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International Journal of Pharmacognosy and Phytochemical Research 2014; 6(3): 477-481
ISSN: 0975-4873
*Author for correspondence: Email: phanisurya163@gmail.com
Research Article
Evaluation of In vitro and In vivo Anti-Inflammatory Activity of
Aqueous Extract of Gliricidia sepium Flowers in Rats
*Kola Phani Kumar1, Vadite Siva Naik, V.Bhuvan Chandra, R. Lavanya, K. Narendra
Kumar, V.Bhagyasree, B.Soumya , Lakshmi Sudeepthi N2
1Department of pharmacology, University College of Pharmaceutical sciences, Acharya Nagarjuna University,
Nagarjuna Nagar, Guntur , Andhra Pradesh-522 510, India.
2 KVSR Siddhartha College of Pharmaceutical Sciences, Vijayawada, Krishna, Andhra Pradesh-520010, India.
Available Online: 1st September 2014
ABSTRACT
The present study was aimed for scientific evaluation of the anti-inflammatory activity of aqueous extract of Gliricidia
sepium linn. (fabaceae) flowers by in-vitro and in vivo models.The anti-inflammtory activity of aqueous extract obtained
by decoction was evaluated by in vitro HRBC membrane stabilization assay and in vivo carrageenan induced paw edema
model in albino wistar rats. Aqueous extract showed dose dependant anti-inflammatory activity in human red blood cell
membarane stabilization method at different concentrations (100-500 µg/kg) with a percentage protection of 7.15, 11.25,
22.71, 24.83 and 26.95 compared to standard diclofenac 32.09% at 10 µg/kg. Diclofenac sodium at 10 mg/kg, aqueous
extract administered at a dose of 250 and 500 mg/kg p.o. at 1, 3, 6 and 8 hours significantly (p<0.05) decreased and
increased the volume of paw edema & % protection compared to carrageenan group and dicclofenac, respectively. The
aqueous extract has shown a significant (p<0.05, p<0.01, p<0.001) percentage inhibition of paw edema 69.81±2.93 and
78.07±3.19 on 8th hour at 250 and 500mg/kg, respectively.These results provide a scientific basis for the use of the flowers
of Gliricidia sepium as an anti-inflammatory agent.
Keywords: Gliricidia sepium, Carrageenan Anti-inflammatory, Diclofenac.
INTRODUCTION
Inflammation is a fundamental pathological process of an
immune system towards tissue damage, tissue malfunction
and infection1.This complex reaction results from the
release of local hormones like Prostaglandins, histamine,
serotonin and cytokines. These biochemical molecules
regulate both homeostatic and pathological reactions such
as inflammation, fever, pain 2. Inflammation is associated
with various diseases. To treat this many of anti-
inflammatory agents like corticosteroids and NSAIDS are
available but these synthetic agents produces adverse
effects such as damage to GIT, gastric erosions and in
extreme case severe hemorrhage and death 3, 4.
Consequently there is a need for development of new anti-
inflammatory agents with minimum side effects. In this
context the value of Herbal medicinal plants, herbs and
spices is priceless as the treatment is 100% natural with no
side effects.
Gliricidia sepium is a medium size, semi deciduous trees,
native to central America which grows about 10m [33ft]
height and belonging to the family fabaceae 5. It is a fast-
growing, nitrogen-fixing tree used throughout the tropics
for the many environmental services. Gliricidia is widely
used to provide crop shade for cacao, coffee, and other
shade loving crops 6,7. The tree is also an important source
of green manure, fodder, and fuel wood. Several
phytochemicals like flavonoids 8, triterpenoid saponins,
stigmastanol glucoside, rhamnogalactoside of kaempferol,
coumarin, coumaric acid and melilotic acid and 12a-
hydroxy retenoids were reported in various parts of the
plant and bark, respectively 9. Furthermore, different parts
of the plants are reported to have viz. antimicrobial 10,
nematicidal 11, larvicidal 12 antioxidant 4 and it’s a folk
remedy for rheumatism, headache, fever, wounds,
fractures and urticaria etc 13. Gliricidia sepium is a folk
remedy for some of inflammation related diseases
fractures, gangrene, head-ache, itch, prickly heat,
rheumatism, urticaria, and wounds 14, till today there is no
scientific report on this plant. Hence, the present study was
aimed to investigate the anti-inflammatory activity of
Gliricidia sepium aqueous extract of flowers.
