Volume: 2: Issue-3: July-Sept -2011 ISSN 0976-4550

EVALUATION OF ANTI-INFLAMMATORY AND MEMBRANE STABILIZING EFFECTS O F

AQUEOUS ROOT EXTRACT OF Boerhavia diffusa LINN IN RATS.

G.M. Oladele1, O.J. Ode1 and M.A. Ogunbodede2

1
Department of Veterinary Pharmacology and toxicology, University of Abuja, Abuja, Nigeria.
2
Department of Theriogenology, University of Abuja, Abuja, Nigeria.
Correspondence address: gbengamike2@yahoo.com

ABSTRACT : Boerhavia diffusa is a widely used plant in traditional medicine for various disease
problems. In this study, the anti-inflammatory and membrane stabilizing effects of the aqueous extract of
its roots was evaluated in rats. Carrageenan-induced rat paw oedema model was used for anti-
inflammatory effect while rat red blood cells were used for membrane stabilizing property. The extract in
doses of 100-400 mg/kg significantly (p<0.05) inhibit carrageenan-induced rat paw oedema in a dose
dependent manner. The concentration of 20-80 mg/ml of the extract also showed a dose dependent
inhibition of the rat red blood cells haemolysis induced by hypotonic solution. It was concluded that the
extract possesses anti-inflammatory as well as membrane stabilizing properties.
Key words: Boerhavia diffusa, root, anti-inflammatory, membrane stabilizing, rats.

INTRODUCTION
Boerhavia diffusa or chicken weed is an herbaceous perennial plant of the family Nyctaginaceae. It is
widely distributed in the tropics and subtropics (CSIR, 1988). The plant is popular with indigenous
people all over the world because of its uses as vegetable and medicinal properties. It was reportedly
used in renal ailments as diuretic (Anand, 1995), treatment of stomach ache, anaemia, cough, and cold,
and as a diaphoretic, laxative, expectorant, and a potent antidote for snake and rat bites (Chopra et al.,
1956). It was also used in the treatment of nephrotic syndrome (Singh and Udupa, 1972), hepatitis, gall
bladder abnormalities, and urinary disorders (Cruz, 1995). While the flowers and seeds are used as
contraceptive (Chopra et al., 1956), the roots have been reportedly used for treatment of asthma
(Ambasta, 1986) and hepatoprotective in action ( Rawat et al, 1997), it has also been shown to be
antispasmodic, anticonvulsant (Borrelli et al, 2006) and pain-reliever (Hiruma-Lima et al, 2000).
The herb is a diuretic that acts on the glomeruli of the kidney and also protects the kidney from being
damaged (Rawat et al, 1997). Barthwal et al, 1991 reported the antihemorrhagic action, and its
antimicrobial effects have also been shown (Agrawal et al, 2004). The cytotoxic and anticancer activities
have been reported by Mehretra et al, (2002), Bharali et al, (2003) and Leyon et al, (2005). The aqueous
leaf extract of Boerhavia diffusa significantly reduced thiobarbutric acid reactive substances and
hydrogen peroxides with a significant increase in reduced glutathione, superoxide dismutase, catalase,
glutathione peroxidase and glutathione-s-transferase in liver and kidney (Satheesh et al, 2004), and its
hypoglycemic antidiabetic effects have been demonstrated (Gholap et al, 2004; Pari et al, 2004). The
plant is a good antihypertensive agent (Hensen et al, 1995) and it is believed to enhance lactation period
and also the amount of milk in cattle (CSIR, 1988). Literature survey of this plant however, revealed very
little information about the use of its root as an anti-inflammatory agent and treatment of inflammation
related diseases. This study therefore aimed at evaluating the anti-inflammatory and membrane
stabilizing properties of the aqueous extracts of the root of the plant, as the root is highly medicinal.

