| SelaginellamoellendorffiiHieron, also called Shibo, Yanbo, Huangdanjuanbo, can be found in most part of southern China and southern provinces of Changjiang. It is an evergreen herb, the overground part of the herb can be harvested most of seasons and the root and residual soil should be removed. SelaginellamoellendorffiiHieron is a traditional Chinese herbal medicine and it can be used to treat Acute Infectious Hepatitis, thrombocytopenia, general dropsy and so on. The extractive of the herb can be prepared by cold-maceration, percolation, ethanol refluxing process et al. The main effective constituent of SelaginellamoellendorffiiHieron is bis-flavonoid, especially Amentoflavone. There are a great amount of flavonoids in pteridophyte and gymnosperm, such as plants in Pinopsida, Ginkgopsida. Flavonoids have various pharmacological activities, for example, anti-inflammatory, antioxidant, antiviral, anticancer, vasodilation. So the research of flavonoids take a lot of attention in domestic and overseas.Skin is the first line to protect human body from the outside harmful environment. When someone is injured, the wound will stop bleeding and close, the skin would regenerate rapidly and restore its protective function. The effective skin wound healing need many kinds of cells to participate in this process and this process is controlled precisely in many different aspects. The skin would healing can be divided in to four steps, haemostasisfO-several hours after injury), inflammation (1-3 days), proliferation (4-21 days) and remodelling (21 days-1 year). Fibroblast is very important in normal skin would healing process, and many excetral cellular matrix components, such as collagen, fibrin, fibronectin, proteoglycan, have the potential to promote the survive, proliferation and migration of fibrobiast. The fibroblast has different populations in different tissues of human, and even though in the same tissue, the populations of fibroblast can be different. For example, in human dermis, different populations of fibroblast reside in different layers of the skin with unique feature and function.Connective tissue growth factor (CTGF, CCN2) is a member of CCN(1-6) family. The study of CCN2 began when we found that it played a role in the excessive synthesis of ECM components and fibrosis disease. CCN2 do not usually expressed in normal skin tissue, but when the skin injured, expression of CCN2 will escalate and promote the expression of collagen I, collagen III, MMP and FGF in fibroblast. Although CCN2 can not affect the differentiation of fibroblast, but it can promote the migration of mesenchymal stem cells into the wound part and differentiate into fibroblast. Actually, CCN2 expression is relate to the enlarged scar formation and fibrosis. And CCN2 can promote keratinocyte migrate to the wound by integrin ? 5 ? 1.SelaginellamoellendorffiiHieron has the effects to stop bleeding, promoting blood circulation and pain relief, detoxification swelling. So our project is designed to extract the water-soluble component and fat-soluble components of SelaginellamoellendorffiiHieron and a series of extraction and purification methods will used to purify the extracts. The structure and function of the components will be studied. The water-soluble components were extracted by hot water, the fat-soluble components were extracted by ethanol refluxing method, and all the components were purified by D101 macroporous resin, RC 18 silicagel column, Sephadex LH-20 and preparation HPLC. The structure of the purified components were analyzed by UV spectrum, FT-IR spectrum, NMR, HPLC-ESI-MS, acid hydrolysis, optical rotation photometry and unsaturation test. The effect of the components on NIH3T3 was studied by cell proliferation, cell migration, qRT-PCR and Western blot?We had successfully extracted and purified the water-soluble component A and fat-soluble component B from SelaginellamoellendorffiiHieron.Component A was yellow amorphous powder. Cut the root of SelaginellamoellendorffiiHieron and dried it. The 15kg herb was powdered and refluxed with ethanol, the remains were extracted by 100 liter distilled water for three times,4 hr a time. The extracting solution was condensed and freeze-dried and 1552g water-soluble component was extracted. Dissolve the water extractive by hot diluted alcohol and loaded onto the D101macroporous resin, gradient eluted with ethanol. The 60% ethanol eluted components was 521g and then dissolved in ethanol, loaded onto RP C18 silicagel column, eluted with methanol and water. We got 22.6g water-soluble component