MATERIALS AND METHODS
Plant collection and identification: Flowers of Gliricidia
sepium was collected from Acharya Nagarjuna University
campus, Guntur district of Andhra Pradesh. The plant was
identified, confirmed and authenticated by comparing with
voucher specimen available at survey of Medicinal plants
and collection unit, Department of Botany.
Preparation of Aqueous Plant Extract: The flowers were
cut into small pieces, powdered and the crude drug is
boiled directly with distilled water at 60º-700 c for 3hourrs.
Then it is cooled and filtered. The extract was concentrated
under reduced pressure and stored in vacuum desiccators,
Kola Phani Kumar et al. / Evaluation of In vitro…
IJPPR, Vol 6, Issue 3, September-November 2014, 477-481
Page478
which was used for in-vitro and in-vivo anti-inflammatory
investigations.
Phytochemical Screening: Preliminary qualitative
phytochemical screening of Gliricidia sepium flower
extract shows a positive test for carbohydrates [Benedict’s
test and Barfoed’s test], proteins [Millons test], flavonoids
[Shinoda test], saponins [froth formation test] and Tannins
[color reaction with ferrous chloride].
Acute Toxicity [LD50] Study: Acute oral toxicity of was
studied as per OECD guidelines 425 using albino Swiss
mice. The extract was found to be safe up to 2000 mg/kg
body weight.
Pharmacological Evaluation-
In vitro Anti-inflammatory activity-
HRBC membrane stabilization assay 15, 16: Fresh human
blood was collected, centrifuged and prepared 10% v/v
Table 1: In vitro anti-inflammatory activity of aqueous extract of Gliricidia sepium
Groups
% Hemolysis
Mean ± SEM
% protection
Diclofenac 10µg/ml
67.9±0.085
32.09
GSAE 100 µg/ml
92.8±0.0236***
7.15
GSAE 200µg/ml
88.7±0.0731***
11.25
GSAE 300 µg/ml
77.3±0.0853***
22.71
GSAE 400 µg/ml
75.2±0.0873***
24.83
GSAE 500 µg/ml
73.0±0.101***
26.95
Results are MEAN±SEM, ***P<0.001 [Dunnett’s post hoc test] significant when compared with standard Diclofenac.
0
10
20
30
40 DICLOFENAC 10µg/ml
GSAE 100 µg/ml
GSAE 200 µg/ml
GSAE 300µg/ml
GSAE 400µg/ml
GSAE 500µg/ml
% Protection
*** ***
*** *** ***
Fig. 1: in vitro anti-inflammatory activity of aqueous extract of Gliricidia sepium and diclofenac.
Table 2: Effect of Gliricidia sepium aqueous flower extract on carrageenan induced rat paw oedema volume
S.No.
1h
3h
4h
8h
Control [Saline]
0.48±0.021
0.56±0.017
0.60±0.035
0.71±0.029
Diclofenac 10 mg/kg
0.31±0.017
0.27±0.019
0.22±0.014
0.11±0.01
Aq.extract 250 mg/kg
0.45±0.023***
0.36±0.015*
0.29±0.026ns
0.21±0.014**
Aq.extract 5oo mg/kg
0.37±0.03*
0.31±0.028ns
0.25±0.032ns
0.16±0.023ns
Results are MEAN±SEM, [n=6] *P < 0.05, **P<0.01 and ***P<0.001 [Dunnett’s post hoc test] significant when
compared with standard Diclofenac.
