888

PHYTOTHERAPY RESEARCH Phytother. Res. 19, 888894 (2005) Published online in Wiley InterScience (www.interscience.wiley.com). S. BASU ET AL. DOI: 10.1002/ptr.1752

Evaluation of the Antibacterial Activity of Ventilago madraspatana Gaertn., Rubia cordifolia Linn. and Lantana camara Linn.: Isolation of Emodin and Physcion as Active Antibacterial Agents
Subhalakshmi Basu, Abhijit Ghosh and Banasri Hazra*
Department of Pharmaceutical Technology, Jadavpur University, Calcutta 700 032, India

The antibacterial activity of the extracts of Ventilago madraspatana stem-bark, Rubia cordifolia root and Lantana camara root-bark, prepared with solvents of different polarity, was evaluated by the agar-well diffusion method. Twelve bacteria, six each of gram-positive and gram-negative strains, were used in this study. Chloroform and ethanol extracts of V. madraspatana showed broad-spectrum activity against most of the bacteria except S. aureus, E. coli and V. cholerae. On the other hand, the activity of the chloroform and methanol extracts of R. cordifolia and L. camara was found to be more specic towards the gram-positive strains, although gram-negative P. aeruginosa was also inhibited by the methanol extracts of both these plants in a dose dependent manner. The water extracts of V. madraspatana and L. camara were found to be inactive, while that of R. cordifolia was signicantly active against B. subtilis and S. aureus compared with streptomycin and penicillin G used as standards. In the course of bio-assay guided fractionation, emodin and physcion were isolated for the rst time from the stem-bark of V. madraspatana. It was noteworthy to nd the MICs of emodin in the range 0.52.0 g/mL against three Bacillus sp. Both the anthraquinonoid compounds inhibited P. aeruginosa, emodin being more effective, showing an MIC of 70 g/mL. Copyright 2005 John Wiley & Sons, Ltd.
Keywords: Ventilago sp.; Rubia sp.; Lantana sp.; antibacterial quinonoids; emodin; physcion

INTRODUCTION The indiscriminate use of antibiotics for the treatment of microbial diseases has led to the alarming emergence of multi-drug-resistant bacterial pathogens, calling for the development of novel antimicrobial agents (American Society of Microbiology, 1995). Traditionally, many drugs have been brought to the clinic by taking the lead from plant-derived natural products (Cragg et al., 1997). Plants are known to produce a variety of quinonoid metabolites, which are characteristically reactive as they undergo facile redox reactions; hence, there is a great potential for the development of some of these compounds into new chemotherapeutic agents (Cowan, 1999). Therefore, in the continuation of our search for bioactive quinonoids, three indigenous plants of ethnopharmacological importance, which are known to contain various quinonoid compounds, were chosen for investigation of their antibacterial property. Thus, selected parts of Ventilago madraspatana Gaertn. (stem bark), Rubia cordifolia Linn. (root) and Lantana camara Linn. (root bark) were studied for the rst time against
* Correspondence to: Dr Banasri Hazra, Department of Pharmaceutical Technology, Jadavpur University, Calcutta 700032, India. E-mail: hazra1@vsnl.com Contract/grant sponsor: University Grants Commission, New Delhi; Contract/grant number: F.3-15/ 2002/SR- II. Copyright 2005 John Wiley & Sons, Ltd. Copyright 2005 John Wiley & Sons, Ltd.

several gram-positive and gram-negative bacteria, and two anthraquinonoid constituents, namely emodin and physcion, isolated from the stem bark of V. madraspatana, were found to show antibacterial activity. The plant genus Ventilago (Rhamnaceae), represented by nearly 40 species, is distributed throughout India and Southern parts of Asia. Ventilago madraspatana (Raktavalli in Sanskrit), indigenous to India, is a large evergreen woody climber with long sarmentose branches. The leaves are oblong-lanceolate; owers are small, densely pubescent and with paniculate spikes; fruits are yellow, shining and globose (Chopra et al., 1956). Traditionally, the root bark of V. madraspatana is used as a carminative, stomachic, tonic and stimulant. The powdered stem bark mixed with gingelly oil is applied externally to treat skin diseases and itch (Chopra et al., 1956). The local tribes in Orissa, India, use it for the treatment of uterine tumour and haemorrhages. In Taiwan, the stem of V. leiocarpa is a folk medicine for treatment of rheumatism, hepatitis and neuralgia (Lin et al., 1996), while V. harmandiana is used in Thailand for the treatment of wounds and chronic inammation. Phytochemical reports on the root bark of V. madraspatana show the presence of various anthraquinones, including ventinone- A, B, chrysophanol, physcion, emodin, islandicin, xanthorin and xanthorin-5-methyl ether (Kesava Rao et al., 1983). Naphthalene derivatives
Received 1 March 2005 Phytother. Res . 19, 888894 (2005) Accepted 23 June 2005

