INTERNATIONAL JOURNAL OF FRONTIERS IN … 1/Issue 2...INTERNATIONAL JOURNAL OF FRONTIERS IN SCIENCE AND TECHNOLOGY Research Article ISSN 2321 – 0494 Received on: 20.3.13, Received

INTERNATIONAL JOURNAL OF FRONTIERS IN SCIENCE AND TECHNOLOGY

Research Article

ISSN 2321 – 0494 www.ijfstonline.org Received on: 20.3.13, Received and Accepted on: 20.5.13

COMPARATIVE STUDY OF BIOACTIVE FRACTION OF TRIGONELLA

FOENUM- GRAECUM L. LEAF AND SEED EXTRACTS FOR

INFLAMMATION

Dr. V.RAVICHANDIRAN and Dr. S.JAYAKUMARI*

Department of Pharmacognosy, School of Pharmaceutical Sciences, VISTAS,

Vels University, Chennai, Tamilnadu, India.

April- June 2013 Volume 1 Issue 2 Page | 128

ABSTRACT:

Fenugreek (Trigonella foeum-graceum L.) has been used in Indian traditional medicine for

treatment of different kinds of inflammation disorders. In the present study, anti-inflammatory

activity of the purified bioactive fraction (Alkaloid enriched fraction) of aqueous extract of

leaf and seed of the plant on support of its folklore were studied by both in-vivo and in-vitro

methods and compared with indomethacine. The aqueous extract of seed and leaves of

Trigonella-foenum-graecum (AETFGS & AETFGL) and its alkaloid rich chloroform fraction

(CFTFGS & CFTFGL) were studied for free radical scavenging activity by DPPH and

Nitric oxide methods. Antiinflammatory activity was carried out by both in –vitro (HRBC

membrane stabilization method ) and in vivo ( Carrageenan induced model ). The

comparative results indicated tha alkaloid rich fraction of the seed exhibited significant

protective effect of inflammation in all the tested models. That effect was may be due to its

potent scavenging of nitric oxide radical, since it is an significant inorganic mediator in

the inflammation pathway. The results of the present study, therefore, support the traditional uses

of seeds of the plant for inflammations. However, more research is needed for its use in clinical

studies.

key words : Antiinflammatory Activity . HRBC membrane stabilization, Carrageenan,

Alkaloid fraction, Trigonella foenum-graecum L. seeds and leaves

INTRDUCTION

Most people especially in rural areas depend on herbal medicines to treat

inflammation associated problems such as rheumatism, muscle swelling, cut wound,

Corresponding author address:Dr.S.Jayakumari. e.mail:[email protected]

Jayakumari.et.al., www.ijfstonline.org


accidental bone fracture, insect bites, pain and burn by fire and hot water. The therapeutic

efficacy of currently available Non-Steroidal Anti-Inflammatory Drugs is significantly limited

by associated gastro-intestinal toxicity, which causes a higher incidence of morbidity in long

term NSAIDS users. Frank (Frank., 1998). Several indigenous herbal drugs have been

described in Ayurveda for the management of the inflammatory diseases. Fenugreek

(Trigonella foenum-graecum; Fabaceae) is one of those plant whose seeds and leaves are

used in traditional medicine. The seed of the plant contains Pyridine type alkaloids, mainly

trigonelline (0.2-0.36%) (Jagdeep Kaur., 2011). Hypoglycemic effect of the plant has

studied , (Handa et al., 1992). Glucose tolerance test was carried out on rabbits after

administered with various extracts of fenugreek seeds , the results revealed that alkaloid rich

fraction showed maximum hypoglycemic effect (Jain et al., (1987) Pandian R.S. et al.,

(2002) reported the gastroprotective effect of fenugreek seeds on experimental gastric ulcer

in rats. But no scientific evidence available against the effect of alkaloid enriched fraction in

inflammation and its associated nitric oxide scavenging activity. These observations are main

important criteria to choose this plant to explore and to provide a scientific support, for the

development of lead molecule.

MATERIALS AND METHODS

The plant specimen for the proposed study was collected from local market of

Chennai, tamilnadu. It was identified and authenticated by Dr. P. Jayaraman, Director, Plant

Anatomy and Research Centre (PARC) Tambaram, Chennai.



