ANTI-INFLAMMATORY, ANALGESIC AND ULCEROGENIC ACTIVITY OF VIGNA MUNGO LINN. LEAVES

Abstract

The anti-inflammatory, analgesic and ulcerogenic activity of extract of leaves of Vigna Mungo linn. (Leguminosae) were investigated as well as the mechanisms of action. The extract significantly (P<0.05) inhibited the formation of paw edema induced by    carrageenan in rat and increased reaction latency to thermal pain in rat in a dose-dependent manner. The extract caused a significant (P<0.05) dose-dependent ulceration of rat gastric mucosa and concentration-dependent inhibition of hypotonicity-induced haemolysis of red blood cell. Also the extract significantly (P<0.05) inhibited the activates of the phospholipase A2 and prostaglandine synthesis in a concentration related manner. These suggest that the leaves posses anti-inflammatory, analgesic and ulcerogenic activities mediated through sequential inhibition of the enzymes responsible for prostaglandine synthesis from arachidonic acid. Phytochemical analysis of the extract revealed the presence of glycoside, tannins, alkaloid, flavonoids and Saponins. Acute toxicity studies established an oral LD50 greater than 3000 mg/kg.

Full text

IJPPVOL1ISSUE2

International Journal of Phytopharmacy Research Article
Vol. 1 (2), pp.55-60, Nov-Dec 2011
©Scholar Science Journals
ANTI-INFLAMMATORY, ANALGESIC AND ULCEROGENIC ACTIVITY OF
VIGNA MUNGO LINN. LEAVES
Mohammed Rageeb Mohammed Usman
1
, S. D. Barhate
2
1
Ph.D Research Scholars JJT University, Jhunjhunu, Rajasthan, India, 333001
2
Shri Suresh Jain Institute of Pharmaceutical Education and Research Center, Jamner,
Maharashtra, India, 424206.
Corresponding Author: rageebshaikh@gmail.com
ABSTRACT
The anti-inflammatory, analgesic and ulcerogenic activity of extract of leaves of Vigna
Mungo linn. (Leguminosae) were investigated as well as the mechanisms of action. The
extract significantly (P<0.05) inhibited the formation of paw edema induced by carrageenan
in rat and increased reaction latency to thermal pain in rat in a dose-dependent manner. The
extract caused a significant (P<0.05) dose-dependent ulceration of rat gastric mucosa and
concentration-dependent inhibition of hypotonicity-induced haemolysis of red blood cell.
Also the extract significantly (P<0.05) inhibited the activates of the phospholipase A
2
and
prostaglandine synthesis in a concentration related manner. These suggest that the leaves
posses anti-inflammatory, analgesic and ulcerogenic activities mediated through sequential
inhibition of the enzymes responsible for prostaglandine synthesis from arachidonic acid.
Phytochemical analysis of the extract revealed the presence of glycoside, tannins, alkaloid,
flavonoids and Saponins. Acute toxicity studies established an oral LD
50
greater than 3000
mg/kg.
Keywords- Vigna Mungo Linn; Pharmacological evaluation; phytochemical investigation
1. INTRODUCTION
Medicinal plants with anti-inflammatory activity are considerably employed in the traditional
treatment of several disorders of inflammation. The inflammatory response involves a
complex array of enzyme activation, mediator release, fluid extravasations, cell migration,
tissue breakdown and repair.
1
these different reactions in the inflammatory response cascade
are therapeutic targets which anti-inflammatory agent including medicinal plants interfere
with to suppress exacerbated inflammatory responses usually invoked in such disorders as
rheumatoid arthritis, in infection or injury. Inhibition of the synthesis of pro-inflammatory
prostaglandins is one of such therapeutic targets to which some of the potent anti-
inflammatory agents of clinical relevance (NSAIDs) owe their activity
2
. several anti-
inflammatory medicinal plants have also demonstrated the ability to inhibit the synthesis of
prostaglandins
3
.
Vigna Mungo Linn. (Leguminosae) is widely distributed in tropical west Africa and
extensively cultivated all over India. It is commonly knows as Black gram, Urad and Udid.
Hot aqueous extracts of the leaves are useds in the treatment of stomach, rhematic pain and