0.0
0.2
0.4
0.6
0.8
CONTROL
DICLOFENAC 10 mg/kg
GSAE 250 mg/kg
GSAE 500 mg/kg
1hr 3hr 4hr 8hr
*** **
**
Change in paw volume (ml)
Fig. 2: Effect of Gliricidia sepium aqueous flower extract on carrageenan induced rat paw volume
Results are MEAN±SEM, [n=6] *P < 0.05, **P<0.01 and ***P<0.001 [Dunnett’s post hoc test] significant when
compared with standard Diclofenac
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IJPPR, Vol 6, Issue 3, September-November 2014, 477-481
Page479
suspension with normal saline. The reaction mixture
consists of total volume 4.5ml i.e. 1ml of different
concentrations of extract [100, 200, 300, 400, 500 µg/ml],
2ml of 0.25% NaCl, 1ml of 0.15M phosphate buffer [pH
7.4] and 0.5ml 10% HRBC 10% v/v was added.
Diclofenac used as standard. The mixtures were incubated
at 560C for 30 minutes and centrifuged at 3000rpm for 20
minutes. The supernatant solution was collected and
subjected to spectrophotometric analysis at 560 nm.
Percentage membrane stabilization was calculated.
% Hemolysis = [Absorbance of Test sample / absorbance
of Control] X 100
% Membrane Stabilisation = 100 – [[Absorbance of Test
sample / Absorbance of Control] X100]
In vivo anti-inflammatory activity
Experimental Animals: Albino wistar rats weighing 150-
250g was procured from Biogen, Bangalore.. Animals
were maintained under controlled condition of temperature
at 27o ± 2o C and 12-h light-dark cycles. They were housed
in polypropylene cages and had a free access to standard
pellets [Amruth] and water ad libitum. All the animal
studies were conducted according to prescribed guidelines
of Committee for the Purpose of Control and Supervision
of Experiments on Animals [Reg No:
1725/GO/a/13/CPCSEA], Govt. of India.
In-vivo anti-inflammatory activity 17,18, 19: Animals were
divided into 4 groups of 6 animals each. Before 1h of
carrageenan injection into rat paw Group I received saline,
Group II received Diclofenac 10 mg/kg, Group III received
GSAE 250 mg/kg and group IV received GSAE 500
mg/kg [Group IV].
Before carrageenan injection, the paw volumes for each rat
were measured separately by water plethysmometer
(INCO INDIA PVT LTD). Edema caused by carrageenan
was measured at 0, 1, 3, 4 and 8 hours. The anti-
inflammatory potency of the extract was determined by
comparing its effect with standard diclofenac sodium.
Then percent inhibition of edema was calculated for each
group with respect to the control group as follows.
Percentage of inhibition of paw edema =
[Vc – Vt / Vc] × 100
Where Vc and Vt represent average paw volume of control
and drug treated animals respectively.
Statistical Analysis: Results were analyzed by using one-
way ANOVA followed by post hoc Dunnett’s test using
Graph pad Prism-5 v software. The results were expressed
as Mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 was
considered as significant.
RESULTS
The aqueous extract of the Gliricidia sepium was studied
for in vitro anti-inflammatory activity by HRBC
membrane stabilization method. The plant extract showed
dose dependant anti-inflammatory activity and %
protection of HRBC in hypotonic solution. Results were
compared with standard diclofenac [Table &Figure 1].
Aqueous extract of flowers 250 & 500 mg/kg and standard
drug were tested for anti inflammatory activity at different
hours in carrageenan induced paw edema model using
water plethysmometer. Diclofenac sodium at 10 mg/kg,
Aqueous extract administered at a dose of 250 and 500
mg/kg p.o. at 1, 3, 6 and 8 hours significantly[*P<0.05,
Table 3: % protection of aqueous extract of Gliricidia sepium flowers against carrageenan induced paw oedems
S.No.
1h
3h
4h
8h
Diclofenac10 mg/kg
33.66±4.37
50.21±4.06
61.73±2.75
84.04±1.54
Aq.extract 250 mg/kg
5.66±1.21***
35.12±4.ns
51.06±5.90ns
69.81±2.93**
Aq.extract 5oo mg/kg
14.81±3.3**
43.15±5.51ns
57.62±6.07ns
78.07±3.19ns
0
20
40
60
80
100 DICLOFENAC 10 mg/kg
GSAE 250 mg / kg
GSAE 250 mg / kg
1hr 3hr 4hr 8hr
*** *
**
% Protection
Fig. 3: % protection of Gliricidia sepium extract on carrageenan induced paw oedema
Results are MEAN±SEM, [n=6], *P < 0.05, **P<0.01, ***P<0.001 [Dunnett’s post hoc test] significant when
compared with standard Diclofenac.
