figure 5: TLC of fruits extracts of Ammi majus and Ammi visnaga obtained by extraction method using silica gel GF254 as adsorbe nt and (S1)as a mobile phase. Detection by UV-light at (1) 254 nm , (2) 366 nm , (3) iodine vapor. ( A.M: Ammi majus , K : kaempferol standard, MEe: methanolic – ethanolic extract, A.V: Ammi visnaga , Q: quercetin standard )


53

Isolation and Quantitative determination of quercetin and Kaempferol by preparative TLC From the investigation of the fruits extracts (fractions) of Ammi majus and Ammi visnaga on TLC plates, it was found that quercetin present in both fruits extracts but higher in Ammi visnaga while kaempferol also present in both fruits extracts but higher in Ammi majus. The percentage of both quercetin and kaempferol were obtained by weighing of the isolated compounds as shown in table (4) Table 4 : Percentages of quercetin and

kaempferol prese nt in the fruits of Ammi majus and Ammi visnaga.

Plant fruits (total)

Ammi majus Ammi visnaga

Quercetin

0.036 % 0.042 %

Kaempferol

0.045 % 0.035 %

Characterization of the isolated kaempferol and quercetin

TLC Both isolated compounds appeared as a single spot having the same color and Rf value as that of reference standards. Measuring melting points The isolated compounds were identified to be quercetin and kaempferol from their sharp melting point. Since one of these compound showed a melting point of 314 – 316 c 0 compared to quercetin melting point 316 c0. The other compound showed a melting point of 275 – 276 c0 compared to melting point 276 -278 c0 for standard kaempferol. IR. The IR spectra of each isolated quercetin and kaempferol were recorded as KBr disc using IR spectra photometer BUCK scientific model 500, gave identical results were compared with authentic standard samples; which confirm that our isolated compounds are quercetin and kaempferol,(17) as shown in figures (6-7).

Figure 6 : IR Spectrum of isolate d Kae mpferol

O

OH

OH

OH

HO

O

K ampf


54

Figure 7 : IR Spectrum of isolated Quercetin HPLC analysis

Generally, the percentage of quercetin and kaempferol is higher in methanolic – ethanolic extract than in methanolic extract and the methanolic extract contains higher amount of quercetin and kaempferol than ethanolic extract. In addition, Ammi majus had shown different percentages of quercetin and kaempferol than Ammi visnaga. Since the percentage of quercetin in Ammi majus was lower than the percentage of quercetin in

Ammi visnaga in all extracts. While the percentage of kaempferol in all Ammi majus extracts was higher than the percentage of kaempferol in Ammi visnaga extracts.The result indicates that the HPLC method was efficient for qualitative identification and quantitative determination of quercetin and kaempferol. as show in table (5) and figures (8-13).

Table 5 : Perce ntage of flavonols in Ammi majus and Ammi visnaga.

Extraction solve nts

Percentage of the que rcetin in the plant fruits.

Pe rcentage of the kaempfe rol in the plant fruits.

methanolic extract of Ammi majus (F1)

0.026 0.037

methanolic extract of Ammi visnaga (F1)

0.033 0.025

ethanolic extract of Ammi majus (F2)

0.011 0.018

ethanolic extract of Ammi visnaga (F2)

0.015 0.012

methanolic – ethanolic extract of Ammi majus (F3)

0.045 0.052

methanolic – ethanolic extract of Ammi visnaga (F3)

0.050 0.043

O

OH

OH

OH

OH

HO

O

Querc


55

Figure 8 :HPLC analysis of kae mpfe rol standard

Figure 9 : HPLC analysis of methanolic-e thanolic extract of Ammi majus

Figure 10 : HPLC analysis of methanolic-ethanolic extract of Ammi visnaga


56

Figure 11: HPLC analysis of Quercetin standard

Figure 12 : HPLC analysis of methanolic-e thanolic extract of Ammi majus

Figure 13 : HPLC analysis of methanolic-e thanolic extract of Ammi visnaga


57

Conclusions Phytochemical investigation of Ammi majus and Ammi visnaga fruits, grown in Iraq revealed the presence of important group of medicinal natural products belong to flavonoid derivatives. Quercetin and kaempferol were isolated and identified in Ammi majus and Ammi visnaga fruits by using simple and reproducible TLC and HPLC method. The flavonoid, quercetin and kaempferol are found in the fruits of Amm majus and Ammi visnaga, were quercetin present in large quantities in the fruits of Ammi visnaga than that of Ammi majus , while kaempferol present in the fruits of Ammi majus in large quantities than in Ammi visnaga . References 1. W.H.O.: WHO Monographs on selected

medicinal plants. 2007; 3: pp. 9-31. 2. Walters; Drink R. and David J.K.:

Vascular plant taxonomy (4 thed). Kendall / Hunt publishing company. Dobuque, Iowa, 1996; pp. 115-116.

3. PDR for herbal medicine (4thed.). Medical Economic Company, New Jersey, 2007; pp. 85-86.

4. Chakravarty H.L.: Plant wealth of Iraq (1st ed.). Baghdad Botany Directorate, Ministry of Agriculture and Agrarian, Republic of Iraq, 1976; pp. 11, pp.27.

5. Elgamal M.H.A.; Shalaby N.M.M.; DuDDeck H. and Hiegemann M.: Coumarins and Coumarin glycosides from the fruits of Ammi species. 1992; pp. 819.

6. Harbone J. and King L.: Flavonoid sulphate in the Umbelliferae. Biochemical systematic and ecology, 1976; 4: 111-115.

7. Singab, A.N.B.: Acetylated flavonol triglycosides from Ammi majus L.. Phytochemistry , 1998; 49: 2177-2180.

8. Bruneton J.: Pharmacognosy, phytochemistry, medicinal plants. Paris, Lavoisler, 1995; pp.98-99.

9. O'Neil M.J.; Heckelman P.E.; Koch C.B. and Roman K.J.: The Merck index, An encyclopedia of chemicals, drugs, and biologicials (14thed). Merck and Co., INC. White house Station, N.J., USA. 2006; pp.5274, pp. 8034.

10. Lamson D.W. and Brignall M.S.: Antioxidant and cancer ш: Quercetin and kaempferol. Alt. Med. Rev., 2000; 5(3): 196-208.

11. Kris– Etherton P.M.; Hecker K.D.; Bonanome A.; Coval S.M.; Binkoshi A.E.; Hilpert, K.F.; Griel A.E. and Etherton T.D.: Bioactive compounds in food: Their role in the prevention of cardiovascular disease and cancer. Am. J. Med.., 2002; 113(9): 71S– 88S.

12. Wagner H. and Bladt S.: Plant Drug analysis, A thin layer chromatography atlas. (2nd ed.). Springer– Velag, Berlin, 1996; pp. 164-166.

13. Reich E.; Schibli A: High– performance thin layer chromatograghy for the analysis of medicinal plants. Thieme, NewYork. Stuttgart, 2006; p. 159, pp. 234-237.

14. Stahl E.: Thin layer chromatography hand book, 1999; pp. 60-128.

15. Nicola E.L.: Phytochemical and biological studies of some falvonoids present in the fruit peels of some Citrus species. M.Sc. thesis. Baghdad University, 2006; pp.36, 38.

16. AL-Maliki E.J.: Phytochemical studies of two wildly grown Iraqi plants of potential economical value. M.Sc thesis, 2001; pp. 69.

17. Pouchert C.J.: The Aldrich Libroray of Infrared spectra (2nd ed.). Aldrich chemical company, USA, 1978; pp. 793D.

Iraqi J Pharm Sci, Vol.19(1) 2010 Effect of glibenclamide on sodium and potassium level

58

The Effect of Long Term use of Glibenclamide on Serum and Urinary Sodium and Potassium Level in Type 2 DM Patients

Ali A. Ali*,1

*Department of Clinical Pharmacy, College of Pharmacy, University of Baghdad, Baghdad, Iraq.

Abstract Long-term use of sulfonylureas including chlorpropamide, is known to potentiate the antidiuretic action of arginine vasopressin (AVP), predisposing to hyponatremia.The present study was designed to evaluate the effect of long term use of glibenclamide on serum and urinary levels of sodium and potassium in Type 2 DM patients in Iraqi DM centers. Ninety eight patients with Type 2 DM who were maintained on different doses of glibenclamide for at least 1 year, attending the centre for Diabetes and Endocrinology in Al-Rusafa, Baghdad, were enrolled in the study, in addition to 15 normal healthy subjects. Patients were allocated into three groups according to the dose of glibenclamide that they received. Blood and urine samples were obtained for evaluation of sodium and potassium levels in these samples by flamephotometry. The results indicated that glibenclamide use resulted in significant elevation in serum levels of sodium and potassium compared to controls, while urinary excretion of these cations was not significantly changed. Stratification of patients according to the dose of glibenclamide revealed that this effect on sodium and potassium was not dose dependent. In conclusion, long term use of glibenclamide impairs normal values of Sodium and potassium independent of the administered dose. Key words: Glibenclamide, sodium, potassium.

الخالصة ن دواء الكلوربروبامايد وهو من مجموعة السلفونيل يوريا يستخدم لعالج مرضى السكري من النوع الثاني غير المعتمد على ( ا

وم ) االنسولين و يعتقد ان هذا الدواء يعمل مباشرة على وهو معروف بتحفيزه لعمل الهرمون المضاد للبول والذي يسبب نقص الصودي المضاد .لللبو مستقبالت هرمون من نفس مجموعة الكلوربروبامايد دواء الكليبينكالمايد وهو تاثير دراسة ود أي (في هذا البحث ن

ونيل يوريا والبوتاسيوم في الدم )السلف وم ساعة في المرضى المصابين 24المجموع خالل البول وكذلك في ) المصل(على تركيز الصوديوقد شارك في هذه 2009من عامالثاني ولغاية شهر كانون 2008في شهر ايلولبداء السكري النوع الثاني وقد اجريت هذا الدراسة

والسكري في الرصافة ببغداد بالتعاون مع الطبيب االستشاري هناك و 98الدراسة شخص 15مريض خارجي من مركز الغدد الصم عينات من المصل وا من 24خالل المجموع لبولا( لبولسليم وقد اخذت وم وقد تم قياس)زلساعة في ال سي وتا تركيز الصوديوم والب

ج ان قياس االلوان في اللهب بواسطة جهاز كليبينكالمايدواظهرت النتائ وم في ال وتاسي يؤثر بشكل ملحوظ على تركيز الصوديوم والبعلى تركيز الصوديوم تاثيره ما القياسية ا المجموعة مقارنة مع الدم المج لبولفي ا والبوتاسيم مصل يكن 24موع خالل لم ساعة

.ملحوظ بشكلIntroduction Glibenclamide is a first line option for treating patients with type 2 diabetes mellitus (DM) who are not overweight or who can not take metformin(1). It is mostly used when diet control and exercise have failed to achieve tight control of plasma glucose level; it also can be used alone or in combination with other hypoglycemic agents to provide better glycemic control (2).Sodium is the important electrolyte in the extracellular space while potassium is the essential one in the intracellular space, this asymmetrical distribution of the electrolytes across the cell membrane requires the active exchange of both cations by the Na+/K+-ATPase .(3)Potassium is the principle electrolyte (cation) of intracellular fluid and the primary buffer within the cell itself. Ninety percent of potassium is contain in blood and bone.

Damaged cells release potassium into the blood .(4) Using free flow micropuncture technique in rats, intravenous infusion of glibenclamide (3mg/hr) evoked a natriuresis and diuresis; while potassium excretion remained unchanged. It has been reported that glibenclamide impaires sodium reabsorption in one or more of the nephron segment that comprise the loop of Henle(5).Moreover, other hypothesis indicates that the natriuretic effect of glibenclamide is a consequence of blockade of potassium channels in the apical membrane of the thick ascending part of Henle loop; or it may inhibited a small secretory potassium flux in the proximal tubule(6).The present study was designed to evaluate the effect of long term use of different doses of glibenclamide on serum and urinary levels of sodium and potassium levels of Iraqi patients with type 2 DM.

1Corresponding author E- mail : [email protected][email protected] Received : 27/6/2009 Accepted : 9/3/ 2010



59

Subjects and methods Ninety eight patients (54 female and 44 males) with type 2 diabetes mellitus, were referred to the specialized center for Endocrinology and Diabetes, Baghdad during the period from September 2008 to January 2009 were enrolled in the study; their age range was 52.26 ± 8.65 years and diagnosed to have type 2 DM for at least five years( Average duration of type 2 DM 5.5 ± 0.4 years) and maintained on glibenclamide 5 mg , 10 mg or 15 mg at least 1 year with adequate glycemic control (Average fasting blood sugar = 6.0 ± 0.94 mmole/L) and no detectable secondary complications .All patients were inflamed about the nature of the study and their signed consents were obtained before participation. Additionally, 15 healthy subjects with comparable age to that of patients were utilized as control. After overnight fasting, venous blood samples were obtained from all subjects without using tournique to avoid leakage of cellular potassium, and serum was prepared for evaluation of serum levels of sodium and potassium using flamephotometer (The flamephotometer used is FP 20 seac from Seag Radim company-Italy). Moreover, 24 hours urine sample was collected for evaluation urinary excretion of sodium and potassium using flamephotometry. For 24 hours urine collection, we instructed the patients to void at 8 AM and discard the specimen. Then collect all urine including the final specimen voided at the end of the 24-hour collection period (i.e, 8 AM the next morning)(7).For evaluation of the effect of glibenclamide on serum and urinary levels of sodium and potassium, all patients data were compared with those of controls; Meanwhile ,for the evaluation of the effect of glibenclamide dose ,patients were classified in three groups, those who are treated with 5 mg/day(n=56), those treated with 10 mg/day(n=29) and those treated with 15 mg/day(n=13). All data were expressed as mean ± standard deviation; un-paired students t-test between patients and control and ANOVA test was performed for evaluation of differences between groups and p-values < 0.05 were considered significant. Flame photometry based on atomic emission method for the routine detection of metal salts, principally Na, K, Li, Ca and Ba. Quantitative

analysis of these species is performed by measuring the flame emission of solution containing the metal salts.Solution is aspirated into the flame. The hot flame evaporates the solvent, atomizes the metal, and excites a valence electron to an upper state. Optical filters are used to select the emission wavelength monitored for the analyte species. Comparison of emission intensities of unknowns to either that of standard solutions, or to those of an internal standard, allows quantitative analysis of the analyte metal in the sample solution.(8, 9) Results In the present study, table 1 shows that serum sodium concentration in total number of patients is significantly different (P<0.05) compared to that in control group. Table 1 also shows that both urine sodium and potassium concentrations in total number of patients are not-significantly different (P>0.05) compared to that in control group. In table 2, the average urinary excretion of sodium and potassium was not significantly different (P>0.05) among patients who were using different doses of glibenclamide. Table 1 : Effect of glibenclamide on serum and urinary leve ls of sodium and potassium in type 2 patients.

Parame ter

Control subjects (n=15)

DM patients treate d with

glibe nclamide (n=98)

Serum sodium (meq/L)

139.95 ± 3.859 142.29 ± 6.67*

Serum potassium (meq/L)

4.13 ± 0.56 4.71 ± 0.663*

Urine sodium (meq/L)

103.14±26.25 100.64 ± 48.8

Urine potassium (meq/L)

62.02±33.2

51.76 ± 25.12

Values were expressed as mean ± SD; n= number of subjects; * = significantly different from control (p< 0.05).


