Bangladesh Journal of Pharmacology Research Article Comparison of terpene components Comparison of terpene components Comparison of terpene components from flowers of from flowers of from flowers of Artemisia annuaArtemisia annuaArtemisia annua BJP Introduction Artemisia annua L. is an annual herb native of China, where it has been used in the treatment of fever and malaria for many centuries. Many secondary meta- bolites of terpene peroxides were isolated from the plant, such as artemisia ketone, artemisinic alcohol, arteannuin B and myrcene hydroperoxide (Bertea et al., 2005; Brown et al., 2003). The most famous terpene peroxide is Artemisinin, which chemical structure is an amorphane-type sesquiterpene endoperoxide, and it has become an important plant-derived compound in the treatment of the chloroquine-resistant and cerebral malarias (Klayman, 1985). The essential oils, another important composition of A. annua, have been subjected to extensive chemical study. It was reported that the oils contained artemisia ketone, 1,8-cineole and cam- phor as key components. Significant variations in the percentage occurrence of different constituents have also been reported. The percentage of artemisia ketone, 1,8-cineole and camphor were reported to vary from 0.0 -63.0, 1.5-31.5 and 5.0-20.0%, respectively, Other major components reported were -, -pinene, borneol, carva- crol, thymol, myrcene, limonene, camphene, copaene, -caryophyllene, -terpineol, -, - and - elemene, sabinine, -guaiene, caryophyllene, caryophyllene oxide, germacrene-D and so on (Fabien et al., 2002; Neetu et al., 2002; Perazzo et al., 2003; Soylu et al., 2005; Rasooli et al., 2003; Flora et al., 2004; Ma et al., 2007; Divya et al., 2007; Hashemi et al., 2007). Chongqing, China, shares eighty percent of A. annua around the world, where an A. annua GAP Cultivation Demonstration Site has been built for three years. But A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2012; 7: 114-119 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index; ISSN: 1991-0088 Abstract Terpene constituents of essential oils obtained by steam distillation from Artemisia annua flowers at the pre-, full- and post-flowering stage was investigated by gas chromatography (GC) and gas chromatography-mass spectrometric detector (GC-MS). The aim was to evaluate change the biosynthesis pathway of terpenes at different flowering stages. The samples studied showed that main components of essential oils were monoterpenes hydrocarbons (48.1%) and oxygenated monoterpenes (41%) in the pre- flowering, oxygenated monoterpenes (35.6%) and sesquiterpenes hydro- carbons (5.0%) in the full-flowering, and oxygenated monoterpenes (29.6%), sesquiterpenes hydrocarbons (32.2%) and oxygenated sesquiterpenes (25.3%) in the post-flowering, respectively. The relative content of monoterpenes decreased from pre-flowering to post-flowering, while that of sesquiterpenes increased. The results indicated that the biosynthesis pathway of terpenes might be changed at different flowering at stages, while the change of content and composition of terpenes might be a self-adaptation of A. annua. Article Info Received: 1 June 2012 Accepted: 12 June 2012 Available Online: 18 June 2012 DOI: 10.3329/bjp.v7i2.10815 Cite this article: Yang ZN, Zhu SQ, Yu ZW. Compari- son of terpene components from flow- ers of Artemisia annua. Bangladesh J Pharmacol. 2012; 7: 114-19. Comparison of terpene components from flowers of Artemisia annua Zhan Nan Yang1,2, Shi Qiong Zhu1 and Zheng Wen Yu1 1School of Life Sciences, Guizhou Normal University, Guiyang, Guizhou, 550 001, Peoples Republic of China; 2Key Laboratory for Information System of Mountainous Area and Protection of Ecological Environment of Guizhou Province, Guizhou Normal University, Guiyang, Guizhou 550 001, Peoples Republic of China. This work is licensed under a Creative Commons Attribution 4.