CN108586560B - A two-aqueous-phase one-step extraction method of momordicoside and polysaccharide with blood glucose lowering activity - Google Patents

A two-aqueous-phase one-step extraction method of momordicoside and polysaccharide with blood glucose lowering activity Download PDF

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CN108586560B
CN108586560B CN201810243490.6A CN201810243490A CN108586560B CN 108586560 B CN108586560 B CN 108586560B CN 201810243490 A CN201810243490 A CN 201810243490A CN 108586560 B CN108586560 B CN 108586560B
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刘杨
王�琦
张杰良
肖湘
伦镜盛
钟名其
赵永杰
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Abstract

The invention relates to momordica saponins and polysaccharide with hypoglycemic activityThe aqueous two-phase one-step extraction method comprises the following steps: mixing dried fructus Momordicae Charantiae powder with isopropanol/(NH)4)2SO4The method comprises the steps of uniformly mixing two aqueous phase systems, extracting by adopting ultrasonic-microwave synergistic extraction, separating an upper phase and a lower phase, wherein the upper phase is a crude extract of the momordica saponins, performing rotary evaporation until no alcohol smell exists, preparing a momordica saponins extract, performing static adsorption and purification by using AB-8 type macroporous resin to obtain a refined extract of the momordica saponins, and performing freeze drying to obtain a finished product of the momordica saponins.

Description

A two-aqueous-phase one-step extraction method of momordicoside and polysaccharide with blood glucose lowering activity
Technical Field
The invention relates to the technical field of deep processing of natural products, in particular to a two-aqueous-phase one-step extraction method of momordica saponins and momordica polysaccharide and in-vitro hypoglycemic activity investigation.
Background
The modern research shows that saponin and polysaccharide in the balsam pear are main active ingredients for playing a role in reducing blood sugar, so that the modern research shows that the extraction process technology of saponin and polysaccharide in the balsam pear is important for guiding the fine and further processing of the balsam pear and the development of functional food thereof, at present, the extraction processes of the balsam pear saponin and the balsam pear polysaccharide are mainly and independently extracted, the simultaneous extraction process of the balsam pear saponin and the polysaccharide is not reported, and the common organic solvent bath or hot alcohol bath extraction, the ultrasonic or microwave extraction, the macroporous adsorption resin extraction, the enzyme extraction and other methods are usually adopted, the methods are mainly used for extracting the balsam pear saponin alone or combined two by two, but the methods have high energy consumption, long extraction time, low extraction purity, complex operation, organic solvent residue and the like, the methods of the hot water bath extraction, the ultrasonic-one-step extraction, the polysaccharide extraction and the other methods are mainly used for carrying out the extraction of the single or combined extraction, the two-step extraction of polysaccharide and the polysaccharide inhibition of the glucose-sugar-reducing activity is more than that the polysaccharide-reducing-glucose-inhibiting animal model is mainly used for researching the balsam pear saponin-polysaccharide-.
Disclosure of Invention
The invention aims to provide a novel extraction method capable of simultaneously extracting and obtaining momordica saponins and momordica polysaccharide, which adopts an ultrasonic-microwave assisted two-aqueous-phase system separation technology, fully utilizes the cavitation effect of ultrasonic vibration, the high-energy effect of microwave and the rapid separation effect of two aqueous phases, particularly provides a mild separation environment and an enrichment effect by the two-aqueous-phase system, and is beneficial to obtaining the high-purity momordica saponins and momordica polysaccharide.
The invention relates to a method for extracting momordica saponins and momordica polysaccharide by two aqueous phases in one step, which comprises the following steps:
(1) preparing a crude extract of momordica saponins and momordica polysaccharide: taking dried balsam pear powder in isopropanol/(NH)4)2SO4In a double aqueous phase system, shaking and mixing uniformly, and performing ultrasonic-microwave synergistic extraction/reactionPerforming extraction by an instrument; after extraction, centrifugally separating the upper phase from the lower phase to obtain an upper phase enriched in saponin, namely a crude extract of the momordica saponins; the lower phase enriched with polysaccharide is the crude extract of polysaccharide from Momordica charantia;
(2) preparing a balsam pear saponin extracting solution: rotary evaporating the crude extract of momordicoside until no alcohol smell exists to obtain momordicoside extract;
(3) preparing a balsam pear saponin refined extract: further subjecting the extract of momordicoside to AB-8 type macroporous resin static adsorption and purification to obtain a refined extract of momordicoside;
(4) preparing a balsam pear polysaccharide refined extract: carrying out absolute alcohol precipitation, acetone washing and dialysis on the crude extract of the momordica charantia polysaccharide to remove salt so as to prepare a refined extract of the momordica charantia polysaccharide;
(5) freeze drying to obtain fructus Momordicae Charantiae saponin and fructus Momordicae Charantiae polysaccharide.