60

Table 2: Effect of 5 mg /day , 10 mg/day and 15 mg/day Glibenclamide on serum and urinary sodium and potassium le vels in type 2 DM patients.

parame te r Patients treated with 5 mg /day glibe nclamide

N=56

Patients treated with 10 mg /day glibenclamide

N=29

Patients treated with 15 mg /day

glibe nclamide N=13

Serum sodium ( meq/L) 142.3 ± 4.5 a 142.7 ± 7.8 a 141.53±4.33a Serum potassium ( meq/L) 4.746 ± 0.659 a 4.648 ± 0.748 a 4.73 ± 0.496 a Urine sodium ( meq/L) 109.08 ± 52.28 a 96.4 ±41.58 a 73.75 ± 46.89 a

Urine potassium (meq / L) 55.39 ± 21.59 a 41.85 ±24.77 a 54.75 ± 35.964 a

Values were expressed as mean ± SD; n= number of subjects; values with identical superscripts (a ) were considered non significantly different (P>0.05) (ANOVA). Discussion Systemic administration of glibenclamide inhibits sodium reabsorption in the loop of Henle (6); this extra-pancreatic effect of glibenclamide reveal natriuretic activity(10) ,which is attributed to elevation of functional sodium delivery to the distal tubules. Moreover ,glibenclamide impairs sodium reabsorption in one or more of the nephron segments that comprise the loop of Henle(5).In the present study, long term use of glibenclamide by DM patients resulted in significant elevation in serum levels of sodium and potassium (table 1) ;this effect may be attributed to enhancement of free water excretion induced by glibenclamide in patients with DM(11).In clinical practice, administration of 5 mg single oral dose of glibenclamide in patients with type 2 DM did not essentially affect sodium and potassium excretion (12).Several agents belong to sulfonylureas group ,including glibenclamide, have diuretic action in well hydrated normal subjects (11), and glibenclamide enhances water excretion in patient with diabetes insipidus (13,14), and diabetes mellitus(11).In this respect, the reported elevation in serum levels of sodium and potassium (in the present study),without affecting urinary levels, may be explained on the bases of improper hydration status of enrolled patients, especially when associated with excessive of water excretion. The data presented in table 2 indicated that there is no dose-related effect for glibenclamide on sodium and potassium homeostasis; this observation may indicate that the reported elevation in serum levels of the cation is beyond the pharmacodynamic activity of glibenclamide .Moreover, the limitation of patients ' sample, duration of follow up multifactorial pathophysiology may have potential interference in this respect. Accordingly, larger patients sample with proper selection criteria could be a proper approach for clear definition of the problems.

Conclusion In this study we conclude that glibenclamide dose not satisfy syndrome of inappropriate antidiuretic hormone secretion (SIADH) criteria because no hyponatremia occure (SIADH criteria are hyponatremia and urinary sodium concentration >20mmol/L) (15,

16).Also we conclude that long-term use of different doses of glibenclamide in type 2 DM may impaire sodium and potassium homeostasis unrelated to the administered dose. Acknowledgments I would like to thank Dr.Khalid Ibraheem , specialist of endocrinology, centre of diabetes and endocrinology in Al-Rusafa-Baghdad . Also I am grateful to the staff of quality control department - central laboratories /Baghdad. References 1. Hensen J; Haenelt M; Gross P. Water

retention after oral chlorpropamide is associated with an increase in renal papillary arginine vasopressin receptor.Eur.J Endocrinolo 1995 Apr;132(4): 459-64.

2. WHO Model list of Essential Medicines, 15th edition, Word Health Organization, p.21.Retrieved on 2007-11-19.

3. Thomas L: Water balance and fluid compartments; Sodium, Clinical lab diagnostics, 1st edition, 1998, vol.1, 289.

4. Frances Fischbach, Potassium, A manual of laboratories and diagnostic test, sixth edition, 2000, chapter 6, 349.

5. Baily M.A and S.J. Walter: Renal effects of Glibenclamide: A micropuncture study: The journal of pharmacology and experimental thrapeutics ; may 1998, vol.285, issue 2,464-467.


61

6. Bailey M.A., Shirley D.G., Stocking CJ. Jonathan M. Slater, Stephen J. Walter . The natriuretic effect of glibenclamide:evidence for a non-luminal site of action. European Journal of physiology. Vol.444, Number 6/ September, 2002, 777-784.

7. Harrington JT and Cohen JJ," Measurement of Urinary Electrolytes-Indications and Limitations", N Engl J Med, 1975,293(24):1241-3.

8. Sawyer, Heineman, Beebe, Chemistry Experiments for Instrumental Methods, Wiley, New York, 1984.

9. D. C. Harris Quantitative Chemical Analysis 4th Ed., W. H. Freeman and Company, New York 1995 Chapter 21.

10. Clark MA, Humphrey SJ ,Smith MP, Ludens JH. Unique natriuretic properties of the ATP-sensitive K1 channel-blocker glyburide in conscious rats. J Pharmacol Exp Ther 1993; 165:933-937.

11. Moses AM, Howantiz J, Miller M. Diuretic action of three sulfonylurea drugs. Ann Intern Med 1973,78:541—544,.

12. Ylitalo P, Oksala H, Pitkäjärvi T . Comparison of acute and prolonged effects of glibenclamide and chlorpropamide in patients with non- Insulin-dependent diabetes. Pubmed. Arzneimittelforschung. 1985; 35 (10) : 1596-9.

13. Rado JP; Borbely L.: Glybenclamide enhancement of polyuria in patients with pituitary diabetes insipidus. Endocrinology 1972, 59:397—402,.

14. Rado JP; Borbely L; Szende L; Fiscner J; Tako J.: Investigation of the diuretic effect of glibenclamide in healthy subjects and in patients with pituitary and nephrogenic diabetes insipidus. Horm Metab Res , 1974,6:289—292.

15. Rose,BD,post,TW. : Clinical Physiology of Acid-Base and Electrolyte Disorders, 5 th ed,McGraw - Hill,New York ,2001 , 707-701.

16. vRohr, Cerny T, Toss RA, Brunner KW. The syndrome of inappropriate ADH secretion (SIADH) in small cell lung cancer. Schweiz Med Wschr, 1991; 121; 1271-82.

Iraqi J Pharm Sci, Vol.19(1) 2010 Design and synthesis of new NSAID

6

Design and Synthesis of New Non-Steroidal Anti-inflammatory Agents with Expected Selectivity toward Cyclooxygenase-2

Inhibition

Nadeem A. Abdul Razzak*,1 , Samera.F.Hussan* and Ahlam J. Qaseer*

* Department of Pharmaceutical Chemistry , College of Pharmacy,University of Baghdad ,Baghdad, Iraq.

Abstract This study includes design and synthesis of new non-steroidal anti-inflammatory agents (NSAIDs) with expected cyclooxygenase-2 (COX-2) selective inhibition to achieve better activity and low gastric side effects. Two series of compounds have been designed and synthesized as potential NSAIDs,these are: Salicylamide derivatives (compounds 3,4,5 ) and Diflunisal derivatives (compounds 10&11). In vivo acute anti-inflammatory effect of one of the synthesized agents (compound 3) was evaluated in the rat using egg-white induced paw edema model of infla mmation. Preliminary pharmacological study revealed that compound 3 exhibited less anti-inflammatory effect compared to that of aspirin after 120 and 210 minutes, which encourage the continuation of the search to demonstrate or identify the preliminary pharmacological activity for the synthesized compounds and to identify their selectivity toward COX-2 isoenzymes. Keywords:nonsteriodal anti-inflammatory drugs(NSAID),aspirin de rivatives ,diflunisal, Cyclooxygenase -1(cox-1),cyclooxygenase-2(cox-2)

الخالصةة متوقعة كمثبطات لالنزيم الدراسةتتضمن ستيرويدية مضادة لاللتهابات ذات فعالي جديدة غير خليق مركبات تصميم وت

واوكسجنيز ى فعالية افضل واعراض جانبيية اقل )cox-2( 2 سايكل عل للحصول ن من المركبات وهي. خليق مجموعتي مشتقات :تم تونيسال و)5,4,3المركبات (السلسيل أمايد أجريت دراسة التقييم الدوائي األولي للفعالية المضادة ).11و10ركبان الم(مشتقات الديفل

ة الجديدة رثوية لواحد من المركبات المخلق جرذ باستخدام زالل ) 3المركب (لاللتهابات غير ال بطريقة استحداث وذمة تحت جلد يد الولوجيةأشارت النتائج )..األلبومين(البيض ة البي ة إن المركب الفعالي ألسبرين بعد من ااقل أظهر تأثيرًا مضادًا لاللتهابات قد 3األولي

ة البحث على إكمال يشجعدقيقة٬ مما 210و 120 ة وكذلك معرفة درجة انتقائها لمعرف ة المركبات المخلق التقييم الدوائي األولي لبقيوأوكسجني سايكل .زالمثبط إلنزيم ال

Introduction Inflammation defined as a complex series of tissue changes that result in pain and fever (1); or it is a normal, protective response to tissue injury caused by physical trauma, noxious, chemical, or microbiologic agents. Inflammation is the body's effect to inactivate or destroy invading organisms, remove irritation, and set the stage of tissue repair(2) . Inflammation can be divided into three phases; acute, chronic and immune response (3).There are two cyclooxygenase (COX) enzymes, COX-1 and COX-2. COX-1 is a constitutive enzyme, involved in tissue homeostasis; while COX-2 is induced in inflammatory cells and produces the prostanoid mediators of inflammation. Also COX-3 has recently been described (4). Although COX-1 and COX-2 have similar structures, there are slight differences that affect the drug binding and lead to different actions (5). Both enzymes have a long narrow channel into which arachidonic acid enters and be converted into PGs, with COX-2 has an additional side pocket.

Selective COX-2 inhibitors have chemical structure with rigid side extension that binds in this side pocket. .NSAIDs have three major pharmacological desirable actions, all of which result mainly from the inhibition of COX-2 in inflammatory cells and the resultant decrease in prostanoid synthesis; these are:

anti-inflammatory action,antipyretic effect and analgesic effect. We have two types of NSAID these are traditional NSAID and selective NSAID(5) .Traditional NSAIDs (TNSAIDs) are mainly carboxylic acid containing compounds that are either aromatics or aliphatics. They include different chemical classes with different physical properties (6).They are frequently prescribed for muscloskeletal complications and are often taken without prescription for minor aches and pains. There are new many different NSAIDs on the market and non of these is ideal in controlling or modifying the sign and symptoms of inflammation (7) .

1Corresponding author E- mail : [email protected][email protected] Received : 16/6/2009 Accepted : 1/11/2009



7

For example salicylic acid derivatives e.g. aspirin, (acetyl salicylic acid) has been in use as a pharmaceutical agent for over 100 years (8). Aspirin is unique among COX-inhibitors because it covalently modifies the protein of enzymes and irreversibly inhibits them (9). Aspirin transfers its acetyl group to serine-530 (Ser530), which prevents proper binding of arachidonic acid in the COX active site .No other residues on either COX-1 or Cox-2 are acetylated (10,11,12).Aspirin suffers from the side effects associated with local gastrointestinal irritation; therefore a number of attempts or modifications have been carried out to overcome this problem such as salt formation, e.g. calcium acetyl salicylate ,buffered aspirin (magnesium or aluminum salts) and such as nuclear modification such as diflunisal (13).Diflunisal ,5-(2',4'-difluorophenyl) salicylic acid, is not converted to salicylic acid in vivo. It is largely devoid of antipyretic effect, perhaps because of poor penetration into the CNS. It is 3-4 times more potent than aspirin (6).The higher potency of diflunisal may be resulted from the existence of two vanderwaals binding sites (aromatic rings) and the ortho fluoro atom which promotes non-coplanarity between the two aromatic rings which is required for effective binding to the COX enzyme. In addition, the presence of para-fluoro atom will retard metabolism, by preventing oxidation of the para-position, hence increase duration of action (13).COX-2 selective inhibitors, or Coxibs, were developed in an attempt to inhibit prostacyclin synthesis by COX-2 isoenzyme induced at the site of inflammation without affecting the action of the constitutively active COX-1 isoenzyme found in the gastrointestinal tract, kidney, and platelets (3).

Preferentially Selective COX-2 Inhibitors for example : Meloxicam which is a novel NSAID acting by preferential inhibition of COX-2 (14). It has a selectivity towards COX-2 up to 100 fold over COX-1 depending on the test system (15).. An isosteric functional groups to 2-amino-5-methyl thiazole moiety in meloxicam are investigated as a possible bioisosteric analogues (16).

Highly Specific COX-2 Inhibitors Since the loss of selectivity is a potential problem with large dosage of NSAIDs that, preferentially inhibit COX-2; thus new agents that are more specific towards COX-2 even at large dose were synthesized, these agents include:celecoxib,roficoxib,valdicoxib,etoricoxib,and imricoxb.

Experimentals A.chemistry Materials: 2-aminobenzothiazole,2-aminopyridine&2-aminopyrimidine (BDH,England ), Acetyl salicylic acid(Judex England),DCCI, Diflunisal(Jordon),Ethanol 95%and 99%,all solvents were of analar type and used without further purification

General procedure melting points(uncorrected) were determined by capillary method on Thomas hoover apparatus (England)and IR spectra were recorded on model 500 scientific IR spectrophotometry ,Buck company(USA) in pharmacy collage ,Baghdad university.Ascending thin layer chromatography (TLC) was run on DC-Kartan SI Alumina 0.2 mm to check the purity and progress of reaction. The CHN analysis was done using an Exeter CE-440 elemental microanalyzer (Germany). The analysis was carried out at micro analytical center, Faculty of Science-Cairo University.The identification of compounds was done using iodine vapor and the chromatograms were eluted by two

solvent systems: A:THF : Diethyl ether : Cyclohexane (40:40:20) (17)

B:Methanol : Acetic acid : Diethyl ether : Benzene (1:18:60:20)(18)

Method of preparation of aspirin anhydride Aspirin (5.0gm, 27.77mmole) was dissolved in methylene chloride (75ml), and dicyclohexyl carbodiimide (DCCI) (2.86gm, 23.84mmole) was added. The reaction mixture was continuously stirred at room temperature for 3.5 hours. A white precipitate of dicyclohexylurea (DCU) was formed, then removed by filtration. The filtrate was evaporated under vacuum and an oily product was formed to yield compound (1) (18).

Synthesis of N-(2-pyridyl)-acetyl salicylamide Compound 1: (4.0gm, 11.69mmole), 2-aminopyridine (1.1gm, 11.69mmole), zinc dust (catalytic amount, 0.01gm), glacial acetic acid (1.12ml, 19.64mmole) and dioxane (32ml) were placed in a flask, equipped with reflux condenser. The reaction mixture was refluxed gently for 90 minutes, the solvent was evaporated under vacuum, the residue was dissolved in the minimum volume of ethyl acetate, washed with NaHCO3 (10%, 3X), HCl (1N, 3X), and distilled water (3X), dried using anhydrous magnesium sulfate. The filtrate was evaporated under vacuum to give a crude product 2. The recrystilization was carried out using ethyl acetate-petroleum ether (60-80oC)


8

mixture, a white crystalline product was obtained compound(2) (19, 20). Compound (2) :melting point (115-117),yield (41.14 white powder),IR in KBR disk :3390 N-H stretching vibration of secondary amide,1768 c=0 stretching vibration of acetate ester ,1604-1577&1532 c=o stretching vibration of aromatic and N-H bending (amide II)is also included in this region. Synthesis of N-(2-pyridyl)-salicylamide Compound 2 (0.5gm, 1.953mmole) was dissolved in a minimum volume of absolute ethanol 99%: tetra hydro furan(THF) (3:1) mixture. The solution was cooled to 18oC, and then sodium hydroxide solution (2N, 1.162ml, 2.325mmol) was added drop wise, with continuous stirring over a period of 30 minutes. Stirring was continued at 18oC for additional three hours. The reaction mixture was neutralelized with equivalent quantity of HCl, excess of cold water was added to precipitate the phenolic compound, which was then filtered and dried to give compound (3)(20).The same method of synthesis has been done for the other salicylamide derivatives but with two different heterocycles (2-amino

benzothaiazole) and (2-amino pyrimidine) to yield the final compounds (4)&(5). Compound (3):melting point (218-220),yield(51% white crystals),Rf value (0.75A&0.79B) ;IR in KBr disk :3000-3500 broad O-H stretching vibration of phenol,3238 N-H stretching vibration of secondary amide , 1675 C=O stretching vibration of secondary amide (amide I), 1611-1604, 1541-1537 C=C stretching vibration of aromatic and N-H bending vibration of secondary amide,1234C-O stretching vibration of phenol (figure 1) . CHN analysis found C 66.71, H 4.63,N 12.74. CHN calculated C 67.28,H 4.67,N 13.08 Compound (4):melting point 290 decomposed ,yield (47% white crystals ). Compound (5):melting point (191-193),yield(56%white crystals). IR in KBr disk for both compounds :3000-3500 broad band stretching vibration of phenol , 3330 &3301 N-H stretching vibration of secondary amide ,1678&1623 c=o stretching vibration of secondary amide (amide I),1245&1224 c-o stretching vibration of phenol.

