Optimization Of Extraction Technology And Phytochemical Diversity Of Essential Oil In Leaves From A Natural Population Of Wintersweets

Wintersweet (Chimonanthus praecox), the only representative in the genus Chimonanthus in the family Calyeanthaeeae, is a deciduous shrub native to China, which has survived from the tertiary period. It is also a famous traditional fragrant flower plant and folk tranditional Chinese herbal medicine in China. But for a long time, because of destruction of the habitats and lacking of awareness of the value of wild C. praecox, wild resources encounters to destruction seriously. It has been recorded as the second-class protection plants of China with limited wild resources. The comparatively intact wild populations of C. praecox, being in a very small minority of areas, mainly distribute in Wanyuan of Sichuan, northeast of Chongqing, Jishou of Hunan, and the Baokang of Hubei in China. However, up to now, less information has been reported about genetic diversity level of wild Chimonanthus germplasm resources, extraction rate and composition characteristics of essential oils, aromatic and medicinal properties, etc.In this paper, the best hydrodistillation extraction conditions for getting higher essential oil yield from leaves of C. praecox were firstly established, the essential oil characteristics of wild C. praecox in Wanyuan City of Sichuan also firstly studied. The contents and chemical constituents of essential oils from wild and cultivated wintersweet leaves in the same habitat were compared, for better understanding the aromatic and medical properties, as well as for providing the scientific reference of development and utilization of wild C. praecox germplasm resources in the fields of essence, medicine, health, and bio-pesticides. These are of importance to better understand the genetic diversity, then further to powerfully protect, rationally develop, and sustainably utilize the wild germplasm resources of C. praecox. The results are as the follows:1. The optimum conditions of extracting essential oil from wintersweet leaves were firstly set up using both single-factor and L9(34) orthogonal tests, which are the leaf comminution degree for0.9mm, the material-liquid ratio for5-fold volume water, the material immersion and distillation times for2and8.5h, respectively. The optimum conditions are economic, convenient, and efficient for extracting the essential oil. After the validation of experiments, the extraction rate under the optimum conditions was higher than those under any other group in the orthogonal experiment, account for0.1210%.2. The extracts obtained from the leaves of the wild and cultivated C. praecox were pale-yellow oils with an aromatic-spicy odour. The yields of essential oil in leaves of wild plants W1and W2were different, accounting for0.0999%and0.0922%, respectively, which were higher than that of cultivated C. praecox for0.0842%. A total of forty-six and fifty-nine components were identified by GC-MS from the oils of the wild plants W1and W2, respectively, while that of cultivated C1only for twenty-five types. Of which, twenty-two like borneol, terpinen-4-ol, bornyl acetate, damascenone,10-(acetylmethyl)-(+)-3-carene, β-elemene, P-caryophyllene, a-guaiene, etc were detected in both the wild and control plants. The wild plants contained the sixty-four special components such as o-cymene, eucalyptol, y-terpinene, linalool,(1s)-(-)-camphor, trans-pinocarveol,(s)-cis-verbenol, and so on. These indicated that wild C. praecox leaves could become a better resource for research and development of plant essential oils.3. The extracts obtained from the leaves of wild C. praecox population were pale-yellow oils with an aromatic-spicy odour, similar to that of the cultivated plant. Average essential oil yields in8tested sampling points ranged from0.0694%to0.1056%. Six sampling points possessed the oil yields about0.08%, of them, the sampling point A1and D3have higher yields than others, up to0.1056%and0.1016%, respectively.A total of ninety-three components were identified by GC-MS from the leaves essential oils in the8sampling points of wild wintersweets. The chemical compositions accounted for100.00%(A1、98.49%(B1)、94.15%(C1)、96.21%(C2)、99.80%(D1)、98.60%(D2)、97.88%(D3) and100.00%(D4) of the whole volatile oils, respectively. Of which, monoterpenes and sesquiterpenes were the main constituent groups.4. Eight major components existed in the leaf essential oils from natural C. praecox population in Wanyuan City of Sichuan. They were bornyl acetate (4.35%～33.62%)、(-)-spathulenol (7.75%～23.71%)、caryophyllene oxide (11.91%～28.24%)、 borneol (1.91%～5.88%)、β-selinene (2.26%～4.79%)、(+)-δ-cadinene (0.57%～5.22%)、elemol (2.34%～6.81%) and phytol (2.53%～4.74%). Their contents had wide variation.These components were clustered into three chemotypes:bornyl acetate-type, bornyl acetate/caryophyllene oxide/(-)-spathulenol-type, caryophyllene oxide/ (-)-spathulenol-type. Of them, bornyl acetate/caryophyllene oxide/(-)-spathulenol-type is the main type, accounting for more than half of the whole individual plants while bornyl acetate-type is quite little. The essential oil compositions of the sampling points from same place and same altitude were very similar owing to each other being highly clustered.5. Partial correlation analysis of the eight main essential oil compounds in leaves from wild C. praecox population showed that bornyl acetate was significantly negatively related with elemol、(-)-spathulenol and caryophyllene oxide at0.01level, and with β-selinene at0.05level, respectively. β-selinene was negatively correlated with phytol at0.05level. Similarly, the significantly negative correlation at0.01level between elemol and (-)-spathulenol was also observed. And, caryophyllene oxide was significantly negatively related with elemol、(-)-spathulenol and phytol at0.05level, respectively. Thus, secondary metabolism of the major components showed the trend of one falling with another rising.