Summary of Thesis written for PhD Award
It was found that the incidence of fungal pathogens is increasing day by day. Aspergillus fumigatus (10.23%) occurred frequently followed by yeasts (Malassezia furfur, Candida albicans (9.3%) and Fusarium solani (8.83%). The yeasts- Candida albicans, C. pseudotropicalis, Malassezzia furfur, Torula herbarum, Cryptococcus neoformans and two yeast-like fungi viz., Blastomyces dermatitis and Sporothrix schenckii were also recovered. It was noticed that the frequency of the yeast infections was relatively high. Exophiala spinifera also produces yeast-like cells and has been recovered in the present studies.
After Aspergillus fumigatus and yeast infections, Fusarium moniliforme and F. oxysporum were the most frequently occuring species. Other important opportunistic fungi causing infections in men were found to be Alternaria alternata, Curvularia clavata, C. verruciformis, Exophiala spinifera, Helminthosporium sp., Cladosporium, Phoma multirostrata and Trichoderma etc.
Not only the opportunistic fungi, some dermatophytes, viz., Trichophyton mentagrophytes (8.3%) and T. rubrum (9.3%) were also isolated. Since there is a greater need to search for suitable antifungal agents for mycotic infections in general and newly emerging opportunistic fungal pathogens in particular, an opportunistic pathogen- Fusarium oxysporum and a potential human pathogen-Trichophyton mentagrophytes were selected to evaluate their sensitivity to different locally available plants of family Asteraceae.
A thorough survey and collection of plants of Asteraceae was made for evaluation of antimycotic potential present in them. There were 60 species belonging to 44 genera of Asteraceae family occurring in Chhindwara District of Madhya Pradesh. Parthenium is found to be the dominant genus, while Blumea is represented by the maximum number of species. On the other hand, Siegesbeckia was recovered from Tamia only. Most of the plants of family Asteraceae are highly medicinal. Nomenclature of the members is updated.
The present floristic study of the district records of family Asteraceae showed that Parthenium hysterophorus occurred most frequently. Most genera were represented by one or two spp. Blumea was found to be the largest genus represented by 5 species, whereas Siegesbeckia was present only in Tamia. Guizotia, Tagetes, Helianthus, Carthamus and Spilanthes are the member cultivated in Chhindwara Distt. Some weeds, viz., Acanthospermum, Ageratum, Parthenium, Xanthium, Pentanema, Cichorium and Caesulia occurred in cultivated as well as waste lands of Chhindwara. Among the medicinal plants, Eclipta alba and Spilanthes calva are cultivated. It was noticed that Parthenium hysterophorus, Xanthium strumarium, Tridax procumbens, Ageratum conyzoides and Acanthospermum hispidum are the common weeds occurred in all areas.
Essential oils of Ageratum conyzoides, Blumea mollis, B. balsamifera, Caesulia axillaris, Chrysanthemum indicum, Cyathocline purpurea, Dahlia pinnata, Eupatorium triplinerve, Spilanthes acmella, Tagetes erecta and T. patula were tested for fungitoxic activity against F. oxysporum and T. mentagrophytes. It was noted that essential oils obtained from flowers of Tagetes erecta and T. patula exhibited the maximum inhibition of F. oxysporum and T. mentagrophytes. Plants like Cyathocline purpurea, whole plant of Tagetes erecta, Eupatorium triplinerve and Tagetes patula (whole plant) showed significant inhibition, while the minimum inhibition was represented by oil of Dahlia pinnata, Ageratum conyzoides and Chrysanthemum indicum. The essential oil of Eupatorium triplinerve was much effective against F. oxysporum and T. mentagrophytes. It was interesting to record that most of the essential oils tested in the present investigation were more effective for T. mentagrophytes as compared to F. oxysporum.
