H.T.T.Hai, C.T.T. Lam, T.D.Thang, N.X.Dung Chemical composition , TR. 42-46 42 Chemical composition of the fruit oil of Michelia sp. from Vietnam - A New Natural Source of Safrole Ha Thi Thanh Hai (a) , Chu Thi Thanh Lam (a) , Tran Dinh Thang (b) , Nguyen Xuan Dung (c) Abstract. The volatile components of the fruit oil of Michelia sp. from Nghean province, were analysed by Capillary Gas chromatograph (GC) and Gas chromatograph/Mass spectroscopy (GC/MS) as well as by background subtraction of coeluting compounds. A total 33 compounds were identified of which the major component was safrole (93.41%). The lesser amount were limonene (1.19%) and linalool (2.46%). This result shows that Michelia sp. from Nghe an as a new source of safrole. 1. Introduction The Magnoliaceae contains about 15 genera and ca. 300 species most of the species occur primarily in East and South-East Asia but also in South-East North America, and tropical America. There are 50 species of the genus Michelia distributed in tropical and subtropical Asia [1]. Michelia species are commonly cultivated for their fragrant flowers and as ornamentals. The flowers of some Michelia are marketed for their scent, used in making garlands, placed between stored clothes, sprinkled in bridal beds and used in the preparation of scented hair lotions. In Thailand an infusion of the flowers is applied as a cosmetic after bathing. In Indonesia an infusion made by steeping the bitter bark in water is given against fever. Several Michelia species, yield a timber of minor economic importance, used for light construction, vehicle bodies, packing cases and for the production of veneer plywood, wood-wood board and as fuel [2]. M. balansae (A.DC.) Dandy is an ornamental tree planted in gardens and growing wild. The flower is beautiful and fragrant and can be used for the extraction of essential oil. Only one report on flower oil components of this species. Chemical constituents of the flower oil of M. balansae (A.DC.) Dandy from South China Botanical Garden, Guangzhou are as follows: butyl acetate (2.18%), ethyl butyrate (0.23%), 1- ethoxyethyl acetate (0.60%), ethyl 2-methylbutyrate (0.43%), 2- methylbutyl acetate (2.80%), 2-metylpropyl 2-methylpropanoate (1.86%), methyl hexanoate (0.21%), α-pinene (0.12%), 1-metylpropyl butyrate (0.34%), β-pinene (0.10%), β-myrcene (0.10%), ethyl hexanoate (61.01%), 2-methylpropyl 3-butyrate (0.17), 2-methylbutyl 2-metyl 2-metylpropanote (0.10%), p-cymene (0.42%), limonene (15.70%) ethyl 2-hexenoate (0.10%), camphor (0.27%), 2-methylpropylhexanoate (1.32%), and carvone (0.10%) [3]. The volatile components of the leaf oil of M. balansae (A.DC.) Dandy from the Nghean province, Vietnam were analyzed by Capillary GC, GC/MS as well as by background substraction of coeluting compounds. The major components in the leaf . NhËn bµi ngµy 29/5/2008. Söa ch÷a xong 28/10/2008. tr−êng §¹i häc Vinh T¹p chÝ khoa häc, tËp XXXVII, sè 4a-2008 43 oil were α-pinene (18.41%), α-, β-phellandrene (17.36% and 7.42%), germacrene D (17.98%) and bicyclogermacrene (7.55%). The lesser amounts were β-myrcene (3.88%), δ-elemene (5.11%) and β-caryophyllene (3.47%) [4]. The leaf oil of M. montana from India were prepared by hydrodistillation and analysed by a combination of GC and GC/MS. Thirty components have been identified. The major constituents were found to be asaricin (81.8%) and safrole (13.0%) [5]. The essential oil in Michelia leaves was extracted by steam distillation. The oil obtained was dried with anhydrous magnesium sulfate. Thirty three peaks were separated by GC, and 27 of them were identified by MS with NBS mass spectral data and Wiley/NBS registry of mass spectral data. The structure of linalool was further verified by GC/FTIR. The identified constituents represent 97% of the peak area of the essential oil on FID. The major chemical constituent of them are linalool, trans-caryophyllene and diethyl-o-phthalate [6]. In the course of the systematic study of Michelia from Indochina, mono-, sesquiterpenes and other components of the fruit oil of Michelia sp. from Nghean province have been investigated. 