Results are MEAN±SEM, [n=6], *P < 0.05, **P<0.01, ***P<0.001 [Dunnett’s post hoc test] significant when
compared with standard Diclofenac.
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IJPPR, Vol 6, Issue 3, September-November 2014, 477-481
Page480
**P<0.01, ***P<0.001] decreased and increased the
volume of paw edema & % protection compared to
carrageenan group and diclofenac respectively. Aqueous
extract at 250 p.o. at 1, 3, 4 and 8 hours prevented the
carrageenan induced paw edema with a percentage
inhibition of 5.66±1.21, 35.12±4.36, 51.06±5.90,
69.81±2.93 at 1, 3, 4, 8 hour, respectively., while
14.81±3.37, 43.15±5.51, 57.62±6.07, 78.07±3.19 at dose
of 500 mg/kg p.o. at 1, 3, 4, 8 hour, respectively.
Diclofenac sodium decreased the carrageenan induced paw
edema with a percentage inhibition of 33.66±4.37,
50.21±4.06, 61.73±2.75, 84.04±1.54 at 1, 3, 4, 8 hour,
respectively [Table 1,2 & Figure 1,2].
DISCUSSION
Inflammation is the common problem in all the age groups
irrespective of gender and the use of NSAIDS to treat
inflammation produces vascular and gastrointestinal
complications.Anti-inflammatory drugs available in the
market produces symptomatic relief with adverse effects.
Now a day’s medicinal plants and their formulations are
used for various disorders in ethno medical practices as
well in the traditional system of medicine in India.
Furthermore, these natural remedies can be used to cure
problems rather than just mask symptoms 20. Herbal
medicines importance is increased in treatment of chronic
and acute diseases with free of adverse effects. Solanum
trilobatum 21, Plumeria acuminate 22, Thesium chinense,
Mexican medicinal plant extracts 23 and Crude Saponin
Extracts 24 also showed the anti-inflammatory effect in
carrageenan induced paw edema.
In the present study the plant extract exhibited dose
dependant membrane stabilization effect by inhibiting
hypotonicity induced lyses of RBC membrane in in vitro
assay. The RBC membrane is analogous to the lysosomal
membrane and its stabilization indicates lysosomal
membrane stabilization. It plays a vital role in reduction of
pathological mechanisms invovlved in inflammation by
augmenting the release of activated neutrophil,
bactericidal enzymes and proteases, which produce further
tissue inflammation and damage 25.
Carrageenan induced inflammation is the most sensitive
and reliable model for evaluating acute phase and orally
active anti-inflammatory agents. The time course of
increase in paw edema is represented as biphasic event.
Development of carrageenan induced inflammation
during 1 hour is due to the release of histamine and
serotonin 26-28 along with trauma of injection, where as the
second phase and third phase is attributed to the release of
kinin like substances and prostaglandins, respectively 29-31
[Cyclooxygenase - 2], protease and lysosome. Mediators
like histamine, serotonin and COX-2 released
prostaglandins increases the vasodilatation, hyperemia,
pain and edema. Table 4 & 5 represent the in vivo anti-
inflammatory activity of extract. Significant [*P<0.05,
P**<0.01,***P<0.001] anti-inflammatory activity was
observed at 4th and 8th hours compared to standard
reference diclofenac. Aqueous extract showed dose
dependant anti-inflammatory activity at third phase of
carrageenan induced paw edema, with maximum anti-
inflammatory activity at 500 mg/kg.
