CN102746412B - Method for extracting momordica charantiap polysaccharide - Google Patents
Method for extracting momordica charantiap polysaccharide Download PDFInfo
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- CN102746412B CN102746412B CN201210213148.4A CN201210213148A CN102746412B CN 102746412 B CN102746412 B CN 102746412B CN 201210213148 A CN201210213148 A CN 201210213148A CN 102746412 B CN102746412 B CN 102746412B
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Abstract
The invention relates to a method for extracting momordica charantiap polysaccharide. The method comprises the following steps of adding hot water into momordica charantiaps as raw materials, carrying out ultrasound/microwave-assistant extraction, collecting a supernatant, carrying out concentration, decoloration and drying to obtain crude momordica charantiap polysaccharide, dissolving the crude momordica charantiap polysaccharide in water, carrying out centrifugal separation to obtain a supernatant, carrying out impurity removal, dialysis, chromatography and alcohol precipitation of the supernatant to obtain precipitates, and carrying out washing and drying to obtain pure momordica charantiap polysaccharide. The method does not produce harmful substances, completely retains active ingredients of momordica charantiap polysaccharide, and does not pollution on the environment. Momordica charantiap polysaccharide extracted by the method has high purity and a high yield. The method fully utilizes momordica charantiap resources and is a resource-saving and environmentally-friendly green production technology.
Description
technical field
The present invention relates to belong to agriculture, secondary product of forestry deep processing field, be specifically related to a kind of extracting method of bitter melon polysaccharide.
background technology
Balsam pear (Momordica charantiap), calls balsam pear, bright and beautiful lichee, leprosy grape, balsam pear, is Curcurbitaceae, the annual fruit of climbing up by holding on to draft balsam pear of Momordica, leaf alternate, palmate 5～7 drastic cracks.Spend little, unisexuality, monoecism, yellow, the long 2cm that is no more than.Fruit fusiform, has wart like structure, orange-yellow when ripe.Bitter, before the flesh bright red ,Wei Tian Ming Dynasty, medical book is not recorded balsam pear, Ming Dynasty < < Herbal for Relief of Famines > >, < < Compendium of Materia Medica > > begin to list in, doubt while travelling to the West for Triratna eunuch, from the South Sea Islands, move to.Ming Dynasty physician LI Shi-Zhen in < < Compendium of Materia Medica > > not, claim balsam pear " bitter; cold, nontoxic, there is evil removing; separate tired, the merit of the improving eyesight that clears away heart-fire, beneficial gas establishing-Yang ".Modern Chinese medicine thinks, balsam pear has heat clearly to wash the illnesss such as heat, improving eyesight removing toxic substances, cute conjunctivitis pain, pyreticosis polydipsia.Balsam pear is a kind of tame renewable green resource, and all there are plantation, aboundresources in all parts of the country.Balsam pear is not only good vegetables, is also good medicine, and it is a kind of dietotherapy good merchantable brand of medicine-food two-purpose, the polysaccharide material extracting from balsam pear, having the multiple effects such as the blood sugar of reduction, antibacterial anti-inflammatory, oxidation-resistance, and can improve immunizing power, is a kind of healthy environmental friendly product that is beneficial to man.Bitter melon polysaccharide product appearance is faint yellow to yellowish brown pressed powder, and easily moisture absorption, dissolves in hot water, is insoluble in the organic solvents such as alcohol, ketone, ether, chloroform.Polysaccharide has the multiple biological activitys such as immunologic function that improve human body, and modern medicine study shows, bitter melon polysaccharide has the multiple effects such as the blood sugar of reduction, antibacterial anti-inflammatory, oxidation-resistance.
summary of the invention
The present invention discloses a kind of extracting method of bitter melon polysaccharide, take balsam pear as raw material, adopts hot water lixiviate Crude polysaccharides, and then, with Sevag method isolating protein, hydrogen peroxide decolouring, ethanol precipitating is separated, then through purification by chromatography, cryodrying, pulverizes, and obtains polysaccharide sterling.The inventive method does not generate objectionable impurities, and the activeconstituents of bitter melon polysaccharide can be saved complete, free from environmental pollution, and the bitter melon polysaccharide purity of extraction is high, and yield is high.