METHODS (Kokate ., 1994)

The leaves and seeds of Trigonella-foenum-graecum were shade dried and coarsely

powdered. About 500 gms of leaves and seed powder of Trigonella-foenum-graecum were

taken and carried out extraction successively with solvents of increasing polarity viz,

petroleum ether, chloroform, methanol and water. Among all the extracts aqueous extract

showed the presence of alkaloid, so aqueous extract was fractionated with solvents of

increasing polarity such as petroleum ether, chloroform and methanol. Among them

chloroform fraction answered for alkaloid . The aqueous extract of leaves and seeds of

Trigonella-foenum-graecum are AETFGL & AETFGS and its chloroform fraction

designated as CFTFGL and CFTFGS.

PHYTOCHEMICAL STUDY ( Harborne., 1998 )

TEST FOR ALKALOID

Both dried extract and its chloroform fraction were in 2 ml of dilute hydrochloric

acid and tested for alkaloid with Mayer’s reagent and Dragendorff’s test

Thin layer chromatography- (Egon Stahl., 1969)

Aqueous extracts of leaves, seeds and its chloroform Fraction of Trigonella-foenum-

graecum L. were subjected to thin layer chromatographic studies, to confirm presence of

alkaloid on support of chemical test. A number of developing solvent systems were tried, but

the satisfactory resolution was obtained in the solvent system, Toluene: Ethyl acetate:

Methanol (6:2:2) for aqueous extract of leaves and Toluene: Ethyl acetate: methanol: water

(7:1:1:1) for aqueous extract of seeds. After development, the plates were air dried and

sprayed with Dragendorff’s reagent. The observations of TLC of Aqueous extract and its



chloroform fraction of leaves and seeds were shown in Tables- I & II and in Figs – I. II and

III.

Fig. I TLC OF AETFGL Fig.II TLC OF CFTFGL

Fig.III TLC of CFTFGL and AETFGL



IN- VITRO ANTI OXIDANT STUDIES

DPPH radical scavenging activity- (Ganapathy et al., 2007)

The free radical scavenging activity was measured in terms of hydrogen donating or

radical scavenging ability, using the stable radical, DPPH. About 0.1 ml solution of DPPH in

methanol was prepared and 1 ml of this solution was added to 3 ml of the different

concentration (25-600µg/ml) of aqueous extract leaf , seed and its alkaloid fraction of

Trigonella-foenum-graecum were used. vitamin E was used as standard at the same

concentration . The mixture was shaken and allowed to stand at room temperature for 30

minutes and the absorbance was measured at 517 nm using a spectrophotometer. The IC50

value (50% of inhibitory concentration in µg/ml) test sample was compared with that of

vitamin E. Decrease in absorbance of the reaction

Table 1 Thin layer chromatography of Total Aqueous extract and its chloroform fraction of

Trigonella-foenum-graecum Leaves

Test

extract

Solvent system

Number of

spots

Rf values

Detecting agent

Aqueous

extract

Toluene:Ethyl

acetate:Methanol

(6:2:2)

1

0.64

Dragendorff’s

reagent

Chloroform

Fraction

Toluene:Ethyl

acetate:Methanol

(6:2:2)

1 0.73 Dragendorff’s

reagent



Table II Thin layer chromatography of Total Aqueous extract and its chloroform fraction of Trigonella-foenum-graecum Seeds

Test

extract

Solvent system

Number of

spots

Rf values

Detecting agent

Aqueous extract

Toluene:Ethyl acetate:Methanol :Water(7:1:1:1)

1

0.70

Dragendorff’s reagent

Chloroform Fraction

Toluene:Ethyl acetate:Methanol :Water(7:1:1:1)

1 0.66 Dragendorff’s reagent

Mixture indicates higher free radical scavenging activity. The percentage inhibition of DPPH

radical was calculated using the formula,

Percentage inhibition (%) = (Absorbance of control -Absorbance of test) × 100

Absorbance of control

The results were given as percentage of inhibition in Table-III.

Nitric oxide scavenging activity (Annie Shirwaikar ., 2006)

Nitric oxide was generated from sodium nitroprusside and measured by Griess reaction. Sodium

nitroprusside (5 mM) in standard phosphate buffer saline

Solution (0.025 M, pH: 7.4) was incubated with different concentrations of aqueous

extract of leaves and seeds of Trigonella-foenum-graecum and its chloroform fraction (25-

600µg/ml), ascorbic acid as reference standard (25-600µg/ml)



Table III DPPH Radical Scavenging activity of Trigonella-foenum-graecum Leaves &Seeds

The absorbance of the chromospheres formed during diazotization of nitrite with

sulphanilamide and its subsequent coupling with napthylethylenediamine was read at 546 nm.

