| Xuanshen is the root of Scrophularia ningpoensis Hemsley, family Scrophulariaceae. Nanchuan district of Chongqing has become the largest national planting base, which is the focus areas of S. ningpoensis(Scrophularia ningpoensis). Huanglian is the root of Coptis chinensis Franch., family Ranunculaceae. Huanglian is Chinese herbs from Shizhu county of Chongqing.In order to develop and utilize the medicinal value of Xuanshen and Huanglian, and develop economy of local herbal industry, we studied chemical constituments and biological activities of two drugs. This paper has seven major parts:1. Extraction and separation experiments1.1Study on chemical constituents from S. ningpoensisA sample of air-dried root of S. ningpoensis was chopped and diacolated with70%ethanol at room temperature. The crude extract was redissolved in water and was loaded on a glass column containing D101macroporous resin, and washed with water, then eluted using a gradient mehtod with30%,60%and90%ethanol fractions. Three fractions were separated by silica gel column and HSCCC to obtain nine compounds. The structures of compounds were identified by1H and13C-NMR spectroscopic data. Compounds were identified as Harpagide、Aucubin、Acteoside、Prim-O-glucosyl-cimifugin、D-(+)-sucrose、Cinnamic acid、Harpagoside、Angroside C and Twenty-seven alkane. The60%ethanol fraction was used for subsequent HSCCC isolation. HSCCC is liquid-liquid distribution chromatography technology. S. ningpoensis was isolated in chloroform-n-butanol-methanol-water (4:1:3:2, v/v/v/v) solvent system by HSCCC. The crude extract was loaded onto a HSCCC column and yielded harpagoside and angroside C with the purity of higher than98%and98.5%resepctively. It is feasible to isolate active compounds harpagoside and angroside C from Scrophularia ningpoensis using HSCCC.1.2Study on chemical constituents from Rhizoma CoptidisExtracts of Rhizoma Coptidis were isolated in CHCl3-MeOH-0.2MHCl (4:1.5:2) solvent system by HSCCC with one step of separation, which were berberine palmatine, coptisine, epiberberine and jatrorrhizine with the purity of80.56%、78%、82%、82%and85.65%resepctively. Each part with single spot respectively from HSCCC separation by Sephadex LH-20column chromatography with eluting in chloroform/methanol system, could obtain5alkaloids with the purity of higher than98%.2. Cell experiments2.1Antihyperglycemic effect of constituents in vitroIn this experiment, we studied antihyperglycemic activity of the chemical constituents of S. ningpoensis and Rhizoma Coptidis by glucose consumption in HepG2cells. The extracts and compounds can promote glucose consumption of HepG2cells in different extent from S. ningpoensis. The60%ethanol fraction was the main active parts of S. ningpoensis in antihyperglycemic activity. Antihyperglycemic activity of each compounds in0.1-2.5μg/ml concentration range was selected, and glucose consumption ability of berberine was highest, and had dose-dependent relationship within test concentration in glucose consumption ability of HepG2cell. The glucose consumption ability of harpagoside and coptisine were higher.2.2Inhibition the level of reactive oxygen in HepG2cellThis experiment established reactive oxygen species model by using the method of glucose stimulation and measured by fluorescence method. According to the results of the fluorescence intensity and inhibition rate of S. ningpoensis and Rhizoma Coptidis, compounds had different degree of suppression. Compared with glucose group, berberine and coptisine (1～10μg/ml) had good antioxidant activity, which could inhibit the level of reactive oxygen by glucose stimulation and had dose-dependent relationship within test concentration. At the same time, compared to the compounds from S. ningpoensis, the antioxidant ability of the alkaloids in Rhizoma coptidis was basically better.2.3The effect of inhibition on ACE activity to chemical constituents of S. ningpoensisAngiotensin converting enzyme (ACE) in the renin-angiotensin system is the role of regulating blood pressure. We measured the content of hippuric acid by HPLC in ACE-HHL-ACEI system and calculated the inhibition rate of ACE activity. In this experiment, we investigated the effect of inhibition on ACE activity of each compound at the same concentration (2mg/ml), with different volume (volume X is30,60and120μl). These results showed that compounds had the effect of inhibition on ACE activity in the same concentration with increasing volume. But compared to the inhibitory effect of captopril, inhibition of ACE activity of each compound of S. ningpoensis was significantly lower at this concentration (P<0.01), which indicated that the effects of compounds in lowering blood pressure were not well.3. Animal experiments3.1Gastroprotective effect of compounds from Rhizoma Coptidis on experimented gastric ulcer in ratsThis paper studied the possible mechanisms underlying the gastroprotective effect of5alkaloids from Rhizoma Coptidis and8-Alkylberberine (8-BBR-Cn) derivatives with a long aliphatic chain (n=4,8,12and16) against gastric mucosal injury in rats.Gastric mucosal injury in rats was induced by ethanol. The ulcer index and percentage inhibition were evaluated. Compared with model control group, the ulcer index of epiberberine, palmatine and jatrorrhizin (25、50、100mg/kg) was not decreased significantly. Additionally, the ulcer index of cimetidine, berberine, coptisine and8-alkyl berberine derivatives was decreased significantly in rats (P<0.001). In this experiment, to test PGE2and NOS, we found the fact that berberine, coptisine and8-BBR-Cn (n=4,8,12and16) had protective effect on gastric mucous membrane, which was related to prostaglandin E2and nitric oxide synthase pathways.8-BBR-C16displayed stronger gastroprotective effect (P<0.001).Coptisine, berberine and8-alkylberberine derivatives (25,50,100mg/kg, ig) could significantly reduce the volume of gastric juice and the total gastric acidity, and decrease activity of pepsin (P<0.001) in gastric mucosal ulcer model by pylorus-ligature, which showed dose-dependent relationship. Coptisine, berberine and8-alkylberberine derivatives Coptisine, berberine and8-alkylberberine derivatives (25,50,100mg/kg, ig) also could significantly inhibit H+K+-ATP enzyme activity (P<0.05, P<0.01), these results indicated that drugs could inhibit excessive secretion of gastric acid by pylorus-ligature operation and had protective effect on gastric ulcer. To NO, each drug was significantly increased NO level (P<0.05, P<0.01). To MDA, coptisine could significantly inhibit MDA level in chemical constituents from Rhizoma Coptidis (P<0.05,P<0.01).3.2Weight regulation and gender preference effects by palmatine in long-term treatment in ratBody weight of both genders was measured at10-day intervals for three months. A significant increase in the female body weight and a decrease in the male body weight were observed in rats after treatment with palmatine for90days. Comparing to the female control group, body weight of female rats by palmatine treatment (100mg/Kg) increased from50day (P<0.05), and body weight of female rats increased significantly from60day to90day (P<0.01). Body weight of male rats relative to the male control group rats decreased from40day to70day (P<0.05) and decreased significantly from80day to90day (P<0.01). So, palmatine had the regulation effect of gender selection in rat weight. This experiment provides evidence that the changes in hematological parameters, estrogen level and uncoupling protein2(UCP2) expression are involved in the effect of the body weight regulated by palmatine. |
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