CN103239435B - Preparation method of gynura divaricata total caffeoylquinic acid and application in antihyperglycemic agent or health-care product - Google Patents
Preparation method of gynura divaricata total caffeoylquinic acid and application in antihyperglycemic agent or health-care product Download PDFInfo
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Abstract
The invention discloses a preparation method of gynura divaricata total caffeoylquinic acid and application of the gynura divaricata total caffeoylquinic acid in an antihyperglycemic agent or a health-care product. The effective part of the gynura divaricata total caffeoylquinic acid takes an overground part of gynura plant gynura divaricata (gynura divaricata) as a material; the preparation method comprises the steps of refluxing and extracting the dry overground part of the gynura divaricata through certain concentration of ethanol; filtering, concentrating, drying and dissolving by a hydrochloric acid solution; filtering, and extracting filtrate by ethyl acetate, redissolving an ethyl acetate extract through low-concentration ethanol, gradiently eluting by macroporous resin water ethanol, gathering phenolic acid ingredients, and recovering a solvent to dry and obtain the effective part of the total caffeoylquinic acid. The gynura divaricata total caffeoylquinic acid prepared by the method has significant inhibited effect on important target-protein-tyrosine-phosphatase 1B for researching intestinal carbohydrate hydrolase alpha-glucosaccharase and diabetes mellitus, and can be further researched and developed as a drug or a health-care product for auxiliary treatment of the diabetes mellitus. The invention provides a certain scientific basis for civil application of the gynura divaricata for treatment of the diabetes mellitus, and provides a new resource for development of a safe and efficient novel antihyperglycemic agent or health-care product.
Description
Technical field
The invention belongs to the field of Chinese medicines, be specifically related to a kind of method and the application of this effective site in treatment diabetes medicament or health product of extracting and preparing total caffeoylquinic acid effective site from Chinese medicine Gynura divaricata.
Background technology
Gynura divaricata (Gynura bicolor DC.) belongs to herbaceos perennial for Compositae (Compositae) Radix Gynurae, has another name called Radix et Rhizoma Gynurae divaricatae, is mainly distributed in south China area.Gynura divaricata in Jiangsu, Zhejiang, Sichuan and Fujian among the people, win its fresh blade or with stem and leaf to boil water the taking of making tea, treatment diabetes had to good curative effect.Within 2000, Institute of Botany starts introducing and planting, and carries out the research work of chemical composition for treating diabetes.
Chemical constitution study shows, Gynura divaricata mainly contains the chemical combination composition of other types such as alkaloids, flavonoid, cerebroside, organic acid and adenosine, uridnine.Animal pharmacological test confirmation, the water extract of Gynura divaricata has significant hypoglycemic activity (Hu Yong etc., Xi'nan College of Forestry journal, 2007,27 (1): 55-58; Horse due east etc., hypoglycemic activity and the mechanism thereof of Radix et Rhizoma Gynurae divaricatae water extract to type 2 diabetes mellitus rat, Chinese herbal medicine, 2010,41 (4), 623-626).Published patent of invention 200710084533.2,200610052502.4,201110372288.1 all points out that Gynura divaricata has diabetes effect.
In the clinical treatment of type 2 diabetes mellitus, glycoside hydrolase inhibitor such as alpha-glucosidase, maltase and saccharase inhibitor are a class reaches treatment diabetes orally-taken blood sugar reducing medicines to delay intestinal carbohydrate absorption.Due to being closely connected of alpha-glucosidase and diabetes, medicine scholar is devoted to find the inhibitor of this fermentoid always, to develop more treatment diabetes medicament.At present, come into the market and clinically widely used alpha-glucosidase inhibitor antidiabetic drug mainly contain: acarbose (acarbose), voglibose (voglibose), miglitol (miglitol) etc.
PTP 1B (protein tyrosine phosphatase-1B, PTP1B) is an important target in diabetes study.As one of member of typical non-receptor type PTPase family, PTP1B in insulin signaling pathway by negative regulation Insulin receptor INSR (insulin receptor), thereby reach the effect of controlling blood glucose.Find the specific inhibitor of PTP1B, improve the sensitivity of insulin signaling pathway by suppressing the activity of PTP1B, the treatment of diabetes is had to important application prospect.