All the determinations were performed in six replicates. Percentage inhibition of nitric oxide

radical was calculated by using the formula,

Percentage inhibition (%) = (Absorbance of control -Absorbance of test) × 100

Absorbance of control

S. No

Concentration (ug/ml)

% inhibition

AETFGL CFTFGL AETFGS CFTFGS Standard

1 25 20.96± 0.54* 17.70±1.21* 16.94± 0.42* 18.64±0.65* 22.58 ±

0.62

2 50 29.03 ± 0.45** 27.41±2.24* 20.33 ± 0.72* 23.72±0.84** 33.25 ±

0.72

3 100 40.32 ± 1.08** 38.70±0.59* 28.81 ± 1.08** 32.20±0.59* 40.72 ±

0.32

4 200 48.38 ± 0.62** 46.77±0.87** 42.37 ± 1.62** 44.06±0.77** 50.00±

0.66

5 400 56.45± 0.51** 54.83±0.63** 47.55± 0.54** 49.15±0.23** 58.06±

0.64

6 600 64.51 ±0.84** 62.90±0.52** 52.54 ±0.81 54.23±0.62** 66.12± 0.91

7 IC50 280 µg/ml 370 µg/ml 570 µg/ml 510 µg/ml 190 µg/ml

Values are mean ± SEM of 6 parallel measurements

Statistical significant test for comparison was done by ANOVA, followed by Dunnet’s ‘t’ test

All the values are significant **P< 0.01 when compared against standard. (n=6)

AETFGL –Aqueous extract of Trigonella-foenum-graecum leaves

CFTFGL- Chloroform fraction Trigonella-foenum-graecum leaves

AETFGS- Aqueous extract of Trigonella-foenum-graecum seeds

CFTFGS-Chloroform fraction of Trigonella-foenum-graecum seeds



The result was exhibited as percentage of inhibition in Table-IV

The test samples were dissolved in phosphate buffer saline (0.025 M, pH: 7.4) and

incubated at 25oC for five hours.After five hours 0.5 ml of incubation solution was removed

and diluted with 0.5 ml of Griess reagent (1% sulphanilamide, 2% O-phosphoric acid and 0.1%

napthyl ethylene diamine dihydrochloride).

Table IV Nitric Oxide Radical Scavenging activity of Trigonella-foenum-graecum Leaves

S. No Concentration

(ug/ml)

% inhibition

AETFGL CFTFGL

AETFGS

CFTFGS

Std.

1 25 15.78± 0.42* 13.15±0.35*

9.30±

0.32*

11.62±0.25* 22.58 ±

0.62

2 50 18.42± 0.42* 15.72±0.54*

13.95±

0.52*

16.27±0.34* 33.25 ±

0.72

3 100 23.37 ± 0.28* 21.05±0.51*

18.60 ±

0.78*

18.60±0.71* 40.72 ±

0.32

4 200 31.57 ± 1.01** 28.94±0.77* 27.90 ±

0.42**

30.23±0.57**

50.00±

0.66

5 400 50.00± 0.54** 47.36±0.53**

48.83±

0.54**

51.16±0.13** 58.06±

0.64

6

600 55.26 ± 0.61 ** 52.3 ± 0.41**

55.81 ±

0.46

58.13±

0.40** 66.12 ±

0.91

IC50 390 µg / ml 570 µg / ml

410 µg

/ml

380 µg / ml 190 µg

/ml



Values are mean ± SEM of 6 parallel measurements.

Statistical significant test for comparison was done by ANOVA, followed by Dunnet’s ‘t’ test

All the values are significant **P< 0.01 when compared against standard. (n=6)

AETFG –Aqueous extract of Trigonella-foenum-graecum leaves

CFTFG- Chloroform fraction Trigonella-foenum-graecum leaves



In-vitro anti-inflammatory activity

Membrane stabilizing activity (Oyedapo ., 2010)

Preparation of erythrocyte suspension: Whole blood was obtained with heparinized syringes

from rats through cardiac puncture. The blood was washed three times with isotonic buffered

solution (154 mM NaCl) in 10 mM sodium phosphate buffer (pH 7.4). The blood was

centrifuged each time for 10 minutes at 3000 g.