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inflammatory disorderds. Indicating that the plants leaves may possess anti-inflammatory,
analgesic and ulcerogenic properties among others. The young leaves are used as vegitable.
4
The antiatherogenic nature of Phaseolus Mungo L. has been reported.
5
The enzymes IAA
oxidase and peroxides are responsible for the production of IAA.
6
black gram fiber exhibits
significant hypoglycemic action in experimental animals.
7
The search for new anti-inflammatory agents from the vast array of medicinal plant sources
in intensifying since they may hold promise for the discovery of therapeutic agents with
beneficial effect not just in suppressing relevant aspects of the inflammatory cascade but also
on diverse disease conditions where the inflammatory response is amplifying the disease
process. This present study carried out to assess the validity of the folkloric uses of this plant
in the management of pain and treatment of inflammatory disorders and establish the possible
mechanisms of pharmacological action.
2. MATERIAL AND METHODS
2.1. Plant Materials: Fresh leaves of Vigna Mungo Linn. Collected in local area of Jalgaon.
The plant was identified and authenticated from the Department of Botany, Rashtrasant
Tukadoji Maharaj University, Nagpur. The Account no. of authentication certificate was
9146. The fresh leaves were cleaned, dried and pulverized. About 200 g of the powdered
leaves was boiled and boiled in distilled water for 2 h. the filtrate was partitioned with
chloroform: methanol (2:1) to remove traces of fatty constituents from the hot water
extraction. The methanol fraction (15.29%) was concentrated and subjected to phytochemical
analysis using the general method and evaluated for biochemical and pharmacological
activity.
2.2. Animal and Tissues: Adult albino rats (150-200) of either sex. The animal were
acclimatized for about 7 days under standard environmental condition and were maintained
on a regular feed and water ad libitum Ox vesicles (OSV) and ox blood were obtained from
healthy animals slaughtered in the local abattoir Nsukka. Strains of Bacillus pulmilum used
for enzyme assay were obtained from the Dept. of Microbiology.
2.3. Chemical: The chemical used for this study included analytical grades of methanol,
hydroquinone, ethyl acetate, sucrose, ethylene diamine tetracetate (EDTA), hydrochloric
acid, sulphuric acid, sodium chloride, chloroform, tri-sodium citrate, alpha naphthol,
glutathione, adenosine 5
1
-diphosphate (ADP) and hemoglobin. Other reagents and solvent
were also of analytical grade.
2.4. Acute toxicity and lethality (LD
50
) test: Acute toxicity study was carried out according
to OECD guidelines (Organization for economic co-operation and development).
8
The acute
toxicity study of various extracts of Vigna Mungo Linn. Leaves shown signs of toxicity like
tremor, convulsion and deep breathing at 2000 mg/kg b.w. 1/10
th
of the same dose for all
these extract were taken as therapeutic dose i.e.200 mg/kg b.w.
2.5. Anti-inflammatory activity test: Anti-inflammatory activity was assessed in rat using a
modification.
9
of the method of Winter et. al.
10
increase in paw volumes was used to assess
inflammation. Four group of rats (n = 5) were deprived of food but not water for 18 h and
then received i.p. injection of the extract (0.5 and 1 mg/kg) thirty minutes later, each animal
received subplantar injection of carrageenan (0.1 ml of 2% suspension) in its right hind paw.
Paw volume was measured by mercury displacement before and at 1.5 and 5.5 h after
carrageenan injection. Control animals received either normal saline (5 ml/kg) or
phenybutazone (150 mg/kg).
2.6. Analgesic activity test: Analgesic activity was tested in rats using the hot plate method.
Twenty five rats of either sex were grouped in five (n = 5 per group). Each group received
one dose of the extract (50, 100, 150 mg/kg), normal saline (5 ml/kg) or acetylsalicylic acid
(100 mg/kg). at 15 and 60 min after extract administration, animal were lowered onto the