ANTIBACTERIAL ACTIVITY OF V. MADRASPATANA

889

and naphthoquinones, such as ventilaginone, ventilagol, maderone, cordeauxione and isocordeauxione, are also reported from the root bark of this plant (Hanumaiah et al., 1985a; Hanumaiah et al., 1985b). Apart from the antifeedant activity of V. madraspatana on insects reported by Krishnakumari et al. (2001), there has been no scientic report on the biological activities of this plant. However, the stem-bark of V. leiocarpa, the Chinese species, was found to possess antiinammatory and hepatoprotective activities (Lin et al., 1995; Chang et al., 1996), while V. harmandiana, a Thai species, has been studied recently for its antiinammatory effect (Panthong et al., 2004). The leaves of V. denticulata from Thailand showed potent antiviral activity against herpes simplex virus type-1 (Lipipun et al., 2003). Rubia cordifolia Linn. (Rubiaceae; Manjistha in Sanskrit) is a perennial prickly climber, common throughout the lower hills of Himalayas and in the Western Ghats of India. This plant also grows in the moist and tropical forests of Japan, Indonesia and Sri Lanka. The roots are very long and cylindrical with a thin red bark. The leaves are ovate-lanceolate; the owers small, greenish white in terminal panicle or cymes; the fruits are globose and dark purplish in colour (Chatterjee and Pakrashi, 1997). Various ethnomedical applications are documented for Rubia cordifolia, e.g. its fruits are used in the treatment of hepatic obstructions (Chatterjee and Pakrashi, 1997); the stems are applied for treating cobra-bite and scorpion-sting (Chopra et al., 1956); the roots possess alterative, astringent and diuretic properties, and are widely used in traditional medicinal preparations to treat amenorrhoea, chronic diarrhoea, dropsy, intestinal atony, renal calculi, jaundice and paralysis. A paste of the root with honey is applied externally to inammation, freckles and other skin diseases; an infusion is given to women after delivery to produce a copious ow of lochia (Chatterjee and Pakrashi, 1997). Phytochemical investigations on this plant have indicated the presence of a wide variety of quinonoids and triterpenoids, only a few of which have been investigated for their bioactive potential (Dosseh et al., 1981; Itokawa et al., 1993; Takeya et al., 1993; Hassanean et al., 2000). Scientic studies were done on the root extracts of R. cordifolia and its constituents for the management of tumour (Itokawa et al., 1993), cancer (Adwankar and Chitnis, 1982) and liver disorders (Pandey et al., 1994). Also, some reports are available on the antiinammatory (Antarkar et al., 1983), antioxidant (Tripathi et al., 1995; Joharapurkar et al., 2003) and anti-hepatitis B (Ho et al., 1996) properties of the same extract. Lantana camara Linn. (Verbenaceae), the most widespread species of this genus, is a native of Southern America, now distributed throughout the tropical and sub-tropical parts of the world (Ross, 1999). It is a woody straggling shrub with prickly stems and owers of various colours yellow, orange, red, pink or white. The leaves are ovate, with a coarse surface and toothed margin; the fruits are dark purple or black, shiny and globose in shape. The aerial parts, leaves and roots of L. camara are widely used in folklore medicine all over the world, particularly in Latin America, South-East Asia, West Indies, Africa and Australia, for various applications in the ailments of skin and stomach, rheumatism, asthma,
Copyright 2005 John Wiley & Sons, Ltd.

convulsion, indigestion and fever (Ross, 1999; Chopra et al., 1956). Phytochemical studies on this plant show the presence of a wide variety of quinonoids, triterpenes, iridoid glycosides and avonoids (Ghisalberti, 2000). Pharmacological studies have been reported, mostly on its leaves and aerial parts, for antibacterial, antifungal, immunosuppressant and insect repellent activities (Ross, 1999). Thus, it transpired that the antibacterial effect of these plants had not been investigated systematically, and was carried out in the present study on the extracts, prepared with solvents of different polarity, from V. madraspatana stem-bark, R. cordifolia root and L. camara root-bark. Further, emodin and physcion were detected for the rst time in the stem-bark of V. madraspatana, and their antibacterial activity was studied in detail.

