| Bruguiera gymnorhizα is also called large leaf orange tree. Its family and genus are Rhizophoraceae and Bruguiera Lam. Its seeds germinate and grow into green rod-like hypocotyl on the tree.is a kind of marine plant with high medicinal value. It is heat-clearing, detoxifying, anti diarrhea and hemostatic. In west-south coast of China, the application of Bruguiera gymnorhiza hypocotyl has long history. People use mashed and boiled hypocotyl to cure diarrhea, and in Qiongshan of Hainan province, people use the hypocotyl to cure diabetes mellitus. At home and abroad, there are numerous studies providing evidences that mangrove plants conent a large amount of leading compounds being used to cure human diseases, which are valuable resources.a-amylase inhibitor is a kind of glycosidase inhibitor, which can effectively inhibit the activity of amylase in saliva and intestine, prevent carbohydrate of food from being degradated, thus reduce the absorption of sugar, lower blood glucose and lipid, and is applied clinically.In this study, Bruguiera gymnorhiza hypocotyl harvested from the north sea pass mangrove nature reserve of of Guangxi Zhuang Autonomous Region’s was set as experimental material. The a-amylase inhibitor in Bruguiera gymnorhiza hypocotyl was extracted and purified, and screened by in vitro model and HePG2 cell model.The main contents and results are as follows:1. Screening hypocotyls of Bruguiera gymnorrhiza pancreatic a-amylase inhibitor active ingredient extraction solventIn order to obtain a large number of active ingredients a-amylase inhibitor from hypocotyls of Bruguiera gymnorrhiza,use petroleum ether 95% ethanol and water as extract, three times by ultrasonic asssted extraction, combined extract. Using burrow method, filtering paper method and bernfeld method, we could determine pancreatic alpha amylase and a-amylase and salivary amylase inhibition of three exacts. The results showed that:three extracts liquid amylase inhibition rate of the order ethanol extract> water extract> petroleum ether extract. The results showed that the pancreatic alpha amylase,a-amylase and salivary amylase inhibition rates of ethanol extract (0.25g of raw material) were 62.14%.They have shown a strong inhibitory effect2. Screening the active site of pancreatic a-amylase inhibitor active ingredient from hypocotyls of Bruguiera gymnorrhizaUltrasonic wave method is used for extract chemical compositions from hypocotyls of Bruguiera gymnorrhiza with 70% alcohol. The extraction process as follows:solid liquid ratio was 1:10, ultrasonic frequency was 45KHz,extraction power was 450W and extraction time was 30 minutes. Using liquid-liquid extraction method, we got 6 parts as petroleum ether, chloroform phase, ethyl acetate phase, n-butanol phase, water phase and mother liquor. Using burrow method and bernfeld method, we could determine a-amylase inhibition of six exacts.The results showed that:the six exacts amylase inhibition rate of the order mother liquor> n-butanol phase> water phase> chloroform phase> petroleum ether phase. Mother liquor,n-butanol phase (0.25gofraw material) were 62.95%,54.84%. In each phase, n-butanol phase is the largest. The result was uniform with that burrow method and bernfeld method. The figures showed hypocotyls of Bruguiera gymnorrhiza had positive pancreatic alpha amylase inhibitors and the active ingredients were collected in n-butanol.3.Separation and purification of pancreatic alpha amylase inhibitorsThe six types of different macroporous resins such as HPD450,D101,HPD300,S-8,DM2 and AB-8 were studied to n-butanol extraction phase adsorbability and desorption. The results showed that the AB-8 resin possessed higher absorption and desorption capacity. N-butanol extracts was separated by macroporous resin. By using water and ethanol as solution gradient elution program.70% alcohol eluent of n-butanol phase inhibition ratio (0.125g of raw material) was 66.32%.70% alcohol eluent of n-butanol phase was further purified by silica column chromato graphy. Firstly, TLC method was used to select the best washing