CN102166274B - Method for extracting alpha-glucosidase inhibitor of small intestines from China soapberry - Google Patents
Method for extracting alpha-glucosidase inhibitor of small intestines from China soapberry Download PDFInfo
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Abstract
The invention discloses a method for extracting an alpha-glucosidase inhibitor of small intestines from China soapberry. The method is characterized by comprising the following steps of: crushing shells of dried China soapberry fruits into powder with the average grain size of 200 to 350 mu m; in a material-to-liquid ratio of 1g:40-50ml, adding 50 to 70 percent methanol, performing shaking extraction at room temperature for 18 to 24 hours, separating supernatant, concentrating and drying the supernatant to obtain methanol extractum of which the yield is 25 to 30 percent; dispersing the methanol extractum in distilled water, in a material-to-liquid ratio of 1g:30-40ml or a volume ratio of 1:2, adding ethyl acetate, performing the shaking extraction for 2 to 3 times, and collecting water phase residue; concentrating the water phase residue to 15 to 20ml, loading to a polystyrene type low pole macroporous absorbent resin Diaion HP-20 chromatographic column, performing gradient elution by using water-methanol, wherein the concentration is 0, 50 percent and 100 percent respectively and the elution flow rate is 1ml/min, and collecting active elution peaks; concentrating the active elution peak component to 4 to 5ml, and loading to a Sephadex LH-2- column, wherein the filling materials are 50cm high and the diameter of the column is 3.2cm; performing water elution, wherein the elution flow rate is 0.5ml/min; and collecting the active elution peaks, and concentrating and drying to obtain active ingredients of the alpha-glucosidase inhibitor of small intestines, wherein the yield is 0 to 15 percent.
Description
Technical field
The present invention relates to from Fructus Sapindi Mukouossi, extract the method for small intestinal alpha-glucosidase inhibitor, belong to Chinese herbal medicine preparation and chemical technology field.
Background technology
Postprandial hyperglycemia is the risk factor of type ii diabetes and metabolic syndrome always, and the control post-prandial glycemia has great importance to diabetics undoubtedly.Under physiological status, all there is alpha-glucosidase (EC 3.2.1.20) at small intestinal in the main absorption site of glucose in the mucosa brush border that the small intestinal upper, middle and lower are three sections, this enzyme is a kind of mammal digestive enzyme.Polysaccharide trans-oral saliva in the food, pancreatic amylase are digested to oligosaccharide, disaccharidase and the trisaccharide that contains a small amount of glucose molecule, get into the small intestine meridian alpha-glucosidase and are hydrolyzed to single glucose, are absorbed into blood by small intestinal, are the main causes that post-prandial glycemia raises.Therefore, be the activity of the inhibitor of target spot with the alpha-glucosidase through this enzyme of adjusting, can brush digestion and the absorption that the edge stops carbohydrate at intestinal villi, thereby weaken the peak value of post-prandial glycemia, prevent postprandial hyperglycemia.Compare with other OHA and insulinize, the notable attribute of alpha-glucosidase inhibitor is effectively to stop the post-prandial glycemia of diabetics to raise, and makes patient's blood glucose steadily and lentamente maintain certain level, improves the glucostasis index.The inhibitor acarbose of typical alpha-glucosidase has been recommended as a line medicine of treatment type ii diabetes at present by domestic clinician; But digestive tract side effects such as flatulence are obvious, are the domestic and international research focuses so from natural plants, extract the alpha-glucosidase inhibitor of high-efficiency low-toxicity always.More existing at present relevant report and patents of extracting about alpha-glucosidase inhibitor, but do not see that relevant report and the patent of from Fructus Sapindi Mukouossi, extracting alpha-glucosidase inhibitor arranged.
Summary of the invention
The objective of the invention is to be directed against the deficiency of prior art and the method for from Fructus Sapindi Mukouossi, extracting the small intestinal alpha-glucosidase inhibitor is provided, be characterized in that raw material sources are wide, simple to operate, suppress the efficient height.
The object of the invention has following technical measures to realize that wherein said raw material umber is parts by weight except that specified otherwise.