Wherein, in the step (1), isopropanol/(NH)4)2SO4Under the conditions of aqueous two-phase extraction, the linear length (T LL) is 65, the volume ratio (R) of the upper phase to the lower phase is 1, and the mass fractions of the components are respectively 15.33 percent (NH)4)2SO431.61% isopropanol, the remainder being made up with 0.01 mol/L PBS buffer (pH = 7.0);
the ultrasonic-microwave synergistic extraction/reaction instrument is of a CW-2000 type, the ultrasonic power is 50W, and the microwave power is 800W;
the mass ratio (material-liquid ratio) of fructus Momordicae Charantiae powder to aqueous two-phase system is 1:70(g/g), extraction time is 15min, and extraction temperature is 70 deg.C;
the centrifugation condition was 4000rpm for 10 min.
Wherein, the macroporous resin purification of the momordica saponins in the step (3) comprises the following specific steps:
static adsorption:
pouring 5-8g of the pretreated macroporous resin into a conical flask containing 100-150m L Momordica saponins extract, and shaking for 8h at 160r/min in a constant temperature oscillator at 25 ℃.
Static desorption:
washing the macroporous resin with double distilled water until no red color appears by a phenol-sulfuric acid method, filtering until the macroporous resin is dry, pouring the macroporous resin into a conical flask filled with 100-150m L absolute ethyl alcohol, oscillating for 1h at 160r/min in a constant temperature oscillator at 25 ℃, filtering, and rotationally evaporating the alcohol solution until no alcohol smell exists (adding a proper amount of double distilled water in the rotary evaporation process), thereby obtaining the refined extract of the momordica saponins.
Adding 4 times volume of absolute ethyl alcohol into the crude extract of the balsam pear polysaccharide in the step (4) for alcohol precipitation, and standing for 12 hours or overnight;
the dialysis desalting steps are as follows: re-dissolving the acetone-washed polysaccharide precipitate in double distilled water, and adding into 3500Da dialysis bag for dialysis for 30-40 h.
Wherein, the step (3) is to prepare a finished product of the balsam pear saponin in light yellow powder and a finished product of the balsam pear polysaccharide in white powder by freeze drying.
The raw material bitter gourd powder is preferably bitter gourd fruits, and is prepared by drying bitter gourd at low temperature and carrying out superfine grinding.
The invention also aims to provide the momordica saponins and the momordica polysaccharide crude extract, the momordica polysaccharide refined extract and the finished products prepared by the method.
The purity (percentage of momordicoside contained in the saponin finished product) of the momordicoside finished product prepared by the invention is measured by applying a vanillin-72 percent sulfuric acid method under an ultraviolet visible spectrum to be 75.24-79.57 percent; the purity of the polysaccharide product (percentage of polysaccharide in the polysaccharide product) is 95.70-99.54% by phenol-sulfuric acid method under ultraviolet visible spectrum.
The yield of the finished product of the momordica saponins (the mass percentage of the finished product of the saponins to the momordica powder) is 1.45 to 1.47 percent; the polysaccharide yield (the mass percentage of the polysaccharide finished product and the balsam pear powder) of the balsam pear polysaccharide finished product is 22.13-27.21 percent.