The present invention adopts following technical scheme for achieving the above object:
A kind of extracting method of bitter melon polysaccharide, it is characterized in that: take balsam pear as raw material, adopt hot water extraction or water extraction film to extract Crude polysaccharides from method, then with Sevag method isolating protein, hydrogen peroxide decolouring, ethanol precipitating is separated, then through purification by chromatography, cryodrying, pulverize, obtain polysaccharide sterling, concrete steps are as follows:
(1) hot water extraction extracts Crude polysaccharides
A, get the raw materials ready: take fresh balsam pear as raw material, clean, dry, pulverized 20 orders, stand-by;
B, water extraction: the balsam pear material through pulverizing is dropped in extraction unit, add distilled water, stirring and evenly mixing, microwave treatment 1～2min or ultrasonication 10～15min, lixiviate 3-4h under the condition of pH value 7-8 and temperature 85-95 ℃ left and right, filtering separation, obtain lixiviate filtrate for the first time, residue adds suitable distilled water again, stirs, lixiviate 1-2h under 95-100 ℃ of condition, filtering separation, obtains lixiviate filtrate for the second time, merges lixiviate filtrate twice, stand-by, during lixiviate, solid-liquid ratio is 1; (18-22);
C, concentrated: lixiviate filtrate is dropped in vacuum concentrator, and decompression, cryoconcentration, to lixiviate filtrate original volume 1/4 left and right, obtain concentrated solution, stand-by;
D, decolouring: balsam pear Crude polysaccharides concentrated solution is dropped in decolouring still, and under the condition stirring, adding a small amount of concentration is 20%～30% hydrogen peroxide, mixes, standing, decolours stand-by;
E, alcohol are analysed: destainer is dropped into alcohol and analyse in still, under the condition stirring, slowly adding concentration is the ethanol of 90-95%, until ethanol volumetric concentration reaches 75%～85% in feed liquid, then in-10～-15 ℃ of hold over night, after alcohol is analysed completely, carrying out centrifugation ， get lower floor precipitating thing is balsam pear Crude polysaccharides again, then uses 95% washing with alcohol balsam pear Crude polysaccharides, collect upper phase and washing lotion, reclaim ethanol;
F, dry: the balsam pear Crude polysaccharides precipitating thing after washing is carried out to cryodrying, then pulverize, obtain balsam pear Crude polysaccharides;
(2) Crude polysaccharides purifying
A, dissolving: balsam pear Crude polysaccharides is dissolved with appropriate distilled water, be placed in supercentrifuge, collect supernatant liquid, discard lower floor's material;
B, removal of impurities: in resulting balsam pear Crude polysaccharides clear liquid, add the concussion of Sevag reagent, standing, then centrifugation, discard lower floor's impurity, collect supernatant liquid, the supernatant liquid of collection is standing after layering, water and organic phase are separated, organic phase reclaims chloroform and propyl carbinol recycles, water is balsam pear Crude polysaccharides clear liquor, stand-by, and the volume ratio of balsam pear Crude polysaccharides clear liquid and Sevag reagent is: 1:(1-1.5);
C, dialysis: the removal of impurities of dialysing of balsam pear Crude polysaccharides clear liquor, after dialysis treatment, take out dialyzed solution, stand-by;
D, chromatography: balsam pear Crude polysaccharides dialyzate process DEAE-32 column chromatography purification, upper Sephadex G-25 post is separated, uses sodium chloride aqueous solution wash-out, collects the elutriant containing polysaccharide, obtains bitter melon polysaccharide sterling elutriant, stand-by;
E, alcohol are analysed: containing the chromatographic solution of bitter melon polysaccharide through suitable concentrated after, carry out alcohol and analyse, then centrifugation, lower floor's precipitating thing of collection is bitter melon polysaccharide, stand-by, the ethanol of liquid phase reclaims;
F, dry: carrying out again cryodrying processing after the washing of bitter melon polysaccharide precipitating thing, then pulverized 80-85 mesh sieve, and obtained bitter melon polysaccharide sterling.