In conclusion, extract showed anti-inflammatory activity
in later phases in dose dependent manner and the inhibitory
effect of aqueous extract may be due to the inhibition of
cyclooxygenase induced prostaglandin synthesis and
neutrophil mobilization. This anti-inflammatory effect of
the extract observed might be due to the presence of
flavonoids and saponins in the plant. The present
investigation has also opened avenues for further research
especially with reference to the isolation and development
of potent phytomedicines from this extract for treatment
of inflammation.
REFERENCES
1. Ashley NT, Weil ZM, and Nelson RJ. Inflamation:
mechanisms, Costs, and Natural Variation. Annu.Rev.
Ecol. Evol.Syst. 2012; 43: 385-406.
2. Miller MJ, Ahmed S, Bobrowski P, Haqqi TM. The
chondroprotective actions of a natural product are
associated with the activation of IGF-1 production by
human chondrocytes despite the presence of IL-1b.
BMC Complement Altern Med. 2006; 6:13.
3. Almekinders LC. Anti-inflammatory treatment of
muscular injuries in sport. An update of recent studies.
Sports Med. 1999; 28:383–388.
4. Akharaiyi FC, Boboye B and Adetuyi FC.
Antibacterial, Phytochemical and Antioxidant
Activities of the Leaf Extracts of Gliricidia sepium
and Spathodea campanulata. World Applied Sciences
Journal. 2012; 16 [4]: 523-530.
5. Medzhitov R. Origin and physiological roles of
inflammation. Nature. 2008; 454:428-435.
6. Chadhokar PA. Gliricidia maculate, Apronising
legume forage plant. World Animal Review. 2010; 44:
36-43.
7. Csurhes S. And Edwards. Potential environmental
weeds in australia; candidate species for preventative
control. Queens land department of natural resources.
1998; 164-168.
8. Rastrelli L, Berger I, Kubelka W, Caceres A,
Tommasi DN, Simone DF. New 12a-
Hydroxyrotenoids from Gliricidia sepium bark. J Nat
prod.1999; 62 [1]:188-190
9. Beena Jose, And Joji Reddy L. Evaluation Of
Antibacterial Activity of The Leaf And Flower
Essential Oils of Gliricidia sepium From South India.
International Journal of Applied Pharmaceutics. 2010;
2[3]:177-179.
10. Nazli R, Sohail T, Nawab B And Yaqeen Z.
Antimicrobial property of Gliricidia sepium plant
extract.Pakisthan J. agric. Res. 2011;24 : 1-4.
11. Nazli R, Akhter M, Ambreen S, Solangi AH and
Sultana N. Insecticidal, Nematicidal and antibacterial
activities of Gliricidia sepium, Pak. J. Bot. 2008;
40[6]: 2626-2629.
12. Kaliyamoorthy Krishnappaa, Shanmugam
Dhanasekarana, Kuppusamy Elumala. Larvicidal,
ovicidal and pupicidal activities of Gliricidia sepium
[Jacq.] [Leguminosae] against the malarial vector,
Kola Phani Kumar et al. / Evaluation of In vitro…
IJPPR, Vol 6, Issue 3, September-November 2014, 477-481
Page481
Anopheles stephensi Liston [Culicidae: Diptera]
Phytochemistry: Asian Pacific Journal of Tropical
Medicine. 2012; 5[8]:598–604.
13. Manners GD, Jurd L. Additional Flavonoids in
Gliricidia sepium. Phytochemistry, 1979;18(6):1037-
1042.
14. Abulude, F.O, and Adebote V.T. Antibacterial
Investigation Of Crude Extracts Of The Root Bark Of
Gliricidia Sepium. Continental J. Microbiology. 2009;
3: 23 – 26.
15. Chatterjee S, Das SN. Anti-arthritic and Anti-
inflammatory effect of a poly-herbal drug [EASE@]:
its mechanisam of action. Indian.J.Pharmacology.
1996; 28:166-119.
16. Rajurkar R, Jain R, Matake N, Aswar P, Khadbadi
SS. Ati-inflammatory Action of Abutilon indicum[L]
Sweet Leaves by HRBC Membrane Stabilization. Res
J. Pharm. Tech. 2009; 2[2]:415-416
17. Mohamed F.Zayed, Memy H. Hassan. Syntesis and
biological evaluation studies of novel quinazoline
derivatives as anti-bacterial and anti inflammatory
agents. Saudi Phar J 2014; 22:157-162.
