|Broad spectrum antimycotic drug for the control of fungal infection in human beings|
Sushil K. Shahi, A. C. Shukla, A. K Bajaj*,
G. Medgely and Anupam Dikshit
Biological Product Laboratory, Botany Department, University of Allahabad, Allahabad 211 002, India
*Moti Lal Nehru Medical College, Allahabad, India
St. Johns Institute of Dermatology, St. Thomas Hospital, London, UK
During antifungal evaluation of the secondary metabolites (essential oil) of some Eucalyptus species, E. citriodora was found to be most effective as an antifungal against human pathogenic fungi Microsporum nanum, Trichophyton mentagrophytes and T. rubrum. While the pure oil (100%) killed the M. nanum in 20 s and T. mentagrophytes, T. rubrum in just 15 s, at their minimum fungicidal concentrations (MFCs) it required 5 h 30 min against M. nanum and 4 h against T. mentagrophytes and T. rubrum. On comparing the minimum effective concentration of the oil with those of prevalent synthetic antifungal drugs, the oil was found to be more effective. More- over, it did not exhibit any adverse side effects on mammalian skin up to 5% concentration. The oil in the form of ointment broad spectrum antimycotic drug (BSAD) was subjected to topical testing on patients attending out patient department of M.L.N. Medical College, Allahabad. All patients showed positive potassium hydroxide (KOH) results at the beginning of the trial. Patients were diagnosed as either tinea pedis or tinea cruris. After the second week of treatment, all patients were KOH-negative. At the end of the third week of treatment, every patient was KOH-negative and remained negative when re-examined after one month of treatment, thereby leaving no scope for relapse. At the end of treatment, while 55.5% of patients recoverd completely, 44.5% showed significant improvement from the disease. The ointment thus not only showed maximum effectivity, but was also found to be cost effective, had long shelf life, and showed absence of any adverse side effects.
INFECTION caused by fungi in man and animal, mycosis, are common throughout the world. Dermatophytoses poses a serious concern to sociologically backward and economically poor populations of India.
Dermatophytoses is the disease caused by a group of fungi known as dermatophytes. It is also known as ringworm or tinea and involves superficial infections of keratinized tissue of the skin of animals and human beings. Clinical surveys carried out in India have shown ringworm as one of the most common dermatomycoses caused by the species of Epidermophyton, Microsporum and Trichophyton. The disease is predominant 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. These drugs are mostly fungistatic. Besides, these drugs have recently been found to possess various side effects1.
In recent years there has been a gradual revival of interest in the use of medicinal plants in developed as well as in developing countries, because herbal medicines have been reported to be safe and without any adverse side effects. Thus, a search for new drugs with better and cheaper substitutes, plant resources are a natural choice. Recently, some products of higher plants origin have been shown to be effective source of chemotherapeutic agents without undesirable side effects and with strong fungicidal activity14. These findings prompted us to explore other plant products which could be exploited as effective antifungals. We report here the detailed antifungal study of the essential oil, obtained from the leaves of E. citriodora in vitro as well as in clinical trials against dermatophytoses in human beings.
For our studies we extracted the essential oil from the fresh leaves of E. citriodora Hook by hydrodistillation, using Clevengers apparatus5. The oil, thus obtained was evaluated for its antifungal properties against the test pathogens, M. nanum, Fuentes, T. mentagrophytes (Robin) Blanchards, and T. rubrum (Castellani) Sabouraud.
For in vitro investigations, the minimum effective concentrations (MECs) of the oil were determined following the poisoned food technique6 with slight modifications1. Minimum fungistatic and fungicidal concentrations of the oil were determined by the method of Garber and Houston7. This was done by reinoculating the inhibited discs on sabouraud dextrose agar (SDA) medium in culture tubes1. Inoculated culture tubes were incubated at 27 ▒ 1░ C and the observations recorded on seventh day. While fungal growth indicated fungistatic activity, its absence denoted fungicidal action.
The minimum killing time (MKT) of the oil was determined by mycelial disc killing technique1. Two treatment sets were maintained, one with pure oil and the other with the minimum fungicidal concentrations (MFCs) of the oil. The treatment set using MFCs of the oil was prepared by mixing the required quantity of the oil in acetone (5% of the total quantity of the treatment solution) and then added to required quantity of distilled water. Simultaneously, controls were maintained using sterilized water (in place of the oil) and acetone was added to the distilled water in required quantities.
