| In this paper, phytochemicals including Volatile components and non-volatile components were isolated from L. polystachyus, and the activity of anti-inflammatory and anticancer were conducted on two animal cell lines including murine macrophage cells(RAW264.7) and mouse hepatoma cells(Hepa1c1c7), determined through the quinone reductase(QR) induction bioassay with inducing quinone reductase(QR) rate is greater than or equal to 2 and the nitric oxide(NO) inhibiting bioassay with inhibiting production of nitric oxide(NO) rate is greater than or equal to 50%, and some functional components which owned activity both in anti-inflammatory and anticancer effect isolated from non-volatile components of L. polystachyus, identified by mass spectrometry(MS) and nuclear magnetic resonance(NMR). In addition, the mechanism of anti-inflammatory and anticancer of isolated component from L. polystachyus were discussed through ELISA and western-blot methods. The main results are as follow: 1. Study on the extraction, identification and anticancer activity of volatile components from L. PolystachyusVolatile components were extracted using simultaneous distillation extraction(SDE) method from L. polystachyus, and analysed with a gas chromatography – mass spectrometry(GC-MS), followed by the evaluation of anticancer activity both on extracted volatile components and its main monomer component by the use of Mouse hepatoma cells(Hepa1c1c7). Results showed a total of 80 volatile compounds were identified by GC-MS, accounting for 92.56 percent of the total volatile. Among them, geranylacetone, ?-ionone, nonanal, spathulenol were the most abundant aroma components. The volatile oils of L. polystachyus exhibited strong anticancer activity with CD values of 27.50±0.10 ?g/m L, while geranylacetone and nonanal, the two main aroma components in the volatile oils, exhibited week anticancer activities compared to volatile oils with CD values of 283.10±0.10 ?g/m L and 297.77±0.12 ?g/m L, respectively. 2. The separation of non-volatile components from L. polystachyus and their activity evaluation on anti-inflammatory and anticancer(1) The crude extract was extracted by 30% ethanol/ethyl acetate,and anti-inflammatory and anticancer activity was conducted using Hepa1c1c7 cell and RAW264.7 cell, and results showed the crude extract exhibited good anti-inflammatory and anticancer activity. The crude extract further isolated by silica gel column chromatography and gained four components marked as F1~F4. Activity analysis showed that F3 owned both anticancer and anti-inflammatory activity with IC50 of 18.36±1.20 ?g/m L and CD values of 12.19±0.08 ?g/m L.(2) Eight fractions marked as F3-1~F3-8 were obtained from F3 by TLC, and all the isolated fractions exhibited bifunctional activity subject to QR induction and NO inhibition bioassay with CD values range from 1.59±0.09 to 28.3±0.02 ?g/m L and IC50 values range from 11.9±1.39 to 51.85±5.25 ?g/m L. High performance liquid chromatography(HPLC) analysis showed that the components of F3-5 and F3-8 fractions is relatively simple.(3) Two monomer components marked as F3-5-1 and F3-8-1 separated from F3-5 and F3-8 by HPLC respectively. F3-5-1 owned anticancer activity with CD values of 22.50±0.39 ?g/m L and anti-inflammatory activity with IC50 of 50.25±1.18 ?g/m L, and F3-8-1 exhibited anti-inflammatory and anticancer with CD values of 47.08±0.21 ?g/m L and IC50 of 30.11±3.13 ?g/m L. 3. Structure identification of isolated monomers which owned bifunctional activity in anti-inflammation and anticancer from L. polystachyusThe structure of F3-5-1 and F3-8-1 which isolated from L. polystachyus were analyzed by high-resolution mass spectrometry and nuclear magnetic resonance, and the results showed that F3-5-1 has a basic nuclear structure of chromogen ketone and presumed as aesculetin; F3-8-1 identified as vomifoliol and belong to sesquiterpene. 4. The mechanism of anti-inflammatory and anti-cancer of active component from L. polystachyusIn the aspect of the anti-inflammatory, the expression of inflammation factor including TNF-? and i NOS whether be affected by vomifoliol with different concentration in LPS induced RAW264.7 cells was conducted, and results showed the protein expression of i NOS and TNF-? decreased markedly when the cells treated with vomifoliol in 20, 30 and 40 ?g/m L for 24 h and exhibited dose-dependent manner. Our results showed vomifoliol exhibite anti-inflammatory activity through inhibiting the expression of inflammatory cytokines TNF-? and i NOS on protein level in RAW264.7 cells.As to the activity of anticancer, NQO1 protein expression was analyzed when Hepa1c1c7 cells treated with different concentrations of vomifoliol, and found the NQO1 protein expression increased in a dosage-dependent pattern when cells treated with 25, 50 and 75 ?g/m L vomifoliol for 48 h, which accordance with the increaded QR activity induced by vomifoliol. Our result showed that vomifoliol exerts anticancer activity through up-regulation of NQO1 protein expression to enhance inducing QR activity in Hepa1c1c7 cells. |
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