isolation, purification and characterization of galactomannans as an excipient from senna tora seeds...

International Journal of Biological Macromolecules 65 (2014) 167175

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l h o mepa ge: www.elsev ier .com

Isolation, purication and characterization of galas an e

Harshal Aa Research Scho Tamilb Professor and lege RNadu, India

a r t i c l

Article history:Received 7 NoReceived in reAccepted 11 JaAvailable onlin

Keywords:Senna toraGalactomannanSeed gum

eroge to guseed ghichund ticatn extr

water dissolution, centrifugation and precipitation with acetone. The polysaccharide obtained from S.tora seeds was characterized by using physicochemical and chromatographic procedures, as well as FTIR,Mass, 13C NMR and 1H NMR spectroscopy. The results indicated that the gum has the basic structure ofgalactomannans with a main chain of (14)-linked -d-mannopyranosyl units to which single -(16)-d-linked galactopyranosyl units are attached through block pattern. The rheological studies indicated that

1. Introdu

Galactomendospermseveral funtomannansbackbone wmannose/ga[2]. They arcous, stablegalactomantose contenbackbone [the galactomof the degrincluding wactions. Gal

CorresponE-mail add

(K.G. Lalitha).1 Tel.: +91 9

0141-8130/$ http://dx.doi.othe S. tora gum (1%, w/w) solution possesses pseudoplastic ow. The viscosity and other rheological prop-erties conrmed its suitability as an excipient in the development of sustained release delivery systems.

2014 Elsevier B.V. All rights reserved.

ction

annans are neutral polysaccharides obtained from the of seeds of some Leguminosae plant and they havections, including reserve of carbohydrates [1]. Galac-

are polysaccharides built up of a -(14)-d-mannanith single d-galactose branches linked -(16). Theirlactose (M/G) ratios differ according to the speciese water soluble hydrocolloids which form highly vis-

aqueous solutions [3]. The main difference betweennans from different plant sources lies in the galac-t as well in its distribution along the mannopyranosyl1,4]. The degree of substitution of galactose differs in

annans extracted from various plants. The differencesee in substitution greatly affect solution properties,ater solubility, thickening ability and synergistic inter-actomannans can often be used in different forms for

ding author. Tel.: +91 8097148638.resses: [email protected] (H.A. Pawar), [email protected]

894893301.

human consumption. Featuring different physicochemical prop-erties, galactomannans are a versatile material used for manyapplications: they are excellent stiffeners and stabilizers of emul-sions, and the absence of toxicity allows their use in the textile,pharmaceutical, biomedical, cosmetics and food industries [5].Most galactomannans used in pharmaceutical technology and cos-metics are usually unpuried gums [6]. When associated with otherpolysaccharides such as xanthan gum and kappa-carrageenan,galactomannans can form gels with new properties [710].

The four major galactomannans of commercial importancein food and non-food industries are guar gum (GG, Cyamopsistetragonolobo, M/G ratio: 2:1), tara gum (TG, Caesalpinia spinosa,M/G ratio: 3:1), locust bean gum (LBG, Ceratonia siliqua, M/G ratio:3.5:1) and Fenugreek (Trigonella foenum-graecum L., M/G ratio: 1:1)[11]. Currently the international trends demand the introduction ofalternative sources of seed gums [12] and it is therefore importantto search for alternative renewable sources for e.g. the productionof edible and biodegradable lms and coating materials. In particu-lar, Latin American sources of galactomannans are not well known,in spite of the rich biodiversity of the local ora and of the favorableclimate for their production [13].

Senna tora (L.) Roxb. belonging to the family Fabaceae is anannual under shrub which grows all over the tropical countries

see front matter 2014 Elsevier B.V. All rights reserved.rg/10.1016/j.ijbiomac.2014.01.026xcipient from Senna tora seeds

. Pawara,, K.G. Lalithab,1

lar, Ultra College of Pharmacy, 4/235, College Road, Thasildar Nagar, Madurai 625020, Head, Department of Pharmaceutical Chemistry, Ultra College of Pharmacy, 4/235, Col

e i n f o

vember 2013vised form 11 January 2014nuary 2014e 20 January 2014

a b s t r a c t

Seed galactomannans are neutral, hetnose/Galactose ratios differ from gumthe various industrial applications of ing and spreading under shrub of wapplications. The seeds have been foinvestigation deals with isolation, purSenna tora (S. tora). The galactomanna/ locate / i jb iomac

actomannans

Nadu, Indiaoad, Thasildar Nagar, Madurai 625020, Tamil

neous polysaccharides widely distributed in nature. The Man-m, resulting in a change in structure, which in turn, determinesalactomannans. Senna tora (Family: Fabaceae) is a fast grow-

seeds, pods and leaves are extensively used for medicinalo be an alternative source of commercial gums. The presention and characterization of galactomannans from the seeds ofaction was based on mechanical separation of the endosperm,

168 H.A. Pawar, K.G. Lalitha / International Journal of Biological Macromolecules 65 (2014) 167175

(throughout India, Pakistan, Bangladesh and west China). It growswell in wasteland as a rainy season weed. It is also known asChakramard in Ayurveda [14,15]. Several studies have been con-ducted throughout the last decade to investigate chemical andbiological pcosides werAntioxidanhave alreadby the scietion, Catholsupplemenbetic subjeceasy availabthe high deties of the sinvestigatedcharacterizin order to sto neutrace