Summary of the invention
The object of the invention is to find and provide the anti-diabetic in Gynura divaricata effective chemical fraction.
In order to achieve the above object, the invention provides the preparation method of Gynura divaricata total caffeoylquinic acid and in the purposes of preparing in antidiabetic medicine or health product.
Gynura divaricata total caffeoylquinic acid effective site provided by the invention, taking the dry aerial parts of Gynura divaricata as raw material, prepares by following operating procedure:
(1) after Gynura divaricata aerial parts is dried and is pulverized, with mass percent concentration be 60-100% alcoholic solution reflux, extract,, 6～10 times of solid-liquid ratios (L/kg), extraction time is 1-3 time, and extraction temperature is 50-80 DEG C, and extraction time is 1-3 hour, merge extractive liquid,, filter, filtrate decompression is concentrated into without alcohol taste, and vacuum drying obtains crude extract powder.
(2) the described alcohol extracts powder of step (1), first dissolves with 2～5% aqueous hydrochloric acid solutions of certain volume, and after filtering, filtrate is extracted with ethyl acetate 2 times, and combined ethyl acetate layer reclaims solvent, obtains acetic acid ethyl ester extract.
(3) the described acetic acid ethyl ester extract of step (2) redissolves through low-concentration ethanol water again, the macroporous resin column of flowing through D101 absorption, first use deionized water rinsing resin, water ethanol gradient elution again, collecting concentration of alcohol is the eluent between 40-60%, concentrating under reduced pressure, dry, obtain total caffeoylquinic acid effective site.
The Gynura divaricata total caffeoylquinic acid effective site preparing with said method, comprises following active component: 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid methyl ester, 3,5-O-dicaffeoylquinic acid methyl ester, 4,5-O-dicaffeoylquinic acid methyl ester.Calculate the quality percentage composition > 50% of total phenolic acid in this effective site with dry product weight.
Above-mentioned Gynura divaricata total caffeoylquinic acid effective site has alpha-glucosidase, protein-tyrosine phosphatase 1B inhibitor effect, can be applied to and prepare antidiabetic medicine or health product.
Described antidiabetic medicine or health product are respectively pharmaceutically acceptable carrier and the health food of Gynura divaricata total caffeoylquinic acid effective site that contains treatment effective dose or functional component.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1:
Get Gynura divaricata dry aerial parts 300g, pulverize, the alcoholic solution 3000mL that is 80% with mass percent concentration reflux, extract, 2 times at 70 DEG C, merge extractive liquid,, filtration, is evaporated to without alcohol taste, and vacuum drying obtains crude extract powder;
By above-mentioned crude extract powder, first dissolve with 2% aqueous hydrochloric acid solution of certain volume, after filtering, filtrate is with 500mL ethyl acetate extraction 2 times, and combined ethyl acetate layer, reclaims solvent, obtains ethyl acetate extract.
Take macroporous adsorbent resin (D101) 600g, add distilled water at room temperature to place and spend the night, make resin fully swelling, wet method dress post, flowing with 95% ethanol, to clean pillar to effluent and the volume ratio of water be the muddiness that is not white in color while mixing at 1: 5, then with the abundant drip washing pillar of distilled water to effluent without ethanol taste, placement is spent the night;
Above-mentioned ethyl acetate extract dissolves through 10% ethanol water (v/v), filter, filtrate is slowly flow through the macroporous resin column that above-mentioned processing and balance are crossed, first use deionized water rinsing, use again the ethanol gradient elution of variable concentrations, collecting concentration of alcohol is the eluent between 40-60%, and effluent launches through TLC, sprays 5% FeCl
3alcoholic solution shows aeruginous; Concentrating under reduced pressure is collected liquid, and lyophilization, obtains total caffeoylquinic acid effective site.Calculate the quality percentage composition > 50% of total phenolic acid in this position with dry product weight.
