antioxidant activity of pongamia pinnata...

International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)

Volume III, Issue 4 April 2017

All rights are reserved 15

ANTIOXIDANT ACTIVITY OF PONGAMIA PINNATA LINN. SEED

EXTRACT IN STREPTOZOTOCIN INDUSED DIABETIC RAT

* Amit Kumar and * * Dr. Anil Middha and

* * * Rajesh Kumar

* Research Scholar, OPJS University, INDIA

* * Department of Pharmacy OPJS University, INDIA

*** Research Scholar, OPJS University, INDIA

Abstract

Several medicinal plants in the modulation of oxidative stress associated with diabetes

mellitus. Effect of 50 % hydroalcoholic extract of plants on serum glucose, lipid profile and

antioxidant status in STZ induced diabetic rats was studied. Based on this, potentiation of

dreaded disease like diabetes mellitus may shows a ray for better protocol for future

treatment. The efficacy of Pongamia pinnata in experiment showed the significant decrease in

the blood glucose level, increase the antioxidant efficacy in streptozotocin induced diabetes.

The oral administrations of the 50% hydroalcoholic extract decrease the blood glucose lavel

and oxidative stress..The present study showed that the 50% hydroalcoholic extract of

Pongamia pinnata not only possesses hyperglycemic properties but also reduced oxidative

stress in diabetes condition

Introduction

A free radical is any atom that there is at least one unpaired electron in the outermost shell (1)

.

These uncoupled electrons are very reactive with adjacent molecules such as lipids, proteins,

and carbohydrates and can cause cellular damage (2)

. Free radicals can also be produced by

many cells as a protective mechanism. Neutrophils produce free radicals to attack and destroy

pathogens, while the liver uses free radicals for detoxification (3)

. However, the presence of

free radicals within the body can also have a significant role in the development and

progression of many disease processes like heart disease, congestive heart failure,

hypertension, cerebrovascular accidents, and diabetic complications (4)

.

Plant profile of Pongamia pinnata Linn. (5)

Botanical name: Pongamia pinnata Linn.Family : Legoseaumin Synonyms:pongamia

glabra (Vent.,),Derris indica(Lamk.,)

Local/common name: English - Indian beechHindi -Dithouri, Karuaini Gujrati - Kanajo,

Karanji Sanskrit -Karaµjaka, Naktam¡la, Nakt¡hva, Gh¤takaraµja Tamil -Pungan,

PonganaTelugu -Lamiga, Kanuga




PLANT MONIGRAM

Occurance & Description:

Pongamia is derived from the temil namePongama or Ponga and pinnata (Latin)Means

leaflets arrange on either side of the stalk .,and Glabera (Latin) means without hairs. Plant is

distributed throughout India.Pongamia pinnata Linn. is a medium sized semi-evergreen

glabrous tree with a shot bole and spreading cropup to 18m or more in height and 1.5m in

girth.The flowers appears in April-June and its pods ripen during March-May of following

years.

Description:Baek-Graish green or brownLeaves-CompoundLeaflets- 5-7 ovate or

elepticFlower-Pinkish- whiteFruit-pod which is thick,woody,smooth, compreshed with a

short curbed beakSeeds-1or2 per pod,smooth or wrinkled testa and reddish brown leathery.

Traditional Uses:Root-bark:Cooling,beneficial in gonorrhea,rheumatoid arthiritis,wound

scabiesBark:Used internally in bleeding pillesBark(decoction):In beri-beriLeaf-Juice:In

flatulent,dyspepsia,diarrhea,cough and leprosy.Leaf(Past):Ointment for leprosy

paronychiaFlower: Cures blood sugarSeed kernle: Benifical in wooping cough and leprosy.

(6)

EXPERIMENTAL

Collection of plant material

Fig.No.1 Plant of Pongamia pinnata Fig No.2 Pongamia pinnata plant seeds




Seeds of Pongamia pinnata Linn. were collected in the month of August. The plant was

authenticated by Dr.Anil Middha, Department of pharmacy,OPJS University Churu

Rajasthan.

