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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 10  |  Issue : 4  |  Page : 190-194  

Antimicrobial activity of a novel polyherbal combination for the treatment of vaginal infection


1 Department of Pharmacognosy, B. S. Anangpuria Institute of Pharmacy, Faridabad, Haryana, India
2 Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India
3 Department of Pharmaceutics, NIET Pharmacy Institute, Greater Noida, Uttar Pradesh, India

Date of Web Publication1-Oct-2019

Correspondence Address:
Gaurav K Jain
Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/japtr.JAPTR_3_19

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  Abstract 

The present study evaluated the antimicrobial activity of Azadirachta indica (AI), Cichorium intybus (CI), and Trigonella foenum-graecum (TFG) against bacterial and fungal pathogens responsible for the vaginal infections. The AI, CI, and TFG were selected to include antimicrobial and antifungal action against wide range of microbes. The different extracts of the herbs were evaluated for antibacterial and antifungal activity by well diffusion assays. Based on the results, the combination was selected and evaluated, “polyherbal antimicrobial (PHA).” The developed PHA extract demonstrated synergistic broad-spectrum antimicrobial activities including antibacterial and antifungal activity (minimum inhibition concentration: 5–7 mg/ml).

Keywords: Antimicrobial, Azadirachta indica, Cichorium intybus, polyherbal combination, Trigonella foenum-graecum


How to cite this article:
Yadav SK, Jain GK, Mazumder A, Khar RK. Antimicrobial activity of a novel polyherbal combination for the treatment of vaginal infection. J Adv Pharm Technol Res 2019;10:190-4

How to cite this URL:
Yadav SK, Jain GK, Mazumder A, Khar RK. Antimicrobial activity of a novel polyherbal combination for the treatment of vaginal infection. J Adv Pharm Technol Res [serial online] 2019 [cited 2020 Nov 28];10:190-4. Available from: https://www.japtr.org/text.asp?2019/10/4/190/268452




  Introduction Top


Vaginal infection is a common problem among women, and about 75% of women will have at least one episode of vaginitis during their lifetime. Vaginal tract can be infected by diverse pathogens, resulting in diverse diseases such as urinary tract infections, bacterial vaginosis, vulvovaginal candidiasis, vaginitis, trichomoniasis, and sexually transmitted diseases.[1] Various antimicrobial compounds have been studied to treat these vaginal infections including antibacterial, antifungal, antiparasitic, and antiviral agents. Most of these agents used to date for treating vaginal infections have myriads of side effects and emergence of problem of drug resistance. Since ancient times, herbal medicines have served as a platform for the prevention and cure of diseases, and to date, many more constituents of these natural sources are yet to be explored.[2],[3],[4] There is a pressing need to develop a natural formulation, which can act against the microorganisms causing vaginal infections.[5] Over the past decade, interest in herbal medicine has increased tremendously. According to the World Health Organization, 60%–80% of population in developing countries depends essentially on plants for primary health-care needs.[6] The resurgence of herbal medicines has increased the international trade enormously, and the global herbal medicine market is expected to reach about USD 117 billion by 2024, driven by the rising popularity of herbal medicine compared to conventional drugs. The literature revealed Azadirachta indica (AI),[7] Cichorium intybus (CI),[8] and Trigonella foenum-graecum (TFG)[9] as herbs that possess antibacterial and antifungal activity. Pharmaceutical companies have established renewed concern in exploring plants as a major source for new lead structures and for the development of standardized phytotherapeutics with promising quality, safety, and efficacy.[10] The aim of the present study is to screen out a novel polyherbal combination for antimicrobial activity with the objective to treat infections of the vagina.


  Materials and Methods Top


Plant raw materials

Three herbs, namely AI, CI, and TFG, were selected and procured from Moonlight Traders, Delhi; Asian Traders, Khari Baoli, Delhi; and Green Earth Products, International Exporters, New Delhi, respectively.