The total caffeoylquinic acid effective site more than obtaining is dissolved with certain volume Chromatographic Pure Methanol, and 45 μ m filtering with microporous membranes are prepared type high performance liquid chromatography (Agilent Prep-HPLC1100) and separate, and chromatographic process is as follows:
Mobile phase: A water (0.1% formic acid), B acetonitrile; Elution process: isocratic elution, 20%B, 0-30mmin; Linear elution, 20-30%B, 30-55min; Linear elution 30-20%, 55-57min; Flow velocity 6mL/min, room temperature, detects wavelength 325nm, the each 250 μ L of sampling volume, running time 57mins, (21.2mm ID × 150mm, 5 μ m) separate C18 post, automatic fraction collector peak trigger mode collection fraction.
Collect respectively fraction, concentrating under reduced pressure through high vacuum dry, obtain 6 coffee mesitoyl quinine acid compounds, according to MS and NMR data and document comparison, the structure of identifying them is respectively: 3,4-O-dicaffeoylquinic acid (1), 3,5-O-dicaffeoylquinic acid (2), 4,5-O-dicaffeoylquinic acid (3), 3,4-O-dicaffeoylquinic acid methyl ester (4), 3,5-O-dicaffeoylquinic acid methyl ester (5), 4,5-O-dicaffeoylquinic acid methyl ester (6); Except compound 3, outside 5-O-dicaffeoylquinic acid (2), other 5 coffeic acyl quininic acid derivatives are first and separate and obtain from Gynura divaricata.The structure of each compound is as follows:
Embodiment 2: total phenolic content of Gynura divaricata total caffeoylquinic acid effective site is measured (colorimetry)
With 3, (the self-control of 4-O-dicaffeoylquinic acid, HPLC detects its purity > 95%) be reference substance bioassay standard curve, standard substance are dissolved in to acetone-water solution and are made into series concentration, in 10mL volumetric flask, add 8mL reference substance solution, add Folin-Ciocalteau reagent 0.5mL, shake up, place 1min.Add again 20%Na
2sO
4solution 1.5mL, shakes up, and places 1h, measures trap, drawing standard curve in spectrophotometer at 750nm place.
Accurately take Gynura divaricata total caffeoylquinic acid effective site sample, as stated above, in 750nm place working sample trap.Calculate according to standard curve, in Gynura divaricata total caffeoylquinic acid effective site, with 3,4-O-dicaffeoylquinic acid meter, the quality percentage composition > 50% of total phenolic acid.
Embodiment 3: the external hypoglycemic activity of Gynura divaricata total caffeoylquinic acid effective site and coffeic acyl quininic acid derivative thereof is measured
(1) the inhibition experiment to alpha-glucosidase
Alpha-glucosidase (α-glucosidase, Type I) is purchased from sigma company, Infinite F50 type microplate reader (Tecan, Switzerland), each monomeric compound sample takes out respectively a certain amount of, is dissolved in pure methanol, and being made into concentration is the high concentration mother solution of 100mg/mL, adopt 96 orifice plate screening systems, the method of reaction system reference literature report, slightly change, reaction system after improving and optimizating is: 28 μ L phosphate buffer (phosphate buffer, PBS, pH=6.8) in, add alpha-glucosidase (1U/mL) 10 μ L, add certain density sample solution 2 μ L, 37 DEG C of constant temperature 10min, then add nitre phenyl-β-D-pyranglucoside (4-nitrophenyl β-D-glucopyranoside, PNPG, 10mM in PBS) 10 μ L, 37 DEG C of isothermal reaction 35min, under 405nm wavelength, measure absorbance (A value).Acarbose (acarbose, acarbose) Gou Bai Bayer A.G, for the positive control of this law, set blank (only with buffer) and negative control (only with buffer and enzyme liquid) simultaneously, inhibition of enzyme activity rate is calculated as follows: inhibition of enzyme activity rate (Inhibitory, %)=(A negative control-A sample)/(A negative control-A blank) × 100%.Positive control medicine acarbose (acarbose).As calculated, the IC of the right alpha-glucosidase of positive control acarbose
50for 1.38mM.