2. Materia

2.1. Collect

The pod Thane, MThe seeds wdry place udra D. ShinCollege, MuSpecimen nin Ultra Col

2.2. Isolatio

The drietreatment fpowder waremove lipipowder of water and stion obtainwas pressewater for 1 hcloth. The mtiple extracwater with continued uviscous soluof 10% trichtate proteinprecipitatedtinuous stirmass was toven at 40

2.3. Purica

The abore-precipitastored in airsolution in wan almost wwas milled

2.4. Characterization of gum

2.4.1. Organoleptic evaluationThe Organoleptic evaluation of the puried gum sample was

out.odor,ture

of th

Ident ideng th

pow cove

leating plling

powd co

seco colorsacc

then mL tion

solu

Physi. Solentinthe srenta 199

. pH.ter. Tmpl

The e ele

met

. LODy of re coing mnt wing w

) = w

. Ashd waacopinat3.4.1d an

eighe of ting the ced toted wroperties of S. tora. Antihepatotoxic naphtha-pyrine gly-e reported to be isolated from the seeds of S. tora [16].t properties and inhibitory effect of the extract of S. toray been reported [17,18]. A recent study was conductedntists of the Department of Food Science and Nutri-ic University of Daegu, Korea who concluded that S. torats can help to improve serum lipid status in type-II dia-ts without signicant adverse effect [19]. In view of theility of the plant, the medicinal value of the seeds andmand of seed gums throughout the world, the proper-eed polysaccharide (gum) obtained from S. tora were. The present investigation was aimed at isolation and

ation of puried polysaccharide from the seeds of S. toraurvey its potential applications as an additive/excipientuticals and pharmaceuticals.

ls and methods

ion of plant material

s of S. tora were collected from Kalyan taluka (Districtaharashtra) in the month of SeptemberOctober 2010.ere manually separated; shade dried and kept in a cool,ntil further use. Plant was authenticated by Dr. Rajen-de, Associate Professor, Blatter Herbarium; St. Xaviersmbai and was identied as S. tora (L.) Roxb (Herbariumo. 8361). The herbarium specimen of S. tora was storedlege of Pharmacy, Madurai for future reference.

n of gum

d seeds were dehusked and de-germed by mechanicalollowed by milling and screening of the endosperm. Thes soaked in benzeneethanol solution (1:1) overnight tods and then it was dried in vacuum oven. The endospermS. tora seeds (10 g) was soaked in 200 mL of distilledtirred under overhead stirrer for 34 h. The viscous solu-ed was passed through the muslin. The marc obtainedd to remove the mucilage and boiled with 200 mL of. Viscous solution obtained was ltered through muslinarc obtained was not discarded but it was sent for mul-

tions with decreasing quantity of extracting solvent, i.e.,the increase of number of extractions. The isolation wasntil the material becomes free from mucilage. All thetions obtained were mixed together. An equal quantityloroacetic acid was added to the mixture to precipi-. The solution was centrifuged and the supernatant was

out by addition of acetone in the ratio 1:0.5 with con-ring. The coagulated mucilage, which formed as a whiteransferred to an evaporating dish and dried in vacuumC, powdered and stored in airtight containers.

tion of gum

ve obtained crude gum was dissolved in warm water,ted using ethanol (1:1), dried at 40 C, powdered andtight container at room temperature. The process of dis-ater and precipitation with alcohol was repeated until

hite precipitate was obtained. The dried polysaccharideand sifted with a 60 mesh for further use.

carriedcolor, and texquality

2.4.2. The

by usin

a. Theandwithblotswe

b. TheparefewThe

c. Polyand

d. To 2solu

e. Gum

2.4.3. 2.4.3.1repres1 g of in diffecopoei

2.4.3.2pH megum sameter.and thon the[23].

2.4.3.3stabilitmoistuon dryconstaon dry

LOD (%

2.4.3.4ash anPharmdeterm

2.4.weigheand wbottomincreasbon. Trepeatcalcula The Organoleptic evaluation refers to the evaluation of shape, taste and special features which include touch. The majority of information on the identity, purity ande material can be drawn from these observations.

ication test for gumtication of the isolated polysaccharide was carried oute following tests [20,21]:

der was mounted on a slide with ruthenium red solutionred with a cover slip. After a few seconds, it was irrigatedd acetate and the excess stain was sucked off with aaper (lead acetate solution was added to prevent undue

of the test solution). The color of the particles was noted.der sample was mounted on a slide with freshly pre-rallin soda solution and covered with a cover slip. After ands it was irrigated with 25% sodium carbonate solution.

of the particles was noted.haride was heated with distilled water for some time

cooled. Formation of gelatinous mass was noted.of polysaccharide solution, 23 drops of N/50 iodinewas added and the color of the particle was noted.tion (35 mL) was treated with 4% borax solution.

cochemical evaluationubility. The solubility is expressed in terms of partsg the number of mililiters (mL) of the solvent in whicholid is soluble. Solubility of powder was determined

solvents at room temperature as per Indian Pharma-6 [22].