Preparation of extracts

The leaves of AI and CI and the seeds of TFG were powdered and extracted with aqueous, alcoholic, and hydroalcoholic solutions prepared using hot extraction method. Soxhlet apparatus was used for carrying out hot extraction process, where temperature was controlled at 75°–80°C for all the three herbs. Various extracts were separately collected and evaporated to dryness under reduced pressure at 40°–45°C using a rotary evaporator. The dried extracts were weighed, and the extraction efficiency (%) was calculated with reference to the air-dried substance. The dried extracts were dissolved in various solvents at varying concentrations, and their comparative antimicrobial activity was evaluated by comparing the zones of inhibition. Chloramphenicol (20, 30, and 40 μg/mL) was used as the standard for Staphylococcus aureus and Streptococcus agalactiae; ciprofloxacin (5, 10, and 15 μg/mL) was used as the standard for  Escherichia More Details coli; and miconazole (15, 20, and 25 μg/mL) was used as the standard for Candida albicans and Aspergillus fumigatus.

Inoculum and culture media

The primary subcultures for each microorganism were procured from authentic sources. The bacterial strains employed were E. coli(Strain No. 8739-8/03/15), S. aureus (Strain No. B-17-DF-EB-IITD), and S. agalactiae (Strain No. T-15-DF-TK-IITD). The fungal strains employed were C. albicans (Strain No. B778-8/03/15) and A. fumigatus (Strain No. F-2/21-DBE3-IITD).

Antimicrobial screening using agar well diffusion method

The medium (25 mL) was poured into presterilized  Petri dish More Detailses and set aside for solidification for 5 h. Cotton swabs charged with 0.1 mL of diluted inoculum (105 cfu/mL) of test microorganisms were inoculated and spread evenly over the surface of agar plates. Uniform sized wells of 8 mm diameter were aseptically punched into the seeded agar with a flamed cork borer to make three holes at equidistant positions on the Petri dishes. In each Petri dish, two out of three holes were filled with 0.2 mL of sample solution with varying concentrations, and the third hole was filled with the standard solution. The solvent blanks were also taken similarly as negative controls.

Incubation

All the plates were incubated at 37°C for 24 h for bacteria cultures and at 28°C for 48 h and 120 h for fungal cultures of C. albicans and A. fumigatus, respectively. The zone of inhibition produced by sample and standard solutions was recorded using sliding calipers and compared for their antimicrobial activity evaluation. The antimicrobial screening study of each sample was carried out in triplicate (n = 3).

Statistical analysis

Statistical analysis of the results for polyherbal formulation was performed using Prism 7 software (GraphPad, USA). Differences were considered to be statistically significant when P < 0.05. The data were presented as mean ± standard deviation and were analyzed by one-way ANOVA.


  Results and Discussion Top


The extraction process for each of the herb was optimized in terms of maximum yield based on solvent system and time duration required for extraction. The percent yields observed for dried extracts of AI, CI, and TFG were found to be 31.19%–37.43%, 21.94%–27.65%, and 18.72%–27.16%, respectively [Table 1]. The data [Table 2], [Table 3], [Table 4] showed that hydroalcoholic and water extracts of AI (30 mg/mL) had comparable antibacterial activity at similar doses against S. aureus, S. agalactiae, and E. coli. The activity was highest for the former extract against S. agalactiae. Hydroalcoholic and ethanol extracts of CI (15 mg/mL) showed the greatest activity against S. aureus, but poor antifungal activity and no activity against E. coli. Water extract of CI showed higher antifungal activity, but no antibacterial activity. Ethyl acetate extract of CI did not show any antifungal activity. Hydroalcoholic and ethanol extracts of TGF showed the highest activity against A. fumigatus and good activity against C. albicans at 6 mg/mL concentration; the hydroalcoholic extract was found to inhibit bacterial growth to a greater extent than the ethanol extract. Based on the results, the hydroalcoholic extracts of AI, CI, and TFG at a concentration of 20 mg/ml, 15 mg/ml, and 5 mg/ml, respectively, were selected to prepare polyherbal antimicrobial (PHA) extract. The PHA extract formed by mixing hydroalcoholic extracts of TFG, CI, and AI in the ratio of 1:3:5 contains 12.5 mg of TFG, 37.5 mg of CI, and 50 mg of AI per 100 mg of PHA extract. The PHA extract was then evaluated for the synergistic antimicrobial activity at four different concentration levels:
Table 1: Percentage yield of various extracts

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Table 2: Results for antimicrobial activity of various extracts of the leaves of Azadirachta indica

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Table 3: Results for antimicrobial activity of various extracts of the leaves of Cichorium intybus

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Table 4: Results for antimicrobial activity of various extracts of the seeds of Trigonella foenum-graecum

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  1. Dose 1: 1% of the actual dose
  2. Dose 2: 10% of the actual dose
  3. Dose 3: 25% of the actual dose
  4. Dose 4: 50% of the actual dose.