Hypotonic solution-induced rat erythrocyte haemolysis:

Membrane stabilizing activity of the extract was assessed using hypotonic solution-

induced rat erythrocyte haemolysis. (Gandhisan, et al, 1991). The test sampleconsisted of stock

erythrocyte (RBC) suspension (0.50 ml) mixed with 5 ml of hypotonic solution (50 mM NaCl)

in 10 mM sodium phosphate buffered saline (pH 7.4) containing the test sample (10, 25 and 50

mg/ml) or indomethacin (0.1 mg/ml). The control sample consisted of 0.5 ml of RBC mixed with

hypotonic-buffered saline solution alone. The mixtures were incubated for 10 min at room

temperature and centrifuged for 10 min at 3000 g and the absorbance of the supernatant was

measured at 540 nm (Shinde et al., 1999). The percentage inhibition of haemolysis or membrane

stabilization was calculated by the formula 100 × (OD1-OD2/OD1)

Where, OD1 = Optical density of hypotonic-buffered saline solution alone



OD2 = Optical density of test sample in hypotonic solution The results was expressed as

percentage of inhibition in Table- V

Table V Anti inflammatory study of Trigonella-foenum-graecum by membrane

stabilization method

Treatment Conc. µg/ml Absorbance % Inhibition

Control - 0.48±0.012 -

Indomethacine 50 0.15±0.002a 68.75

AETFGL

25

50

100

200

0.35±0.001 a

0.33±0.002 a

0.28±0.01 a

0.20 ±0.2

a

27.08

31.25

41.66

58.33

CFTFGL

25

50

100

200

0.37±0.05 a

0.34±0.04 a

0.33±0.003 a

0.23±0.02 a

22.91

29.16

37.50

52.08

AETFGS

25

50

100

200

0.38±0.002 a

0.36±0.002 a

0.30±0.05 b

0.24 ±0.02b

20.84

25.00

37.50

50.33

CFTFGS

25

50

100

200

0.37±0.001 a

0.34±0.02 a

0.29±0.01 a

0.21±0.31 a

22.91

29.16

39.58

56.26

a P<0.01,

bP<0.05,compared to control group.

Data was analysed by one way ANOVA followed by Dunnet’s‘t’ test n=6

AETFGL- Aqueous extract of Trigonella-foenum-graecum leaves

CFTFGL- Chloroform fraction of Trigonella-foenum-graecum leaves





In-vivo anti-inflammatory activity

Carrageenan induced Paw oedema in rats (Adeyemi et al ., 2002 )

The anti-inflammatory test was evaluated by the carrageenan-induced paw edema test in

the rat, according to the method of Winter et al., (1962). Male Wistar rats (190-230 g) were

briefly anesthetized with ethyl ether and injected subplantarly into the right hind paw with 0.1

mL of suspension of carrageenan (200 μg/ mL) in isotonic saline. The left hind paw was injected

with 0.1 mL of saline and used as a control. Paw volume was measured prior and 3 and 5 h after

carrageenan administration, using a mercury plethysmograph (Ugo Basile, Italy). Aqueous

extract and its chloroform fraction of Leaves and seeds of the plant at the dose of 200 mg/kg

were orally administered 30 minutes prior to carrageenan injection. The control group received

an equivalent volume of water. Indomethacin (5 mg/kg, p.o.) was used as the reference drugs.

Results were expressed as percentage of inhibition of edema was calculated by the formula (1 −

Vt /Vc) × 100 where Vt and Vc are the mean paw volume in the treated and controlled groups,

respectively The result was given as percentage of inflammation of Carrageenan induced edema

in Table- VI and VII.

RESULTS

About 500 gms of Trigonella-foenum-graecum leaf and seed powder were separately

extracted with distilled water and the yield of aqueous extract was about 6.4 and 7.3 % w/w.It

was fractionated with solvens of increasing polarity. Among the tested fractions , chloroform

fraction of leaves and seeds showed positive test for alkaloids.