The specific method for determining the purity of the momordica saponins by the vanillin-72% sulfuric acid method is as follows:
preparing a mother solution:
preparing standard ginsenoside Rg1 solution by accurately weighing 5.0mg of ginsenoside Rg1 in a test tube, dissolving with methanol, and metering to 10m L with a volumetric flask to obtain 0.5mg/m L ginsenoside Rg1 standard solution;
preparing 8% vanillin solution by accurately weighing 8.0g vanillin in a 100m L conical flask, dissolving with glacial acetic acid, and diluting to 100m L with a volumetric flask to obtain 8% vanillin solution;
and (3) preparing a 72% sulfuric acid solution, namely accurately measuring 87m L double distilled water in a 500m L beaker, slowly adding 163m L concentrated sulfuric acid into the beaker, stirring the mixture by using a glass rod while adding the concentrated sulfuric acid, cooling the temperature to room temperature, and then fixing the volume to 250m L by using a volumetric flask to prepare the 72% sulfuric acid solution.
Drawing a standard curve of the ginsenoside Rg 1:
respectively sucking 30, 60, 90, 120, 150, 180 and 210 mu L ginsenoside Rg1 standard solutions into a test tube with a plug, filling the solutions in each test tube to 210 mu L by using methanol, taking 210 mu L methanol as a blank control, adding 0.3m L8% vanillin solution and 3m L72% sulfuric acid solution, shaking uniformly, carrying out water bath at 60 ℃ for 15min, carrying out ice bath for 10min, standing at room temperature for 2-3min, measuring the ultraviolet-visible spectrum absorbance value of each test tube at 560nm, and drawing a standard curve by taking the mass (mu g) of ginsenoside Rg1 as an abscissa and the absorbance value (OD) as an ordinate.
And (3) determining the purity of the finished product of the momordica saponins:
weighing a certain mass of finished balsam pear saponin product, preparing a saponin solution with a certain concentration, sucking 210 mu L saponin solution into a test tube with a plug, adding 0.3m L8% vanillin solution and 3m L72% sulfuric acid solution, shaking up, carrying out water bath at 60 ℃ for 15min, carrying out ice bath for 10min, standing at room temperature for 2-3min, measuring the absorbance luminosity value at 560nm, and calculating the purity of the finished balsam pear saponin product according to a drawn standard curve.
The specific method for measuring the purity of the momordica polysaccharide by using the phenol-sulfuric acid method comprises the following steps:
preparing a mother solution:
preparing a glucose standard solution, namely putting a proper amount of standard glucose into a 105 ℃ oven until the mass is not reduced any more, taking out the standard glucose until the mass is not reduced, accurately weighing 10.0mg of glucose into a 100m L conical flask, dissolving the glucose in double distilled water, and fixing the volume to 100m L by using a volumetric flask to prepare a 10mg/m L glucose standard solution;
preparing 80% phenol solution by melting phenol in a 50 deg.C water bath, accurately weighing 40.0g phenol in a 100m L beaker, adding 10.0g double distilled water, mixing, pouring into a brown reagent bottle, storing in a 4 deg.C refrigerator, and diluting the mother liquor to 5% when in use.
Drawing a glucose standard curve:
respectively sucking 0.0m, 0.1 m, 0.2m, 0.3m, 0.4 m and 0.5m L glucose standard solutions into a glass test tube, filling the solutions in each tube to 0.5m L by using double distilled water, taking 0.5m L double distilled water as a blank control, adding 0.5m L5% phenol solution, shaking uniformly, standing for 2min for full reaction, adding 2.5m L concentrated sulfuric acid solution, fully mixing uniformly, standing at room temperature for 20min, measuring the ultraviolet visible spectrum absorbance value of each tube at 490nm, and drawing a standard curve by taking the glucose concentration (mg/m L) as an abscissa and the absorbance value (OD) as an ordinate.
And (3) determining the purity of the momordica polysaccharide finished product:
weighing a certain mass of a momordica polysaccharide finished product, preparing a polysaccharide solution with a certain concentration, sucking 0.5m L polysaccharide solution into a glass test tube, adding 0.5m L5% phenol solution, shaking uniformly, standing for 2min for full reaction, adding 2.5m L concentrated sulfuric acid solution, standing for 20min at room temperature after fully mixing uniformly, measuring the ultraviolet visible spectrum absorbance value of each tube at 490nm, and calculating the purity of the polysaccharide finished product according to a drawn standard curve.
In addition, the invention also aims to prepare the finished products of the momordica saponins and the momordica polysaccharide with certain biological and hypoglycemic activities by establishing a novel aqueous two-phase one-step extraction process technology of the momordica saponins and the momordica polysaccharide, thereby comparing and evaluating the hypoglycemic effects of the momordica saponins and the momordica polysaccharide.
