CN111171172A - Preparation method of pueraria polysaccharide extract - Google Patents
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- CN111171172A CN111171172A CN202010101046.8A CN202010101046A CN111171172A CN 111171172 A CN111171172 A CN 111171172A CN 202010101046 A CN202010101046 A CN 202010101046A CN 111171172 A CN111171172 A CN 111171172A
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Abstract
The invention relates to a preparation method of a pueraria polysaccharide extract, which comprises the steps of crushing a pueraria medicinal material into filaments, washing with cold water, filtering to obtain the filaments, decocting the filaments in water, extracting, filtering, concentrating the filtrate, removing impurities by an adsorbent, removing protein, precipitating with alcohol, centrifuging to obtain precipitate, and drying to obtain the pueraria polysaccharide extract with the content of more than 40%. The extraction and purification method provided by the invention is simple and convenient to operate, and the yield of the pueraria polysaccharide is high.
Description
Technical Field
The invention relates to a preparation method of an extract, in particular to a preparation method of a pueraria polysaccharide extract, belonging to the field of preparation of traditional Chinese medicine extracts.
Background
Kudzu root is the hypertrophic root of perennial deciduous vine of the genus leguminosae (Pueraria. DC). The pueraria plants are nearly 18 in the global range, are mainly distributed in subtropical and temperate regions, and are about 9 and 2 varieties in China, and are intensively distributed in Yunnan and adjacent provinces. The radix Puerariae mainly comprises radix Puerariae starch, cellulose and isoflavone compounds. The kudzu root starch is the main component of kudzu root, fresh kudzu root contains about 20-25% of kudzu root starch, and the kudzu root starch is unique in nutrition and is a green health food with good compatibility of medicine and food. The kudzu root starch has the effects of clearing away heat and toxic materials, nourishing and nourishing, preventing heatstroke and lowering temperature, beautifying and protecting skin, and removing in-vivo garbage, has obvious curative effects on wind-fire toothache, oral ulcer, sore throat, heat cough, high fever, headache, scar and rash, skin tumor and itching, hemorrhoids, dysentery with heat, diarrhea, drunkenness, prostatitis and the like, and has special effects on preventing and treating hypertension, hyperlipidemia, coronary heart disease, angina, diabetes, cancer and the like.
Besides the pueraria starch, the research on pueraria extract at present at home and abroad mainly focuses on pueraria flavone and isoflavone, and the research on pueraria polysaccharide is less reported. The traditional method generally adopts ethanol as a solvent to extract the kudzuvine root, the yield of the extract is more than 8 percent, the flavone content in the alcohol extract is more than 40 percent, the polysaccharide content of the kudzuvine root is more than 5 percent, and the extract mainly takes the pharmacological action of the flavone.
The polysaccharide compound is mainly used for enhancing the immunologic function of a human body, resisting aging, inhibiting tumors, promoting blood circulation, resisting thrombus, reducing blood pressure, blood fat and blood sugar, protecting main organs such as liver and kidney and the like. The pueraria polysaccharide is favored by people due to unique physiological activity, is mainly used as food, health care products and the like, has good curative effect on resisting tumors, and relevant reports show that the pueraria polysaccharide has an injury effect on the proliferation of pheochromocytoma cell PC 12.
The existing extraction method of the pueraria polysaccharide is complex and has complicated steps, so the cost is high, the obtained finished product quantity is less, and new impurities are easy to be mixed, thereby limiting the batch production and use.
Disclosure of Invention
The invention provides a preparation method of a pueraria polysaccharide extract, which comprises the following steps:
(1) pulverizing radix Puerariae into filaments, washing with cold water, and filtering to obtain filaments;
(2) decocting radix Puerariae in water, and filtering to obtain filtrate;
(3) concentrating the filtrate, and removing impurities by using an adsorbent;
(4) removing protein by a Sevag method;
(5) precipitating with ethanol, and centrifuging to obtain precipitate;
(6) and (5) drying the precipitate to obtain the pueraria polysaccharide extract.
