Antibacterial and Phytochemical Studies on the Stem of Suregada angustifolia. from Peninsular India

Abstract

Phytochemical analysis of the stem bark of Suregada angustifolia. (Baill. ex Muell. Arg.) Airy Shaw resulted in the isolation of six known compounds: friedelin, epi-friedelinol, n.-octacosanol, α.-amyrin, β.-sitosterol, and β.-sitosterol-3 β.-D-glucopyranoside. The aqueous and various solvent (methanol, chloroform, and hexane) extracts of stem bark were tested by the agar well-diffusion method against 12 human pathogenic bacteria. Maximum antibacterial activity was observed in the order of chloroform, hexane, and methanol extracts. Aqueous (room temperature, boiled, and autoclaved) extracts did not demonstrate any activity. The results of the current investigation correspond positively with the claims of the ethnomedicinal uses.

Keywords:

• Antibacterial activity
• ethnomedicine
• Euphorbiaceae
• human pathogenic bacteria
• Peninsular India
• phytochemistry
• skin infections
• stem
• Suregada angustifolia

Introduction

The genus Suregada. Roxb. ex Rottl. (Euphorbiaceae) is represented by about 40 species, mainly distributed in Old World tropics (Philcox, 1997). Two species occur in Peninsular India (Hooker, 1887; Gamble, 1925; Henry et al., 1987) wherein one species—Suregada angustifolia. (Baill. ex Muell. Arg.) Airy Shaw (Gelonium angustifolium. Baill. ex Muell. Arg.; G. lanceolatum. auct. non Willd.)—a small tree commonly found in deciduous and evergreen forests of Peninsular India, is known as kaatusirumarapoopachilai. by the tribals, namely Kanikars or Kanis in the Tirunelveli district of Tamil Nadu state in Peninsular India. Stem bark (30 g) of Suregada angustifolia. is crushed with 50 ml of water and the prepared paste is applied to the body for skin infections. Stem bark (10 g) boiled with 50 ml of water and 20 g of salt is used as a mouthwash to treat toothache (twice a day for 3 days).

Of the 40 species, phytochemical reports are available only for two species, G. bifarium. Roxb. (Mishra et al., 1973) and G. multiflorum. A. Juss. (Barbieri et al., 1987; Talapatra et al., 19891998; Chakravarthy et al., 1991; Sairam & Marcil, 1991; Das & Chakravarthy, 1993; Das et al., 1994). Molecular studies such as anti-HIV protein of MAP 30 and GAP 31 (Lee-Huang et al., 19911993; Bourinbaiar & Lee-Huang, 1996), anti-HIV and ribosome inactivation studies (Endo et al., 1988; Lee-Huang et al., 1994a,b), specifically on gelonin (Montanaro et al., 1985; Montecucchi et al., 1989; Alam et al., 1992; Singh & Kar, 1992; Singh et al., 1999; Better & Carroll, 2000), inhibitor of protein synthesis (Stirpe et al., 1980), antitumor (Lee-Huang et al., 1994a) and human breast cancer (Lee-Huang et al., 2000), cytotoxity studies (Rosenblum, 1991), and DNA topoisomerase inhibitor, RNA N-glycosidase activity, and multiple antiviral actions (Lee-Huang et al., 1994b) have been reported from G. multiflorum.. There has been no report of phytochemical or biological studies on Suregada angustifolia., hence it is reported here.

Materials and Methods

Plant material

Stem material of Suregada angustifolia. was collected from the Tirunelveli district of Tamil Nadu during September 2002, and a voucher specimen (MBV and MV, 2377), identified by Dr. M.B. Viswanathan, has been deposited in the herbarium of the Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi, for reference. The air-dried stem coarse powder (100 g) of the plant was successively extracted with hexane, chloroform, and methanol using a Soxhlet apparatus. The extracts so collected were distilled off on a water bath at atmospheric pressure, and the last trace of solvents was removed in vacuo..

Phytochemical studies

The solvent extracts were tested for preliminary phytochemical screening, and the plant constituents were isolated and purified by chromatographic techniques (Harborne, 1998).

Antibacterial studies

Fresh stem material (30 g) was weighed, chopped, and divided into three portions. Each portion was crushed by grinding in a mortar and transferred to a suitable glass bottle, and 50 ml of distilled water were added. The first bottle was autoclaved at 10 lb. for 20 min, the second was boiled (100°C) for 20 min and the third was mechanically shaken (200 rpm) at room temperature (25°C) for 2 h. The extracts were filtered using cheesecloth and 0.45-µm filter papers and transferred into sterile closed containers and considered as 100% extracts. By adding distilled water, 50% of the extracts were prepared (Sen & Nandi, 1951). Various solvent extracts of stem prepared at different concentrations (20, 10, and 5 mg/ml) were used for antibacterial activity.

Test microorganisms

Microorganisms procured from the Microbial Type Culture Collection (MTCC) at the Institute of Microbial Technology (IMTECH), Chandigarh, for testing were Aeromonas hydrophila. (646), Enterobacter aerogenes. (111), Escherichia coli. (724), Klebsiella pneumoniae. (432), Proteus vulgaris. (426), Pseudomonas aeruginosa. (741), Salmonella typhi. (733), and V. vulnificus. (1146) (Gram-negative bacteria) and Bacillus subtilis. (441) and Staphylococcus aureus. (96) (Gram-positive bacteria). Another two Gram-negative bacteria, Vibrio cholerae. and V. parahaemolyticus., were taken from the microbial cultures deposited in the Department of Environmental Sciences, Bharathiar University (Coimbatore, Tamil Nadu, India).

