Evaluation of the Antibacterial Activity of Ventilago Madraspatana Gaertn., Rubia Cordifolia Linn. and Lantana Camara Linn.- Isolation of Emodin and Physcion as Active Antibacterial

8/12/2019 Evaluation of the Antibacterial Activity of Ventilago Madraspatana Gaertn., Rubia Cordifolia Linn. and Lantana Ca

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888 S. BASU ET AL.

Copyright 2005 John Wiley & Sons, Ltd. Phytother. Res . 19, 888894 (2005)

Copyright 2005 John Wiley & Sons, Ltd.

PHYTOTHERAPY RESEARCHPhytother. Res. 19, 888894 (2005)Published online in Wiley InterScience (www.interscience.wiley.com). DOI : 10.1002/ptr.1752

Evaluation of the Antibacterial Activityof Ventilago madraspatana Gaertn., Rubiacordifolia Linn. and Lantana camara Linn.:Isolation of Emodin and Physcion as ActiveAntibacterial 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 dif-fusion 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 thebacteria except S. aureus , E. coli and V. cholerae . On the other hand, the activity of the chloroform andmethanol extracts of R. cordifolia and L. camara was found to be more specic towards the gram-positivestrains, although gram-negative P. aeruginosa was also inhibited by the methanol extracts of both these plantsin a dose dependent manner. The water extracts of V. madraspatana and L. camara were found to beinactive, while that of R. cordifolia was signicantly active against B. subtilis and S. aureus compared withstreptomycin and penicillin G used as standards. In the course of bio-assay guided fractionation, emodin andphyscion were isolated for the rst time from the stem-bark of V. madraspatana . It was noteworthy to nd theMICs of emodin in the range 0.52.0 g/mL against three Bacillus sp. Both the anthraquinonoid compoundsinhibited P. aeruginosa , emodin being more effective, showing an MIC of 70 g/mL. Copyright 2005 JohnWiley & Sons, Ltd.

Keywords: Ventilago sp .; Rubia sp .; Lantana sp. ; antibacterial quinonoids; emodin; physcion

Received 1 March 2005 Accepted 23 June 2005

* Correspondence to: Dr Banasri Hazra, Department of PharmaceuticalTechnology, Jadavpur University, Calcutta 700032, India.E-mail: [email protected]/grant sponsor: University Grants Commission, New Delhi;Contract/grant number: F.3-15/ 2002/SR- II.

INTRODUCTION

The indiscriminate use of antibiotics for the treatmentof microbial diseases has led to the alarming emergenceof multi-drug-resistant bacterial pathogens, calling forthe development of novel antimicrobial agents (Ameri-can Society of Microbiology, 1995). Traditionally, manydrugs have been brought to the clinic by taking the leadfrom plant-derived natural products (Cragg et al ., 1997).Plants are known to produce a variety of quinonoidmetabolites, which are characteristically reactive as they

undergo facile redox reactions; hence, there is a greatpotential for the development of some of these com-pounds into new chemotherapeutic agents (Cowan,1999). Therefore, in the continuation of our searchfor bioactive quinonoids, three indigenous plants of ethnopharmacological importance, which are knownto contain various quinonoid compounds, were chosenfor investigation of their antibacterial property. Thus,selected parts of Ventilago madraspatana Gaertn. (stembark) , Rubia cordifolia Linn. (root) and Lantana camaraLinn. (root bark) were studied for the rst time against

several gram-positive and gram-negative bacteria, andtwo anthraquinonoid constituents, namely emodin andphyscion, isolated from the stem bark of V. madra-

spatana , were found to show antibacterial activity.The plant genus Ventilago (Rhamnaceae), repre-

sented by nearly 40 species, is distributed throughoutIndia and Southern parts of Asia. Ventilago madra-

spatana (Raktavalli in Sanskrit), indigenous to India, isa large evergreen woody climber with long sarmentosebranches. The leaves are oblong-lanceolate; owers aresmall, densely pubescent and with paniculate spikes;fruits are yellow, shining and globose (Chopra et al .,

1956).Traditionally, the root bark of V. madraspatanais used as a carminative, stomachic, tonic and stimu-lant. The powdered stem bark mixed with gingelly oilis 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 haemor-rhages. In Taiwan, the stem of V. leiocarpa is a folkmedicine for treatment of rheumatism, hepatitis andneuralgia (Lin et al. , 1996), while V. harmandiana isused in Thailand for the treatment of wounds andchronic 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-methylether (Kesava Rao et al ., 1983). Naphthalene derivatives


