| V.amoena Fisch.var.angusta Freyn.(Vicia L.) as folk medicine is a plant mainly distributed in Northeast, North and Northwest of China, growing in riverside, shore, hillsides, forest margins and shrub wetlands. The dried stem leaves of V.amoena Fisch.var. angusta Freyn. is used as medicine called "Tougucao" and harvested in summer and autumn. It is used to expel pathogenic wind and dampness, alleviate pain, and promote immunity. The prescriptions of Chinese patent medicines such as "Kanlisha", "Yangxuerongjin pill", "Shutongantumoji", all contain "Tougucao" which was recorded in the 2015 year edition of "Chinese Pharmacopoeia"(one part) as V.amoena Fisch., V.cracca L.,V.pseudorobus Fisch.et Mey., V.amoena Fisch.var.sericea Kitag. and V.amoena Fisch.var.angusta Freyn.(Ch.P, four part).In Liaoning traditional Chinese medicine standards (2014), the thin layer chromatography qualitative identification of Tougucao only tested compound sitosterol,and no quantitative method was added. At present, study of V.amoena Fisch.var.angusta Freyn. was seldom reported, though it is widely used, there is no scientific standard for quality control.Therefore, we studied the chemical composition of V.amoena Fisch.var.angusta Freyn. and established a quality standard.Preliminary screening immune activity of V.amoena Fisch.var.angusta Freyn.Immunoregulation is one of pharmacology effects of material medicas which can dispel wind and dampness by tonifying liver and kidney. Priliminary work showed the extract of V.amoena Fisch.var.angusta Freyn. played an important role in the activation of Toll-like receptors (TLRs), increased the mRNA expression of Cytokines and that of NF-κBp65 protein in macrophages by TLR signaling pathway.Chemical separation results initially confirmed flavonoids,polysaccharides and other compounds probably are active portion.Study on chemical composition of V.amoena Fisch.var.angusta Freyn.Study on chemical composition of 70%(v/v) ethanol extract of the stem leaves of V.amoena Fisch.var.angusta Freyn. was carried out. The 14 compounds were isolated and purified by means of macroporous resin D101,reiterant siliga gel,Sephadex LH-20,ODS column chromatographies, preparative TLC and preparative HPLC isolation methods. On the basis of their physic-chemical properties and spectral data, their structures were elucidated as kaempferol(1),quercetin(2), tricin(3), kaempferol-3-O-a-L-rhamnoside(4),kaempferol-3-O-α-L-arabinoside(5),quercetin-3-O-a-L-rhamnoside(6),quercetin-3-O-β-D-glucoside(7),kaempferol-3,7-O-α-L-dirha-mnoside(8), wogonin(9), hyperoside(10), myricetrin(11),chlorogenic acid(12), daucosterol(13), (3-sitosterol(14).And compounds 1-14 are isolated from this plant for the first time.Study on the identification of crude drug V.amoena Fisch.var.angusta Freyn.In this chapter, the characters of V.amoena Fisch.var.angusta Freyn.was described; Leaves and power surface slices were made to observe the microscopic characters of the leaves and powder of V.amoena Fisch.var.angusta Freyn.. A thin-layer chromatography analysis method of V.amoena Fisch.var.angusta Freyn. was developed,which could show main component. The best condition for thin-layer separation effect were as follows:Silica gel G thin-layer plate, hyperoside, quercetin-3-O-α-L-rhamnoside, kaempferol-3,7-0-α-L-dirhamnoside as control, ethyl acetate-formic acid-water(10:1:1)as developing solvent; 1%AlCl3 ethanol solution as colour developing agent,105℃ heating to spot color clear and examined under UV lamp at 365nm. The applicability of the system was examined, and 12 batches herbs was investigated by the TLC method. The result showed that the method was stable and reliable.Water, total ash,acid insoluble ash and alcohol-soluble extractive of 12 batches of V.amoena Fisch.var.angusta Freyn. was inspected. Preliminarily drawn up, the water content was not more than 15.0%, the total ash was not more than 12.0%, The acid insoluble ash was not more than 3.0%, the alcohol-soluble extractive was not less than 14.0%.Study on total flavonoids