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A novel polyherbal microbicide with inhibitory effect on bacterial, fungal and viral genital pathogens

A novel polyherbal microbicide with inhibitory effect on bacterial, fungal and viral genital pathogens
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  International Journal of Antimicrobial Agents 32 (2008) 180–185 Short communication A novel polyherbal microbicide with inhibitory effect on bacterial,fungal and viral genital pathogens G.P. Talwar a , ∗ , Sajad A. Dar a , Mahendra K. Rai a , b , K.V.R. Reddy c , Debashis Mitra d ,Sujata V. Kulkarni d , Gustavo F. Doncel e , Christopher B. Buck  f  , John T. Schiller f  ,Sumathi Muralidhar g , Manju Bala g , S.S. Agrawal b , Kavita Bansal a ,Jitendra K. Verma a a Talwar Research Foundation, E-8, Neb Valley, Neb Sarai, New Delhi 110068, India b  Delhi Institute of Pharmaceutical Sciences and Research, University of Delhi, New Delhi, India c  National Institute for Research in Reproductive Health (ICMR), Mumbai, India d  National Centre for Cell Science, Pune, India e CONRAD, Eastern Virginia Medical School, Arlington, VA, USA f   National Cancer Institute/National Institute of Health, Bethesda, MD, USA g  Regional STD Teaching, Training and Research Centre, Safdarjung Hospital, New Delhi, India Received 21 January 2008; accepted 11 March 2008 Abstract A polyherbal cream (Basant) has been formulated using diferuloylmethane (curcumin), purified extracts of   Emblica officinalis  (Amla),purified saponins from  Sapindus mukorossi , Aloe vera and rose water along with pharmacopoeially approved excipients and preservatives.Basant inhibits the growth of WHO strains and clinical isolates of   Neisseria gonorrhoeae , including those resistant to penicillin, tetracycline,nalidixic acid and ciprofloxacin. It has pronounced inhibitory action against  Candida glabrata ,  Candida albicans  and  Candida tropicalis isolated from women with vulvovaginal candidiasis, including three isolates resistant to azole drugs and amphotericin B. Basant displayeda high virucidal action against human immunodeficiency virus HIV-1NL4.3 in CEM-GFP reporter T and P4 (Hela-CD4-LTR-  Gal) celllines with a 50% effective concentration (EC 50 ) of 1:20000 dilution and nearly complete (98–99%) inhibition at 1:1000 dilution. It alsoprevented the entry of HIV-1(IIIB) virus into P4-CCR5 cells (EC 50  ∼ 1:2492). Two ingredients, Aloe and Amla, inhibited the transductionof human papillomavirus type 16 (HPV-16) pseudovirus in HeLa cells at concentrations far below those that are cytotoxic and those used inthe formulation. Basant was found to be totally safe according to pre-clinical toxicology carried out on rabbit vagina after application for 7consecutive days or twice daily for 3 weeks. Basant has the potential of regressing vulvovaginal candidiasis and preventing  N. gonorrhoeae ,HIV and HPV infections.© 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords:  Basant;  Neisseria gonorrhoeae ;  Candida ; HIV; HPV 1. Introduction According to the World Health Organization (WHO), 340millioncasesofnewsexuallytransmittedinfectionsoccurred ∗ Corresponding author. Tel.: +91 11 2953 1039/2953 6769;fax: +91 11 2953 6768.  