Figure 1 : IR spectrum of compound( 3) in KBr disk. Synthesis of 5-(2 ',4 '-difuorophenyl)-acetylsalicylic acid, Compound (6) In a 250ml boiling flask, equipped with reflux condenser, diflunisal (5.0gm, 20mmol) and acetic acid anhydride (15ml, 159mmole) were placed and 3 drops of concentrated sulfuric acid were added drop wise. The reaction mixture was refluxed gently for 1 hour, then allowed to cool with occasional stirring. Ice-water was then added until a precipitate was formed, which was filtered using pump, washed with cold distilled water several times, the crude product was collected. The recrystalization was carried out using ethanol 95% to give compound( 6) (19) .

Compound (6): melting point (172-174),yield (89% white crystals ) IR values in KBr disk 3000-2676 O-H stretching vibration (H-bonded ) of carboxylic acid ,1768 c=o stretching vibration of acetate ester ,1700 stretching vibration of carboxylic acid ,1319 O-H bending vibration of carboxylic acid ,1274 c-o stretching vibration of carboxylic acid. Synthesis of 5-(2',4'-difluorophenyl)-acetyl salicylic acid Anhydride compound(7): compound 6 (5.0gm, 17.1mmole) was dissolved in THF (30ml), then DCCI (1.75gm, 8.55mmole) was added. The reaction mixture was continuously stirred at room temperature for 4 hours. A


9

white precipitate of DCU was formed which was then removed by filtration. The solvent was evaporated under vacuum and a solid product of compound (7) was obtained (18). Compound (7): melting point (149-150),yield (80% white powder ) IR value in KBr disk :1815&1743 c=o stretching vibration of anhydride (asymmetric &symmetric bands),1277&1173 c-(c=o)-o(c=o)-c stretching vibration of anhydride.

Synthesis of 5-(2,4-difluorophenyl)-N-(2-pyridyl) acetyl salicylamide compound(8) Compound 5 (2.5gm, 4.4mmole), 2-aminopyridine (0.418gm, 4.4mmole), zinc dust (0.004gm), glacial acetic acid (0.425ml) and dioxane (25ml) were placed in round-bottom flask, equipped with reflux condenser. The reaction was carried out as in the synthsis of compound (2). The recrystilization was carried out using ethyl acetate-petroleum ether (60-80oC) mixture, a white crystalline product was obtained( (compound 8) (19, 20). Compound (8): melting point (180-181),yield (42.2 white powder) . The same method of preparation has been done with 2-amino benzothiazole to yield compound (9) compound (9):melting point (161-164),yield(39.5% white crystals) . IR values in KBr disk for compounds (8&9): 3335&3333 N-H stretching vibration of

secondary amide ,1774&1753 c=o stretching vibration of acetate ester ,1696&1686 c=o stretching vibration of secondary amide (amide I).

Synthesis of 5-(2,4-difluorophenyl)-N-(2-pyridyl) salicylamide, Compound (10) To a cooled solution(18 oC) of compound (3) (0.4gm, 1.085mmole) in absolute ethanol 99%: THF (3:1) mixture, sodium hydroxide (2N, 0.651ml, 1.302mmol) was added drop wise, with continuous stirring over a period of 30 minutes. Then the reaction mixture was worked up as described previously in preparation of compound (3).The same method of preparation has been done for diflunisal anhydride with (2-amino benzothiazole) to yield the final product compound (11).

Compound (10):melting point (230-232),yield (48,%of white powder),RF value (0.53 A&0.69B),IR in KBR disk: 3376-3227 O-H stretching vibration of phenol, 3304 N-H stretching vibration of secondary amide,1660 C=O stretching vibration of secondary amide (amide I), 1598 and 1530 C=C stretching vibration of aromatic and N-H bending of secondary amide (amide II), 1262 C-O stretching vibration of phenol ( figure 2). CHN analysis found :C 64.93,H 3.80, N 8.33 CHN calculated : C 66.26,H 3.68, N 8.59.

Figure 2 : IR spectrum of compound 10 in KBr disk.

Compound (11): melting point (251-253),yield 42% faint yellow crystals).IR value in KBr disk :3450-3100 O-H stretching vibration of phenol and N-H stretching vibration of amide is buried within this band ,1677 C=O stretching vibration of secondary amide (amide I),1604-1534 C=C stretching vibration of aromatic and bending vibration of secondary amide (amide II).

B.Pharmacology Acute anti-inflamma tory activity of one of the chemically synthesized compounds, compound( 3) was evaluated in vivo using egg-white to induce paw edema in rats. The decrease in paw thickness is the basis of screening the newly synthesized compound for its anti-inflammatory activity.Eighteen albino rats of either sex, weighing 300 ± 10 gm supplied by the animal house of the College of


10

Pharmacy, University of Baghdad were used in this study. Animals were kept under standardized conditions (12 light-12 dark cycle) for 7 days for acclimatization; and were fed commercial chaw and had provided with water. Rats were brought 1 hour before performing the experiment to the laboratory, and were allocated into 3 groups (each of 6 rats) as follows: A/ Six rats served as control; they received drug vehicle (0.5ml propylene glycol in water 50% v/v). B/ Six rats received aspirin as a reference substance in a dose of (100mg/kg, i.p.) in propylene glycol (21).C/ Six rats received compound 3 suspended in propylene glycol (118.8mg/kg, i.p.) (22). The anti-inflammatory activity of the tested compound was studied using egg-white induced edema model. Acute inflammation was produced by a subcutaneous injection of 0.05ml of undiluted fresh egg-white into the planter side of the left hind of the rats; 30 minutes after intraperitoneal injection of the drug or the control. The paw thickness was measured by vernea at eight time intervals (0, 30, 60, 90, 120, 150, 180 and 210 minutes) after the drug administration.

Statistical Analysis Students t-test was used to make comparisons with respect to baseline, while comparisons between different groups at specified time was done using analysis of variance (ANOVA). P values less than 0.05 were considered significant.

Result and conclusion The designed compounds have been synthesized successfully as shown in scheme (1) & (2) and their structures were confirmed, using elemental microanalysis (CHN), infrared spectroscopy (IR spectra) and their purity was confirmed by their physical data (melting points and R f values).The conversion of carboxylic acid group of aspirin and diflunisal to corboxamide group by conjugating the selected moiety of heterocyclic compound may produce new non-steroidal anti-inflammatory agents with expected selectivity toward COX-2 inhibition and hence less gastric irritation. Preliminary pharmacological evaluation has been done for one of the designed compounds (compound 3) and it has been found that this compound exhibit slightly less potent anti-inflamma tory effect than aspirin as shown in (figure 3 and table 1).

Sche me (1): Synthesis of compounds (3,4 ,5).

H3C

O

OH

O

O

DCC

H3C

O

O

O

H3C

O

O

O

O

NH2N

H3C

O

O

O

NNH

Methylen chloride

aspirin1

Reflux

2

(2) HCl

(1) NaOH

OH

O

NNH

3

N

SH2N

Reflux

H3C

O

O

O

N

SNH

(2) HCl

(1) NaOH

OH

O N

SNH

4

N

NH2N

Reflux

H3C

O

O

O

N

NNH

(2) HCl

(1) NaOH

OH

O N

NNH

5

2

DCC


11

4

4 . 2

4 . 4

4 . 6

4 . 8

5

5 . 2

5 . 4

5 . 6

5 . 8

6

6 . 2

0 1 2 0 2 1 0

T i m e ( m i n )

Paw

th

ick

nes

s (m

m)

P r o p y l e n e g ly c o lA s p i r i nC o m p o u n d 3 5a

a

a

a

b

ba

b

b

Sche me (2): Synthesis of compounds (10 and 11).

Figure 3: Effect of vehicle (propyle ne glycol), aspirin and compound 3 on paw ede ma in rat afte r 120 and 210 minutes of egg-white injection. Results are expressed as mean ± SEM (n=6/group). Time zero is the time of egg-white injection. Non-ide ntical supe rscripts (a,b) among different groups represent significant diffe re nce (P<0.05).

Acetic anhydride

Reflux

diflunisal

OH

HO

OF

F

HO

OF

F O

H3C

O

THF

O

OF

F O

H3C

O

DCC

OF

F O

H3C

O

O

HNF

F O

H3C

O

R-NH2

Reflux

R

(1) NaOH

(2) HCl

O

HNF

F OH

R

N

6

7

N

N

S

22

Rcompound no.

10

11

compound no. R

8

9 N

S

Paw

th

ickn

ess

(mm

)

DCC

Acetic anhydride

RNH2 (1) NaOH

(2)HCl

Compound no. Compound no.


12

Table 1:Effect of control (propylene glycol), reference drug (aspirin) and tested compound (compound 3) on egg-white induced paw edema in rats.

Time (min) Paw thickness (mm)

Control (n=6) Aspirin (n=6) Compound 3 (n=6) 0 5.45 ± 0.12 a 5.39 ± 0.11 a 5.54 ± 0.12 a

30 6.15 ± 0.15 5.85 ± 0.13 5.79 ± 0.09 * 60 6.41 ± 0.07 * 5.64 ± 0.14 * 5.85 ± 0.11 * 90 6.56 ± 0.11 * 5.48 ± 0.12 * 5.68 ± 0.09 * 120 5.85 ± 0.12 * a 5.18 ± 0.12 * b 5.43 ± 0.07 * b 150 5.41 ± 0.09 * 4.95 ± 0.15 * 5.21 ± 0.11 * 180 5.30 ± 0.12 * 4.70 ± 0.11 * 4.98 ± 0.09 * 210 5.25 ± 0.07 * a 4.58 ± 0.06 * b 4.75 ± 0.13 * b

Data are expressed as mean ± SEM. n= number of animals. Control, reference drug and tested compound were given 30 minutes before the injection of egg-

white. Time (0) is the time of injection of egg-white (induction of paw edema). *P<0.05 with respect to time (0). Non-identical superscripts (a, b) among different groups represent significant difference (P<0.05). References 1. Guyton, A.C. and Hall, J.E. (EDs.):

Resistance of body to infection: Leuckcytes, graneulocytes, the monocyte-macrophase system and inflammation. In: Textbook of medical physiology (10th ed.). Harcourt Asia PTELTD, Philadelphia, 2000; pp. 397.

2. Harvey, R.A. and Champe, P.C. (EDs.): Anti-inflammatory drugs and autocoids. In: Lippincott’s illustrated review pharmacology (3rd ed.), Lippincott Williams and Wilkins, Philadelphia, 2006; pp. 495-498.

3. Waghner, W.; Katzung, B.G. and Furst, D.E.: Nonsteroidal anti-inflammatory drugs, disease-modifying antirheumatic drugs, non-opioid analgesic and drugs used in gout. In: Basic and clinical pharmacology (9th ed.). Katzung, B.G (ED). McGraw Hill, New York, 2004; pp. 573-576.

4. Rang, H.P., Dale, M.M. and Ritter, J.M. (EDs.): Anti-inflammatory and immunosuppressant drugs. In: Pharmacology (4th ed.). Churchill Livingstone, London, 2003; pp. 246-248.

5. Dubois, R.N.; Abramson, S.B. and Grofferd, L.; et al.: Cyclooxygenase 2 in biology and disease. FASEB J. 1998; 12: 1063-1073.

6. Roberts II, L.J.; Marrow, J.D.: Analgesic-antipyretic and anti-inflammatory agents and drugs employed in the treatment of gout. In: Goodman and Gilman’s the pharmacology basis of therapeutics (10 th ed.), Hardman, J.G.; Limird, L.E. and

Molinoff, P.B. (EDs.): McGraw-Hill, New York, 2001; pp. 688, 703.

7. Bowman, W.C. and Rands, M.J. (EDs.): The immune system and inflammatory mechanisms: immunosuppressant and anti-infla mmatory drugs. In: Textbook of pharmacology (2nd ed.). Black well scientific publication, London, 1984; pp. 13.15.

8. Vane, D.R.; Flower, R.; Botting, R.M.: New insight into the mode of action of anti-infla mmatory drugs. Inflamm. Res. 1995; 44: 1-10.

9. Rome, L.H.; Lands, W.E.M.; Roth, G.J.; Majerus, R.W.: Prostaglandins 1976; 11: 23-30.

10. Dewitt, D.L.; El-Harith, E.A.; Kraemer, S.A.; et al.: The aspirin and heme-binding site of ovine and murine prostaglandin endoperoxides synthases. Biol. Chem. 1990; 265: 5192-5198.

11. Lecomte, M.; Laneuville, O.; Ji, C.; Dewitt, D.L. and Smith, W.L.J.: Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin. Biol. Chem. 1994; 269: 13207-13215.

12. Rowlinson, S.W.; Crews, B.C.; Goodwin, D.C.; Schneider, C.; Gierse, J.K. and Marnett, L.J.J.: Spatial requirements for 15-(R)-hydroxy-57, 8Z, 11Z, 13E-eicosatetraenoic acid synthesis within the cyclooxygenase active site of murine COX-2, Why acetylated COX-1 does not synthesize 15-(R)-hete. Biol. Chem. 2000; 274: 6586-6591.


13

13. Muhi-Eldeen, Z.: Rheumatoid arthritis and anti-inflammatory agents. Essentials of medical chemistry. Esraa, Jordan, 2005; pp. 370-385.

14. Patoia, L.; Santucci, L.; Furon, P. and Dionisi, M.S.: A 4 weeks, double-blind, parallel-group study to compare the gastrointestinal effects of meloxicam 7.5mg, meloxicam 15mg, proxicam 20mg and placebo by means of focal blood loss, endoscopy and symptom evaluation in healthy volunteers. Br. J. Rheumatol. 1996; 35(1): 61-67.

15. Seibert, K.; Zhang, Y.; Leahy, K.; et al.: Pharmacological and biochemical demonstration of the role of cyclooxygenase in inflammation and pain. Proc. Natl. Acad. Sci. USA 1994; 91: 12013-12017.

16. Engelhardt, G. and Pairet, M.; Meloxicam: A new NSAID with an important safety profile through preferential inhibition of COX-2. European Rheumatology . 1995; 24: 272.

17. AL-Mikhlafi Sadik, A.S.: Synthesis and evaluation of novel nonsteroidal anti-

inflammatory agents. Ph.D. Thesis, College of Pharmacy, University of Baghdad, Baghdad, 2004.

18. Pradip, K.; Baner, J. and Gordon, L.: J. Pharm. Sci. 1981; 70: 1299.

19. Furniss, B.S.; Hannaford, A.J.; et al.: Vogel’s textbook of practical organic chemistry (4th ed.). Longman, London, 1978; pp. 651, 501.

20. Khorana, H.G.: Carbodiimides. Part V.1: A novel synthesis of adenosine di- and triphosphate and P1 and P2 diadenosine-5 -́pyrophosphate. J. Am. Chem. Soc. 1954; 76: 3517.

21. Chandrashekher, S.P.; Naveen, K.J.; Amarjit, S. and Shinivas, K.K.: Modulatory effect of COX inhibitors on sildenafil-induced antinociception. Pharmacology 2003; 69: 183-189.

22. Chakraborty, A.; Devi, R.K.B.; Rita, S. and Sharatchandra, K.H.: Preliminary studies on anti-inflammatory and analgesic activities of spilanthes acmella in experimental animal models. Indian J. Pharmacol. 2004; 36(3): 148-150.

Iraqi J Pharm Sci, Vol.19(1) 2010 Detection of CA19-9 in benign cases

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Detection of Carbohydrate Antigen CA19-9 Levels in Sera and Tissues' Homogenate of Breast and Thyroid Benign Cases

Gheid H. Alubaidi*,1 , Zeyan A. Ali** and Abdulrahman R. Mahmood*

* Department of Chemistry,College of Education Ibn-Alhaitham,University of Baghdad,Baghdad,Iraq. ** Department of Che mistry, Science Education College, University of Salahaddine,Erbil, Iraq. Abstract The aims of the present study are to evaluate the levels of CA19-9 in sera and tissues' homogenate of breast and thyroid benign patients in order to assess its use as an early diagnostic parameter in differentiation between malignant and benign cases. The study was conducted on 8 patients with breast benign tumor and 8 patients with thyroid benign tumor, by the enzyme linked immunosorbent assay (ELISA) technique. The results of CA19-9 levels in sera were (15 ±1.58 and 10.67 ±2.08)U/ml respectively compared with serum CA19-9 levels of control group which was 7.74 ±4.92 U/ml, the results were found to be highly significantly in breast tumor patients and non significantly in thyroid tumor patients than control group. The results of CA19-9 levels in tissues' homogenate were (356.2 ±173.75 and 20 ±14.4)U/ml respectively. The results were found to be highly significant in tissues' homogenate of breast tumor patients and non significant in thyroid tumor patients of higher compare with the it's serum levels of the same patients groups. Key words: Carbohydrate antigen CA19-9, Benign Cases and CA19-9, Breast tumor and CA19-9, Thyroid tumor and CA19-9.