2. Experimental Source: Fruits of Michelia sp. were collected in Pu Huong National Park, Nghean province. A voucher specimen (NLN5) is deposited at the Herbarium of the Institute of Ecology and Biological Resources, Vietname Academy of Science and Technology. Dried fruits were shredded and their oil were obtained by steam distillation for 3h at normal pressure, according to the Vietname Pharmacopoeia [7]. The yield of the fruit dried oil is 0.20%. GC- About 15mg of oil, which was dried with anhydrous sodium sulfate, was dissolved in 1ml of n-hexane (for spectroscopy or chromatography). GC analysis was performed on a Agilent Technologies HP 6890 Plus Gas chromatograph equipped with a FID and fitted with HP-5MS column (L=30m, ID=0.25mm, film thickness =0.25µm). The analytical conditions were: carrier gas H 2 , injector temperature (PTV) 250 o C, detector temperature 260 o C, column temperature programmed 60 o (2 min hold) to 220 o (10 min hold) at 4 o C/min. GC/MS- An Agilent Technologies HP 6890N Plus Chromatograph was fitted with a fused silica capillary column HP-5 MS column (L=30m, ID = 0.25mm, film thickness =0.25µm). The conditions of use were the same as described above with He as carrier gas, and interface with a mass spectrometer HP 5973 MSD (70eV). The temperature was programmed as reported above. Components identification was carried out by comparing MS data with those reported in Library Willey on Chemstation HP [8, 9]. 3. Results and discussion Thirty-three compounds identified in the fruit oil of Michelia sp. from Vietnam are listed in Table 1 together with their experimental Kovats indices and H.T.T.Hai, C.T.T. Lam, T.D.Thang, N.X.Dung Chemical composition , TR. 42-46 44 relative peak areas. Table 1: The chemical composition of the fruit oil of Michelia sp. from Vietnam No Compounds KI %FID 1 α-pinene 939 trace 2 fenchene 953 trace 3 β-pinene 980 0.10 4 myrcene 990 0.23 5 δ-3-carene 1011 0.50 6 p-cymene 1026 0.13 7 limonene 1032 1.19 8 (Z)-β-ocimene 1043 0.69 9 (E)-β-ocimene 1052 trace 10 (Z)-linalool oxide (furanoid) 1063 trace 11 α-terpinolene 1088 trace 12 rosefuran 1091 trace 13 linalool 1100 2.46 14 allo-ocimene 1132 trace 15 p-mentha-1,5-dien-8-ol 1166 trace 16 p-cymen-8-ol (1167-1183) 1176 trace 17 α-terpineol 1179 trace 18 safrole 1287 93.42 19 piperonal 1332 trace 20 eugenol 1359 0.16 21 α-copaene 1376 trace 22 methyleugenol 1407 trace 23 α-gurjunene 1410 trace 24 β-caryophyllene 1419 0.10 25 γ-elemene 1443 trace 26 β-selinene 1473 trace 27 germacrene D 1480 trace 28 δ-cadinene 1525 trace 29 α-calacorene 1530 trace 30 spathulenol 1576 0.18 31 caryophyllene oxide 1581 0.12 32 isopathulenol 1638 trace 33 vulgarol B 1647 trace The results of the analysis demonstrate that approximately (99.00%) (33 compounds) of the essential oil were identified. The fruit oil of Michelia was composed mainly of oxygenated compounds (96.34 %). The major component was safrole (93.42%) other compounds present in significant amounts were limonene (1.19%) and linalool (2.46%). The monoterpene hydrocarbons and sesquiterpene hydrocarbons contain small amounts (2.84%, 0.10%). The mixture of the oxygenated compounds such as (Z)-linalool oxide (trace), rosefuran (trace), linalool (2.46%), allo ocimene (trace), p- tr−êng §¹i häc Vinh T¹p chÝ khoa häc, tËp XXXVII, sè 4a-2008 45 metha-1,5-dien-8-ol (trace), p-cymen-8-ol (trace), α-terpineol (trace), safrole (93.42%) piperonal (trace), eugenol (0.16%), methyleugenol (trace), spathulenol (0.18%), caryophyllene oxide (0.12%), isospathulenol (trace) and vulgarol B (trace) furnisches the typical odor impressions. Safrole is an important raw material for the chemical industry because of two derivatives: heliotropin, which is widely used as a fragrance and flavoring agent, and piperonyl butoxide, a vital ingredient of pyrethroid insecticides. Safrole has many fragrance applications in household products such as floor waxes, polishes, soaps, detergents, and cleaning agents. The source of safrole in the 20 th century was Ocotea pretiosa from Brazil, Colombia and Paraguay [10]. This was based on a discovery in l939 that wood distillation from a large tree in the state of Santa Catarina yielded a rich source of sassafras oil, containing 84.0% safrole. At the end of last century (in the early 1990s), certain forest shrubs of the Piperaceae such as P. hispidinervum, P. callosum and P. callosum, indigenous to the humid forests of Central America and Greater Amazonia, were found to contain high levels of safrole in their leaves. The safrole content of the oil in unselected stock is about 85%, but a improvement to more than 90% is possible through selection. Leaves of P. hispidinervum in experimental plots contained 