The momordica saponins and momordica polysaccharide finished products obtained by the invention have half-inhibitory concentration IC to α -glucosidase501.05mg/m L and 6.01mg/m L, respectively, IC of acarbose500.21mg/m L, the inhibition effect of momordica saponins is obviously better than that of momordica polysaccharide, but the inhibition activity of momordica saponins to α -glucosidase is lower than that of positive control acarbose, wherein the inhibition activity of momordica saponins to α -glucosidase is equivalent to 1/5 of acarbose.
The semi-inhibitory concentration IC of the obtained finished product of the momordica saponins to α -amylase506.70mg/m L, no significant inhibition of Momordica charantia polysaccharide, IC of acarbose500.16mg/m L, Momordica saponins inhibition of α -amylaseThe activity corresponds to 1/42 for acarbose.
The aqueous two-phase one-step extraction method of the momordica saponins and the momordica polysaccharide has the following advantages:
(1) the ultrasonic-microwave assisted double-water-phase system separation technology adopts a three-strong combination method, and fully utilizes the cavitation effect of ultrasonic vibration, the high-energy effect of microwave and the quick separation effect of double water phases. The defects of low biological activity, low yield, high cost and the like of the traditional extraction technology are overcome, the extraction process is simple, the cost is low, and the method is suitable for intermittent and large-scale production and processing of the high-purity and high-yield momordica saponins and momordica polysaccharide.
(2) According to the invention, the finished products of the momordica saponins and the momordica polysaccharides with certain biological and hypoglycemic activities are prepared by establishing a novel double aqueous phase one-step extraction process technology of the momordica saponins and the momordica polysaccharides, so that the hypoglycemic effects of the momordica saponins and the momordica polysaccharides are compared and evaluated, and a foundation is laid for further reasonably utilizing the momordica resources.
Drawings
FIG. 1 is a flow chart of the present invention for preparing momordicoside and momordicoside by a two-aqueous phase one-step method;
FIG. 2 is a standard graph of ginsenoside Rg1 and glucose; wherein, fig. 2(a) is a standard curve of ginsenoside Rg 1; FIG. 2(b) is a glucose standard curve;
FIG. 3 shows the purity and yield of momordicoside and momordicoside; wherein, figure 3(a) shows the purity of momordica saponins and momordica polysaccharides; FIG. 3(b) shows the yield of momordicoside and momordicoside;
FIG. 4 is a graph showing the inhibition rate of Momordica saponins, Momordica polysaccharides and acarbose on the activity of α -glucosidase;
FIG. 5 is a graph showing the inhibition rate of Momordica saponins, Momordica polysaccharides and acarbose on the activity of α -amylase.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.
Example 1
Preparing fructus Momordicae Charantiae saponin and fructus Momordicae Charantiae polysaccharide
A two-aqueous phase one-step method for preparing momordicoside and momordicoside is shown in figure 1. The method mainly comprises the following steps:
(1) preparing isopropanol/(NH)4)2SO4Aqueous two-phase system: weighing 53.655g (NH)4)2SO4To a 500m L reaction flask, 185.71g of 0.01 mol/L PBS buffer (pH =7.0) was added and shaken (NH)4)2SO4Dissolving, adding 110.635g of isopropanol, shaking, uniformly mixing, standing and layering;
(2) preparing a crude extract of momordica saponins and momordica polysaccharide: weighing 5g of dried bitter gourd powder in 350g of isopropanol/(NH)4)2SO4In the aqueous two-phase system, the mixture is shaken and mixed evenly and extracted by using a CW-2000 type ultrasonic-microwave synergistic extraction/reaction instrument. Ultrasonic power of 50W, microwave power of 800W, extraction time of 15min, extraction temperature of 70 deg.C, centrifuging at 4000rpm for 10min after extraction is finished, and separating upper and lower phases to obtain crude extract of momordicoside (upper phase) and crude extract of momordicoside (lower phase);