The pH of the mucilage was determined using a digitalhis was done by shaking a 1% (w/v) dispersion of the

e in water for 5 min and the pH determined using a pHpH meter was set to neutral (7.4) at a room temperaturectrode was immersed into the dispersion. The readinger was recorded. Triplicate measurements were made

. The inherent moisture in additives may inuence thedosage form containing moisture sensitive drugs, so thentent of the isolated gum sample was detected by lossethod. The gum sample (1 g) was heated at 105 C until

eight in a hot air oven and percentage loss of moistureas calculated using the formula:

eight of water in sampleweight of dry sample

100

content. Ash values such as total ash, acid insolubleter-soluble ash were determined according to Indianoeia [22,24]. The following procedures were used forion of ash values.. Total ash. About 3 g of sample was accuratelyd taken in a silica crucible, which was previously ignitedd. The powder was spread as a ne, even layer on thehe crucible. The crucible was incinerated gradually byemperature to make it dull red hot until free from car-rucible was cooled and weighed. The procedure was

get constant weight. The percentage of total ash wasith reference to air dried sample.

H.A. Pawar, K.G. Lalitha / International Journal of Biological Macromolecules 65 (2014) 167175 169

2.4.3.4.2. Acid insoluble ash. The ash obtained as describedabove was boiled with 25 mL of 2 N HCl for 5 min. The insoluble ashwas collected on an ash less lter paper and washed with hot water.The insoluble ash was transferred into a silica crucible, ignited andweighed. Thpercentagethe air-drie

2.4.3.4.3the determwater. The and washedinto silica cwas repeateter was subof weight wwater-solubsample.

2.4.3.5. Spedeterminedusing standwas lled wat 20 C to stant tempedried and wdividing the25 C determ

2.4.3.6. Swemilliliters) specied cowere determ

Accuratereduced to tstoppered madded and mwas then alvolume occportion waand the me

Swellingaccording t

Swelling in

where X0 isand Xt deno

2.4.3.7. ViscOstwalds vpreparing thsolved in thup to 100 mously for apand homog

2.4.4. DifferThermal

DifferentialNitrogen

of powderefrom 30 C cooling cyc

2.4.5. X-ray diffractionDiffraction pattern of powdered S. tora polysaccharide was

recorded with an X-ray diffractometer. X-ray diffraction was per-formed at room temperature (30 C) with a diffractometer; target,

1.540 mm

Scanntudyken. ecord.

Gel p S. tpha

-seriuted

of ped byon vo

Estim totaf Dub

mL deders ct tubakenisibldard

0 to 1cg/m

Identled 2. The, mixe 7. Faportain

in 1men

of th 254 pyl ae Phion w

metvaluef valulitera

Detennosined

arat mg of thme pn.aratg oarate procedure was repeated to get a constant weight. The of acid insoluble ash was calculated with reference tod sample.. Water-soluble ash. The ash obtained as described forination of total ash was boiled for 5 min with 25 mL ofinsoluble matter was collected on ash less lter paper

with hot water. The insoluble ash was then transferredrucible, ignited for 15 min, and weighed. The procedured to get a constant weight. The weight of insoluble mat-tracted from the weight of the total ash. The differenceas considered as water-soluble ash. The percentage ofle ash was calculated with reference to the air dried

cic gravity. Specic gravity of the gum isolates was following the procedure of Skoog and West (1963)ardized pycnometer [25]. A dried and clean pycnometerith 1% gum solution that was previously equilibratedthe indicated level, capped and placed in 25 C con-rature bath for 30 min. Afterwards, the pycnometer waseighed. Specic gravity of the solution was obtained by

weight of the gum solution by the weight of water atined previously.

lling index. The swelling index is the volume (intaken up by the swelling of 1 g of test material undernditions. The swelling indices of the selected hydrogelsined by the method described by WHO [26].

ly weighed quantity of the gum (1 g), previouslyhe required neness, was introduced into a 25 mL glass-easuring cylinder. Twenty-ve milliliters of water wasixture was shaken thoroughly every 10 min for 1 h. It

lowed to stand for 3 h at room temperature. Then theupied by the gum, including any sticky mucilaginouss measured. The same procedure was repeated thricean value was calculated.

index (SI) was expressed as a percentage and calculatedo the following equation

dex (SI) = 100(

Xt X0X0

)

the initial height of the powder in graduated cylindertes the height occupied by swollen gum after 3 h.

osity. Viscosity measurements were carried out withiscometer tubes after 24 h of hydration of the gum. Fore gum solutions (0.05%, w/v), 50 mg of the gum was dis-e minimum quantity of water and then made volumeL with water. The prepared solution was agitated vigor-proximately 15 min until the solution become viscouseneous.

ential scanning calorimeter (DSC) properties of S. tora gum powder were studied using

Scanning Calorimeter., at the rate of 50 mL/min, was used as purge gas; 2 mgd material was sealed in aluminium pan and heatedup to 500 C at the rate of 10 C/min, followed by ale back to 30 C at the same rate.

