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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20

Antimicrobial activity of plant extracts against sexually transmitted pathogens a

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b

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Nutan Jadhav , Sangeeta Kulkarni , Arati Mane , Roshan Kulkarni , d

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Aparna Palshetker , Kamalinder Singh , Swati Joshi , Arun Risbud

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& Smita Kulkarni a

Department of Virology, National AIDS Research Institute, Pune, India b

Department of Microbiology, National AIDS Research Institute, Pune, India c

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Division of Organic Chemistry, CSIR-National Chemical Laboratory, Pune, India d

SNDT Women's University, C.U. Shah College of Pharmacy, Mumbai, India Published online: 27 Nov 2014.

To cite this article: Nutan Jadhav, Sangeeta Kulkarni, Arati Mane, Roshan Kulkarni, Aparna Palshetker, Kamalinder Singh, Swati Joshi, Arun Risbud & Smita Kulkarni (2014): Antimicrobial activity of plant extracts against sexually transmitted pathogens, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.983919 To link to this article: http://dx.doi.org/10.1080/14786419.2014.983919

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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.983919

SHORT COMMUNICATION Antimicrobial activity of plant extracts against sexually transmitted pathogens

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Nutan Jadhava, Sangeeta Kulkarnib, Arati Maneb, Roshan Kulkarnic, Aparna Palshetkerd, Kamalinder Singhd, Swati Joshic, Arun Risbudb and Smita Kulkarnia* a

Department of Virology, National AIDS Research Institute, Pune, India; bDepartment of Microbiology, National AIDS Research Institute, Pune, India; cDivision of Organic Chemistry, CSIR-National Chemical Laboratory, Pune, India; dSNDT Women’s University, C.U. Shah College of Pharmacy, Mumbai, India (Received 21 July 2014; final version received 29 October 2014)

Comprehensive management of sexually transmitted infections (STIs) using vaginal or rectal microbicide-based intervention is one of the strategies for prevention of HIV infection. Herbal products have been used for treating STIs traditionally. Herein, we present in vitro activity of 10 plant extracts and their 34 fractions against three sexually transmitted/reproductive tract pathogens – Neisseria gonorrhoeae, Haemophilus ducreyi and Candida albicans. The plant parts were selected; the extracts/fractions were prepared and screened by disc diffusion method. The minimum inhibitory and minimum cidal concentrations were determined. The qualitative phytochemical analysis of selected extracts/fractions showing activity was performed. Of the extracts/ fractions tested, three inhibited C. albicans, ten inhibited N. gonorrhoeae and five inhibited H. ducreyi growth. Our study demonstrated that Terminalia paniculata Roth. extracts/fractions inhibited growth of all three organisms. The ethyl acetate fraction of Syzygium cumini Linn. and Bridelia retusa (L.) Spreng. extracts was found to inhibit N. gonorrhoeae at lowest concentrations. Keywords: anti-STI activity; Candida albicans; Haemophilus ducreyi; microbicide; Neisseria gonorrhoeae; plant extracts/fractions

1. Introduction Sexually transmitted infections (STIs) and reproductive tract infections (RTIs) continue to be a global public health concern (WHO 2011). Genital herpes, gonorrhoea, trichomoniasis, candidiais and bacterial vaginosis dominate the list of commonly encountered STIs/RTIs. It is known that individuals with STIs/RTIs have a significantly higher chance of acquiring and

