AP967A

AP967A - Phytodrug for treating skin fungal infections and methods of preparing and using same.

Info

Publication number: AP967A
Application number: APAP/P/1998/001205A
Authority: AP
Inventors: Charles O N Wambebe; Nkechi M Enwerem; Shingu K Gamaniel; Ibrahim Kolo
Original assignee: United Nations Dev Programme Undp; National Institute For Pharmaceutical Res And Development Niprd
Priority date: 1998-01-05
Filing date: 1998-01-05
Publication date: 2001-05-18

Abstract

A pharmaceutical composition for treating skin fungal infections comprises an extract of Mitra villosus plant leaves, the extract haying beenprepared using an organic solvent such as hexane or methanol. The extract is preferably formulated with an ointment base to produce an ointment.

Description

BACKGROUND OF THE INVENTION

This invention relates to a pharmaceutical composition for treating skin fungal infections comprising an extract of ,· MfTRAOARPUS VILLOSUS and to methods of preparing the extract.

The need to develop new antifungal agents has become relevant in the light of significant toxicity, high cost and non-availability of existing antifungal drugs. In particular, there is a desire on the part of some to develop and commercialize new herbal remedies for dermatomycoses utilizing a blend of existing and new knowledge derived from collaborations between modem scientists and traditional African healers.

AP 00967

Nigeria has a very strong traditional medical practice where the traditional healers play a major role as health care providers in both urban and rural settings. These traditional practitioners are knowledgeable regarding the use of plants and plant extracts for healing purposes. Moreover, there is a large reservoir of potentially medicinal plants in Nigeria. This combination is valuable in the search for new drug remedies based on ethnomedical information.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided a pharmaceutical composition for treating skin fungal infections comprising an extract of Mitra villosus (family Rubiaceae), preferably Mitra villosus plant leaves, the extract having been prepared using an organic solvent.

It is to be noted that the extract is prepared using an organic solvent. However, thereafter, the solvent may be removed by any suitable method to produce a dried powder which may then be formulated as required into a pharmaceutical composition.

The organic solvent used to prepare the extract is preferably selected from hexane, methanol and mixture thereof. A preferred extraction procedure is a hot hexane soxhlet procedure.

According to a second aspect of the invention there is provided an ointment for treating skin fungal infections comprising an extract of Mitra villosus, the extract having been prepared using an organic solvent, and an ointment base.

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AP 00967

The ointment base is preferably an emulsifying ointment base, more preferably comprising a mixture of an emulsifying wax, white soft paraffin and liquid paraffin.

For example, the emulsifying ointment base may comprise about 30% by weight of the emulsifying wax, about 50% by weight of the white soft paraffin and about 20% by weight of the liquid paraffin.

The ointment preferably comprises about 1% to about 5% by weight of the ointment, of the extract.

According to a third aspect of the invention there is provided a method of preparing an extract of Mitra villosus, including the steps of:

(i) drying Mitra villosus material;

(ii) reducing the dried material to a powder; and (iii) subjecting the powder to an extraction process utilizing an organic solvent.

The drying step, step (i), may consist of air drying the Mitra villosus material, for example using a fan to circulate air over the leaves. Preferably, however, the drying step comprises drying the Mitra villosus material in an open environment using atmospheric air.

The dried material may be reduced to a powder, generally a coarse powder, by grinding, for example using a wooden mortar and pestle or utilising a hammer mill.

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The pharmaceutical composition or ointment of the invention is of particular use in treating humans afflicted with skin fungal infections.

AP 00967

DESCRIPTION OF EMBODIMENTS

In accordance with the invention, a pharmaceutical composition for treating and managing skin fungal infections is provided. The composition is prepared by extracting effective antifungal phytochemicals from the dried and ground leaves of Mitra villosus plant. In a preferred form of the invention, the dried, ground leaves are subjected to a hot soxhlet extraction process using n-hexane as an extraction solvent. The extract may be combined with an excipient ointment carrier material to prepare an ointment. The ointment, which may preferably contain from about 1 to about 5% of the extract by weight of the ointment, is preferably applied topically and directly to areas of the skin which are infected by fungus.