MATERIALS AND METHODS Plant material. Ventilago madraspatana stem-bark was collected from Bolangir district, Orissa, Rubia cordifolia root was purchased locally from a dealer in traditional medicinal plants, and Lantana camara roots were procured from the Salt Lake neighbourhood in Calcutta. All the plant materials were authenticated by the Botanical Survey of India, Calcutta, and a voucher specimen is preserved in our laboratory for future reference. The samples were dried in shade and pulverized before use in the following studies. Preparation of plant extracts for antibacterial assay. The plant samples (5 g each) were reuxed separately with chloroform, alcohol and water for 2 h. The extracts were evaporated to dryness under reduced pressure and the following extracts (percentage yields in parenthesis) were obtained: chloroform (3.8%) and ethanol (3.1%) extracts of V. madraspatana stem-bark; chloroform (2.0%), methanol (2.4%) and water (8.7%) extracts of R. cordifolia root; chloroform (17.8%), methanol (20.9%) and water (10.1%) extracts of L. camara rootbark. Either sterile normal saline or DMSO was used to reconstitute the extracts and prepare the solutions for antibacterial assay. Microbial organisms. The microorganisms employed in this study consisted of six gram- positive (Bacillus cereus, Bacillus pumilus, Bacillus subtilis, Micrococcus luteus, Mycobacterium luteum, Staphylococcus aureus) and six gram-negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella dysenteriae, Vibrio cholerae) bacteria. The test organisms were obtained from stock cultures of the Departments of Microbiology, Bose Institute, and Food Technology and Bio-Chemical Engineering, Jadavpur University, Calcutta. The Mueller-Hinton (M-H) agar medium (0.4% beef infusion solids, 1.75% casein hydrolysate, 1.5% agar, 0.15% starch and 1% sodium chloride, in w/v; pH 7.4) was used for the maintenance of bacterial strains in slants as well as for the study of antibacterial activity. The rst three reagents were procured from Himedia, Mumbai and the rest from Merck, Mumbai, India.
Phytother. Res. 19, 888894 (2005)

890

S. BASU ET AL.

Determination of antibacterial activity. Antibacterial assays of the samples against the selected bacterial cultures were carried out in vitro by the agar well diffusion method. A number of colonies were picked up from the bacterial stock culture and transferred to 5 mL of M-H broth and shaken in a water-bath at 37 C for ~18 h. 200 L of this culture containing the logarithmic phase cells was resuspended in sterile saline for adjustment of cfu/mL spectrophotometrically at 600 nm. It was then seeded into sterile molten M-H agar (48 50 C), and poured into sterilized petri dishes to give a nal inocula of 1 107 cfu/mL. Wells of uniform diameter (9 mm) were made on the solidied agar plate using a sterile borer. Solutions of the plant samples and standard drugs, streptomycin and penicillin G (Sigma, USA), as well as the solvents used (saline/DMSO) were placed separately (0.1 mL of each) in each well under aseptic conditions. The extracts and the pure compounds were tested over the concentration ranges 1.010.0 mg/mL, and 0.10.5 mg/mL, respectively. The plates were then maintained at room temperature for 2 h allowing the solution to diffuse into the medium. After incubation at 37 C for 24 h (48 h in the case of S. dysenteriae and V. cholerae) the zones of inhibition around each well were measured. Each experiment was performed in triplicate and an average of three independent determinations for each zone was recorded. Determination of minimum inhibitory concentration (MIC). The MIC of the pure compounds against each of the bacteria was determined by the agar well diffusion method. Different concentrations of the compound (0.1500 g/mL) were introduced into the wells on the petri dishes containing sterilized M-H agar medium with 1 107 cfu/mL prepared as before. The plates were incubated at 37 C for 24 h. The results were noted on the basis of the presence or absence of inhibition zones. The MIC was determined as the lowest concentration of the sample showing a zone of inhibition. Instrumentation. UV/VIS spectra were measured using a Pharmacia Biotech Ultrospec 2000 UV/Visible spectrophotometer. IR spectra were recorded in a Perkin Elmer Model 783 IR spectrophotometer. 1HNMR were taken in deuterochloroform or deuteromethanol with trimethylsilane (TMS) as an internal standard using a Brucker AM 300L instrument. The MS were recorded in a JEOL JMS600 spectrophotometer. Isolation of emodin and physcion. The chloroform extract of V. madraspatana stem-bark was analysed by chromatography on a silica gel (60120 mesh) column and eluted with solvents of increasing polarity. Among the several fractions thus obtained by elution with mixtures of petroleum ether (bp 6080 C) and chloroform, one prominent yellow band at a ratio of 60:40, v/v, followed by another orange-yellow one with the same solvent system at a ratio of 50:50, v/v, were found to appear as major products. These semi-pure fractions were collected and subjected to bacteriological assay, and were found to show positive antibacterial activity. The rst fraction was then puried through preparative thin layer chromatography (PTLC) using a mixture of petroleum ether (bp 6080 C) and chloroform (80:20; v/v), which gave a yellow compound as the major
Copyright 2005 John Wiley & Sons, Ltd.