The extracting method of described bitter melon polysaccharide, is characterized in that: during raw sugar purifying in Sevag reagent used the volume ratio of chloroform and propyl carbinol for=(4-5): 1.
The extracting method of described bitter melon polysaccharide, is characterized in that: the dialysis concrete operation method described in during raw sugar purifying is: first with the tap water 45-50h that dialyses, then with distilled water or deionized water dialysis 22-25h.
The extracting method of described bitter melon polysaccharide, is characterized in that: the concrete steps that described water extraction film extracts Crude polysaccharides from method are as follows:
A, get the raw materials ready: take fresh balsam pear as raw material, clean, dry, pulverized 20 orders, stand-by;
B, water extraction: the fresh balsam pear of pulverizing adds 20%～50% distilled water of its weight, homogenate in hollander, microwave treatment 10min then, suitable heating, lixiviate 1H left and right, filters, and reclaims residue and is used; Obtain supernatant liquor, stand-by.
C, decolouring: supernatant liquor is dropped in decolouring still, and under the condition stirring, adding a small amount of concentration is 20%～30% hydrogen peroxide, mixes, standing, and decolouring is stand-by.
D, film from; Get the supernatant liquor taking off after decolouring, by it as in membrane separation apparatus, under the condition that is 0.05～0.150MPa at pressure, make supernatant liquor cross the hollow-fibre membrane of molecular weight cut-off >=6000, obtain the concentrated solution of balsam pear Crude polysaccharides, stand-by, adopt the identical method alcohol of hot water extraction to analyse, be dried to obtain dry Crude polysaccharides.
Beneficial effect of the present invention is:
The technological process that the inventive method adopts hot water formulation to extract bitter melon polysaccharide from balsam pear is physical separating process, does not generate objectionable impurities, and technological process can realize cleaner production; Using water as digestion agent, can save a large amount of organic solvents, and reduced the destruction of organic solvent to bitter melon polysaccharide physiologically active, improved the quality of product, reduced the pollution to environment.At water extraction film, in method, can obtain two kinds of active substances of bitter melon polysaccharide and momordica saponins simultaneously, balsam pear resource is fully utilized.Therefore, this technique is a kind of resource-conserving, environmentally friendly green production process.
accompanying drawing explanation
Fig. 1 is process flow sheet of the present invention.
embodiment
embodiment 1hot water extraction method extracts Crude polysaccharides
(1) get the raw materials ready: 20 orders are cleaned, dry, pulverized to fresh balsam pear, stand-by;
(2) water extraction: the balsam pear material through pulverizing is dropped in extraction unit, add the distilled water of 20 times of amounts, stirring and evenly mixing, microwave treatment 1～2min(or ultrasonication 10～15min) lixiviate 3H under the condition of pH value 7.5 and 90 ℃ of left and right, filtering separation, obtains lixiviate filtrate for the first time, and residue adds the distilled water of 10 times of amounts, stir, lixiviate 1h under 100 ℃ of conditions, filtering separation, collects residue and fully utilizes separately, merge lixiviate filtrate twice, stand-by;
(3) concentrated: lixiviate filtrate is dropped in vacuum concentrator, and decompression, cryoconcentration, to feed liquid original volume 1/4 left and right, obtain concentrated solution, stand-by;
(4) decolouring: balsam pear Crude polysaccharides concentrated solution is dropped in decolouring still, and adding a small amount of concentration under the condition stirring is 30% hydrogen peroxide, mixes, standing, decolours stand-by;
(5) alcohol is analysed: destainer is dropped into alcohol and analyse in still, under the condition stirring, slowly by 95% appropriate ethanol, until alcohol concn is got to 75%～85%(V/V in feed liquid), be statically placed in refrigerator, in-10～-15 ℃, spend the night, allow alcohol analyse completely, centrifugation, obtaining precipitating thing is balsam pear Crude polysaccharides, use again 95% washing with alcohol 2 times, stand-by; Collect liquid phase and washing lotion, reclaim ethanol;
(6) dry: the balsam pear Crude polysaccharides precipitating thing after washing to be placed in to vacuum-drying and under decompression and cold condition, to be dried; Or move in freeze drier lyophilize under decompression and the condition of-57 ℃.Then pulverize, obtain balsam pear Crude polysaccharides.