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. systemic study about the essential oils of the herb has not been previously mentioned in literature. The effect of flowering on terpenes content of A. annua flower essential oils at pre-, full- and post-flowering stage was investigated. In the present paper we report the analytical results of the essential oils at different flower developing stages. Materials and Methods A. annua cultivar Wuling-3938 plants were grown in the Artemisia GAP Cultivation Demonstration Site of Holleypharm, Chongqing, China. The flowers were harvested at pre-, full- and post-flowering stage in September to November 2007 on a same plant. The flowers were separated from other capitula organs, leaves and stem of A. annua, and identified by Mr. Rongchang Luo of Holley Natural Resource Exploiture Co. Ltd, Chongqing, China and deposited in the Herba- rium, College of Bioengineering, Chongqing University, Chongqing, China. Shade-dried plant materials (50.0 g) were hydro- distill- ed separately in Clevenger-type equipment for 4h. The oils were collected and dried over anhydrous sodium sulfate and stored in a refrigerator at 4°C for analysis. GC analyses were performed using a Shimadzu GC- 2010 gas chromatograph equipped with an FID and an HP-5 fused silica column (30 m × 0.32 mm i.d., 0.25 μm film thickness) with a 5% phenyl-substituted methyl- polysiloxane phase. The oven temperature was pro- grammed at 40°C for 4 min and then increased to 240°C at a rate of 4°C/min. Injector and detector temperatures were 250 and 265°C, respectively. The carrier gas, helium (99.999%), was adjusted to a linear velocity of 43 cm/s. The essential oil samples were diluted 5-fold, and 1 μL of a diluted solution was injected into the GC/MS in the split mode with a split ratio of 1/20. MS analyses were performed using a Shimadzu MS- QP2010 with ionization energy of 70 eV, a scan time of 0.5 s and a mass range of 33–450 amu. The percentages of compounds were calculated by the area normalization method without considering response factors. The components of the oil were identified by comparison of their mass spectra with those of the spectrometer database using the NIST147 mass spectral database and also with those of authentic compounds. The identifications were confirmed by comparison of the fragmentation patterns and Retention index with those reported in the literature (Divya et al., 2007a; Flora et al., 2007; Divya et al., 2007b). The retention index was found with a standard mixture of C8 to C22 compounds under chromatography conditions, consistent with those of the chromatography conditions of the samples analyzed. Results and Discussion The flower essential oils were obtained from A. annua at pre-, full- and post-flowering stage with 2.2, 1.4 and 1.3% yield (relative to dried weight), respectively. Three oils were pale yellow, and the results of the analysis of the essential oils are given in Table I. A total of sixty-three compounds were identified in three essential oil samples. The oils contain mainly monoterpenes and sesquiterpenes, representing 96.2, 98.9 and 99.3% of the oils in A. annua at pre-, full- and post-flowering stage, respectively. Monoterpenes hydrocarbons and oxygenated monoterpenes content was greater in the preliminary florescence oil, they were 48.1 and 41.0%. The main compounds in the oil were - myrcene (37.7%), 1,8-cineole (16.1%) and camphor (15.0%). The oxygenated monoterpenes and sesquiterpenes hydrocarbons were dominants in the flourishing