*Corresponding author. Email: [email protected] q 2014 Taylor & Francis

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transmitting HIV (Fleming & Wasserheit 1999). Furthermore, STIs/RTIs may lead to complications such as chronic infection, infertility, still-births, pre-term delivery and congenital anomalies, emphasising the need to develop efficacious biomedical interventions for controlling STIs (Robyn & Workowsk 2014). Approaches for controlling STIs comprise – syndromic management, improving the clinical management of STIs, availability of effective and affordable drugs, accessible and acceptable health services and behavioural interventions for preventing new infections (Vuylsteke 2004). As the emergence of drug resistance in STI/RTI organisms (Department of AIDS Control 2014) and potential side effects demands the discovery of newer drugs, the exploration of newer antimicrobial substances from natural sources may serve as promising alternatives. Traditional medicine has a long history of use worldwide. A large number of herbal agents are available for the symptomatic treatment of STIs, suggesting that herbs can be used as a source for developing new microbicide candidates. Several herbal derivatives have been reported to possess activity against STIs including HIV (Vermani & Garg 2002). Polyphenon E ointment, a proprietary extract of green tea, has been approved by the FDA as a topical treatment for genital warts (Tyring 2012). A polyherbal vaginal tablet Praneemw was found to be safe and cleared human papilloma virus infection in 30 days (Shukla et al. 2009). With a mandate of exploring Indian medicinal plants as microbicide candidates, the study was undertaken to evaluate the activity of plant extracts/fractions against STIs/RTIs causing pathogens using in vitro methods. Herein, we present the data on 10 plant extracts and their 34 fractions against Candida albicans, Neisseria gonorrhoeae and Haemophilus ducreyi, causing candidiasis, gonorrhoea and chancroid which are considered as important cofactors in HIV transmission (Fleming & Wasserheit 1999; Hester & Kennedy 2003). 2. Results and discussion The details of plants selected, identified and authenticated by the Botanical Survey of India, Western Center, Pune, India along with the voucher specimen numbers are mentioned in Table S1. The phytochemical analyses of the extracts/fractions are given in Table S2. It was observed that Terminalia paniculata (TP), Terminalia crenulata (TC), Cuscuta reflexa (CR), Bridelia retusa (BR) and Syzygium cumini (SC) plant extracts/fractions inhibited growth of either of the organisms by agar disc diffusion (ADD) method (Table 1). The inhibition of microbial growth by some representative extracts/fractions is shown in Figure S1. Extracts/ fractions of Polygonum glabrum, Rhus mysurensis, Cassytha filiformis, Polygonum chinesis and Scutia myrtina did not show activity against any of the organisms tested. An important feature of the study is the evaluation of activity against more number of well characterised C. albicans and N. gonorrhoeae strains, compared with earlier studies reporting activity against single strain (Tatiya et al. 2011; Kalita & Saikia 2012; Mohamed et al. 2013). The minimum inhibitory concentration (MIC) and minimum cidal concentration (MCC) of the extracts/fractions showing activity against C. albicans were determined. The MIC and MCC of the two extracts (TP and TC) and one fraction (TCF-3) for sensitive as well as resistant C. albicans strains did not differ and were found to be 312 mg/mL (data not shown) and 1250 mg/mL (Table 1), respectively [MIC of fluconazole: 0.25 – 0.5 mg/mL (sensitive strains), . 32 mg/mL (resistant strain), dimethyl sulfoxide (DMSO): 1250 mg/mL]. The MIC of N. gonorrhoeae could not be determined due to the inability of the organism to grow in broth. The MCC for the sensitive and resistant strains of N. gonorrhoeae differed. The BR extract (16.25 – 312.5 mg/mL) and fraction SCF-4 (39.6 –1250 mg/mL) were found to have maximum activity, followed by TPF-1 and SCF-2. Some fractions were found to have more activity than their crude extracts, e.g. fractions TCF-3, CRF-1 and CRF-2 inhibited all the four strains of N. gonorrhoeae but the respective extracts inhibited only two strains. Likewise, some extracts did not show activity but their

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Table 1. Determination of anti-microbial activity. C. albicans ATCC Botanical name

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T. paniculata Roth

T. crenulata Roth

C. reflexa Roxb

Code no TP

Method ADD

90028 (Fu-S)

N. gonorrhoeae

Lab isolate 71 (Fu-S)

68 (Fu-R)

