Antifungal Activity and Qualitative Phytochemical Analysis of Some Medicinal Plants in Jaffna (Sri Lanka)

Many members of the Labiatae family are used in traditional and folk medicine and also used as culinary and ornamental plants. Leaves are the most used plants parts of this family. Ethanolic extract of the leaves, stem, seeds of Leucas zeylanica, Ocimum canum, Ocimum sanctum and leaves of Mentha arvensis, Ocimum basilicum were subjected to phytochemical screening and antifungal assays against Aspergillus sp., Penicillium sp., Trichoderma sp., Mucor sp., Rhizopus sp. was determined by using the agar streaking assay method after 48 and 72 hours of incubation. All parts of the plants were found to contain flavonoid/s and alkaloid/s except for the absence of alkaloids in seeds of L. zeylanica and stem of O. sanctum respectively. Tannins were present in all parts of plants such as L. zeylanica, O. canum, M. arvensis and absent in O. sanctum and O. basilicum. Phlobatannins were only present in leaves of L. zeylanica and saponins were present only in leaves of O. basilicum. The leaves of L. zeylanica, O. basilicum, M. arvensis, O. sanctum and seeds of O. sanctum and O. canum showed the presence of steroids. Terpenoids were present in all parts of O. sanctum and O. canum than the other plants. The cardiac glycosides were present in all parts of O. sanctum than the other plants tested. Leaves of O. sanctum and M. arvensis exhibited strong positive antifungal activity against Aspergillus sp. Leaves of O. canum, O. basilicum and M. arvensis and stem of O. canum showed strong positive activity against Mucor sp. L. zeylanica only exhibited the antifungal activity against Mucor sp. Penicillium sp. was inhibited by the leaves and seeds extracts of O. sanctum. Degree of activity was low in L. zeylanica compared with other plant extracts. Most of these plant parts did not show any activity against Trichoderma sp. and Rhizopus sp. This study revealed that the antifungal activity of leaves of these plants was high than other plant parts against tested fungi.


Introduction
Many medicinal plants are a source of great economic value all over the world. Nature has bestowed on us a very rich botanical wealth and a large number of diverse type of plants grow in different parts of the country [1]. The Labiatae family (Lamiaceae) is one of the largest and most distinctive families of flowering plants. In Sri Lanka, there are 63 species belonging to 12 genera of these 51 are indigenous and 12 being endemic [2]. Leucas zeylanica (muditumpai-T; geta-thumba-S), Ocimum canum (ganjankorai-T; heentala-S), Ocimum sanctum (karunthulasi-T; madurutala-S), Ocimum basilicum (tirnutpachi-T; suwanda-tala-S) and Mentha arvensis (pudina-T; odutalan-S), are used in herbal medicine in Sri Lanka and other countries. Bioactive compounds with antimicrobial activity play an important role in herbal medicine. Objective of this study is the extraction of different plant parts in ethanol, identifying the phytochemicals qualitatively and screening for their antifungal activity.

Materials and Methods
The whole plant of L. zeylanica, O. canum, and O. sanctum were collected from uncultivated farmlands located at meesalai (chavakachcheri), Jaffna in the Northern Province of Sri Lanka. O. basilicum and M. arvensis were collected from farmlands at Thirunelvely (Jaffna) in the Northern Province of Sri Lanka.
The taxonomic identities of these plants were confirmed by using herbarium samples preserved in the department of Botany, University of Jaffna.

Method of Extraction of Plant Materials
The plant materials were air dried and these samples were ground into uniform powder with electric blender. 100 g of each ground samples were taken into a container separately and adequate amount of ethanol was added to an each container with occasional shacking. Each extract was filtered after 24 hours period.
This procedure was repeated and both filtrates were combined. The solvent was evaporated by using rotatory evaporator and the weights were recorded.

Phytochemical Screening Tests for Ethanol Extract
The phytochemical tests have carried out on the crude of the ethanol extract (0.2 g) using standard phytochemical procedures [3]. The compounds interests are alkaloids, saponins, terpenoids, steroids, flavanoids, tannins, phlobatannins, and cardiac glycosides.  Test for tannins: 0.2 g of crude of the sample was boiled with 20 ml of distilled water and it was filtered. Then few drops of 0.1% FeCl 3 were added.
Test for phlobatannins: 0.2 g of crude of the sample was boiled with 1% aqueous hydrochloric acid.
Test for saponins: 0.2 g of crude of the sample was boiled with 20 ml of distilled water in a water bath and it was filtered. Then filtrate was mixed with 5 ml of distilled water and it was shaken vigorously for a stable persistent froth.
Test for flavonoids: 0.2 g of crude of the sample was heated with 10 ml of ethylacetate over a steam bath for 3 minutes. The filtrate was shacked with 1 ml of dil NH 3 solution.
Test for steroids: 2 ml of acetic anhydride to 0.2 g of crude sample with 2 ml of con H 2 SO 4 were added.
Test for terpenoids: 3 ml of con H 2 SO 4 was added carefully to the mixture of 5 ml of extract and 2 ml of CHCl 3 .
Test for cardiac glycosides: Treat 0.2 g of crude with 2 ml of glacial acetic acid which contains one drop of FeCl 3 solution and this underplay with 1 ml of con H 2 SO 4 .

Anti Fungal Activity Assay for the Ethanol Extracts
All plants extracts were tested for their antifungal activity against different fungi such as Pencillium sp., Aspergillus sp., Rhizopus sp., Mucor sp. and Trichoderma sp. Antifungal activity of the extracts was determined by using the agar streaking assay method [4]. The extracts of L. zeylanica (seeds, stem and leaves) were streaked on Potato Dextrose Agar (PDA) in the Petridish at equal distance among them. A fungal disc (5 mm) (Penicillium sp.) was taken with the help of sterile cork borer and it was placed on the centre of above PDA medium. The antifungal activity was determined in terms of inhibition/antagonism (strong positive, weak positive and no activity) after 48 hours, 72 hours of incubation periods. All these test plates were compared with control plate. The above procedure was repeated to fungi such as Aspergillus sp., Mucor sp., Trichoderma sp. and Rhizopus sp. and other plants.

Results and Discussion
The phytochemical analysis of five medicinal plants is summarized in Table 1.      This study revealed that the extract of O. basilicum and O. sanctum and M. arvensis contained significant amount of antifungal compounds and most of these compounds were present in leaves than other parts of these plants.

Conclusion
The present study of medicinal plants in family Labiatae showed that distribution of phytochemicals such as alkaloids, terpenoids, steroids, flavanoids, tannins and cardiacglycosides were rich compared to the distribution of phlobatannins, and saponins. Degree of antifungal activity varied among plants as well as among plant parts. Growth of Aspergillus sp., Mucor sp. and Penicilium sp. were strongly inhibited by O. sanctum, O. basilicum and M. arvensis extracts while Trichoderma sp. and Rhizopus sp. growth was only inhibited by O. canum, O. sanctum. Antifungal compounds were rich in leaves than the other parts except L. zeylanica. Leaves of Labiatae family members except L. zeylanica could be used to treat fungal diseases in herbal medicine. Further studies could be carried out to purify these bioactive compounds.