Cu ( =stant 1

2.4.6. To s

was tawere rJapan)

2.4.7. The

onto Sewith Tand elweightobtain eluti

2.4.8. The

dure oin 100was admillilitthe teswas sha UVv

Stanfrom 51000 m

2.4.9. Boi

for 3 hbonatebecamtory evwas obsolvedbelow

TLCgel GFisopro(MobilDetecttion inThe Rf with Rin the

2.4.10.Ma

determbelow:

Prep100

10 mL The sasolutio

Prep10 mPrepA), lter, Ni; voltage, 40 kV; current 30 mA; time con-/s; scanning rate 2/min; measured from 5 to 50.

ing electron microscope (SEM) the surface characteristics of gum, SEM of the powderThe SEM photomicrographs of native and swelled gumed by using a scanning electron microscope (JEOL 5400,

ermeation chromatography (GPC)ora gum solution (1 mL, 0.5% in water) was loadedrose CL-4B column (bed volume 180 mL) pre-calibratedes dextran standards of known molecular weight (Mw)with water at a ow rate of 18 mL h1. The molecularolysaccharide was determined from the standard graph

plotting the log Mw of the standards versus Ve/Vo (Velume of the standards and Vo void volume).

ation of total sugar contentl sugar content was determined according to the proce-ois et al. [27]. About 10 mg of S. tora gum was dissolveddistilled water. One milliliter of 5% phenol solution

to 1 mL gum solution in acid-washed test tube. Fiveoncentrated H2SO4 was directly and rapidly added toes. The solution was allowed to stand for 10 min and. Absorbance of the solution was read at 490 nm usinge spectrophotometer.

calibration curve was prepared using solutions ranging00 mcg/mL. Solutions were prepared in triplicates fromL stock glucose solution.

ication of gum components by TLC00 mg of the gum sample in 20 mL of 10% sulfuric acid

solution was cooled and added excess of barium car-ing with a magnetic stirrer until the pH of the solutioniltered the solution and evaporated the ltrate in a rota-ator at 3050 C under vacuum until a syrupy residueed (Gum hydrolysate). The obtained residue was dis-0 mL of 40% methanol and TLC was performed usingtioned procedure.e gum hydrolysate was performed on precoated silicaplates (Stationary Phase) using mixture of n-butanol,lcohol and water in the ratio 11:6:3 as solvent systemase). Galactose and mannose were used as standard.as done by spraying the plate with 10% H2SO4 solu-

hanol and the plate was kept in oven at 110 C for 1 h.s for the separated spots were calculated and comparede of pure galactose and mannose and values reportedture [28,29].

rmination of mannose/galactose ratio (M/G ratio)e and galactose content in the polysaccharide were

in triplicate using Cleggs method (1958) as described

ion of dilute galactose and mannose solution:of galactose was dissolved in 100 mL distilled water.is solution was diluted to 100 mL with distilled water.rocedure was repeated to make the dilute mannose

ion of sample solution:f S. tora gum was dissolved in 100 mL distilled water.ion of Anthrone reagent (0.1%):


Table 1Organoleptic features of the puried S. tora seed gum.

Color Odor Taste Shape Touch and texture

Dull brown Odorless Tasteless Irregular Hard and rough

100 mg (270 mL cowater).

ProcedurOne mill

mannose wtively. Five mtube. The soeach solutiotometer. Thfollowing fo

Galactose o

where, B is t(or mannos

2.4.11. InveNo one m

the questionIn additionto obtain inthan one m

An IR sFourier tranpolysaccharpotassium blets for FTIR4000 cm1.

Mass spelectrospraMass SpectrAPI 4000 LCsolved in so

Nuclear a Bruker AvMA). The sD2O for 1H to internal

2.4.12. RheRheolog

tive informget the knowand aging pis the mainrheologicalPharmaceu

The rheostudied at d5, time: 5 m

3. Results

The gumowing powof the Orga

With rutpink and a g

hemical evaluation of S. tora seed gum.

eter Results

lity Soluble in hot water forming viscouscolloidal solution but insoluble inmethanol, ethanol, acetone, DMSO andether6.57.3 (near to neutral, less irritatingand hence suitable for uncoatedtablets)

n drying 6.7% (w/w)ntent Total 1.2% (w/w)

Acid insoluble 0.26% (w/w)Water soluble 0.34% (w/w)

c gravity (1%, w/v,tion)

0.9929

ng index 84%ity (0.

witholysin bllutioman

resu. Thelightirritastinaal drerenn, re

as aermuggee of y theseedra gu

her ahaint about 80 C (Glass transition temperature, Tg) due to loss/bound water present in the S. tora seed polysaccharide. TheAnthrone was dissolved in 100 mL sulphuric acidncentrated H2SO4 was dissolved in 300 mL distilled

e:iliter from each dilute solution of sample, galactose andere pipetted into a series of test tubes 1, 2, and 3, respec-illiliter of the Anthrone reagent was added to each test

lutions were allowed to cool for 12 min. Absorbance ofn was read at 360 nm using a UVvisible spectropho-e galactose and mannose contents were found out usingrmula and M/G ratio was determined.

r mannose content (%) = 25 BS A

he reading of sample, A is the reading of dilute galactosee) and S is the weight of origin sample.

stigation of the structure of the polysaccharideethod of determining structure will give answers to alls for determination of the structure of polysaccharides.