embodiment 2water extraction film extracts Crude polysaccharides from method
(1) get the raw materials ready: 20 orders are cleaned, dry, pulverized to fresh balsam pear, stand-by;
(2) water extraction: the fresh balsam pear of pulverizing is added to 20%～50% distilled water of its weight, homogenate in hollander, microwave treatment 10min then, suitable heating, lixiviate 1H left and right, filters, and reclaims residue and is used; Obtain supernatant liquor, stand-by;
(3) decolouring: supernatant liquor is dropped in decolouring still, and adding a small amount of concentration under the condition stirring is 20%～30% hydrogen peroxide, mixes, standing, and decolouring is stand-by;
(4) film from: get the supernatant liquor taking off after decolouring, by it as in membrane separation apparatus, under the condition that is 0.05～0.150MPa at pressure, make supernatant liquor cross the hollow-fibre membrane of molecular weight cut-off >=6000, obtain the concentrated solution containing balsam pear Crude polysaccharides, stand-by, collect and see through liquid, for extracting momordica saponins;
(5) alcohol is analysed: balsam pear Crude polysaccharides concentrated solution is dropped into alcohol and analyse in still not under the condition of stirring, slowly add 95% appropriate ethanol of people, until alcohol concn reaches 75%～85%(V/V in feed liquid), be statically placed in-10-15 ℃ spends the night, and allows alcohol analyse completely, centrifugation, obtain my ikugua Crude polysaccharides of precipitating thing, then use 95% washing with alcohol 2 times, stand-by, collect liquid phase and washing lotion, reclaim ethanol;
(6) the balsam pear Crude polysaccharides precipitating thing after washing is placed in to vacuum-drying is dried under decompression and cold condition; Or move in freeze drier lyophilize under decompression and the condition of-55 ℃.Then pulverize, obtain balsam pear Crude polysaccharides.
embodiment 3balsam pear Crude polysaccharides purifying
(1) dissolve: embodiment 1 and 2 gained balsam pear Crude polysaccharides are dissolved with appropriate distilled water, be placed in supercentrifuge, centrifugation 0.5h left and right, to remove a small amount of insolubles, obtains clear liquid, stand-by;
(2) removal of impurities: in resulting balsam pear Crude polysaccharides clear liquid, add the Sevag reagent (chloroform: propyl carbinol=4:1, V/V) of approximately 1 times of amount, concussion 0.5h left and right, standing, allow the impurity such as floating preteins fully separate out, then centrifugation, remove the impurity such as albumen, qualified rear standing, the layering of the reason that Clicks here, separates water and organic phase, and organic phase recycles for reclaiming chloroform and propyl carbinol, water is balsam pear Crude polysaccharides clear liquor, stand-by;
(3) dialysis: balsam pear Crude polysaccharides clear liquor is packed in dialysis machine dialysis tubing, with the tap water 48h that dialyses, then with distilled water or deionized water dialysis 24h to remove inorganic salt, low molecular weight impurities, after dialysis treatment, take out dialyzed solution, stand-by;
(4) chromatography: balsam pear Crude polysaccharides dialyzate process DEAE-32 column purification, go up again Sephadex G-25 post separated, use sodium chloride aqueous solution wash-out, detect polysaccharide elution peak, with automatic segmentation collector, collect the elutriant containing polysaccharide, obtain bitter melon polysaccharide sterling elutriant, stand-by, collect other elutriants and fully utilized;
(5) alcohol is analysed: containing the chromatographic solution of bitter melon polysaccharide after suitable concentrated of vacuum and low temperature, drop in precipitating still, under the condition of normal temperature, stir, slowly add 95% appropriate ethanol, make in feed liquid containing alcohol concn 75%～85%(V/V) till, be statically placed in-10～-15 ℃ and spend the night, allow bitter melon polysaccharide fully separate out, then carry out centrifugation, liquid phase send reduction vaporization to reclaim ethanol, and precipitating thing is bitter melon polysaccharide, stand-by;
(6) dry: bitter melon polysaccharide precipitating thing is respectively washed 2 times with dehydrated alcohol, acetone, ether successively, organic washing lotion send reduction vaporization to reclaim organic solvent, balsam pear Crude polysaccharides precipitating thing after cleaning is placed in to vacuum drier, dry under decompression and cold condition, then pulverize 80 mesh sieves, obtained bitter melon polysaccharide sterling.