الخالصةويات حالية هو قياس مست في مصول وأنسجة مرضى أورام الثدي والغدة الدرقية الحميدة CA19-9الغرض من الدراسة ال

ستخدامه كمؤشر تشخيصي مبكر يمكن من خالله ا ةلغرض تحديد امكانية ا 8اجريت الدراسة على .لتمييز بين الحاالت الحميدة والخبيثن باورام الثدي الحميدة و عي االنزيمي 8مرضى مصابي ة االمتزاز المنا .)ELISA(مصابين باورام الغدة الدرقية الحميدة بواسطة تقني

ج لمستويات مقارنة مع مستوياته في مجموعة السيطرة U/ml)2.08±10.67و 1.58±15(في المصول CA19-9وقد كانت النتائوقيمة معنوية غير عالية في اورام , U/ml)4.92±7.74(والتي كانت ة جدا في مصول اورام الثدي وية عالي و أظهرت النتائج قيمة معن

ة مقارنة بمجموعة االصحاء ه .الغدة الدرقي ة لمجانس انسجة المرضى فقد كانت مستويات و 173.75±356.2(اما بالنسب20±14.4(U/ml ة جدا في مجانس انسجة اورام الثدي وقيمة معنوية غير عالية في اورام . بالتعاقب أظهرت النتائج قيمة معنوية عالي

ة مع مصول نفس المرضى .الغدة الدرقية مقارنIntroduction The term "polyp" is clinical description of any evaluated tumor which may be found in tissues as a projection in the lumen as polyp referred as benign tumor.(1) T umor markers in general are substances present in or produced by a tumor or by the tumor host in response to the tumor's presence that can be used to differentiate a tumor from normal tissue or to determine the presence of a tumor based on measurements in the blood or secretions.(2) Although increased levels of serum tumor markers are often associated with the presence of cancer, marker concentrations may also rise in a number of benign conditions.(3) Chemically, CA19-9 is a tetrasaccharide derived from Lewis blood group antigens that are not exclusive to erythrocytes, but they can be found in different tissues and organs. Carbohydrate antigen CA19-9 after its discovery was first described as tumor associated antigen and used as tumor marker for colon and pancreas caner.(4) In addition, a previous study demonstrated that the level of

serum CA19-9 is dependent on the severity of the bile duct obstruction and the degree of cholangitis. An increase in the serum level CA19-9 can be detected even in benign bile duct diseases.(5) It has been reported that CA19-9 was elevated in breast malignant cases only, while the level of Ca19-9 was within normal value (37U/ml) in the benign cases.(6) A recent study claimed that CA19-9 levels in sera of thyroid cancer was found to be significantly higher than that for control group.(7) The aims of the present study are to investigate CA19-9 levels in sera and tissues homogenate of patients with benign breast and thyroid tumors by enzyme linked immunosorbent assay ELISA technique, then to assess the differences in the levels of CA19-9 in the serum of benign tumor patients and control, also to correlate the serum and tissue homogenate levels of CA19-9 in patients with breast and thyroid, such determinations might be useful to predict whether or not the tumor marker is related to the benign tumor.

1Corresponding author E- mail : [email protected][email protected] Received : 29/12/2009 Accepted : 6 / 4/ 2010



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Materials and methods Patients The patients included in this study with age ranged 17-75 years, were classified into three groups as follows:

1. The first group included 8 patients with benign breast tumor (G1).

2. The second group included 8 patients with benign thyroid tumor (G2).

3. The last group included 30 healthy subjects considered as control group (G3).

The patients were selected, according to the histopathological investigation. They were admitted for treatment at Medical City Hospitals (Baghdad Teaching Hospital and Nursing Home Hospital) and all surgical operations for all patients were carried out under the supervision of surgeons. Preparation of Blood Samples Five milliliters (mls) of venous blood were drawn from each patient by veinpuncture just before surgery, left to clot, and then centrifuged at 4000 r.p.m. for 30 min. Serum was separated and stored at -20˚C until time of analysis. Collection of Specimens The tumor tissue was surgically removed from patients. The specimens were immediately kept in normal saline solution and stored at -20˚C until the time of homogenizing process. Homogenization of Tumor Tissues The frozen tissue was sliced finely scalped in Petridish standing on ice, and then homogenized with three fold volumes of phosphate buffer pH7.4 by the homogenizer. The homogenate was filtered through nylon gauze to eliminate fiber connective tissues. The filtrate was centrifuged at 4000 r.p.m for 30 min at 4˚C in order to precipitate the remaining intact cells and the intact nucleus. The supernatant and precipitate fraction were separated and frozen at -20˚C until use. Determination of (CA19-9) Antigen Using ELISA Assay A basic ELISA work follows simple steps according to included manufacturer’s instructions of CA19-9 Kit supplied by Dia Metra Company, Italy. Statistical analysis Comparison between variables was performed by using student's t-test. P values ≤ o.o5 considered significant and ≤ o.oo1 considered highly significant.

Results and discussion The mean ± standard deviation (SD) for serum levels of CA19-9 of control group was found to be 7.74 ± 4.92 U/ml from table ( 1 )

Table 1: CA19-9 values in se ra of patients groups

Group

Mean ± SD P Value

Breast benign patients

15 ±1.58 0.0027

Thyroid benign patients

10.67 ±2.08 0.32

Control 7.74 ± 4.92

A highly significant elevation in the level of CA19-9 in sera of benign breast patients, but a non- significant elevations were found in sera of thyroid patients compared to control was observed.The results are in agreement with other reported data stated that elevated serum CA19-9 levels were found in 61% of benign cases, while CA19-9 serum levels remain within normal range in 50% of malignant cases.(8)A study performed on 91 adults to determine validity of CA19-9 among other cancer antigens as tumor markers, which claimed that CA19-9 showed statistical differences in the serum and ascitic fluids both of malignant and benign compared with control.(9)A research conducted to evaluate the etiology of elevated CA19-9 serum level in 353 subjects, 2.8% were diagnosed with malignancies, 27.5% with benign and 69.7% were non tumor patients, the authors concluded that CA19-9 should not be used as a screening tool, also in cases of a persistently elevated CA19-9 levels, further work-up for determining the etiology should be done.(10)A recent study has confirmed the role of estrogens as the cause of endometrial thickening through hormonal imbalance leading to elevated CA19-9 among other tumor markers in the serum of benign gynecological conditions may be a source of misdiagnosis of malignant diseases.(11) Table 2 shows the results of the mean ± SD of serum and tissue homogenate of breast and thyroid benign patients. A highly significantly elevation were found in tissue homogenate compared to serum in both studied groups.A study conducted on patients with different malignant diseases and benign conditions postulated that CA19-9 may be used as fairly reliable diagnostic tool, but cannot be used to predict survival.(12) Some authors have reported that the elevation in CA19-9 could be provided by differences in the cellular and humoral immune response to hydatidosis. The theories about these findings include substance that may be synthesized by the host in response to infection which lead then to conclude that the possibility of a hydatidosis should be born in mind in


64

differential diagnosis of palpable mass and elevated CA19-9 levels in the breast especially in endemic areas.(13) Table (2):- CA19-9 value in se ra and tissues of patie nts groups

Group Serum

Mean ± SD Tissues Mean

± SD P Value

Breast benign patients

15 ±1.58 356.21 ±173.75 4.7*10 -4

Thyroid benign patients

10.67 ±2.08 20 ±14.4 0.187

Conclusion A conclusion could be drawn that the tumor marker CA19-9 was elevated in benign conditions like breast and thyroid benign tumors, so it couldn't be used as peculiar detective, screening and diagnostic tool for malignant cases.

References 1. Schwartz F C, Bunicardi MD, and

Andersen DM, Philadelphia, "Small intestine colon, rectum and anus principles of surgery'', (2005), 8th ed, 1038-1083.

2. Bishop ML, Fody EP, Schoeff L, Philadelphia, "Clinical Chemistry", (2005), 3rd ed, 608-614.

3. Guo Q, Zhang B, Dong X, Xie Q, Guo E, Huang H and Wu Y, Elevated levels of plasma fibrinogen in patients with pancreatic cancer: possible role of a distant metastasis predictor" Pancreas, (2009), 38(3): e75-79.

4. Ugorsks M and Laskowsk A, Sialy Lewis(a): a tumor associated carbohydrate antigen involved in adhesion and metastatic potential of cancer cells, Acta Bioch Polonica, (2002), 49(2): 303-310.

5. Leelawwt K, Sakchinabut S, Narong S and Wannaprasert J, Detection of serum MMP-7 and MMP-9 in cholangiocarcinoma

patients: elevation of diagnostic accuracy, BMC Gastroenterology, (2009), 9: 30-38.

6. Shitrit D, Zingerma B and Kramer MR, Diagnstic value of CYFRA 21-1, CEA, CA19-9, CA15-3 and CA125 assay in pleural effusions: Analysis of 116 cases and review of the literature, The Oncologist, (2005), 10: 501-507.

7. Aldujaili AH, Altaei WF, Turkey KM, Alubaidi GH, Comparative study of CA19-9 levels as tumor marker in sera and tissues homogenate of breast, prostate and thyroid cancer patients, Ibn Al-Haitham J for Pure and Appl SCI, (2009), 22(1): 88-96.

8. Harrelli D, Caruso S, pedrazzani C and Roviello F, CA19-9 serum levels in obstructive jaundice: clinical value in benign and malignant conditions, Am J Surg, (2009), 16-19.

9. Tuzun Y, Celik Y, Bavan K and Canoruc F, Correlation of TMS in ascitic fluid and serum: Are measurements asitic TMS a futile attempt? , J Int Med Res, (2009), 37(1): 79-86.

10. Kim BJ, Lee KT, Moon TG and Rhee J C, How do we interpret an elevated carbohydrate antigen 19-9 levels in asymptomatic subject?, Dig Liver Dis, (2009), 41(5): 364-369.

11. Cecchi E, Lapi F, Vannacci A and Mugelli A, Increase levels of CA125 andCA19-9 serum tumor markers following cyclic combined hormone replacement therapy, J Clin Pharm Ther. (2009), 34(1): 129-132.

12. Sandlom G, Granroyh S and Rasmussen IC, TPS, CA19-9, VEGF-A and CEA as diagnostic and prognostic factors in patients with mass lesions in the pancreatic head, Ups J Med Sci, (2008), 113(1): 57-64.

13. Yuksel BC, Ozel H, Akin T, Avsar FM and Hengirmen S, Primary hydatid cyst of the breast with elevated CA19-9 levels, Am J Med Hyg, (2005), 73(2): 368-370.

Iraqi J Pharm Sci, Vol.19(1) 2010 Chronic renal failure on thyroid hormones

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The Effect of Chronic Renal Failure on Thyroid Hormones Layla K. Ali*,1

*Nursing Department , Koya Technical Institute Abstract Chronic renal failure (CRF) affects thyroid function in multiple ways, including low circulating thyroid hormone concentration, altered peripheral hormone metabolism, disturbed binding to carrier proteins, possible reduction in tissue thyroid hormone content, and increased iodine store in thyroid glands.The target of study is to find a relationship between chronic renal failure and thyroid function.In addition, we tried to study the effect of CRF on serum creatinine dependent on the level of thyroid hormones (T3 and T4) and thyroid stimulating hormones(TSH). Forty patients with chronic renal failure (20 male, 20 female) were enrolled in this study in addition to forty healthy individual as control group (20 male, 20 female). The age ranged from (25 -65) years. T4, T3, TSH, urea, uric acid and creatinine were measured in each of the two groups. The results revealed statistically significant reduction in T3 and T4 while there is elevation in TSH, urea,uric acid and creatinine in the patients group compared to the control group. Key word : Chronic Renal Failure,Thyroid Hormones.

الخالصةر ا ؤث ة في الدم٬ ي ة في تركيز هرمون الغدة الدرقي ة الهرمونات الدرقية بطرق متعددة تتضمن قل لقصور الكلوي المزمن على فعالي

ي ة ـف ات الدرقـي اض مسـتوى الهرموـن خـف ة ان ل٬ أحتمالـي ن الناـق البروتي تبدل أو تغير ايض الهرمون في االنسجة المحيطية٬ وإرتباطه ـبةاالنسجة المحيطية والبالزما وكذل ود في الغدة الدرقي الهدف من الدراسة ايجاد عالقة بين القصورالكلوي المـزمن . ك زيادة خزن الي

ة ة والهرمون المحفز للدرقي على مستويات الهرمونات الدرقي ة الى دراسة تاثير الفشل الكلوي المزمن اعتمادا ة باالضاف . والغدة الدرقيعمار المجاميع .من االصحاء كمجموعة سيطرة" شخصا 40الكلوي باالضافة الى بالفشل " مصابا" مريضا 40جمعت النمادج من ا

ة ) 25 65(تتراوح بين ة . سن والثايروكسين(تم قياس هرمونات الغدة الدرقي ن وديد الثايروني ٬الهرمون المحفز للدرقية٬اليوريا ) ثالثي يوالكرياتنين ن وجـد بينت النتائج وجود انخفاض معنوي .٬ حامض اليوريك حي في كل من ثالثي يوديد الثايرونين والثايروكسين في

ة وي مقارـن ي مرضـى القصـور الكـل اتنين ـف ك والكرـي ا٬ حـامض اليورـي ة ٬اليورـي ز للدرقـي ن الهرمـون المحـف ارتفاع ملحوظ في كـل مـ .باالصحاء

Introduction The thyroid gland produces two major related hormones thyroxine and triiodothyronine, commonly called T4 and T3, respectively, which play essential roles in the complete lack in the processes of metabolism, growth and development in most vertebrate tissue. Complete lack of thyroid secretion usually causes the basal metabolic rate to fall 40 to 50 percent below normal and extreme excesses of thyroid secretion can increase the basal metabolic rate to 60 to 100 percent above normal [1, 2] . Thyroid secretion is controlled primarily by thyroid stimulating hormone (TSH) secreted by the anterior pituitary gland. Small amount of reverse triiodothyronine (rT3) and other compounds are also found in venous blood [1]. The synthesis of the thyroid hormones requires (150 -200 µg) of iodine daily. Most dietary iodide is reduced to iodine before absorption. Iodine after conversion to iodide in stomach is rapidly absorbed from the gastrointestinal tract and distributed in the extra cellular fluid [3].There are three thyroid hormones binding proteins in human plasma:

albumin, with high capacity and low affinity; thyroxine binding globulin (TBG), with low capacity and high affinity, and transthyretin (TTR) with an intermediate capacity [4]. Thyroid hormone levels are under strict control; this is achieved mainly by feed-back inhibition through: Hypothalamic-pituitary – thyroid axis (HPTA); thyrotrpin – releasing hormone (TRH).l.Thyroid hormone regulation ,thyroid stimulating hormone (TSH) secretion or negative – feed back system on pituitary secretion of (TSH); and factors altering (TSH) secretion ,such as somatostatins dopamine and /or glucocorticoids [5]. Some changes in thyroid function test results are observed in most of the acute and chronic illnesses (i.e. renal diseases, cardiovascular diseases, inflammatory conditions and pulmonary diseases). Alteration in thyroid function test findings may reflect changes in production of thyroid hormone by effects on the thyroid itself, on the hypothalamic - pituitary thyroid – axis, on peripheral tissue metabolism of the hormones, or a combination of these effects [6] .