The extracts of plants of family Asteraceae of Chhindwara were also screened to search for their fungitoxic potential. These include: Acanthospermum hispidum, Ageratum conyzoides, Blainvillea acmella, Blumea balsamifera, B. mollis, Caesulia axillaris, Carthamus tinctorius, Cichorium intybus, Cyathocline purpurea, Dahlia pinnata, Echinops echinatus, Eclipta alba, E. prostrata, Elephantopus scaber, Emilia sonchifolia, Erigeron asteroides, Eupatorium triplinerve, Galinsoga perviflora, Gnaphalium albo-lutium, G. purpureum, Launaea acaulis, Parthenium hysterophorus, Spilanthes acmella, Synedrella nodiflora, Tagetes erecta, T. patula, Tithonia diversifolia, Tridax procumbens, Vernonia cineria, V. divergens and Xanthium strumarium. It was recorded that both the species of Tagetes, viz., T. erecta and T. patula checked the mycelial growth of F. oxysporum and T. mentagrophytes most efficiently. It was also noted that extract of T. erecta was more fungitoxic than T. patula. In the former, extract of the flowers showed the maximum inhibition, whereas the leaves exhibited the least growth. It is probably due to presence of more bio-active principles in flowers as compared to other parts of the plant. The extracts of plant species other than Tagetes which were found to be effective inhibitor of mycelial growth of F. oxysporum include: Cyathocline purpurea followed by Parthenium hysterophorus, Blumea balsamifera, B. mollis, Eupatorium triplinerve, Tithonia diversifolia, Eclipta prostrata, Ageratum conyzoides, and Galinsoga parviflora, whereas the minimum inhibition was exhibited by extracts of Spilanthes acmella followed by Emilia sonchifolia and Vernonia divergens. Other plant extracts remarkably effective against Trichophyton mentagrophytes, were found to be Eupatorium triplinerve followed by Parthenium hysterophorus, Gnaphalium albo-luteum, Blumea balsamifera, B. mollis, Cyathocline purpurea and E. alba, while the minimum inhibition was exhibited by extracts of Emilia sonchifolia, Tridax procumbens and Cichorium intybus. It was interesting to note that extracts of Elephantopus scaber and Launea acaulis stimulated the growth of T. mentagrophytes.
The screening of seven Asteraceous plants, viz., Caesulia axillaris, Ageratum conyzoides, Tagetes erecta, T. patula, Eupatorium triplinerve, Blumea mollis and B. balsamifera in combination was carried out against two human pathogenic fungi viz., Trichophyton mentagrophytes and Fusarium oxysporum. Oxyconazole nitrate was taken as control. The combination of essential oils of Tagetes erecta with T. patula and T. patula with Eupatorium triplinerve showed highest fungitoxic potential against both the test fungi. Tagetes erecta with Eupatorium triplinerve showed the same efficacy against F. oxysporum but this combination showed less inhibition of Trichophyton mentagrophytes. Further, relatively less activity was observed in the combination of T. erecta with B. mollis against both human pathogenic fungi. Combinations of Caesulia axillaris with Tagetes patula and Caesulia axillaris with Eupatorium triplinerve showed relatively less efficacy against Fusarium oxysporum. No remarkable antifungal activity was observed in the combinations of B. mollis, B. balsamifera and Ageratum conyzoides. Oxyconazole nitrate, taken as control, showed better antimycotic activity against Fusarium oxysporum as compared to Trichophyton mentagrophytes.
The highest inhibition against Fusarium oxysporum and Trichophyton mentagrophytes was shown by the combination of Tagetes erecta with Eupatorium triplinerve followed by combinations of Tagetes erecta with T. patula and T. patula with Eupatorium triplinerve. Combination of Tagetes erecta with B. mollis showed similar results as compared to oxyconazole nitrate. There were no remarkable results shown by the other combination of essential oils we studied. Fusarium oxysporum was more sensitive than Trichophyton mentagrophytes. Oxyconazole nitrate was more effective against F. oxysporum. Oils of Tagetes erecta, T. patula and Eupatorium triplinerve, in combination, were more efficaceous than those of azoles.
Antimycotic activity of essential oils of 11 Asteraceous plants singly and in combination with azoles was also investigated. The test plants include: Ageratum conyzoides, Blumea balsamifera, B. eriantha, B. mollis, Caesulia axillaris, Chrysanthemum indicum, ahlia pinnata, Eupatorium triplinerve, Guizotia abyssinica, Tagetes erecta and T. patula. The synthetic drugs, viz., miconazole nitrate, ketoconazole nitrate and oxyconazole nitrate were combined with those of Asteraceous essential oils. Encouragingly, essential oils of Tagetes erecta and T. patula were found to be remarkably active against both the human pathogens. Trichophyton mentagrophytes and Fusarium oxysporum were more sensitive to Tagetes erecta followed by T. patula and Eupatorium triplinerve. Tagetes erecta completely inhibited Trichophyton mentagrophytes when tested singly. On the otherhand, oxyconazole nitrate, when treated singly, showed complete inhibition of Fusarium oxysporum. However, Guizotia abyssinica, Chrysanthemum indicum and Dahlia pinnata did not show inhibiting properties.