11(^ 2.6) 10(^ 0.9) 10(^ 2.0)

ATCC

Lab isolate

H. ducreyi ATCC

49226 (P-S)

49926 (P-R)

1511 (P-S)

1562 (P-R)

33921 (T-S)

NA

NA

NA

NA

NA

MCC TPF-1 ADD MCC TPF-2 ADD MCC TC ADD

1250 1250 1250 ND ND ND ND NA NA NA 10(^ 0.5) 8(^ 0.6) 10(^ 0.8) 10(^ 1.2) ND ND ND 312.5 312.5 312.5 156 NA NA NA 10(^ 0.5) 10(^ 0.6) 10(^ 1.6) 10(^ 1.4) ND ND ND 1250 2500 2500 2500 15(^ 2.4) 10(^ 1.5) 10(^ 1.1) NA 8(^ 0.6) 8(^ 0.6) NA

ND 8(^ 0.7) ND NA ND NA

MCC TCF-3 ADD MCC CR ADD

1250 1250 1250 ND 2500 625 ND 11(^ 2.2) 10(^ 0.9) 10(^ 2.0) 10(^ 1.2) 10(^ 0.5) 11(^ 0.9) 10(^ 0.4) 1250 1250 1250 312.5 1250 312.5 625 NA NA NA NA 7 (^ 6.5) 7 (^ 0.6) NA

ND NA ND NA

MCC CRF-1 ADD MCC CRF-2 ADD MCC B. retusa (L.) BR ADD Spreng MCC S. cumini Linn SCF-2 ADD MCC SCF-4 ADD MCC Positive ADD control MCC Solvent control ADD (DMSO) MCC

ND NA ND NA ND NA

ND NA ND NA ND NA

ND NA ND NA ND NA

ND 9(^ 0.7) 1250 9(^ 1.0) 5000 9(^ 0.6)

2500 1250 10(^ 0.8) 11(^ 1.2) 1250 1250 10(^ 1.0) 11(^ 1.2) 5000 1250 8(^ 0.44) 9(^ 0.87)

ND 9(^ 0.7) 1250 9(^ 0.7) 2500 NA

ND 8(^ 0.3) ND 8(^ 0.5) ND 8(^ 0.5)

ND ND NA NA ND ND NA NA ND ND 36(^ 2.0) 33(^ 3.0)

ND NA ND NA ND NA

312.5 16.25 156 ND 10(^ 1.2) 7(^ 1.0) 10(^ 0.5) 9(^ 0.5) 312.5 312.5 312.5 312.5 10(^ 0.6) 7(^ 0.5) 10(^ 0.7) 9(^ 0.8) 39.6 1250 39.6 39.6 41(^ 1.2) 10(^ 3.4) 37(^ 1.1) 13(^ 1.4)

ND 8(^ 0.7) ND NA ND 36(^ 0.6)

. 23 NA

. 32 NA

. 32 NA

10U NA

. 100U NA

100U NA

. 100U NA

ND NA

2500

2500

2500

2500

2500

5000

2500

ND

Note: ADD, agar disc diffusion: mean of zone inhibition in mm (^SD); MCC, minimum cidal concentration: is the lowest concentration of a drug required to kill a microorganism (mg/mL). Assays carried out in triplicate on three separate occasions. P-S, penicillin sensitive; P-R, penicillin resistant; T-S, tetracycline sensitive; Fu-S, fluconazole sensitive; FuR, fluconazole resistant; NA, no activity; ND, not done.