Preparation of the extract

The Seeds of Pongamia pinnata Linn. was dried under shade and powdered mechanically

and sieved using a mesh No.60.

Solvents used for extraction Hexane, Chloroform, Ethanol(50%v/v),Distilled water

Method of Extarction

The extraction of seeds were carried out from less polar to more polar solvents by using a

Percorater apparatus for continuous cool percolation procedure.

Ethanolic extract

The marc was dried and then extracted with Ethanol 50% v/v for 72 hrs , ethanol is more

polar solvent , active constituents of the seed were extracted in this extraction. The filtered

extract was dried under redused pressur 400c using a rotary evaporator.The dried ethanolic

extract was transferred into air tight container.expressed in Table No.1

Physico-chemsical standardization(7)

Estimation of sugar/starch(7)

Shown in table no.3

Estimation of total alkaloid: (7)

Shown in table no.3

Estimation of Total Phenols (7)

Shown in table no.3

Phytochemical screening

Chemical evaluation comprises of different chemical tests (8)

are shown in table no.2

Test for carbohydrates and glycosides

A small quantity of the extract was dissolved separately in 4 ml of distilled water and filtered.

The filtrate was subjected to the following testes to detect the presence of Carbohydrate and

glycosides.

(a) Molisch’s test




The filtrate was treated with 2-3 drops of 1% alcoholic α-napthol solution and 2 ml of

concentrated H2SO4 was added along the sides of the test tube. Appearance of brown ring at

the junction of two liquids shows the presence of carbohydrates.

(b) Fehling’s test

The filtrate was treated with 1 ml of Fehling’s solution A and B and heated on the water bath.

A reddish precipitate was obtained shows the presence of carbohydrate.

Test for fixed oils and fates

(a) Spot test

Small quantity of extract was pressed between two filter papers. Appearance of oil stain on

the paper indicates the presence of fixed oil.

(b) Saponification test

Few drops of 0.5% alcoholic potassium hydroxide were added to a small quantity of various

extracts along with a drop of phenolphthalein. The mixture was heated on the water bath for

1-2 hours. Formation of soap pr partial neutralization of alkali indicates the presence of fixed

oils and fats.

Test for proteins and free amino acid

Small quantity of the extract was dissolved in few ml of distilled water and treated with

following reagents.

(a) Millon’s test – Appearance of red color shows the presence of proteins and free amino

acids.

(b) Ninhydrin reagent –Appearance of purple color shows the presence of proteins and free

amino acids.

(c) Biuret test – Equal volumes of 5% sodium hydroxide solution and 1% copper sulphate

solution were added, appearance of pink or purple color shows the presence of proteins and

free amino acids.

Test for saponins

Foam test – The extract was diluted with 20 ml of distilled water and it was agitated in a

graduated cylinder for 15 minutes. The formation of 1 cm layer of foam shows the presence

of saponins.




Test for phenolic compounds and tannins

Small quantity of the extract was taken in distilled water and test for the presence of phenolic

compounds and tannins was carried out with the following reagents.

(a) Dilute ferric chloride solution (5% w/v) - Violet color.

( b)10% lead acetate solution-White precipitate.

Test for phytosterols

Small quantity of the extract was dissolved in 5 ml of chloroform separately. Then

this chloroform solution was subjected to the following tests to detect the presence of

phytosteroles.

(a) Libermann-Burchard’s test

The above preapared chloroform solution was treated with few drops of concentrated

sulphuric acid followed by few drops of diluted acetic acid, 3 ml of acetic anhydride. A

bluish green color appeared indicates the presence of phytosterols.

(b) Salkowski reaction

To 1 ml of the above prepared chloroform solution, few drops of concentrated sulphuric acid

was added. Brown color produced shows the presence of phytosterols.

Test for Alkaloids

Small quantity of the extract was treated with few drops of diluted hydrochloric acid and

filtered. The filtrate was used for the following tests.