, each method has limitations so it is always desirableformation on particular aspects of structure using moreethod.pectrum of the polysaccharide was taken using asform infrared spectrometer (FTIR, SHIMADZU). Theide (S. tora gum) was ground with spectroscopic graderomide (KBr) powder and then pressed into 1 mm pel-

measurement in the wavenumber range of 400 and

ectrum of the puried gum sample was taken by usingy ionization (ESI) technique with Waters Quadrapoleometer (Switzerland). ESI-MS/MS was performed using/MS/MS (Applied Biosystem). The gum sample was dis-lvent mixture of dimethylsulfoxide and water (1:1).magnetic resonance (NMR) spectra were obtained usingance 300 MHz NMR spectrometer (Bruker Co., Billerica,ample (Gum hydrolysate) solutions were prepared inNMR and 13C NMR. 1H chemical shifts were referencedD2O.

ological study of S. tora gumical properties are much useful in behavior and predic-ation for various pharmaceutical products, as well as to

ledge of the effects of processing, formulation changeshenomena. In particular to the gum materials, viscosity

parameter to predict the quality of the material and behavior is a useful tool for its applicability in varioustical dosage forms as an excipient.logical behavior of 1% (w/v) solution of S. tora gum wasifferent RPM using Brookeld viscometer (Spindle no.in, 100 mL beaker).

and discussion

obtained from S. tora seeds was an amorphous freeder with dull brown color (yield = 35%w/w). The results

Table 2Physicoc

Param

Solubi

pH

Loss oAsh co

Specisolu

SwelliViscos

heatedlated pnot stagum sogalacto

TheTable 2to be sis non-trointeand or

Diffor gaisampleendothcurve sbecausto studS. tora

S. toand otmain cpeak aof freenoleptic evaluation are summarized in Table 1.henium red and corallin soda, the particles did not stainelatinous mass was not formed when the powder was05%, w/v, in water) 1.714 cP

distilled water. All these tests indicated that the iso-accharide is gum. In the iodine test, the particles didue, indicating the absence of starch. With borate ions,n gave rise to cohesive gels indicating the presence ofnans in gum.lts of physicochemical evaluation are summarized in

pH values of 1% solution of the S. tora gum was foundly acidic or near neutral, which indicated that the gumting to the mucous membrane of buccal cavity and gas-l tract, and could be used for the development of buccalug delivery systems.tial scanning calorimetry (DSC) measures the heat losssulting from physical or chemical changes within a

function of temperature. A sharp symmetric melting can indicate relative purity, whereas broad asymmetricsts impurities or more than one thermal process. DSC,its sensitivity and accuracy, has been extensively used

phase transitions of polymers. The DSC thermogram of polysaccharide is shown in Fig. 1.m shows distinct feature in DSC having one endotherm

t relatively higher temperature due to decomposition of. The seed polysaccharide exhibited broad endothermicFig. 1. DSC thermogram of S. tora seed polysaccharide.


0

1000

2000

3000

6040200

2

Fig. 2. X-ray diffraction pattern of S. tora seed polysaccharide.

latter step to cleavage325.83 C isbroad naturphous in naThe X-ray dshow any cpolysacchar

To studyder was takpowder (Nanative gummicrographirregular-shof the powdpores and crms the rebe retardedand crevicetain the druit was also

y = 0 .0083 xR = 0.9949

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

120100806040200

Abs

orba

nce

Concentraon in g/mL

Fig. 5. Calibration curve of glucose for Phenol Sulphuric Acid Assay.

uniform and the size distribution was not within a narrow range.The powder contains larger to ultra-ne particles. This might be thereason for the heavy nature of the powder. The molecular weightof puried S. tora gum was found to be 198 kDa by gel permeationchromatogr

5 replphum isound coeftrati) ofte. Th

usiTota(mea

of ths galrds olloweatio)

6 rectra re re

cpyraFig. 3. SEM of native S. tora seed polysaccharide.

is followed by depolymerization which proceeds due of glycosidic linkage. The small endothermic peak at

due to melting/decomposition of polysaccharide. Thee of S. tora gums thermogram shows that it is amor-ture even though it looks crystalline to unaided eyes.iffraction pattern (Fig. 2) of S. tora seed gum did notharacteristic peak, which indicates that the obtainedide is completely amorphous.

the surface characteristics of gum, SEM of the pow-en. The scanning electron micrographs of S. tora gumtive and Swelled) are shown in Figs. 3 and 4. SEM of

shows distinct particles of fairly dened shape. The of S. tora gum at 50 magnication (Fig. 3) shows someaped particles which points to the amorphous natureer. The SEM of S. tora gum exhibited rough surface withrevices on it (Fig. 3). Thus this micrograph further con-

Fig.nol Suthe guwas folation concen(% RSDaccuraculatedcurve. (w/w)

TLCtied astandarst fo(M/G r

Fig.IR spethat a( andgalactosults from DSC. If the surface is rough, drug release will because of the entrapment of drug particles in the poress. Hence, it can be concluded that S. tora gum can sus-g release because of its rough surface. From the SEM,evident that the particle size of the powder was not

Fig. 4. SEM of swelled S. tora seed polysaccharide.