florescence oil (35.6 and 5.0%). The oil contained predominantly caryophyllene (19.4%), germacrene D (18.1%), camphor (15.8%), 1,8-cineole (10.6%), (Z)- -farnesene (9.4%). The terminal florescence oil contains mainly oxygenated monoterpenes (29.6%), sesquiterpenes hydrocarbons (32.2%) and oxygenated sesquiterpenes (25.3%). The major constituents identified in the oil were camphor (16.6%), caryophyllene (16.3%), -caryophyllene oxide (15.8%), -farnesene (9.0%), (-)-spathulenol (7.2%). A significant difference was the absence of oxygenated sesquiterpenes in preliminary florescence, while the monoterpenes hydrocarbons were lacked in the terminal florescence oil. It is reported (Liu et al., 1988) that the inflorescence oil of A. annua from Changchun, China, contained Artemisia ketone (63.1%), 1,8-cineole (1.5%), -pinene (1.5%) and caryophyllene (1.9%). Another literature (Divya Goel et al., 2007) reported the major compounds of the cultivar A. annua petal oil were trans-sabinol (10.2%), paramentha-1, 4 (8)-dien-3-ol (10.1%) and 1,8- cineole (6.8%). The influence of transplanting time on inflorescence essential oil yield and composition has been studied (Flora Haider et al., 2007). Their result shows that oil yield was found to range from 0.5 to 1.6% (w/v). Camphor (23.30-57.00%), 1,8-cineole (5.60- 21.40%) and -caryophyllene (2.5-8.7%) were key compounds. The percentage occurrence of rest of the compounds was found to vary with different transplanting time. Our results were more similar to that of Flora Haider. The content of the monoterpenes hydrocarbons were decreased sharply with flower developing, especially for -myrcene, it from 37.7% in the preliminary florescence oil to 0.2% in the flourishing florescence oil, the compound was lacked in the terminal florescence oil (Figure 1A). The oxygenated sesquiterpenes were Bangladesh J Pharmacol 2012; 7: 114-119 115 Table I Chemical composition of the flower essential oils at pre-, full- and post-flowering stage of A. annua No TR RIa Components Content (%) Pre-flowering Full-flowering Post-flowering 1 5.0 816 (3-Methyl-2-oxiranyl)methanol － － 0.5 2 5.1 820 2-Ethoxypropane － 1.7 0.5 3 7.3 928 Origanene 0.3 － － 4 7.5 937 -Pinene 0.9 － － 5 7.8 955 Camphene 3.1 0.4 － 6 8.2 976 Sabinene 3.8 0.4 － 7 8.3 981 2,2-Dimethylhexanal － － 0.2 8 8.3 983 -Pinene 1.5 － － 9 8.5 991 -Myrcene 37.7 0.2 － 10 8.6 995 Yomogi alcohol － － 0.7 11 8.6 995 2,3-Dehydro-1,8-cineole － 0.6 － 12 9.1 1019 (+)-4-Carene 0.1 - － 13 9.1 1021 No － 0.1 － 14 9.3 1029 No － 0.1 － 15 9.4 1032 Limonene 0.5 － － 16 9.5 1037 1,8-cineole 16.1 10.6 0.3 17 9.9 1057 Artemisia ketone 0.1 0.2 2.4 18 9.9 1060 Tricyclene 0.3 － － 19 10.0 1062 -Terpinen － 0.3 － 20 10.3 1076 cis- -Terpineol 0.4 0.5 － 21 10.4 1081 No － － 0.5 22 10.6 1092 5-(2-Methylenecyclopropyl)-1-pentanol 0.7 － － 23 10.8 1100 (3E,5E)-2,6-Dimethyl-3,5,7-octatrien-2-ol 4.0 1.4 2.6 24 10.9 1104 No － 0.2 － 25 10.9 1106 Nonanal － － 0.4 26 10.9 1107 Plinol C 0.6 0.6 － 27 11.4 1128 trans-p-Mentha-2,8-dienol － 0.2 － 28 11.7 1140 ND 0.3 0.3 0.4 29 11.8 1143 Ipsdienol － － 0.4 30 11.9 1149 Pinocarveol 0.3 0.2 0.4 31 12.0 1152 Berbenol 0.2 － － 32 12.1 1157 Camphor 15.0 15.8 16.6 33 12.3 1165 Nerol 0.3 － － 34 12.3 1165 Lavandulol － － 0.4 35 12.3 1167 (-)-cis-Myrtanol － 0.2 － 36 12.3 1168 Isogeraniol － 0.5 0.2 37 12.4 1171 ND － － 0.2 38 12.5 1176 Myrcenol 0.2 0.4 － 39 12.6 1180 Borneol 0.5 1.1 3.9 116 Bangladesh J Pharmacol 2012; 7: 114-119 Table I Chemical composition of the flower essential oils at pre-, full- and post-flowering stage of A. annua (Cont.) No TR RIa Components Content (%) Pre-flowering Full-flowering Post-flowering 40 12.8 1187 4-Terpineol 0.6 1.2 1.0 41 12.9 1194 iso-Amyl tiglate 0.5 0.5 0.4 42 13.0 1199 