Further:
in the step (2), the water adding amount is 8-10 times of the weight of the radix puerariae medicinal material, the first decoction is carried out for 0.5-1.5h, and the second decoction is carried out for 0.5-1 h;
the concentration in the step (3) is to be concentrated to 2-3 times of the weight of the kudzu root medicinal material, and the impurity removal of the adsorbent is as follows: weighing an adsorbent with the volume of 15-30% of the concentrated solution, pouring into the concentrated decoction, stirring uniformly, placing in a 80-82 deg.C constant temperature water bath kettle, keeping the temperature for 40-50min, hot filtering, and cooling the filtrate to room temperature;
the sevag method protein removal in the step (4) is as follows: mixing the concentrated solution with chloroform and n-butanol according to a volume ratio of 12-20: 3-6: 0.5-2, oscillating for 15-30min, standing, retaining a water layer, repeating for 1-3 times, and standing, retaining the water layer;
the alcohol precipitation in the step (5) is as follows: adding ethanol into the water layer obtained in the step (4) to ensure that the mass percentage of the ethanol reaches 30-40%, stirring, standing, centrifuging, and collecting precipitates; adding ethanol into the supernatant to make the mass percent of the ethanol reach 45-55%, stirring, standing, centrifuging, and collecting the precipitate; continuously adding ethanol into the supernatant to enable the mass percentage of the ethanol to reach 60-80%, stirring, standing, centrifuging, and collecting precipitate;
the step (6) is as follows: mixing the collected precipitates, and oven drying to obtain radix Puerariae polysaccharide extract.
In one embodiment, in the step (2), the amount of water added is 8 times of the weight of the radix puerariae medicinal material, the first decoction is carried out for 0.5h, and the 2 nd decoction is carried out for 0.5 h; in the step (3), the concentration is carried out until the concentration is 2 times of the weight of the kudzu root medicinal material.
Further, the adsorbent in the step (3) is selected from at least one of diatomite and activated carbon.
In one embodiment, the adsorbent in step (3) is diatomaceous earth, and is used in an amount of 20% by volume of the concentrate.
In one embodiment, the step (4) is: mixing the concentrated solution with chloroform and n-butanol at a volume ratio of 15:4:1, oscillating for 20min, standing, retaining the water layer, repeating for 2 times, and standing, retaining the water layer.
In one embodiment, the step (4) is: mixing the concentrated solution with chloroform and n-butanol at a volume ratio of 15:4:1, oscillating for 25min, standing, retaining the water layer, repeating for 1 time, and standing, and retaining the water layer.
In one embodiment, the step (5) is: adding ethanol into the water layer obtained in the step (4) to enable the mass percentage of the ethanol to reach 30%, stirring, standing, centrifuging, and collecting precipitates; adding ethanol into the supernatant to enable the mass percentage of the ethanol to reach 45%, stirring, standing, centrifuging, and collecting precipitate; and continuously adding ethanol into the supernatant to ensure that the mass percentage of the ethanol reaches 70 percent, stirring, standing, centrifuging, and collecting the precipitate.
In one embodiment, the step (5) is: adding ethanol into the water layer obtained in the step (4) to enable the mass percentage of the ethanol to reach 35%, stirring, standing, centrifuging, and collecting precipitates; adding ethanol into the supernatant to enable the mass percentage of the ethanol to reach 50%, stirring, standing, centrifuging, and collecting precipitate; and continuously adding ethanol into the supernatant to enable the mass percentage of the ethanol to reach 75%, stirring, standing, centrifuging, and collecting the precipitate.