Figure 1. Structures of physcion and emodin.

product. This was collected and the compound thus obtained [checked by TLC; Rf = 0.53 with petroleum ether (bp 6080 C): chloroform = 20:80; v/v; yield 0.22%] was crystallized from methanol into yellow plates (mp 201 C). Spectroscopic analysis by IR, UV, NMR and MS conrmed it to be physcion (Fig. 1). The second fraction collected from the column was also puried through PTLC using chloroform:ethyl acetate (80:20; v/v) to get the major product (Rf = 0.67 with chloroform:ethyl acetate = 90:10; v/v; yield 0.31%) which was crystallized from methanol into orange needles (mp 255 C). This compound was found to match with an authentic sample of emodin (Sigma, USA), and analysed spectroscopically for necessary conrmation (Fig. 1). Characterization of physcion. UV/VIS (EtOH): max (log ) 224 nm (4.46); 256 nm (4.18); 264 nm (4.19); 286 nm (4.17); 436 nm (3.99). IR (KBr): max(cm1) 2918, 1629, 1566, 1479, 1450, 1367, 1296, 1163, 1035. 1H NMR (CDCl3; 300 MHz): 2.45 (3H,s, CH3-6); 3.94 (3H, s, OCH3-3); 6.69 (1H, d, J = 2.6 Hz, H-2); 7.09 (1H, bs, H-7); 7.38 (1Hd, J = 2.6 Hz, H-4); 7.64 (1H, bs, H-5); 12.12 (1H, s, peri-OH-1); 12.32 (1H, s, peri-OH-8). EI-MS (70 ev): m /z 284(M+), 256, 241. Characterization of emodin. UV/VIS (EtOH): max (log )) 222 nm (4.47); 253 nm (4.38); 266 nm (4.29); 289 nm (4.36); 438 nm (4.16). IR (KBr): max(cm1) 3377, 2925, 1629, 1481, 1456, 1336, 1211, 1103, 1033. 1H NMR (CDCl3; 300 MHz): 2.46 (3H,s, CH3-6); 6.67 (1H, d, J = 2.3 Hz, H-2); 7.09 (1H, bs, H-7); 7.29 (1Hd, J = 2.3 Hz, H-4); 7.63 (1H, d, J = 0.9 Hz, H-5); 9.88 (1H, s, OH-3); 12.12 (1H, s, peri-OH-1); 12.29 (1H, s, peri-OH-8). EI-MS (70 ev): m/z 270(M+), 242, 217, 214, 213, 189.

RESULTS In the present study, the plant extracts and the isolated pure compounds were screened over a range of doses (110 mg/mL and 0.10.5 mg/mL, respectively) against six gram-positive (Bacillus cereus, Bacillus pumilus, Bacillus subtilis, Micrococcus luteus, Mycobacterium luteum, Staphylococcus aureus) and six gram-negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella dysenteriae, Vibrio cholerae) bacteria. The solvent vehicles, i.e. normal saline and DMSO, were also studied similarly and were found to be ineffective against all the tested strains (data not shown). Streptomycin and penicillin G were used as positive controls. Streptomycin showed a broad-spectrum activity, while penicillin G,
Phytother. Res. 19, 888894 (2005)

ANTIBACTERIAL ACTIVITY OF V. MADRASPATANA

891

Table 1. Antibacterial activity (inhibition zone in mma) of the extracts of the stem bark of Ventilago madraspatana
Streptomycin (mg/mL) 10.0 0.1 Penicillin G (mg/mL) 0.01

EtOH extract (mg/mL) Bacteria Gram-positive Bacillus cereus Bacillus pumilus Bacillus subtilis Micrococcus luteus Mycobacterium luteum Staphylococcus aureus Gram-negative Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Salmonella typhimurium Shigella dysenteriae Vibrio cholerae
a

CHCl3 extract (mg/mL) 10.0 1.0 2.5 5.0 7.5

1.0

2.5

5.0

7.5

14 12

11 19 12 12

12 22 14 11 14 12 12 11

12 25 14 13 14 12 13 12

14 25 15 14 15 13 14 12

10 11 12 12

11 11 11 10 13 16

12 13 13 11 13 16

13 16 13 13 10 16 22

16 17 16 14 12 16 24 10

20 24 19 22 22 23 20 17 15 16 15 20

23 22 15 28 19

Including the diameter of well (9 mm); No activity (diameter of the inhibition zone less than 10 mm).