Thin Layer chromatography (TLC)

To support phytochemical screening, total aqueous extract and its alkaloid rich

fraction ( chloroform fraction ) were subjected to thin layer chromatography. The aqueous

extract of leaf and chloroform fraction of seed showed clear well separated spot with Rf

value of 0.63- 0.70 in Toluene: Ethyl acetate: Ethanol (70% v/v ) in the ratio of 7: 1: 0.5 as

solvent system and and gave orange with Dragon dorffs reagent as detecting agent (Table

1,II & Figure 1,II & III).



Inhibition of DPPH radical

The potential decrease in the concentration of DPPH radical due to the scavenging

ability of AETFGL and CFTFGL showed significant free radical scavenging activity of

about 64.51 ±0.84 % and 62.90±0.52 % respectively at higher concentration with the IC 50

value of 279 and 380 µg / ml. ( Table III )

Nitric oxide scavenging activity

The scavenging property of nitric oxide by the test sample and standard were

concentration dependent. There was moderate inhibition of nitric oxide formation . The

maximum was 58.13 ± 0.40 % produced by CFTFGS at the concentration of 600 µg / ml

The results was expressed as percentage of inhibition in Table- IV

In-Vitro Anti-inflammatory activity

Effect on erythrocyte membrane stability

The in-vitro anti-inflammatory activity carried out the aqueous extract of leaf and seed of

Trigonella-foenum-graecum with its chloroform fraction using the HRBC method. The aqueous

extract of leaf showed good anti-inflammatory property. The extract shown 58.33% inhibition

and chloroform fraction 52.08% inhibition at 200µg/ml concentration, with respective to the

standard, which exhibited 68.75%i nhibition. Aqueous extract of leaves having more significant

by comparison to chloroform fraction. In case of seed the chloroform fraction of Trigonella-

foenum-graecum showed 56.26% inhibition where as aqueous extract shown 50.33% at

200µg/ml concentration. So the chloroform fraction was more potent than aqueous extract. The

P<0.01 noted that more significant. The result was given in Table V.



Acute toxicity study

Ahmadiani et al ., (2000) reported acute toxicity studies of aqueous extract of leaves and seeds

Trigonella foenum graecum L. Acute toxicity study Was conducted using standard protocol. Up

to 2000 mg/kg, body weight the sample was found to non toxic. So according to OECD

guidelines 1/10th

of maximum dose was taken for the present pharmacological study .

In-vivo anti-inflammatory activity

Carrageenan induced Paw oedema in rats

Carrageenan-induced rat paw oedema is used widely as a working model of inflammation in the

searchor new anti-inflammatory drug. The anti inflammatory activity of the aqueus extract and

its alkaloid fraction of Trigonella-foenum-graecum Leaves and seeds were evaluated by

carrageenan-induced rat paw oedema method and the result is shown in Table VI . The

administration of aqueous extract and chloroform fraction of leaves of Trigonella-foenum-

graecum at the dose of 200mg/Kg significantly (aP<0.01) reduce the inflammation. The oral

administration of aqueous extract of Trigonella-foenum-graecum leaves at the dose of

200mg/Kg showed 26.38%, 42.85%, and 54.28% inhibition respectively after 1st,

3 rd

and 4th

hour as compared to Indomethacine (5mg/Kg) which showed 55.71%, 67.14%,and 74.12%

inhibition respectively Though both the aqueous extract Trigonella-foenum-graecum seed and

its chloroform fraction showed good anti-inflammatory activity but chloroform fraction was

found more significant (** P<0.01) .



Table VI Anti inflammatory activity of Aqueous extract and Chloroform fraction of Trigonella-foenum-graecum Leaves on Carrageenan induced paw edema in rats

Treatment Dose

(mg/kg)

Mean Oedema volume (ml)

% of inflammation at time

1hr 2hr 3hr 5hr 1hr 2hr 3hr 5hr

Vehicle control

2%

CMC

0.12

0.14

0.22

0.26 - - - -

Standard

(Indomethacine)