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surface of a hot plate (50±2
0
C) enclosed with cylindrical glass and the time for the animal to
raise or lick the fore limb was noted as the reaction time (RT). Cut off time in the absence of
a response was 90 sec to prevent the animal from being burnt.
11
2.7. Gastric ulcerogenic activity test: The ulcerogenic activity of the extracts was
investigated using the method of Cashin et. al.
12
rat of either sex were fasted for 18 h with
access to water. At the end of the fasting the animal received the extracts (250 or 500 mg/kg;
n = 5) orally control animal received indomethacin (30 mg/kg) or normal saline (5 ml/kg).
Eight hours after drug administration, animals were scarified and stomachs opened along the
greater curvature. The stomach mucosa was examined for ulcer lesion using a hand lens (x20
magnification). The length of lesions on the gland at portion were estimated and summed up
to calculate the ulcer index using the method.
2.8. Phospholipase A
2
activity test: The preparation of Phospholipase A
2
from B. pulmilus
and assay of the extract on its activity were performed using the method of Vane.
13
aliquots
(0.5 ml)
of re-suspended erythrocytes were mixed with normal saline containing 2 Mm
calcium chloride and the enzyme preparation and incubated either in the absence or presence
of the extract (0.37, 0.74, 1.10 mg/kg) at 37
0
C for 1 h. the incubated reaction mixture was
centrifuged at 3,000 g for 10 min and the absorption of the supernatant read against the blank
at 418 nm. Prednisolone, a known inhibitor of the enzyme was used as control.
2.9. Prostaglandine synthesis activity test: Prostaglandine synthesis was isolated from ox-
seminal vesicle using the method of Nugteren et. al.
14
the enzyme activity was assayed using
a modification method of Yoshimoto et. al.
15
and Flower et. al.
16
the enzyme activity was
monitored at 278 nm due to the formation of PGB from PGE
2
by concentrated alkali
treatment. The reaction mixture consisted of 1.5 ml cofactor solution and 0.5 ml arachidonic
acid as substrate. After incubating at 37
0
C for 2 min, the reaction was stopped by adding 1.5
ml of 0.2 M citric acid, extracted twice with 5 ml ethyl acetate and centrifuged at 2,500 g for
10 min. each time 4 ml aliquots of the top organic layer were pipette into a clean test tube.
2.10. Membrane stabilization activity test: The membrane stabilization effect of the extract
was evaluated using hypotonicity induced haemolysis of red blood cells. Briefly, citrated ox-
blood sample were centrifuged at 3,000 g for 10 min. the pellets containing the red blood
cells were re-suspended in volumes of saline equal to those of the plasma. An aliquot (0.5 ml)
of the red cell suspension was added to 4 ml water and incubated at 37
0
C for 1 h.
3. STATISTICAL ANALYSIS
Data was analyzed using ANOVA, further subjected to Fischer LSD post hoc test and
expressed as Mean ± SEM. Differences between means were regarded significant at P<0.05.
4. RESULTS AND DISCUSSION
4.1. Phytochemical tests: Phytochemical tests on the extract have positive reaction for
glycosides, tannins, alkaloids, flavonoids and Saponins.
4.2. Acute toxicity: The acute toxicity studies revealed an oral LD
50
greater than 2000 mg/kg.
4.3. Effect on carrageenan induced paw edema formation: The extract significantly
(P<0.05) inhibited the formation of paw edema in rats. The magnitude of inhibition increased
with time with the effect of the extract comparing well with that of phenybutazone (Table
No.1)
4.4. Effect on reaction latency to thermal induced pain in rat: The extract caused a dose-
dependent increase in reaction latency to thermal pain. At 15 min, the extract (50 mg/kg)
significantly (P<0.05) evoked a longer reaction latency than aspirin and also significantly
(P<0.05) prolonged the reaction latency at 60 min (Table No.2)

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4.5. Effect on gastric ulcerogenic activity: The extract caused a significantly (P<0.05) dose-
dependent ulceration of the rat gastric mucosa. The ulcerogenic effect of the higher dose was
comparable to indomethacin (Table No.3)
4.6. Effect on phospholipase A
2
activity: The extract significantly (P<0.05) inhibited
phospholipase A
2
activity in a concentration related manner provoking inhibition comparable
to that pf Prednisolone (Table No.4)
4.7. Effect on prostaglandine synthesis activity: The extract evoked a significant (P<0.05)
concentration related inhibition of prostaglandine synthesis activity (Table No.5)
4.8. Effect on hypotonic-induced haemolysis: The extract significantly (P<0.05) inhibited
hypotonicity-induced haemolysis of red blood cells in a concentration-dependent manner
(Table No.6)
REFERENCES
1. Vane JR, Bolting RM, New insight into the mode of action of anti-inflammatory drugs,
Inflamm. Res., 1995, 44(1), 1-10.
2. Flower RJ, Vane JR, Inhibition of prostaglandin synthesis, Biochem. Pharmacol., 1974,
23, 1439-1450.
3. Jager AK, Hutching A, Van SJ, Screening of Zulu medicinal plants for prostaglandin
synthesis inhibition, J. Ethnopharmacol., 1996, 52, 95-100.
4. Asima C, Satyesh C, The Treatise of Indian Medicine, CSIR, New Delhi, 1992, 2,
128,129.
5. Srivastava A, Joshi LD, Effect of feeding black gram (Phaseolus Mungo) on serum
lipids of normal and diabetic guinea pigs, Ind. J. Med. Res., 1990, 92,383-386.
6. Ghosh S, Basu PS, Production and metabolism of indol acetic acid in roots and root
nodules of Phaseolus Mungo Linn., Microbial. Res., 2006, 161(4), 362-366.
7. Boby RG, Leelamma S, Black gram fiber (Phaseolus Mungo): mechanism of
hypoglycemic action, Plant Food Human Nutrition., 2005, 60(4), 173-80.
8. Guidelines Document on Acute Oral Toxicity Environmental Health and Safety
Monograph Series on Testing and Assessment no. AOT 425.
9. Ezekwesili C, Nwodo OF, Anti-inflammatory activity of Vignia ungiculata seeds
extract, J. Med. Lab. Sci., 2000, 9, 141.
10. Winter CA, Risley EA, Nuss GW, Carrageenan-induced edema in hind limp paw of the
rat as an assay for anti-inflammatory drugs, Proc. Soc. Exp. Biol., 1962, 111, 544-557.
11. Sharma KK, Khanna T, Sen P, Current status of centrally acting peptides. In:
(Dhawan, B. N., ed) Advance in Biosciences, 1982, 38,142.
12. Cashin CH, Darson W, The pharmacology of bernoxaprofen, a new compound with
anti-inflammatory activity apparently unrelated to inhibition of PG synthesis, J. Pharm.
Pharmacol., 1979, 29, 330-336.
13. Vane JR, Inhibition of prostaglandin synthesis as a mechanism of action for aspirin like
drug. Nat. New Biol., 1971, 231, 232-235.
14. Nugteren DH, Beerthius RK, Enzymic conversion of all-8, 11, 14-eicosatrienoic acid
in to prostaglandin E, Rec. Trav. Chin., 1966, 85, 1757-1765.
15. Yoshimoto A, Tomita k, cofactor requirements of the enzyme synthesizing
prostaglandin in bovine serninal vesicles. J. Biochem., 1970, 68, 487-499.
16.
Flower RK, Vane JR, Inhibition of prostaglandin synthesis, Biochem. Pharmacol., 1974,
23, 1439-1450.