1,5-Menthadien-7-ol － 0.1 － 43 13.1 1201 -Terpineol 1.3 0.3 0.2 44 13.2 1204 Myrtenol 0.5 0.2 － 45 13.5 1217 trans-3(10)-Caren-2-ol 0.2 0.3 0.5 46 13.9 1234 (E)-3(10)-Caren-4-ol － 0.2 － 47 14.3 1247 (2E)-2,7-Dimethyl-2,6-octadien-1-ol 0.1 0.1 － 48 14.3 1248 ND － － 0.2 49 14.6 1259 4,6,6-Trimethylbicyclo[3.1.1]hept-3-en-2- yl acetate － 1.6 － 50 15.1 1279 Nerol acetate 0.2 － 0.4 51 16.0 1310 Hydroxy- -terpenyl acetate － 0.6 － 52 18.6 1377 Copaene － 1.1 1.4 53 19.2 1392 -Elemen － － 1.5 54 20.8 1420 -Caryophyllene 2.3 19.4 16.3 55 22.4 1445 -Farnesene 2.6 9.4 9.1 56 22.9 1454 -Caryophyllene － 1.0 － 57 23.9 1470 Chamigren 1.8 － － 58 24.5 1479 Germacrene D 1.9 18.1 4.0 59 25.5 1495 -Elemene 0.3 － － 60 25.7 1498 Germacrene B － 0.9 － 61 33.1 1570 (-)-Spathulenol － 1.8 7.2 62 33.6 1575 -Caryophyllene oxide － 3.0 15.8 63 47.7 1681 Aromadendrene oxide-(2) － 2.8 2.2 64 62.9 1904 ND － － 1.4 65 63.0 1907 -Cadinol － 1.1 － 66 63.5 1919 (10Z,12Z)-9-Methyl-10,12-hexadecadienyl acetate － － 1.1 67 64.4 1942 ND 0.4 － － 68 65.2 1961 ND － － 0.8 69 65.4 1967 n-Hexadecanoic acid － － 4.4 70 65.8 1977 ND 0.3 － － 71 65.9 1979 9,12,15-Octadecatrienal － － 0.3 72 70.6 2106 trans-Phytol － － 0.4 73 71.4 2131 Stearolic acid － － 0.5 74 76.9 2297 2,6,10,14-Tetramethylheptadecane － － 0.6 75 96.2 Total identified 76 Monoterpenes hydrocarbons － 77 Oxygenated monoterpenes 29.6 78 Sesquiterpenes hydrocarbons 32.2 79 Oxygenated sesquiterpenes 25.3 80 Fatty acids and aliphatic esters 7.2 TR = retention time; ND = not identified; (-) Not detected; aRI is the Retention index relative to C8-C22 n-alkanes on the HP-5ms column Bangladesh J Pharmacol 2012; 7: 114-119 117 increased quickly with flower developing, the (-)- spathulenol and -caryphyllene oxide were nice examples, both the compounds weren’t detected in the preliminary florescence oil, there were only 1.8% and 3.0% in the flourishing florescence oil, they were added rapidly to 7.2 and 15.8% in the terminal florescence oil (Figure 1B). In general, the oxygenated monoterpenes were declined slowly with flower developing, among major com- pounds, artemisia ketone, camphor, borneol and trans-3 (10)-Caren-2-ol were raised tendency, while 1,8- cineole was declined (Figure 1c). Interestingly, the sesqui- terpenes hydrocarbons increased sharply from 8.9% at pre-flowering stage to 5.0% (at the top of content) at full -flowering stage, followly by, which decreased slowly to 32.2% at post-flowering stage. Among of these com- pounds, the change of - caryophyllene and -far- nesene was good examples. The content of former from 2.3% added to 19.4%, then decreased to 16.3%; and that of the later from 2.6% added to 9.4%, then decreased to 9.0% (Figure 1D). As to the change of all terpenes in flower oils of A. annua at different flowering stage, the content of the monoterpenes hydrocarbons and oxygenated mono- terpenes were the highest at the pre-flowering stage, and the sesquiterpenes hydrocarbons arrive to the top 40 30 20 10 0 C on te nt 20 10 0 -Caryophyllene oxide (-)-Spathulenol Aromadendrene oxide-(2) Compounds C on te nt Fre-flower Full-bloom Post-flower -Myrcene Sabinene Camphene C on te nt 20 10 0 C on te nt 1,8-cineole Compounds trans-3(10) Caren-2-o1 -Terpineol Myrtenol Camphor Borneol A.ketone 20 10 0 -Caryophyllene -Farnesene Germacrene D Compounds C on te nt 60 40 20 Terpenes Monoterpens Oxygenated Sesquiterpenes Oxygenated Figure 1: Content of monoterpene hydrocarbons (A), oxygenated sesquiterpenes (B), oxygenated monoterpenes (C), sesquiterpene hydrocarbons (D) and terpenes (E) of A. annua flower at different flowering stages A B C D E 118 Bangladesh J Pharmacol 2012; 7: 114-119 at the full-flowering stage, while the oxygenated sesquiterpenes in flower