E-mail addresses:, Talwar). intheyear1999worldwide,ofwhichanestimated92millioninfections were  Chlamydia trachomatis  and 62 million were  Neisseriagonorrhoeae [1].Cervicalcancercausedbyhumanpapillomavirus (HPV) is the second commonest cancer andthefifthcauseofcancerdeathsinwomen[2].Accordingtothe United Nations Joint Programme on HIV/AIDS (UNAIDS),approximately 40 million people worldwide are living withhumanimmunodeficiencyvirus/acquiredimmunedeficiencysyndrome(HIV/AIDS),withanestimated4.3millionpeople 0924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.doi:10.1016/j.ijantimicag.2008.03.004  G.P. Talwar et al. / International Journal of Antimicrobial Agents 32 (2008) 180–185  181 acquiring HIV in 2006. Amongst reproductive tract infec-tions, vulvovaginal candidiasis (VVC) is the second mostcommon vaginal infection [3].Safe sex is promoted, but the use of condoms by malesis not consistent. Women’s groups are strongly advocat-ing the development of safe and effective microbicidesthat would be under their control. Research in academiaand industry has led to many candidate microbicides,but none has so far successfully completed Phase IIItrials.A microbicide in the form of a cream ready for vaginaluse was formulated. This formulation, named ‘Basant’, usesingredientsofmeritofplantorigin.Theaimofthisstudywasto determine the inhibitory action of this formulation on  N.gonorrhoeae , various types of   Candida  and HIV. The activeingredients of the formulation have also been tested againsttheinfectivityofHPVtype16(HPV-16)pseudovirusinHeLacells. 2. Material and methods Basant was formulated using 0.36% (w/v) purified cur-cumin, 2.5% (w/v) purified extracts of Amla, 1% (w/v)purified saponins from  Sapindus mukorossi , 2.5% (w/v)purified Aloe vera powder and rose extract water. Qual-ity control criteria were developed for each ingredient toassure reproducibility from batch to batch. The ingredientswere formulated in pharmacopoeially approved excipients:sodiumalginateasgellingagent,xanthangumasbioadhesiveagent, lactic acid, citric acid and potassium sodium tartrateasbufferingagents,benzoicacidaspreservativeandglycerolas humectant. A batch of Basant was prepared under GMP(GoodManufacturingPractice)conditionsbyalicensedcom-pany M/s Gufic Biosciences.The clinical isolates were from patients attending theRegional STD Centre at Safdarjung Hospital, New Delhi,India. Vaginal swabs were taken and cultured in gonococcalagar (HiMedia Laboratories Pvt Ltd., Mumbai, India) for  N.gonorrhoeae andSabourauddextroseagar(SDA)(HiMedia)for Candida .Identificationoftheorganismswasdonebybio-chemicaltests,andtheirantibioticsensitivitywasdeterminedby standard criteria. 2.1. Testing against Neisseria gonorrhoeae The effect of Basant on  N. gonorrhoeae  was tested asdescribed elsewhere [4] with the following modifications.The inoculum was prepared in normal saline to turbid-ity equivalent to a 0.5 McFarland standard (ca. 1.5 × 10 8 colony-forming units (CFU)/mL) and was swabbed onMueller–Hinton blood agar (HiMedia Laboratories PvtLtd.) containing 5% sheep blood and different concentra-tions (v/v) of Basant. The plates were incubated for 24hat 37 ◦ C under a 5% CO 2  atmosphere and growth wasobserved. 2.2. Evaluation of antifungal activity A fungal suspension (10  L) in normal saline withturbidity equivalent to a 0.5 McFarland standard (ca.1.5 × 10 8 CFU/mL) was mixed in microtitre plate wells with100  L of RPMI-1640 broth (HiMedia) with 2% glucose[5]. Basant was added at concentrations of 5%, 2%, 1% and0% (v/v) to different wells and mixed. Following overnightincubation of the mixture, 10  L was spread on SDA andincubated for 24h at 37 ◦ C. Investigations were carriedout three times. The minimal fungicidal concentration wasdefinedasthelowestconcentrationofBasantthatcompletelykilled the fungi. 