Anti-fungal formulation active against a broad spectrum of dermatophytoses

FIELD

The present invention provides a formulation based on the essential oil of plant Rabdosia melissoides, useful as a fungicide against a wide spectrum of dermatophytic fungi.

The formulation of the present invention has fungitoxic action against different dermatophytes and this property of the formulation is attributed to the fungicidal action of the volatile oil hydrodistilled from the medicinal plant Rabdosia melissoides and this is the first report of its utilization in these types of potent antimicrobial activities.

BACKGROUND

Dermatophytoses is a disease caused by a group of fungi known as dermatophytes. It is also known as ringworm or tinea and involves superficial infection of keratinized tissue of the skin of animals and human beings. Various species of three anamorpohic (asexual or imperfect) genera Epidermophyton, Microsporum and Trichophyton are commonly involved in these mycoses. The disease is prominent in tropical and subtropical countries due to their prevailing moisture and temperature regimes, and poses a therapeutic problem despite several antimycotic drugs available in the market.

Treatment of dermatophytoses is by use of topical or oral antifungal agents. There are 17 synthetic chemical compounds which are generally utilized (Majumdar 1999 Indian drugs 36(1): 1-14) for various superficial fungal infections but many of them produce adverse effect with fungistatic action only. Since herbal medicines have been reported to be comparatively safe and without any adverse side effect there has been revival of interest in the use of medicinal plants in developed and developing countries.

During the course of research, in an attempt prepare fungitoxic formulation from natural products the applicants have found that volatile oil of Rabdosia melissodies can be employed for controlling a number of dermatophytes.

The plant Rabdosia melissodies Benth. Syn. Plactranthus melissoides (Lamiaceae) is an important source of thymol. For the first time this species was introduced for cultivation by Central Institute of Medicinal and Aromatic Plants (Singh et al. PAFAI Journal, January March, 67-69, 1986). It is an herbaceous plant found to grow scattered in nature especially in Tarai region of Uttar Pradesh, India. The leaves and inflorescence of the plant on hydrodistillation yield essential oil which has been reported to exhibit repellent and fumigant properties for storage grain insects (Singh & Singh 1991, Insect Science Application 12(4) 487-491.

The basis on which the invention has been developed is the applicant's own finding that the essential oil of Rabdosia melissoides acts as a potent antidermatophytic agent. The oil showed broad range of antifungal activity completely inhibiting the mycelial growth of fungi such as Microsporum abudouiniis, M. nanum, Trichophyton mentagrophytes var interdigitale, T. mentagrophytes var mentagraphytes, T. tonsurans, T. violaceum, Epidermophyton onvious floccosum, Microsporum gypseum and Trichophyton rubrum, considered to be the most important dermatophytes (Mehrotra et al 1978, Ind. J. Pathol. Microbiol 21: 131-134; Banerjee Pasricha 1987, Ind. J. Med. Microbiol, 5: 207-212).

Twelve constituents have been identified in the essential oil of leaves of Rabdosia melissoides [Singh et al 1983, Planta Medica 47(4) 256]. They are .varies.-pinene, camphene, .beta.-pinene, myrcene, .varies.-phellandrene, d-limonine, carene, .varies.-terpinene, p-cymene (25.4%), T-terpinene, thymol (19.8%) and carvacrol (15.4%). Thus thymol, p-cymene and carvacrol are the major constituents of the oil. There are quite a number of other plants viz. Origanum vulgare, O. majorana, Thymus serpyllum, Thymus vulgaris and Trachyspermum ammi syn Carum copticum Satureja montana and some Ocimum sp. which have been reported to contain thymol as a major constituent along with p-cymene and carvacrol. Among the oils of above plants, the oil of Origanum vulgare sp. hirtum has been reported to exhibit antifungal properties in vitro against human pathogens Malassezai furfur (yeast), Trichophyton rubrum (contains ginalool also) and Trichosporum beigelli [Konstantia et al 1998 J. Agric. Food. Chen. 46(5) 1739-1745). The oil showed promising results when tested on T. rubrum infected rats. The oil of Trachyspermum ammi contains 3 major constituents, thymol, carvacrol, and p-cymene, and has been reported to check growth of Epidermophyton floccosum, Microsporum canis, Trichophyton mentagrophytes in vitro. [Sing et al 1986; MyKosen 29(1) 37-40]. The oil of T. ammi has also been evaluated in the form of an ointment in human beings but it has been found to produce some adverse effects on the mammalian skin when applied locally. (Dixit et al. 1994 Traditional Medicinal Plants in Skin care CIMAP Publication, Lucknow, India p 12). The oils of Thymus serphyllum and T. vulgaris are reported to check the growth of Candida albicans. [Menghini et al 1987, Plantes Medicinales et Phytotherapie 21(1) 36-42].