In one embodiment, the step (5) is: adding ethanol into the water layer obtained in the step (4) to enable the mass percentage of the ethanol to reach 40%, stirring, standing, centrifuging, and collecting precipitates; adding ethanol into the supernatant to enable the mass percentage of the ethanol to reach 55%, stirring, standing, centrifuging, and collecting precipitate; and continuously adding ethanol into the supernatant to enable the mass percentage of the ethanol to reach 80%, stirring, standing, centrifuging, and collecting the precipitate.
The content of the pueraria polysaccharide is determined by adopting the following method:
adopting a phenol-sulfuric acid method to draw a glucose standard curve, taking glucose concentration x as an abscissa (mu g/mL) and a light absorption value Y as an ordinate, measuring the absorbance of the glucose after the reaction with a sulfuric acid phenol solution at 490nm, and fitting a regression equation that Y is 0.0088x +0.0071 and R is20.9980, and the linearity is good in the range of 0 to 50. mu.g/mL.
0.1g of the finished product of pueraria polysaccharide of the above examples and comparative examples was taken, 3ml of water was added, ground into a homogenate, transferred into an Erlenmeyer flask, and the mortar was washed 3 times with 12ml of distilled water, and the eluate was transferred into the Erlenmeyer flask. Adding 10ml of 6mol/L hydrochloric acid into the Erlenmeyer flask, uniformly stirring, hydrolyzing in a boiling water bath for 30min, cooling, neutralizing with 10% NaOH solution, and keeping the pH value neutral. Then distilled water is used for fixing the volume to 100ml, filtration is carried out, 10ml of filtrate is taken, distilled water is used for fixing the volume to 100ml, and the total sugar hydrolysate is diluted into 1000 times. Taking 1ml of total sugar hydrolysate, and determining the content of reducing sugar: sucking 1ml total saccharide solution, placing in a test tube, soaking in ice bath for cooling, adding 4ml anthrone reagent, heating in boiling water bath for 10min, taking out, cooling, performing color comparison, and calculating the absorbance value by standard curve to remove the saccharide content in the sample solution.
Mass of reducing sugar (C)1V1/m)×100％
Total sugar ═ C2V2/m)×0.9×100％
Wherein, C1: mass concentration of reducing sugar (mg/mL);
C2: mass concentration of reducing sugar after hydrolysis (mg/mL);
V1: volume of reducing sugar extract (mL) in the sample;
V2: volume of total sugar extract (mL) in the sample;
m: sample mass (mg).
The invention has the beneficial effects that:
(1) according to the method, firstly, the kudzu root medicinal material is smashed into filaments and then is washed by cold water, so that impurities such as water-soluble flavone, protein and monosaccharide of the kudzu root medicinal material can be removed in advance, the obtained filaments of the kudzu root are more beneficial to extraction of kudzu root polysaccharide, the extraction time is shortened, and excessive impurities are prevented from being extracted.
(2) The color of the finished product polysaccharide can be obviously improved by using the adsorbent for decolorization treatment.
(3) The Sevag method can effectively remove free protein in polysaccharide and avoid anaphylactic reaction caused by taking pueraria polysaccharide by people with protein allergy physique.
(4) The multi-stage gradient alcohol precipitation method adopted by the invention realizes the extraction of the pueraria polysaccharide in the largest range, thereby improving the yield.
In conclusion, the preparation method of the pueraria polysaccharide extract is simple and convenient to operate and high in polysaccharide yield.
Detailed Description
The foregoing summary of the invention is described in further detail below with reference to specific embodiments. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples. Various substitutions and alterations can be made without departing from the technical idea of the invention as described above, according to the common technical knowledge and conventional means in the field, and the scope of the invention is covered.