Table 2. Antibacterial activity (inhibition zone in mma) of the extracts of the root of Rubia cordifolia
Streptomycin (mg/mL) 0.1 Penicillin G (mg/mL) 0.01

H2O extract (mg/mL) Bacteria 1.0 2.5 5.0 7.5 10.0

MeOH extract (mg/mL) 1.0 2.5 5.0 7.5 10.0

CHCl3 extract (mg/mL) 1.0 2.5 5.0 7.5 10.0

Gram-positive BC BP BS 13 MIL MYL SA 12 Gram-negative EC KP PA ST SD VC

15 15

16 18

17 19

18 19

12 10 13 20 13

15 11 15 11 20 13

16 12 17 12 24 13

17 12 17 12 10 25 14

18 13 19 13 11 26 14

10 11 13

10 11 12 12 14

18 12 14 12 17

19 12 16 13 10 18

22 15 18 13 10 20 10

20 24 19 22 22 23 20 17 15 16 15 20

23 22 15 28 19

BC, Bacillus cereus; BP, Bacillus pumilus; BS, Bacillus subtilis; MIL, Micrococcus luteus; MYL, Mycobacterium luteum; SA, Staphylococcus aureus; EC, Escherichia coli; KP, Klebsiella pneumoniae; PA, Pseudomonas aeruginosa; ST, Salmonella typhimurium; SD, Shigella dysenteriae; VC, Vibrio cholerae. a Including the diameter of well (9 mm); No activity (diameter of the inhibition zone less than 10 mm).

as expected, was found to be effective against most of the gram-positive organisms (Tables 14). The extracts of V. madraspatana stem-bark exhibited antibacterial activity against both gram-positive and gram-negative strains (Table 1). The ethanol extract showed strong inhibition towards B. pumilus, B. subtilis, K. pneumoniae and S. typhimurium, and moderate inhibition against M. luteus, P. aeruginosa and S. dysenteriae. The chloroform extract inhibited the growth of gram-negative K. pneumoniae and P. aeruginosa, and most of the gram-positive bacteria tested. Both the extracts were inactive against S. aureus, E. coli and V. cholerae. The methanol and chloroform extracts of R. cordifolia root were found to be active against all the grampositive bacteria used in this study. P. aeruginosa was the only gram-negative strain which was inhibited by the methanol extract in a dose dependent manner
Copyright 2005 John Wiley & Sons, Ltd.

(Table 2). However, the water extract was found to be active only against B. subtilis and S. aureus, which were highly susceptible to inhibition by all the three extracts in comparison with the standard drugs. Similarly, the methanol and chloroform extracts of L. camara root-bark were moderately active against most of the gram-positive strains tested, while P. aeruginosa was the only gram-negative bacterium which was strongly inhibited by the methanol extract. However, this extract was found to be inactive against B. cereus at the maximum dose (10 mg/mL) tested (Table 3). The anthraquinonoid emodin isolated from V. madraspatana stem-bark was active against the grampositive B. cereus, B. pumilus, B. subtilis, S. aureus and gram-negative P. aeruginosa in a concentration dependent manner. The analogous compound physcion was found to inhibit M. luteus, K. pneumoniae and P. aeruginosa (Table 4).
Phytother. Res. 19, 888894 (2005)

892

S. BASU ET AL.

Table 3. Antibacterial activity (inhibition zone in mma) of the extracts of the root bark of Lantana camara
Streptomycin (mg/mL) 10.0 0.1 Penicillin G (mg/mL) 0.01

MeOH extract (mg/mL) Bacteria Gram-positive Bacillus cereus Bacillus pumilus Bacillus subtilis Micrococcus luteus Mycobacterium luteum Staphylococcus aureus Gram-negative Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Salmonella typhimurium Shigella dysenteriae Vibrio cholerae
a

CHCl3 extract (mg/mL) 10.0 1.0 2.5 5.0 7.5

1.0

2.5

5.0

7.5

11 11 10

12 11 10

13 12 11 12

14 15 10 12 14

14 16 10 13 16

11 12 11 10

12 14 11 11

12 15 12 12

13 15 13 14

14 15 13 11 14

20 24 19 22 22 23 20 17 15 16 15 20

23 22 15 28 19

Including the diameter of well (9 mm); No activity (diameter of the inhibition zone less than 10 mm).