Mycelial discs of 5 mm diameter, cut out from the periphery of 7-day-old cultures of the test pathogens, were aseptically placed inside the culture tubes of different treatment and control sets. These mycelial discs were taken out of the tubes at different time intervals and washed immediately in the washing solution (containing acetone and sterilized distilled water in 1:2 ratio) to remove the treatment solution. These washed mycelial discs were aseptically transferred upside-down to the SDA medium (pH 5.6) in the petri plates. The same procedure was followed with the control sets. The inoculated petri plates were incubated at 27 ▒ 1░ C and the observations recorded on seventh day. Percentage of fungal growth inhibition (FGI) was calculated according to the formula1.
where dc = colony diameter of control disc, and dt = colony diameter of treated disc.
The effect of inoculum density (increased progressively up to 30 discs in multiples of 5 and each of 5 mm diameter) of the test pathogens on toxicity of the oil was determined according to the procedure outlined by Dikshit and Dixit8, using the agar-free medium as adopted by Shahi et al.9. The expiry of toxicity of the oil was determined by storing them at room temperature and testing their antifungal activity at minimum effective concentrations (MECs) at regular intervals of 60 days up to 60 months following the usual poisoned food technique6.
For determination of the effect of temperature, five lots of the oil were kept in small vials, each containing 5 ml of oil separately. These were exposed to different temperatures, 40, 60, 80 and 100░ C in the incubator for one hour. The antifungal activity of the oil was then tested at their MECs by the usual poisoned food technique6.
Antifungal spectrum of the oil was determined at
various concentrations, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 Ál/ml, against some other
test pathogens by the usual poisoned food technique6. Nature of toxicity, i.e.
fungicidal of the oil was determined by the method of Garber and Houston7.
The comparative study of the oil with some synthetic antifungal drugs was carried out by comparing MECs. The antifungal activity was assayed following the poisoned food technique6 against the test pathogens. The nature of toxicity (i.e., fungistatic/fungicidal, at their MECs) of the antifungal compounds was ascertained by the method of Garber and Houston7.
For in vivo investigations, experiments were carried out to find the maximum tolerable concentrations (MTCs) for irritant activity, if any, of the oil by their topical application on human skin, following the patch test method of Roxburgh and Borrie10. 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 areas of 5 cm2 on upper hairy and lower glabrous surface of palms and 3 cm2 of neck region of each individual were first washed with distilled water followed by 70% ethyl alcohol and then allowed to dry for five min. 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 observation have been recorded after 24 h.
For the clinical response of ointment
BSAD prepared from the oil, 1% concentration v/v of BSAD was applied topically
on human patients for the control of fungal infections (dermatophytoses)1. The
patients were treated with the ointment. Medication was administered twice a day for 3
weeks. The patients were not allowed
Table 1. Minimum effective
concentrations of the oil of Eucalyptus
citriodora against test pathogens
Table 2. Minimum killing time of the oil of E. citriodora against
Table 4. Patients showing clinical response of ointment prepared
drugs for dermatophytoses in guinea pig model may not be the same as in human dermatophytoses. As such, following the clinical trials conducted by Shahi et al.4 the present study was conducted for the topical testing of the oil of E. citriodora in the form of ointment BSAD on human patients perhaps for the first time. Although the undiluted oil of E. globulus has been shown to have some side effects when administered orally13, in the present investigation the oil of E. citriodora interestingly did not show any irritation or adverse side effects up to 5% concentration on human skin. The ointment BSAD effectively cured dermatophytoses at concentration of 1%. Reactions of drugs vary with partients, being dependent on individual age, sex, and their immune system. Therefore, if the drugs respond favourably in 50% patients, it can be used as an alternative to synthetic drugs. Hence, the oil of E. citriodora, owing to its strong antifungal activity, inhibiting heavy doses of inocula, with long shelf life, having fungicidal properties, and with no irritation on human skin, can be used successfully in the form of broad spectrum antimycotic drug for the control of fungal infection in human beings. The commercial viability of the oil can be determined after successful multicentre clinical trials, which is in progress.
ACKNOWLEDGEMENTS. Thanks are due to Head, Department of Botany, University of Allahabad, for providing the facilities, to Dr Uma Banerjee, Division of Microbiology, All India Institute of Medical Sciences, New Delhi, India, for providing the culture of dermatophytes; and to Council of Scientific and Industrial Research, New Delhi, for financial assistance.
Received 3 October 1998; revised accepted 24 December 1998