Example 1 preparation of Pueraria polysaccharide extract
Taking 100Kg of kudzu root medicinal material, crushing into shreds, kneading and washing with 300Kg of cold water twice, then filtering out the shreds, adding into an extraction tank, adding 800Kg of water into the extraction tank, decocting for 0.5h, discharging decoction, adding 800Kg of water into the extraction tank, decocting for 0.5h, combining the two decoctions, and concentrating to 200 Kg; weighing 40L of diatomaceous earth, adding into the concentrated decoction, stirring, placing in 80-82 deg.C constant temperature water bath, keeping the temperature for 40-50min, hot filtering, and cooling the filtrate to room temperature; adding chloroform 53.3L and n-butanol 13.3L into the filtrate, mixing, and stirring for 25 min; standing, collecting water layer, adding chloroform 53.3L and n-butanol 13.3L into the water layer, mixing, and stirring for 25 min; standing, collecting water layer, adding ethanol to make ethanol content be 30%, standing, centrifuging, and collecting precipitate; adding ethanol into the supernatant to make the ethanol content be 45%, standing, centrifuging, and collecting precipitate; continuously adding ethanol into the supernatant to make the ethanol content reach 70%, standing, centrifuging, and collecting precipitate; mixing the three obtained precipitates, and oven drying to obtain radix Puerariae polysaccharide extract 26.3Kg with polysaccharide content of 43.8%.
Example 2 preparation of Pueraria polysaccharide extract
Taking 100Kg of kudzu root medicinal material, crushing into filaments, kneading and washing with 500Kg of cold water twice, then filtering out the filaments of kudzu root, adding the filaments of kudzu root into an extraction tank, adding 1000Kg of water into the extraction tank, decocting for 1.5h, discharging decoction, adding 1000Kg of water into the extraction tank, decocting for 1h, combining the two decoctions, and concentrating to 300 Kg; weighing 90L of active carbon, adding into the concentrated water decoction, stirring, placing in 80-82 deg.C constant temperature water bath, keeping the temperature for 40-50min, hot filtering, and cooling the filtrate to room temperature; adding 80L of chloroform and 20L of n-butanol into the filtrate, and stirring for 25 min; standing, collecting water layer, adding 80L of chloroform and 20L of n-butanol into the water layer, mixing, and stirring for 25 min; standing, collecting water layer, adding ethanol to make ethanol content reach 35%, standing, centrifuging, and collecting precipitate; adding ethanol into the supernatant to make the ethanol content reach 50%, standing, centrifuging, and collecting precipitate; continuously adding ethanol into the supernatant to make the ethanol content reach 75%, standing, centrifuging, and collecting precipitate; mixing the three obtained precipitates, and oven drying to obtain radix Puerariae polysaccharide extract 28.1Kg with polysaccharide content of 41.1%.
Example 3 preparation of Pueraria polysaccharide extract
Taking 100Kg of kudzu root medicinal material, crushing into shreds, kneading and washing with 400Kg of cold water twice, then filtering out the shreds, adding the kudzu root into an extraction tank, adding 900Kg of water into the extraction tank, decocting for 1h, discharging decoction, adding 900Kg of water into the extraction tank, decocting for 1h, combining the decoctions, and concentrating to 250 Kg; weighing 37.5L of diatomite, adding into the concentrated decoction, stirring, placing in 80-82 deg.C water bath, keeping the temperature for 40-50min, hot filtering, and cooling the filtrate to room temperature; adding chloroform 66.7L and n-butanol 16.7L into the filtrate, mixing, and stirring for 25 min; standing, collecting water layer, adding chloroform 66.7L and n-butanol 16.7L into the water layer, mixing, and stirring for 25 min; standing, collecting water layer, adding ethanol to make ethanol content 40%, standing, centrifuging, and collecting precipitate; adding ethanol into the supernatant to 55%, standing, centrifuging, and collecting precipitate; continuously adding ethanol into the supernatant to make the ethanol content reach 80%, standing, centrifuging, and collecting precipitate; mixing the three obtained precipitates, and oven drying to obtain radix Puerariae polysaccharide extract 26.9Kg with polysaccharide content of 42.0%.