Table 4. Antibacterial activity (inhibition zone in mma) of emodin and physcion

Streptomycin (mg/mL) 0.5 0.1 Penicillin G (mg/mL) 0.01 MIC (g/mL) Emodin Physcion

Emodin (mg/mL) Bacteria 0.1 0.2 0.3 0.4 0.5 0.1

Physcion (mg/mL) 0.2 0.3 0.4

Gram-positive BC 16 BP 14 BS 13 MIL MYL SA 10 Gram-negative EC KP PA 13 ST SD VC

17 14 13 11 13

17 15 14 14 14

18 16 14 15 15

18 16 15 15 17

11 10

13 11 11

15 12 11

15 13 12

20 24 19 22 22 23 20 17 15 16 15 20

23 22 15 28 19

0.5 2.0 1.5 >500 >500 90.0 >500 >500 70.0 >500 >500 >500

>500 >500 >500 200.0 >500 >500 >500 250.0 200.0 >500 >500 >500

BC, Bacillus cereus; BP, Bacillus pumilus; BS, Bacillus subtilis; MIL, Micrococcus luteus; MYL, Mycobacterium luteum; SA, Staphylococcus aureus; EC, Escherichia coli; KP, Klebsiella pneumoniae; PA, Pseudomonas aeruginosa; ST, Salmonella typhimurium; SD, Shigella dysenteriae; VC, Vibrio cholerae. a Including the diameter of well (9 mm); No activity (diameter of the inhibition zone less than 10 mm). > indicates the highest concentration tested which did not inhibit the bacterial growth.

Minimum inhibitory concentrations of emodin and physcion The MICs of emodin and physcion against grampositive and gram-negative bacteria were determined using a concentration of 107 cfu/mL inocula. The MICs of emodin for B. cereus, B. subtilis and B. pumilus were 0.5, 1.5 and 2.0 g/mL, respectively, while the value was found to be 70.0 g/mL against P. aeruginosa. S. aureus was susceptible to a minimum concentration of 90.0 g/mL of emodin. The MIC of physcion against both M. luteus as well as P. aeruginosa was found to be 200 g/mL, and it was 250 g/mL against K. pneumoniae.
Copyright 2005 John Wiley & Sons, Ltd.

DISCUSSION The samples selected from the three plants, namely V. madraspatana, R. cordifolia and L. camara, exhibited varying degrees of inhibitory activity against the bacterial strains chosen for the present study. The extracts of V. madraspatana were found to possess a broadspectrum activity against both gram-positive and -negative strains, lending support to the traditional uses of this plant in the treatment of unspecied skin diseases. The other two plants were specically active against the gram-positive ones only, with the exception of Pseudomonas aeruginosa which was
Phytother. Res. 19, 888894 (2005)

ANTIBACTERIAL ACTIVITY OF V. MADRASPATANA

893

inhibited by all the three samples. In fact, the inhibition zones produced by 10 mg/mL of the semi-pure alcohol extracts against P. aeruginosa was nearly equivalent to that shown by 0.1 mg/mL of streptomycin, while only 2.5 mg/mL of the chloroform extract of V. madraspatana produced the same effect (Tables 13). This observation on the strong inhibitory power of all the three plant samples against a gram-negative bacterium is noteworthy. Again, it is worth mentioning that the activity was located, by and large, in the chloroform and alcohol extracts of the samples; out of the three extracts prepared with water, that of R. cordifolia responded only against B. subtilis and S. aureus (Table 2), while the water extracts of the other two plants were totally inactive (data not shown). This is signicant from the phytochemical perspective since all analytical reports on Ventilago sp. had shown it to be a rich source of hydroxyanthraquinonoid compounds, which would be extracted better with chloroform rather than with the more polar solvents (Sanyal et al., 2003; Hazra et al., 2004). In the present study also, done for the rst time on the stem-bark of this species, two major products isolated from the bioactive fractions were found to be hydroxyanthraquinonoids, namely emodin and physcion (Fig. 1). Emodin was matched with an authentic sample from Sigma, USA, which also helped to verify the structure of physcion (the methoxy analogue) through comparison of their NMR spectra. The peaks appearing in the range of 12.112.3 conrmed the presence of peri-hydroxy groups in both compounds, while the peak at 9.88 in the spectrum of emodin represented its third hydroxy group. Obviously, this peak was found to be absent in physcion; instead, a new peak appeared at 3.94 for the aromatic methoxy group. The rest of the protons of the two analogues appeared more or less at similar positions. Emodin is known to occur in at least 17 plant families worldwide, and has been investigated for antifungal, antiparasitic, antioxidant, immunosuppressive, antiulcer and antiinammatory activities (Lin et al., 1996; Izhaki, 2002). Studies are going on to evaluate its anticancer activity and related mechanism of action (Hazra et al., 2004). However, antimicrobial studies on emodin were found to be rather scanty (Kitanaka and Takido, 1986; Izhaki, 2002). Physcion, isolated from the rhizomes of Rheum emodi, was reported to inhibit some fungal species (Agarwal et al., 2000). The antibacterial effects of emodin and physcion against some of the bacterial strains, particularly that of emodin showing MICs in the range 0.52.0 g/mL against the three different Bacillus sp., were noteworthy

