CN102266360B

CN102266360B - Method for extracting alpha-glucosaccharase inhibitor from Fructus Terminaliae Immaturus

Info

Publication number: CN102266360B
Application number: CN201110161831A
Authority: CN
Inventors: 高鸿; 邓鹏翅; 黄毅娜; 钟凯; 祝瑞雪; 曾维才
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2011-06-16
Filing date: 2011-06-16
Publication date: 2012-07-04
Anticipated expiration: 2031-06-16
Also published as: CN102266360A

Abstract

The invention discloses a method for extracting an alpha-glucosaccharase inhibitor from Fructus Terminaliae Immaturus, and the method is characterized in that the method comprises the following steps: crushing dried Fructus Terminaliae Immaturus to achieve average particle size of 200-350 mu m, adding 50-100wt% ethanol as an extractant in a material-to-liquid ratio of 1 g:(20-30 mL), shaking at room temperature for 30-36 h, separating supernatant, concentrating, and drying to obtain ethanol extract with a yield of 35-40%; dispersing the ethanol extract in distilled water in a material-to-liquid ratio of 1 g:(20-30 mL), adding n-hexane in a volume ratio of 1:2, performing shaking extraction for 2-3 times, and collecting water-phase extract; and concentrating the water-phase extract to 15-20 mL, separating with macroporous absorbent resin Diaion HP-20 chromatography column, performing gradient elution with 25wt%, 80wt% and 100wt% water-methanol at an elution flow rate of 1 mL/min, collecting elution peak (eluate) with activity, concentrating, and drying to obtain a small intestine alpha-glucosaccharase inhibitor effective component with a yield of 10-20%. The alpha-glucosaccharase inhibitor disclosed by the invention, at a concentration of 0.4 mg/mL, has an inhibitory rate to the hydrolysis activity of small intestine alpha-glucosaccharase up to 76.2%, and has an IC50 (half maximal inhibitory concentration) of 51.7 mu g/mL.

Description

A kind of method of from FRUCTUS TERMINALIAE IMMATURUS, extracting alpha-glucosidase inhibitor

Technical field

The present invention relates to a kind of method of from FRUCTUS TERMINALIAE IMMATURUS, extracting alpha-glucosidase inhibitor, belong to Chinese herbal medicine preparation and chemical technology field.

Background technology

The glucose toxicity effect of postprandial hyperglycemia has following 2 points: reduce insulin sensitivity on the one hand, reduce β cell insulin secretion, quicken its MSOF; Acute on the other hand blood glucose peak directly causes oxidative stress status.To cardiovascular system, post-prandial glycemia increases the general detrimental effect that not only has hyperglycemia, still the independent hazard factor of numerous cardiovascular events.So, solve the prevention and the treatment problem of postprandial hyperglycemia, have very important significance for the generation and the progress of control of cardiovascular disorders.Alpha-glucosidase inhibitor can block disaccharidase hydrolytic enzyme such as being present in surperficial maltase of small intestinal mucosa microvillose membrane and saccharase; The process of polysaccharide, oligosaccharide and disaccharidase digestion the becoming monosaccharide such as glucose, fructose of picked-up is obstructed; Thereby reducing the post-prandial glycemia peak value, is a kind of medicine that the treatment diabetes of broad prospect of application are arranged.Compatriots' dietary habit is main with carbohydrate, so alpha-glucosidase inhibitor can better bring into play hypoglycemic effect, thereby diabetes, cardiovascular disease and metabolic syndrome are played tangible preventive and therapeutic effect.Be used for clinical alpha-glucosidase inhibitor at present and have three kinds: acarbose (Acarbose), voglibose (Voglibose) and miglitol (Miglitol); But it is more single to originate, and is the bacterial fermentation product, and all there is the gastrointestinal side effect in kind very little, does not have more choice clinically.Therefore, exploitation more many, more effectively, side effect alpha-glucosidase inhibitor still less supplies the selection of clinical use just to seem particularly important.China's natural resources of Chinese medicinal materials is abundant, from natural plants and medicine the new low toxicity of searching, alpha-glucosidase inhibitor is the domestic and international research focus always efficiently.Do not see at present other relevant patents and non-patent literature report are arranged.

Summary of the invention

The objective of the invention is to provide the method for preparing of alpha-glucosidase restraining agent effective ingredient in a kind of dry fruit of FRUCTUS TERMINALIAE IMMATURUS, have advantages such as technology is simple, easy to operate to the deficiency of prior art.

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 TERMINALIAE IMMATURUS, extracting the small intestinal alpha-glucosidase inhibitor may further comprise the steps:

(1) dry fruit of FRUCTUS TERMINALIAE IMMATURUS is pulverized, particle mean size is 200～350 μ m, is that the ethanol of 50～100wt% is as extractant by solid-liquid ratio 1g: 20～30ml adding concentration then; At room temperature shook 30～36 hours; Separation of supernatant obtains ethanol extract behind the concentrate drying, yield is 35～40%.

(2) above-mentioned ethanol extract is scattered in the distilled water, solid-liquid ratio is 1g: 20～30ml, adds normal hexane concussion extraction 2～3 times then in 1: 2 by volume, collects the water extract.

(3) above-mentioned water extract is concentrated to 15～20ml; Last macroporous adsorbent resin Diaion HP-20 chromatographic column is used the water-methanol gradient elution, and concentration is 25wt%, 80wt% and 100wt%; Elution flow rate is 1ml/min; Collection has active eluting peak, obtains the small intestinal alpha-glucosidase restraining agent effective ingredient behind the concentrate drying, and yield is 12～20%.

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, has the R of high activity eluting peak _fValue 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 0.4mg/ml concentration, the extract of this experiment reaches 76.2%, IC to the suppression ratio of small intestinal alpha-glucosidase maltose hydrolysing activity ₅₀Value is 51.7 μ g/ml.