2.3. Antimicrobial activity of individual ingredients of  Basant  Anagardiffusionbioassay[6]wasperformedtostudytheeffect of the ingredients of Basant on  N. gonorrhoeae  andvariousspeciesof  Candida ,withthefollowingmodifications.Mueller–Hinton agar plates with methylene blue were inoc-ulated with suspensions containing ca. 1.5 × 10 8 CFU/mL of the pathogen. Basant ingredients were dissolved in water atthe concentration used in the total formulation, except cur-cumin that was dissolved in dimethyl sulfoxide (DMSO).Basant cream was used as 10% suspension in water. Aplacebo cream suspension was used as a negative control.All plates were incubated overnight at 37 ◦ C, providing a 5%CO 2  atmosphere for  N. gonorrhoeae.2.4. Anti-HIV testing Basant was investigated for possible anti-HIV activity intwo laboratories. At the National Centre for Cell Sciences,Pune, India, the activity of Basant was tested in CEM-GFPand Hela-CD4-LTR-  Gal (P4) cell lines using NL4.3 HIVvirus (kindly made available by the NIH AIDS ReagentProgram, Germantown, MD) at 0.1 multiplicity of infection(MoI). The virus was pre-incubated with different dilutionsof Basant at 37 ◦ C, after which the control or treated viruswas added to CEM-GFP or P4 cells in serum-free mediaand incubated for a further 4h. The cells were washed withserum-freemediatoremovecell-freevirus,followedbyincu-bation in complete media. CEM-GFP cells were monitoredby green fluorescent protein (GFP) expression, and culturesupernatant was collected on Day 7 post infection for p24antigen analysis [7]. In the case of P4, cells were stained for  -galactosidase after 48h to identify infected cells, and theculture supernatant was assayed for virus production usingp24 antigen capture enzyme-linked immunosorbent assay(ELISA) (Perkin Elmer, Boston, MA). Percentage of inhibi-tion of virus replication was calculated with respect to cellstreated with placebo cream.At CONRAD Laboratory, the inhibitory action of Bas-ant on HIV-1 entry into P4-CCR5 cells (NIH AIDSReagent Program), which stably express CD4 and HIV  182  G.P. Talwar et al. / International Journal of Antimicrobial Agents 32 (2008) 180–185 Table 1Effect of different concentrations of Basant on standard strains and clinical isolates of   Neisseria gonorrhoeae  and various  Candida  spp. a Standard WHO strains and clinical isolates Antibiotic resistance Growth at different concentrations (v/v) of Basant after 24h5% 2% 1% 0%  Neisseria gonorrhoeae b  N. gonorrhoeae  WHO–C None  − −  + +  N. gonorrhoeae  WHO–G Tetracycline (TRNG), nalidixic acid  − − −  +  N. gonorrhoeae  WHO–K Penicillin (PPNG), nalidixic acid,ciprofloxacin, ceftriaxone lesssensitive − − −  +  N. gonorrhoeae  WHO–L Penicillin (PPNG), nalidixic acid,ciprofloxacin, ceftriaxone lesssensitive − − −  +  N. gonorrhoeae  1586 Nalidixic acid, ciprofloxacin  − − −  +  N. gonorrhoeae  1669 Nalidixic acid, ciprofloxacin  − −  + +  N. gonorrhoeae  1794 Nalidixic acid, ciprofloxacin  − −  + +  N. gonorrhoeae  2182 Nalidixic acid, ciprofloxacin  − − −  +  N. gonorrhoeae  2436 Penicillin (PPNG), tetracycline(TRNG), nalidixic acid, ciprofloxacin − − −  +  N. gonorrhoeae  2482 Penicillin (PPNG), nalidixic acid,ciprofloxacin − − −  +  N. gonorrhoeae  2676 Penicillin (PPNG), tetracycline(TRNG), nalidixic acid,ciprofloxacin less sensitive − −  + + Candida  spp. c C. glabrata  ATCC 90030 None 0  d 0 0–0.064 × 10 8 CFU/mL 1.38–1.49 × 10 8 CFU/mL C. glabrata  (7 clinical isolates) None 0 0 0–0.09 × 10 8 CFU/mL 1.3–1.5 × 10 8 CFU/mL C. glabrata  (2 clinical isolates) Fluconazole, itraconazole,ketoconazole, voriconazole andamphotericin B0 0 0–0.075 × 10 8 CFU/mL 1.4–1.5 × 10 8 CFU/mL C. albicans  ATCC 36082 None 0 0 0–0.077 × 10 8 CFU/mL 1.4–1.47 × 10 8 CFU/mL C. albicans  (5 clinical isolates) None 0 0 0.05–0.08 × 10 8 CFU/mL 1.37–1.5 × 10 8 CFU/mL C. albicans  Fluconazole, itraconazole,ketoconazole, voriconazole andamphotericin B0 0 0–0.067 × 10 8 CFU/mL 1.33–1.4 × 10 8 CFU/mL C. tropicalis  (2 clinical isolates) None 0 0 0.04–0.07 × 10 8 CFU/mL 1.35–1.41 × 10 8 CFU/mL − , no growth observed; +, growth observed; TRNG, tetracycline-resistant  N. gonorrhoeae ; PPNG, penicillinase-producing  N. gonorrhoeae ; CFU, colony-forming units. a All investigations were performed in triplicate. b Testing was done by agar dilution method and growth was observed after 24h. c Testing was done by broth dilution method after which 10  L was spread on Sabouraud dextrose agar and colonies were counted after 24h. d No colonies seen. chemokine co-receptors, was tested in a single-round infec-tion assay by incubating the HIV-1 virus (IIIB) andP4-R5 cells concomitantly with various dilutions of Bas-ant for 2h [8]. The virus and compound were then removed and cells were cultured in fresh medium for anadditional 48h. Infection was quantitated by chemilumines-cent detection of HIV long terminal repeat (LTR)-drivengalactosidase expression (Galacto-Star TM ; Tropix, Bedford,MA). 2.5. Anti-HPV testing SerialdilutionsofBasantandsomeofitscomponentswereadded to HeLa cell cultures in Dulbecco’s Modified Eagle’sMedium/10% foetal bovine serum in a 96-well plate. HPV-16 pseudovirus expressing GFP was added to each well at aMoI of ca. 0.1. The cells were incubated for 2 days and thensubjected to FACS analysis. Infectivity was scored based onthepercentageofcellspositiveforGFPandthepercentageof inhibition was calculated relative to a ‘no inhibitor’ control[9]. 2.6. Pre-clinical toxicology of Basant  Pre-clinical toxicology of Basant was performed at theNational Institute for Research in Reproductive Health(NIRRH), Mumbai, India, in 24 rabbits according to the pro-tocolrecommendedbytheUSFoodandDrugAdministration(FDA)forproductsmeantforvaginaluse[10].Animalswere dividedintwogroups:GroupIcomprised12animalsdividedinto two groups of 6 animals each, treated with Basant orplacebo once daily for 7 days; and Group II comprised thesame number treated twice daily for 21 days. Vaginal cellmorphology,vaginalhistologyandcytokinelevelsofvaginallavage were studied in addition to haematology and serumbiochemistry.  G.P. Talwar et al. / International Journal of Antimicrobial Agents 32 (2008) 180–185  183Table 2Anti-HIV evaluation, viral entry inhibition of Basant and its ingredientsCompound Solvent stock solution Diluent Cytotoxicity EC 50  (  g/mL) a Viral entry inhibition EC 50  (  g/mL) b Basant R10 c R10 278 dil. d 2492 dil.Placebo R10 R10 10 dil. 443 dil.Curcumin DMSO R10 >10 >10Saponins (reetha) dH 2 O R10 >10 >10Amla dH 2 O R10 >50 42Aloe dH 2 O R10 <2 dil. 2126 dil.C-2 e PBS R10 >1000 1.81% ethanol R10 >10 f  >10 f  DMSO R10 >10 f  >10 f  HIV, human immunodeficiency virus; EC 50 , 50% effective concentration; DMSO, dimethyl sulfoxide. a Cytotoxicity assay (MTS). b Viral entry inhibition assay (IIIB). c RPMI medium supplemented with 10% foetal bovine serum. d Fold dilution. e Viral entry inhibition control. f  Soluble equivalent concentration. 3. Results 3.1. Activity against Neisseria gonorrhoeae and VVC-causing organisms Four WHO strains and seven clinical isolates of   N. gon-orrhoeae  were tested for their growth and survival followingexposuretoBasant.Itwasobservedthatataconcentrationof 2%(v/v)andabove,Basantinhibitedthegrowthofallstrainsof   N. gonorrhoeae  susceptible or resistant to two or moreantibiotics (Table 1). The formulation was also active against two ATCC  Candida  strains ( Candida glabrata  and  Candidaalbicans ) as well as against 17 clinical isolates (amongstwhich nine were  C. glabrata , six were  C. albicans  and twowere  Candida tropicalis ). Killing of all the isolates, includ-ing two multidrug-resistant strains of   C. glabrata  and one of  C. albicans , was partial at 1% (v/v) but complete at 2% (v/v)concentration of Basant (Table 1). 