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and naphthoquinones, such as ventilaginone, ventilagol,maderone, cordeauxione and isocordeauxione, are alsoreported from the root bark of this plant (Hanumaiahet al ., 1985a; Hanumaiah et al ., 1985b). Apart fromthe antifeedant activity of V. madraspatana on insectsreported by Krishnakumari et al . (2001), there has beenno scientic report on the biological activities of this plant. However, the stem-bark of V. leiocarpa , theChinese species, was found to possess antiinammatoryand hepatoprotective activities (Lin et al ., 1995; Changet al ., 1996), while V. harmandiana , a Thai species, hasbeen studied recently for its antiinammatory effect(Panthong et al ., 2004). The leaves of V. denticulatafrom Thailand showed potent antiviral activity againstherpes simplex virus type-1 (Lipipun et al ., 2003).

Rubia cordifolia Linn. (Rubiaceae; Manjistha inSanskrit) is a perennial prickly climber, commonthroughout the lower hills of Himalayas and in theWestern Ghats of India. This plant also grows in themoist and tropical forests of Japan, Indonesia and SriLanka. The roots are very long and cylindrical with athin red bark. The leaves are ovate-lanceolate; theowers small, greenish white in terminal panicle orcymes; the fruits are globose and dark purplish incolour (Chatterjee and Pakrashi, 1997).

Various ethnomedical applications are documentedfor Rubia cordifolia , e.g. its fruits are used in the treat-ment of hepatic obstructions (Chatterjee and Pakrashi,1997); the stems are applied for treating cobra-bite andscorpion-sting (Chopra et al ., 1956); the roots possessalterative, astringent and diuretic properties, and arewidely used in traditional medicinal preparations totreat amenorrhoea, chronic diarrhoea, dropsy, intestinalatony, renal calculi, jaundice and paralysis. A paste of the root with honey is applied externally to inamma-tion, freckles and other skin diseases; an infusion isgiven to women after delivery to produce a copiousow of lochia (Chatterjee and Pakrashi, 1997).

Phytochemical investigations on this plant haveindicated the presence of a wide variety of quinonoidsand triterpenoids, only a few of which have been invest-igated for their bioactive potential (Dosseh et al. , 1981;Itokawa et al. , 1993; Takeya et al ., 1993; Hassaneanet al. , 2000). Scientic studies were done on the rootextracts of R. cordifolia and its constituents for themanagement of tumour (Itokawa et al ., 1993), cancer(Adwankar and Chitnis, 1982) and liver disorders(Pandey et al ., 1994). Also, some reports are availableon the antiinammatory (Antarkar et al. , 1983), anti-

oxidant (Tripathi et al. , 1995; Joharapurkar et al ., 2003)and anti-hepatitis B (Ho et al. , 1996) properties of thesame extract.

Lantana camara Linn. (Verbenaceae), the mostwidespread species of this genus, is a native of Southern America, now distributed throughout thetropical and sub-tropical parts of the world (Ross, 1999).It is a woody straggling shrub with prickly stems andowers of various colours yellow, orange, red, pinkor white. The leaves are ovate, with a coarse surfaceand toothed margin; the fruits are dark purple or black,shiny and globose in shape.

The aerial parts, leaves and roots of L. camara arewidely used in folklore medicine all over the world,

particularly in Latin America, South-East Asia, WestIndies, Africa and Australia, for various applications inthe ailments of skin and stomach, rheumatism, asthma,

convulsion, indigestion and fever (Ross, 1999; Chopraet al ., 1956).

Phytochemical studies on this plant show the pre-sence of a wide variety of quinonoids, triterpenes,iridoid glycosides and avonoids (Ghisalberti, 2000).Pharmacological studies have been reported, mostly onits leaves and aerial parts, for antibacterial, antifungal,immunosuppressant and insect repellent activities (Ross,1999).

Thus, it transpired that the antibacterial effect of theseplants had not been investigated systematically, andwas carried out in the present study on the extracts,prepared with solvents of different polarity, fromV. madraspatana stem-bark, R. cordifolia root andL. camara root-bark. Further, emodin and physcionwere detected for the rst time in the stem-bark of V. madraspatana , and their antibacterial activity wasstudied in detail.

