| Objective:The ferns show the diversity in terms of germplasm and metabolism which are a special branch of plants in the history. And ferns are a group between bryophytes and spermatophytes in plant taxonomy. The fern plant family is widely distributed, and there are about 12000 species around the world. As one of the countries who have the most fern species, China have about 2600 species including more than 396 species which are available for medicinal. Ferns have extensive application areas, especially in the pharmaceuticals, foods, ornamental, greening, industry and so on. More and more people pay attention to the economic value of ferns. Medicinal ferns are rich in natural active constituents which have a variety of bioactivities. Activities such as anti-bacteria, antivirus, antioxidant and antitumor have attracted quite attention. In fact, active constituents in many species of ferns have not been fully known and used in clinic, so it is of important significance to research the chemical constituents and bioactivities of medicinal ferns.Lepidogrammitis drymoglossoides(Bak.) Ching is a fern plant belonging to the genus Lepidogrammitis of Polypodiaceae. It widely distributes in the south of Yangtze River basin and other provinces in southern China and has been used as a folk medicine to treat snakebites, bruises, bleeding, boils and sores. Now, as one of the main drugs, L. drymoglossoides has been prepared into therapeutic agent to treat snakebites and paronychia. But there are few researches about active constituents of L. drymoglossoides. So we examined the constituents and bioactivity of L.drymoglossoides. Meanwhile, this research will also provide a basis for the quality control and further development of L. drymoglossoides.Method:In this thesis, we examined the chemical constituents of L. drymoglossoides collected from Xinhui City of Guangdong province and evaluated the antioxidant activity of 29 compounds and 5 extracted parts in vitro. In addition, we determined the content of a new compound (ZLD-1) by HPLC analysis.The dried whole plant of L. drymoglossoides (21 kg) was extracted with 95% ethanol two times (each for 2 h) by using heating reflux method. The 95% extract was concentrated under reduced pressure to afford a residue (1.2 kg), which was further suspended in water and partitioned successively with petroleum ether, chloroform, ethyl acetate and butanol to yield 239.5 g,132.0 g,56.0 g and 122.5 g of the corresponding extracts, respectively. The four extractions mentioned above were separated and purified by silica gel column chromatography (CC), Sephadex LH-20, macroporous resin, HPLC, recrystallization and some other techniques to get compounds. Their structures were determined on the basis of physicochemical properties and spectral data (UV, IR, MS,1H-NMR,13C-NMR, DEPT, 1H-1H COSY, HSQC, HMBC, NOESY).We evaluated the antioxidant activity of 29 compounds and 5 extracted parts in vitro by the method of exogenous radical scavenging and cellular experiments. In the exogenous radical scavenging experiments,3 kinds of extraneous radicals (DPPH, ABTS+,·OH) were used. In the part of cellular experiments, the immortalized mouse hippocampal neurons HT22 cells were selected. To evaluate the antioxidant activity of some compounds from L. drymoglossoides, the MTT assay was carried out to detect the inhibitory activity for the HT22 cells injury induced by glutamate (Glu).We determined the content of the new compound (filic-3-ene-28 oic acid, ZLD-1) by HPLC analysis with the following conditions:Agilent 1260 Infinity HPLC system, DiamonsilTM C18 (250 mm×4.6 mm,5 μm) chromatogram column, mobile phase (acetonitrile-0.5%phosphorous acid,87:13), flow rate (1.0 mL·min-1), sample size (20 μL), detection wavelength (205 nm).Results:31 compounds were isolated from the whole plant of L. drymoglossoides and 29 of them were identified. Their structures were elucidated as filic-3-ene-28 oic acid (ZLD-1), β-ecdysterone (ZLD-2), stigmasterol (ZLD-3), β-sitosterol (ZLD-4),β-daucosterol (ZLD-5), diploterol (ZLD-6), ursolic acid (ZLD-7), physcion (ZLD-8), emodin (ZLD-9), umbelliferone (ZLD-10), scoparone (ZLD-11), aesculetin (ZLD-12), caffeic acid (ZLD-13), 4-0-β-D-glucopyranosyl caffeic acid (ZLD-14), chlorogenic acid (ZLD-15), protocatechuic acid (ZLD-16), pyrocatechualdehyde (ZLD-17),4-hydroxybenzoic acid methyl ester (ZLD-18), gallic acid (ZLD-19), ferulic acid (ZLD-20), (-)-syringaresinol-4-O-β-D-glucopy-ranoside (ZLD-21), lyoniresinol (ZLD-22), (-)-pinoresinol-4,4’-di-O-β-D-glucopyranoside (ZLD-23), n-butyl-O-β-D-fructopyranoside (ZLD-24), docosanyl tetracosanoate (ZLD-25), montanic acid hexacosyl ester (ZLD-26), hentriacontanoic acid, ethyl ester (ZLD-27), hexadecanoic acid (ZLD-28), stearic acid (ZLD-29).In the research of radical scavenging, extracts of ethanol, CHCI3, EtOAc and n-BuOH showed obvious effect in scavenging extraneous radicals (DPPH, ABTS+,·OH), and there was dose-effect relation within a certain concentration range; aesculetin (ZLD-12), caffeic acid (ZLD-13),4-O-β-D-glucopyranosyl caffeic acid (ZLD-14), chlorogenic acid (ZLD-15), protocatechuic acid (ZLD-16), pyrocatechualdehyde (ZLD-17),4-hydroxybenzoic acid methyl ester (ZLD-18), gallic acid (ZLD-19), ferulic acid (ZLD-20) had significant function of scavenging extraneous radicals (DPPH, ABTS+, ·OH), and the IC50 value of compounds 15-17,19 was less than Vc. In cellular experiments, compounds 1,3,8,13,17 (10 μM) showed inhibitory activity for the HT22 cells injury induced by Glu.On the basis of systematical study on the chemical constituents and activity, we furthur analyzed the content of filic-3-ene-28 oic acid by HPLC assay. The results showed that the linear range of filic-3-ene-28 oic acid was 37.5-290 μg·mL-1, and the average recovery was 99.96% (RSD=1.61%, n=6) with an average content of 0.086 mg·g-1.Conelusion:31 compounds were isolated from the whole plant of L. drymoglossoides and 29 of them were identified based on physicochemical properties and spectral data analysis. These compounds were classified as three triterpenoids (1,6,7), three coumarins (10-12), two anthraquinones (8,9), four steroids (2-5), three lignans and glycosides (21-23) and fourteen other compounds (13-20,24-29). Among them, one compound (ZLD-1) is a new compound, and compounds 3,7,9-11,14-15,18-22,24-27,29 were isolated from L. drymoglossoides for the first time. This research result shows that phenolic acids and steroids are the main chemical components.The extracts and compounds from L. drymoglossoides were screened on antioxidant activity. The results showed that extracts of EtOAc and n-BuOH were the main active parts. And the main constituents for antioxidant activity were the phenolic acids, the triterpenoids and steroids also showed antioxidant activity with a certain degree.We developed a HPLC method for the determination of filic-3-ene-28 oic acid from L. drymoglossoides. The methodological evaluation and results indicated that this method was simple, specific and good separation effect. This provides a reference for the further research and development of L.drymoglossoides.Recent studies on pharmacological activities about phenolic acids in natural medicines are reviewed in this thesis. |
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