Evaluation of anti-inflammatory activity of methanol extract of Barleria Cristata leaves by in vivo and in vitro methods
M Gambhire, A Juvekar, S Wankhede
inflammation, membrane stabilization, protein denaturation
M Gambhire, A Juvekar, S Wankhede. Evaluation of anti-inflammatory activity of methanol extract of Barleria Cristata leaves by in vivo and in vitro methods. The Internet Journal of Pharmacology. 2008 Volume 7 Number 1.
The methanol extract of
Source of financial support received
This work was fully supported by grants from University Grants Commission
Medicinal plants are believed to be an important source of new chemical substances with potential therapeutic effects. The research into plants with alleged folkloric use as anti-inflammatory agents should therefore be viewed as a fruitful and logical research strategy in the search for new anti-inflammatory drugs.
The chemical constituents of the plant have been identified as flavonoid type phenolic compounds especially apigenin, quercetin, quercetin-3-O-β-D-glucoside, naringenin and apigenin glucuronide. Different parts of
Materials And Methods
Fresh leaves of the plant
The BCM extract was screened for the presence of various phytochemical constituents i.e. steroids, alkaloids, tannins, flavonoids, glycosides, etc by employing standard screening tests .
Chemicals and drugs
Histamine, Serotonin, Egg albumin and Evan’s blue were obtained from Himedia Lab [(Mumbai) India], Indomethacin [Recon, (Bangalore) India], Cyproheptadine [Fleming Laboratories Limited, (Hydrabad), India] and all other chemicals used were of analytical grade.
Wistar albino rats of either sex weighing 180–200 g and Swiss albino mice of either sex weighing 18–22 g were used for animal studies. The animals were grouped in polyacrylic cages and maintained under standard laboratory conditions (temperature 25 ± 2 °C) and relative humidity (50±5%) with dark and light cycle (14/10 h). They were allowed free access to standard dry pellet diet and water
Acute toxicity test
The animals were divided into six groups containing six animals each. BCM was dissolved in distilled water and administered orally as a single dose to mice at different dose levels viz. 500, 750, 1000, 1250, 1500 and 2000 mg/kg of body weight (b.w.). The Mice were observed periodically for symptoms of toxicity and death within 24 h and then daily for next 14 days .
Histamine and serotonin-induced rat paw edema
The paw edema was induced by subplantar administration of 0.1 ml of a 0.1% freshly prepared solution of histamine or serotonin into the right hind paw of rats. The paw volume was measured immediately i.e. at 0 h and after 1 h of histamine or serotonin injection . Different groups of animals were pretreated with BCM (125, 250 and 500 mg/kg) or with 5 ml/kg of distilled water (vehicle control) or 10 mg/kg cyproheptadine (standard drug). The drugs were administered orally 1 h before eliciting paw edema. Percent inhibition of paw edema was calculated by following equation,
Anti-inflammatory activity (%) inhibition = (1-D/C) × 100,
where D represents the percentage difference in increased paw volume after the administration of test drugs to the rats and C represents the percentage difference of increased volume in the control groups.
Acetic acid-induced vascular permeability in mice
This test was followed by the method described by Whittle (1964)  with some modifications. Five groups of six mice per group were used for the study. Group I served as vehicle control, groups II, III and IV were treated orally with 125, 250 and 500 mg BCM extract/kg respectively while group V received indomethacin 10 mg/kg orally. One hour after the treatment, 0.2% Evan’s blue in normal saline was injected intravenously through tail vein at a dose of 0.1 ml/10 g b.w. Thirty minutes later, each mouse was injected intraperitoneally with 0.2 ml of 0.6% acetic acid in normal saline solution. After 1 h, the mice were sacrificed and the abdominal wall was cut to expose the entrails. The abdominal cavity was washed with 5ml of normal saline to collect pigments in a test tube. After centrifuging the contents of the tube to eliminate contaminants, the solution was subjected to colorimetric analysis using a spectrophotometer at a wavelength of 590 nm. The vascular permeability effects were expressed as the absorbance (A), which represented the total amount of dye leaked into the intraperitoneal cavity.