The method of from Fructus Sapindi Mukouossi, extracting the small intestinal alpha-glucosidase inhibitor may further comprise the steps:
(1) shell of dry Fructus Sapindi Mukouossi fruit is pulverized, particle mean size is 200～350 μ m, and adding concentration by solid-liquid ratio 1g: 40～50ml is 50～70% methanol; Concussion was at room temperature extracted 18～24 hours; Separation of supernatant, concentrate drying get methanol extractum, and yield is 25～30%;
(2) above-mentioned methanol extractum is scattered in the distilled water, solid-liquid ratio is 1g: 30～40ml, adds ethyl acetate then in 1: 2 by volume, and the water raffinate is collected in concussion extraction 2～3 times;
(3) above-mentioned water raffinate ` is concentrated to 15～20ml; Last polystyrene type low pole macroporous adsorbent resin DiaionHP-20 chromatographic column is used the water-methanol gradient elution, and concentration is 0,50%, 100%; Elution flow rate is 1ml/min, collects to have active eluting peak;
(4) above-mentioned component with activated water eluting peak is concentrated to 4～5ml, last Sephadex LH-20 post, the high 50cm of filler; Column diameter 3.2cm; Use water elution, elution flow rate is 0.5ml/min, collects to have active water elution peak; Get the small intestinal alpha-glucosidase restraining agent effective ingredient behind the concentrate drying, yield is 10～15%.
Performance test
The detection method of active eluting peak: eluting is partly launched with the thin-layer silicon offset plate of 0.2mm, and developing solvent is a chloroform: methanol: water=6: 4: 1.With the colour developing of 5% sulphuric acid spraying post-heating, the Rf value with high activity eluting peak is 0.2～0.3.
The present invention has following advantage:
(1) technology is simple, easy to operate;
(2) suppress active strong.Experiment showed, that when 0.2mg/ml concentration, the extract of this experiment arrives 92.5% and 90.2% respectively, IC to the suppression ratio of small intestinal alpha-glucosidase sucrose and maltose hydrolysing activity
50Value is respectively 31.3 μ g/ml and 33.1 μ g/ml.
The specific embodiment
Through embodiment the present invention is carried out concrete description below, be necessary to be pointed out that at this present embodiment only is used for the present invention is further specified, can not be interpreted as restriction protection domain of the present invention.The person skilled in the art in this field can make some nonessential improvement and adjustment to the present invention according to the content of the invention described above.
Embodiment 1:
(1) get the powder 10g of dry Fructus Sapindi Mukouossi fruit, particle mean size is 200 μ m, and adding concentration by solid-liquid ratio 1g: 40ml is 50% methanol 400ml, and concussion was at room temperature extracted 18 hours, separation of supernatant, and concentrate drying gets methanol extractum 2.6g;
(2) press solid-liquid ratio 1g: 30ml, above-mentioned methanol extractum is scattered in the 78ml distilled water, added 156ml ethyl acetate concussion extraction 2 times again in 1: 2 by volume, extract is separated with the water extract;
(3) above-mentioned aqueous portion is concentrated to 16ml, last macroporous adsorption resin chromatography post, and the high 50cm of filler, column diameter 4cm uses the water-methanol gradient elution, and flow velocity is 1ml/min, and concentration is 0,50%, 100%.Collect eluent, detect, getting the strongest active position is 100% water elution peak;
(4) above-mentioned component with activated water eluting peak is concentrated to 5ml, last Sephadex LH-20 post, the high 50cm of filler, column diameter 3.2cm uses water elution, and elution flow rate is 0.5ml/min.The fraction collection eluent, collection has active water elution peak, gets white solid 1.2g behind the concentrate drying.
Embodiment 2:
(1) get the powder 10g of dry Fructus Sapindi Mukouossi fruit, particle mean size is 300 μ m, and adding concentration by solid-liquid ratio 1g: 50ml is 60% methanol 500ml, at room temperature shook 20 hours, and separation of supernatant, and with obtaining methanol extractum 2.9g after the supernatant concentration drying;
(2) press solid-liquid ratio 1g: 40ml, above-mentioned methanol extractum is scattered in the 116ml distilled water, added 232ml ethyl acetate concussion extraction 3 times again in 1: 2 by volume, extract is separated with the water extract;
(3) above-mentioned aqueous portion is concentrated to 18ml, last polystyrene type low pole macroporous adsorbent resin Diaion HP-20 chromatographic column, and the high 50cm of filler, column diameter 4cm uses the water-methanol gradient elution, and elution flow rate is 1ml/min, and concentration is 0,50%, 100%.Collect eluent, detect, getting the strongest active position is 100% water elution peak;
(4) above-mentioned component with activated water eluting peak is concentrated to 6ml, last Sephadex LH-20 post, the high 50cm of filler, column diameter 3.2cm uses water elution, and elution flow rate is 0.5ml/min.The fraction collection eluent, collection has active water elution peak, gets white solid 1.3g behind the concentrate drying.
