WO2018051363A1

WO2018051363A1 - Antifungal formulations from medicinal plants for vaginal candidiasis

Info

Publication number: WO2018051363A1
Application number: PCT/IN2017/050393
Authority: WO
Inventors: Dr Suja Kizhiyedath POLACHIRA; Anu Geetha VIDYA; Reshmi Nair Rajagopal JAYALEKHA
Original assignee: Hll Lifecare Limited
Priority date: 2016-09-19
Filing date: 2017-09-12
Publication date: 2018-03-22

Abstract

The present invention relates to an antifungal herbal formulation for treatment of vaginal candidiasis comprising three medicinal plant extracts namely Curcuma longa, Ventilago madaraspetana and Terminalia chebula. The herbal formulation of the present invention is effective against three different species of Candida namely Candida albicans (ATCC No.90028), Candida tropicalis and Candida glabrata (both are clinical strains). The present invention provides an herbal formulation useful as a vaginal gel, cream, vaginal film and pessary.

Description

ANTIFUNGAL FORMULATIONS FROM MEDICINAL PLANTS FOR VAGINAL CANDIDIASIS

FIELD OF INVENTION: The present invention generally relates to methods and formulations for treating vaginal candidiasis. More particularly, it relates to an antifungal vaginal gel formulation comprising hydro alcoholic extracts of three medicinal plants for treatment of vaginal candidiasis.

BACKGROUND OF THE INVENTION:

Vaginal candidiasis is caused by excessive growth of yeast in female vagina which results in irritation, vaginal itching, burning with urination, white and thick vaginal discharge etc. Vaginal yeast infection generally caused by the yeast species Candida albicans. Candida albicans also commonly present in human mouth, throat and intestinal tract. About 20% of non-pregnant women aged between 15 to 55 harbours Candida albicans in their vagina. Most of them have no symptoms and it is harmless to them. Only overgrowth of Candida albicans causes a heavy white curd-like vaginal discharge, a burning sensation in the vagina etc. Younger and older people are less common for vaginal infections due to lack of oestrogen in the vagina. Oestrogen causes the lining of the vagina to mature and to contain glycogen, a substrate on which Candida albicans survives.

Overgrowth of Candida albicans most commonly occurs in pregnant women, people taking higher dose of combined oral contraceptive pill and oestrogen-based hormone replacement therapy, people under broad spectrum antibiotics, diabetes patients, HIV and Iron deficiency anaemia patients. However, 3 out of 4 women will experience vaginal infection at one point in their lives.

There are treatments available for vaginal candidiasis based on the severity. Oral agents and intravaginal agents are available to treat the infections. Generally, azole based antifungal drugs are used in the treatment of vaginal infections. However, they have their own side effects and also lead to drug resistance. There are a few natural therapies available for treatment of vaginal infections. United States Patent 7,014,873 to West, et al. entitled "Method and formulation for treating candidiasis using morinda citrifolia" deals with nutraceutical formulations comprising one or more of a processed Morinda citrifolia fruit juice, puree juice, oil or oil extract, dietary fiber, alcohol extract, etc., for inhibiting and preventing the overgrowth of Candida fungus and for treating candidiasis and its associated symptoms.

Reference is made to publication by Sudha Salhan and Nivedita Sarda, Department of Obstetrics and Gynecology, Safdar Jang Hospital, New Delhi, India and Kala Suhas Kulkarni Medical Advisor, R&D Center, The Himalaya Drug Company, Bangalore, India titled "Efficacy of V-Gel, a polyherbal vaginal gel, in the management of vaginitis" for Indian Journal of Clinical Practice (2002): (13), 7, 25-28 deals with vaginal gel comprising active ingredients such as Triphala and Curcuma longa for treating vaginitis.

Another reference made to publication by Umadevi, K. and Asha Swarup, Department of Obs and Gynae, M.S. Ramaiah Medical Teaching Hospital & College, Gokula Extension, Bangalore, India titled "Efficacy of PD-959 Gel in Abnormal Vaginal Discharge" for Asian Journal of Obs. & Gynae Practice (Dec.98 - Feb.99): (3), 1, 68 relates to V-gel comprising active ingredients like Triphala and Curcuma longa for treatment of abnormal vaginal discharge. Though many drugs are available for treatment of vaginal infections but with drug resistance and acute toxicity, there is still need for herbal formulations that particularly useful in the treatment of vaginal candidiasis, with complete cure and negligible side effects.