18. Alam K, Pathak D, Ansari SH. Evaluation of anti -
inflammatory activity of Adhatoda Zeylanica
[MEDIC.] leaves extract. International Journal of
Pharma and Bio Sciences. 2011; 2[1]:157-162.
19. Igbe I, Ching FP, Eromon A. Anti-inflammatory
activity of aqueous fruit pulp extract of Hunteria
umbellata K. Schum in acute and chronic
inflammation. Acta Pol Pharm. 2010; 67[1]:81-85.
20. Mukherjee PK, Venkatesh P and Ponnusankar S.
Ethnopharmacology and integrative medicine – Let
the history tell the future. J Ayurveda Integr Med.
2010; 1[2]: 100–109.
21. Ravi V, Saleem TSM, Maiti PP, Gauthaman K and
Ramamurthy J. Phytochemical and pharmacological
evaluation of Solanum nigrum Linn. African Journal
of Pharmacy and Pharmacology. 2009; 3[9]: 454-457.
22. Gupta M, Mazumder UK, Gomathi P, Thamilselvan
V. Antiinflammatory evaluation of leaves of Plumeria
acuminate. BMC Complementary and alternative
medicine [Internet]. 2006; 36:1472-6882.
23. Meckes M, David-Rivera AD, Nava-Aguilar V,
Jimenez A. Activity of some Mexican medicinal plant
extracts on carrageenan-induced rat paw ; edema.
Phytomedicine. 2004; 11[5]:446-51.
24. Halimatu S.H, Muhammad I.S, Muhammad
A.M,Andrew A.E, Hajara I, Ali S.H, and Abdullahi
H.Y. Analgesic and anti-inflammatory activities of the
saponins extract of Carissa edulis root in rodents. Int.
J. Biol. 2014; 4[4]: 1310-1317.
25. Govindappa M, Nagasravya S, Poojashri MN,
Sadananda TS, Chandrappa CP, Gustavo S,
Sharanappa P and Anil Kumar NV. Antimicrobial,
antioxidant and in vitro anti-inflammatory activity and
phytochemical screening of water extract of Wedelia
trilobata[L.] Hitchc. Journal of medicinal plants
research. 2011; 5[24]:5718-5729.
26. Sarika A, Purabi R, Vinod S, Avnish K, Rambir S,
and Poonam S. Anti-Inflammatory Activity of
Lactobacillus on Carrageenan-Induced Paw Edema
inMaleWistar Rats International Journal of
Inflammation, Volume [2012], Article ID 752015, 6
pages, doi:10.1155/2012/752015.
27. Anuja GI, Latha PG, Shine VJ, Suja SR, Shikha P,
Satheesh Kumar K, and Rajasekharan S.
Antiedematous and Analgesic Properties of Fertile
Fronds of Drynaria quercifolia. Hindawi Publishing
Corporation ISRN Inflammation. [2014]Article ID
302089.
28. Anuradha M, Kushwha P, Murthy N.Evaluation of
diclofenac potassium microsphere for anti-
inflammatory Activity. Asian journal of
pharmaceutical and clinical research. 2012; 5[2]:19-
22.
29. Nayak BS, Patel KN. Anti-Inflammatory Screening
Of Jatropha Curcas Root, Stem And Leaf In Albino
Rats. Rom. J. Biol. – plant biol.2010; 55: 9-13.
30. Rajendran V and Lakshmi KS. In vitro and in vivo
anti-inflammatory activity of leaves of Symplocos
cochinchnensis [Lour] Moore ssp laurina. Bangladesh
Journal of Pharmacology. 2008; 3:121-124.
31. Valiollah H, Hossein S, Mohsen M. Effect of
Fluvoxamine on Carrageenan-Induced Paw Edema in
Rats Evaluation of the Action Sites. Services. Iranian
Journal of Pharmaceutical Research 2011; 10 [3]: 611-
618.