CN102477105A - Method for combined preparation of polysaccharide and flavone by using pumpkin stems and leaves as raw material - Google Patents

Abstract

The present invention discloses a method for combined preparation of polysaccharide and flavone by using pumpkin stems and leaves as raw material. The method comprises pulverizing pumpkin stems and leaves, performing microwave extraction with hot water, performing microwave extraction with 50-85% ethanol, mixing the extractive solutions, concentrating under a reduced-pressure to 1/3-1/5 of the original volume, separating and purifying the obtained concentrated solution via macroporous resin, and eluting with a water-alcohol system; removing proteins in the raffinate by an enzyme method or a trichloroacetic acid (TCA) method, dialyzing to remove small molecular components, precipitating with an alcohol, and drying to obtain pumpkin polysaccharide; and concentrating the eluate into an extract, dissolving in an alkali, filtering via an ultrafiltration membrane, precipitating the filtrate with an acid, refluxing with hot ethanol for dissolution and crystallization, and drying to obtain pumpkin flavone. The method has simple operation, remarkable economic benefit and little environmental pollution. The obtained products have high contents of active components.

Description

Climing with Cushaw stem is that raw material is united the method for producing polysaccharide and flavones

Technical field:

The present invention relates to a kind of climing with Cushaw stem be that raw material is united novel method, the novel process of producing polysaccharide and flavones.

Background technology:

Mellitus (Diabetes mellites is called for short DM) are a kind of comparatively common endocrine metabolism obstacle diseases, and research shows that the beta Cell of islet apoptosis plays important effect in the incidence and development of mellitus.Type i diabetes is the cell-mediated autoimmune disorder of a kind of T, is characteristic with the beta Cell of islet selective destruction.And in type ii diabetes animal model people islet amyloid polypeptide transgenic mouse and fat (ZDF) mouse body of Zucker mellitus, also find to exist the β apoptosis.Therefore, protect impaired beta Cell of islet, the apoptosis that reduces beta Cell of islet is an important channel of treatment mellitus.Recent study finds that various plants and natural drug are playing an important role aspect the protection beta Cell of islet.(pumpkin polysaccharide PP) is the effective constituent of extracting in the natural phant pumpkin to squash polyoses, found to have the lowering blood glucose effect, yet hypoglycemic mechanism is not clear.

The existing technology of extracting squash polyoses:

1) after one Chinese patent application number 200310111322.5 employing fragmentations, mill juice, the filtration; With the film of microfiltration membrane, different molecular weight, and the reverse osmosis concentration membrane sepn go out squash polyoses, lyophilize obtains squash polyoses, this method cost is high; Protein content is high, and impurity is many;

2) one Chinese patent application numbers 200310116848.2 is used hot-water extraction with pumpkin powder, and gained filtrating is used ethanol sedimentation, and the gained throw out is washed; Add Tricholroacetic Acid, place in the refrigerator and spend the night, centrifugal; The filtrating after drying of dialysing against the current, the polysaccharide product, this method is simple; But quality is relatively poor, and product quality is bad, and impurity is more;

3) one Chinese patent application 200710017285.X adopts the hot water lixiviate to obtain pumpkin water extract; Organic solvent deposit, deposition is used water dissolution, supernatant concentration; Remove albumen; Obtain three kinds of components, the 50% ethanol sedimentation thing of squash polyoses through ethanol precipitation, 70% ethanol sedimentation thing and 80% ethanol sedimentation thing, this method gained squash polyoses purity is not high;

4) one Chinese patent application 200810302942.X extracting in water 0.5-3h after treatment, it is centrifugal to filtrate, and supernatant is crossed resin column and is carried out column chromatography; Elutriant concentrates back adding ethanol and precipitates; Throw out adds the centrifugal deproteinated of Tricholroacetic Acid, the dry squash polyoses that gets of supernatant concentration, and this method gained polysaccharide content is high; But from raw material, only extract a kind of product, cost is higher relatively.

Pumpkin (Cucurbita moschata Duch.) is the annual herbage that overgrows of Curcurbitaceae Cucurbita.Claim in the Compendium of Material Medica that it has the effect of " bowl spares, tonifying liver gas, the beneficial motive, beneficial lung qi, beneficial vital essence ".Warm in nature, it is sweet, nontoxic to distinguish the flavor of, and goes into spleen, stomach two warps, ability moistening lung qi-benefitting, the apocenosis of reducing phlegm, expelling parasite detoxifcation, diseases such as treatment cough, asthma, lung carbuncle, constipation.In recent years, more research having been carried out in pumpkin chemical ingredients, pharmacological action and clinical application both at home and abroad, proved that pumpkin has multiple pharmacological component, is the precious resources of various healthcare products of exploitation and medicine.Result of study is found; The Chinese pumpkin nutritive ingredient is moisture 90.24%, protein 0.65%, fat 0.13%, glucide 6.08%, fiber 2.15%, ash content 0.73%, also has useful physiologically active substances such as abundant Serlabo, VITAMINs, amino acid, mineral substance and vegeto-alkali, cucurbitine, trigonelline etc. simultaneously.The pumpkin rattan mainly contains polysaccharide and flavonoid compound, and flavones has multiple pharmacological effect, comprises antioxygenation, anti-inflammatory action, anticancer and anti-gene mutagenesis.Xie Zongbo etc. show that through experiment pumpkin stem flavones has stronger anti-oxidant activity to lard.

