1. Combined Effect of Heat, Essential Oils and Salt on the Fungicidal Activity against Trichophyton mentagrophytes in Foot Bath Shigeharu Inouye Katsuhisa Uchida Yayoi Nishiyama Yayoi Hasumi Hideyo Yamaguchi Shigeru Abe Instructor ：藍清隆 Speaker ：黃于庭

2. Abstract  Determine the effective conditions of essrntial oils against Trichophyton mentagrophytes in vitro for the treatment of tinea pedis in a foot bath.  Most fnugal mycelia were killed after treatment at 42°C for 20 min without oil.  The fungicidal activity of essential oils was 42°C>27°C

3. Introduction  Tinea pedis is one of the most prevalent superficial infections.  Foot baths are frequently used in aromatherapy, spa therapy and balneotherapy for treating various skin disorders.

4.  Treatment conditions, especially the thermal and salt effects of a foot bath using a variety of essential oils were the main target in this study.

5. Materials and Methods

6. Fungal strain  T. mentagrophytes TIMM 2789 is a  stock culture of Teikyo University  Institute of Medical Myocology.  T. rubrum TIMM 5497 was a clinical  stain freshly isolated.

7. Essential oils and chemicals

8. Counting of Trichophyton cells

9. 100ml microconidia suspension Incubate at 27°C Agar block 含有菌絲的 medium Incubate at 27°C For 5 days 10 7, 10 6, 10 5, 10 4, 10 3 and 10 2 /dish

10. MCF  The minimum fungicidal concentration

11. Determination of the thermal stability of T. mentagrophytes hyphae and fungicidal activity of essential oils in a water bath

12. A DMSO solution of essential oils at 640, 160, 40 mg/ml diluted with 0.1% aqueous agar test sample with oil concentrations of 0.32, 0.16, 0.08, 0.04, 0.02, 0.01 and 0.005%

13. essential oils 256, 64, 32 mg suspended in 0.1% aqueous agar suspensions with concentrations of 5.12, 2.56, 1.25 and 0.64%

14. A DMSO solution of Clotrimazole (1 mg/ml) diluted with 0.1% aqueous agar suspensions of 0.008, 0.004, 0.002, 0.001, and 0.0005%

15. 10 7 /ml agar block 5ml 0.1% aqueous agar 10%sodium chloride-0.1% aqueous agar 剛剛配置好不同濃度的精油 27 ° C, 37 ° C, 42 ° C and 47 ° C in a water bath for 10 and 20min

16. washed with sterile saline excess saline was removed The number of surviving cells on the agar blocks was determined by the standard curve.

17. Determination of the thermal stability of conidia of T. mentagrophytes in the presence or absence of the oils

18. 5ml aqueous conidia suspension 0.1% agar containing 0.05% DMSO heated at 27 ° C for 20 min 42 ° C for 20 min and 40 min

19. 5ml aqueous conidia suspension 0.02% and 0.2% oregano oil or 0.02% and 0.2% patchouli oil containing 0.05% DMSO heated at 27 ° C for 20 min 42 ° C for 20 min and 40 min +

20. 200  l conidia suspension diluted 10-fold and 100-fold incubated at 27 ° C for 5 and 7 days

21. Preparation of dried mycelia of T. mentagrophytes TIMM 2789

22. 100ul aqueous conidia suspension added broth cultured at 30 ° C for 1 week under shaking at 150 rpm

23. The mycelia were collected by centrifugation washed three times with ddH2O suspendedin 200ml of ddH 2 O to sonicate with a gel sonicator washed with ddH 2 O lyophilized for 3 days The dried power was 1.89 g

24. Determination of adsorption of oil constituents on mycelia of T. mentagrophytes

25. The dried mycelia was suspended at 42 ° C in 0.1% aqueous agar or 10%sodium chloride-0.1% aqueous agar 0.16% tea tree 0.08% lavender 0.02% thyme thymol shaking at 42 ° C for 20 min 3500 rpm 離心後用濾紙除去多餘的水分

26. The mycelia were extracted with ethyl actate, and the extract was dried over anhydrous sodium sulfate 氣相色譜分析法

27. Scanning electron microscopy

28. 27 ° C or 42 ° C for 20min with or without oil fixed cacodylate buffer at 4 ° C(pH7.2) after 1~16hr fixed 1% osmium tetroxide in cacodylate buffer at 4 ° C(pH7.4) 丙酮脫水後使用二氧化碳臨界乾燥機乾燥 鍍上鋨後使用 SEM 觀察

29. Results and Discussion

30. Effect of temperature and treatment time on the viability of T. mentagrophytes in 0.1% aqueous agar with or without 10% sodium chloride

31. `

32. The sodium chloride didn't effect the viability.

33. Anti-Trichophyton activity of tea tree oil and thyme thymol oil at various temperatures and treatment times in 0.1% aqueous agar with or without 10% sodium chloride

35. Treatment time could be saved by raising temperature.

36. Effect of temperature and essential oils on the viability of T. rubrum and T. mentagrophytes in 0.1% aqueous agar

38. The conidia of T. mentagrophytes were heat-labile, and more than 90% of the conidia did not germinate after heating at 42°C for 20 min and 40 min.

39. Anti-Trichophyton activity of oregano, cinnamon, bark, lemongrass, clove, palmarosa, peppermint, lavender, geranium "Bourbon", and thyme geraniol at 42°C for 20 min in 0.1% aqueous agar with or without 10%sodium chloride

41. Effect of sodium chloride on adsorption of oil constituents to the mycelia of T. mentagrophytes

43. Effect of heat and essential oils on the hyphal morphology of T. mentagrophytes

47. Cellular damage at 42°C caused by essential oils was comparable to or less than that at 27°C, and no heat effect was recognized on the morphology.

48. Thank you