[31,32]. Thfrom the stcosidic boncompositiovibrational the broad aphy.resents the standard curve of glucose used in the Phe-

ric Acid Assay to determine the total sugar content oflate. Using the proposed method, the calibration curveto be linear in the range of 50100 g/mL. A corre-cient of 0.9949 indicates good linearity between theon and absorbance. The % relative standard deviation

0.40 indicates that the used method is precise ande total polysaccharide content of S. tora gum was cal-

ng regression equation obtained from the calibrationl sugar content in S. tora gum was found to be 92.06%n of three determinations).e hydrolysate showed two spots. The spots were iden-actose and mannose by comparing their Rf values withf pure galactose and mannose. Galactose was liberatedd by mannose. The average mannose to galactose ratio

was found to be 5.1.presents FTIR spectrum of S. tora polysaccharide. Theof polysaccharide shows peaks at 813 and 875 cm1

lated with the presence of anomeric congurationsonformers) and glycosidic linkages, attributed to -d-nose units and -B-mannopyranose units, respectivelye broad band between 1198 and 983 cm1 resultsretching vibration of C O in C O H bonds (e.g. gly-ds) and is related with the galactomannans sugarn. The peak at 1149 cm1 corresponds to bendingmodes of C O, present in the pyranose ring, while

band between 1134 and 983 cm1 is a characteristicFig. 6. FTIR spectrum of the S. tora polysaccharide.


ra gum.

contributiobetween 28stretching athe anomercharacteristmannose [3

The analeven when any manipu

An ESI-Mm/z: 591.4,344.3, 313.2

Fig. 7 shof 90 Da) an120 Da) corpathway almannose unresidue (1to two manFig. 7. ESI-MS spectra of S. to

n of C OH bending [33]. The broad bands ranging003000 and 31003500 cm1 are attributed to C Hnd to O H stretching vibration, respectively [34]. Inic region (950700 cm1), gum exhibited the obviousic absorption at 813 cm1, suggesting the existence of5,36].ysis of ESI-MS allows studying intact oligosaccharides,present in mixtures and with low abundances withoutlation/derivatization being required [37].S spectrum of gum shows characteristic fragments at

527.5, 506.5, 490.5, 467.5, 428.5, 420.3, 392.5, 371.5,, 265.4, 217.3, 195.3, 175.4 and 145.4.

ows the ion at m/z 527.5 (loss of 60 Da), m/z 467.5 (lossd, with higher abundance, the ion at m/z 428.5 (loss ofresponding to a 16 type linkage. This fragmentation

lowed to identify polysaccharide as being composed ofits linked by a (14) linkage and an additional galactose

6) linked. The ion at m/z 344.2 (C12H24O11) correspondsnose units linked through (14) linkage [5].

Fig. 8. Fragme

Fig. 9. ESI-MSMS spectra of S. tora gntation pattern (cleavage of two bonds within the sugar ring) of sugar.

um.


Fig. 10. 13C NMR spectra of galactomannan from the Senna tora seeds, in D2O.

In order to conrm the proposed oligosaccharide structuresobserved in the ESI-MS spectra, the sample was further analyzed byLCESI-MS/ditions are residues anthe sugar ri

LCMSment at: 51(loss of 3 Hose units 138.9, 113 (out cyclizat

The 13C Well resolvd-galactopy

Fig. 11. 1H NM

Table 3Chemical shifts (in ppm) and the corresponding carbon atoms for 1H NMR signalsof S. tora gum.

Chemical shift (ppm) Corresponding C-atom (moiety)

4.899 H-1 () {-d-sugar moieties}4.683 H-2 () {-d-sugar moieties}3.999

H2 to H63.8543.621

of the galactomannan is shown in Fig. 11. In the anomeric regiontwo distinct signals corresponds to -d-mannopyranose and -d-galactopyranose were observed. The high resolution 13C NMRspectra of isolated polysaccharide contained two main signalsin the anomeric region at 101.379 ppm and 99.932 ppm, whichwere assigned to the -d-mannopyranosyl and -galactopyranosylresidues, respectively [3840]. The ratio of galactose and mannosecan also be obtained directly from the relative areas of the signalsfor H1 for both of them, and the ratio was 1:5.17 which conrmsthe previous obtained results. The chemical shifts of galatomannansummarized in Tables 35 are in accordance with those reportedin the literature [41].

The above results obtained indicated that the polysaccharideisolated from seeds of S. tora was a galactomannan with a chainof ve d-mannopyranosyl residue linked through (14) linkageand a -d-galactopyranosyl residue at O-6 of mannose unit. Theproposed structure is shown in Fig. 12.

rheological property of the gum solution was studied byg the rheogram. Fig. 13(Graph-I) represents rheogram oftress versus shear rate. It was found that the curve obtainedeasurement at decreasing shear rate was super imposed

t obtained from measurements taken at increasing shearg. 14s theses. Teudo

l shift gum.

cal sh

9 MS. Oligosaccharide cleavages under ESI-MS/MS con-the result of glycosidic cleavages between two sugard of cross ring cleavages (cleavage of two bonds withinng as shown in Fig. 8).MS spectrum (Fig. 9) of gum shows characteristic frag-4.9, 485.3 (loss of 3 hexose units 2H2O), 448.9, 3932O), 378.9 (two hexose units + H2O), 325.2 (two hex-2H2O), 311.0, 248.9, 232.9, 180.9 (hexose unit), 175.0,hexose unit 2H2O-HCHO), 102.9 (loss of water with-ion, 103 Da), 96.9, 91.0 (pyranose), 81.0 and 62.0.NMR spectrum of galactomannan is shown in Fig. 10.ed signals correspond to -d-mannopyranose and -ranose residue were observed. The 1H NMR spectrum

Theplottinshear sfrom mon tharate. Firate. Adecreaited ps

Table 4Chemicaof S. tora

Chemi

101.37R spectra of galactomannan from the Senna tora seeds, in D2O.