MATERIALS AND METHODS

Plant material. Ventilago madraspatana stem-bark wascollected from Bolangir district, Orissa, Rubia cordifoliaroot was purchased locally from a dealer in traditionalmedicinal plants, and Lantana camara roots were pro-cured from the Salt Lake neighbourhood in Calcutta.All the plant materials were authenticated by theBotanical Survey of India, Calcutta, and a voucherspecimen is preserved in our laboratory for futurereference. The samples were dried in shade andpulverized before use in the following studies.

Preparation of plant extracts for antibacterial assay. Theplant samples (5 g each) were reuxed separately withchloroform, alcohol and water for 2 h. The extracts wereevaporated to dryness under reduced pressure and thefollowing 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%) extractsof R. cordifolia root; chloroform (17.8%), methanol(20.9%) and water (10.1%) extracts of L. camara root-bark. Either sterile normal saline or DMSO was usedto reconstitute the extracts and prepare the solutionsfor 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 sixgram-negative ( Escherichia coli , Klebsiella pneumoniae ,Pseudomonas aeruginosa , Salmonella typhimurium ,Shigella dysenteriae , Vibrio cholerae ) bacteria. The testorganisms were obtained from stock cultures of theDepartments of Microbiology, Bose Institute, and FoodTechnology and Bio-Chemical Engineering, JadavpurUniversity, Calcutta. The Mueller-Hinton (M-H) agarmedium (0.4% beef infusion solids, 1.75% caseinhydrolysate, 1.5% agar, 0.15% starch and 1% sodiumchloride, in w/v; pH 7.4) was used for the maintenanceof bacterial strains in slants as well as for the study

of antibacterial activity. The rst three reagents wereprocured from Himedia, Mumbai and the rest fromMerck, Mumbai, India.


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Determination of antibacterial activity. Antibacterialassays of the samples against the selected bacterialcultures were carried out in vitro by the agar well dif-fusion method. A number of colonies were picked upfrom the bacterial stock culture and transferred to 5 mLof M-H broth and shaken in a water-bath at 37 C for~18 h. 200 L of this culture containing the logarithmicphase cells was resuspended in sterile saline for adjust-ment of cfu/mL spectrophotometrically at 600 nm. Itwas then seeded into sterile molten M-H agar (4850 C), and poured into sterilized petri dishes to give anal inocula of 1 107 cfu/mL. Wells of uniform diam-eter (9 mm) were made on the solidied agar plate usinga sterile borer. Solutions of the plant samples and stand-ard drugs, streptomycin and penicillin G (Sigma, USA),as well as the solvents used (saline/DMSO) were placedseparately (0.1 mL of each) in each well under asepticconditions. The extracts and the pure compounds weretested over the concentration ranges 1.010.0 mg/mL,and 0.10.5 mg/mL, respectively. The plates were thenmaintained at room temperature for 2 h allowing thesolution to diffuse into the medium. After incubationat 37 C for 24 h (48 h in the case of S. dysenteriaeand V. cholerae ) the zones of inhibition around eachwell were measured. Each experiment was performedin triplicate and an average of three independentdeterminations for each zone was recorded.

Determination of minimum inhibitory concentration(MIC). The MIC of the pure compounds against eachof the bacteria was determined by the agar well dif-fusion method. Different concentrations of the com-pound (0.1500 g/mL) were introduced into the wellson the petri dishes containing sterilized M-H agarmedium with 1 107 cfu/mL prepared as before. Theplates were incubated at 37 C for 24 h. The resultswere noted on the basis of the presence or absenceof inhibition zones. The MIC was determined as thelowest concentration of the sample showing a zone of inhibition.

Instrumentation. UV/VIS spectra were measuredusing a Pharmacia Biotech Ultrospec 2000 UV/Visiblespectrophotometer. IR spectra were recorded in aPerkin Elmer Model 783 IR spectrophotometer. 1H-NMR were taken in deuterochloroform or deutero-methanol with trimethylsilane (TMS) as an internalstandard using a Brucker AM 300L instrument. The MSwere recorded in a JEOL JMS600 spectrophotometer.