Thymol, carvacrol and p-cymene have been reported to exhibit fungicidal activity (CRC Hand book of Medicinal Mints (Aromathematics) Phytochemicals and Biological activity, Stephen M. Beckstrom--Sternberg James A. Duke, CRC Press 1996 Printed in USA pp 419, 386, 409]. On further detailed literature survey it was found that thymol exhibits antifungal activity in vitro [Myers 1927, J. Am. Med Asso. 89 1834] and is effective as antifungal preservative [Dersarkission and Goodberry 1980, Studies Conserv 25-28] and checks the growth of honey bees pathogens, [Calderone et al. 1994, Journal of Essential Oil Research 6(3) 279-287]. p-Cymene has been found to check the growth of wood-habiting fungi Trichoderma viridi (De Groot 1972, Mycologia 64 862-870). While the carvacrol has been found to inhibit the germination and growth of vegetable post harvest pathogenic fungi (Caccioni et al 1994, Journal of Essential Oil Research 6(2) 173-179]. The essentials oil of Kunzea ericoides var. liniaris containing 31% p-cymene has weak antifungal activity (Perry et al 1997. Phytochem 45 (8) 1605-16012) which reveal that p-cymene is not useful for dermatophytoses and carvacrol up to 40% present in the oil of Saturega montana was found to inhibit the growth of only one species of Trichophyton and the nature of toxicity is also fungistatic (Menphini et al 1993 Riv. Ital FPOS, 4, SP. No. 566-71) for dermatophytoses further the Satureja montana oil was effective in 24-48 hour in 0.1 u ml/l while the oil of Rabdosia melissoides is effective in 80 minutes at the same concentration. The studies have shown the superiority of Rabdosia melissoides oil over thymol (Table 6).

So, it is very clear that p-cymene, carvacrol or thymol are not shown to be a good agent against dermatophytic fungi separately or in combinations present in various known oils but the combination of these, present in R. melissoides oil along with certain unidentified constituents played a synergistic fungicidal action, so the oil from the plant--R. melissoides is a potential novel source for cure of dermatophytoses and its formulation development is also a novelty.

Thus available information about the essential oils and plants having thymol, carvacrol and p-cymene as major constituents or in isolation have not been found to act as antifungal agents effective against a broad spectrum of cutaneous mycoses/dermatophytoses.

OBJECTS

The main object of present invention is to develop a herbal formulation(s) active against a broad spectrum of dermatophytic fungi.

Another object of the invention is to provide a formulation useful as a topical cream which smoothens the skin and allows slow absorption of the contents, thereby causing effective action which completely eliminates the infection by killing the fungi.

Yet another object of the invention is to provide an anti-fungal formulation containing the essential oil extracted from plant Rabdosia melissoides as its main active principle.

Yet another object of the invention is to provide an anti-fungal formulation which is commercially viable.

A further object of the invention is to provide a formulation which is stain-free.

Yet another object of the invention is to provide a cheap anti-fungal formulation compared to other synthetic preparations available in market.

Yet another object of the invention is to provide a formulation having quick action.

Yet another object of the invention is to provide a formulation in which the activity of ingredient is thermostable.

SUMMARY

The invention provides novel formulation containing the essential oil of Rabdosia melissoides effective as an anti-fungal agent against a wide spectrum of decmatophytic fungi. Further, the invention also provides a method for the treatment of skin disorders using the said novel formulation.

DETAILED DESCRIPTION

Accordingly, the invention provides novel anti-fungal formulation comprising about atleast 1% of oil extracted from Rabdosia melissoides and one more vegetable oils, solvents and additives. Preferably, the oil extracted from Rabdosia melissoides is in the range of 1 to 5% by wt. of the formulation.

The formulation is prepared by drop wise drop wise absorption of 1-5 ml of essential oil of the plant Rabdosia melissoides in 95-99 ml of the base material at 40-60.degree. C. with constant stirring and cooling down to room temperature and further curing the preparation for 48 hrs. in closed glass vials.

