Phytochemical, Antibacterial and Antinociceptive Studies of Hoya parasitica

MATERIALS AND METHODS

Preparation of Plant Extract
H. parasitica was collected from the district of Satkhira, Bangladesh in October 2005 before its flowering stage and was taxonomically identified by the experts at Bangladesh National Herbarium (Voucher No. 30224). About 500 g of powdered leaves were taken in a clean, flat-bottomed glass container (4 L) and soaked in 2200 mL of 90% ethanol. The container with its contents was sealed and kept for a period of 10 days accompanying occasional shaking and stirring state. The whole mixture then underwent a coarse filtration by a piece of clean, white cotton material followed by a filtration through Whatmann filter paper and the filtrate thus obtained was air-dried to get the crude extract.

Preliminary Phytochemical Screening
The crude extract was subjected to preliminary phytochemical testing for the detection of phytochemical compounds such as alkaloids, flavonoids, gums, reducing sugars, saponins, steroids and tannins (Evans, 1989; Ghani, 1998).

Microorganisms
Both gram positive and gram-negative bacterial species were used for antibacterial test. The bacterial strains used for the investigation are shown in Table 2. These bacterial species were collected from the Microbiology Laboratory of Square Pharmaceutical Limited, Pabna, Bangladesh.

Animals
Young Swiss-albino mice of either sex, weighing 20-25 g, purchased from the Animal Research Branch of International Center for Diarrhoeal Disease and Research, Bangladesh (ICDDR, B) were used for antinociceptive activity. The animals were kept at animal house (Pharmacy Discipline, Khulna University) for adaptation after their purchase under standard laboratory conditions (Relative humidity 55- 65%, room temperature 25.0±2°C and 12 h light: dark cycle), fed with standard diets (ICDDR, B formulated) and had free access to tap water.

Antibacterial Activity
Antibacterial activity of H. parasitica was tested by using agar diffusion method (Rios et al., 1988). Measured amount of the test samples were dissolved in definite volumes of solvent to give solutions of known concentration (10 μg mL^-1). Three wells were made through the media of the seeded plates (for the sample, blank and the standard Gentamycin) by using sterile cork borer (4 mm). Solution of sample, blank and Gentamycin were poured through the holes by using micropipette with desired amount. All the plates were kept in refrigerator (4°C) for 2 h to ensure proper diffusion. Finally the plates were incubated upside down at 37°C for 18-24 h. After proper incubation, the antibacterial activity of the test agent was determined by measuring the diameter of zone of inhibition (millimeter) with a slide calipers.

Antinociceptive Activity
Antinociceptive activity of the ethanol extract of H. parasitica was tested using the model of acetic acid induced writhing in mice (Whittle, 1964; Ahmed et al., 2004). The experimental animals were randomly divided into three groups, each consisting of five animals. Group I was treated as 'control group' and received 1% (v/v) Tween-80 in water at the dose of 10 mL kg^-1 of body weight; group II was treated as 'positive control' and was given the standard drug diclofenac sodium at the dose of 25 mg kg^-1 of body weight; group III was test group and was treated with ethanol extract of H. parasitica at the dose of 500 mg kg^-1 of body weight. Control vehicle, standard drug and extract were administered orally 30 min prior to the intraperitoneal injection of 0.7% acetic acid (10 mL kg^-1 body wt.); after interval of 15 min, the number of writhes (squirms) were counted for 5 min.

Statistical Analysis
Student’s t-test was used to determine a significant difference between the control group and experimental groups. A p-value of <0.01 and <0.001 were considered statistically significant.

RESULTS

Phytochemical Screening
The ethanolic extract of H. parasitica revealed the presence of flavonoids, reducing sugars, tannins, gums and saponins (Table 1).

Table1:	Phytochemical screening of ethanolic leaves extract of H. parasitica
+: Indicates positive result; -: Indicates negative result

Table 2:	Antibacterial activity of ethanolic leaves extract of H. parasitica
Values greater than 6 mm indicates some activity

Table 3:	Effect of ethanolic leaves extract of H. parasitica on acetic acid induced writhing in albino mice
Values are expressed as mean±SEM (No. of animals, n = 05); *: Indicates p<0.01, **: Indicates p<0.001, vs. control; po: per os (orally)

Antibacterial Activity
The ethanolic extract of the plant demonstrated strong antibacterial activity against Staphylococcus aureus, Proteus sp., Enterococci sp. and Shigella dysenteriae with zone of inhibition of 23, 19, 15 and 20 mm, respectively. This was comparable to the values of Gentamycin 44, 43, 39 and 32 mm, respectively. The extract produced moderate antibacterial activity against Hafinia sp., E. coli and Shigella flexneri where the zone of inhibition were 9, 10 and 8 mm, respectively (Table 2).

Antinociceptive Activity
At the oral dose of 500 mg kg^-1 of body weight, the extract produced 50% writhing inhibition in test animals. The result was statistically significant (p<0.01) and was comparable to the standard drug diclofenac sodium, which showed 69% writhing inhibition at the dose of 25 mg kg^-1 of body weight (Table 3).

DISCUSSION

Antibacterial activity of H. parasitica was tested by using the agar diffusion method. Agar diffusion method is widely acceptable for the preliminary screening of antimicrobial activity. It is essentially a qualitative or semi qualitative test indicating the sensitivity or resistance of microorganisms to the test materials (Pelczar et al., 1993). The extract showed strong antibacterial activity against all the test microorganisms with exception of Shigella sonnei. As the extract showed antibacterial activity, this plant extract may be used in remedy for different microbial diseases such as diarrhea, dysentery and skin diseases. The antibacterial activity of the plant may be attributable to the presence of the phytochemical constituents observed. The presence of tannins, flavonoids, saponins and reducing sugars has earlier been associated with antibacterial activity (Hostettman and Nakanishi, 1979; Sodipo et al., 1991; Okwute and Mann, 1999).

Antinociceptive activity of the ethanol extract of H. parasitica was tested by acetic acid induced writhing model in mice. Acetic acid induced writhing model represents pain sensation by triggering localized inflammatory response. Acetic acid, which is used to induce writhing, causes algesia by liberation of endogenous substances, which in turn excite the pain nerve endings (Taesotikul et al., 2003). Increased levels of PGE₂ and PGF_2α in the peritoneal fluid have been reported to be responsible for pain sensation caused by intraperitoneal administration of acetic acid (Derardt et al., 1980). The ethanol extract of H. parasitica produced significant writhing inhibition comparable to the standard drug diclofenac sodium. On the basis of this result it can be concluded that the extract of H. parasitica possesses antinociceptive activity and this may be due to the phytochemical constituents.

In conclusion, it can be suggested that the ethanol extract of H. parasitica possess antibacterial and antinociceptive effects which support the traditional uses of this plant as antirheumatic. Therefore, further research is essential to find out the active principles responsible for these activities.