(Table 4). These MIC values compare favourably with those reported for the standard antibiotics such as chloramphenicol, penicillin, streptomycin and erythromycin against Bacillus sp. (Garrod and OGrady, 1972). Anke et al. (1980) had reported emodin to be effective against Bacillus bravis (106 cfu/mL) with a minimum inhibitory concentration of 5 g/mL. Emodin was found to be moderately effective against S. aureus also (with MIC at 90 g/mL), although the crude chloroform extract of V. madraspatana did not inhibit this strain at the highest concentration tested. Our results were more or less consistent with the report of Hatano et al. (1999) who found emodin to be effective on four strains of methicillin-resistant as well as methicillin-sensitive strains of S. aureus, and physcion was found to be ineffective. Physcion was found to be moderately active against Micrococcus luteus and Klebsiella pneumoniae, which did not respond to emodin. Again, both the compounds were effective against P. aeruginosa, emodin being more potent (MIC 70.0 g/mL) than physcion (MIC 200.0 g/mL). Thus, these two quinonoid compounds together presumably contributed signicantly to the broad-spectrum antibacterial effect of the chloroform extract of V. madraspatana. Recently, there has been a surge of interest in the diverse mode of action of bioactive anthraquinonoids derived from plants, e.g. emodin, physcion, chrysophanol, etc. (Chen et al., 2002). Quinones, in addition to providing a source of stable free radicals, are known to form irreversible complexes with nucleophilic amino acids, often leading to a loss of function of vital proteins in the microbial organism. Thus, probable targets in the microbial cell could be surface-exposed adhesions, cell-wall peptides and membrane-bound enzymes. Further, quinones may also render substrates unavailable for proliferation of the microorganism (Cowan, 1999). Hence, the observations on plants with quinonoid constituents would be relevant to the development of novel antimicrobial agents in future.

Acknowledgements
The present study received nancial support from the UGC, New Delhi. One of us (A.G.) received a Senior Research Fellowship from the CSIR, New Delhi. Thanks are accorded to Professor A. Patra, University College of Science, Calcutta, for providing the facility for NMR spectroscopy, and Dr L. Roy, Department of Food Technology and Biochemical Engineering Jadarphur University, Calcutta for supplying bacterial strains. The authors are grateful to Professor A. C. Ghose, Emeritus Scientist (ICMR), National Institute of Cholera and Enteric Diseases, Calcutta, for necessary advice and encouragement in course of the investigation.

REFERENCES

Adwankar MK, Chitnis MP. 1982. In vivo anti-cancer activity of RC-18: a plant isolate from Rubia cordifolia, Linn. against a spectrum of experimental tumour models. Chemotherapy 28: 291293. Agarwal SK, Singh SS, Verma S, Kumar S. 2000. Antifungal activity of anthraquinone derivatives from Rheum emodi. J Ethnopharmacol 72: 4346. American Society for Microbiology (ASM) Task Force on Antibiotic Resistance. 1995. Report of the ASM Task Force on
Copyright 2005 John Wiley & Sons, Ltd.

Antibiotic Resistance. Antimicrob Agents Chemother 39 (Suppl.): 223. Anke H, Kolthoum I, Laatsch H. 1980. Metabolic products of microorganisms. 192. The anthraquinones of the Aspergillus glaucus group. II. Biological activity. Arch Microbiol 126: 231236. Antarkar SS, Chinwalla T, Bhatt N. 1983. Antiinammatory activity of Rubia cordifolia Linn. in rats. Indian J Pharmacol 15: 185188.
Phytother. Res. 19, 888894 (2005)