fractions did, e.g. TP and SC extracts did not inhibit the growth of N. gonorrhoeae; however, their fractions (TPF-1, TPF-2, SCF-2 and SCF-4) inhibited the growth. Similarly, fractions TPF1, CRF-1, CRF-2 and SCF-2 inhibited the growth of H. ducreyi but the extracts TP, CR and SC did not inhibit (Table 1). This could probably be attributed to the separation of active constituents during fractionation. The phytochemicals are known active components of the plant extracts, responsible for various activities (Khandelwal 2004). Comparison of the bioactivity with the phytochemical analyses revealed that the activity was higher in the extracts/fractions where flavonoids were present and was further enhanced in the presence of tannins (Table S2). Furthermore, it was observed that the anti-gonococcal activity was mainly due to tannins, alkaloids and/or flavonoids, as observed in BR and SCF-4. It was also noted that the other phytochemicals such as glycosides, phytosterols and triterpenes enhanced the activity. Our findings are in corroboration with the published literature which suggests that majority of the antimicrobial activities are attributed to the tannins and flavonoids which complex with the extracellular and soluble proteins of microbial cell wall and membrane-bound enzymes (Tatiya et al. 2011). Selected plant species are popular medicinal plants in Aryuveda, which have been claimed to have several medicinal uses (Table S1). However, to our knowledge, most of them were not

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screened for activity against sexually transmitted pathogens. Our study for the first time reports the anti-STI/RTI activity of fruits of T. paniculata against all the three organisms tested and activity of the fruits of T. crenulata against C. albicans and N. gonorrhoeae, indicating the potential use of Terminalia species in microbicide development. Leaves of S. cumini have been used in folk medicine. One of the reports shows that the S. cumini leaf extract has shown activity against N. gonorrhoeae (Mondal et al. 2011) at higher concentration (500 mg/mL); however in our study, the extract was found to inhibit four gonococcal strains at a concentration of 200 mg/disc. Studies by Kalita and Saikia (2012) and Tatiya et al. (2011) have shown that the stem bark extract of C. reflexa and B. retusa are reported to have activity against C. albicans ($ 500 mg/mL). Contrary to these reports, we did not observe activity against C. albicans; however, activity against N. gonorrhoeae and H. ducreyi was demonstrated at a concentration of 200 mg/disc. To the best of our knowledge, we report here the inhibitory potential of C. reflexa aerial parts and S. cumini leaf extract against H. ducreyi for the first time. Earlier study reports that decoction of roots and leaves of S. myrtina are traditionally used for treating gonococcal infections (Ramanathan et al. 2011). Also, previous reports demonstrate activity of P. glabrum, P. chinesis and C. filiformis plant extracts against C. albicans (Maharajan et al. 2012; Adonu et al. 2013; Meera et al. 2013). However, in our study, the extracts of these plants did not show activity against N. gonorrhoeae and C. albicans. This may be attributed to the variation in the area and time of the plant collection and the extraction process.

3. Conclusion To summarise, our data reveal that the plant extracts of T. paniculata, T. crenulata, C. reflexa, B. retusa and S. cumini have activity against STIs/RTIs causing pathogens and can be used as potential sources for the development of anti-STI agents. Further efforts in purifying the active principles from these extracts might help in improving their bioactivity profile and would yield promising leads that can be further used in combination for the development of more effective microbicides.

Supplementary material Experimental details relating to this paper are available online, alongside Tables S1 and S2 and Figure S1.

Acknowledgements Authors acknowledge Director, NARI, Pune; Director, NCL, Pune; Principal, CUSCP, Mumbai; Dr S. Sinha, Advisor DBT and Dr N. Chandhiok, Sr DDG, ICMR for extending their support. Authors also appreciate the help of the staff members of the Virology and Microbiology Laboratories.

Funding This work was supported by the Department of Biotechnology (DBT), [grant number BT/PR7965/ Med/14/1203/2006] and the Indian Council of Medical Research (ICMR) [grant number 5/7/180/2006RHN] under the HIV/AIDS and Microbicide programme.

Conflict of interest The authors have no conflicts of interest to disclose.

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Antimicrobial activity of plant extracts against sexually transmitted pathogens.

Comprehensive management of sexually transmitted infections (STIs) using vaginal or rectal microbicide-based intervention is one of the strategies for...
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