International Journal of Applied Biology and Pharmaceutical Technology Page:84

Available online at www.ijabpt.com
Oladele et al ISSN 0976-4550

MATERIALS AND METHODS

Laboratory animals: Wistar albino rats (170-200 g) of both sexes were used for the study. The animals
were kept in the Faculty of Veterinary medicine animal house in a well ventilated rat cages, with free
access to food and water ad libitum.
Preparation of the plant material: The roots of Boerhavia diffusa used for this study were obtained
from a local farm in Ibadan, the plant was authenticated at the Department of Botany, Faculty of Science,
University of Ibadan, Ibadan. The roots were cut into smaller pieces and air dry, the dried roots were then
ground in a domestic grinding machine to almost fine powder. Two hundred and fifty grams (250 g) of
this material was soaked in 500 ml of distilled water for 72 hours with intermittent shaking. The solution
was filtered and the filtrate concentrated with vacuum rotary evaporator maintained at 40 oC. The
percentage yield of the extract was 9.75%. A fresh 10% solution of the extract was prepared with distilled
water to make appropriate dosages required for the studies.
Experimental procedures
Acute toxicity study: Five groups of albino rats (6 per group) were used. The first 4 groups received oral
doses of 100,200, 400 and 800 mg/kg of the extract. The fifth group received distilled water (10 ml/kg)
orally. The animals were observed for toxic symptoms and mortality was determined 24 hours post
administration.
Antiinflammatory study: The anti-inflammatory activity of the extracts was evaluated with carrageenan
induced oedema model (Winter et al., 1962). Rats (6 per group) were divided into five groups. Group 1-3
received oral doses of 100, 200 and 400 mg/kg of the extract. Group 4 was treated orally with
indomethacin (10 mg/kg) as standard drug, while group 5 was administered with distilled water (10
ml/kg) as control. After thirty minutes, 0.1 ml of 1.0% carrageenan was injected into the right hind paw
of each rat. The linear circumference of the injected paw was measured immediately after injection and
also 3 hours after injection and the percentage inhibition of oedema was calculated as described by Jain
and Khanna, (1981).
% Inhibition of oedema = l0 – l1/l0
Where l0 = change in paw circumference in control group, l1 = change in paw circumference in treated
groups.
Membrane stabilizing activity: The method use was as described by Shinde et al., (1999). Whole blood
was obtained with heparinized syringes from rats through cardiac puncture, centrifuged and supernatant
was carefully pipetted. The remaining packed cells were washed four times with equal volume of isotonic
buffered solution (154 mM NaCl in 10 mM sodium phosphate buffer (pH 7.4), the packed cells were
been centrifuged each time at 1000 rpm. 10% rat erythrocytes suspension was prepared with normal
saline and kept in refrigerator at 40C as stock erythrocytes.
The test sample consisted of 2 ml of hypotonic solution (50 mM Nacl in 10 mM sodium phosphate
buffer, pH 7.4) and varying concentrations of the extract (20, 40 and 80 mg/ml) or indomethacin (0.1
mg/ml) in normal saline to make 4.0 ml, this was then mixed with 0.5 ml of stock erythrocytes to make a
total of 4.5 ml. The control sample consisted of 0.5 ml of stock erythrocytes mixed with hypotonic-
buffered saline alone. The mixture was incubated for 10 minutes at room temperature, centrifuge for 10
minutes at 1000 rpm and the absorbance of the supernatant was measured at 540 nm. The percentage
inhibition of haemolysis or membrane stabilization was calculated according to modified method
described by Shinde et al, (1999).
% Inhibition of haemolysis = 100 x (OD1 – OD2) / OD1
Where OD1 = optical density of hypotonic buffered-saline solution alone (control) and OD2 = optical
density of test sample in hypotonic solution.

Statistics: The data collected were statistically analysed using one-way Analysis of variance (ANOVA)
and Duncan New multiple range post hoc test, mean differences at p<0.05 were considered significant.

International Journal of Applied Biology and Pharmaceutical Technology Page: 85

Available online at www.ijabpt.com
Oladele et al ISSN 0976-4550

RESULTS AND DISCUSSION

Acute toxicity study: The extract was safe in rats at the tested doses (40-320 mg/kg), there was no
mortality within the studied period. However, there were behavioural changes such as depression,
reduced motor activity and ataxia.
Antiinflammatory Effects: The extract significantly (p<0.05) inhibited the oedema induced by
carrageenan in dose-dependent manner as inhibition of 400 mg/kg is 45.3%, 200 mg/kg is 37.7 while 100
mg/kg is 26.4%. Indomethacin also significantly (p<0.05) inhibited oedema of the rat paw (Table 1).

Table 1: Effect of aqueous extract of the root of Boerhavia diffusa on carrageenan-induced hind
paw oedema in rats.
Sample Dose Difference in paw % inhibition
(mg/kg) circumference (cm) of oedema
Root Extract 100 0.39±0.032* 26.4
Root Extract 200 0.33±0.019* 37.7
Root Extract 400 0.29±0.015* 45.3
Indomethacin 10 0.21±0.009* 60.4
Distilled water 10ml/kg 0.53±0.130 -
Each value represents the mean ± SEM from 6 rats in each group. All values are significant at p<0.05 compared
with control group (distilled water).