3% essential oils of which 93% is safrole [11]. Since 90 th decace of the 20 century, China and Vietnam has been exporters of sassafras oil, from wild and cultivate trees of Cinnamomum camphora and C. parthenoxylon (Jack) Nees [12]. Forest preservation in developing countries is a controversial issue that involves many different interests. The worldwide demand for raw materials, such as safrole, offers an opportunity to these countries that have a source of products in their natural forests. Forest preservation is best linked to local populations maintenance. In this context, Michelia sp. is more than a new safrole source. Since this species occurs in low populations in several areas, we have plant to cultivate this species for sustainable demand of safrole and assist in the maintenance of the rainforest of Vietnam. An integrated project based on this species among research institutes, universities, governmental, and non- governmental institutions could produce a sustainable alternative crop for the tropical rainforest. Acknowledgements: The Authors wish to thank Dr. B. M. Lawrence, Editor in Chief of the Journal of Essential Oil Research (USA) for his support in literature. References [1] Wu. Z. & Peter H. R. (eds). Flora of China. Vol. 7 (Berberidaceae through Capparaceae). Science Press, Beijing, and Missouri Botanical Garden Press, St. Louis. In Preparation. H.T.T.Hai, C.T.T. Lam, T.D.Thang, N.X.Dung Chemical composition , TR. 42-46 46 [2] Oyen L.P.A. and Dung N. X. (Editors), Plant Resources of South East Asia, N o 19, Essential Oil Plants, Backhuys Publishers, Leiden, the Netherlands, 1999. [3] Zhu Liangfeng et al., Aromatic Plants and Essential Constituents, Hai Feng Publishing Co., Hong Kong, 1993. [4] Dung N. A, Thang T. D. and Dung N. X., Chemical Composition of the Leaf Oil of Michelia balansae (A.DC.) Dandy from Vietnam, Journal of Essential Oil Bearing Plants, 8 (1), 2005, 11-15. [5] Genderen V., Marcel H.P., Leclercq P. A., Delgado H. S., Kanjilal P. B., Singh R. S., Compositional analysis of the leaf oils of Piper callosum Ruiz & Pav. from Peru and Michelia montana Blume from India, Spectroscopy, 14 (2), 1999, 51-59. [6] Qin C., Lu Z. Chen K., Study on chemical constituents of essential oil of Michelia leaves by gas chromatography-mass spectrometry, SePu, 17(1), 1999, 40-42. [7] Vietnamese Pharmacopoeia, Medical Publishing House, Hanoi (1997). [8] Adams R. P., Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectrometry. Allured Publishing Corp. Carol Stream, IL, 2001. [9] Joulain D. and Koenig W. A., The Atlas of Spectral Data of Sesquiterpene Hydrocarbons. E. B. Verlag, Hamburg, 1998. [10] Rizzini, C.T. and W.B. Mors, Botancia economica brasileira. 2nd ed. Ambito Cultural, Rio de Janeiro, RJ, 1995. [11] Rocha S. F. R. and Ming L. C., Piper hispidinervium: A Sustainable source of safrole. In: Janick, J. (ed.), Perspectives on New Crops and New Uses. ASHS Press, Alexandria, Virginia, 1999, 479481. [12] Dung N. X., Moi L. D, and Leclercq P. A., Consituents of the Essential Oils of Cinamomum parthenoxylon (Jack) Ness (Family Lauraceae) from Vietnam, J. of Essential Oil Research (USA) 7, 1995, 53-57. Tóm tắt Thành phần hoá học của tinh dầu quả loài giổi (Michelia sp.) ở Việt Nam - một nguồn cung cấp safrol Thành phần hoá học của tinh dầu quả loài giổi (Michelia sp.) ở Nghệ An, đợc phân tích bằng phơng pháp sắc ký khí (GC) và sắc ký khí-khối phổ (GC-MS). Ba mơi ba hợp chất đợc xác định, với thành phần chính là safrol (93,41%). Các hợp chất có hàm lợng tơng đối lớn là limonen (1,19%) và linalool (2,46%). Kết quả này cho thấy loài giổi (Michelia sp.) ở Nghệ An là một nguồn mới cung cấp safrol. (a) Faculty of Agriculture, Forestry and Fisheries, Vinh University (b) Faculty of Chemistry, Vinh University (c) Faculty of Chemistry, College of Natural Science, Hanoi National University. . sp. ) ở Việt Nam - một nguồn cung cấp safrol Thành phần hoá học của tinh dầu quả loài giổi (Michelia sp. ) ở Nghệ An, đợc phân tích bằng phơng pháp sắc ký khí (GC) và sắc ký khí-khối phổ (GC-MS) Cinamomum parthenoxylon (Jack) Ness (Family Lauraceae) from Vietnam, J. of Essential Oil Research (USA) 7, 1995, 5 3-5 7. Tóm tắt Thành phần hoá học của tinh dầu quả loài giổi (Michelia sp. ). (trace), rosefuran (trace), linalool (2.46 %), allo ocimene (trace), p- tr−êng §¹i häc Vinh T¹p chÝ khoa häc, tËp XXXVII, sè 4a-2008 45 metha-1,5-dien-8-ol (trace), p-cymen-8-ol (trace),