05

0.07

0.08

0.12

0.06 55.71±0.03 62.85±0.2 67.14±0.1 74.12±0.1

AETFGL

200

0.09

0.11

0.15

0.08 26.38±0.05a

* 30.00±0.02a*

42.85±0.2

3a**

54.28±0.01

a**

CFTFGL

200

0.08

0.07

0.16

0.045 25.71±1.21

a* 30.00±0.09 a*

41.42±0.1

2 a

51.42±0.09

a**

Data was analysed by one way ANOVA followed by Dunnet’s’ t test, n=6

Comparison made between Control against test, a P<0.01,

b P<0.05

Comparison made between Standard against test, ** P<0.01, * P<0.05

AETFGL- Aqueous extract of Trigonella-foenum-graecum leaves

CFTFGL-Chloroform fraction of Trigonella-foenum-graecum leaves

The chloroform fraction of Trigonella-foenum-graecum L showed 53.28% inhibition after 4th

hour. The most significant reduction in inflammation in rats may be due to presence of the

alkaloid. .The result showed that the aqueous extract of leaves of Trigonella-foenum-graecum



and chloroform fraction of seed were co-relate in all the models viz antioxidant activity and

anti-inflammatory activity, may be due to the presence of alkaloid. Results were shown in

Tables VI & VII

Table VII Anti inflammatory activity of Aqueous extract and Chloroform fraction of

Trigonella-foenum-graecum Seeds on Carrageenan induced paw edema in rats

Treatme

nt

Dose

(mg/

kg)

Mean

Oedema

volume

(ml)

% of inflammation at time

1hr 2hr 3hr 5hr 1hr 2hr 3hr 5 hr

Vehicle

control

2%

CMC

0.17

0.18

0.26

0.26 -- - -- -

Standard

(Indomet

hacine)

05

0.8

0.09

0.12

0.09 55.71±0.01 62.85±0.05 67.14±0.1 74.12±0.3

AETFGS

200

0.09

0.11

0.15

0.18 22.12±0.4b* 29.32±0.22a* 43.35±1.09 a** 51.88±0.61 a**

CFTFGS

200

0.10

0.17

0.13

0.04 24.77±0.81 a*

31.61±0.09 a 46.22±0.01

a** 53.28±0.001

a**

Data was analysed by one way ANOVA followed by Dunnet’s’t test, n=6

Comparison made between Control against test, a P<0.01,

b P<0.05

Comparison made between Standard against test, ** P<0.01, * P<0.05

AETFG- Aqueous extract of Trigonella-foenum-graecum seeds

CFTFG-Chloroform fraction of Trigonella-foenum-graecum seeds



.DISCUSSION

The traditional medicinal plant Trigonella-foenum-graecum belongs to the family

Leguminosae, commonly known as ‘Vendayam’ in Tamil. Earlier folklore claims reported that

the whole plant is used in Diuretic, fever, and used as laxative, Antibacterial, Antifungal,

Antidiabetic, Antiarthritic, and Anti-inflammatory. Alkaloids represent a group of natural

product that had a great influenceing in inflammatory associated diseases such as cancer, arthritic

and diabetic So the present work was focussed to isolate of alkaloid rich fraction from aqueous

extract of leaves and seeds of Trigonella-foenum-graecum and evaluated for In-vitro (HRBC

model) and in vivo (Carrageenan induced paw oedema method) anti-inflammatory activity with

its Antioxidant study.

In-vitro Antioxidant study

Nitric Oxide (NO) is a free radical produced in mammalian cells, involved in the

regulation of various physiological processes. However, excess production of NO is associated

with several inflammatory diseases (Gibananda, 2002). Nitric oxide is a very unstable species

under aerobic conditions. It reacts with O2 to produce stable product nitrate and nitrite through

intermediates. It was estimated by using Griess reagent and in presence of test compound which

was the scavenger. In this study the nitrite produced by the incubation of solutions of sodium

nitro prusside in standard phosphate saline buffer at 25o C was reduced by the aqueous extract of

leaves of Trigonella-foenum-graecum (AETFGL) and Chloroform fraction of seeds of

Trigonella-foenum-graecum (CFTFGS). The aqueous extract of leaves and chloroform fraction

of seeds produced a very significant free radical scavenging property which may be due to the

presence alkaloid.



DPPH assay is considered a valid method to evaluate scavenging activity of

antioxidants, since the radical compound is stable and does not have to generate as in other

radical assays. DPPH radicals react with suitable reducing agents and then electrons become

paired off and the solution loses colour stoichiometrically with the number of electrons taken up.

Such reactivity has been widely used to test the ability of plant extract to act as free radical

scavengers. Reduction of the DPPH radicals can be observed by the decrease in absorbance at

517 nm. (Kishore et al, 2001) DPPH assay of AETFG and CFTFG of leaf and seed showed a

dose dependant increase in the percentage of inhibition of free radicals. Here the aqueous extract

of leaf and chloroform fraction of seed of Trigonella-foenum-graecum showed a well marked

activity.

