Nature’s Gold

1. Nature’s Gold Essential Oils and Cancer Research Possibilities Nicole Stevens

2. What is an Essential Oil? The Essence of a Plant! Defense Chemicals Regulatory Chemicals Aromatic Chemicals Life & Death Chemicals

3. Ancient Knowledge Egyptians (prior to 1500 B.C.) • Frankincense and Myrrh considered sacred • Sandalwood, clove, lemon used for cleansing, antibiotics Middle Eastern Peoples (~1500 B.C.) • Frankincense and Myrrh mentioned in many religious texts • Modern steam distillation methods developed by Persian doctor Greeks (~400 B.C) • Hippocrates and Diodes encourage essential oil use for health Romans (~50 A.D.) • “De Materio Medica”: book on healing properties of many herbs Europeans (12th Century A.D.) • Perfumers and spice traders escape the Black Plague by topically applying essential oils

4. Modern Rediscovery René-Maurice Gattefossé, Ph.D. (1910) • Hand badly burned in a laboratory fire—saved by application of Lavender essential oil • Develop essential oils for clinical applications Jean Valnet, M.D. (1941) • Saves many World War II soldiers in the battlefield using essential oils Margaret Maury (1960) • Developed methods of applying essential oils along the body’s nerve pathways

5. Current Research • Stevens 2002: Many essential oils can inhibit growth of cancer cells in culture • Worked with fairly high concentrations of oil for purposes of screening • Stevens 2005: Lower concentrations of active essential oils • Photodynamic Therapy (PDT)

6. How PDT Works: Sensitization 2. Drug can stimulate production of cell’s own photosensitive molecules (porphyrins) Photosensitizer 1. Photosensitive molecules themselves can build up in cell Cancer Cell

7. How PDT Works: Treatment 1. Light excites sensitive molecules 2. Excited molecules damage DNA and proteins Light 3. Sufficient damage causes cell death Cancer Cell

8. Traditional PDT: Drawbacks • Non-specificity of sensitization • Variable stability of photosensitizers in vivo • Toxicity of photosensitizers • Limited adaptability

9. The Big Question Can Essential Oils be used in Anti-Cancer Photodynamic Therapy?

10. Essential Oil-based PDT • Some essential oils have been shown to have a photosensitizing effect following exposure to light • Only in sensitive individuals • Lower incidence of toxicity • Highly adaptable (topical application, oral administration, inhalation, injection) • Possible synergistic effects • Kill cancer cells while simultaneously protecting normal cells, stimulating immune system, etc. • Very little research has been done on the use of essential oils in photodynamic therapy against cancer cells

11. The Starting Lineup Why?

12. Traditional and Current Uses • Bergamot (Citrus bergamia) • Traditional uses: bloating and stomachache, bladder and blood problems, antimicrobial, relaxant, anti-depressant • Current uses: flavoring and perfumes, cleansing and refreshing, relaxant, skin ailments (acne, cold sores) • Recent Research: Kawaii et al (1999): Bergamot extract causes differentiation of HL-60 human leukemia line Romano et al (2005): Bergamot oil active against Candida yeast infections Shao 2003: Bergamot may play a role in skin and hair growth (Source: www.darbycreeks.org/ Bergamot.jpg )

13. Traditional and Current Uses • Dill (Antheum graveolens) • Traditional uses: analgesic, sleep-inducer, halitosis, antispasmodic, relief of intestinal spasms, flatulence and cholic • Current uses: flavoring, anti-microbial, digestive ailments, intestinal muscle relaxant, possible blood pressure reductant • Recent Research: Souri et al (2004): Dill extract showed strong antioxidant activity Hosseinzadeh et al (2002): Dill extract protects and soothes intestinal tract in ulcer-induced mice Zheng et al (1992): Dill induces detoxifying enzyme GST, may play important role in chemoprevention (Source: www.milkandhoneyfarm.com/ garden/images/dill.jpg)

14. Traditional and Current Uses • Grapefruit (Citrus x paradisi) • Traditional uses: antiseptic, flavoring and perfume, fluid retention, cleansing (both internal and external), astringent • Current uses: dietary supplement and flavoring, aromatherapy (for stress and depression), antiseptic, disinfectant • Recent Research: Negi et al (2001): Grapefruit extract active against Gram+ and Gram- bacteria Ortuno et al (1997):Coumarins from grapefruit give insect and fungal resistance Robbins et al (1998): Grapefruit lowers elevated hematocrit levels, raises low levels (Source: www.botany.hawaii.edu)

15. Traditional and Current Uses • Lemon (Citrus limon) • Traditional uses: antiseptic, antimicrobial, improve immune function, improve circulation, external cleansing • Current uses: dietary supplement, stress and digestion problems, antidepressant, cleanser and stain remover, antimicrobial • Recent Research: Masahiro et al (2002): Lemon oil inhibits elastase, an enzyme which degenerates dermal elastin Vigushin et al (1998): Limonene from lemon oil showed low cytotoxicity in Phase I clinical trials Lis-Balchin and Dean (1997): Lemon essential oil showed activity against Listeria (Source: www.illustratedgarden.com, MBG Rare Books)

16. Traditional and Current Uses • Lemongrass (Cymbopogon citratus) • Traditional uses: antiseptic, cough, antifungal, expectorant, analgesic, antimicrobial, heacaches, edema, digestion • Current uses: support proper blood glucose levels, external cleanser, fever reducer, inflammation, diuretic • Recent Research: Formigioni et al (1986): Lemongrass extract did not produce cytotoxicity in rats Melo et al (2001): Lemongrass extract helps protect cells from induced oxidative damage Oyedele et al (2002): Lemongrass extract is an effective mosquito repellent Ohno et al (2003): Lemongrass extract kills Helicobacter pylori (Source: http://davesgarden.com/pf/showimage/1036/)