The present invention provides an effective treatment for vaginal candidiasis with fewer side effects using a novel antifungal vaginal gel formulation comprising herbal extracts and its method of preparation.

SUMMARY OF THE INVENTION An object of the present invention is to treat vaginal candidiasis caused by species namely Candida albicans, Candida glabrata, and Candida tropicalis using an antifungal herbal formulation comprising medicinal plant extracts.

The present invention relates to an antifungal herbal formulation comprising three medicinal plant extracts namely Curcuma longa, Ventilago madaraspetana and Terminalia chebula. The present herbal formulation is effective against three different species of Candida namely Candida albicans (ATCC No.90028), Candida tropicalis and Candida glabrata (both are clinical strains). The present invention provides an herbal formulation useful as a vaginal gel, the composition comprising at least three plant extracts in the form of oil or powder or mixtures thereof.

According to the present invention, the herbal formulation can also be made in the form of cream, vaginal film and pessary The herbal formulation is also effective against candidiasis (caused by all species) with no toxicity and no drug resistance.

These objectives and advantages of the invention will become more evident from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS:

Fig. 1 shows antifungal MIC value of plant extract against Candida spp. at 48 hours; and Fig. 2 shows cytotoxicity of plant extract against HeLa Cell Lines at 48 hours. DETAILED DESCRIPTION OF THE INVENTION:

The present invention relates to an antifungal herbal formulation comprising at least three medicinal plants for treatment of vaginal candidiasis. The invention provides an herbal formulation useful as a vaginal gel, cream, vaginal film and pessary. The herbal formulation of the present invention comprises three medicinal plant extracts namely Curcuma longa, Ventilago madaraspetana and Terminalia chebula and is effective against three different species of Candida namely Candida albicans (ATCC No.90028), Candida tropicalis and Candida glabrata (both are clinical strains). The composition comprising at least two or more plant extracts in the form of oil or powder or mixtures thereof, the plant extracts being selected from the group consisting of Curcuma longa hydro alcoholic extract; Ventilago madaraspetana and Terminalia chebula hydro alcoholic extract. The invention is further demonstrated with the following illustrated examples.

Example 1

Preparation of plant extracts for herbal formulation

In the present invention, the antifungal herbal gel formulation is prepared by using extracts of medicinal plants namely Curcuma longa, Ventilago madaraspetana and Terminalia chebula. According to the invention, 80% of hydro alcoholic extract of the plants are taken for the antifungal studies. The plant extracts are prepared using the following method. The plant materials are cleaned, dried under shade and powdered. Then 24gram of the powder is taken and subjected to pressurized sequential extraction using Accelerated Solvent Extractor (ASE 150, Dionex Inc., USA). The material is mixed with diatomaceous earth at 4: 1 ratio. The mixture is placed into a sample cell (100ml) and loaded onto the ASE 150 system. Extraction is performed using 80% ethanol under pressure (1500 psi) at 60°Cwith a flush volume of 60% using 2 static cycles. The solvent is then evaporated in a rotary evaporator (Buchi, Switzerland). The dried extracts are weighed and dissolved in DMSO to obtain the appropriate concentrations and were assayed for antifungal activity.

Antifungal Studies of the plant extract

Antifungal activity of the plant extracts (prepared in Example 1) are tested by Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard- second edition by CLSI. The MIC (Minimum Inhibitory Concentration) values are calculated. Cytotoxicity of these extracts is also tested. The extract and formulation is found to be effective against three different species of Candida namely Candida albicans (ATCC No.90028), Candida tropicalis and Candida glabrata (both are clinical strains).

Results of Antifungal Assay: The growth inhibition was measured at different concentrations (12.5, 25, 50, 100, 200 μg/ml) in DMSO. The results are shown in Table. 1 and Fig. 1. The ethanolic extract of Terminalia chebula was found to be active at a concentration of 25μg/ml whereas Curcuma longa and Ventilago madaraspetana showed activity at 12^g/ml against C. albicans. The MIC values of the plant extracts against C. tropicalis were reported as 50μg/ml for Terminalia chebula and Ventilago madaraspetana, 25μg/ml for Curcuma longa. Similarly antifungal activity for C. glabrata was also examined and was reported as 25μg/ml for Curcuma longa and Terminalia chebula and 50μg/ml for Ventilago madaraspetana. The growth control showed significant growth of the organisms. Table. 1 shows antifungal MIC ^g/ml) of plant extract against Candida spp. at 48 hours

">" implies no inhibition.