(2) the inhibition experiment to protein tyrosine phosphatase esterase 1B
PTP1B enzyme is purchased from Enzo Life Science company, and all compounds are mixed with the methanol solution of 10mg/mL after weighing, and adds 2 μ l and do not affect enzymatic activity in every hole.Adopt 96 orifice plate screenings, the method of reaction system reference literature report, slightly change, add following reagent to be mixed with the reaction solution of 100 μ L: 0.05 μ g PTP1B enzyme, 1.5mM Insulin receptor INSR β residues (IR5) and 100mM MES buffer (300mM sodium chloride, 2mM EDTA, 2mM DTT, 0.1%NP-40, pH=6.0); At 37 DEG C, be incubated 30min, finally use certain density phosphate detectable (purchased from Enzo Life Science company) end reaction, change (Infinite F50 microplate reader by measuring its absorption value at 620nm place, Tecan company of Switzerland), calculate its suppression ratio.Suppression ratio is calculated as follows: [1-(Asample/Acontrol)] × 100%.Adopt PTP1B enzyme inhibitor SURAMIN (purchase white Enzo Life Sciences company) as positive control.
Gynura divaricata total caffeoylquinic acid effective site and wherein separate the compound monomer composition that obtains to the inhibition activity of alpha-glucosidase and protein tyrosine phosphatase esterase 1B in table 1, table 2.
The inhibition activity of table 1 sample to alpha-glucosidase
The inhibition activity of table 2 sample to protein tyrosine phosphatase esterase 1B
External alpha-glucosidase activity is measured and is found: Gynura divaricata total caffeoylquinic acid effective site with therefrom separate the di-coffee mesitoyl quinine acid compounds monomer that obtains especially 3,4-O-dicaffeoylquinic acid methyl ester and 4,5-O-dicaffeoylquinic acid methyl ester has all shown significant alpha-glucosidase and has suppressed active, their IC
50value is all significantly lower than positive control medicine acarbose.External protein tyrosine phosphatase esterase 1B determination of activity is found: Gynura divaricata total caffeoylquinic acid effective site and 3,5-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid monomer have showed stronger protein tyrosine phosphatase esterase 1B and have suppressed active.Infer that thus coffee mesitoyl quinine acid compounds is the hypoglycemic active substance of Gynura divaricata basis, for the application among the people of illustrating Gynura divaricata treatment diabetes provides scientific basis.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification made under essence of the present invention and principle, substitutes, combination and simplification; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (1)
1. the preparation method of a Gynura divaricata total caffeoylquinic acid, it is characterized in that, comprise following operating procedure: by after dry Gynura divaricata aerial parts and pulverizing, with alcoholic solution reflux, extract,, the mass percent concentration of described alcoholic solution is 60-100%, and consumption is 6-10 times of quality of medicinal material volume, and extraction time is 1-3 time, extraction temperature is 50-80 DEG C, and extraction time is 1-3 hour; Extracting liquid filtering, is evaporated to without alcohol taste, and vacuum drying obtains crude extract powder; Crude extract powder first uses 2 ~ 5% aqueous hydrochloric acid solutions of certain volume to dissolve, and filters, and filtrate is extracted with ethyl acetate 2 times, and combined ethyl acetate layer reclaims solvent, obtains ethyl acetate extract; Redissolve through low-concentration ethanol water, the macroporous resin column of flowing through D101 absorption, first uses deionized water rinsing resin, then water ethanol gradient elution again, and collecting concentration of alcohol is the eluent between 40-60%, and concentrating under reduced pressure is dry, obtains total caffeoylquinic acid.
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| CN101040851A (en) * | 2006-03-20 | 2007-09-26 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | The use of dicaffeoylguinic acid ramification and the analog in the treatment of diabetes and the corresponding disease |
| CN102690324A (en) * | 2012-06-18 | 2012-09-26 | 江苏省中国科学院植物研究所 | Gynura bicolor protein extract, and preparation method and application thereof |
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| CN101040851A (en) * | 2006-03-20 | 2007-09-26 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | The use of dicaffeoylguinic acid ramification and the analog in the treatment of diabetes and the corresponding disease |
| CN102690324A (en) * | 2012-06-18 | 2012-09-26 | 江苏省中国科学院植物研究所 | Gynura bicolor protein extract, and preparation method and application thereof |
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| 季芳等.药用植物来源的α-葡萄糖苷酶抑制剂研究进展.《中国中药杂志》.2010,第35卷(第12期),1633-1640. |
| 药用植物来源的α-葡萄糖苷酶抑制剂研究进展;季芳等;《中国中药杂志》;20100630;第35卷(第12期);1633-1640 * |
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