99.932

73.030371.2276 71.1204 70.4660 70.0551 69.8123 68.7786 66.3245

61.805261.4983

56.011555.5820 55.2552 (Graph-II) represents rheogram of viscosity versus shear rate of shear increases, the viscosity of gum solutionhis indicates that 1% (w/v) solution of S. tora gum exhib-plastic ow and the system was shear thinning.

s (in ppm) and the corresponding carbon atoms for 13C NMR signals

ift (ppm) Corresponding C-atom (moiety) Intensity

C1() 0.74

C1() 0.44

C-2 to C-5

0.750.550.810.880.420.440.400.79

CH2OH0.450.86

O-CH34.760.400.65Fig. 12. Structure of S. tora seed polysaccharide.


Table 5Assignment of 13C NMR signals of S. tora gum in D2O.

Units C-1 C-2 C-3 C-4 C-5 C-6

-d-Galacto 69.8123 71.2276 61.4983-d-Mannop

0

10

20

30

40

50

60

70

80

90

0

Shea

r St

ress

Fig.

0

2000

4000

6000

8000

10000

12000

Vis

cosi

ty in

Cps

Fig

Viscositygums. The viscosity anused in slowcertain chastrength anThe resultsseeds posseseed polysaa thick viscrequired forological prosustained re

4. Conclus

The phafrom differeextensivelypotential inbased prod

tionnansets ople

modl, as ave sontaties cf susgatearmatic oarma

ncespyranosyl 99.9316 68.7787 70.0551 yranosyl 101.3790 70.4660 71.1204

12010080604020

Shear Rate

13. Rheogram of shear stress versus shear rate (Graph-I).

applicatomanas tablthe simin the mentathe aboseeds cproperment oinvestient phsynthethe ph

Refere120100806040200

Shear Rate

. 14. Rheogram of viscosity versus shear rate (Graph-II).

is the main parameter to assess the quality of naturalapplications of any natural gum are dependent on itsd other rheological properties. For any polymer to be

release hydrophilic matrix systems, it should possessracteristics like fast hydration of the polymer, high geld should be stable during the shelf life of the product.

indicate that the polysaccharide isolated from S. torass pseudoplastic ow and good swelling capacity. S. toraccharide hydrates quickly and swells rapidly and formsous gel around it. This is the most important criterion

hydrophilic matrix tablets. The viscosity and other rhe-perties conrmed its suitability in the development oflease delivery systems.

ion

rmaceutical applications of galactomannans obtainednt commercial and noncommercial sources have been

studied over the past decade. Galactomannans show the global trend toward the use of more plant-

ucts for ecological motives, and their production and

[1] J.S.G. ReidApplicati

[2] M.S. Kok,[3] H. Neuko

Wissensc[4] I.C.M. Dea

31 (1975[5] C.A. Migu

179185[6] M. Uner, [7] C.T. Andr

Macromo[8] E.G. Azer

844850[9] T.M.B. Br

Macromo[10] M.J. Hern

tional 7 ([11] V.D. Praj

wadi, B.C8392.

[12] H. Joshi, V[13] E.G. Azer[14] I. Anita, R

cognosy [15] C.S. Nara[16] S.M. Wo

276280[17] C.H. Wu,

49 (2001[18] G.C. Yen,

276027[19] S.H. Cho, [20] P.K. Lala, [21] F. Smith,

hold Pub[22] Indian Ph

ernment [23] E.E. Jarald

and Rese[24] S.S. Roho

Drug Del[25] D.A. Skoo

Rinehart [26] World He

rial, WHO[27] M. Duboi[28] E.L. Hirst[29] S.A.I. Rizv[31] S.D. Figue

313320[32] B.M. Prad

53 (200573.0303 67.3245 61.8052

do not cause pollution or disturb the ecosystem. Galac- are used in various pharmaceutical dosage forms suchr capsules, hydrogels and lms as an excipient. Besidesuse as inert excipient, these polysaccharides play roleication of drug release, especially in colonic environ-

matrix or coating material. It has been concluded fromtudy that the polysaccharide (gum) isolated from S. torains galactomannans. The viscosity and other rheologicalonrmed its suitability as an excipient in the develop-tained release delivery systems. Thus there is need to

further S. tora polysaccharide as an excipient in differ-ceutical dosage forms. It may provide an alternative tor semisynthetic excipients/polymers currently used inceutical industry.

, M.E. Edwards, in: A.M. Stephen (Ed.), Food Polysaccharides and Theiron, Marcel Dekker Inc., New York, 1995, pp. 155186.

