| Smilax china L. is known as Jinggangteng and belongs to the Lillacaee family. It is a perennial deciduous climbing shrub whose root commonly was used as traditional Chinese medicine. Smilax china L. was recorded in the Chinese pharmaeopoeia (2010version) and has various pharmacological effects including relieve sickness wet, disinioxieation and so on. The traditional Chinese medicine preparations, such as Jingangteng capsule and Jinggangteng tablet, that are made from the main ingredient of rhizome of Smilax china L. have good therapeutic effects against variouse gynecological inflammation, especially the effect on chronic pelvic inflammatory disease. In this paper, with the bioassay guiding, the anti-chronic pelvic inflammatory disease effective fraction and the chemical components of Smilax china L. were studied. The aim of this study is to provide material foundation and quality assurance of the second research of Smilax china L.The domestic and international literatures of Smilax china L, including botany, phytochemisty, pharmacological and clinical literatures showed that the flavonoids and saponins might be the effective components of anti-chronic pelvic inflammatory disease in Smilax china L. In this study, the system solvent method was adopted to get three extracted parts, ethyl acetate extract, n-butanol extract and water extract, respectively. Then the induced chronic pelvic inflammatory disease mice were used to evaluate the activity of the three extracted parts. The process of the experiment as follows:0.06mL of25%phenol mucilage was injected into the right uterus of rats to induce CPID in rats and observed the effects of high, medium and low doses of extracted parts of Smilax china L.(corresponding to4,2and1times of the clinical equivalent dose, respectively) on changes in hematology of CPID model rats in order to investigate the treatment of the three extracts from Smilax china L. The results showed that the high, middle doses of the ethyl acetate fraction of Smilax china L. could significantly reduce the increased hemodynamic indexes of rats, there was significant difference (P<0.01). The high, middle doses of the n-butanol fraction of Smilax china L. could reduce the increased hemodynamic indexes of rats, but without the significant effect of the ethyl acetate fraction. There was no significant difference between the water-soluble fraction group of Smilax china L. and the model control group on the hemodynamic indexes. The results indicated that the ethyl acetate fraction of Smilax china L. showed significant effect on the CPID, so the ethyl acetate fraction was regarded as the active fraction of Smilax china L. on the CPID.It is generally believed that the effective compounds were flavonoids in ethyl acetate fraction. Combining with the conclusion of the literatures, the extraction process of total flavonoids from Smilax china L. was investigated. In the process of experiments, the content of astilbin in the extract was considered as the index to investigate the factors influencing the extraction of total flavonoids from Smilax china L. The single factor test result showed that ethanol concentration, extraction time, extraction times and ethanol consumption were the four main factors influencing the flavonoid extraction yield, especially the effect of the ethanol concentration. Then, according to the content of astilbin, neoisoastilbin and quercetin-3-O-a-L-rhhamnoside in the extract of Smilax china L. by orthogonal test, selected out the best way to extract the total flavonoids from Smilax china L. Combined with the pharmacodynamics experiment results, ultimately the extraction process was that the extraction solvent was70%ethanol,1kg of medicinal materials with10L ethanol, extract2times, extracting time2h,2h, respectively. Verification experiment showed that the extraction technology was stable, reliable and could be used for the subsequent extraction of total flavonois from Smilax china L.The total flavonoids obtained by the extraction process of total flavonoids from Smilax china L. were too crude and had many kinds of impuritys. So the purification process of total flavonoids was carried in this paper. In this study, polyamide (PA) was suitablely used to purify the total flavonoids from Smilax china L. through investigating the static and dynamic adsorption of different resin. The purification parameters of total flavonoids were also investigated, such as the concentration of sample, loading amount of the sample, concentration of ethanol, consumption of ethanol, velocity of eluent. Astilbin, a flavonoid glycosides isolated from the effective fraction of Smilax china L. with high purity was used as reference substance. The elution quality and purity of the total flavonoid were regarded as the indexes to optimize the purification process. The results showed that when the concentration of the sample solution was0.25g·mL-1and the polyamide was80-100mesh, the sample solution adsorbed on the polyamide about2h, then the polyamide column was eluted with6BV water and5BV15%ethanol, discarded the eluent. Finally, the polyamide