(a) Mayer’s reagent – cream precipitate

(b) Dragendroff’s reagent – Orange brown precipitate

(c) Hager’s test – yellow precipitate

Test for flavonoids

(a) With aqueous NaOH solution

Small quantity of the extract was dissolved in aqueous sodium hydroxide. Appearance of

yellow colour indicates the presence of flavonoids.

(b) With conc. sulphuric acid

To a small portion of extract, concentrated sulphuric acid was added. Yellow orange color

was obtained shows the presence of flavonoids.

PHARMACOLOGICAL SCREENING

Animals

Sprague-Dawley rats (150-200g) same age, procured from listed supplies of OPJS

university were used for the study .The animal were exposed to alternated cycle of 12 hrs of




dark and light each, before each test, the animal were fasted for at lest 12 hrs.The toxicity

study was according to Guideline 423(9)

Antioxidant studies(10)

The ethanolic (50%) extract of

seeds of Pongamia pinnata Linn. was used for the evaluation of antioxidant activity.

INVITRO STUDIES

DPPH scavenging activityDPPH scavenging activity or the Hydrogen donating capacity was

quantified in presence of stable DPPH radical on the basis of Blois method (Blois, 1958).

Briefly, to a methanolic solution of DPPH (100 M, 2.95 ml), 0.05 ml of test compounds

dissolved in methanol was added at different concentration (2 -10 mg/ml). Reaction mixture

was shaken and absorbance was measured at 517nm at regular intervals of 30 seconds for 5

minutes, and the reading was taken till 20 min. Ascorbic acid was used as standard. The

degree of discoloration indicates the scavenging efficacy of the extract. Shown in table no.5

Scavenging effect(%)=(1-B/A)x100

Where A = Absorbance of DPPH control with solvent (517nm)

B = Absorbance of decolorized DPPH in presence of test sample(517 nm)

Total antioxidant capacity

Total antioxidant capacity was measured according to spectrophotometric method of Preito ,

0.1 ml of the extract (10 mg/ml) dissolved in water was combined in an eppendorf tube with

1 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM

ammonium molybdate). The tubes were capped and incubated in a thermal block at 95 0C for

90 min. After cooling to room temperature, the absorbance of the aqueous solution of each

was measured at 695 nm against a blank. Ascorbic acid was used as the standard and the total

antioxidant capacity is expressed as equivalents of ascorbic acid. Shown in table no.5

Photochemiluminescence

For the determination of the integral antioxidative capacity (AC) of the water soluble

substances in extract the method of photochemiluminscence (PCL) was used. A new method

for the application of the photochemiluminescence for the estimation of the radical

scavenging activity of the plant extracts have been developed. This method is quick, sensitive

and accurate and may serve the purpose of biomarker in the standardization of the plant

extracts and their formulations.

Apparatus: Photochem (Analytic Jena AG, Germany)

Standard kit: ACW (Analitik jena AG) – where the luminol plays a double role of

photosensitizer as well as the radical detecting agent




The total antioxidative capacity of the water soluble compounds (ACW) were measured by

chemiluminescence assay using Photochem. The extracts (1 mg/mL in water) were mixed

with reagent containing luminol. The antioxidants were quantified by their inhibitory effects

on luminescence generation by comparison with ascorbic acid used as standard and the

antioxidative capacity was calculated as equivalents units of ascorbic acid (from the

calibration curve of ascorbic acid) and the results were expressed as n moles ascorbic acid/ g

equivalents. Shown in table no.3

IN VIVO STUDIES

Material requirements

Aniaml : Sprague-Dawley rats (150-300g)

Chemical: Ascorbic acid (20%v/v)

Dose: Ethanolic(50%) extract of seeds of Pongamia pinnata Linn.

Ascorbic acid(used as standard drug)

Experimental procedure

Effect of Ethanolic (50%)extract of pongamia pinnata Linn. on straptozotocin indused

oxidative stress

Group I – Normal control

Group II – STZ induced control

Group III – Ethanolic (50%) extract of seed of Pongamia pinnata Linn. (1.4 g/kg p.o) in STZ

indused rat.