Pumpkin is wide, wide in variety in China's plantation, resource is very abundant, is a kind of urgency desirable natural health-care products resource leaved for development.But people plant the pumpkin major part and obtain the Semen Cucurbitae seed at present, and pulp is used for food-processing with edible, and the pumpkin rattan is then abandoned in the field, causes a large amount of wastes of resource, and easy contaminate environment.Therefore, utilize the pumpkin resource of China's abundant, improve economic value added, turn waste into wealth, fully excavate its new utility value and Application Areas, have great importance.

Summary of the invention:

The technical problem that the present invention will solve provide a kind of climing with Cushaw stem be that raw material is united the method for producing polysaccharide and flavones, this method has been carried out abundant extraction to pumpkin tendril effective active composition, has improved the utilization ratio of pumpkin, can obtain good economic benefit.

The object of the invention is realized through following technical proposals:

Climing with Cushaw stem is that raw material is united the method for producing polysaccharide and flavones, it is characterized in that may further comprise the steps:

(1) extracts: the pumpkin tendril is pulverized, add 5-20 times of calorimetric water microwave extraction earlier, doubly measure the ethanolic soln microwave extraction of 50-85% again with 5-10, filtered while hot, united extraction liquid;

(2) macroporous resin adsorption is separated: the 1/3-1/5 that extracting solution is evaporated to original volume; Liquid concentrator is used 60% ethanol, 85% ethanol elution successively through the saturated absorption of macroporous resin, collects fluid injection and elutriant down respectively;

(3) polysaccharide purification: will descend fluid injection to adopt enzyme process or TCA method to remove albumen, the small molecules composition is removed in dialysis, separates out with ethanol sedimentation again, and lyophilize promptly gets the Folium Cucurbitae polysaccharide;

(4) flavones purifying: elutriant is condensed into medicinal extract, adds alkali dissolution, ultra-filtration membrane filters, and sees through liquid and adds Acid precipitation, and hot ethanol refluxes dissolving crystallized, is drying to obtain the Folium Cucurbitae flavones.

Macroporous resin can adopt a kind of among D4020, D103, HP20, the HZ80 in the said step (2).

Principle of the present invention is: microwave has very strong penetration power, can evenly inside and outside raw material, rapid heat, and breaks through cytolemma and cell walls; Make the extracellular solvent get in the cell more easily; Accelerate dissolution also discharges effective constituent, has shortened extraction time greatly, has improved extraction efficiency; Macroporous resin has stronger adsorptive power to flavones ingredient, and to the polysaccharide adsorptive power a little less than, reach flavones and the effective of polysaccharide component separates.

Embodiment:

Below in conjunction with embodiment the present invention is described in further detail, but embodiment of the present invention is not limited in this.

Embodiment 1:

Get 1kg pumpkin tendril and pulverize, add 8L hot water microwave extraction 2 times, filter; Filter residue is measured 60% ethanolic soln microwave extraction 2 times, filtered while hot, united extraction liquid with 5 times again; Extracting solution is evaporated to 1/5 of original volume, and liquid concentrator is crossed D103 macroporous resin column to saturated absorption, collects fluid injection down; Water, 60% ethanol, 85% ethanol carry out wash-out successively again, collect the flavones elutriant.

To descend fluid injection to be concentrated into 5L, add isopyknic 30% Tricholroacetic Acid, stir, under 4 ℃ of conditions, leave standstill 3-4h; Under the 5000rpm rotating speed centrifugal 15 minutes, remove albumen precipitation, repeatable operation 3 times will be removed the proteic solution dialysis tubing of packing into; Distill water dialysis is used in the flowing water dialysis again, adds edible ethanol to determining alcohol and reaches 85%, stirs 10 minutes, staticly settles; Centrifugal collecting precipitation, absolute ethanol washing, lyophilize gets Folium Cucurbitae polysaccharide 16g, content 78%.

Elutriant is condensed into medicinal extract, adds the sodium hydroxide solution dissolving of pH10, ultra-filtration membrane filters; Add dilute hydrochloric acid solution to solution through liquid and be neutral, collecting precipitation refluxes dissolving crystallized with hot ethanol, leach; Spraying drying gets Folium Cucurbitae flavones 4.57g, content 57% (in rutin).

Embodiment 2:

Get 1kg pumpkin tendril and pulverize, add 5L hot water microwave extraction 2 times, filter; Filter residue is measured 70% ethanolic soln microwave extraction 2 times, filtered while hot, united extraction liquid with 10 times again; Extracting solution is evaporated to 1/3 of original volume, and liquid concentrator is crossed D4020 macroporous resin column to saturated absorption, collects fluid injection down; Water, 60% ethanol, 85% ethanol carry out wash-out successively again, collect the flavones elutriant.