PHA extract exhibited synergistic antimicrobial activity, and the extract at 25% of the actual dose (1.25 mg TFG, 3.75 mg CI, and 5.0 mg AI per mL) demonstrated enhanced and broad-spectrum antibacterial and antifungal activity [Table 5]. The microbiological activity of D3 against all the tested microorganisms, except E. coli, was significantly higher (P < 0.05) than D2. However, the microbiological activity of D3 is not significantly different (P > 0.05) from that of D4. Representative photographs for comparative zones of inhibition, as observed for PHA extract, are shown in [Figure 1]. This PHA extract could further be developed into the formulation for effective application and treatment of local vaginal infections.
Table 5: Results for antimicrobial activity of polyherbal antimicrobial extract

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Figure 1: Antimicrobial activity of polyherbal antimicrobial extract against (a) Staphylococcus aureus, (b) Streptococcus agalactiae, (c) Escherichia coli, (d) Candida albicans, and (e) Aspergillus fumigatus

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  Conclusion Top


The developed PHA extract using a novel combination of antimicrobial agents from polyherbal sources demonstrated enhanced antibacterial and antifungal activity and could be an effective formulation for the treatment of vaginal infections.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Olowe OA, Makanjuola OB, Olowe R, Adekanle DA. Prevalence of vulvovaginal candidiasis, trichomoniasis and bacterial vaginosis among pregnant women receiving antenatal care in Southwestern Nigeria. Eur J Microbiol Immunol (Bp) 2014;4:193-7.  Back to cited text no. 1
    
2.
Parasuraman S, Thing GS, Dhanaraj SA. Polyherbal formulation: Concept of ayurveda. Pharmacogn Rev 2014;8:73-80.  Back to cited text no. 2
    
3.
Kim KJ, Liu X, Komabayashi T, Jeong SI, Selli S. Natural products for infectious diseases. Evid Based Complement Alternat Med 2016;2016:9459047.  Back to cited text no. 3
    
4.
Mukherjee S. Emerging infectious diseases: Epidemiological perspective. Indian J Dermatol 2017;62:459-67.  Back to cited text no. 4
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5.
Mulu W, Yimer M, Zenebe Y, Abera B. Common causes of vaginal infections and antibiotic susceptibility of aerobic bacterial isolates in women of reproductive age attending at felegehiwot referral hospital, Ethiopia: A cross sectional study. BMC Womens Health 2015;15:42.  Back to cited text no. 5
    
6.
Sachan AK, Vishnoi G, Kumar R. Need of standardization of herbal medicines in modern era. Int J Phytomed 2016;8:300-7.  Back to cited text no. 6
    
7.
Alzohairy MA. Therapeutics role of Azadirachta indica (Neem) and their active constituents in diseases prevention and treatment. Evid Based Complement Alternat Med 2016;2016:7382506.  Back to cited text no. 7
    
8.
Rehman A, Ullah N, Ullah H, Ahmad I. Antibacterial and antifungal study of Cichorium intybus. Asian Pac J Trop Dis 2014;4 Suppl 2:S943-5.  Back to cited text no. 8
    
9.
Omezzine F, Bouaziz M, Daami-Remadi M, Simmonds MSJ, Haouala R. Chemical composition and antifungal activity of Trigonella foenum-graecum L. Varied with plant ploidy level and developmental stage. Arab J Chem 2017;10:S3622-31.  Back to cited text no. 9
    
10.
Herbal Medicine Market Size, Forecast and Trend Analysis, 2014 – 2024. Report ID: 5583962; 2017. p. 70. Available from: https://www.reportbuyer.com/product/5583962/herbal-medicine-market-size-forecast-and-trend-analysis-2014-2024.html. [Last accessed on 2018 Aug 22].  Back to cited text no. 10
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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