Comparative example 1
The cold water kneading step in example 1 was removed: taking 100Kg of kudzu root medicinal material, crushing into 46-60 meshes, adding into an extraction tank, adding 800Kg of water into the extraction tank, decocting for 0.5h, discharging decoction, adding 800Kg of water into the extraction tank, decocting for 0.5h, combining the two decoctions, and concentrating to 200 Kg; weighing 40L of diatomaceous earth, adding into the concentrated decoction, stirring, placing in 80-82 deg.C constant temperature water bath, keeping the temperature for 40-50min, hot filtering, and cooling the filtrate to room temperature; adding chloroform 53.3L and n-butanol 13.3L into the filtrate, mixing, and stirring for 25 min; standing, collecting water layer, adding chloroform 53.3L and n-butanol 13.3L into the water layer, mixing, and stirring for 25 min; standing, collecting water layer, adding ethanol to make ethanol content be 30%, standing, centrifuging, and collecting precipitate; adding ethanol into the supernatant to make the ethanol content be 45%, standing, centrifuging, and collecting precipitate; continuously adding ethanol into the supernatant to make the ethanol content reach 70%, standing, centrifuging, and collecting precipitate; mixing the three obtained precipitates, and oven drying to obtain radix Puerariae polysaccharide extract 33.4Kg with polysaccharide content of 24.5%.
The polysaccharide extract prepared in comparative example 1 was 7.1Kg more than that prepared in example 1, but the polysaccharide content was greatly reduced, indicating that the cold water kneading step exerts a significant impurity removing effect.
Comparative example 2
The three-level gradient alcohol precipitation method in the example 1 is changed into a first-level alcohol precipitation method: taking 100Kg of kudzu root medicinal material, crushing into shreds, kneading and washing with 300Kg of cold water twice, then filtering out the shreds, adding into an extraction tank, adding 800Kg of water into the extraction tank, decocting for 0.5h, discharging decoction, adding 800Kg of water into the extraction tank, decocting for 0.5h, combining the two decoctions, and concentrating to 200 Kg; weighing 40L of diatomaceous earth, adding into the concentrated decoction, stirring, placing in 80-82 deg.C constant temperature water bath, keeping the temperature for 40-50min, hot filtering, and cooling the filtrate to room temperature; adding chloroform 53.3L and n-butanol 13.3L into the filtrate, mixing, and stirring for 25 min; standing, collecting water layer, adding chloroform 53.3L and n-butanol 13.3L into the water layer, mixing, and stirring for 25 min; standing, collecting water layer, adding ethanol to ethanol content of 80%, standing, centrifuging, collecting precipitate, and oven drying to obtain radix Puerariae polysaccharide extract 18.3Kg with polysaccharide content of 40.7%.
The polysaccharide extract prepared in the comparative example 2 is 8.0Kg less than that prepared in the example 1, which shows that the method for improving the yield of the pueraria polysaccharide extract by adopting a multi-stage gradient alcohol precipitation method is remarkable.
Claims (10)
1. A preparation method of a pueraria polysaccharide extract is characterized by comprising the following steps:
(1) pulverizing radix Puerariae into filaments, washing with cold water, and filtering to obtain filaments;
(2) decocting radix Puerariae in water, and filtering to obtain filtrate;
(3) concentrating the filtrate, and removing impurities by using an adsorbent;
(4) removing protein by a Sevag method;
(5) precipitating with ethanol, and centrifuging to obtain precipitate;
(6) and (5) drying the precipitate to obtain the pueraria polysaccharide extract.