17. Traditional and Current Uses • Orange (Citrus aurantium) • Traditional uses: antiseptic, antimicrobial, improve immune function, rickets, digestive problems, fluid retention, lowering cholesterol • Current uses: dietary supplement, stress and digestion problems, antidepressant, cleanser and stain remover, antimicrobial • Recent Research: Ramadan et al (1996): Orange oil as a potent topical anti-fungal agent Vargas et al (1999): Orange oil has antimicrobial and antioxidant properties Bodake et al (2002): Orange oil has chemopreventive effects on the development of induced tumors Reddy et al (1997): Perillyl alcohol from orange oil in colon cancer chemoprevention (Source: www.illustratedgarden.com, MBG Rare Books)

18. Materials and Methods • Cell lines • Cancer lines • C6 = Brain glioma • DC4 = Breast ductal carcinoma • HeLa = Cervical adenocarcinoma • Jurkat = T-cell leukemia • Essential Oil Delivery Vehicle • DMSO = Dimethylsulfoxide (Source: http://www.priondata.org/data/A_DMSO.html)

19. Materials and Methods • Essential Oil concentrations tested • 0.01% (100ppm) • 0.001% (10ppm) • 0.0001% (1ppm) • Light wavelength: UVA (320-400nm) • Viability assay: AlamarBlue 1. Non-fluorescent blue solution is added to cells 2. Live cells will change the blue substrate into pink fluorescent solution 3. Fluorescence is read on a spectrophotometer

20. Results

21. Results

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23. Results

24. Results

25. Results

26. Results HeLa Bergamot No UV Bergamot + UV

27. Results C6 Bergamot No UV Bergamot + UV

28. Results Signs of Death

29. Conclusions • Cell lines respond differently to treatment • Some essential oils appear to function as photosensitizing agents • While some essential oils alone can kill cancer cells, in some cases there is a significant increase in cell death when treated with light as well • Generally, response is dose-dependent, and cancer cells respond to very small doses

30. Future Research • Optimize essential oil concentration • More effective oils? • May depend on cancer type • Minimize non-specific activation • Optimize wavelength of PDT treatment • May depend on cancer type • Most recent research indicates multiple wavelengths may be effective • Optimize time of treatment • Toxicity in non-cancer cells • Efficacy in a living system

31. References • Girard, J., J. Unkovic, J. Delahayes, and C. Lafille. 1979. [Phototoxicity of Bergamot oil. Comparison between humans and guinea pigs.] Dermatologica 158(4): 229-43. • Kavil, G., and G. Volden. 1984. Phytophotodermatitis. Photodermatol. Apr 1(2): 56-75. • Yasui, Y., and T. Hirone. 1994. Action spectrum for bergamot-oil phototoxicity measured by sunburn cell counting. J Dermatol May 21(5):319-22. • Naganuma, M., S. Hirose, Y. Nakayama, K. Nakajima, and T. Someya. 1985. A study of the phototoxicity of lemon oil. Arch Dermatol Res. 278(1): 31-6. • Zaynoun, S.T., B.E. Johnson, and W. Frain-Bell. 1977. A study of oil of bergamot and its importance as a phototoxic agent. I. Characterization and quantification of the photoactive component. Br J Dermatol. 96(5): 475-82. • Zaynoun, S.T., B.E. Johnson, and W. Frain-Bell. 1977. A study of oil of bergamot and its importance as a phototoxic agent. II. Factors which affect the phototoxic reaction induced by bergamot oil and psoralen derivatives. Contact Dermatitis 3(5): 225-39. • Lowe, N.J. 1986. Cutaneous phototoxicity reactions. Br J Dermatol. Aug 115(Suppl 31): 86-92. • Allen, J.E. 1993. Drug-induced photosensitivity. Clin Pharm. Aug 12(8): 580-87. • Karbownik, M., D. Tan, L.C. Manchester, and R.J. Reiter. 2000. Renal toxicity of the carcinogen delta-aminolevulinic acid: antioxidant effects of melatonin. Cancer Lett. 161(1): 1-7. • Lemberkovics, E., A. Kery, G. Marzcal, B. Simandi, and E. Szoke. 1998. Acta Pharm Hung. 68(3): 141-9. • Egan, C.L., and G. Sterling. 1993. Phytophotodermatitis: a visit to Margaritaville. Cutis. 51(1): 41-2. • Kawaii S, Tomono Y, Katase E, Ogawa K, and Yano m. 1999. HL-60 Differentiating Ativity and Flavonoid contents of the readily extractable fraction prepared from citrus juices. J Agric Food Chem 47: 128-135. • Romano L, Battaglia F, MasucciL, Sanguinetti M, Posteraro B, Plotti G, Zanetti S, Fadda G. 2005. In vitro activity of bergamot natural essence and fucocoumarin-free and distilled extracts, and their associations with boric acid, against clinical yeast isolates. J Antimicrob Chemother 55(1) 110-114) • Shao, LX. 2003. Effects of the extract from bergamot and boxthorn on the delay of skin aging and hair growth in mice. Zhongguo Zhong Yao Za Zhi 28(8): 766-9. • Souri E, Amin G, Farsam H, Andaji S. 2004. The antioxidant activity of some commonly used vegetables in Iranian diet. Fitoterapia. 75(6): 585-8. • Hosseinzadeh H, Karimi GR, Ameri M. 2002. Effects of Anethum graveolens L. seed extracts on experimental gastric irritation models in mice. BMC Pharmacol. 2(1): 21. • Zheng GQ, Kenney PM, Lam LK. 1992. Anethofuran, carvone, and limonene: potential cancer chemopreventive agents from dill weed oil and caraway oil. Planta Med. 58(4) 338-41.