Membrane stabilizing activity
This test was followed by the method described by Shinde
% Inhibition of haemolysis = 100 x (A1-A2/A1)
A1 = Absorption of hypotonic buffered saline solution alone
A2 = Absorption of test sample in hypotonic solution
Effect on protein denaturation
Test solution (1ml) containing different concentrations (50 - 250 μg/ml) of plant extract or indomethacin (100 μg/ml) was mixed with 1ml of egg albumin solution (1mM) and incubated at 27 ±1 °C for 15 min. Denaturation was induced by keeping the reaction mixture at 70 °C in a water bath for 10 min. After cooling the turbidity was measured spectrophotometrically at 660 nm . Percentage inhibition of denaturation was calculated from control where no drug was added. Each experiment was carried out in triplicate and the average was taken.
The experimental data was expressed as mean ± SEM, the significance of difference among the various treated groups and control group were analyzed by means of one-way ANNOVA followed by Dunnett’s t-test using Graphat Instat Software (San Diego, CA, USA).
In this study anti-inflammatory activity of methanol extract of
Preliminary phytochemical screening of the methanol extract of
Inhibition of histamine and serotonin-induced rat paw edema
The BCM extract (125, 250 and 500 mg/kg) significantly (
Inhibition of acetic acid-induced vascular permeability in mice
Effect of BCM extract (125, 250 and 500 mg/kg), indomethacin (10 mg/kg) and control vehicle on acetic acid-induced increased vascular permeability in mice was studied. Results of the activity showed that BCM at dose (125 and 250mg/kg) moderately inhibited the vascular permeability (23.73% and 42.19% respectively), whereas BCM at a dose of 500 mg/kg and indomethacin 10 mg/kg significantly (
Membrane stabilizing activity
In the study of membrane stabilization activity the BCM extract at concentration range of 0.60-1.0 mg/ml protect significantly (
Inhibition of protein denaturation
The inhibitory effect of different concentrations of BCM on protein denaturation is shown in Table 4. BCM extract (50-250 μg/ml) showed significant (
Inflammation is the response of living tissues to injury. It involves a complex array of enzyme activation, mediator release, extravasations of fluid, cell migration, tissue breakdown and repair .
The early phase of inflammation mainly mediated by histamine and serotonin, its significant inhibition by BCM extract revealed that the anti-inflammatory activity is possibly backed by its anti-histamine and anti-serotonin activity.
Increased vascular permeability occurs as a result of contraction and separation of endothelial cells at their boundaries to expose the basement membrane, which is freely permeable to plasma proteins and fluid . Histamine and other mediators of inflammation increases vascular permeability at various times after injury. Chemical induced vascular permeability (acetic acid) causes an immediate sustained reaction that is prolonged over 24 h  and its inhibition suggests that the BCM extract may effectively suppress the exudative phase of acute inflammation.
The vitality of cells depends on the integrity of their membrane, exposure of RBC’s to injurious substances such as hypotonic medium results in lysis of its membrane accompanied by haemolysis and oxidation of haemoglobin . The haemolytic effect of hypotonic solution is related to excessive accumulation of fluid within the cell resulting in the rupturing of its membrane. Such injury to RBC membrane will further render the cell more susceptible to secondary damage through free radical induced lipid peroxidation . It is therefore expected that compounds with membrane-stabilizing properties, should offer significant protection of cell membrane against injurious substances . Compounds with membrane-stabilizing properties are well known for their ability to interfere with the release of phospholipases that trigger the formation of inflammatory mediators . BCM has shown significant (
Denaturation of proteins is a well documented cause of inflammation and rheumatoid arthritis . Several anti-inflammatory drugs have shown dose dependent ability to inhibit thermally induced protein denaturation . Ability of BCM extract to bring down thermal denaturation of protein is possibly a contributing factor for its anti-inflammatory activity.
The anti-inflammatory activity of BCM extract found may be due to the presence of therapeutically active flavonoids i.e. apigenin, quercetin and quercetin-3-O-β-D-glucoside, naringenin and apigenin glucuronide and which was predicted. The therapeutic applications of flavonoids on inflammation have previously been reported .
The data obtained from the present study indicated that several factors may contribute to the anti-inflammatory action of BCM. Firstly, BCM significantly inhibited histamine and serotonin induced rat paw edema showing it’s anti-histaminic and ant-serotonin ability. Secondly, BCM reduced the increased peritoneal vascular permeability in mice, indicating the suppression of the vascular response in the process of acute inflammation. Finally, BCM exhibited significant membrane-stabilizing property and inhibition of protein denaturation
The data of our studies suggests that BCM showed significant anti-inflammatory activity in both the
This work was supported by grants from University Grants Commission