content of V.amoena Fisch.var.angusta Freyn.Determination of total flavonoids in V.amoena Fisch.var.angusta Freyn. by colorimetric methodTo establish a method for determination of total flavonoids content of V.amoena Fisch.var.angusta Freyn. by UV. The sample was the 70%(v/v) ethanol extract of stem and leaves of V.amoena Fisch.var.angusta Freyn..Then using rutin as control, sodium nitrite-aluminum nitrate-sodium hydroxide as colour developing agent, the 70%(v/v) ethanol extract of the stem and leaves of V.amoena Fisch.var.angusta Freyn. and rutin were tested to get the max wavelength 500nm. As a blank control rutin solution determined, absorb degree (A) and concentration(mg/mL) were draw as standard curve of rutin at the wavelength 500nm. The sample in 0.0080～0.0400 mg·mL-1 range show a good linear relationship. The RSD was not more than 3.0% in precision test, reproducibility test and stability test. The total flavonoids content of V.amoena Fisch.var. angusta Freyn. was 1.350 Omg·g-1.Determination of five flavonoids from Vicia amoena var. angusta and Vicia amoena by HPLCDiamonsil C18 column(200 mm×4.6 mm,5 mm)was used. Acetonitrile and 0.1 % phosphoric acid water solution was used as mobile phase, the volume flow was at 1.0 mL·min-1;Ultraviolent wavelength was 350 nm; Column temperature was at 25 ℃, and injection volume was 10 μL. Hyperoside; isoquercitrin; kaempferitrin; quercitrin; kaempferol-3-O-a-L-arabinoside show a good linear relationship,and the average recoveries were 98.10%-102.30%. The RSD values were less than 3%. The average contents of hyperoside; isoquercitrin; kaempferitrin; quercitrin; kaempferol-3-O-a-L-arabinoside in V.amoena. were 0.165 9,0.078 1,0.7615,0.600 2 and 0.1958 mg·g-1,respectively. The average contents of hyperoside; isoquercitrin; kaempferitrin; quercitrin; kaempferol-3-O-α-L-arabinoside in V.amoena.var.angusta. were 0.734 7,0.918 5,0.5255,1.311 8 and 0.173 2 mg·g-1,respectively.Determination of four flavonoids from Vicia amoena var. angusta and Vicia amoena by HPLCDiamonsil C18 column(200 mm×4.6 mm,5μm)was used. Acetonitrile and 0.1 % phosphoric acid water solution was used as mobile phase, the volume flow was at 1.0 mL·min-1;Ultraviolent wavelength was 350 nm; Column temperature was at 25 ℃,and injection volume was 10 μL. show a good linear relationship,and the average recoveries were 98.12%-100.08%. The average contents of kaempferol-3-O-α-L-rhamnoside, quercetin, tricin and kaempferol in V.amoena. were 0.094 2,0.071 0, 0.050 0 and 0.055 5 mg·g-1,respectively. The average contents of kaempferol-3-O-a-L-rhamnoside, quercetin, tricin and kaempferol in V.amoena.var.angusta. were 0.105 4,0.101 3,0.101 8 and 0.096 0 mg·g-1,respectively. The method is believable for determining the contents of kaempferol-3-O-a-L-rhamnoside, quercetin, tricin and kaempferol with good simplicity, sensibility, repeatability, and recovery rate.Optimization of total flavonoids of Vicia amoena Fisch.var.angusta Freyn.Through the static adsorption and static desorption experiments, the best macroporous resin was selected from 10 kinds of macroporous resin, and the best macroporous resin was HPD-826 macroporous resin. Results are as follows:the concentration of the sample was 0.1g·mL-1, HCl(1moL·mL-1) adjusted sample solution pH 5-6, sample volume for 5 times of the resin column bed volume, adjusting the flow rate was 2 mL·min-1 of dynamic adsorption, the eluent was 70% ethanol solution, with a flow rate of 3 mL·min-1 were eluted,elution volume is the 10 times of resin bed volume. The HPD-826 type of macroporous resins for total flavonoids of Vicia amoena Fisch.var.angusta Freyn. enrichment, the result is 3.289 6 mg·g-1.This paper first gives a systematic chemical separation,improvement of quality standards and enrichment of macroporous resins for total flavonoids to Vicia amoena Fisch.var.angusta Freyn..The above studies will provide date support for research, development and utilization of Vicia amoena Fisch.var.angusta Freyn.. |
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