In a preferred embodiment, the formulation of the present invention comprises 1% (v/w) of the oil obtained from aerial parts of Rabdosia melissoides wherein 1 ml oil is absorbed over the base made by mixing 25 gm stearyl alcohol and 74 gm propylene glycol at 60.degree. C. over a water bath and cooling down the preparation with constant stirring upto the ambient temperature and finally curing it for 48 hours in closed glass vials.

In an embodiment of the present invention the base used may be prepared by mixing different concentrations of liquid paraffin to the melted mixture of petroleum jelly and natural wax like bees wax or carnauba wax.

In yet another embodiment, the formulation is in the form of a cream, gel, lotion, capsule or combinations thereof.

In an embodiment, the oil from Rabdosia melissoides is absorbed over mineral oil, animal oil or vegetable oil selected from white oil, cord liver oil, tallo oil, whale oil, seal oil, linseed oil, poppy oil, soya oil, sunflower oil, mustard oil and coconut oil to prepare the formulation in a lotion form.

In an embodiment, the cream, gel or lotion is prepared by absorbing the oil of Rabdosia melissoides over solvents selected from polyalcohols, alkylglycols such as ethylene glycol, propylene glycol and glycerol.

In yet another embodiment of the present invention the said composition has shelf life of more than 48 months.

In still another embodiment of the invention there is no need to add any antioxidant in the formulation as the active ingredient is rich in phenolic compounds.

In still another embodiment of the invention the oil in the preparation does not loose its activity upto temperature of 100.degree. C.

In still another embodiment of the formulation may act as a moisturiser.

In still another embodiment of the invention, the formulation does not leave any stain on clothes.

In still another embodiment of the formulation the said preparation is ecofriendly and biodegradable.

Thymol carvacrol and p-cymene have been reported to have fungicidal activity against a broad spectrum of cutaneous mycoses/dermatophytoses.

Fungicidal is a word which is used to describe all compounds which kill fungus, either plant pathogen or animal pathogen. Since, oil containing compounds thymol, carvacrol and p-cymene have not been reported for killing the fungi causing dermatophytoses i.e. superficial infection of keratinised tissue of skin of animals and human beings, our invention for use of the natural oil having these constituents along with some unidentified compounds present in the oil is, novel. However, the oil of Trachyspermum ammi which have these three compound have been reported once for use in an ointment but produce some adverse effects. Whereas this oil i.e. of R. melissoides does not produce adverse effects and is utilized for this type of preparation for the first time. Further, other oils have been tested only with two species are general fungus while in this invention we have included a wide range of dermatophytic fungi which is essential to get the total picture of a product.

The invention is illustrated by the following examples which should not be construed to limit the scope of the present invention.

Development and Testing of Different Formulations

EXAMPLE 1

Essential oil of Rabdosia melissoides 1 ml Glycerol IP 99 ml

The essential oil obtained by hydrodistillation of herbs of Rabdosia melissodies (1 ml) was absorbed drop wise over previously warmed (40.degree. C.) glycerol over water bath with constant stirring, after the addition of total oil the mixture was kept at ambient temperature in closed glass vial for curing.

The preparation was effective.

EXAMPLE 2

Essential oil Rabodosia melissodies 1 ml Linseed oil 99 ml

The essential oil of Rabdosia melissodies 1 ml was absorbed over warmed linseed oil as described in experiment example 1.

The formulation was very effective. However, the only drawback was that the smell of the linseed oil was dominating.

EXAMPLE 3

Essential oil of Rabdosia melissodies 1 g Bees wax. 55 g Petroleum jelly white 30 g Liquid paraffin 14 g

The essential oil obtained from hydrodistillation of herbs of Rabdosia melissodies (1 gm) was absorbed over the previously prepared base by melting mixture of bees wax (55 gm), petroleum jelly (30 gm) and liquid paraffin (14 gm). The oil was absorbed drop wise over a water bath at 60.degree. C. with occasional stirring. The mixture was kept in a closed glass vial and left for 48 hours for curing.

The ointment was very effective and it produces a feeling of having a sticky layer on the surface of the skin so its use is good preferably for the exposed parts of the skin which are thicker and where the ointment is absorbed slowly in a little long duration, further, if in case of severe infection such formulation will form a thin protective layer from the water and dust. In case of tinea mannum and T. badis infections.