Following fluid injection adds 0.5% papoid, and ℃ go out enzyme 5 minutes of enzymolysis 4h under 55 ℃ of conditions, reheat to 100 is removed albumen precipitation; Repeatable operation 32 times will be removed the proteic solution dialysis tubing of packing into, and distill water dialysis is used in the flowing water dialysis again, add edible ethanol to determining alcohol and reach 80%; Stirred 10 minutes, and staticly settled centrifugal collecting precipitation; Absolute ethanol washing, lyophilize get Folium Cucurbitae polysaccharide 18g, content 74.2%.

Elutriant is condensed into medicinal extract, adds the sodium hydroxide solution dissolving of pH9, ultra-filtration membrane filters; Add dilute hydrochloric acid solution to solution through liquid and be neutral, collecting precipitation refluxes dissolving crystallized with hot ethanol, leach; Spraying drying gets Folium Cucurbitae flavones 4.35g, content 56.8% (in rutin).

Embodiment 3:

Get 2kg pumpkin tendril and pulverize, add 24L hot water microwave extraction 2 times, filter; Filter residue is measured 75% ethanolic soln microwave extraction 2 times, filtered while hot, united extraction liquid with 8 times again; Extracting solution is evaporated to 1/4 of original volume, and liquid concentrator is crossed HP20 macroporous resin column to saturated absorption, collects fluid injection down; Water, 60% ethanol, 85% ethanol carry out wash-out successively again, collect the flavones elutriant.

To descend fluid injection to be concentrated into 5L, add isopyknic 30% Tricholroacetic Acid, stir, under 4 ℃ of conditions, leave standstill 3-4h; Under the 5000rpm rotating speed centrifugal 15 minutes, remove albumen precipitation, repeatable operation 3 times will be removed the proteic solution dialysis tubing of packing into; Distill water dialysis is used in the flowing water dialysis again, adds edible ethanol to determining alcohol and reaches 85%, stirs 10 minutes, staticly settles; Centrifugal collecting precipitation, absolute ethanol washing, lyophilize gets Folium Cucurbitae polysaccharide 33g, content 68.1%.

Elutriant is condensed into medicinal extract, adds the sodium hydroxide solution dissolving of pH10, ultra-filtration membrane filters; Add dilute hydrochloric acid solution to solution through liquid and be neutral, collecting precipitation refluxes dissolving crystallized with hot ethanol, leach; Spraying drying gets Folium Cucurbitae flavones 9.1g, content 56.4% (in rutin).

Embodiment 4:

Get 2kg pumpkin tendril and pulverize, add 40L hot water microwave extraction, filter; Filter residue is again with 6 times of amount 80% ethanolic soln microwave extraction, filtered while hot, united extraction liquid; Extracting solution is evaporated to 1/5 of original volume, and liquid concentrator is crossed HZ80 macroporous resin column to saturated absorption, collects fluid injection down; Water, 60% ethanol, 85% ethanol carry out wash-out successively again, collect the flavones elutriant.

Following fluid injection adds 0.3% papoid, and ℃ go out enzyme 10 minutes of enzymolysis 2h under 50 ℃ of conditions, reheat to 100 is removed albumen precipitation; Repeatable operation 3 times will be removed the proteic solution dialysis tubing of packing into, and distill water dialysis is used in the flowing water dialysis again, add edible ethanol to determining alcohol and reach 90%; Stirred 15 minutes, and staticly settled centrifugal collecting precipitation; Absolute ethanol washing, lyophilize get Folium Cucurbitae polysaccharide 34g, content 72.5%.

Elutriant is condensed into medicinal extract, adds the sodium hydroxide solution dissolving of pH9, ultra-filtration membrane filters; Add dilute hydrochloric acid solution to solution through liquid and be neutral, collecting precipitation refluxes dissolving crystallized with hot ethanol, leach; Spraying drying gets Folium Cucurbitae flavones 9.3g, content 58.2% (in rutin).

Claims (2)

1. climing with Cushaw stem is that raw material is united the method for producing polysaccharide and flavones, it is characterized in that may further comprise the steps:

(1) extracts: the pumpkin tendril is pulverized, add 5-20 times of calorimetric water microwave extraction earlier, doubly measure the ethanolic soln microwave extraction of 50-85% again with 5-10, filtered while hot, united extraction liquid;

(2) macroporous resin adsorption is separated: the 1/3-1/5 that extracting solution is evaporated to original volume; Liquid concentrator is used 60% alcohol, 85% ethanol elution successively through the saturated absorption of macroporous resin, collects fluid injection and elutriant down respectively;

(3) polysaccharide purification: will descend fluid injection to adopt enzyme process or TCA method to remove albumen, the small molecules composition is removed in dialysis, separates out with ethanol sedimentation again, and lyophilize promptly gets the Folium Cucurbitae polysaccharide;

(4) flavones purifying: elutriant is condensed into medicinal extract, adds alkali dissolution, ultra-filtration membrane filters, and sees through liquid and adds Acid precipitation, and hot ethanol refluxes dissolving crystallized, is drying to obtain the Folium Cucurbitae flavones.

2. the method for producing polysaccharide and flavones according to claim 1 is characterized in that macroporous resin in the said step (2) can adopt a kind of among D4020, D103, HP20, the HZ80.