Fresh leaves of the plant Barleria Cristata Linn were collected from Mumbai region, India. The plant material was taxonomically identified by Dr. Ganesh Iyer, Prof. in Botany, Ramnarain Ruia college, Mumbai, India. A voucher specimen (No. 9-1/08) has been preserved in our laboratory for future reference. The leaves were dried under shade and then powdered with a mechanical grinder and stored in airtight container. The dried powder material of the leaves was defatted with petroleum ether (60-80) and subsequently extracted with methanol in a soxhlet apparatus. The solvent was completely removed under reduced pressure and methanol extract of Barleria Cristata leaves (BCM) was obtained (yield 12.4%). Solution of BCM was prepared freshly in distilled water and used for the studies.

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 [₃].

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 ad libitum. The rats and mice were acclimatized to laboratory condition for 10 days before commencement of experiment. The Institutional Animal Ethics Committee had approved the experimental protocols and care of animals was taken according to CPCSEA guidelines.

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 [₄].

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 [₅]. 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,

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.

This test was followed by the method described by Whittle (1964) [₆] 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.

This test was followed by the method described by Shinde et al (1999) [₇] with some modifications. Whole human blood was obtained from a healthy human volunteer and transferred to heparinized centrifuge tube. The blood was washed three times with isotonic buffered solution (154 mM NaCl) in 10 mM sodium phosphate buffer (pH 7.4) for 10 minutes at 3000g. The test sample consisted of stock erythrocyte (RBC) suspension (0.5 ml) mixed with 5 ml of hypotonic solution (50 mM NaCl) in 10 mM sodium phosphate buffered saline (pH 7.4) containing the BCM extract (0.2- 1.0 mg/ml) or indomethacin (0.1 mg/ml). The control sample consisted of 0.5 ml of RBC suspension mixed with hypotonic buffered saline solution alone. The mixtures were incubated for 10 min at room temperature and centrifuged for 10 min at 3000g and the absorbance of the supernatant was measured at 540 nm. Each experiment was carried out in triplicate and the average was taken. The percentage inhibition of haemolysis or membrane stabilization was calculated by following equation.

% Inhibition of haemolysis = 100 x (A₁-A₂/A₁)

Where:

A₁ = Absorption of hypotonic buffered saline solution alone

A₂ = Absorption of test sample in hypotonic solution

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 [₈₉]. 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).

The BCM extract (125, 250 and 500 mg/kg) significantly (p<0.01) and dose-dependently inhibited histamine-induced rat paw edema (19.82, 30.23 and 46.33 %, respectively) and cyproheptadine (55.08 %) produced significant (p<0.01) inhibition of histamine-induced rat paw edema when compared with control group after 1 h of histamine injection (Table 1). Also the BCM extract (125, 250 and 500 mg/kg) dose-dependently and significantly (p<0.01) inhibited serotonin-induced rat paw oedema. BCM at dose of 500 mg/kg exhibited maximum inhibition (41.64 %) and cyproheptadine produced inhibition of 51.48 % in serotonin-induced rat paw edema after 1 h of the serotonin injection (Table 1).

Figure 1

Table 1: Effect of methanol extract of leaves on histamine and serotonin induced rat paw edema

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 (p<0.01) inhibited vascular permeability (63.39% and 68.45% respectively) when compared with vehicle control group (Table 2).

Figure 2

Table 2: Effect of methanol extract of leaves on acetic acid-induced vascular permeability in mice

In the study of membrane stabilization activity the BCM extract at concentration range of 0.60-1.0 mg/ml protect significantly (p<0.01) the erythrocyte membrane against lysis induced by hypotonic solution. Also indomethacin (0.10 mg/ml) offered a significant (p<0.01) protection of the RBC’s against the damaging effect of hypotonic solution. At a concentration of 1 mg/ml, the BCM extract showed 91.36 % where as indomethacin at 0.1 mg/ml showed 54.79 % inhibition of RBC haemolysis when compared with blank (Table 3).

Figure 3

Table 3: Effect of methanol extract of leaves on erythrocyte haemolysis

The inhibitory effect of different concentrations of BCM on protein denaturation is shown in Table 4. BCM extract (50-250 μg/ml) showed significant (p<0.05) inhibition of denaturation of egg albumin in concentration dependent manner. BCM extract at concentration of 250 μg/ml and indomethacin at concentration of 100 μg/ml showed significant (p<0.01) inhibition (61.24% and 82.67% respectively) of protein denaturation when compared with control.

Figure 4

Table 4: Effect of methanol extract of leaves on protein denaturation

This work was supported by grants from University Grants Commission, India. The authors gratefully acknowledge for financial assistance and fellowship from University Grants Commission, India.