In-Vitro Antimycotic Potential Of Thuja-occ against Curvularia-lunata causing Phaeoyphomycosis In Human
NATIONAL JOURNAL OF HOMOEOPATHY 2002 May / Jun VOL 4 NO 3.
Dr Girish Gupta

Abstract
Various potencies of a homoeopathic drug, Thuja-occidentalis-Q 30, 200, 1M, 10M and 50M and Ketoconazole (a synthetic dioxolane imidazole compound-a broad spectrum antifungal) as a positive control were evaluated by following Poison food method. Thuja 30 and 200 was found highly effective even more than positive control and vehicle. Linear diametric growth, sporulation and exudation were taken as parameters of assessment.

Introduction
Curvularia is a common soil saprophyte and plant pathogen that has been isolated from cases of mycotic keratitis, endocarditis and PhaeHoyphomycosis 1, 6, 7,10,11,13,16,17, 19, 20, 24. Mycotic infarcts were voided in the heart, spleen, left kidney and right cerebral hemisphere. Curvularia geniculata were grown from all infected material. Harris and Dawnham described a disseminated case involving Curvularia that occurred in a football player⁵, Rohwedder et al. recorded a 25 year old immuno-competent man with a 10 year history of progressive disseminated infection caused by Curvularia lunata¹⁴. Phillips et al reported on sinusitis caused by Curvularia lunata¹². Monte et al. also described a disseminated infection of Curvularia. In experimental infection⁸, Whitecomb et al. found that Curvularia lunata was capable of producing infection in mice, but other species of Curvularia were non-infective23. In mice infected with Curvularia lunata, lesions were found in liver and spleen. The drugs amphotericin B, miconazole and ketoconazole were found effective but toxicity and treatment duration are limiting factor for their use.
Thuja-occidentalis, (American Arbor-vita; white cedar) a tall tree belongs to the family of cupressaceae, introduced in India from North America and are being cultivated in plains of India. Thuja is considered as antisycotic homoeopathic drug used mainly for wart like excrescences upon mucus and cutaneous surface, vegetative condylomata and spongy tumors. Thuja is known to contain oil of thuja, a thujol, flavone glycoside thujin and an acid called thujin. £-Thujone, d-iso-thujone and I-fenchone are the major constituents of the essential oil from leaves and terminal branches. Others include £ and â pinene, sabinene, myrcene, £ phellandrene, terpinene, limonene, 1, 8 - coneole, ___ terpinene, p-cymene, terpinolene, £ fenchylacetate, camphor, linalool, isothujyle acctate, terpinene-4-ol and borneol. Antifungal efficacy of Thuja-occidentalis against Candida albicans, Trichophyton-rubrum, Trichophyton-mentagrophytes, Aspergillus-flavus and Aspergillus-niger has been reported². Present paper explores in-vitro antifungal potential of Thuja-occidentalis in Q, 30, 200, 1M, 10M, 50M potencies against Curvularia-lunata known to cause Phaeohyphomycosis (check MS for the correct spelling) in human.

MATERIALS AND METHODS: FUNGAL ISOLATE
Curvularia-lunata was isolated from a patient complaining of hyperpigmented, dry, scaly skin lesions on face, neck, and forearms since 6 months. MIC value of Ketoconazole calculated for Curvularia-lunata was 0.25 mg/ml by following the method of Wahab et al^21-22.
MEDICINE
Thuja-occidentalis in various potencies viz. Q, 30, 200, 1M, 10M, 50M was used in the present study. Quantity of drug was standardised to be 0.5ml/petridishes.

CONTROLS
Three controls were taken, one of sterile water (autoclaved), second of vehicle i.e. rectified spirit (90% v/v) and third of Ketoconazole (Ranbaxy, India) as positive control. Quantity of Ketoconazole was taken as per MIC calculated.