(3) preparing a balsam pear saponin extracting solution: performing rotary evaporation on the crude extract of momordicoside at 83 deg.C with a rotary evaporator until no alcohol smell exists (adding appropriate amount of double distilled water during rotary evaporation) to obtain momordicoside extract;
(4) preparing a momordica saponins extract, namely performing static adsorption on 5g of pretreated macroporous resin, pouring the macroporous resin into a 250m L conical flask filled with 150m L momordica saponins extract, oscillating the macroporous resin for 8 hours at 160r/min in a constant temperature oscillator at 25 ℃, performing static desorption, namely flushing the macroporous resin with double distilled water, performing suction filtration until the macroporous resin is not red by a phenol-sulfuric acid method, drying the macroporous resin, pouring the macroporous resin into a conical flask filled with 150m L absolute ethyl alcohol, oscillating the macroporous resin for 1 hour at 160r/min in a constant temperature oscillator at 25 ℃, filtering to remove the macroporous resin, and performing rotary evaporation on the alcoholic solution at 80 ℃ until the alcoholic smell is absent (adding a proper amount of double distilled water in the rotary evaporation process), thereby preparing the momordica saponins extract;
(5) preparing fructus Momordicae Charantiae polysaccharide refined extract by precipitating fructus Momordicae Charantiae polysaccharide crude extract with 4 times volume of anhydrous alcohol overnight, washing with 100m L acetone, dissolving polysaccharide precipitate in double distilled water, dialyzing in 3500Da dialysis bag for desalting, and dialyzing for 40 hr to obtain fructus Momordicae Charantiae polysaccharide refined extract;
(6) freeze drying to obtain yellowish powdered momordicoside and white powdered momordicoside.
Measuring absorbance value of the finished product of the momordica saponins at 560nm by applying a vanillin-72% sulfuric acid method to obtain the yield and purity of the finished product of the momordica saponins; the phenol-sulfuric acid method is used for measuring the absorbance value of the momordica polysaccharide finished product in ultraviolet visible spectrum at 490nm, so that the yield and the purity of the momordica polysaccharide finished product can be obtained.
The standard curve of ginsenoside Rg1 and glucose is shown in FIG. 2, wherein FIG. 2(a) is standard curve of ginsenoside Rg 1; FIG. 2(b) is a glucose standard curve.
The purity and yield of momordicoside and momordicoside are shown in fig. 3, wherein fig. 3(a) shows the purity of momordicoside and momordicoside, and fig. 3(b) shows the yield of momordicoside and momordicoside.
The conditions in the steps of the above-described embodiment can be adaptively changed according to the content of the specification, and the following data can be obtained:
the purity of the finished product of the momordica saponins is 75.24-79.57%, and the yield (the mass ratio of the finished product of the saponins to the momordica powder) is 1.45-1.47%; the purity of the polysaccharide product is 95.70-99.54%, and the yield (mass ratio of polysaccharide product to fructus Momordicae Charantiae powder) is 22.13-27.21%.
Example 2
In-vitro blood glucose-reducing activity comparison evaluation of momordica saponins and momordica polysaccharide
In the embodiment, the samples are acarbose, momordica saponin finished products and momordica polysaccharide finished products, and the in-vitro hypoglycemic activity determination indexes of the momordica saponin and the momordica polysaccharide are evaluated by the inhibition rates of the momordica saponin and the momordica polysaccharide on the activities of α -glucosidase and α -amylase.
1.α -glucosidase system
The enzyme activity unit is defined as the amount of PNPG which is required for hydrolyzing PNPG to release 1 mu mol of PNP within 1min by α -glucosidase under the conditions of 37 ℃ and pH =6.8, and the inhibitor activity unit is the inhibitor dosage which is required for reducing 1 enzyme activity unit under the same conditions.