2. The method of claim 1, wherein:
in the step (2), the water adding amount is 8-10 times of the weight of the radix puerariae medicinal material, the first decoction is carried out for 0.5-1.5h, and the second decoction is carried out for 0.5-1 h;
the concentration in the step (3) is to be concentrated to 2-3 times of the weight of the kudzu root medicinal material, and the impurity removal of the adsorbent is as follows: weighing an adsorbent with the volume of 15-30% of the concentrated solution, pouring into the concentrated decoction, stirring uniformly, placing in a 80-82 deg.C constant temperature water bath kettle, keeping the temperature for 40-50min, hot filtering, and cooling the filtrate to room temperature;
the sevag method protein removal in the step (4) is as follows: mixing the concentrated solution with chloroform and n-butanol according to a volume ratio of 12-20: 3-6: 0.5-2, oscillating for 15-30min, and standing to keep a water layer; repeating for 1-3 times, and retaining water layer;
the alcohol precipitation in the step (5) is as follows: adding ethanol into the water layer obtained in the step (4) to ensure that the mass percentage of the ethanol reaches 30-40%, stirring, standing, centrifuging, and collecting precipitates; adding ethanol into the supernatant to make the mass percent of the ethanol reach 45-55%, stirring, standing, centrifuging, and collecting the precipitate; continuously adding ethanol into the supernatant to enable the mass percentage of the ethanol to reach 60-80%, stirring, standing, centrifuging, and collecting precipitate;
the step (6) is as follows: mixing the collected precipitates, and oven drying to obtain radix Puerariae polysaccharide extract.
3. The method of claim 2, wherein: in the step (2), the water adding amount is 8 times of the weight of the radix puerariae medicinal material, the first decoction is carried out for 0.5h, and the 2 nd decoction is carried out for 0.5 h; in the step (3), the concentration is carried out until the concentration is 2 times of the weight of the kudzu root medicinal material.
4. The method of claim 1 or 2, wherein: the adsorbent in the step (3) is at least one selected from diatomite and activated carbon.
5. The method of claim 4, wherein: the adsorbent is diatomite, and the volume of the adsorbent is 20% of the volume of the concentrated solution.
6. The method of claim 2, wherein: in the step (4), the concentrated solution is mixed with chloroform and n-butanol according to the volume ratio of 15:4:1, the mixture is shaken for 20min, the water layer is reserved after standing, the operation is repeated for 2 times, and the water layer is reserved after standing.
7. The method of claim 2, wherein: in the step (4), the concentrated solution is mixed with chloroform and n-butanol according to the volume ratio of 15:4:1, the mixture is shaken for 25min, the water layer is reserved after standing, the operation is repeated for 1 time, and the water layer is reserved after standing.
8. The method of claim 2, wherein: the alcohol precipitation in the step (5) is as follows: adding ethanol into the water layer obtained in the step (4) to enable the mass percentage of the ethanol to reach 30%, stirring, standing, centrifuging, and collecting precipitates; adding ethanol into the supernatant to enable the mass percentage of the ethanol to reach 45%, stirring, standing, centrifuging, and collecting precipitate; and continuously adding ethanol into the supernatant to ensure that the mass percentage of the ethanol reaches 70 percent, stirring, standing, centrifuging, and collecting the precipitate.
9. The method of claim 2, wherein: the alcohol precipitation in the step (5) is as follows: adding ethanol into the water layer obtained in the step (4) to enable the mass percentage of the ethanol to reach 35%, stirring, standing, centrifuging, and collecting precipitates; adding ethanol into the supernatant to enable the mass percentage of the ethanol to reach 50%, stirring, standing, centrifuging, and collecting precipitate; and continuously adding ethanol into the supernatant to enable the mass percentage of the ethanol to reach 75%, stirring, standing, centrifuging, and collecting the precipitate.
10. The method of claim 2, wherein: the alcohol precipitation in the step (5) is as follows: adding ethanol into the water layer obtained in the step (4) to enable the mass percentage of the ethanol to reach 40%, stirring, standing, centrifuging, and collecting precipitates; adding ethanol into the supernatant to enable the mass percentage of the ethanol to reach 55%, stirring, standing, centrifuging, and collecting precipitate; and continuously adding ethanol into the supernatant to enable the mass percentage of the ethanol to reach 80%, stirring, standing, centrifuging, and collecting the precipitate.
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