Isolation of emodin and physcion. The chloroformextract of V. madraspatana stem-bark was analysed bychromatography on a silica gel (60120 mesh) columnand eluted with solvents of increasing polarity. Amongthe several fractions thus obtained by elution with mix-tures 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 samesolvent system at a ratio of 50:50, v/v, were found toappear as major products. These semi-pure fractionswere 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

Figure 1. Structures of physcion and emodin.

product. This was collected and the compound thusobtained [checked by TLC; R f = 0.53 with petroleumether (bp 6080 C): chloroform = 20:80; v/v; yield0.22%] was crystallized from methanol into yellow plates(mp 201 C). Spectroscopic analysis by IR, UV, NMRand MS conrmed it to be physcion (Fig. 1). Thesecond fraction collected from the column was alsopuried through PTLC using chloroform:ethyl acetate(80:20; v/v) to get the major product ( R f = 0.67 withchloroform:ethyl acetate = 90:10; v/v; yield 0.31%) whichwas crystallized from methanol into orange needles(mp 255 C). This compound was found to matchwith an authentic sample of emodin (Sigma, USA), andanalysed 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(cm 1) 2918, 1629,1566, 1479, 1450, 1367, 1296, 1163, 1035. 1H NMR(CDCl 3; 300 MHz): 2.45 (3H,s, CH 3-6); 3.94 (3H, s,OCH 3-3); 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(cm 1) 3377, 2925,1629, 1481, 1456, 1336, 1211, 1103, 1033. 1H NMR(CDCl 3; 300 MHz): 2.46 (3H,s, CH 3-6); 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 isolatedpure compounds were screened over a range of doses(110 mg/mL and 0.10.5 mg/mL, respectively) againstsix gram-positive ( Bacillus cereus , Bacillus pumilus ,Bacillus subtilis , Micrococcus luteus , Mycobacteriumluteum , Staphylococcus aureus ) and six gram-negative(Escherichia coli , Klebsiella pneumoniae , Pseudomonasaeruginosa , Salmonella typhimurium , Shigella dysen-teriae , Vibrio cholerae ) bacteria. The solvent vehicles,i.e. normal saline and DMSO, were also studied simi-larly and were found to be ineffective against all the

tested strains (data not shown). Streptomycin and peni-cillin G were used as positive controls. Streptomycinshowed a broad-spectrum activity, while penicillin G,


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Table 1. Antibacterial activity (inhibition zone in mm a) of the extracts of the stem bark of Ventilago madraspatana

Streptomycin Penicillin GEtOH extract (mg/mL) CHCl 3 extract (mg/mL) (mg/mL) (mg/mL)

Bacteria 1.0 2.5 5.0 7.5 10.0 1.0 2.5 5.0 7.5 10.0 0.1 0.01

Gram-positiveBacillus cereus 11 12 12 14 10 11 12 13 16 20 23

Bacillus pumilus 14 19 22 25 25 11 13 16 17 24 22Bacillus subtilis 12 12 14 14 15 11 11 13 13 16 19 15Micrococcus luteus 11 13 14 10 11 13 14 22 28Mycobacterium luteum 10 12 22 Staphylococcus aureus 23 19

Gram-negativeEscherichia coli 20 Klebsiella pneumoniae 12 14 14 15 12 13 13 16 16 17 Pseudomonas aeruginosa 12 12 13 12 16 16 22 24 15 Salmonella typhimurium 12 13 14 10 16 Shigella dysenteriae 11 12 12 15 Vibrio cholerae 20

a 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 mm a) of the extracts of the root of Rubia cordifolia

Streptomycin Penicillin GH2O extract (mg/mL) MeOH extract (mg/mL) CHCl 3 extract (mg/mL) (mg/mL) (mg/mL)

Bacteria 1.0 2.5 5.0 7.5 10.0 1.0 2.5 5.0 7.5 10.0 1.0 2.5 5.0 7.5 10.0 0.1 0.01

Gram-positiveBC 12 15 16 17 18 10 10 18 19 22 20 23BP 10 11 12 12 13 11 12 12 15 24 22BS 13 15 16 17 18 13 15 17 17 19 12 14 16 18 19 15MIL 11 12 12 13 11 12 12 13 13 22 28MYL 10 11 10 10 22 SA 12 15 18 19 19 20 20 24 25 26 13 14 17 18 20 23 19

Gram-negativeEC 20 KP 17 PA 13 13 13 14 14 10 15 ST 16 SD 15 VC 20

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 exhibitedantibacterial activity against both gram-positive andgram-negative strains (Table 1). The ethanol extractshowed strong inhibition towards B. pumilus , B. subtilis ,K. pneumoniae and S. typhimurium , and moderateinhibition against M. luteus , P. aeruginosa and S.dysenteriae. The chloroform extract inhibited the growthof gram-negative K. pneumoniae and P. aeruginosa , andmost of the gram-positive bacteria tested. Both theextracts were inactive against S. aureus , E. coli andV. cholerae .