Anti-inflammatory activity

Membrane stabilization model

Leaf and seed extract of Trigonella-foenum-graecum with its chloroform fraction were

subjected for the in-vitro anti-inflammatory activity. The venthayam extract with membrane-

stabilizing properties are well known for their ability to interfere with the early phase of

inflammatory reactions, namely the prevention of release of phospholipases that trigger the

formation of inflammatory mediators (Umukoro et al ., 2006). The aqueous extract of leaves of

Trigonella-foenum-graecum (AETFG) and Chloroform fraction of seeds of Trigonella-foenum-

graecum (CFTFG) demonstrate significant membrane stabilizing property, which suggests that

its inflammatory events ,namely the release of chemical mediators by HRBC membrane

stabilization against hypotonicity induced haemolysis was found to be effective. The extracts and

its fraction exhibited membrane stabilization effect by inhibiting the hypotonicity induced lyses



of erythrocyte membrane. The erythrocytes membrane is analogus to the liposomal membrane

and its stabilization implies that extract and its fraction may as well stabilize liposomal

membrane. (Rajendran Vadiru et al ., 2008). It posses the significant anti-inflammatory

activity may be due to presence of alkaloid.

Carrageenan induced paw edema model

The anti-inflammatory activity carried out for aqueous extract of leaves of Trigonella-

foenum-graecum and its fraction as well as aqueous extract of seed of Trigonella-foenum-

graecum with its fraction. For the anti-inflammatory activity it is important to estimate the

activity in the acute phase as well as chronic phase of inflammation. The inflammatory condition

induced by carrageenan involves step-wise release of vasoactive substances such as histamine,

bradykinin and serotonin in the early phase and prostaglandins in the acute late phase .These

chemical substances produced increase in vascular permeability , thereby promoting

accumulation of fluid in tissues that accounts for the oedema (Appleton et al ., 1995). These

inflammatory mediators are released endogenously and contribute to the various phases of paw

edema. (Mujumdar et al, 2000).

The aqueous extract of leaves of Trigonella-foenum-graecum and chloroform fraction of

seeds of Trigonella-foenum-graecum showed the dose dependent anti-inflammatory activity,

which was found to be statistically significant at higher concentration in acute Carrageenan

induced rat paw oedema model. This activity appears to be significant in early phase of

inflammation in which very biochemicals like Histamine, 5-HT and various Bradykinin

involved.The all parameters has been carried out, we concluded that the aqueous extract of

leaves of Trigonella-foenum-graecum and chloroform fraction of seed of Trigonella-foenum-



graecum were effective against antioxidant activity and anti-inflammatory activity may be due to

presence of more alkaloid. The results of the study showed that Leaves aqueous extract and seed

chloroform fraction was correlate with each other possessed anti-inflammatory property ,as it

significantly inhibited edema induced by carrageenan, in rats. and it also showed significant protection of

the erythrocyte against lysis induced by hypotonic solution It has been demonstrated in a recent

study that fenugreekadministration to diabetes obese KK ay miceinhibits macrophage infiltration

into adipose tissuesand decreased the mRNA expression levels ofinflammatory genes, which is

responsible for itsanti-inflammatory action. ( Uemura ., et al 2010 ) enugreek has beenreported

to accelerate the process of wound healingvia its antioxidant potential in rats injured in

theposterior neck area (Abdullah et al., 2007 )

CONCLUSION

Thus, the results of the present study was concluded that the aqueous extract of leaves of

Trigonella-foenum-graecum and chloroform fraction of seed of Trigonella-foenum-graecum

were effective against anti-inflammatory activity.by both the tested methods. The result may be

concluded that the reduction of oedema in acute inflammatory condition may be due to its

scavenging property of the alkaloids. However , a more extensive study is necessary to

determine the exact mechanism of specific alkaloid of conflict of interest statement.

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INTERNATIONAL JOURNAL OF FRONTIERS IN … 1/Issue 2...INTERNATIONAL JOURNAL OF FRONTIERS IN SCIENCE AND TECHNOLOGY Research Article ISSN 2321 – 0494 Received on: 20.3.13, Received