Group IV- Ascorbic acid (200 mg/kg p.o) in STZ indused rat.

The experimental group was subdivided in such a manner that all sub groups concurrentiy

received STZ (55mg/kg) and either the Ethanolic (50%) extract of seeds of Pongammia

pinnata Linn. (1.2g/kg body wt, orally) or the Ascorbic acid (200 mg/kg orally) or vehicle.

The The above treatment was given daily for 4 weeks. The control group receive vehicle in

stead of STZ.After compleating the treatment of 4 weeks. The animal was anesthetize by

diethylether and then sacrificed.The blood was collected by cardio puncture and the liver was

dissected out immediately and transferred into Tris-HCL buffer (0.1 M; pH 7.5) and a small

part of liver preserved in 10% formalin for further studies.The ice cold Tris-HCL buffer




soaked liver were studied for the assessment of antioxidant parameters like TBARS, LPO,

SOD, CAT and GSH. Shown in table no.6

RESULT & DISCUSSION

The phytoconstituents were extracted by using different solvent of increasing polarity like

Hexane, Chloroform, Ethanolic(50%), Aqueous.The extractive values were presented in

Table-1

Percentage yield of different extracts of dried seed of Pongamia pinnata Linn.:-

Phytochemical evaluation

The phytoconstituents were identified by chemical test which showed the various

phytoconstituents ( Table No.2) mainly in the following extract

Plant used Part

used

Method of

extraction

% Yield (w/w)

Hexane Chloroform 50% ethanol Water

Pongamia

pinnata

(Linn.)

Seeds

Cold

Percolation

20.5

10.0

36.0

15.5




Table:2 Phytochemical screening of different extract of dried seed of Pongamia pinnata

Linn.:-

S. No. Constituents Tests Hexane Chloroform 50%

Ethanolic

Aqu.

1. Carbohydrate Molish’s test + + + +

Anthrone test + + + _

Fehling’s test _ _ + +

2. Glycoside Legal’s test + + + _

Keller killanis test _ _ + +

3. Fixed oil & fats Spot test _ + − +

Saponification test _ _ − _

4. Proteins & amino acids Million’s test _ _ _ _

Ninhydrin test _ + + _

Biuret test _ + _ _

5. Saponins Foam test _ _ + +

6. Phenolic compunds &

tannins

FeCl3 test + _ + +

Lead acetate test + + + _

7. Phytosterol Salkowiski test _ + + +

Libermann burchard test _ _ _ +

8. Alkaloids Dragendroff’s test _ _ + _

Mayer’s test _ + + +

Hager’s test _ _ − _

9. Gum & mucilage Swelling test + _ − _

10. Resin Resin _ + + +

11. Flavonoids Aq. NaOH test _ _ + _

Shinoda’s test _ + + _

Where:- ( +) = Presence, ( −) = Absence




Table No:3 Quantitative analysis of dried seeds of Pongamia pinnata Linn.:

Table No.4 LD 50 vaiue of 50% hydroalcoholic extract of dried seed of Pongamia

pinnata Linn.

Animal - Rat Weight of animal- 150-300 No of animal per group - 3

Rout- Oral LD 5O valus = 2000 mg/kg ED 50 value = 200 mg/k

DPPH radicals react with suitable reducing agents, then losing colour stoichometrically with

the number of electrons consumed, which is measured spectrophotometricallty at 517 nm.17)

As shown in Table No.5, Pongamia pinnata Linn. extract strongly scavenged DPPH radical

with the IC50 being (0.4157mg/ml). The scavenging was found to dose dependent. The total

antioxi omolybdenum complex which was measured spectrophotometrically at 695 nm. The

total antioxidant dant capacity of the extract was calculated based on the formation of the

phosph capacity of the extract was found to be 0.3299 nmol/g ascorbic acid

Parameters Range (%) Mean ± SD (%)

Total sugar 0.560 - 0.571 0.565 ± 0.007

Total starch 1.237 - 1.242 1.24 ± 0.003

Total phenolics 0.189 -0.186 0.187± 0.002

Total alkaloid 0.33 - 0.28 0.305 ± 0.035

Sl.No No. of animal per

group

Dose(mg/kg No. of death of

animal

1 3 5 0

2 3 50 0

3 3 300 0

4 3 2000 2




Table No:5

Antioxidant activity of different extract of dried seeds of Pongamia pinnata Linn.