Claims (3)
1. the extracting method of a bitter melon polysaccharide, it is characterized in that: take balsam pear as raw material, adopt hot water extraction or water extraction film to extract Crude polysaccharides from method, then with Sevag method isolating protein, hydrogen peroxide decolouring, ethanol precipitating is separated, then through purification by chromatography, cryodrying, pulverize, obtain polysaccharide sterling, concrete steps are as follows:
(1) hot water extraction extracts Crude polysaccharides
A, get the raw materials ready: take fresh balsam pear as raw material, clean, dry, pulverized 20 orders, stand-by;
B, water extraction: the balsam pear material through pulverizing is dropped in extraction unit, add distilled water, stirring and evenly mixing, microwave treatment 1～2min or ultrasonication 10～15min, lixiviate 3-4h under the condition of pH value 7-8 and temperature 85-95 ℃, filtering separation, obtain lixiviate filtrate for the first time, residue adds suitable distilled water again, stirs, lixiviate 1-2h under 95-100 ℃ of condition, filtering separation, obtains lixiviate filtrate for the second time, merges lixiviate filtrate twice, stand-by, during lixiviate, solid-liquid ratio is 1; (18-22);
C, concentrated: lixiviate filtrate is dropped in vacuum concentrator, and decompression, cryoconcentration, to lixiviate filtrate original volume 1/4, obtain concentrated solution, stand-by;
D, decolouring: balsam pear Crude polysaccharides concentrated solution is dropped in decolouring still, and under the condition stirring, adding a small amount of concentration is 20%～30% hydrogen peroxide, mixes, standing, decolours stand-by;
E, alcohol are analysed: destainer is dropped into alcohol and analyse in still, under the condition stirring, slowly adding concentration is the ethanol of 90-95%, until ethanol volumetric concentration reaches 75%～85% in feed liquid, then in-10～-15 ℃ of hold over night, after alcohol is analysed completely, carrying out centrifugation ， get lower floor precipitating thing is balsam pear Crude polysaccharides again, then uses 95% washing with alcohol balsam pear Crude polysaccharides, collect upper phase and washing lotion, reclaim ethanol;
F, dry: the balsam pear Crude polysaccharides precipitating thing after washing is carried out to cryodrying, then pulverize, obtain balsam pear Crude polysaccharides;
(2) Crude polysaccharides purifying
A, dissolving: balsam pear Crude polysaccharides is dissolved with appropriate distilled water, be placed in supercentrifuge, collect supernatant liquid, discard lower floor's material;
B, removal of impurities: in resulting balsam pear Crude polysaccharides clear liquid, add the concussion of Sevag reagent, standing, then centrifugation, discard lower floor's impurity, collect supernatant liquid, the supernatant liquid of collection is standing after layering, water and organic phase are separated, organic phase reclaims chloroform and propyl carbinol recycles, water is balsam pear Crude polysaccharides clear liquor, stand-by, and the volume ratio of balsam pear Crude polysaccharides clear liquid and Sevag reagent is: 1:(1-1.5);
C, dialysis: the removal of impurities of dialysing of balsam pear Crude polysaccharides clear liquor, after dialysis treatment, take out dialyzed solution, stand-by;
D, chromatography: balsam pear Crude polysaccharides dialyzate process DEAE-32 column chromatography purification, upper Sephadex G-25 post is separated, uses sodium chloride aqueous solution wash-out, collects the elutriant containing polysaccharide, obtains bitter melon polysaccharide sterling elutriant, stand-by;