3.2. Activity of individual ingredients It was of interest to determine whether the four activeingredients of Basant, namely curcumin, Amla, Aloe veraand saponins, each had antimicrobial action against thepathogens tested with the total formulation. It was observedthat saponins from  S. mukorossi  inhibited the growth of allmicroorganismstested;theirmainactionwasagainst  N.gon-orrhoeae  and  C. glabrata . Curcumin inhibited the growth of   N. gonorrhoeae  and  C. albicans  but showed marginal inhibi-tion of   C. glabrata . It had no action against  C. krusei . Aloevera had marginal antimicrobial activity against  C. albicans buthadhighinhibitoryactionagainstHIV(Table2)andHPV (Table 3). Amla extracts acted on  N. gonorrhoeae  but not onthe three species of   Candida . It had high inhibitory action ontransduction of HPV-16 in HeLa cells (Table 3). The combi- nation of these ingredients, with pharmacopoeially approvedexcipients,extendedthespectrumofantimicrobialandantivi-ralactionoftheformulation.Moreover,thezoneofinhibition Table 3Inhibition of HPV-16 infectivity by Basant and its ingredientsCompound Cytotoxicity(  g/mL) a Infectivity inhibition(EC 50 ) (  g/mL)Basant 300 dil. b ∼ 2700 dil. c Aloe >100 dil. 1800 dil.Amla 300 4Curcumin 5 >5Saponins 100 >100HPV, Human papillomavirus; EC 50 , 50% effective concentration. a Cytotoxicity is defined as a >50% decrease in WST-1 substrate (Roche)development. b Fold dilution. c Inhibition curve could not be fitted. obtained with the total formulation was significantly largerfor all microorganisms than that obtained with any singleingredient (data not shown). 3.3. Anti-HIV activity Results in Table 4 show that in CEM-GFP reporter T-cell line and P4 (Hela-CD4-LTR-  Gal) cell line, 50% inhibitionof NL4.3 HIV virus was obtained at 1:20000 dilution of Basant. Near total inhibition of the virus was observed withBasant diluted 1000 times. The 50% effective concentration Table 4Percent inhibition of virus production by Basant in HIV-1 NL4.3-infectedCEM-GFP and P4 cellsBasant dilution % inhibition by p24 assay  a CEM-GFP cells P4 cells1:1000 98 991:5000 89 N.C.1:10000 81 641:20000 51 531:40000 22 451:80000 N.C. 32HIV, human immunodeficiency virus; N.C, not calculated. a Results presented are the mean of three independent experiments.  184  G.P. Talwar et al. / International Journal of Antimicrobial Agents 32 (2008) 180–185 (EC 50 ) of Basant for inhibiting entry of HIV-1 (IIIB) virus inP4-CCR5 cells was 1:2492. On testing the individual ingre-dients of Basant, it was found that Aloe vera had the highestinhibitory activity and selectivity index (>1000), with littleor no cytotoxicity (EC 50  =1:2126) (Table 2). 3.4. Action on HPV transduction The potential inhibitory effect of Basant and its com-ponents on transduction of HPV-16 using a standard cellculture-based pseudovirus infection system [9] was studied.Results in Table 3 show that Aloe completely inhibited theinfectivity of the HPV-16 pseudovirus, with negligible cyto-toxicity, at a dilution of 1:100. Its EC 50  was at a dilutionof 1:1800. Amla extracts had an EC 50  of 4  g/mL, nearly100 times lower than its cytotoxic dose. These results showthat Aloe and Amla exercise potent action on transduction of HPV-16 in HeLa cells at concentrations far below those usedin Basant. 3.5. Safety of Basant  Basant did not cause any abnormalities in the structuralintegrity of the vaginal epithelium of mature NZW rabbitsand did not have any systemic effect as evidenced by bloodchemistry and haematology after 7 consecutive days use ortwice-daily application for 3 weeks in the vagina. No sig-nificant change was found in cytokines (interleukin (IL)-1b,IL-2, IL-6, IL-10, IL-12, tumour necrosis factor-alpha andgranulocyte–macrophage colony-stimulating factor) of rab-bitsafterintravaginaladministrationofBasant(dataavailablefrom NIRRH). 