Embodiment 3:
(1) get the powder 10g of dry Fructus Sapindi Mukouossi fruit, particle mean size is 350 μ m, and adding concentration by solid-liquid ratio 1g: 50ml is 70% methanol 500ml, at room temperature shook 24 hours, and separation of supernatant, and with obtaining methanol extractum 3.0g after the supernatant concentration drying;
(2) press solid-liquid ratio 1g: 30ml, above-mentioned methanol extractum is scattered in the 90ml distilled water, added 180ml ethyl acetate concussion extraction 3 times again in 1: 2 by volume, extract is separated with the water extract;
(3) above-mentioned aqueous portion is concentrated to 20ml, last polystyrene type low pole macroporous adsorbent resin Diaion HP-20 chromatographic column, and the high 50cm of filler, column diameter 4cm uses the water-methanol gradient elution, and elution flow rate is 1ml/min, and concentration is 0,50%, 100%.Collect eluent, detect, getting the strongest active position is 100% water elution peak;
(4) above-mentioned component with activated water eluting peak is concentrated to 5ml after, last Sephadex LH-20 post, the high 50cm of filler, column diameter 3.2cm uses water elution, elution flow rate is 0.5ml/min.The fraction collection eluent, collection has active water elution peak, gets white solid 1.5g behind the concentrate drying.
Application example 1
Following by the Fructus Sapindi Mukouossi extract that obtains in the foregoing description to small intestinal alpha-glucosidase inhibition activity test method and result:
Order rat small intestine acetone extraction powder from SIGMA company, (pH7.0) homogenized is removed supernatant after centrifugal for PBS, 0.1M with phosphate buffer; The mixed liquor homogenized of reuse PBS+10%Triton, centrifugal back obtains supernatant.After dialyzer dialysis in 24 hours, to obtain the thick alpha-glucosaccharase enzymatic solution of rat small intestine.The sucrose of the thick enzymatic solution of rat small intestine alpha-glucosidase and maltose hydrolysing activity are respectively 0.38U/ml and 1.85U/ml.Wherein enzyme activity unit is defined as: under 37 ℃, pH6.8 condition, under the effect of enzyme, decomposed 1 μ mol sucrose or maltose in 1 minute, be defined as an enzyme activity unit (U).
The active enzyme reaction mixed solution of sucrose hydrolysis of measuring the small intestinal alpha-glucosidase comprises: the thick solution of rat small intestine alpha-glucosidase (0.2ml), Fructus Sapindi Mukouossi extract (0.1ml; Water-soluble), sucrose solution (0.2ml, 56mM sucrose is dissolved in the phosphate buffer of 0.1M pH6.3).
The enzyme reaction mixed solution of measuring the maltose hydrolysing activity of small intestinal alpha-glucosidase comprises: the thick solution of rat small intestine alpha-glucosidase (0.05mL), Fructus Sapindi Mukouossi extract (0.1ml; Water-soluble), maltose solution (0.35ml, 3.5mM maltose is dissolved in the phosphate buffer of 0.1M pH6.3).
Above reaction mixture was reacted 15 minutes in 37 ℃ of incubators, stop the hydrolysis of enzyme with the Tris-Hcl solution of 0.75ml.With glucose oxidase method (GOD method), be determined at the OD value under the 490nm wavelength, active with the cubage alpha-glucosidase inhibition of glucose.
Suppress percentage rate=[1-(the blank light absorption value of extract light absorption value-extract)/enzyme light absorption value alive] * 100%.
Test result is as shown in table 1:
Table 1 Fructus Sapindi Mukouossi extract suppresses active to the small intestinal alpha-glucosidase
Claims (1)
1. from Fructus Sapindi Mukouossi, extract a kind of method of small intestinal alpha-glucosidase inhibitor, it is characterized in that this method may further comprise the steps: `
(1) shell of dry Fructus Sapindi Mukouossi fruit is pulverized, particle mean size is 200～350 μ m, and adding concentration by solid-liquid ratio 1g: 40～50ml is 50～70% methanol; Concussion was at room temperature extracted 18～24 hours; Separation of supernatant, concentrate drying get methanol extractum, and yield is 25～30%;
(2) above-mentioned methanol extractum is scattered in the distilled water, solid-liquid ratio is 1g: 30～40ml, adds ethyl acetate then in 1: 2 by volume, and the water raffinate is collected in concussion extraction 2～3 times;
(3) above-mentioned water raffinate ` is concentrated to 15～20ml; Last polystyrene type low pole macroporous adsorbent resin DiaionHP-20 chromatographic column is used the water-methanol gradient elution, and concentration is 0%, 50%, 100%; Elution flow rate is 1ml/min, collects to have active 100% water elution peak;
(4) above-mentioned component with activated water eluting peak is concentrated to 4～5ml, last Sephadex LH-20 post, the high 50cm of filler; Column diameter 3.2cm; Use water elution, elution flow rate is 0.5ml/min, collects to have active water elution peak; Get the small intestinal alpha-glucosidase restraining agent effective ingredient behind the concentrate drying, yield is 10～15%.
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