Membrane stabilizing activity: The extract at concentration range of 20-80 mg/ml significantly (p<0.05)
protect the rat blood cells membrane against lysis induced by hypotonic solution. The inhibition is
comparable with that of indomethacin as shown in table 2.

Table 2: Effect of aqueous extract of the root of Boerhavia diffusa on rat erythrocytes hemolysis
Sample concentration optical density (OD) % Inhibition of haemolysis
Hypotonic soln 50 mM 0.498±0.002 -
Root Extract 20 mg/ml 0.311±0.004* 37.55
Root Extract 40 mg/ml 0.268±0.002* 46.18
Root Extract 80 mg/ml 0.218±0.005* 55.62
Indomethacin 0.1 mg/ml 0.213±0.002* 57.23
Each value represents the mean ± SEM 6 experiments. All values are significant at p<0.05 compared with control
group (hypotonic solution).

The result of the study showed that the aqueous root extract of Boerhavia diffusa possesses anti-
inflammatory property, as the extract significantly inhibited carrageenan induced oedema in rat paws. The
carrageenan induced rat paw model is the basic model for screening agents with anti-inflammatory effect
(Bamgbose and Noamesi, 1981). During inflammation, lysosomal hydrolytic enzymes are released to the
sites and these causes damages of the surrounding organelles and tissues with attendance variety of
disorders (Sadique et al, 1989). The oedema developed after injection of carrageenan into the rat paws
has been attributed to the inflammatory mediators released. These mediators include vasoactive
substances such as histamine, bradykinin, serotonin and prostaglandins (Sadique et al, 1989; Heller et al,
1998). The increase in vascular permeability produced by these chemicals promote accumulation of fluid
in tissues and thus the oedema (White, 1999). The mode of action of this extract could be due to the
prevention of liberation of proinflammatory mediators and inhibition of prostaglandin synthesis which
thereby resulted in inhibition of inflammation.

International Journal of Applied Biology and Pharmaceutical Technology Page:86

Available online at www.ijabpt.com
Oladele et al ISSN 0976-4550

The extract also showed membrane stabilizing effect as it significantly stabilized the rat erythrocytes
membrane. A possible explanation could be connected with binding to the erythrocytes membrane with
subsequent alteration of the surface charges of the cells which might have prevented physical interaction
with aggregating agents or promote dispersal by mutual repulsion of like charges which are involved in
the haemolysis of red blood cells (Oyedapo et al, 2010).
CONCLUSION
The aqueous extract of the root of Boerhavia diffusa possesses anti-inflammatory properties which can be
attributed to its cell membrane stabilizing effect which therefore inhibit the lysis and release of the pro-
inflammatory mediators.