solution, ethyl acetate:methyl alcohol (20:1â†'15:1â†'10:1â†'8:1â†'6:1â†'4:1â†'2:1â†'3:1â†'1:1â†'0:1) was used as eluent system for gradient elution.Using bernfeld method, we could determine pancreatic alpha amylase inhibition of ten exacts, results showed that the proportion of ethyl acetate:methyl alcohol elution agent for 2:1 and 1:1 flow points of pancreatic alpha amylase inhibition rate was highest, at 70.56%,70.03%.Compared with additional gradient, there was a significant difference (P<0.01).Combinationof 2:1 and 1:1, sample 1 was obtained.Sample 1 can be further isolated, TLC method was used to select the best washing solution, ethyl acetate:methyl alcohol:water (5:3:1â†'3:1:1â†'0:1:1) was used as eluent system for gradient elution, finally, used methanol to wash column. We collected eluent, with a total of 120 compositions. Upon TLC testing, we got 6 compositions. The pancreatic alpha amylase inhibition rate was highest of component 4 (0.075mg of raw material) was 89.76%, which was higher than 0.25mg acarbose. The 4 component was denoted as sample 2.4.Nature of the substance has been identified for pancreatic a-amylase inhibitionThe nature of sample 2 was identificated by UV absorption spectroscopy, TLC and HPLC. we used electrospray ionization mass spectrometry (ESI-MS) to meacure relative molecular mass of it. The results showed that:Sample 2 were shown a good single point in each of the developing agent. Wavelength of UV absorption spectroscopy were 235nm,275nm HPLC analysis conditions is:the Waters 515 highly effective liquid phase color spectrometer, Waters 2487 double-channel ultraviolet detector, Sun FireTMC18 column (4.6x250 mm), sample quantity:20μl, column temperature:room temperature, flow rate:1.0 ml·min-1, detection wavelength of 235nm It showed the mainpeak curve after 1.388 minutes and area was 94%. The results showed that the sample 2 was the screening of pancreatic alpha-amylase inhibitors.Preliminary showed that the substance for flavonoids. The sample 2 inhibiting pancreatic alpha-amylase optimum reaction condition was:temperature of 35℃, pH6.5,inhibition ratio was 92.50%,respectively, the inhibition of enzyme activity was 1076.74U·100mL-1. Sample 2 shows that the conditions still act as a disincentive for the human body conditions.we used electrospray ionization mass spectrometry (ESI-MS) to meacure relative molecular mass of it. The conditions of mass spectrometry:ion source:ESI; checking out pattern:positive and negative ions;scanning range:50-1000m/z.The result showed that relative molecular mass of this material could be 413.5.Effect of pancreatic alpha-amylase inhibitors on glucose consumption glycogen content in HepG2 cellsGlucose consumption and intracellular glycogen content in HepG2 cells at normal and stress state were respectively determined after the cells were incubated using different concentrations of pancreatic alpha-amylase inhibitors for 24h, MTT assay was used to monitor the proliferation of the HepG2 cells. Results:the concentration of sample 2 was 0.1 mg·mL-1,0.25 mg·mL-1,0.5 mg·mL-1 could obviously increase cell glucose consumption. Compared with additional the blank control group, there was a significant difference (P<0.01). MTT assay showed that the concentration of sample 2 was 0.1 mg·mL-1,0.25 mg·mL-1,0.5 mg-·L-1 could obviously increase cell the proliferation. The results showed that:sample 2 could increase glucose consumption.In conclusion,70% ethanol was used as the extraction agent.First extract was isolated and purified by macroporous resin, and then further purification by silica gel chromatography column. The active substance of pancreatic alpha-amylase inhibitors extracted from hypocotyls of Bruguiera gymnorrhiza. The nature of sample 2 was identificated by UV absorption spectroscopy, TLC and HPLC. The results showed that the sample 2 was the screening of pancreatic alpha-amylase inhibitors. Preliminary showed that the substance for flavonoids and relative molecular mass of this material could be 412. |
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