oils were peak at the post- flowering phase (Figure 1E). The present results have shown that the content of terpenes in the A annua oils has close relationship with flower developing. The flowering effect on monoterpenes content of plant oil has been reported elsewhere (Dudai et al., 1992). So flowering may be change the biosynthesis pathway of terpenes in the A. annua flower oil, in turn, the change of content of terpenes in the oils may be the self-adaptation for the physiological phenomenon of flowering. Acknowledgement This work was financially supported by grants from the National Natural Science Foundation of P. R. China (No. 31060056). References Bertea CM, Freije JR, Van DWH, Verstappen FW, Perk L, Marquez V, De Kraker JW, Posthumus MA, Jansen BJ, de Groot A, Franssen MC, Bouwmeester HJ. Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 2005; 71: 40-47. Brown GD, Liang GY, Sy LK. Terpenoids from the seeds of Artemisia annua L. Phytochemistry 2003; 64: 303-23. Dudai N, Putievsky E, Ravid U, Palevitch D, Halevy AH. Monoterpene content in Origanum syriacum as effected by environmental conditions and flowering. Phys Plant. 1992; 84: 453–59. Goel D, Singh V, Ali M, Mallavarupu GR, Kumar S. Essential oils of petal, leaf and stem of the antimalarial plant Artemisia annua. J Nat Med. 2007a; 61: 187-91. Goel D, Singh V, Ali M, Mallavarupu GR, Kumar S Composi- tion of the essential oil from the root of Artemisia annua. J Nat Med. 2007b; 61: 458-61. Haider F, Dwivedi P, S Singh, Naqvi AA, Bagchi G. Influence of transplanting time on essential oil yield and composition in Artemisia annua plants growth under the climatic conditions of sub-tropical north India. Flavour Fragrance J. 2004; 19: 51-53. Hashemi P, Abolghasemi MM, Fakhari AR, Ebrahimi SN, Ahmadi S Hydrodistillation–solvent microextraction and GC –MS identification of volatile components of Artemisia aucheri. Chromatographia 2007; 66: 283-86. Iraj Ri, Rezaee MB, Moosavi ML, Kamkar J. Microbial sensitivity to and chemical properties of the essential oil of artemisia annua L. J Essent Oil Res. 2003; 15: 59-62. Jain N, Srivastava SK, Aggarwal KK, Kumar S, Syamasundar KV, Jain N, Kumar S. Essential oil composition of Artemisia annua L. 'Asha' from the plains of Northern India. J Essent Oil Res. 2002; 14: 305-07. Juteau F, Masotti V, Bessière JM, Dherbomez M, Viano J Antibacterial and anti-oxidant activities of Artemisia annua essential oil. Fitoterapia 2002; 73: 532-35. Klayman DL. Qinghaosu (Artemisinin): An antimalarial drug from China. Science 1985; 228: 1049-55. Liu Q, Yang ZY, Deng ZB, Sa GH, Wang XJ. Preliminary analysis on chemical constituents of essential oil from inflorescence of A. annua L. Acta Botanica Sinica 1988; 30: 223 -25 Ma CF, Wang HH, Lu X, Li HF, Liu B, Xu GW. Analysis of Artemisia annua L. volatile oil by comprehensive two- dimensional gas chromatography time-of-flight mass spectrometry. J Chromatogr. A 2007; 1150: 50–53. Perazzo FF, Carvalho JCT, Carvalho JE, Rehder VLG. Central properties of the essential oil and the crude ethanol extract from aerial parts of Artemisia annua L. Pharmacol Res. 2003; 48: 497-502. Soylu EM, Yigitbas H, Tok FM, Soylu S, Kurt S, Baysal Ö, Kaya AD. Chemical composition and antifungal activity of the essential oil of Artemisia annua L. against foliar and soil- borne fungal pathogens. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz. 2005; 112: 229-39. Bangladesh J Pharmacol 2012; 7: 114-119 119 Author Info Zheng Wen Yu (Principal contact) e-mail: yuzhengwen2001@126.com physiological phenomenon of flowering: 31060056: Author Info Zheng Wen Yu Principal contact email yuzhengwen2001126com: DatePrinted: This article was downloaded by you on: Nov 26, 2017