at the methanol:H20=50:50 and by using the preparation HPLC, we finally got water-soluble component A 25.2mg.Fat-soluble component B was white amorphous powder. Cut the root of the herb and dried it The 15kg herb was powdered and refluxed with 100 liter ethanol for three times,4 hours a time. The extracting solution was condensed and freeze-dried, 1223g ethanol extracts was acquired. The ethanol extracts was dissolved in petroleum ether to degrease. The remains was dissolved in ethyl acetate and loaded onto the D101macroporous resin, eluted with ethyl acetate. The ethyl acetate extracting solution was condensed and freeze-dried and ethyl acetate extracts 112.7g was acquired. Dissolved the ethyl acetate extracts in ethyl acetate and loaded onto the RP C18 silicagel column, eluted with petroleum ether and ethyl acetate. Collected the elution buffer and when petroleum ether-.ethyl acetate=1:6, we got fat-soluble component 12.5g, this extracts was redissolved in ethyl acetate and loaded onto the Sephadex LH-20 column. Eluted with methanol and H20, we got 2.2g extracts at 100 methanol elution buffer. Fat-soluble component B 30.3mg was acquired by preparation HPLC at last.We analyzed the structure of the water-soluble component A and fat-soluble component B from SelaginellamoellendorffiiHieron by a series methods. The results were listed below:Water-soluble component A had a molecular weight of m/z 815.2252[M-H]-by HPLC-ESI-MS test. Together with the NMR results, we speculated that the molecular formula of A was C35H43022. FT-IR spectrum revealed that component A had aromatic ring, hydroxyl and carbonyl. UV spectrum revealed that component A had a flavonol backbone by the absorbance at 259nm and 338nm. The cid hydrolysis of component A was detected by HPLC and D-glucosyl and the 6-D-galactosyl moiety were found and the ratio of them was 2:1. Together with the results of 1H NMR and 13C NMR, we speculated that the structure of water-soluble component A was:7,3'-di-O-methylquercetin-4'-O-[B-galactosyl-(1?3)-B-glucoypranactosyl]-3-O-B-glucopyranosideFat-soluble component A had a molecular weight of m/z251.0933[M-H]-by HPLC-ESI-MS test. Together with the NMR results and unsaturation test, we speculated that the molecular formula of component B was C13H16O5. Component B could react to FeCl3-K3[Fe(CN]6], and together with the absorbance at 232 and 291 nm in UV spectrum and 1735,1612,1427,1358,866 cm-1 absorbance in FT-IR spectrum, we speculated that component B had aromatic ring and unsaturated ketone. And the structure of component B was deduced by NMR data as below:(3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-oneSelaginellamoellendorffiiHieron can be used to treat bleeding, hematemesis, hematochezia, hemorrhoidal bleeding, burn and scald. So we speculated that SelaginellamoellendorffiiHieron had the effects on skin wound healing. We used mouse embryonic fibroblast NIH3T3 as research model to study the effect of component A and B on the proliferation and migration of NIH3T3.We found that water-soluble component A could promote the proliferation of NIH3T3 and this was related to the concentration. The effect was significant lower when we used a low concentration (1uM). And compared to the positive control dermatan sulfate, component A had a similar effect on the cell proliferation of NIH3T3. And at the same time, component A was found to promote the cell migration of NIH3T3. This effect was dose dependent too. Compared to the positive control dermatan sulfate, they had a similar effect too. This indicated that component A could promote cell proliferation and migration of NIH3T3 efficiently. The results of qRT-PCR revealed that component A could promote the CTGF mRNA expression of NIH3T3, and the effect was dose dependent. And correlated to this, the protein expression of CTGF was elevated too.In conclusion, we had extracted and purified two new components, water-soluble component A and fat-soluble component B from SelaginellamoellendorffiiHieron. The molecular formula and structure of component A and B were analyzed. At the same time, we had found that component A could promote the cell proliferation and migration of NIH3T3, and this was dose dependent. The qRT-PCR and Western blot results also revealed that component A could promote the mRNA and protein expression of CTGF in NIH3T3 with a dose dependent manner. Depending on the results, we speculated that water-soluble component A from SelaginellamoellendorffiiHieron had potential on promoting skin wound healing. |
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