E, alcohol are analysed: containing the chromatographic solution of bitter melon polysaccharide through suitable concentrated after, carry out alcohol and analyse, then centrifugation, lower floor's precipitating thing of collection is bitter melon polysaccharide, stand-by, the ethanol of liquid phase reclaims;
F, dry: carrying out again cryodrying processing after the washing of bitter melon polysaccharide precipitating thing, then pulverized 80-85 mesh sieve, and obtained bitter melon polysaccharide sterling;
The concrete steps that described water extraction film extracts Crude polysaccharides from method are as follows:
A, get the raw materials ready: take fresh balsam pear as raw material, clean, dry, pulverized 20 orders, stand-by;
B, water extraction: the fresh balsam pear of pulverizing adds 20%～50% distilled water of its weight, homogenate in hollander, microwave treatment 10min then, suitable heating, lixiviate 1H, filters, and reclaims residue and is used; Obtain supernatant liquor, stand-by;
C, decolouring: supernatant liquor is dropped in decolouring still, and under the condition stirring, adding a small amount of concentration is 20%～30% hydrogen peroxide, mixes, standing, and decolouring is stand-by;
D, film from; Get the supernatant liquor taking off after decolouring, by it as in membrane separation apparatus, under the condition that is 0.05～0.150MPa at pressure, make supernatant liquor cross the hollow-fibre membrane of molecular weight cut-off >=6000, obtain the concentrated solution of balsam pear Crude polysaccharides, stand-by, adopt the identical method alcohol of hot water extraction to analyse, be dried to obtain dry Crude polysaccharides.
2. the extracting method of bitter melon polysaccharide according to claim 1, is characterized in that: during raw sugar purifying in Sevag reagent used the volume ratio of chloroform and propyl carbinol for=(4-5): 1.
3. the extracting method of bitter melon polysaccharide according to claim 1, is characterized in that: the dialysis concrete operation method described in during raw sugar purifying is: first with the tap water 45-50h that dialyses, then with distilled water or deionized water dialysis 22-25h.
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| CN102936295A (en) * | 2012-12-11 | 2013-02-20 | 青岛海洋新材料科技有限公司 | Extraction method of bitter gourd polysaccharides |
| CN104311691B (en) * | 2014-11-11 | 2016-12-14 | 重庆市路迪机械厂 | A kind of extracting method of bitter melon polysaccharide |
| CN105440151A (en) * | 2016-01-25 | 2016-03-30 | 铜仁学院 | Technology for ultrasonic-assisted extraction of valerian polysaccharide |
| CN105820262A (en) * | 2016-03-31 | 2016-08-03 | 唐云龙 | Momordica polysaccharide extracting method |
| CN106490531A (en) * | 2016-10-21 | 2017-03-15 | 桂林淮安天然保健品开发有限公司 | The preparation method of balsam pear health-care product |
| CN109223851A (en) * | 2018-07-20 | 2019-01-18 | 三峡大学 | A kind of application of aqueous extract of Momordica Charantia Linn |
| CN109206532B (en) * | 2018-08-28 | 2021-05-07 | 华南师范大学 | Method for extracting, separating and purifying polysaccharide from myrtle fruits |
| CN109221837A (en) * | 2018-11-06 | 2019-01-18 | 武汉工程大学 | A kind of fast method for preparing of Bitter Melon Juice drink |
| CN111214406B (en) * | 2020-02-21 | 2021-10-29 | 重庆医药高等专科学校 | Bitter melon seed active ingredient, extraction method thereof and acne-removing repairing facial mask mud |
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| JP5481036B2 (en) * | 2008-03-24 | 2014-04-23 | 国立大学法人北海道大学 | Gagome-derived immunostimulant and method for extracting gagome-derived viscous polysaccharides |
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