EXAMPLE 4

Essential oil of Rabdosia melissodies 1 ml Propylene glycol IP 99 ml

The essential oil obtained by hydrodistillation of herbs of Rabdosia melissodies (1 ml) was absorbed drop-wise over previously warm (50.degree.) propylene glycol (99 ml) over a water bath with constant stirring after addition of total oil the mixture was left over water bath further for one hour in a tightly closed vial. Then the preparation was kept at ambient condition for 48 hrs. for curing.

The formulation was very effective and can be applied preferably for tinea capits and tinea unguia for skull and nail infection.

EXAMPLE 5

Essential oil of Rabdosia melissoides 1 ml Propylene glycol IP 74 gm Stearyl alcohol 25 gm

The essential oil obtained by hydrodistillation of herbs of Rabdosia melissoides (1 ml) was absorbed dropwise with constant stirring in warm (60.degree. C.) mixture of Propylene glycol (74 gm) and stearyl alcohol (25 gm). After addition of all Rabdosia melissoides oil the mixture was taken off from the waterbath and left to cool down to room temperature with constant stirring till the mixture become viscous. The preparation was kept for curing for 48 hrs.

This formulation gave very good effect in controlling the infection and produced smoothness to the skin. On applying slowly with finger it get absorbed in the skin leaving a creamy shining appearance to the skin. The preparation when clinically compared with other ointments commonly available in the market its efficacy was found to be better (Table 4).

For clinical response this cream was applied topically on human patients for the control of fungal infection (dermatophytoses) for three weeks two times daily, the patients were not allowed to take any other systemic or topical medicine. Patients of age group 8 to 40 years selected randomly and a group of 30 individuals was formed, Table 5 reveals the efficacy of the present formulation.

The similar creams were prepared by increasing concentrations of oil of Rabdosia melissoides but it was found that the effect was similar to the cream prepared by mixing 1% oil, however cream having upto 5% R. melissoides oil did not show any irritant activity on human beings (Roxburgh and Borie's method), further the cream with higher concentrations of R. melissoides oil may be useful to the infections of hard superficial surfaces like skull and nail.

The curative properties of oil of Rabdosia melissoides is due to some specific concentrations of the constituents present in the oil along with many minor unidentified constituents.

In vitro Studies of Antifungal Activities

For in vitro investigations, the minimum effective concentrations (MECs) of the oil were determined following minor modifications of poisoned food technique [Grover and Moore 1962, Phytopathalogy 52, 876-880); Shahi et al 1999, Current Science 76 (6) 836-839]. Minimum fungistatic and fungicidal concentrations of the oil were determined by the method of Garber and Houston 1959 [Phytopathalogy 49 449-450]. This was done by reinoculating the inhibited discs on sabouraud dextrose agar (SDA) medium in culture tubes. Inoculated culture tubes were incubated at 27.+-.1.degree. C. and the observations recorded on seventh day. While fungal growth indicated fungistatic activity, its absence denoted fungicidal action. The nature of toxicity of the oil was fungistatic at its minimum effective concentrations of 0.1 .mu.l/ml, 0.2 .mu.l/ml and 0.1 .mu.l/mL against E.floccosum, M. gypseum and T. rubrum respectively (Table 1). The oil also inhibited heavy doses of inoculum. The minimum killing time was found to be 10, 20 and 10 seconds respectively with the pure oil against the three test pathogens (Table 2). However, at minimum fungicidal concentrations of 0.3 .mu.l/mL against E. floccosum, 0.4 .mu.l/mL against M. gypseum and 0.3 .mu.l/mL against T. rubrum it required 80, 90 and 80 minutes respectively to kill the pathogens. The minimum killing time (MKT) of the oil was determined by mycelial disc killing technique (Shahi et al. 1999, Current Science 76 (6) 836-839]. Oil having 0.05 .mu.l/ml to 0.2 .mu.l/ml concentration produces fungi-static effect i.e. it stops the growth of fungus but when application is stopped, it reappears. While higher concentration i.e. 0.3 .mu.l/ml to 0.4 .mu.l/ml and above produces fungicidal effects i.e. it kills the fungus and reoccurrence of the disease do not take place. The oil was found to be thermostable up to 100.degree. C., and it retained its fungicidal even after more than 48 months of storage. The oil exhibited wide range of activity inhibiting the mycelial growth of seven other fungi viz. Microsporum, audouinii, M. canis, M. nanum, Trichophyton mentagrophytes, Var. interdigitale, T. mentagrophytes, Var. mentagrophytes, T. tonsurans and T. violaceum (Table 3). On comparing the minimum fungistatic as well as fungicidal concentrations of the oil with that of the prevalent synthetic antifungal drugs, the oil was found to be more effective. Moreover, the oil did not exhibit any adverse effects on the mammalian skin up to 5% of concentration.