Description of drawings

Fig. 1: the efficient liquid phase chromatographic analysis of FRUCTUS TERMINALIAE IMMATURUS extract

Analysis condition is following: ODS-3 chromatographic column (4.6 * 250mm, 5 μ m); Flow velocity, 1ml/min; Column temperature, 25 ℃; Detect wavelength, 280nm; Gradient eluent: 0min, 10% methanol; 10min, 20% methanol; 20-30min, 30% methanol; 50min, 40% methanol; 70-90min, 50% methanol; 100min, 100% methanol.Terchebin (retention time is 31.20min), Chebulagic acid (retention time is 40.97min) and chebulinic acid (retention time is 56.29min).

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 the restriction to protection domain of the present invention, the person skilled in the art in this field can content according to the present invention make some nonessential improvement and adjustment.

Embodiment 1:

(1) the dry fruit 5g of FRUCTUS TERMINALIAE IMMATURUS is pulverized after, particle mean size is 200 μ m, use then concentration as 50wt% ethanol 150ml as extractant; Concussion at room temperature 36 hours; Separation of supernatant, and with obtaining ethanol extract 1.97g after the supernatant concentration drying, yield is 39.4%.

(2) above-mentioned ethanol extract is scattered in the 40ml distilled water, with 80ml normal hexane concussion extraction 2 times, extract is separated with the water extract then.

(3) above-mentioned water extract is concentrated to 15ml after, last polystyrene type low pole macroporous adsorbent resin DiaionHP-20 (weight is 100g) chromatographic column is used the water-methanol gradient elution, concentration is 25wt%, 80wt% and 100wt%, elution flow rate is 1ml/min.Collection has active 80% methanol-eluted fractions peak, obtains faint yellow solid 0.65g behind the concentrate drying, and yield is 13%.

Embodiment 2:

(1) the dry fruit 5g of FRUCTUS TERMINALIAE IMMATURUS is pulverized after, particle mean size is 350 μ m, use then concentration as 80wt% ethanol 120ml as extractant; Concussion at room temperature 34 hours; Separation of supernatant, and with obtaining ethanol extract 1.76g after the supernatant concentration drying, yield is 35.2%.

(2) above-mentioned ethanol extract is scattered in the 44ml distilled water, with 88ml normal hexane concussion extraction 3 times, extract is separated with the water extract then.

(3) above-mentioned water extract is concentrated to 20ml after, last polystyrene type low pole macroporous adsorbent resin DiaionHP-20 (weight is 100g) chromatographic column is used the water-methanol gradient elution, concentration is 25wt%, 80wt% and 100wt%, elution flow rate is 1ml/min.Collection has active 80% methanol-eluted fractions peak, obtains faint yellow solid 0.96g behind the concentrate drying, and yield is 19.2%.

Embodiment 3:

(1) the dry fruit 5g of FRUCTUS TERMINALIAE IMMATURUS is pulverized after, particle mean size is 300 μ m, use then concentration as 100wt% ethanol 100ml as extractant; Concussion at room temperature 30 hours; Separation of supernatant, and with obtaining ethanol extract 1.81g after the supernatant concentration drying, yield is 36.2%.

(2) above-mentioned ethanol extract is scattered in the 54ml distilled water, with 108ml normal hexane concussion extraction 2 times, extract is separated with the water extract then.

(3) above-mentioned water extract is concentrated to 20ml after, last polystyrene type low pole macroporous adsorbent resin DiaionHP-20 (weight is 100g) chromatographic column is used the water-methanol gradient elution, concentration is 25wt%, 80wt% and 100wt%, elution flow rate is 1ml/min.Collection has active 80% methanol-eluted fractions peak, obtains faint yellow solid 0.85g behind the concentrate drying, and yield is 17%.

Application example 1

Following by the FRUCTUS TERMINALIAE IMMATURUS extract that obtains in the foregoing description to 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 thick enzyme maltose of rat small intestine alpha-glucosidase hydrolysing activity is 1.85U/ml.Wherein enzyme activity unit is defined as: under 37 ℃, pH6.8 condition, under the effect of enzyme, decomposed 1 μ mol maltose in 1 minute, be defined as an enzyme activity unit (U).

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), tried thing (0.1ml; Be dissolved in 25% dimethyl sulfoxide), maltose solution (0.35ml; 3.5mM maltose is dissolved in 0.1M, the phosphate buffer of 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 TERMINALIAE IMMATURUS extract suppresses active to the rat small intestine alpha-glucosidase

Claims

1. method of from FRUCTUS TERMINALIAE IMMATURUS, extracting the small intestinal alpha-glucosidase inhibitor is characterized in that this method may further comprise the steps:

(1) dry fruit of FRUCTUS TERMINALIAE IMMATURUS is pulverized, particle mean size is 200～350 μ m, is that the ethanol of 50～100wt% is as extractant by solid-liquid ratio 1g: 20～30ml adding concentration then; At room temperature shook 30～36 hours; Separation of supernatant obtains ethanol extract behind the concentrate drying, yield is 35～40%;

(2) above-mentioned ethanol extract is scattered in the distilled water, solid-liquid ratio is 1g: 20～30ml, adds normal hexane concussion extraction 2～3 times then in 1: 2 by volume, collects the water extract;

(3) above-mentioned water extract is concentrated to 15～20ml; Last macroporous adsorbent resin Diaion HP-20 chromatographic column is used the water-methanol gradient elution, and methanol concentration is 25wt%, 80wt% and 100wt%; Elution flow rate is 1ml/min; Collection has active 80wt% methanol-eluted fractions peak, obtains the small intestinal alpha-glucosidase restraining agent effective ingredient behind the concentrate drying, and yield is 12～20%.