Total Antioxidant

The total antioxidant capacity of the Ethanolic(50%) and water extract measured

spectrophotometrically at 695 nm based on the formation of the phosphomolybdenum

complex was found to be 0.3299 nmol/ g ascorbic acid in 50%Hydro aicoholic & 4.5950

nmol/g of ascorbic acid.in Water.

Photochemiluminescence

The antioxidant capacity of Ethanolic(50%) extract was found 0.99557 nmol/g ascorbic acid.

IN VIVO ANTIOXIDANT

50% ethanolic extract of Pongamia pinnata seed at a dose of 100, 200 mg and 400 mg (O.D x

28 days) were subjected for per se effect by studying LPO, SOD, CAT and GPx in liver

homogenate of rats. There is significant decrease (0.44-0.31, p<0.001) in liver of lipid

peroxidation product malondialdehyde (LPO), and dose dependently increased the level of

superoxide dismutase (SOD) (109.45-156.45, p<0.01, p<0.001), catalase (CAT) (25.67-

41.18, p<0.001) showed the high significant at 200 mg/kg dose, and glutathione peroxidase

(GPx) (3.33-4.76, p<0.05, p<0.001) (TableNo.6 ).

Table 6. Per se effect of 50% ethenolic extract of Pongamia pinnata Linn. on

Concentraction

µg/ml

DPPH Scavengeing%

Butanol Chloroform Water 50%Hydroaicohol Ascorbic

Acid

100 3.0612 3.9115 11.9528 14.7108 20.4081

200 15.4761 17.9421 13.2996 17.7721 25.8503

400 26.8707 20.8333 25.4208 57.4829 69.8129

800 32.0578 48.3843 56.4625 88.1802 93.9625

I.C50(mg/ml)

(at20min.)

0.9945 0.9823 0.7080 0.4157 0.396884




lipid peroxidation, superoxide dismutase, catalase and glutathione peroxidase

Values are expressed as Mean ± SEM of 6 rats in each group

P value: a<0.05,

b<0.01,

c<0.001 compared to control group

Fig. No.3 Per se effect of 50% ethenolic extract of Pongamia pinnata Linn. on

lipid peroxidation, superoxide dismutase, catalase and glutathione peroxidase

0

50

100

150

200

250

300

I II III IV

CAT

SOD

Pro

tein

(un

it/m

g)

Group Treatment Dose(m

g/kg)

LPO

(nmoles/g of

protein)

SOD (unit/mg

of protein)

CAT

(units/mg of

protein)

GSH(mmole/

g tissue)

I Control 10 ml 0.44 ± 0.01 109.45 ± 5.14 25.67 ± 1.43 3.33 ± 0.15

II STZ+Ethanoloc(50%)

Extract of Pongamia

pinnata Linn.

55+100 0.40 ± 0.02

128.95 ± 3.12b

27.17 ± 1.65 3.58 ± 0.19

III STZ+Ethanoloc(50%)

Extract of Pongamia

pinnata Linn.

55+200 0.38 ± 0.02b

140.42 ± 4.22c

34.50 ± 1.52c

4.10 ± 0.16a

IV STZ+Ethanoloc(50%)

Extract of Pongamia

pinnata Linn.

55+400 0.31 ± 0.01c

156.45 ± 2.97c

41.18 ± 1.09c

4.42 ± 0.19b

One-way ANOVA 0.0001 <0.0010 <0.0001 <0.0001




CONCLUSION

In spite of overwhelming influence of modern medicine and tremendous advances made

in the production of synthetic drugs, traditional medicaments referred to now-a-days as herbal

drugs in different places in the literature, have retained their place in therapy. Their

effectiveness, low cost and comparative freedom from serious toxic effects make these

medicaments not only popular but also an accepted mode of treating disease even in

developed countries.