4. Discussion Two important requisites of a microbicide, in addition toefficacy, are: (i) safety and lack of damage to the mucosalvaginal epithelium on long-term use; and (ii) acceptabilitynot only to women but also to the male partner for voluntaryuse.ThesafetyofBasantisevidentfromthetoxicologystudiescarriedoutonrabbitvagina,whichistheexperimentalmodelrecommended by the FDA for vaginal use products. Dailyapplication for 7 days or twice-daily application for 3 weeksdid not cause any abnormality to the vaginal mucosa.Aloeveraisextensivelyemployedincosmeticsandinfor-mulationsforskin.Ithasasoothingeffectonmucosallinings.Aloe gel is reported to have wound healing properties [11].It has a considerable inhibitory effect on HIV (Table 2) and HPV(Table3).Numerousscientificstudieshaveshownanti- inflammatory and anticancer properties of curcumin [12,13].Theseattributesaredesirableinamicrobicidetobeappliedinthe vagina and cervix. Curcumin is highly safe and its intakeat as high as 8g every day for 3 months by humans causes noill effects [14].An attractive feature of our Basant formulation isthat, in contrast to many others currently in develop-ment where a single active compound is used, we haveemployed a combination of compounds. The combina-tion has widened the spectrum of infections inhibited byBasant.Basant demonstrated considerable action not only against C. albicans  but also against  C. glabrata  and  C. tropicalis (Table 1). Although  C. albicans  has been found to be respon-sible for the majority of symptomatic episodes of VVC,casesofsporadicandrecurrentVVCcausedbynon- albicans species are increasing [3]. Basant was effective against all standard strains and clinical isolates of   C. albicans  and  C.glabrata  tested, including those resistant to azole drugs andamphotericin B. Besides  Candida , Basant has also shownpre- and post-infection inhibitory effect on  C. trachomatis [15].The data communicated in this paper show a high viruci-dal action of Basant against HIV (Tables 2 and 4). Clinical trials in high-risk patients are intended to assess its ability toprevent the transmission of HIV/AIDS by the heterosexualroute.HPV, which is transmitted sexually, is the second largestcause of female cancer worldwide [2]. Basant is amongst the few microbicides tested for their action against HPV.With the approval of the Drugs Controller General of IndiaandInstitutionalEthicsCommittees,multicentrePhaseIIb trials are in progress at the Institute of Cytologyand Preventive Oncology, NOIDA, India, at Chittaran- jan National Cancer Institute, Kolkata, India, and at TataMemorial Hospital, Mumbai, India, for elimination of HPV-16 and -18 by Basant in the early stages of cervicaldysplasia.Basant is a cream with a pleasant fragrance that is sooth-ing to the skin, free from local and systemic side effectsand endowed with a wide spectrum of activity against bac-teria, fungi and viruses infecting the female genital tract. Ithas utility for the regression of vaginal infections as estab-lished by clinical trials on another polyherbal formulationPraneem [16]. By virtue of its microbicidal activity and viral entryinhibitionproperties,itcouldalsobeusefulforprevent-ing the transmission of the tested pathogens by the vaginalroute. Acknowledgments The authors thank Dr Vinod Arora, Dr Simrata Bedi andMsNeetaGuptaofRanbaxyLaboratoriesfortheirassistanceingettingalargebatchofBasantmadeunderGMPconditionsfrom M/s Gufic Biosciences Ltd. They also thank CONRADfor evaluating Basant against HIV-1. Funding : This study was supported by a research grantfromtheDepartmentofBiotechnology,GovernmentofIndia. Competing interests : None declared.  Ethical approval : Not required.
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