Cytotoxicity Studies:

Cytotoxicity studies of these plant extracts were carried out against HeLa lines by 3-[4, 5- dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay. Stock solutions of the extract were prepared in 100% DMSO and used for the viability assay such that the final amount of DMSO did not exceed 0.1% Cells were seeded at cell density of 10,000 cells per well. Various concentrations of the extract and compounds in RPMI medium containing 10% fetal bovine serum (FBS) were added to the 96-well plate. Control wells did not contain the extract or any compound. The cells were incubated at 37°C in the presence of 5% C02. After 24h, 100 μΐ MTT (2.5 mg/ml stock) dissolved in RPMI was added and again incubated at 37 °C. After two hours, 100 μΐ of lysis buffer (20% SDS in 50% DMF) was added and incubated at 37 °C. As a measure of cell viability, the conversion of MTT into a tetrazolium salt was determined by measuring the absorbance at 570 nm. The results showed that Ventilago madaraspetana was having highest cytotoxicity and Terminalia chebula had the least cytotoxicity. The results are shown in Table. 2 and Fig. 2.

Table. 2 shows cytotoxicity of plant extract against HeLa cell lines at 48 hours.

Example 2

Method of preparation of aqueous gel formulation for vaginal application (100 gram)

Table. 3 shows the amount of ingredient in the formulation. Ingredient Name %W/W

A 1 Carbopol 974P NF Polymer 2.00

2 Deionized water 78.60

B 3 Sodium methylparaben 0.18

4 Sodium Propylparaben 0.02

5 Deionized water 4.00

C 6 Glycerin,USP 15.00

7 Triethanalomine 0.2 gram

D 8 T. chebula, C. longa, V. madaraspetana 400 milligram

(plant extracts prepared in example 1) each

1. Disperse carbopol 974 NF polymer in water and mix thoroughly (part A).

2. Dissolve the sodium methyl paraben and sodium propyl paraben (part B) in water and add this salt solution to the part A.

3. Add glycerine and Triethanalomine (part C) to the part A mixture and mix thoroughly.

a. Gel base is prepared by combining A+B+C

b. Gel formulation is prepared in such a way that 4 milligram of each plant extract is present in lgram of gel. (A+B+C +D)

The prepared antifungal herbal formulation is effective against Candida species and it has no toxicity and no drug resistance. Thus, the present invention provides an effective antifungal herbal formulation as vaginal gel for the treatment of vaginal candidiasis.

Although the foregoing invention has been described in some detail by way of examples for purposes of clarity and understanding, it will be apparent to those of ordinary skill in the art in light of the disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WE CLAIM: 200

1. An antifungal herbal formulation for treatment of vaginal candidiasis comprising hydroalcoholic extracts of medicinal plants consists of Curcuma longa, Ventilago madaraspetana and Terminalia chebula, wherein said hydroalocoholic extracts of medicinal plants present in an amount of about 80% by weight of the total.

205

2. The formulation according to claim 1, effective against Candida species selected from the group consisting of Candida albicans, Candida tropicalis and Candida glabrata.

3. The formulation according to claim 1, is in a form of a gel, cream, vaginal film or 21 0 pessary.

4. The formulation according to claim 1, is formulated for vaginal application.

5. The formulation according to claim 1, wherein said each plant extract is present in an 21 5 amount of 1 to 5 milligram per 1 gram of said formulation.

6. A method for treating vaginal candidiasis in a subject in need thereof, the method comprising the step of vaginally administering an antifungal herbal formulation of claim 1 to said subject.

220

7. The method according to claim 6, wherein said antifungal herbal formulation is administered in a form of a gel, cream, vaginal film or pessary.

8. A process for the preparation of antifungal herbal formulation, as substantially described

225 herein with reference to forgoing Examples 1 and 2.