Both the National Institutes of Health (NIH) and the Centers for Disease Control and

Prevention (CDC) are involved in prevention activities. . Effect of 50 % hydroalcoholic

extract of plants on serum glucose, lipid profile and antioxidant status in STZ induced

diabetic rats was studied. Based on this, potentiation of dreaded disease like diabetes mellitus

may shows a ray for better protocol for future treatment. The efficacy of Pongamia pinnata in

experiment showed the significant decrease in the blood glucose level, increase the

antioxidant efficacy in streptozotocin induced diabetes. The oral administrations of the 50%

hydroalcoholic extract decrease the blood glucose lavel and oxidative stress..

The present study showed that the 50% hydroalcoholic extract of Pongamia pinnata not only

possesses hyperglycemic properties but also reduced oxidative stress in diabetes condition,

which was confirmed by glucose level and pancreatic secreation. These observation and

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

I II III IV

LPO

GSH

Pro

tein

(n m

ole

/g)




description of mechanism of Pongamia pinnata, which interplay with diabetes biology and

pharmacology lead to rapid development in diabetes treatment. In addition to this, studies on

molecular aspect of diabetic therapy will give mechanistic information in diabetes therapy

and also critical balance should be there between the animal model and clinical research. This

holds great promise for future research in human beings.

REFERENCES

1. Schmeltz, I. Grover, J.K., Rathi, S.S., Vats,.. Naturally occurring insecticides

(eds) jaconbson, M. and cros, D.G. Dekker, New Yark. . systems newly developed

that monitor blood glucose levels V. Journal of Experimental Biology, 40 (3) 273-

276 (1971)

2. Kuhn MA. Matsui, T., Matsumoto, K Oxygen free radicals & antioxidants.

American Journal Nurs;103(4):58-62(2003)

3. Lunec J, Holloway KA, Cooke MS, Faux S, Griffiths HR, Evans MD. Urinary 8-

OXO-2-deoxyguanosine: redox regulation of DNA repair in vivo? Free Radic.

Biol Med 33(7):875-85. (2002)

4. Yadav, P., Sarkar, S., Bhatnagar, D., 1997a. Action of Capparis decidua against

alloxan-induced oxidative stress and diabetes in rat tissues. Pharmacological

Research 36 (3), 221-228..

5. Glossary of Indian Medicinal plant vol.3 p.no.123(2003)

6. Maher Abdel-Barry, J.A., Tariq Mohammeda Guha bakshe ,sensarma Study of

transesterification of Karanja oil with methanol for the production of biodiesel A

lexicon of medicinal plant in India 453:pp 32((2000)

7. Lowry.H. Balandarin M.F, kloke, J.A., Wurtelel, E. S roesbrough N.J, farr.A.L

randall.R.I.Protein determination using folin ciocalteau reagent.(j.of biological

chemistry 193”438-448) (1951)

8. Basset,J.Denny.,.J, and Mendhan J.,Vogels test book of quantitative inorganic

analysis 4th

edition ELBS Londmal, Essex.UK,1985,

9. www.o.e.c.d org.com(google.com)

10. R.govindrajan.Dherand pratap ,Comperative Study of different antioxidant

activity methods .. Biotechnology Letters, 28 (9), pp. 637-640. (2006)

http://www.o.e.c.d/




11. Ohkawa,H,Ohishi,N. and Yogi assay for lipid peroxidase in animal tissues by thio

barbituric acid reaction AnalBiochem., ,95:351-358(1979)

12. Ellman, G.L Tissu sulfhysryl groups…Arch biochemical Biophysis.,2:70-

77(1959)

13. Sinha K.A. colourimetry assay of catalase ..Anal Biochem.,47:389-394(1972).

antioxidant activity of pongamia pinnata...

Documents