The methanol and chloroform extracts of R. cor-difolia root were found to be active against all the gram-

positive bacteria used in this study. P. aeruginosa wasthe only gram-negative strain which was inhibitedby the methanol extract in a dose dependent manner

(Table 2). However, the water extract was found to be

active only against B. subtilis and S. aureus , which werehighly susceptible to inhibition by all the three extractsin comparison with the standard drugs.

Similarly, the methanol and chloroform extracts of L. camara root-bark were moderately active against mostof the gram-positive strains tested, while P. aeruginosawas the only gram-negative bacterium which wasstrongly inhibited by the methanol extract. However,this extract was found to be inactive against B. cereusat the maximum dose (10 mg/mL) tested (Table 3).

The anthraquinonoid emodin isolated from V.madraspatana stem-bark was active against the gram-positive B. cereus , B. pumilus , B. subtilis , S. aureusand gram-negative P. aeruginosa in a concentration

dependent manner. The analogous compound physcionwas found to inhibit M. luteus , K. pneumoniae andP. aeruginosa (Table 4) .


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Table 3. Antibacterial activity (inhibition zone in mm a) of the extracts of the root bark of Lantana camara

Streptomycin Penicil lin GMeOH extract (mg/mL) CHCl 3 extract (mg/mL) (mg/mL) (mg/mL)

Bacteria 1.0 2.5 5.0 7.5 10.0 1.0 2.5 5.0 7.5 10.0 0.1 0.01

Gram-positiveBacillus cereus 11 12 12 13 14 20 23

Bacillus pumilus 11 12 13 14 14 12 14 15 15 15 24 22Bacillus subtilis 12 15 16 11 11 12 13 13 19 15Micrococcus luteus 10 10 11 22 28Mycobacterium luteum 22 Staphylococcus aureus 11 11 11 12 13 10 11 12 14 14 23 19

Gram-negativeEscherichia coli 20 Klebsiella pneumoniae 17 Pseudomonas aeruginosa 10 10 12 14 16 15 Salmonella typhimurium 16 Shigella dysenteriae 15 Vibrio cholerae 20

a 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 mm a) of emodin and physcion

Streptomycin Penicillin GEmodin (mg/mL) Physcion (mg/mL) (mg/mL) (mg/mL) MIC ( g/mL)

Bacteria 0.1 0.2 0.3 0.4 0.5 0.1 0.2 0.3 0.4 0.5 0.1 0.01 Emodin Physcion

Gram-positiveBC 16 17 17 18 18 20 23 0.5 >500BP 14 14 15 16 16 24 22 2.0 >500BS 13 13 14 14 15 19 15 1.5 >500MIL 11 13 15 15 22 28 >500 200.0MYL 22 >500 >500SA 10 11 14 15 15 23 19 90.0 >500

Gram-negativeEC 20 >500 >500KP 11 12 13 17 >500 250.0PA 13 13 14 15 17 10 11 11 12 15 70.0 200.0ST 16 >500 >500SD 15 >500 >500VC 20 >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 andphyscion

The MICs of emodin and physcion against gram-positive and gram-negative bacteria were determinedusing a concentration of 10 7 cfu/mL inocula. The MICsof emodin for B. cereus , B. subtilis and B. pumiluswere 0.5, 1.5 and 2.0 g/mL, respectively, while the valuewas found to be 70.0 g/mL against P. aeruginosa .S. aureus was susceptible to a minimum concentrationof 90.0 g/mL of emodin. The MIC of physcion

against both M. luteus as well as P. aeruginosa wasfound to be 200 g/mL, and it was 250 g/mL againstK. pneumoniae .