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A general conviction exists that patients with thyroid function test abnormalities do not have hypothyroidism despite the low serum hormone levels in blood and low T3 in most of the tissues. Many patients with non- thyroid illness (NTI) also receive drugs-that affect thyroid hormone regulation and metabolism. This discussion does not consider pharmacological interference an intrinsic part of the spectrum of changes in hypothalamic – pituitary thyroid function that occur in (NTI)[6]

. Assessment of thyroid function in patients with non- thyroid illness is difficult. Many of them have low serum concentrations of both T4 and T3 and their serum (TSH) concentration may also have been low. Previously, these patients were thought to be eurthyroid, and the term eurthyroid – sick syndrome was used to describe the laboratory abnormalities. It is possible that the changes in thyroid function during severe illness are protective in that they prevent excessive tissue catabolism [7]. There are two important general principles in laboratory assessment of thyroid: thyroid function should not be assessed in seriously ill patients unless there is strong suspicion of thyroid dysfunction. When Thyroid dysfunction is suspected uncritically ill patients, measurement of serum(TSH) alone is inadequate for the evaluation of thyroid function [7]. Chronic kidney disease is defined as either kidney damage or a decreased kidney glomerular filtration rate(GFR) of less than 60 ml / min / 1.73 m2 for 3 or more months [8]. The causes of the chronic kidney disease could be due to primary and secondary glomerular disease, tubulointerstial disease and vascular disease [9]. Previous studies on thyroid function tests indicate lower thyroid hormone concentration (T3, T4) with normal TSH in heaemodialysed patients compared with normal subjects [10] . Thyroid gland produces T4 but only 20% of the most metabolically active thyroid hormone T3 and 5% to 8% as the calorgenically inactive reverse t3 (rT3) hormone and T4 in tissues such as liver,kidneys and muscles [11]. Haemodialysis employs the process of diffusion across a semi permeable membrane to remove toxic products and excess fluid from the blood, while adding desirable components [12] .The aim of this work is to evaluate thyroid gland function in chronic renal failure patients as an attempt to find a relationship between chronic renal failure and thyroid dysfunction.

Materials and Methods -Selection of Subjects &Blood Collection Forty patients with chronic renal failure (20 male, 20 female) were enrolled in this study in addition to forty healthy individual as control group (20 male, 20 female). The age ranged from (25 -65) years.To compare the significance of the difference in the mean values in comparison groups, student t - test was applied; P ≤ 0.05 was considered statistically significant. Patients with ischemic heart disease, diabetes mellitus and thyroid disease (such as hypothyroidism, hyperthyroidism and goiter) were excluded [13]

. Five ml of venous blood were aspirated from control group and CRF patients at 8:00 - 9: 00 am. Blood samples were collected into plain test tubes and centrifuged after 30 minutes of collection for 10 minutes at 3000 rpm.Serum was frozen at -20 C0 till used in determination of T3, T4, TSH ,urea , uric acid and creatinine. - Determination of T3, T4 and TSH Total triiodothyronine (tT3), total thyroxin (tT4) and thyroid stimulating hormone (TSH) were evaluated using VIDAS (T3) REF, 30403, VIDAS (T4) REF 304041 and VIDAS (TSH) REF. 30400 from biomerix (France) respectively.The principle of the quantitave determination of T3, T4 and TSH combines an enzyme immunoassay competition method with a final fluorescence detection (EIFA). -Determination of Urea Urea concentration levels were determined in serum by using enables end point enzymatic (Urease – modified Berthelot reaction) in which urease hydrolyzes urea in an alkaline medium.The ammonium ions react with the salicylate and hypochlorite to form a green colored indophenol.The reaction is catalyzed by the sodium nitroprusside [4]. -Determination of Uric Acid Uric acid concentrations were determined by using uricase- peroxidose - chromogen sequence in which hydrogen peroxide is formed and reacts as Tinder type reaction[4] . -Determination of Creatinine Creatinine levels were evaluated according to Jaffes method.The production of orange color after the addition of alkaline picrate. The color is proportional to the concentration of creatinine[4] .


67

Results and Discussion Table (1) shows the (mean ± SD) of T3, T4, TSH, urea, uric acid and creatinine concentrations in sera of patients w ith chronic renal failure and control group,in which P ≤ 0.05 was considered significant . Table 1: leve ls of T3, T4, TSH, urea, uric acid and creatinine concentrations in se ra of patients with renal failure and control group.

This study shows highly significant reduction in T3 and T4 concentration in patients serum with CRF compared to control group (P ≤0.05) . Intensive studies revealed that renal insufficiency affects thyroid function in multiple ways, including altered peripheral hormone metabolism, disturbed binding to proteins, reduction in tissue thyroid hormone content, and iodine accumulation in thyroid gland [14,15].Another explanation is that the reason for the decrease in T4 could be attributed to multiple factors such as deficiency of thyroxine binding - globin (TBG) [16,17]. In the present study serum TSH was measured in CRF and control groups.CRF group had TSH above normal range. Some authors interpret this TSH elevation as a sign of recovery from a hypothyroid state despite the distortion of TSH in some euthyroid patients with NTI, who have significant elevation of TSH due to underlying primary hypothyroidism[18] . Highly significant elevation was found in urea, uric acid and creatinine in serum of CRF patients compared to control group.Increase in urea in renal failure are caused by impaired ability to excrete proteinaceous catabolites because of marked reduction in glomerular filtration rate(GRF).Increases in serum creatinine are also a result of decreased renal excretion[19] .

References 1. Guyton A.C. and Hall J. E.: "Text Book

of Medical Physiology",11 th ed, Sanders, Elsevier Inc 1600 John F. kenedy Blvd, Philadelphia,2006, 2-9.

2. Iwasaki y., Morishita M., Asai M. , Onish: A., Yoshida M., Oiso y. and Inoue k. :Effects of hormones targeting nuclear receptors on transcriptional regulation of the growth hormone gene in the MtT/S rat somatotrope cell line . Neuroendocrinology, 2004, 79, 229 – 236.

3. Andreoli, Bennett, Carpenter:" Cecil Essentials of Medicine", 4 th ed, W. B. Saunders Company, New York,1997,111-116.

4. Benvenga S.: "The Thyroid, Fundamental of Clinical Text Books", 9 th ed. Le,Utiger RD(Eds),Lippincot Williams and Willkins, Philadelphia ,2005, 97.

5. Zoeller R. T., Tan S.W. and Tyl R. W:"General background on the hypothalamic-pituitary-thyroid(HPT)axis", Crit Rev Toxical. ,2007 ,37 (1-2),11 - 53.

6. Serhat Aytug MD., Lawrence E and Shapiro MD :Euthyroid Sick Syndrome,EMedicine Specialties,Endocriology,Thyroid,2007,18,55.

7. Douglas S. Ross:Thyroid Function in non –thyroid Illness,: Up to Date CME, 2007,134,1-7.

8. Levey As, Coresh J and Bake E.:National kidney foundation practice guidelines for chronic kidney disease:evaluation,classification,and stratification, Ann Intern Med.,2003, 139(2),137-147.

9. Chobanian AV, Bakris GL, Black HR, Cushman WC: Green LA and Izzo JL:The seventh report of the joint national committee on prevention, detection, evaluation and treatment of high blood pressure, JAMA,2003, 289 (19),2560 – 2572.

10. Goffin E., Oliveira DBG, Raggatt P. and Evans DB:Assessment of the thyroid function of patients undergoing regular haemodialysis, Nephron.,1993,65, 568–572.

11. Shamsadini S. , Darvish and Abdollah H.:Blood urea nitrogen and thyroid hormone levels before and after haemodialysid, Health Journal,2006,12,55.

12. Hakim RM and Lazarus JM. :Medical aspects of haemodialysis,In:Brenner BM,Rector FC,eds. The

Subjects

Control (n=40)

Patients (n=40)

t -Test

T3(ng/ml) 1.55 ± 0.54 0.63±0.05 P ≤ 0.05

T4(µg/ml) 9.13±2.84 3.32±1.99 P ≤ 0.05

TSH ( µIU/ml)

2.41± 0.52 6.22±3.31 P ≤ 0.05

Urea (mg/dl) 35.27±5.94 120.81±20.62 P ≤ 0.05

Uric acid (µmol/L)

300.7± 85.05 1220.6±24.1 P ≤ 0.05

Creatinine (mg/dl) 0.94 ± 0.19 7.42 ±1.94 P ≤ 0.05


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kidney.Philadelphia, Saunders ,1986 ,5,1791.

13. Lisandro lrizarry MD. , Nadine A. and Jeffry G.:Toxicity,Thyroid Hormone ,Medicine specialties, Emergency Medicine,Toxicology,2008,65,44-50.

14. Lim Vs:Thyroid function in patients w ith chronic renalfailure, Am J. kidney Discase,2001,38(1), S80 – S84.

15. Kaptein EM , Quion – Verde H., ChoolJian CJ, Tany ww , Friedman PE , Rodriquez HJ and Massry SG:The thyroid in end –stage renal diseases, Medicine (Baltimore),1988,67,187 – 197.

16. Biff F. palmer : Metabolic disturbances in chronic renal failure,Saudi Journal of Kidney disease and Transplantation,2002,13(3),273-280.

17. Boelen A, Mass MA, Lowik CW., Platvoet MC. and Wiersinga WM:Induced interleukin-6 (IL-6) knock-outmice:A causal role of IL-6 in the Development of the low 3,5,3-triiodothyronine syndrome, Endocrinology,1996,137,5250 – 5254.

18. Michalak M. , Vagenakis AG. , and Makri: M:Dissociation of the early decline in serum (T3) concentration and serum IL-6 rise and TNFalpha in nonthyroidal illness syndrome induced by abdominal Surgery,J. Clin. Endocrinol Metab.,2001,86 (9),4198 – 4205.

19. Shivananda Nayak B.: "Manipal Manual of Clinical Biochemistry", 3 rd ed., Medical publishers LTD., New Delhi,2007,182 – 186.

Iraqi J Pharm Sci, Vol.19 (1) 2010 Effect of cadmium chloride in thyroid gland

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Effect of Chronic Exposure of Cadmium Chloride in Drinking Water on Structural and Functional Aspects of Thyroid Gland in Mature

Male Rabbits#

Samir H. Cheyad*,1 , Kalisa K. Khudier** and Khtan A. AL-Mzain**

* Ministry of Industry ,General Commission for Industrial Research and Development AL-Razi Center for Research and Medical Diagnostics.

** Department of Physiology , College of Veterinary Medicine ,University of Baghdad , Baghdad, Iraq.

Abstract The effect of chronic exposure to two different levels of cadmium chloride (CdCl2) 30 ppb and 40 ppb in drinking water for 12 weeks on thyroid function of mature male rabbits was studied. Eighteen mature male rabbits were randomly divided into three groups (each of six ) , control group (group I ): were offered ordinary tap water , and treated groups (II and III ) were offered tap water containing 30ppb and 40 ppb respectively for 12 weeks .Serum concentration of thyroxin (T4 ) and triiodothyronine (T3) were measured every six weeks ,as an index of thyroid function , further more , section of thyroid gland were prepared for histological studies. The results showed that chronic exposure of male rabbits to two different levels of CdCl2 caused significant decrease (p<0.05) in serum T3 and T4 in both treated groups in compared with control group, in addition, there were histological changes in thyroid gland of treated groups manifested by hyperplasia w ith presence of large number of varying size of microfollicels and hypertrophic thyrocytes, small colloid with little secretion . Key wards: Cadmium, Thyroid gland, Thyroxin, Triiodothyronin, Thyrocyt

الخالصةوم ن من كلوريد الكادمي ة تأثير التعرض المزمن لمستويين مختلفي في ماء ) جزء بالبليون ٬40 30( اجريت هذه الدراسة لمعرف

ولمدة ة الغدة الدرقية في ذكور االرانب البالغة 12الشرب خدام 0اسبوع على وظيف ست رنبا بالغا قسمت عشوائيا الى ثالث 18تم ا ا مجاميع متساوية group I(مجموعة السيطرة : ) II group( استلمت ماء عادي طيلة فترة التجربة ومجموعتي المعالجة )

وم بتركيز ا ستلمت ماء الشرب مضافا اليه ) III group( و ) ٬ جزء بالباليون 30(ستلمت ماء الشرب المضاف اليه كلوريد الكادمي ان 12جزء بالبليون ولمدة 40كلوريد الكادميوم بتركيز ة الثايروكسي T4اسبوعا تم قياس هرموني الغدة الدرقي وتراي ايودو

T3ثايرونين ة خذ المقاطع النسيجية للغدة الدرقي كل ست اسابيع فضال عن ا اظهرت النتائج ان تعرض ذكور االرانب المزمن .ويا خفاضا معن ن مختلفين من كلوريد الكادميوم في ماء الشرب قد سبب ان ويي ي في مصل الدم ف T4و T3في تركيز هرموني لمست

ة فرط النسيج مقارنة مع مجموعة السيطرةمجاميع المعالجة ة حدوث حال ة للغدة الدرقي كما اظهرت الفحوصات النسيجي )hyperplasi (ة من الخاليا الطالئي وباحجام مختلفة محاطة بطبق ة hypertrophic( ة ــــتمثلت بوجود عدد كبير من الخاليا الدرقي

thyrocyte ( ة الغروان ة في افرازاته) colloid (فضال عن وجود نقص واضح في كمي خل الجريب مع قل . دا

Introduction Cadmium is modern toxic metals (discovered in 1817) . Its main use in electroplating because of its anticorrosive properties, it also used as color pigment of paints, plastics and as cathode material for nickel –cadmium batteries, cadmium is an important source of environment pollution (1). Hazardous waste disposal sites are large source of Cd concentration found in soil and water . Tobacco smoke is the main reason for cadmium accumulation in our body (2),on other hand , the major route of cadmium intake ( for non smoker ) is ingestion , this is largely due to the presence of Cd ( 2-40 ppb ) in food staff

of natural origin e.g. cereals beans , carrots , beverage, coffee and tea (3) ,or by the ingestion of contaminated food especially fish (4, 5) . The acute toxic effect of Cd are generally restricted to the lung ,where as the effects following chronic Cd exposure in human are multisystemic and include nephropathies emphysematous alteration in the lung , cardiovascular disease , and bone damage possibly ( osteomalacia and osteoperosis ) (6) . Wealth of evidence suggested that heavy metals including cadmium exert profound toxic effects on the activities of a number of endocrine gland including thyroid gland (7,8) .

# Based on oral presentation in the seventh scientific conference of the College of Pharmacy /University of Baghdad held in 26-27 November 2008. 1Corresponding author E- mail : [email protected]@yahoo.com Received :14/4/2009 Accepted : 9/5/2010



70

Cadmium intoxication has been reported to reduce the thyroidal iodide uptake (7) and inhibits hepatic conversion of thyroxin (T4) to triiodothyronine (T3) in rats (9) and mice (10). It has been previously reported that subcutaneous injection of cadmium chloride (CdCl2 ) (mg/kg B.W ) for ten weeks to male rabbits resulted in a significant reduction in serum T3 concentration and hepatic T4 production (11) . The direct relationship between heavy metals poisoning and thyroid dysfunction were studied in male rabbits by Ghosh and Battacharya ,1992 , within 24 hours of intramascular injection of CdCl2 (15mg/kg B.W) a significant increase in thyroid activity over the control with a concomitant rise in T3 titer were observed .. The toxic effect of cadmium on thyroid gland functions has been studied , yet i ts effect at above the permissible level and below the toxic one are questioned , to this aim the present study dedicated . Materials and Methods Number of mature male rabbits 800- 1000 g of local breed were acclimated to holding facilities for one week prior to commencement of dosing . Animals in all stages of experiment were housed in clear plastic cages in conditioned room (22-25 c◦ ) with controlled lightening ,eighteen rabbits were randomly and equally divided into three groups and were treated for three months as fallow : group I, rabbits in this group were received tap water and served as a control group , the group II were received 30 ppb cadmium chloride in drinking water , while the animals of group III were received 40 ppb of cadmium chloride in drinking water . Fasting blood samples were collected at zero, 6, and 12 th weeks of experiment via cardiac puncture technique , then serum was separated and frozen at – 20 c◦ for hormonal analysis ,tetraiodothyroninT4 and triiodothyronin T3 were determined by using immuno assay detection (12). For histological studies rabbits were killed and a portion of trachea with intact thyroid gland was dissected free of connective tissue and preserved in 10% formaline buffer solution till the preparation of histological sections . Tissues were embedded in paraffin and several tissues sections were prepared, and stained with Hematoxylin- Eosin stain (13).Statistical analysis of data was done on the basis of two – way analysis of variance (ANOVA) , using a significant level of p < 0.05 (14) . Results The effect of the two different levels of cadmium chloride on mean values of serum T3 concentration of male rabbits are shown in

table (1) , serum T3 concentration showed a significant decrease (p< 0.05 ) at the 6 th week of exposure to cadmium chloride in group III and at the 12th week of experiment in group II as compared with control . In cadmium treated groups , In table(2) thyroxin serum concentration significantly decreased (p<0.05) compared to control , such decrement was observed at the 6th –12th week of experiment. Within groups , the values tended to decrease significantly (p<0.05 ) with time (at 6 th-12th week ) as compared with the pretreated period . The histological structure of thyroid gland of un treated rabbits was shown in figure (1) , the thyroid follicles containing colloid of uniform color , oval in shape , lined by cuboidal thyroidal follicular cells , figures 2,3 illustrated the histological changes in thyroid gland of rabbits following 30 and 40 ppb of CdCl2 ( group II and group III ),the figures showed a case of hyperplasia manifested by presence of thyroid microfollicels of varying size lined by high columnar thyroidal follicular cells (hypertrophic thyrocyte ) , small colloid with little secretion in follicular lumen ( figure 2) , while the figure 3 showed a large area of irregular and varying size microfollicels with hypertrophic thyrocyte with little colloid secretion . Table 1 : Se rum triiodothyronine (T3) concentration (nmol/l) in rabbits treated with two different levels of cadmium chloride in drinking wate r.