Essential oils mixed with synthetic drugs showed more efficacy as compared to that of tested singly. The combination of Tagetes erecta and all three synthetic drugs viz., miconazole nitrate, ketoconazole nitrate and oxyconazole nitrate checked the growth of Fusarium oxysporum completely. It also inhibited the mycelial growth of Trichophyton mentagrophytes completely when tested in combination with ketoconazole nitrate and oxyconazole nitrate. It showed less activity with miconazole nitrate. Similarly, oxyconazole nitrate mixed with Eupatorium triplinerve and Tagetes patula inhibited Fusarium oxysporum completely. Eupatorium triplinerve and Tagetes patula did not show complete inhibition in combination with miconazole nitrate and ketoconazole nitrate, but the results were significant. Trichophyton mentagrophytes was less sensitive when treated with the combination of Blumea mollis and azoles, while, Fusarium oxysporum was more sensitive against the combination of B. mollis and azoles. Combination of Ageratum conyzoides with azoles showed less efficacy against both the test fungi as compared to other plants tested. No significant results were observed when azoles were mixed with Guizotia abyssinica.
Oxyconazole nitrate showed maximum antifungal activity among the azoles. It was more effective against Fusarium oxysporum followed by miconazole nitrate and ketoconazole nitrate. Miconazole nitrate showed significant activity against Trichophyton mentagrophytes. Combination of Tagetes erecta with oxyconazole nitrate showed highest efficacy against Fusarium oxysporum and Trichophyton mentagrophytes followed by the combination of T. patula and oxyconazole nitrate against F. oxysporum and T. mentagrophytes. After Tagetes erecta and T. patula, the mixture of Eupatorium triplinerve and oxyconazole nitrate showed significant antimycotic results against both the fungi. The efficacy of combination of three Asteraceous essential oils viz., Tagetes erecta, T. patula and Eupatorium triplinerve mixed with azoles enhanced the antifungal efficacy of synthetic drugs. Minimum efficacy was observed in combination of B. mollis followed by B. eriantha, B. balsamifera, Ageratum conyzoides and Guizotia abyssinica. The remarkable activities of essential oils extracted from Tagetes erecta, T. patula and Eupatorium triplinerve on Fusarium oxysporum and Trichophyton mentagrophytes suggests that they are excellent antimycotic agents. The synthetic drugs did not inhibit fungal growth completely when tested singly, while, in the combination of azoles, essential oils of Tagetes erecta, T. patula, Eupatorium triplinerve inhibited growth fully. Therefore, combination of natural drugs with synthetic drugs should be studied in detail.
Efficacy of different concentrations of Asteraceous oils of Tagetes erecta, T. patula and Eupatorium triplinerve was also studied to evaluate the antimycotic potential. Pure oil of Tagetes erecta completely checked the growth of the test fungi followed by Eupatorium triplinerve and T. patula. All the essential oils tested were more efficaceous than oxyconazole nitrate at all concentrations. It is interesting to note that 50% concentration of Tagetes erecta oil was more effective than 100% concentration of oxyconazole nitrate. The 50% concentration of oils of Tagetes erecta and Eupatorium triplinerve inhibited the growth of test fungi completely while at the same concentration less inhibition was shown by the oxyconazole nitrate. At 12.5% concentration, Tagetes erecta and Eupatorium triplinerve showed highest inhibition followed by T. patula and oxyconazole nitrate.
It can be concluded that the essential oils of Tagetes erecta, T. patula and Eupatorium triplinerve can be used as natural antimycotics. Even at the low concentration, essential oils of these plants showed very significant antimycotic activity against human pathogenic fungi. Combination of the extracts and essential oils of these plants can be prepared for developing plant derived antifungal drugs. The volatile fractions of the plants can be mixed with the synthetic drugs for enhancing the antimycotic efficacy of the azoles.
In a nutshell, Tagetes erecta, T. patula, Eupatorium triplinerve, Cyathocline purpurea, Parthenium hysterophorus and Blumea can be utilized for the preparation of topical antimycotics. However, a detailed bio-chemical and in vivo studies are needed on experimental animals before their utilization as antimycotic drugs.