The reaction principle is as follows:
Figure DEST_PATH_BDA0001605923880000081
Figure DEST_PATH_BDA0001605923880000082
the reaction process comprises preparing Momordica saponins solution (concentration of 0.25, 0.50, 1.00, 2.00, 4.00mg/m L) and Momordica polysaccharide solution (concentration of 1.25, 2.50, 5.00, 10.00, 20.00mg/m L) with 50% dimethyl sulfoxide as solvent, selecting acarbose as positive control (concentration of 0.05, 0.10, 0.20, 0.40, 0.80mg/m L0), accurately transferring 50 μ L167 mmol/L2 phosphate buffer solution (pH 6.8), adding 96-well plate, sequentially adding 10 μ L α -glucosidase solution (0.5U/m L) and 20 μ L sample solution, preheating the mixed solution in 37 deg.C constant temperature incubator for 5min, adding 20 μ L substrate (PNPG 1 mmol/L), shaking for 20min, adding 50 μ L1 mol/52 CO 1/L3The solution stops the reaction. And measuring the absorbance value of the enzyme-labeled sample at 405nm by using an enzyme-labeled instrument, and calculating the inhibition rates corresponding to different concentrations according to the following formula, wherein the blank group does not contain enzyme and PNPG, and the sample control group does not contain enzyme.
Inhibition ratio (%) ═ aBlank space-(ASample (I)-ASample controls)]/ABlank space×100％
2.α -Amylase System
Preparing a mother solution:
preparing a phosphate buffer solution, namely measuring 0.2 mol/L potassium dihydrogen phosphate solution 250m L in a 500m L beaker, adding 0.2 mol/L sodium hydroxide solution 118m L, uniformly mixing, fixing the volume to 1000m L by double distilled water, shaking uniformly, and preparing the phosphate buffer solution with the pH value of 6.8;
1% starch solution preparation, namely accurately weighing 1.0g of soluble starch, boiling with a proper amount of double distilled water, and fixing the volume to 100m L to prepare 1% starch solution;
the DNS solution is prepared by accurately weighing 10.0g of 3, 5-dinitrosalicylic acid in a 1000m L beaker, slowly adding 100m L5 mol/L NaOH solution after dissolving with a proper amount of double distilled water, stirring uniformly, then adding 200.0g of sodium potassium tartrate, stirring while adding double distilled water in a 50 ℃ water bath kettle, adding 2.0g of crystalline phenol and 0.5g of anhydrous sodium sulfite after completely dissolving, continuously heating and stirring until completely dissolving, cooling with running water, diluting with double distilled water to a constant volume of 1000m L, storing in a brown reagent bottle, and using for one week.
The reaction process comprises the steps of taking a phosphate buffer solution with the pH value of 6.8 as a solvent, respectively preparing sample balsam pear saponin solutions (with the concentrations of 1.00, 2.00, 4.00, 6.00, 8.00 and 10.00mg/m L) and balsam pear polysaccharide solutions (with the concentrations of 8.00, 12.00, 16.00, 20.00 and 50.00mg/m L), selecting acarbose as a positive control (with the concentrations of 0.05, 0.10, 0.20, 0.40, 0.60, 0.80 and 1.00mg/m L), sequentially and accurately transferring 125 mu L α -amylase solution (2U/m L) and 125 mu L sample solutions into a glass test tube, shaking, incubating for 10min in a constant-temperature incubator at the temperature of 37 ℃, then adding 250 mu L1% starch solution into the constant-temperature incubator at the temperature of 37 ℃, reacting for 10min, then adding 500 mu L DNS reagent, terminating the reaction in a boiling water bath 5min, cooling, adding double distilled water to the test tube, uniformly mixing, calculating the absorbance values of the absorbance values at different concentrations of L nm and determining the absorbance values according to the different light absorption rates.
The kit comprises a blank group (buffer solution + enzyme + buffer solution), a sample control group (sample + enzyme + buffer solution), a control group (buffer solution + enzyme + substrate), and a sample group (sample + enzyme + substrate).
Inhibition rate (%) ([ 1- (A) ]Sample (I)-ASample controls)/(AControl-ABlank space)]×100％
As shown in figure 4, momordicoside and momordicoside have certain inhibition effect on α -glucosidase activity, wherein the half-inhibition concentration IC of momordicoside on α -glucosidase50Is 1.05mg/m L, IC of Momordica charantia polysaccharide50At 6.01mg/m L, IC of acarbose500.21mg/m L, the inhibitory action of momordica saponins is obviously stronger than that of momordica polysaccharide, but the inhibitory activities of momordica saponins and momordica polysaccharide to α -glucosidase are both lower than that of acarbose as a positive control, and the inhibitory action of momordica saponins to α -glucosidaseThe activity corresponds to 1/5 for acarbose.