Determination of antibacterial activity

The agar well-diffusion method (Perez et al., 1990) was followed. Nutrient agar (NA) plates were swabbed (sterile cotton swabs) with 8-h-old broth culture of the respective bacteria. A sterile cork borer was used to prepare two wells, each measuring 8 mm in diameter, in each of the plates. About 0.3 ml each of 100% and 50% aqueous extracts and different concentrations of solvent extracts were added into the wells using sterilized dropping pipettes and allowed to diffuse at room temperature for 2 h. Controls of solvent extracts were maintained. The plates were incubated at 37°C for 18–24 h. Diameter of the inhibition zone was recorded. The experiment was repeated three-times and average values for antibacterial activity were calculated.

Results

Isolation yielded friedelin [hexane (C₆H₆): 1:1 column earlier fraction], epi-friedelinol (hexane (C₆H₆): 1:1 column later fraction), n.-octacosanol (C₆H₆ column earlier fraction), α-amyrin (C₆H₆ column later fraction), β.-sitosterol (C₆H₆-EtoAc 9:1) from combined hexane and chloroform extracts, and β.-sitosterol-3 β.-D-glucopyranoside (CHCl₃-MeOH; 9:1) from the methanol extract. These compounds were identified by melting point (m.p.), mixture melting point (m.m.p.), superimposable IR spectrum, and co-TLC of an authentic sample. Antibacterial activity was recorded for all the solvent extracts except against S. aureus. in the hexane extract. Aqueous extracts were devoid of any activity at 50% and 100% concentrations. Maximum zone of inhibition recorded was 40 mm against A. hydrophila. and E. coli., 37 mm against K. pneumoniae., and 32 mm against S. aureus. at 20 mg/ml in chloroform extract, whereas the results at 10 mg/ml differed slightly such as 33 mm against A. hydrophila., 32 mm against E. coli., 30 mm against K. pneumoniae., and 25 mm against S. aureus.. Hexane extract showed inhibition of 30 mm against A. hydrophila., 29 mm against K. pneumoniae., and 25 mm each against P. vulgaris., V. cholerae., V. parahaemolyticus., and V. vulnificus. at 20 mg/ml. The methanol extract gave a zone of inhibition of 32 mm against S. aureus., 26 mm against E. aerogenes. and E. coli., and 25 mm against P. vulgaris. at 20 mg/ml (Table 1). There was no activity expressed in aqueous (cold, boiled, and autoclaved) extracts. The standard disk controls produced zones of inhibition ranging from 14 mm (V. parahaemolyticus.) to 45 mm (S. aureus.).

Table 1.. Antibacterial activity of aqueous and solvent extracts of stem in Suregada angustifolia.^a.

	Methanol extract (mg/ml)			Chloroform extract (mg/ml)			Hexane extract (mg/ml)
Microorganisms	20	10	5	20	10	5	20	10	5	Standard
Aeromonas hydrophila.	18	14	12	40	33	29	30	24	18	20 (Tr)
Enterobacter aerogenes.	26	22	16	20	16	14	15	12	16	22 (Tr)
Escherichia coli.	26	20	16	40	32	27	24	17	14	30 (K)
Klebsiella pneumoniae.	20	16	12	37	30	26	29	23	19	30 (K)
Proteus vulgaris.	25	20	14	25	20	16	25	18	14	20 (K)
Pseudomonas aeruginosa.	14	12	10	20	15	12	15	12	10	25 (E)
Salmonella typhi.	24	20	16	25	19	15	16	12	10	20 (Na)
Vibrio cholerae.	22	18	11	26	20	16	25	18	14	31 (Tr)
Vibrio parahaemolyticus.	18	12	10	28	23	17	25	20	16	14 (K)
Vibrio vulnificus.	22	14	12	29	25	20	25	22	17	16 (R)
Bacillus subtilis.	22	16	12	27	21	15	20	16	12	30 (A)
Staphylococcus aureus.	32	27	22	32	25	20	—	—	—	45 (M)

Ampicillin (A) 10 µg/disk; erythromycin (E) 10 µg/disk; kanamycin (K) 30 µg/disk; methicillin (M) 5 µg/disk; nalidixic acid (Na) 30 µg/disk; rifampicin (R) 30 µg/disk; Trimethoprin (Tr) 10 µg/disk.

^a.Values (mean of three replicates) are diameter of zone of inhibition (mm); —, no inhibition.

Discussion

The current study clearly indicated maximum activity in the chloroform extract. This is followed by hexane and methanol extracts. The aqueous extracts did not show any activity. The results are positively correlated with the earlier reports of Bridelia crenulata. and Drynaria quercifolia. (Ramesh et al., 2001ab). When comparing methanol and hexane extracts, maximum activity was recorded against A. hydrophila., E. coli., and K. pneumoniae. in chloroform extract. Interestingly, similar activity was observed against S. aureus. both in chloroform and methanol extracts. This organism is responsible for skin and toothache infections. The results of the current investigation provide scientific support for the claims of the ethnic uses of the medicinal plant. Discovery of new drugs for various diseases such as skin, toothache, diarrhea, and other microbial infections is possible if active principles from solvent extracts are isolated and tested pharmacologically and clinically.

The authors are grateful to Dr. A. Saraswathy, Assistant Director in-charge, Captain Srinivasamurti Drug Research Institute, and to Dr. K. Balakrishna, Scientist, for their help. The first author is thankful to Mr. A.C. Tangavelou.