REFERENCES

Agrawal, A., Srivastava, S. and Srivastava, M.M. (2004). Inhibitory effect of the plant
Boerhavia diffusa L. against the dematophytic fungu Mircosporium fulvum. J Environ Biol. 25 (3) : 307-
311.
Ambasta, S.P. (1986). The useful plants of India. PID. CSIR, New Delhi.
Anand, R.K. (1995). Biodiversity and tribal association of Boerhavia diffusa in India-Nepal
Himalayan Terai Region. Flora & Fauna VI(2):167–170.
Bamgbose, S.O.A. and B.K. Noamesi (1981). Studies on cryptolepine II: Inhibition of
carrageenan induced rat paw oedema. Planta Medica 42: 392-396.
Barthwal. M. and Sriastavas, K. (1991). Management of IUD-associated in female rhesus
monkeys (Macaca mulatta). Adv. Contracept 7 (10):67-76.
Borreli, F., Ascione, V., Capasso, R., Izzo, A.A. and Fattorusso,E.(2006). Spasmolytic effects
of nonprenylated rotenoid constitutions of Boerhavia roots. J Prod. (6): 903-906.
Bharali,R., Azad, M.R. and Tabassum, J. (2003). Chemopreventive action of Boerhavia
diffusa on DMBA-induced skin carcinogenesis in mice. Indian J Physiol Pharmacol 47 (4): 459-464.
Chopra, R.N., Nayar, S.L., and Chopra, I.C. (1956). Glossary of Indian Medicinal Plants.
Council of Scientific and Industrial Research (CSIR), New Delhi, India. p. 39.
Cruz, G.L. (1995). Dicionario Das Plantas Uteis Do Brasil. 5th Edition. Bertrand, Rio de
Janeiro, Brazil.
CSIR. (1988). The Wealth of India: Raw Materials Vol. VII B. CSIR, New Delhi, India. p. 174.
Gholap S. and Kar, A. (2004). Hypoglycaemic effects of some plant extracts are possibly
medicated through inhibition in corticosteroid concentration. Pharmazies. 59 (59): 876-878.
Hansen, K.,Nymann, U., Smith, U. W. and Adsersen, A. (1995). In vitro screening of traditional
medicines for antihypertensives effect based on inhibitionof angiotensin converting enzyme
(ACE). Ethnopharcol. 48(1); 43-51.
Heller, A., Koch, T., Schmech, J. and Acker, V.K. (1998). Lipid mediators in inflammatory
disorders. Drugs 55: 487-496.
Hiruma-Lima, C.A., Gracioso, J.S., Bighetti, E.J., Germonensen, R. L. and Souza Broto, A.R. (2000).
The juice of fresh leaves of Boerhavia diffusa, L(nytaginaceae) markedly reduced pain in mice. J
Ethnopharcol. 71(1-20; 267-274.
Jain, P. and khanna, N.K. (1981). Evaluation of anti-inflammatory and analgesic properties of
L-glutamine. Agents and Actions 11: 243-249.
Leyon, P.V., Lini, C.C, and Kuttan, G. (2005). Inhibitory effect of Boerhavia diffusa on
experiental metastsis by B16F10 melanoma C57BL/6mice. Life Sci 76 (12): 1339- 1349.
Mehretra, S., Singh, V.K., Agarwal, S.S. Maurya, R. and Srima,R.C.(2002).
Antilymphoproliferative activity of Ethanolic extract of Boerhavia diffusa roots. Exp Mol Pathol. 72 (3):
236-242.
Oyedapo, O.O., B.A. Akinpelu, K.F. Akinwumi, M.O. Adeyinka and F.O. Sipeolu (2010). Red blood cell
membrane stabilizing potentials of extracts of Lantana camara and its fractions. Inter. J. Plant Physiol.
Biochem. 2 (4): 46-51.

International Journal of Applied Biology and Pharmaceutical Technology Page: 87

Available online at www.ijabpt.com
Oladele et al ISSN 0976-4550

Pari, L. and Armanath Satheesh, M. (2004). Antidiabetic activity of Boerhavia diffusa L:

effect on hepatic key enzymes in experimental diabetes. J Ethnopharmacol. 91 (1): 109 – 113.
Rawat, A.K.S., Mehrotra, S., Tripathi, S.K., and Shama, U. (1997). Hepatoprotective activity
in punarnava – a popular Indian ethnomedicine. Journal of Ethnopharmacology 56(1):61–68.
Sadique, J., W.A. Al-Rqobah, M.F. Bughaith and A.R. El-Gindy (1989). The bioactivity of
certain medicinal plant on the stabilization of RBC membrane system. Fitoterapia,60: 525-532.
Satheesh, M.A. and Pari, L. (2004). Antioxidant effect of Boerhavia diffusa L. in tissues of
alloxan induced diabetic rats. India J Exp Biol. 42 (10): 989-992.
Shinde,U.A, X. Fu and M. Inonye (1999).A pathway for conformational Diversity in protein
mediated by intramollecular chaperones. J. Biol. Chem. 22: 15615-15621.
Singh, R.H. and Udupa, K.N. (1972). Studies on the Indian indigenous drug punarnava
(Boerhaavia diffusa Linn.). Part IV: Preliminary controlled clinical trial in nephrotic
syndrome. Journal of Research in Indian Medicine 7:28–33.
White, M. (1999). Mediators of inflammation and inflammatory process. Journal of Allergy and
Clinical Immunology. Nature 264: 215-217.
Winter, C.A., E.A. Risley and C.W. Nass (1962). Carrageenan induced oedema in the hind paw
of the rat as an assay for anti-inflammatory drugs. Proc. Soc. Exp. Biol. Med. 111: 544-547.

International Journal of Applied Biology and Pharmaceutical Technology Page: 88

Available online at www.ijabpt.com