Groups Time ( weeks)

I

II

III

Pre-treatment

0 0.43± 0.01 A a

0.44±0.56 A a

0.44±0.01 A a

Du

rin

g tr

eatm

ent 6

0.45±0.01 A a

0.43±0.01 A a

0.41±0.02 B b

12 0.44±0.01 A a

0.40±0.02 B b

0.40±0.02 B b

Values are expressed as mean ±SE n = 6/group Groups: I = control, II = rabbits received 30 ppb of cadmium chloride in drinking water, III = rabbits received 40 ppb of cadmium chloride in drinking water. Capital letters denote between group differences, P< 0.05 vs. control. Small letters denote within same group differences, P<0.05 vs. pretreated values.


71

Table 2: Se rum tetraiodothyronine ( T4) conce ntration (nmol/l) in rabbits treate d with two diffe re nt levels of cadmium chloride in drinking water. Groups

Time ( weeks)

I

II

III

Pre - treatment

0 58.17±0.7 A a

57.42±0.56 A a

57.12±0.42 A a

Du

rin

g tr

eatm

ent 6

58.5±1.25 A a

51.0±2.4 B b

49.2±1.6 B b

12 58.35±1.3 A a

44.5±1.9 B c

41.7±0.4 B c

Values are expressed as mean ± SE n = 6/ group Groups: I = control, II = rabbits received 30 ppb of of cadmium chloride in drinking water, III = rabbits received 40 ppb of cadmium chloride in drinking water. Capital letters denote between group differences, P< 0.05vs. control. Small letters denote within group differences, P< 0.05 vs. pretreated values.

Figure 1:.Section in thyroid gland of untreated rabbit (group I), note thyroid follicles with thin e pithelial lining cells (thyrocyte) (arrow), fille d within colloid secretion (c). H&E, 40x

Figure 2: Section in thyroid gland fro m cadmium treated rabbit (group II), note follicular- cells of varying size, high hypertrophic thyrocytes (arrow), with a little area of micro colloid secre tion.(C ) H &E. 40x

Figure 3 :Section in thyroid gland from cadmium treate d rabbit (group III), showing large area of microfollice ls of varying size with hype rtrophic thyrocyte (arrow), and hardly little secretion in the lumen. (C) H&E. 40x

Discussion This study showed that exposure of rabbits to cadmium bring the animal to the case of hypothyroidism manifested by significant decrease in serum T3 and T4 concentration associated with biological changes . To the best of our knowledge , the effects of chronic exposure to CdCl2 at level 30 and 40 times above the permissive level on thyroid gland feature , has not been studied yet , however considerable body of evidence how exist attesting the involvement at heavy metals including cadmium in the toxicity of a number of endocrine glands including thyroid gland (8,9) . Many studies suggested the mechanisms including affecting hepatic thyroxin metabolism through reduction in the activity of hepatic “ Outer Ring Deiodinase ORD” an

C


72

enzyme which responsible for conversion of major circulating form of thyroid hormone (T4) to more biologically active form(T3) with disruption of T3 signaling leading to reduction in T4 conversion (15). In addition cadmium may cause reduction of thyroidal I uptake by damages the structure and function of both follicular and parafollicular cells of thyroid (9,10). Long term exposure to cadmium may induce the activity of hepatic microsomal enzyme especially UDP-GT (Uridine DiPhosphate Glucournyl Tranferase ) (16) and phenol sulftransferase (17) resulting in rapid clearance of T3 and T4 , on other hand , accumulation of cadmium in mitochondria of thyroid follicular epithelial cells might disturb the oxidative phosphorylation of this organelle with subsequent loss of energy supply leading to inhibition in the synthesis and release of thyroid hormone (18) . The suspected selenium deficiency caused by cadmium treatment may lead to histological changes by activation fibrotic process in which inflammatory reaction and excess transforming growth factor B play a role in thyroid gland morphology . (19) Gupta P and Kar A (1999) study the relation between cadmium and selenium , vitamin E and thyroid dysfunction in chicken, in this study they suggested that cadmium decrease T3, probably by inhibiting hepatic 5-monodeiodinase (5D-I)activity, which is a selenium dependent function. Cadmium is known selenium antagonist while vitamin E facilitate selenium metabolism .Vitamin E was shown to protect against cadmium toxicity and maintain 5D-I activity and T3 levels, while the experimenters concluded that the metal –induced inhibition in hepatic 5 D-I activity is mediated through lipid piroxidation my conclusion is that the cadmium inhibited 5D-I activity by decreasing selenium.While vitamin E does decrease lipid peroxidation it does this by facilitating selenium metabolism and selenium is the key metal in glutathione peroxidase which is a potent inhibitor of lipid peroxidation (9) Accordingly we can speculate that selenium deficiency may occur in this study following cadmium exposure resulting in the damage of thyroid gland , decrease the concentration of both (T3 and T4) and inhibit monodeiodinase activity leading to state of hypothyroidism . Acknowledgment The author is grateful to Dr.Barrh N. Al-Oqaily for his helps.

References 1. Flanoga I , Tusek K , Stengar M .

Mercury , selenium and cadmium in human autopsy from idrijaresidents and mercury mine workers . Environ. Res. 2000; 84(3): 211-8 .

2. IARC . IARC Monographs on evaluation of carcinogenic risk of chemical to humans: Beryllium, cadmium , mercury and exposure in the glass manufacturing industry ,Vol.58World Health Organization , International Agency for Research on Cancer , Lyon , France 1993 ; pp: 119-146 , 210- 236 .

3. Satarage S , Haswell, E. and Moore , M R . Safe levels of cadmium intake to prevent renal toxicity . Br. J. Nutr. 2000 ; 84(6):791-802 .

4. Hooth M j , Deongelo , George A B , Gaullord E T. Subchronic sodium chloride exposure in drinking water result in concentration independent in rat thyroid follicular cell hyperplasia . Toxicol. Pathol. 2001 ; 17: 250-295 .

5. Al-Taai , M.S.. Trace metals in water , sediments , fishes , and aquatic plants of Shatt- Al- Hilla . ph. D. thesis, college of science . University of Babylon . 1999.

6. Morimotol U K , Kai K , Okazaki Y . Uncoupling between bone formation and chronic resorption in overiectomized rats with cadmium exposure . Toxicol.Appl. phrmacol.2000 ; 164(3): 264-72 .

7. Khalil Badiei , Pegah Nikghadam . Effect of cadmium on thyroid function in sheep . Comp Clin Pathol .2009 ; 18: 255-259.

8. Susan C , Tlton Ch, Faran , M. Effect of cadmium on reproductive axis of Japanese medaka , Comparative Biochemistry and physiology part .2003 ; C136:265-267.

9. Gupta P, Kar A. Cadmium induced thyroid dysfunction in chicken ; hepatic type I iodothyronine 5-D-I activity and role of lipid peroxidation . comp. Biochm .Physiolo. Pharmacol. Endocrinol.1999 ; 123(1): 39-44.

10. Barbara P. Structure and function of thyroid follicular cells in female rats chronically exposed to cadmium .Bull.Vet.Inst.Pulawy 2003; 47:157-163.

11. Yoshida K , Sugihira N , Suzuki . Effect of cadmium on T4 outer ring monodeiodination by rat liver . Environment Research1987; 40: 400- 405.

12. Birsak H J , Hotz A. The chemical and the thyroid J.Nucl.Med.1991; 18:761-778.

13. Luna L G. Manual of Histological Staining Methods of the Armed Forces institute of Pathology .1968 ; (3rd ed) .


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McGrow - Hill Book Company . NewYork .

14. Snedecor G W, Cochran W G. Statistical Methods,1980; 7th ed . The Lowa State University Press, Ames.

15. Amma L L , Wong C Z , Venstrom B , Forrest D . Distinct tissue specific roles for thyroid hormone receptors β and α – in regulation of type 1- deiodinase expression . Mol. Endocrinol. 2001; 15(3) : 467-475.

16. Wade M G , Sophi P , Kenneth W, Edward Y. Thyroid toxicity due to Subchronic exposure to a complex mixture of 16 organochlorines ,lead ,and

cadmium . Toxicological Scienes. 2003; 67,207-18 .

17. Ghosh N, Bhattacharya S. Thyrotoxicity of chlorides of cadmium and mercury in rabbits . Biomed. Environ. Sci. 1992 ; 5(3) : 236-40 .

18. Yoshizuko M , Mori N , Hamasaki K . Cadmium toxicity in throid gland of pregnant rats. Exp. Mol. Pathol. 1991; 55(1)97-104 .

19. Ruze M , Juna C, Jose G. Single and multiple selenium –zinc-iodine deficiencies affect rat thyroid metabolism and ultra structure .J. Nutr.1999 ; 129:174-180.

Iraqi J Pharm Sci, Vol.19(1) 2010 Helicobacter pylori

74

Study the Prevalence of Helicobacter pylori Infection by Different Diagnostic Methods

Ibtihal N. Saeed*,1 and Aseel I. Ibrahim* * Department of Clinical Laboratory Science,College of Pharmacy,University of Baghdad, Baghdad, Iraq .

Abstract A total of 41 patients with gastro duodenal symptoms (show signs of inflammation with or without duodenal ulcer) . 21 males (51.2%) and 20 female (48.8%) with an average age 0f (20 – 80) years old under going gastrointestinal endoscopy at Baghdad teaching hospital in internal disease clinical laboratory , between (February – June) 2009 . Biopsies specimen of antrum , gastric fundus ,& duodenal bulb were examined by the following methods (rapid urease test , Giemsa stain section to detect bacteria , & Haematoxilin and Eosin stained section for pathological study which are considered the gold standard methods , sera or plasma from these patients were tested by immunochromotography (ICM),serological method for IgG antibodies to H. pylori. History picture are( use of certain medication , tobacco , alcohol, and current infection are taken). The results showed that the percentage of prevalence (positive results)were (83%) by histopathological method while it gave only(73%) by serological method and(66%) by rapid urease test, and the prevalence in males was more than in females (44%), (39%)respectively ,and also the prevalence increase with age (40 – 60) 14 out of 15, most patients show gastritis and duodenal ulcer, 25 (60%) by endoscopy diagnosis and 7 (17%) show malignant cancer ,while 9(22%) without any symptoms. The sensitivity of urease test (82%) and specificity (88.1%) and by ICM sensitivity (86%) and specificity (67%) comparing with gold standard methods 100% . The aim of this study is to compare the different diagnostic techniques of Helicobacter pylori infection by using invasive methods (histological examination of gastric & duodenal biopsies stained by Giemsa &Haematoxilin & Eosin methods , & rapid urease test which is considered the gold standard methods & non-invasive serological methods such as ICM rapid test , all these tests provide information about the incidence and prevalence of H. pylori in population , diagnostic value for each test also the eradication of person. Keywords: Serology, Helicobacter pylori , gastric ulcer , Diagnosis

الخالصةريا البحث دراسة انتشار بكت ة من Helicobacter pylori يتضمن عينة عشوائي واالثنى عشري في المعدة لقرحة المسببة

منها مختلفة طرق تشخيصية وذلك باستخدام في بغداد الطب التعليمي مستشفى مدينة الناظور في إلى شعبة وا ادخل المرضى الذينواجد البكتريا في النسيج الفحص النسيجي باستخدام صبغة الهيماتوكسيلين وصبغة الكمزا وذلك لدراسة التغيرات النسيجية وف , حص ت

ى دليل الفينول األحمر عل باالعتماد على وسط اليوريا السائل الحاوي إنتاج إنزيم اليورياز التحقق من كذلك فحص العينات بطريقةسير ولوجي لألجسام المضادة ة السريعة IgGوكذلك بالطريقة الغير مباشرة بالفحص ال وجد ان نسبة وقد , ICMباالعتماد على الطريق

ة بالرجال 4560االنتشار واإلصابة تزداد مع تقدم العمر وخاصة بين األعمار وان اإلصاب ة . %39نسبيا أعلى من اإلناث %44سن المعدة واالثنى عشري هم حوالي ه المرضى المصابين بقرحة ان يبين التشخيص األولي بالناظور المصابين بسرطان %60ان بينما

ن وال %22المعدة ة %17مرضى العاديي ة الدراسة النسيجي سبة لالنتشار شخصت بطريق بالطريقة %73بينما كانت %83وان اعلى ن %66السيرولوجية و اليورياز الطرق فتتراوح بين , عن طريق فحص انزيم ة هذه سي كفاءة وحسا بالدراسة النسيجية و %100اما

ة على الت 82%, 86% .واليزبالطرق السيرولوجية واالنزيميIntroduction In 1983 Warren and Marshal (1) isolated a new curved gram negative bacillus from gastric mucosa of patients with active chronic gastritis , this bacteria was first named Campylobacter pyloris then C. pylori and finally Helicobacter pylori (2) , establishing an association between the bacteria , gastritis and peptic ulcer disease . H. pylori is the most important cause of chronic gastritis (3,4,5 ) , i t is also the most important etiological factor responsible for duodenal ulcer (3,4,5) ,gastric ulcer (3,4,5) , and has an important role in the pathogenesis of gastric cancer (6,8 ) . H. pylori is also responsible for dyspeptic patients, and screening for H. pylori in those patients improve selectivity for gastroscopy (5). The

identified virulence factors of H. pylori include the flagella used for motility through the mucus , the urease activity used for neutralizing the acid from the stomach . The cytotoxin activity which vascuolize the epithelial cells (10,11) and this examined by histopathological study . Since Marshal and Warren established the association between H. pylori , gastritis ,& peptic ulcer, a great number of diagnostic techniques have been developed (12).The first rapid and simple test developed for the diagnosis of H. pylori infection was urease test based on the capacity of the organism to produce great quantities of this enzyme (13,14,15,16) .