Mitracarpus villosus was selected as a possible candidate for scientific investigation and its effectiveness against fungi was determined. Antifungal activity was assessed against Candida albicans. Tricophyton mentagrophytes. Microsporum canis. Epidermophvton flocossum, and Tricophyton rubrum. using the Agar dilution method. These organisms were chosen because they are usually responsible for superficial mycoses in man. The results indicate that Mitra villosus plant extract has potential to be very useful in treating and managing fungal infections of the skin. -For example, the product inhibited growth of all the organisms tested in vitro.

Anti-inflammatory and antimicrobial activities of the extract were also studied. From the results, the product may have anti-inflammatory properties but no antibacterial effects were observed. Acute toxicity studies in laboratory animals showed an LD₅₀ of 980 mg/kg intraperitoneally in mice suggesting that it may be safe even when taken orally by accident.

AP/P/ 9 8/01205

AP 00967

The Mitra villosus plant extract may be prepared using an extraction process as described hereinafter. The plant material is first dried in an open atmosphere using a fan to circulate atmospheric air. The dried plant material is then reduced to a coarse powder using a hammer mill (1kg) or a wooden mortar and pestle (700g). The powdered material is loaded into a 2L soxhlet extractor mounted on a 2L quick fit distillation flask containing the desired solvent. At the top of the extractor is fixed a double surface condenser through which tap water (22+3°C) is circulated. The solvent in the flask is heated on a steam bath (or a carefully regulated heating mantle). The solvents vaporizes and rises up the arm of the extractor into the cold condenser and drops as a liquid onto the packed plant material.

The solvent then percolates through the plant material. It extracts soluble components and rises up in the extractor until the last air bubble is pushed out of the inner siphon tube. The solution of extract now siphons into and collects in the distillation flask with continued heating. The process is repeated as the extract leaves the plant material and is concentrated in the flask. After extraction is completed (16-24 hours with hexane; 10-14 hours with methanol), the solvent is distilled off and the crude extract concentrate is decanted into a weighed, labelled beaker (100 ml, 250 ml or 400 ml, as desired) and allowed to dry completely in air or a dessicator. The solvent may be effectively removed using a rotary evaporator.

The plant material meanwhile is removed from the extractor and placed on a polyethylene sheet on top of the lab bench to dry completely. It is then loaded again and extracted with the next solvent of choice (of higher polarity). Extraction is usually first done with hexane (or petroleum spirit, 60-80°C) followed by ethylacetate or dichloromethane. Hexane/petroleum ether extracts metabolites that are non-polar (defatting).

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AP 00967

Dichloromethane/ethyl acetate removes slightly polar components.

With a IL or 250 ml soxhlet extractor, siphoning is more frequent and less extraction time is required. With a 250 ml extractor, for example, extraction with hexane may be complete in 10-12 hours while extraction with methanol may last for 6 - 8 hours.

The approximate ranges of yield percentages are as follows:

Hexane = 0,05-0,1%

Methanol + 0,07-0,13%

The phytochemical analysis of the plant extract revealed the presence of alkaloids, saponins, flavonoids, glycosides, tannins, volatile oil and general anthraquinones.

The dried extract may be formulated into an ointment. The preferred excipient base is an emulsifying ointment base. The composition of the excipient may preferably be as follows:

Emulsifying wax - about 30% by weight

White soft paraffin - about 50% by weight

Liquid paraffin - about 20% by weight

The concentration of the dried extract of M. villosus in the antifungal ointment or cream may preferably range from about 1 % by weight to about 5 % by weight, of the weight of the ointment.

The ointment is applied to the clean surface of skin in two divided doses, i.e morning and evening.

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AP 00967

The effectiveness of the extract against organisms which are known to be responsible for superficial mycoses, such as Epidermophyton flocossum ATCC 10227, (EF); Microsporum canis ATCC 11622, (MC); Tricophyton rubrum ATCC 28941, (TR); Tricophyton mentagrophytes ATCC 4808 (TM), was tested as follows. The dried and ground M. villosus plant leaf material was subjected to extraction using four different extraction solvents, namely n-hexane, methanol, cold water and hot water.