The expiry of toxicity of the oil was determined by storing them at room temperature and testing their antifungal activity at minimum effective concentrations (MCs) at regular intervals of 60 days up to 48 months following the usual poisoned food technique (Grover and Moore 1962). For determination of the effect of temperature, vials, containing 5 ml of oil each were kept separately. These were exposed to different temperatures viz. 40, 60, 80 and 100.degree. C. in the incubator for one hour. The antifungal activity of the oil was then tested by poisoned food technique. Antifungal spectrum of the oil was determined at various concentrations viz 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 .mu.l/ml, against other test pathogens by the usual poisoned food technique. Nature of toxicity of the oil whether fungistatic or fungicidal was determined by the method of Garber and Houston (Phytopathology 1959, 49 449-450) (Table 3).

The comparative study of the oil with some synthetic antifungal drugs was carried out by comparing MECs. The antifungal activity and nature of toxicity (i.e., fungistatic/fungicidal at their MECs) assayed following usual method as described earlier.

Clinical Trials

For in vivo investigations all the clinical trials were conducted on the patients (Out Patient Department) at Motilal Nehru Medicinal College, Allahabad (UP) by the method of Shahi et al. (1999) Current Science 76(6):836-39. Patients of either sex were diagnosed for either tinea pedis, tinea corporis or tinea cruris based on site of infection, the diagnosis was further confirmed by microscopic examinations of the scraping (from infected area) treated with 10% KOH. Patients showing mycelium and/or conidia were designated KOH positive. Only KOH positive cases were enrolled. Patients were examined just before the therapy was initiated and at the end of each week of 3 weeks of treatment. Although when the cutaneous fungal disease manifested itself in several body areas, all affected areas could be treatment but only one was selected and designated as the reference lesion. At each visit of the patient, the same reference lesion was scraped for fungal culture to identify the organism and for demonstration of presence of hyphae by microscopic examination of the scrapings which were covered with 10% KOH preparations. Signs and symptoms of inflammation as erythema, scaling, itching, maceration, vesiculation and pustulation were recorded as absent, mild, moderate, or severe. At each visit of the patient, overall clinical improvement was rated as worse, none, partial, significant or completely clear by comparing the conditions with those existing at the time of initial visit. Any adverse systemic or local reaction was noted at each visit and recorded as mild, moderate or severe. Satisfactory response with KOH-negative cases after third week were re-examined after one month relapse rate if any. To find out maximum tolerable concentrations (MTCs) for irritant activity, if any, of the oil by their topical application of human skin following the patch test method of Roxburgh and Borrie (The English Language Book Soc. H. K. Lewis and Co. Ltd. XII edition 1973). People of different sex in the age group of 10 to 30 years were selected randomly and a group of 30 individuals of each sex was constituted. Circular area sof 5 cm.sup.2 on upper hairy and lower glabrous surface of palms and 3 cm.sup.2 of neck region of each individual were first washed with distilled water followed by 70% ethyl alcohol and then allowed to dry for five minutes. Five drops of the graded concentrations of testing solution was applied to each individual separately. The volunteers were not allowed to wash the applied area. The qualitative observations were recorded after 24 hours of application.

TABLE 1 Minimum effective concentrations of the oil of Rabdosia milissoides against test pathogens (Poisoned food technique). Mycelial Growth Inhibition (MGI %) Concentrations (.mu.l/mL) E. floccosum M. gypseum T. rubrum 0.4 100.sup.c 100.sup.c 100.sup.c 0.3 100.sup.c 100.sup.s 100.sup.c 0.2 100.sup.s 100.sup.s 100.sup.s 0.1 100.sup.s 71.0 100.sup.s 0.05 78.2 59.3 74.0 .sup.s fungistatic .sup.c fungicidal