As shown in figure 5, the momordica saponins have certain inhibition effect on α -amylase activity, and the momordica saponins finished product obtained by the invention has half-inhibition concentration IC of α -amylase50At 6.70mg/m L, IC of acarbose50The content of the momordica saponins is 0.16mg/m L, the inhibition activity of the momordica saponins to α -amylase is equivalent to 1/42 of acarbose, and the absorbance photometric value of each concentration group of the momordica polysaccharides measured at 540nm by using an enzyme-labeling instrument shows that the momordica saponins have no obvious inhibition effect on the activity of α -amylase.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (7)

1. An aqueous two-phase one-step extraction method of momordica saponins and momordica polysaccharide is characterized by mainly comprising the following steps:
(1) preparing a crude extract of momordica saponins and momordica polysaccharide: taking dried balsam pear powder in isopropanol/(NH)4)2SO4In a double aqueous phase system, uniformly mixing by shaking, and extracting by using an ultrasonic-microwave synergistic extraction/reaction instrument; after extraction, centrifugally separating the upper phase from the lower phase to obtain an upper phase enriched in saponin, namely a crude extract of the momordica saponins; the lower phase enriched with polysaccharide is the crude extract of polysaccharide from Momordica charantia;
(2) preparing a balsam pear saponin extracting solution: rotary evaporating the crude extract of momordicoside until no alcohol smell exists to obtain momordicoside extract;
(3) preparing a balsam pear saponin refined extract: further subjecting the extract of momordicoside to AB-8 type macroporous resin static adsorption and purification to obtain a refined extract of momordicoside;
(4) preparing a balsam pear polysaccharide refined extract: carrying out absolute alcohol precipitation, acetone washing and dialysis on the crude extract of the momordica charantia polysaccharide to remove salt so as to prepare a refined extract of the momordica charantia polysaccharide;
(5) freeze drying to obtain fructus Momordicae Charantiae saponin and fructus Momordicae Charantiae polysaccharide.
2. The extraction method according to claim 1, wherein the isopropanol/(NH) in step (1)4)2SO4The conditions of aqueous two-phase extraction are as follows: the linear length is 65, the volume ratio of the upper phase to the lower phase is 1, and the mass fraction of each component is 15.33 percent (NH)4)2SO431.61% isopropyl alcohol, and the balance was made up with 0.01 mol/L PBS buffer.
3. The extraction method according to claim 1, wherein the ultrasonic-microwave synergistic extraction/reaction apparatus of step (1) is of the CW-2000 type, the ultrasonic power is 50W, and the microwave power is 800W.
4. The extraction method according to claim 1, wherein the bitter gourd powder and isopropanol/(NH) in step (1)4)2SO4The mass ratio of the aqueous two-phase system is 1:70(g/g), the extraction time is 15min, the extraction temperature is 70 ℃, and the centrifugation condition is 4000rpm for 10 min.
5. The extraction method according to claim 1, wherein the purification of momordicoside by macroporous resin in step (3) comprises the following steps:
static adsorption, namely pouring 5-8g of the pretreated macroporous resin into a conical flask filled with 100-150m L bitter gourd saponin extracting solution, and oscillating for 8 hours at 160r/min in a constant temperature oscillator at 25 ℃;
and (2) performing static desorption, namely flushing the macroporous resin by using double distilled water until the macroporous resin is not red when detected by using a phenol-sulfuric acid method, performing suction filtration until the macroporous resin is dried, pouring the macroporous resin into a conical flask filled with 100-150m L absolute ethyl alcohol, oscillating the macroporous resin in a constant temperature oscillator at 25 ℃ for 1h at 160r/min, filtering, and performing rotary evaporation on an alcohol solution until no alcohol smell exists, thus obtaining the balsam pear saponin refined extract.
6. The extraction method according to claim 1, wherein the absolute ethanol precipitation in the step (4) is mainly as follows: adding 4 times volume of absolute ethyl alcohol into the crude extract of the momordica charantia polysaccharide for alcohol precipitation, and standing for 12h or overnight.
7. The extraction method according to claim 1, wherein the dialysis desalting step in step (4) is: re-dissolving the acetone-washed polysaccharide precipitate in double distilled water, and adding into 3500Da dialysis bag for dialysis for 30-40 h.
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