EXPERIMENTAL PROTOCOL
Antimycotic activity of homoeopathic drug Thuja-occidentalis Q, 30, 200, 1M, 10M, 50M prepared in rectified spirit was tested by poison food technique (because of mycelial fungi)^18,19 against Curvularia lunata. 0.5 ml of each drug was mixed with 10 ml of Sabourauds dextrose agar (Himedia B No 8h181- Mycological peptone 10 g/l, dextrose 40.01 g/l, Agar 15.0 g/l, PH 5.6) supplemented with Chloramphenicol (Makers laboratories Ltd. India) to prevent bacterial growth just before solidification in 5 cm diameter Petri dishes. SDA plates with 0.5 ml sterile water, 0.5 ml rectified spirit and 0.5 ml containing 0.5 mg Ketoconazole (200 mg Tab, dissolve in 20 ml water) as per MIC of Curvularia lunata, were kept as controls. All the plates including controls were inoculated centrally with 1.5 mm diameter of disc of Curvularia lunata aseptically from 8 to 10 days old inoculum - original isolate of patients. All the experimental Petri dishes were used in triplicates and incubated at 37^oC + 1^oC. The linear diametric growth of colonies were measured (in cm.) on 3^rd, 6^th and 9^th days of post inoculation. Percent growth inhibition was calculated as per formula.

RESULTS
Antimycotic potential of homoeopathic drug Thuja-occidentalis Q, 30, 200, 1M, 10M and 50M against Curvularia-lunata have been presented in table-1 and plate 1. Thuja 30 and 200 were found to inhibit the growth of Curvularia-lunata by 93.33 percent in each as compared to control 1.91.03 percent in each as compared to control II (RS), but if it is compared by control I, Control II and Control III Thuja 30, Thuja 200 and Thuja 10M showed significant inhibition in linear diametric growth of drug treated colonies of Curvularia-lunata (Table-1, Plate 1 - Figures 5, 6 and 8).

Percent inhibition in the linear growth of Curvularia-lunata in Ketoconazole was 66.66 percent as compared to control I and 56.52 percent compared to Control II. It is highly significant that Thuja 30 and 200 have shown very high antifungal potential even more than the positive control - ketoconazole (Figured 10 and 11). Thuja 10 M was little higher to the positive control but Thuja 50M was almost equal to ketoconazole. Sporulation and exudation have shown no significant variations on the basis of naked eye observation. There was slight reduction in the spore count of Thuja Q but exudation (observed by change in colour of media) was prevented in all the petridishes except control I (Plate I - figure 1) and positive control Ketoconazole (Plate 1 - figure 3). There was no considerable changes in the morphology of fungal mycelium and spores size.

DISCUSSION
Mycoses particularly opportunistic infections have significantly increased^9,11,18 in the recent past. Eukariotic nature of fungal cells gives a barrier towards the development of non-toxic antifungal agent. Thus treatment available may not cause complete cure and recurrence of infection often takes place. Curvularia sp is one of the common soil saprophyte that has been isolated from several patients having the history of local injury or surgery. The genus has rarely been reported fatal⁶. Among all the reported species, Curvularia lunata has been found capable to produce infection in mice²³. Keeping this in view we have tried to explore In-vitro potential of homoeopathic drug. Thuja-occidentalis against Curvularia lunata. In our previous papers, activity of Thuja-occidentalis was reported against Aspergillus-flavus and Aspergillus-niger, Dermatophytes and Penicillium^2,3,4,16. The most controversial aspect of Homoeopathic dilution is that succussion and trituration actually increases the power of a medicine. Clinical efficacy of homoeopathic drug in human is well established and need not be discusses here but in fungal system there is no increase in activity of higher potency against Curvularia-lunata. As it is shown in the results that Thuja 30 and 200 showed high antifungal efficacy but 50M which is expected to show higher activity has not shown better effects. Thus vital force (unknown factor) of homoeopathic drugs to all living groups (Micro-organisms to higher animals and plants) are different and even varies from genera to genera and species to species. Thus the present finding does not support the concept of high dilution and high energy. Similar results were also observed by Singh and Gupta¹⁵ who reported in-vitro antiviral activity of homoeopathic drugs against animal viruses. In conclusion Thuja 30, 200 and 10M are very effective against pathogenic isolate Curvularia-lunata however other dilutions are not much effective. It is evident from Table -1 that no definite co-relation exists between various potencies of the same drug with regards to their antimycotic properties. Due to lack of experimental animals in our laboratory, in-vivo experiment could not be conducted which are needed for further investigations.

Continue Reading...

Back