TABLE 2 Minimum killing time of the oil of Rabdosia milissoides against test pathogens (Mycelial disc killing technique, Shahi et al. 1999 Current Science 76 (6) 838-839). Fungal Growth Inhibition (FGI %) Minimum killing E. floccosum M. gypseum T. rubrum time (MKT) PO MCC PO MCC PO MCC 120 min. 100 100 100 100 100 100 90 min. 100 100 100 100 100 100 80 min. 100 100 100 82.1 100 100 70 min. 100 90.2 100 64.2 100 91.0 60 min. 100 -- 100 -- 100 81.3 50 min. 100 -- 100 -- 100 61.9 40 min. 100 -- 100 -- 100 -- 30 min. 100 -- 100 -- 100 -- 60 Sec. 100 -- 100 -- 100 -- 30 Sec. 100 -- 100 -- 100 -- 20 Sec. 100 -- 100 -- 100 -- 10 Sec. 100 -- 73.2 -- 100 -- 2 Sec. 63.2 -- 39.2 -- 73.1 -- 1 Sec. 23.2 -- 15.0 -- 52.2 -- PO = pure oil MCC = minimum fungicidal concentration

TABLE 3 Antidermatophytic spectrum of the oil of Rabdosia milissoides Mycelial growth Inhibition at various concentration (.mu.l/ml.) Fungi 0.05 0.1 0.2 0.3 0.4 0.5 Microsporum audouinii 56.2 76.2 100.sup.s 100.sup.s 100.sup.c 100.sup.c M. canis 46.3 82.3 100.sup.s 100.sup.s 100.sup.c 100.sup.c M. nanum 50.2 91.3 100.sup.s 100.sup.s 100.sup.c 100.sup.c Trichophyton mentagrophytes 83.2 100.sup.s 100.sup.s 100.sup.c 100.sup.c 100.sup.c var. interdigitale T. mentagrophytes 51.0 76.3 100.sup.s 100.sup.s 100.sup.c 100.sup.c var. mentagrophytes T. tonsurans 76.9 100.sup.s 100.sup.s 100.sup.c 100.sup.c 100.sup.c T. violaceum 83.2 100.sup.s 100.sup.s 100.sup.c 100.sup.c 100.sup.c .sup.c = fungicidal .sup.s = fungistatic

TABLE 4 Comparative efficacy of the formulation of Rabdosia milissoides with commercial antifugal drugs Oil and trade name Minimum effective concentration (.mu.l/ml.) of antifungal Active E. floccosum M. gypseum T. rubrum agents ingredients MSC MCC MSC MCC MSC MCC Cream of Essential oil 0.1 0.3 0.2 0.4 0.1 0.3 present formulation Dactrine Miconazol 6.0 * 5.0 * 4.0 * Nitrate Nizral Ketoconazole 0.5 * 5.0 * 4.0 * Tenaderm Tolnaftate 1.5 * 0.4 * 0.7 * Batrafine Ciclopirox 0.3 1.0 0.1 0.5 0.3 0.5 alamine solution MSC = minimum fungistatic concentration MCC = minimum fungicidal concentration * = remained static

TABLE 4 Comparative efficacy of the formulation of Rabdosia milissoides with commercial antifugal drugs Oil and trade name Minimum effective concentration (.mu.l/ml.) of antifungal Active E. floccosum M. gypseum T. rubrum agents ingredients MSC MCC MSC MCC MSC MCC Cream of Essential oil 0.1 0.3 0.2 0.4 0.1 0.3 present formulation Dactrine Miconazol 6.0 * 5.0 * 4.0 * Nitrate Nizral Ketoconazole 0.5 * 5.0 * 4.0 * Tenaderm Tolnaftate 1.5 * 0.4 * 0.7 * Batrafine Ciclopirox 0.3 1.0 0.1 0.5 0.3 0.5 alamine solution MSC = minimum fungistatic concentration MCC = minimum fungicidal concentration * = remained static

The Main Advantages of the Present Invention are

1. Most of the topical creams available in the market are fungistatic and there is recurrence of the infection after discontinuance of the medicines. However, the present formulation was highly effective with fungicidal action where infection is completely eliminated and there is no recurrence of the infection.

2. The formulation has very long shelf life (>48 months).

3. There is no need of adding any antioxidant or preservative as the active ingredients in the oil are rich in phenolic components.

4. The formulation acts as a moisturiser and smoothens the skin surface thereby checking the itching of the infected part.

5. The activity of the oil is thermostable upto 100.degree. C. so there is no need of storing under low temperature.

6. The formulation can be washed from the infected surface with water only so there is no need of soap or solvent to clean the infected part as the use of these may cause some adverse effects.