DISCUSSION

The samples selected from the three plants, namely V.madraspatana , R. cordifolia and L. camara , exhibitedvarying degrees of inhibitory activity against the bac-terial strains chosen for the present study. The extractsof V. madraspatana were found to possess a broad-spectrum activity against both gram-positive and-negative strains, lending support to the traditionaluses of this plant in the treatment of unspecied

skin diseases. The other two plants were specicallyactive against the gram-positive ones only, with theexception of Pseudomonas aeruginosa which was


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(Table 4). These MIC values compare favourably withthose reported for the standard antibiotics such aschloramphenicol, penicillin, streptomycin and erythro-mycin against Bacillus sp. (Garrod and OGrady, 1972).Anke et al . (1980) had reported emodin to be effectiveagainst Bacillus bravis (106 cfu/mL) with a minimuminhibitory concentration of 5 g/mL. Emodin was foundto be moderately effective against S. aureus also (withMIC at 90 g/mL), although the crude chloroformextract of V. madraspatana did not inhibit this strain atthe highest concentration tested. Our results were moreor less consistent with the report of Hatano et al . (1999)who found emodin to be effective on four strainsof methicillin-resistant as well as methicillin-sensitivestrains of S. aureus , and physcion was found to be in-effective. Physcion was found to be moderately activeagainst Micrococcus luteus and Klebsiella pneumoniae ,which did not respond to emodin. Again, both thecompounds were effective against P. aeruginosa , emodinbeing more potent (MIC 70.0 g/mL) than physcion(MIC 200.0 g/mL). Thus, these two quinonoid com-pounds together presumably contributed signicantlyto the broad-spectrum antibacterial effect of the chloro-form extract of V. madraspatana .

Recently, there has been a surge of interest in thediverse mode of action of bioactive anthraquinonoidsderived from plants, e.g. emodin, physcion, chrys-ophanol, etc. (Chen et al. , 2002). Quinones, in additionto providing a source of stable free radicals, are knownto form irreversible complexes with nucleophilic aminoacids, often leading to a loss of function of vital pro-teins in the microbial organism. Thus, probable targetsin the microbial cell could be surface-exposed adhe-sions, cell-wall peptides and membrane-bound enzymes.Further, quinones may also render substrates unavail-able for proliferation of the microorganism (Cowan,1999). Hence, the observations on plants with quinonoidconstituents would be relevant to the development of novel antimicrobial agents in future.

Acknowledgements

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

inhibited by all the three samples. In fact, the inhibi-tion zones produced by 10 mg/mL of the semi-purealcohol extracts against P. aeruginosa was nearly equiva-lent 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-negativebacterium is noteworthy.

Again, it is worth mentioning that the activity waslocated, by and large, in the chloroform and alcoholextracts of the samples; out of the three extracts pre-pared with water, that of R. cordifolia responded onlyagainst B. subtilis and S. aureus (Table 2), while thewater extracts of the other two plants were totallyinactive (data not shown). This is signicant from thephytochemical perspective since all analytical reportson Ventilago sp. had shown it to be a rich source of hydroxyanthraquinonoid compounds, which would beextracted better with chloroform rather than withthe more polar solvents (Sanyal et al ., 2003; Hazraet al ., 2004). In the present study also, done for therst time on the stem-bark of this species, two majorproducts isolated from the bioactive fractions werefound to be hydroxyanthraquinonoids, namely emodinand physcion (Fig. 1). Emodin was matched with anauthentic sample from Sigma, USA, which also helpedto verify the structure of physcion (the methoxy ana-logue) through comparison of their NMR spectra. Thepeaks appearing in the range of 12.112.3 conrmedthe presence of peri-hydroxy groups in both compounds,while the peak at 9.88 in the spectrum of emodinrepresented its third hydroxy group. Obviously, thispeak was found to be absent in physcion; instead, anew peak appeared at 3.94 for the aromatic methoxygroup. The rest of the protons of the two analoguesappeared more or less at similar positions.

Emodin is known to occur in at least 17 plant fam-ilies worldwide, and has been investigated for antifungal,antiparasitic, antioxidant, immunosuppressive, antiulcerand antiinammatory activities (Lin et al ., 1996; Izhaki,2002). Studies are going on to evaluate its anticanceractivity and related mechanism of action (Hazra et al. ,2004). However, antimicrobial studies on emodin werefound to be rather scanty (Kitanaka and Takido, 1986;Izhaki, 2002). Physcion, isolated from the rhizomesof Rheum emodi , was reported to inhibit some fungalspecies (Agarwal et al ., 2000).

The antibacterial effects of emodin and physcion

against some of the bacterial strains, particularly thatof emodin showing MICs in the range 0.52.0 g/mLagainst the three different Bacillus sp. , were noteworthy

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Documents

Evaluation of the Antibacterial Activity of Ventilago Madraspatana Gaertn., Rubia Cordifolia Linn. and Lantana Camara Linn.- Isolation of Emodin and Physcion as Active Antibacterial