S.E. Hill, J.R. Mitchell, Food Hydrocolloids 13 (1999) 535542.m, Galactomannans: Properties and Applications, 22, Lebensmmitelhaft und Technologie, London, 1989, pp. 4145., A. Morrison, Advances in Carbohydrate Chemistry and Biochemistry

) 241.e, W.S. Bartolomeu, S. Joana, et al., Carbohydrate Polymers 83 (2011).T. Altnkurt, IL Farmaco 59 (2004) 567573.ade, E.G. Azero, L. Luciano, et al., International Journal of Biologicallecules 27 (2000) 349353.o, C.T. Andrade, Journal of the Brazilian Chemical Society 17 (2006).esolin, M. Milas, M. Rinaudo, et al., International Journal of Biologicallecules 26 (1999) 225231.andez, J. Dolz, M. Dolz, et al., Food Science and Technology Interna-2001) 383391.apati, G.K. Jani, N.G. Moradiya, N.P. Randeria, B.J. Nagar, N.N. Naik-. Variya, International Journal of Biological Macromolecules 60 (2013)

.P. Kapoor, Carbohydrate Research 338 (2003) 19071912.o, C.T. Andrade, Polymer Testing 21 (2002) 551556.. Pranjali, L. Abhijeet, et al., International Imperial Journal of Pharma-& Natural Products 2 (2012) 1423.yan, S. Rangaswami, Current Science 25 (1956) 559.ng, M.M. Wong, O. Selingmann, et al., PlantaMedica 55 (1989).C.L. Hsieh, J.Y. Song, et al., Journal of Agricultural and Food Chemistry) 25792586.

D.Y. Chuang, Journal of Agricultural and Food Chemistry 8 (2000)65.T.H. Kim, N.H. Lee, et al., Journal of Medicinal Food 8 (2005) 311318.Practical Pharmacognosy, Lina Guha, Calcutta, 1981.R. Montagomery, The Chemistry of Plant Gums and Mucillages, Rein-lishing Corporation, New York, 1959.armacopoeia, vol. 556, Ministry of Health and Family Welfare, Gov-of India, Controller of Publication, New Delhi, 1996, pp. A100A111., S. Sumati, S. Edwin, et al., Indian Journal of Pharmaceutical Educationarch 46 (2012) 211216.kale, Y.D. Dhanorkar, V. Pahuja, et al., Journal of Chronotherapy andivery 3 (2012) 4154.g, D.M. West, Fundamentals of Analytical Chemistry, 4th ed., Holt-and Winston, Philadelphia, NY, 1963, pp. 880.alth Organization, Quality Control Methods for Medicinal Plant Mate-, Geneva, 1998, pp. 45.s, C. Gileska, J.K. Hamilton, Analytical Chemistry 28 (1956) 350356., J.K.N. Jones, Discussions of Faraday Society 7 (1949) 268.

i, P.C. Gupta, R.K. Kaul, Planta Medica 20 (1971) 2428.ir, J.C. Ges, R.A. Moreira, et al., Carbohydrate Polymers 56 (2004)

.o, S. Kim, B.F. zen, et al., Journal of Agricultural and Food Chemistry) 28232829.


[33] M.R.S. Prashanth, K.S. Parvathy, N.S. Susheelamma, et al., Food Hydrocolloids20 (2006) 11981205.

[34] S.N. Yuen, S.M. Choi, D.L. Phillips, et al., Food Chemistry 114 (2009) 10911098.[35] Y. Peng, L. Zhang, F. Zeng, et al., Carbohydrate Polymers 54 (2003) 297303.[36] L. Zhang, L. Yang, Q. Ding, et al., Carbohydrate Research 270 (1995) 110.[37] F.M. Nunes, M.R. Domingues, M.A. Coimbra, Carbohydrate Research 340 (2005)

16891698.

[38] G.M. Gu-bitza, B. Laussamauer, M. Schubert-Zsilavccz, et al., Enzyme and Micro-bial Technology 26 (2000) 1521.

[39] C.L.O. Petkowicz, M.R. Sierakowski, M.S. Joana La, et al., Phytochemistry 49(1998) 737743.

[40] S.E.C. Whitney, J.E. Brigham, A.H. Darke, et al., Carbohydrate Research 307(1998) 299309.

[41] H.N. Cheng, N.G. Thomas, Polymer Reviews 52 (2012) 81114.

Isolation, purification and characterization of galactomannans as an excipient from Senna tora seeds1 Introduction2 Materials and methods2.1 Collection of plant material2.2 Isolation of gum2.3 Purification of gum2.4 Characterization of gum2.4.1 Organoleptic evaluation2.4.2 Identification test for gum2.4.3 Physicochemical evaluation2.4.3.1 Solubility2.4.3.2 pH2.4.3.3 LOD2.4.3.4 Ash content2.4.3.4.1 Total ash2.4.3.4.2 Acid insoluble ash2.4.3.4.3 Water-soluble ash

2.4.3.5 Specific gravity2.4.3.6 Swelling index2.4.3.7 Viscosity

2.4.4 Differential scanning calorimeter (DSC)2.4.5 X-ray diffraction2.4.6 Scanning electron microscope (SEM)2.4.7 Gel permeation chromatography (GPC)2.4.8 Estimation of total sugar content2.4.9 Identification of gum components by TLC2.4.10 Determination of mannose/galactose ratio (M/G ratio)2.4.11 Investigation of the structure of the polysaccharide2.4.12 Rheological study of S. tora gum

3 Results and discussion4 ConclusionReferences

isolation, purification and characterization of galactomannans as an excipient from senna tora seeds...

Documents