The percent yields of the different extracts of the fresh leaves of M. villosus are indicated below:

SOLVENT	% YIELD
n-Hexane	1,56
Methanol	2,3
Hot water	1,69
Cold water	0,55

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Agar dilution method was employed for the plant extracts. Double dilutions of the extracts were used in all cases starting with 1000 μζ per milliliter of Sabouraud Dextrose Agar. The following tables illustrate the effectiveness of the extract against the organisms and compares the efficiency of the different extraction solvents.

AP 00967

I. HEXANE EXTRACTION

	Organisms
Concentrations (Ug/ml)	TM	MC	EF	TR
1000	-	-	-	-
500	-	.-	-	-
250	-	+	-	+
125	+	+	+	+

II. METHANOL EXTRACTION

	Organisms
Concentrations (Ug/ml)	TM	MC	EF	TR
1000	-	-	-	-
500	+	+	+	+
250	+	+	+	+
125	+	+	+	+

AP/P/ 98/01205

III. COLD WATER EXTRACTION

	Organisms
Concentrations (Ug/ml)	TM	MC	EF	TR
1000	+	+	+	+
500	+	+	+	+
250	+	+	+	+
125	+	+	+	+

AP 00967

Similar results of no activity was also recorded for the hot water extraction. KEY: - = No growth, i.e growth of organism was inhibited.

+ = Growth of organism.

Two separate formulations of ointment based on M. villosus plant leaf extract were developed at laboratory scale and these were tested on patient volunteers. Twelve patients participated in the limited clinical trials to assess the efficacy and safety of M. villosus extract against fungal skin infections. The ointment was applied topically to the infected skin area twice daily. Ten of the patients recovered fully. In those that recovered, itching stopped and pain disappeared. Some of the patients were able to wear shoes comfortably. No adverse side effects have been observed thus far.

Claims

1 A pharmaceutical composition for treating skin fungal infections comprising an extract of Mitra villosus, the extract having been prepared using an organic solvent.

2 A pharmaceutical composition according to claim 1 comprising an extract of Mitra villosus plant leaves.

3 A pharmaceutical composition according to claim 1 or claim 2 wherein the organic solvent is selected from the group consisting of hexane, methanol, and mixtures thereof.

4 A pharmaceutical composition according to any one of claims 1 to 3 comprising an extract of Mitra villosus. the extract having been prepared using a hot hexane soxhlet procedure.

5 A pharmaceutical composition according to any one of claims 1 to 4 formulated as an ointment.

6 An ointment for treating skin fungal infections comprising an extract of Mitra villosus. the extract having been prepared using an organic solvent, and an ointment base.

7 An ointment according to claim 6 comprising an extract of Mitra villosus plant leaves.

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AP 00967

8 An ointment according to claim 6 or claim 7 wherein the organic solvent is selected from the group consisting of hexane, methanol and mixtures thereof.

9 An ointment according to any one of claims 6 to 8 comprising an extract of Mitra villosus. the extract having been prepared using a hot hexane soxhlet procedure.

10 An ointment according to any one of claims 6 to 9 wherein the ointment base is an emulsifying ointment base.

11 An ointment according to claim 10 wherein the emulsifying ointment base comprises a mixture of an emulsifying wax, white soft paraffin, and liquid paraffin.

12 An ointment according to claim 11 wherein the emulsifying ointment base comprises about 30% by weight of an emulsifying wax, about 50% by weight of a white soft paraffin and about 20% by weight of a liquid paraffin.

13 An ointment according to any one of claims 6 to 12 which comprises about 1 % to about 5 % by weight of the ointment, of the extract.

14 A method for preparing an extract of Mitra villosus comprising the steps of:

(i) (ii)

AP/P/ 9 8 / 0 1 2 0 5 drying Mitra villosus material;

reducing the dried material to a powder; and

AP 00967 (iii) subjecting the powder to an extraction process using an organic solvent.

15 A method according to claim 14 wherein the Mitra villosus material is Mitra villosus plant leaves.

16 A method according to claim 14 or claim 15 wherein the organic solvent is selected from the group consisting of hexane, methanol and mixtures thereof.

17 A method according to any one of claims 14 to 16 wherein in step (i) the Mitra villosus material is air dried.

18 A method according to any one of claims 14 to 17 wherein in step (iii) the powder is subjected to a hot hexane soxhlet procedure.