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Table No. 1: Effect of extract on carrageenan induced paw edema in rats.
Extract Dose
(mg/kg)
Edema (ml)
1.5 h 5.5h
Inhibition of edema (%)
1.5 h 5.5 h
Control _ 0.48±0.01 0.41±0.01 _ _
Methanol
extract
500
1000
0.02±0.01 0.07±0.01
0.42±0.01 0.07±0.01
58.3 82.9
12.5 82.9
Phenybutazone 150 0.03±0.01 0.02±0.01 37.5 51.2
*P<0.05 compared to control (ANOVA; LSD post hoc) n = 5.
Table No. 2: Effect of extract on latency of pain reaction in rats.
Extract Dose (mg/kg) Reaction latency (T) (s)
15 min 60 min
Control _ 6.3±0.6 6.1±0.25
Methanol extract
50
100
150
5.1±0.8
#
6.1±0.25
7.0±1.40 13.8±1.83
*
8.0±1.00 15.3±0.58
*
Acetylsalicylic acid 100 8.30±0.90 12.0±0.60
*
#
*P<0.05 compared to acetylsalicylic acid and control respectively (ANOVA; LSD post hoc).
Table No. 3: Gastric ulcerogenic activity of extract.
Extract Dose (mg/kg) Ulcer index (mm)
Control _ 0±0.00
Methanol extract
250
500
8±1.15
*
14±1.73
*
Indomethacin 30 15±1.15
*
Values of ulcer index shown are Mean ± SEM., *P<0.05 compared to control (ANOVA; LSD post
hoc) n = 5.
Table No. 4: Effect of extract on phospholipase A
2
activity.
Values of absorbance shown are Mean ± SEM of triplicate determination.
*P<0.05 compared to control (ANOVA; LSD post hoc).
Table No. 5: Effect of extract on prostaglandine synthesis activity.
Extract Concentration
(mg/kg)
Absorbance Enzyme
activity
Inhibition of
enzyme activity (%)
Control _ 1.570±0.040 7.15 0.00
Methanol
extract
0.1
0.5
1.0
5.0
0.110±0.005
*
0.055±0.003
*
0.004±0.001
*
0.020±0.006
*
5.54
3.07
1.08
0.54
22.52
57.06
84.89
92.45
Indomethacin 4.0 0.030±0.003
*
2.00 72.02
Values of absorbance shown are Mean ± SEM of triplicate determination.
*P<0.05 compared to control (ANOVA; LSD post hoc). Percent inhibition enzyme activity was
calculated relative to control.
Extract Dose (mg/kg) Absorbance Inhibition of enzyme
activity (%)
Control _ 1.33±0.06 00.0
Methanol extract
0.37
0.74
1.10
0.64±0.01
*
0.14±0.00
*
0.02±0.01
*
51.88
89.47
98.49
Prednisolone 1.00 0.11±0.01
*
99.13

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Table No. 6: Effect on extract on hypotonic-induced haemolysis.
Extract Concentration
(mg/kg)
Absorbance Inhibition of
haemolysis (%)
Control _ 0.46±0.02 0.00
Methanol extract
0.1
0.2
0.4
0.21±0.01
*
0.20±0.01
*
0.15±0.00
*
54.35
56.52
67.39
Indomethacin
0.2
0.4
0.6
0.15±0.01
*
0.13±0.01
*
0.16±0.01
*
67.39
71.74
65.22
Distilled Water _ 0.65±0.28 _
Values of absorbance shown are Mean ± SEM of triplicate determination.
*P<0.001 compared to control (Normal saline) and distilled water (ANOVA; LSD post hoc).
Percent inhibition of haemolysis was calculated relative to control.