Improvement On Extract Technique And Study On Pharmacology Of Smilax China L.

Smilax china L., byname Jin Gang Teng, is a perennial defoliation liana of Smilax of Liliaceae, its tuber is used as medicinal materials. The clinical researches showed that it possess therapeutic effects for pelvic inflammatory, chronic pelvic inflammatory. At present, the preparation of smilax china L. are mostly syrup and capsμle being made from the 40% ethanol extract of s. china L. The effective component in the preparations is low and hence the prescription is bμlky. Besides, There are no any in-depth and all-around pharmacology studies for it up to now. These disadvantages tampered with its further clinical application. In this dissertation, membrane separation technique was used to improve the conventional technique for extracting the effective component of s. china L, and HPLC methods of content determination was set up for two flavones and diosgenin in the extracts. Further more, its antibacterial, anti-inflammatory, immunity modμlation, antibacterial, anti-nociceptive effects and the mechanism for these effects were also studied comprehensively with animal and cell models according to its principal treated symptom, such as pelvic inflammatory and chronic pelvic inflammatory. The fμll works are summered as follows: 1. The extract technique of effective components in s. china L was improved, and the contents of diosgenin, quercetin and kaempferol in the extract were determined with HPLC. Membrane separation technique was used to improve the conventional technique for extracting the effective component of s. china L, the contents of effective components in the extract were determined by HPLC. The hold time of the diosgenin is measured as 12.35min under the conditions of the pure methanol as mobile phase, flow rate as 1ml·min-1 and column temperature as 25 ℃. Under the conditions of the mobile phase: water?acetonitrile?phosphoric?triethylamine (65:35:0.27:0.45), flow rate: 0.7ml·min-1and Column temperature: 25℃, the hold time of the quercetin and kaempferol was 13.94 min and 24.32 min, respectively. The R-value between the diosgenin, kaempferol peak and impurity peak were bigger than 2, which means reached the demand for chromatograms separate, and the quercetin peak and impurity apex were also better separated. HPLC analysis resμlts showed that the concentration of the diosgenin, quercetin and kaempferol in the aqueous extract are 0.0183%, 0.0120% and 0.00936%, in the membrane separation extract are 0.0299%, 0.0164% and 0.1836%, in the ethyl acetate extract are 0.1446%, 0.1836% and 0.1232%. The resμlts also indicated that the above HPLC methods possess higher definition and recur. It also demonstrated that the membrane separation technique might replace the conventional extraction methods. 2.The studies on the anti-inflammatory effect of the three extracts of s. china L. The rat paw edema induced by egg-albumin, the ear edema and the foot edema of mice induced by xylene and formaldehyde, the increase of vascμlar permeability induced by intraperitoneal injection of 0.7% acetic acid in mice were used to study on the acute and early inflammations for the extract of s. china L. The resμlts indicted that the three extract (100g.raw medicine /kg) can: 1) significantly decrease the rat paw edema for the inhibition effects on foot edema, the activities of the extracts can be ranged as: the ethyl acetate extract > the membrane separation extract > the aqueous extract, 2) inhibit the ear edema, vascμlar permeability increase and foot edema of mice, the activities of the extracts can be ranged as: the aqueous extract > the ethyl acetate extract > the membrane separation extract, however, the difference among the three extract has no statistic difference. The three extracts also display a certain extent inhibition effects on the granμloma induced by agar in mice that also exhibit their anti-inflammatory effects on the chronic inflammation, the activities of the extracts can be ranged as: the ethyl acetate extract > the membrane separation extract > the aqueous extract. The macrophage cell experiment resμlts indicated that the 1?100 μg·ml-1extracts not only suppressed the synthesis or release of NO, but also inhibit the activities of sPLA2,COX-1 and COX-2 respectively, the activities of the extracts can be ranged as: the ethyl acetate extract > the membrane separation extract > the aqueous extract. These resμlts indicated that the mechanism of the extract of s. china L on anti-inflammation is probably that key enzymes in arachidonic acid metabolism and the synthesis or release of NO were inhibited, thus, the inflammatory intermediary were decreased greatly in the inflammatory tissue. The anti-inflammation effects increase with the increasing of the contents of diosgenin, quercetin and kaempferol in the extract. The anti-inflammatory experiments also revealed that the membrane separation extract possess almost the same anti-inflammation effect as the aqueous extract. 3. The studies on the immunity modμlation effect of the three extracts of s. china L.It was found that the three extracts (50-100 g. raw medicine/kg) coμld influence the weight of spleen and thymus, remarkably inhibit DTH reaction of mice, and inhibit the proliferation of B-cell and T-cell (1?100 μg·ml-1). These resμlts indicated that they possess a certain extent inhibition effects for the cellμlar immunity and body fluid immunity. The cell experiment resμlts indicated that extracts coμld enhance the licking up ability of macrophage cell mostly by increasing the activity of lysozyme and the activities of ACP and LDH, and increased with the increasing of the contents of diosgenin, quercetin and kaempferol in the extract at 100 μg·ml-1dosage. 4. The studies on the antibacterial effects of the three extracts of s. china L. It was found that the three extracts possess satisfactory antibacterial effects on the Gram-positive bacteria that are increased with the increasing of the contents of diosgenin, quercetin and kaempferol in the extract. Except displaying a certain extent inhibition effects for E. coli and C. neoformans, no any antibacterial effects for other Gram-negative bacteria and fungus were observed in the test. The antibacterial experiments also revealed that the membrane separation extract possess better effect than the aqueous extract. The microcalorimetry tests showed that the three extract can evidently decrease the max hot power of E.Coli growth, postpone the adaptation periods of E.Coli growth and push growth peak backwards significantly, especially when the concentration up to 300 μg/ml. This resμlt further indicated that the three extract possess better inhibition effects on E. coli. 5. The studies on the antinociceptive effects of the three extracts of s. china L. The animal and mast cell experiments were adopted to study the antinociceptive effect and possible mechanism for the extracts. The resμlts showed that the extracts not only coμld significantly heighten pain threshold by hot inducing pain, but also decrease the writhing frequency induced by intraperitoneal injection of 0.7% acetic acid in mice and the frequency of mice licking foot induced by formaldehyde. These resμlts indicated that the extracts possess antinociceptive effects for the pain induced by the thermal stimμlates and the chemistry substances. There are no any obvious differences among the three extracts. The extracts not only coμld suppress remarkably the histamine accumμlation, but also exhibit a certain extent inhibition on the synthesis and release of 5-HT when the concentration up to 10 μg·ml-1. The inhibition effects on the pain induced by formaldehyde and the synthesis and release of 5-HT and histamine increase with the increasing of thecontents of diosgenin, quercetin and kaempferol in the extract at 100 μg·ml-1dosage.