894

S. BASU ET AL. ecological functions in higher plants. New Phytol 155: 205 217. Joharapurkar AA, Zambad SP, Wanjari MM, Umathe SN. 2003. In vivo evaluation of antioxidant activity of alcoholic extracts of Rubia cordifolia Linn. and its inuence on ethanol induced immunosuppression. Indian J Pharmacol 35: 232 236. Kesava Rao B, Hanumaiah T, Rao CP, Rao GSR, Rao KVJ, Thomson RH. 1983. Anthraquinones in Ventilago species. Phytochemistry 22: 25832585. Kitanaka S, Takido M. 1986. Studies on the constituents in the roots of Cassia obtusifolia L. and the antimicrobial activities of constituents of the root and the seeds. Yakugaku Zasshi 106: 302306. Krishnakumari GN, Bhuvaneswari B, Raja Swapna I. 2001. Antifeedant activity of quinones from Ventilago madaraspatana. Fitoterapia 72: 671675. Lin CC, Chang CH, Yang JJ, Namba T, Hattori M. 1996. Hepatoprotective effects of emodin from Ventilago leiocarpa. J Ethnopharmacol 52: 107111. Lin CC, Lin WC, Chang CH, Namba T. 1995. Anti-inammatory and hepatoprotective effects of Ventilago leiocarpa. Phytother Res 9: 1115. Lipipun V, Kurokawa M, Suttisri R et al. 2003. Efcacy of Thai medicinal plant extracts against herpes simplex virus type 1 infection in vitro and in vivo. Antiviral Res 60: 175 180. Pandey S, Sharma M, Chaturvedi P, Tripathi YB. 1994. Protective effect of Rubia cordifolia on lipid peroxide formation in isolated rat liver homogenate. Indian J Exp Biol 32: 180183. Panthong A, Kanjanapothi D, Taesotikul T, Phankummoon A, Panthong K, Reutrakul V. 2004. Anti-inammatory activity of methanolic extracts from Ventilago harmandiana Pierre. J Ethnopharmacol 91: 237242. Ross IA. 1999. Medicinal Plants of the World Chemical Constituents, Traditional and Modern Medicinal Uses. Humana Press: New Jersey. Sanyal U, Bhattacharyya S, Patra A, Hazra B. 2003. Liquid chromatographic separation of derivatives of diospyrin a bioactive bisnaphthoquinonoid plant product and analogous naphthyl compounds. J Chromatogr A 1017: 225232. Takeya K, Yamamiya T, Morita H, Itokawa H. 1993. Two antitumour bicyclic hexapeptides from Rubia cordifolia. Phytochemistry 33: 613615. Tripathi YB, Shukla S, Sharma M, Shukla VK. 1995. Antioxidant property of Rubia cordifolia extracts and its comparison with vitamin E and parabenzoquinone. Phytother Res 9: 440443.

Chang CH, Lin CC, Yang JJ, Namba T, Hattori M. 1996. Antiinammatory effects of emodin from Ventilago leiocarpa. Am J Chin Med 24: 139142. Chatterjee A, Pakrashi SC. 1997. The Treatise on Indian Medicinal Plants (vol. 5). National Institute of Science Communication CSIR: New Delhi. Chen YC, Shen SC, Lee WR et al. 2002. Emodin induces apoptosis in human promyeloleukemic HL-60 cells accompanied by activation of caspase 3 cascade but independent of reactive oxygen species production. Biochem Pharmacol 64: 17131724. Chopra RN, Nayar SL, Chopra IC. 1956. Glossary of Indian Medicinal Plants. CSIR: New Delhi. Cowan MM. 1999. Plant products as antimicrobial agents. Clin Microbiol Rev 12: 564582. Cragg GM, Newman DH, Snader KM. 1997. Natural products in drug discovery and development. J Nat Prod 60: 5260. Dosseh C, Tessier AM, Delaveau P. 1981. Racines de Rubia cordifolia II. Nouvelles Quinones. Planta Med 43: 141147. Garrod LP, OGrady F. 1972. Antibiotics and Chemotherapy. Churchill Livingstone: London. Ghisalberti EL. 2000. Lantana camara L., (Verbenaceae). Fitoterapia 71: 467486. Hanumaiah T, Marshall DS, Rao BK et al. 1985a. Benzisochromanquinones in Ventilago species. Phytochemistry 24: 23732378. Hanumaiah T, Rao BK, Rao CP et al. 1985b. Naphthalene and naphthoquinones from Ventilago species. Phytochemistry 24: 18111815. Hassanean HA, Ibraheim ZZ, Takeya K, Itorawa H. 2000. Further quinoidal derivatives from Rubia cordifolia L. Pharmazie 55: 317319. Hatano T, Uebayashi H, Ito H, Shiota S, Tuchiya T, Yoshida T. 1999. Phenolic constituents of Cassia seeds and antibacterial effect of some naphthalenes and anthraquinones on methicillin-resistant Staphylococcus aureus. Chem Pharm Bull (Tokyo) 47: 11211127. Hazra B, Das Sarma M, Sanyal U. 2004. Separation methods of quinonoid constituents on plants used in Oriental traditional medicines. J Chromatogr B 812: 259275. Ho LK, Don MJ, Chen HC, Yeh SF, Chen JM. 1996. Inhibition of hepatitis B surface antigen secretion on human hepatoma cells. Components from Rubia cordifolia. J Nat Prod 59: 330333. Itokawa H, Ibraheim ZZ, Ya-Fang Q, Takeya K. 1993. Anthraquinones, naphthohydroquinones and naphthohydroquinone dimers from Rubia cordifolia and their cytotoxic activity. Chem Pharm Bull (Tokyo) 41: 18691872. Izhaki I. 2002. Emodin a secondary metabolite with multiple

Copyright 2005 John Wiley & Sons, Ltd.

Phytother. Res. 19, 888894 (2005)