1Corresponding author E- mail : [email protected][email protected] Received : 15/2/2010 Accepted : 26/5/2010



75

The urease catalyzes the degradation of urea to ammonia and bicarbonate. This reaction produces an increase in the pH of the medium that can be detected by an acid – base indicator such as phenol red , that changes color from yellow to pink (15) .The velocity of the change of color depends on the urease concentration according to the numbers of bacteria present (17) .The great advantage of the urease test in the diagnosis of H. pylori is that the result can be obtained before the patient leaves the endoscopy room. The result were comparable in sensitivity and specificity with the histological and culture techniques and staining section by Giemsa stain which are considered the gold standard methods (gastric biopsy is required to perform the test) (18,19) . McNulty and Wise (15) were the first ones to use this test to detect H. pylori infection . Serological tests are useful in H. pylori infection because virtually all patients colonized with this organism under a local antibody response directed against antigens covering the surface and flagella of the organism and this antibody response detected in the serum(23,24,25). Also serological methods used to diagnose H. pylori in which no upper gastrointestinal endoscopy is required . Maastrich 1996 working group cited by Anon suggested that screening dyspeptic patient under 45 years of age for H. pylori might reduce the need of endoscopy (20,21) , but blood must be obtained to detect H. pylori antibodies (22) .H. pylori serology is alterative in comparison with other methods because it is simple , inexpensive , & less of a burden for the patient .Several kits for detection H. pylori by serology have become commercially available since the discovery of H. pylori by Warren in 1983 (1), most of these kits are based on various antibody preparations and different techniques , this lead to an increase in the number of studies that have evaluated kit characteristics . Different studies for comparison between kits to account for the different reference standards and designs used by various investigators (22,23,24,25) . Serological diagnosis simplest and least expensive , non – invasive method for IgG and or IgA antibodies , latex agglutination methods are quick tests , useful for screening purposes .ELISA based tests accurately quantities the amount of antibody (titer) present and are promising tool for assessing the efficacy of H. pylori eradication treatment (27) , also for rapid office – based serologic test , using immunochromotography (ICM) , and the immunoblott for the diagnosis of H. pylori (26) . C13 / C 14 , urea breath test are reliable non –

invasive methods for diagnosis of on going H. pylori infection (30,31) . Material and methods Samples : 41 gastric and duodenal biopsies from patients of the endoscopy department of Baghdad teaching hospital – Baghdad / Iraq , were analyzed between (February – June) 2009 , at least two biopsies were taken from the antrum of each patient for histological study send to histopathological laboratory of the hospital stained by Giemza method (Luna 1968) (32) & Haematoxilin & Eosin method (Modified m. of Guyer ,1953 ) (33)by (Gram Wegurt) to study the histological change and detecting rod shaped H. pylori. Phenol red rapid urease test A solution of urea 10% and solution of phenol red 1% were prepared for the working solution , 0.1ml of phenol red solution were mixed in 1 ml of the urea solution . The reagent is stable for two weeks of 4 – 8 °C each biopsy was embedded in 0.2 ml of the reagent and incubated at room temperature (22°C) for 1 min. Serological diagnosis by (ICM) immunochromotography method of (ACON H. pylori one step –rapid test Devise) Serum / plasma is a sample test that utilized a combination of H. pylori antigen coated particles and anti – human IgG to qualitatively and selectively detect H. pylori antibodies in serum or plasma in10 minutes after serum or plasma specimen is placed in the specimen well., it reads with H. pylori antigen coated particles in the test .The mixture migrate chromatographically along the length of the test strip and interacts with the immobilized anti – human IgG , if the specimen contain H. pylori antibodies , a colored line appear in the test line region , indicating a positive result , if the specimen dose not contain H. pylori antibodies a colored line will not appear in this region , indicating a negative result comparing with positive control – test , the result should be read at 10 min.( ACON lab. Inc – 4/08 Sorrento Valley Boulevard .San Diego ,CA 9212,USA). Personal information about past infection , treated use of certain medication , alcohol and tobacco , this result were analyzed according to age , sex ,race and another characteristics . Calculation of sensitivity and specificity Positive and negative predictive values were made using the following formula :


76

HHHHXhhhhhhhh

x xxx ˆ Ș

Results A total of 41 patients were investigated, 21(51.2%) male and 20(48.8%) female with mean age 45 years old (range 20-80year), these patients under examination showed by endoscopy diagnosis that 14(34%) of them have gastric ulcer, 11(27%) duodenal ulcer, 7(17%) with gastric cancer and 9(22%) non ulcer dyspepsia. Tables 1&2 show that the percentage of infection with Helicobacter pylori or the prevalence of infection which studed by histopathological and Giemsa staining section methods increase in males 18(44%) more than in females 16(39%) and also the percentage of infections increase with age between (40-60) years old 14(34%) out of 15(36.5%) patients, the percentage of infections more than in younger and older patients. Table (3) shows the relation between the endoscopy diagnosis with positive and negative result of infection done by different diagnostic methods , in which histopathology and Giemsa staining methods gives 34(83.0%) positive, 7(17%) negative, by serodaignosis (ICM) test give 30(73%) positive, 11(27%) patients negative while by rapid urease test 27(66%) positive, 14(34%) patients negative. The positive value of serodignosis and urease test consist 88%,79% respectively from the true positive value by histopathological study (34+) patients.From these result the high prevalence of infections were obtained first by histopathological study then by serodiagnosis methods and later the lowest value by urease test. In all test used the prevalence over 75% considered high prevalence of infection in population (6). Also if we determined the positive value of diagnosis in relation with disease or endoscopy finding, histopathological study gives 90% positive in duodenal ulcer and 80% with gastric ulcer comparing with serodiagnosis (82%),(72%) and urease test (72%),(54%) irrespectively.

Table (5) show the sensitivity& specificity for each test depending on true positive, true negative, false positive, false negative values determined in table (4), comparing with a gold standard method of diagnosis and this gives the sensitivity & specificity of histopathology & Giemsa staining methods 100% , by serodiagnosis (ICM) method (86%), (67%) and by urease broth test (82%), (87%), means that the first method gives more accuracy result than others, with many disadvantage, and the other methods give less accuracy with many advantages discussed later in discussion. Table 1 : Show the pre vale nce of H. pylori infection in male & fe male.

Percentage

of infection

Number o f positive H. pylori

Percentage Total number

44% 18 51.3% Male (21)

39% 16 48.7% Female

(20)

Table 2 : Show the prevalence of infection in diffe rent age groups.

Result

(%) Number

of positive

Perce ntage (%)

Total number

(41)

Age in years

14.6 6 21.95 9 20 – 30 12.5 9 29.2 12 31 – 40 17.0 7 17 7 41 – 50 17.0 7 19.5 8 51 – 60 7.3 3 7.3 3 61 – 70 4.8 2 4.8 2 71 - 80


77

Table 3 : Show the percentage of positive and negative H. pylori infection diagnose d by urease , se rological kit and e ndoscopy biopsies.

Sum.

Non-

Ulcer Dyspe psia No.(%)

Gastric cancer

No. (%)

Duodenal ulce r

No.(%)

Gastric ulce r

No.(%)

Total number (41)

41(100%)

34(83%)

30(73%)

27(66%)

9 (22%)

6(14.63)

7(17%)

5 (12.19)

7 (17%)

7(17%)

4(9.75%)

5 (12.19)

11 (26 %)

10(24.4%)

9(21.95%)

8 (19.5%)

14 (34%)

11(26.8%)

10(24.4%)

9(21.95%)

Histopatholog Giemsa stain

Sero.Kit

Urease

H.

pylori (+)

7(17%)

11(27%)

14(34%)

3(7.3%)

2 (4.8% )

4 (9.75 )

0

3 (7.31%)

2 (4.87% )

1(2.43%)

2 (4.87%)

3 (7.3%)

3(7.3%)

4 (27%)

5 (12.2% )

Histopatholog Giemsa stain

Sero. kit

Urease

. H pylori

(-)

Table 4 : Number of true positive , true negative ,false positive ,false negative by different diagnostic methods.

Table 5 : values of differe nt diagnostic methods .

N.P.V. %

P.P.V. %

Spe cificity %

Se nsitivity %

Test

100 100 100 100 Histopath. 100 100 100 100 Giemsa stain

54 96 88 82 Rapid Urease test 45 94 67 86 Rapid serodiagnosis (ICM)

If you know the prevalence of Helicobacter pylori in your population you can make a

judgment about the predictive value of a positive or negative test from the table(6).

Table 6 : Pre dictive value of a test with 85% se nsitivity and 79 % specificity

Probability of disease with negative result(%)

Probability of dise ase with positive result(%)

Pre valence of disease

2 31 10 16 80 50 63 97 90

Test True positive False positive True negative False negative

Histological changes 34 0 7 0 Geimza stain 34 0 7 0

Urease 27 1 7 6

ICM (Kit) serology 30 2 4 5


78

Discussion The aim of this study is to determine the prevalence of infection with H. pylori by different diagnostic methods in random population enter the endoscopy department for gastric and duodenal examination with or without symp toms of inflammation. From the 41 patients under investigations results in table (1) showed that prevalence of infection in males more than in females and these results agree with other studies done by Nicholas et al (1992), in which they found that 47% males out of 82 patients, (44%) 0f them were infected by H. pylori(25). Table (2) showed that prevalence of infection increase with age between (40-60) years old agreed with Nulty (1999) which found that the more likely age of infection in patients over 50 years old (42%) than in younger patients (21%) (22), another group of Liston R, et al (1996) cited by Nulty(1999), found that (31.7%) of elderly patients with seropositive result had no evidence of active infection determined by endoscopy and urease test. Older patients are more likely to have developed atrophic gastritis and H. pylori can not readily colonize this type of gastric mucosa ( 22). It was recognized that prevalence of H. pylori infection increase with age in a symptomatic persons in developed countries and this tend to plateau at around the age of 60 years, related to socioeconomic status and ethnicity (5,23,25).Table(3)which showed high prevalence of infection given by histopathological study which are considered invasive gold standard methods (22,25,26) , (83%) of patients gave positive result ,while by serodiagnosis (ICM) method (73%) and (65%) by urease test method which means that these methods gives lowest value of prevalence comparing with histopathological study and this because many factors were affecting on the value of the result such as for example.

- Negative values in biopsy methods histopathology & Giemsa staining section depend on patient that may be under treatment or past proven H. pylori infection treated with a course of antimicrobial drugs with proton pump inhibitors , that patient give negative and clearance of the disease in biopsy specimen but can give positive result with serodiagnosis, and so give false positive result and high prevalence than biopsies(23,25). - A negative value in urease test depend on non homogeneous distribution of the microorganism in the stomach and this situation is overcomed by use of several specimen from (3-5) for the same patient

(34,35,36) so we minimize the specimen error and this explain the 13 patients which give negative result by this method, which lowering the percentage of infection comparing with other methods. -In serodiagnostic method the percentage of infection (73%) which gives positive result and 11(27%) patients comparing with histopathological study , 4 patients only were true negative and 5 patients were false negative and only 2 patients gave false positive result , this can be explained by: Patients who are in acute case of

infection before an IgG response has developed gave false negative serological result ,means that it may be positive result in biopsy method, also negative result may be due to patient not produce circulating antibody response which detectable only with complex type of antigen (Preez-Preez, et al. cited by Nicholas(1992) (25) .

False positive result according to cross reaction antigen (3-9%) of patient have false positive result with H. pylori that might produce antibodies such as Gastrospirillum hominis and this also depend on type of antigen used in test (23,25) or it may be that patient w ith past infection that gives false positive result with slowly return antibody, it may give positive test for over 6 months from clearance of the disease (25,26,34).

Table (5)show the sensitivity & specificity of each test depend on the true positive ,true negative , false positive ,& false negative value in table (4), each positive value in histopathological study considered true positive value and each negative value considered true negative value (gold standard methods) and so sensitivity & specificity (100%) and this also agreed with other study which find that these methods gives sensitivity & specificity between (98-100%) (34), and for serodiagnosis (86% ) , ( 67%) while urease test gives sensitivity & specificity (82%),(87%).The sensitivity & specificity of serological test reported by many workers varies from (76-96%) and half of the patients with false positive result were over 50 years age. Other group found that elderly patient with positive serology had no evidence of active infection determined by endoscopy and urea breath test (14,22), other workers for the same methods (ICM) test find that the sensitivity is (96%) with(94%) specificity (26) which used this test to diagnose H. pylori


79

infections in thai children between(1.5-16 years old), other study compare different serological kits for H. pylori infection and also found that the sensitivity and specificity around (67%-86%) (22), other outhers (25) find that sensitivity of commercial ELISA kits had an accuracy between (89%- 95%).The sensitivity of urease test according to Eugenia (34) is record to be (97%) by phenol red broth test with (100%) specificity also he mention that other authors have reported that urease test with specificity between (98%-100%) and sensitivity between(64%-98%),and this agreed by Vaira(37,38), Thillainayagam (39), Mal.fertheiner(40), McNulty (15) , Arvid, Morris(42), &Westblom(14) reports specificities of (86%,98%,92%,100%) and sensitivities of (84%,92%,100%,88%) respectively, only Hernandez reports a sensitivity of (72%) and specificity of (83%) for Christensen's urea broth (43).The presence of false negative and false positive result may be explained by the patchy distribution that H. pylori present in gastric mucosa ,especially in the body and fundus of the stomach, so the microorganism can be present in one biopsy and absent in another from the same patient (34, 44,45). So the false negative value by this test were caused by the patchy distribution of the bacteria.

Conclusion It has been proposal that patient with dyspepsia could be screened for H. pylori status before it is recommended (25,22,23) and as H. pylori occurs in over (90%) of patient with chronic duodenal ulceration and up to (80%) of patient with chronic gastric ulceration (25,16,21), i t has been proposal that such an approach would help to reduce the need for endoscopy as well as cost, if such a policy were adopted only seropositive patient would undergo endoscopy and over 45 years of age or those taking anti inflamma tory drugs, this would avoid up to (23%) of endoscopies. However ,further large prospective clinical studies are needed before such an approach can be accepted. Also serological methods can be used for monitoring treatment and successful eradication of infection by detecting the fall in level of IgG antibodies in serum after 3 months of treatment. The great advantage of the urease test in the diagnosis of H. pylori is that the result can be obtained before the patient leaves the endoscopy room, making clinical management easier. The studies suggest that urease result comparable in sensitivity and specificity with histological and culture techniques, being more economic and faster (12,34), Nevertheless an endoscopy is always necessary because a gastric biopsy is

required to perform the test and also culture can be required for evaluating the sensitivity to antibiotics, so urease test should be done jointly with another diagnostic test as histology or culture. Some authors and reports go to connect in a table between prevalence and sensitivity & specificity of different methods (Table6). References 1. Warren ,J.R., and Marshall .1983.

Unidentified curved bacilli on gastric epithelium in active chronic gastritis . Lancet ii :1273- 1275. (letter.) .

2. Hernandez F., Rivera P., Sigaran , Aguilar M., Miranda J., Rodriguez D. , et al. ,1990. The first cases of Helicobacter pylori (Campylobacter pylori) reported from costa Rica . Rev. Biol. Trop. 38 : 481-2.

3. Anderson ,L., S. Holck, C. Povlsen, L. , Elsborg and T. justesen. 1987. Campylobacter pyloridis in peptic ulcer disease .Scand. J. Gastroenterol. 22 : 219-224.

4. Buck, G.E. 1990. Campylobacter pylori and gastroduodenal disease . Clin. Microbiol. Rev . 3 : 1-12.

5. Dooley C. P., H. Cohen ,P.L., Fitz gibbons , et al. 1989. prevalence of Helicobacter pylori infection and histological gastritis in a sympto matic persons.N.Engl. J. Med. 321 :1562-1566.

6. Muller, L.B.,Fagundes, R.B., Moraes, C.C., Rampazzo, A.2007. Prevalence of Helicobacter pylori infection and gastric cancer precursor lesions in patients with dyspepsia .Arg. Gastroenterol. Apr. Jun ; 44:93-8.

7. Jaskiewicz, K., H.D. Ouwrens, C.W. Woodruff, M.J. Van Wik, and S.K. Pricee. 1989. The association of Campylobacter pylori with mucosal pathological changes in population at risk for gastric cancer. S.Afr. Med.J. 75:417-419.

8. Tatsuta M., Tishi H., Okuda S., Taniguchi H., Vokota Y.1993. The association of Helicobacter pylori with differentiate type gastric cancer .Cancer ; 72 :1841-5.

9. Hu. P. Mitchell H., Li. Y.,Znou M.,Hazell S.1994.Association of Helicobacter pylori with gastric cancer and observations on the detection of this bacterium in gastric cancer cases. Am.J. Gastroenterol. ; 89:1806-10.

10. Clyne M.,Labigne A., Drumm B.1995. Helicobacter pylori requires and acidic


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environment to survive 4 n the presence of urea. Infect. Immunol.; 63: 1669-73.

11. Xiang Z., Censini S., Bayeli P., Telford J., Figura N., Rappuoli R., et al.1995. Analysis of expression of Cag A and Vac A virulence factors in 43 strains of Helicobacter pylori reveals that clinical isolates can be divided in to two major types and Cag A is not necessary for expression of the vacuolating cytotoxin.Infect. Immunol. ;36:49-8.

12. Glupczynski Y.,Diagnostico microbiologico de la infection por H. pylori . En lopez-Brea .Med.1995. Helicobacter pylori. Microbiologia ,clinica Y tratamiento. Madrid :Ed. Mosby / Doyma Libros; P. 241-59.

13. Langenberg M.,Tytgat G., Schipper M.1984. Campylobacter like- organisms in stomach of patients and healthy individuals. Lancet . ;I:1348-49.

14. Westblom T.,Madan E., Kemp J., Subik M.1988. Evaluation of rapid urease test to detect Campylobacter pylori infection .J. Clin. Microbiol .; 26:1393-4.