column was eluted with7BV50%ethanol. Collected the50%ethanol elution fluid and recycled the ethanol. The water solution was dryed by lyophilization and got the total flavonoids fraction. The content of total flavonoids fraction could be purified to51%, while the content of total flavonoids in the ethanol extract was16%. It showed that the purification process was stable and reliable and could be used for purification of total flavonoids from Smilax china L. The pharmacological activity showed that the purified total flavonoids had significant effect on the CPID. So the effective fraction of Smilax china L. on the CPID was obtained by the purification process of total flavonoids.To investigate the effective compounds of anti-CPID in the Smilax china L. and lay a material base for the effective fraction Smilax china L, the active fraction was isolated and some components were obtained. During the process of the experiment, firstly, the active fraction was subjected to silica gel column chromatography and eluted with the mixed solvent of petroleum ether and ethyl acetate at different ratios. Different polarity of eluate was obtained. Then the high-speed counter-current chromatography (HSCCC) was used to separate the eluate. Experimental conditions as follows:N-hexane-ethyl acetate-methanol-water (1:10:1:10, v/v/v/v) was used as the two-phase solvent system. The upper phase was used as the stationary phase, while the lower phase was used as the mobile phase. The apparatus was rotated at850rpm, flow rate was at1.5mL·min-1, the separation temperature was set at25℃and the detection wavelength was280nm. The results showed that four compounds were isolated from the eluate (petroleum ether:ethyl acetate=1:3) and two compounds were isolated from the eluate (petroleum ether:ethyl acetate=1:4). The purity of the isolated compounds was calculated by the area normalization method of HPLC. The purity of the six compounds was more than98%. Combined with the literatures, the six compounds were identified as astilbin, neoisoastilbin, quercetin-3-O-a-L-rhamnos-ide, epicatechin, catechin-[8,7-e]-4β-(3,4-dihydroxyphenyl)-dihydro-2(3H)-pyrano-ne and resveratrol by ESI-MS,1H-NMR and13C-NMR spectra. The neoisoastilbin was firstly separated from Smilax china L.To explore the effective compounds in the anti-CPID active fraction, observed the inhibitory effect of the six compounds obtained in this paper at different concentrations on human umbilical vein endothelial cells (HUVEC). The result showed that five compounds suggested the different degree of inhibition on human umbilical vein endothelial cells (HUVEC). These five compounds were astilbin, neoisoastilbin, quercetin-3-O-a-L-rha, resveratrol, and catechin-[8,7-e]-4P-(3,4-dihyd-roxyphenyl)-dihydro-2(3H)-pyranone. These compounds have revealed a potential for basic biomedical research as possible anti-inflammatory components. The action mechanism needs further investigation.In this study, in order to better control the quality of Smilax china L. the content of the four flavonoids were determined in Smilax china L. The content of the astilbin, neoisoastilbin, quercetin-3-O-a-L-rhamnoside, engeletin were simultaneously determined by external standard method of HPLC. At the same time, the quantitative analysis on multi-components by single marker (QAMS) method was used to determine the content of the four flavonoids. The results showed that the content of the astilbin, neoisoastilbin, quercetin-3-O-a-L-rhamnoside, engeletin were8.0mg·g-1,5.7mg·g-1,3.5mg·g-1,4.5mg·g-1, respectively determined by external standard method. No significant difference between the contents of QAMS method that astilbin was used as the internal standard and external standard method was observed. The present research established an HPLC method proved to be easy and accurate to determine the contents of the four flavones in Smilax china L. There is no significant difference between the contents of the four flavones in Smilax china L. in the results of QAMS method and external standard method. The QAMS method could replace the external standard method to determine the contents of the four flavones in Smilax china L and could be used for the quality control of Smilax china L.The content of the total flavonoids in the effective fraction purified by the polyamide resin was51%determined by UV spectrophotometry. But there are some disadvantages by the UV method, such as the accuracy is not enough. In order to determine the content of the flavonoids in the effective fraction more accurately, the content of the four flavonoids were determined by HPLC method in this paper. The results showed that the content of the astilbin, neoisoastilbin, quercetin-3-O-a-L-rham-noside, engeletin were5.80%,2.05%,2.25%,3.80%, respectively in the effective fraction and the total content of the four flavonoids was13.90%. The quality of the anti-CPID effective fraction of the Smilax china L. could be more accurate controlled by determining the content of the compounds in the effective fraction of Smilax china L. |
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