15. McNulty C., Wise R.1985. Rapid diagnosis of Campylobacter associated gastritis . Lancet ;I:1443-4.

16. 16Hemandez F., Rivera P., Sigaran M., Miranda J.1991.Diagnosis of Helicobacter pylori comparison of an urease test, histological visualization of curved bacteria and culture. Rev. Inst. Med.Trop Sao Pablo ; 33: 80-2.

17. Lamouliatte H., Cayla R.,Daskalopoulos G. Upper digestive tract endoscopy and rapid diagnosis of Helicobacter pylori infection .En Lee A. and Megraud F.Helicobacter pylori : Techniques for clinical diagnosis and basic research .London :W.B Saunders.

18. Goodwin CS., Blincow EW., Warren JR., Waters TE., Sanderson CR. Easton L .1985 . Evaluation of culture techniques for isolation Campylobacter pyloridis from endoscopy biopsies of gastric mucosa.J.Clin.Pathol.;38: 1127-31.

19. Gray SF., Wyatt JT., Rathbone BJ.1986. Simplified techniques for identifying Campylobacter pyloridis.J.Clin.Pathol. :39:1279.

20. Patel P., Khulusi S., Mendell MA., Lioyd R., Jazrawi R., Maxwell TD., et al.1995. Prospective screening of dyspeptic patients by Helicobacter pylori serology .Lancet;346: 1315-8.

21. Anon. Current European concepts in the management of Helicobacter pylori infection. The Maastrich Consensus Report .Gut;41: 8-13.

22. CAM MucNulty,P. Nair, BE. Watson, Jsuff, RMValori.1999.A comparison of six commercial kits for Helicobacter pylori detection. Communicable disease and public Health.

23. Evans, D.J.,D.G. Evans, D.Y. Graham, and P.D. Klein.1989.A sensitivity and specific serologic test for detection of Campylobacter pylori infection. Gastroenterology 96: 1004-1008.

24. Marshall, B.J., D. B. McGechle, G.J. Francis nd P.J. Utley. 1984. Pylori Campylobacter . Serology . Lancetii :281 . (Letter to the editor).

25. Talley, J.N., Kost, L.,Haddad,A., and Zinsmeister, R.A. 1992 . Comparison of commercial serological tests for detection of Helicobacter pylori antibodies. Journal of Clin.Microbiol.Des.Vol.30,No. 12 , 3146-3150.

26. Treepongkaruna, S., Nopchinda, S., Taweewongsoun, A.A rapid serologic test and immunoblotting for the detection of H. pylori infection in children.Journal of tropical pediatrics ,Oxford Journals. Medicin, Vol. 52, No. 4, PP:267-271.

27. LE. Crabtree,TM. Shallcross, RV. Heatley, and JI. Wyatt. 1991. Evaluation of commercial ELISA for serodiagnosis of Helicobacter pylori infection. Jour.of Clini. Path .;44:326-328.

28. Yukio bata, Shogo Kikuchi, Hiroto Miwa, Kiyoko Yagyu, Yingsong Lin, and Atsushi Ogihara. Diagnostic accuracy of serological kits for Helicobacter pylori infection with the same assay system but different antigens in a Japanese patient population.

29. R.J.F. Lahiej, H. Straatman, J.B.M.J. Jansen, and A.L.M. Verbeek. 1998. Evaluation of commercially available H. pylori serology kits: a Review. Journal of Clinic. Microbiology,October, Vol. 36, No. 10,P:2803-2809.

30. Bell GD, Harrison G, Morden A, Jones PH, Gant PW, et al. 1987. C13-urea breath analysis, a non-invasive test for Campylobacter pylori in the stomach. Lancet; I: 1367-8.

31. Rauws EAJ, Van Royens EA, Langenberg W, Woensel JV, Trij AA, Tytgat GN. 1989. C14-urea breath test in C. pylori gastritis. Gut ;30:798-803.

32. Luna, L.G.,1968.Manual of histological staining methods of armed Forces institute of pathology 3th edition.The black stone


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division,Mcgyos, Hill book Co., New York.

33. Guyer, M.F.,1953. Animal micrology 5 th edition .The university of Chicago press. Chicago.

34. Eugenia Ma.,Quintana-Guzman,Karl Schosinky-Nevermann, Maria L. Arias-Echandi, Henry Davidovich-Rose, 1999.Comparative study of urease tests for Helicobacter pylori detection In gastric biopsies.Rev. Biomed.;10:145-151.

35. Kjoller M,Fischer A,Justesen T,1992. Transport conditions and Number of biopsies necessary for culture of Helicobacter pylori,Eur. J. Clin. Microbiol. Infect. Dis.;10:166-7.

36. Van der Hulst R, Verheuls S, Weel J, Gerrits Y, Ten F, Dankert J, 1996. Effect of specimen collection techniques, transport media and Incubation of cultures on the detection rate of Helicobacter pylori ,Eur. J. Clin. Microbiol. Infect. Dis .;15:211-5.

37. Varia D, Holton J, Cairns S,1996. Urease test for Campylobacter Pylori infection. Eur. J. Gastroenterol. Hepatol;8:53-6.

38. Varia D, Holton J, Cains S, Polydorou A, Falzon M, Dowsett J, Salmon P,1988. Urease test for Campylobacter pylori :Care in Interpretation .J. Clin. Pathol.;41:812-3.

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40. Malfertheiner P, Dominguez-Munoz J, Hekenmuller H, Neubrand M, Fisher H, Sauer P. 1996. Modified rapid urease test for detection of Helicobacter pylori infection. Eur. J. Gastroenterol. Heptol.;8:53-6.

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43. Hernandez F, Rivera P, Sigaran M, Miranada J.1991. Daignosis of Helicobacter pylori: Comparison of an urease test ,histological Visualization of curved bacteria and culture.Rev. Inst. Med. Trop Sao Pablo; 33:80-2.

44. Barthel J, Everett D.1990. Diagnosis of Campylobacter pylori Infections The"Gold standard" and the alternatives. Rev Infect. Dis .;12:5107-14.

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Iraqi J Pharm Sci, Vol.19(1) 2010 Atorvastatin Dissolution

82

Enhancement of Atorvastatin Tablet Dissolution Using Acid Medium Kahtan j.Hasson*,1

* Department of Pharmaceutical Chemis try ,College of Pharmacy,Al-Mustansyria,Univers ity.Baghdad .Iraq .

Abstract In this study some generic commercial products of Atorvastatin tablets were evaluated by dissolution test in acid medium by comparing with that of parent drug Lipitor of Pfizer Company. Some of solubilizing agents were studied in formulation of Atorvastatin tablet including; surface active agent and PEG 6000 .The most effective factor was the use of PEG6000 in formulation of Atorvastatin tablet which improved the dissolution and the results of dissolution profile of formulated tablet in this work was bioequivalent to that of Lipitor .The quantitative analysis of this work was performed by using reversed phase liquid chromatography and a proper mixture of mobile phase which give a retention time for Atorvastatin about 6 minutes .

خالصة حامضي قي راء فحص االنحاللية في هذا البحث تم أج ن المنتجة من قبل عدة معامل دوائية الوسط ال على حبوب االتورفاستاتية مع قوقد تم أج ة االنحالل لحبوب االتورفاستا راء دراسة مقارن وفي )حبوب لبيتور(ركة فايزروالمنتجة من الشركة األم وهي شتين ابلي

تعتبر مساعدة للذوبان مثل صوديوم لوريل سلفيت مواد بإضافةتجارب إجراءعلى كما ان هذا البحث قد احتوى, مضي الوسط الحاولي اثيل غاليكول نوع ب فعال 6000ومادة ذات تاثير ت في دحيث ساعاالتورفاستاتين صيغة حبوب على وقد كانت المادة االخيرة

درجة ن ستاتين وأصبح تحسي االتورفا ة البحث انحاللي في هذا المصنع االصلي ستحضرمال الى ءمكافي المستحضر لبيتور( ان )ة حليالت الكمي وباستعمال خليط مناسب للطبقة المتحركة أجريتفي هذا البحث قد الت ة باستعمال الطور العكسي للكروماتوغرافيا السائل

ة تقريبا 6ليعطي وقت الفصل .دقيق Introduction Atorvastatin calcium is a synthetic lipid-lowering agent. It is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. This enzyme catalyzes the conversion of HMG-CoA to mevalonate, an early and rate-limiting step in cholesterol biosynthesis.The absolute bioavailability of atorvastatin is approximately 14%. The low systemic availability is attributed to hepatic first-pass metabolism or mucosa gastrointestinal presystemic clearance (1). The plasma concentrations of Atorvastatin and other related compounds which are metabolized by cytochrome P450 isoenzyme CYP3A4 were increased when these drugs are taken with repeated use of grape fruit juice due to inhibition of this enzyme which is located in the gastro intestinal tract (2). Atorvastatin calcium is practically insoluble in aquouse medium below pH4 (3). This physical property of insolubility in water, obviously, will lead to low dissolution in stomach medium. The first study of best dissolution formula which held by Pfizer Co. was carried on gastric medium ,and the use of alkalizing agent (Calcium Carbonate) and the surfactant ( sodium lauryl sulphate ) were recommended in formula of Atorvastatin tablet(4).Several studies were reported in attempt to enhance the dissolution of atorvastatin in its solid dosage forms; Complexation of Atorvastatin by Cyclodexetrin significantly enhanced its

dissolution, but it is rather tedious procedure (5). Atorvastatin calcium was prepared as a redispersible dry powder for emulsion which was formulated by using dextran as a carrier and poloxamer 188 as a surfactant, its dissolution showed a 2.33 fold increase compared to the pure Atorvastatin calcium powder in vitro gastrointestinal medium (6) In this present study, Lipitor tablet( 20 mg) , the patent drug formulation of Atorvastatin Calcium tablet is tested for dissolution in acid medium and the result was about 92 % dissolved Atorvastatin to the labeled amount. Different generic products of Atorvstatin tablet (commercial products) are evaluated by determination of their dissolution profiles in acidic medium .In addition,some experimental formulations of Atorvastatin Calcium were made in this work and the using of sodium lauryl sulfate in different concentrations and PEG 6000 have been evaluated by their influences on the rate of dissolutions .Determinations of the dissoluted amounts of Atorvastatin in acid medium are carried in this work by using a reversed phase HPLC method, prescribed below. Several previous methods of analysis have been used for quantitative determination of Atorvastatin in tablet including spectrophotometry (7), and HPLC method for separation of Atorvastatin and Amlodipine in pharmaceutical formulation (8).




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Experimental work Materials Atorvastatin Calcium crystalline powder ( supplied by Cadila Healthcare Limited Company ,India ), Sodium Lauryl Sulphate ( Shouguang pharm. Ind.Co.Ltd,China ), poly Ethylene glycol 6000 ( Sasol Germany GmbH. ), potassium dihydrogen phosphate (Reagent grade).Acetonitrile and Methanol (HPLC grade). Apparatus Uv.vis.spectrophotometer , specord 40, analytikjena.HPLC; knauer, Dual piston pump, UV detector and computerized recorder,tablet Dissolution tester USP Procedure 6 tablets of each product were tested, one tablet in each of six vessels of the dissolution apparatus of paddle system and 50 RPM ,using 900ml of 0.1M HCL as a medium .the filtered samples were taken at intervals; 10,15,20,25,30,35,40,45 minutes and subjected to HPLC analysis to determine the percent of Atorvastatin Calcium dissolved in medium. Method of analysis An HPLC method for the analysis of Atorvastatin Calcium was carried by using the following chromatographic conditions. Column: ODS, 25 cm x 4.6 mm Mobile phase: Acetonitrile: 0.01M potassium Dihydrogen phosphate solution (40:60), adjusted to pH 4.0.with phosphoric acid. Detection: by U.V at 240 nm. Flow rate: 1.0 ml/minutes.Atorvastatin peak appeared in 6 minutes and the analysis accomplished in 8 minutes (Figure 1)

Figure 1: HPLC chromatogram of Atorvastatin tablet in which The peak of Atorvastatin appeare d in 6 minute s and the Peaks of the excipie nts are eluted at the first minute Which indicated that there is no interfe re nce with the Assay of Atorvastatin .

The accuracy and precision of this HPLC method were proved since the relationship of different concentrations of Atorvastatin with their peak area ratio have shown a straight line with correlation coefficient of value 0.999.

Figure 2: the standard curve of Atorvastatin dilutio ns with pe rcent recovery peak area Using the reverse d-phase HPLC method. Dissolution profile Three commercial products of Atorvastatin tablet were tested for dissolution in acid medium and compared with that of patent product (Lipitor). Most of the generic formulas of Atorvastatin tablet showed low bioequivalence to Lipitor, (see figure 3 )

Figure 3 :dissolution profile s of some commercial products of Atorvastatin tablets; whe re product A ( Avas-20,MICRO Lab.India) was bioe quivalent to Lipitor table t ,however the product B (Atorvatin ,Alfaris Co.Syria) and product C (Atorvast 20,Avanzor,Syria) showed low dissolution .


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In my atte mpt to achieve good Atorvastatin tablet formulation ,different experiments were carried in this work considering the acceptable physical properties ;hardness,friability and disintegration time in addition to the main requirement; the high dissolution .The first experimental product is termed as (Product D) which contained atorvastatin calcium,calcium carbonate ,microcrystalline cellulose ,lactose,colloidal silicone dioxide ,PVD,magnesium stearate and talc .The dissolution profile of this first formula of product D was too low comparing to Lipitor tablet ( figure 4 ) Figur4: dissolution profile of Product D (the first experime ntal batch in this work) which showed very low dissolution. Therefore, product D formula was subjected to improvement and several experiments were made to evaluate the addition of solubilzing agents 1. Effect of surfactant In addition to the ingredients of Atorvastatin tablet mentioned above, different concentrations of sodium lauryl sulphate( SLS) were added in an attempt to increase the dissolution of Atorvastatin . Figure (5) shows that there is significant increase in dissolution of Atorvastatin tablet but it is lower than 80% and there is no significant differences between the added concentrations of 10,20and 40 mg of SLS in enhancement o f dissolution .

Figure 5: effect of SLS on dissolution of Atorvastatin tablet in product D. 2. Effect of poly ethylene glycol (PEG 6000) As it is demonstrated in step 1 the minimum effective concentration of SLS was 10 mg per tablet ,in addition to this substance ,PEG6000 ( finely sieved powder) was incorporated in different concentrations( 10 and 20mg ) with the active constituent of Atorvastatin tablet formulations and the dissolutions profile for each formula was tested ( see figure -6- ) . The attempts to increase the concentration of PEG6000 more than 20mg per tablet were failed because the other physical properties of tablet (hardness and friability) were affected. Figure 6 : effect of PEG 6000 on dissolution of Atorvastatin tablet in product D


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Conclusion This study indicated that the formulation of Atorvastatin 20mg tablet with the presence of SLS (10 mg) and PEG6000 (20 mg) per tablet has enhanced the dissolution of Atorvastatin to a value equivalent to that of parent product (Lipitor). References 1. Martindale, drug reference, 2007.

pharmaceutical, Press, London. 2. Effects of grape fruit juice on

pharmacokinetics of atorvastatin and ravastatin in Japanese , Ichiro Fukazawa ,et al , British journal of Clinical Pharmacology, v57, 2004, page 448-455.

3. Clark s̀ Analysis of Drugs and Poisons, 3rd Edit., Pharm. Press London ,Atorvastatin Monograph .

4. U.S.patent issued on April 18, 2006.

5. Solubility and stability enhancement of Atorvastatin by Cyclodextrin Complexation, by china Reddy et al, J.pharm.Sci.and technol.,May 2009, 63; 217-225.

6. Preparation, characterization and in vitro intestinal absorption of a dry emulsion formulation containing atorvastatin calcium ,by Yong-Mei Yin,et al, Drug Delivery ,Vol.16,jan 2009,page 30-36 .

7. Simultaneous spectrophotometric determination of Atorvastatin and Amlodipine besylate in tablet, Khan MR and Jain D. Indian J.pharm.Sci.2006; 68; 546-458.

8. Stability indicating RP-HPLC estimation of atorvastatin and amlodipine besylate in pharmaceutical formulations , DA Shahi et al ,Indian journal of pharmaceutical sciences ,2008,70.page 754-760 .

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