CN102600245A - Purification method and application of water-soluble alkaloid with alpha-glycosidase activity inhibition function in mulberry branch bark - Google Patents
Purification method and application of water-soluble alkaloid with alpha-glycosidase activity inhibition function in mulberry branch bark Download PDFInfo
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- CN102600245A CN102600245A CN2012101184728A CN201210118472A CN102600245A CN 102600245 A CN102600245 A CN 102600245A CN 2012101184728 A CN2012101184728 A CN 2012101184728A CN 201210118472 A CN201210118472 A CN 201210118472A CN 102600245 A CN102600245 A CN 102600245A
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Abstract
The invention provides an extraction and purification method of water-soluble total alkaloid with an alpha-glycosidase activity inhibition function from mulberry branch bark and application of the water-soluble total alkaloid. The purification method comprises the following steps: based on the mulberry branch bark as a raw material, adding an extraction solvent (water and ethanol at a ratio of 100-20 percent to 0-80 percent), extracting, heating and refluxing, filtering, repeatedly extracting twice, combining two filtrates, and concentrating; adding 75-100 percent methanol or ethanol by volume, and performing alcohol precipitation at a temperature of between 4 and 30 DEG C; centrifuging or filtering to remove residues and insoluble substances, collecting the supernatant after alcohol precipitation and concentration, purifying through a cation exchange resin column, washing non-adsorbed impurities with deionized water, and eluting with 0.2 to 2mol of ammonia water; passing the eluate through an anoin exchange resin, washing with deionized water, collecting the non-adsorbed part, and regulating the pH to 8; filtering with a membrane, drying the filtrate to prepare powder, namely light-yellow water-soluble total alkaloid extract, wherein the extract can inhibit the activity of alpha-glycosidase, and the content of DNJ (1-deoxynojirimycin) is between 50 and 70 percent.
Description
Technical field
The present invention relates to the process for separation and purification and the application thereof of water-soluble alkaloid in a kind of Ramulus Mori with hypoglycemic activity.
Background technology
Diabetes are because a group of causing of hypoinsulinism and/or impaired insulin action is the metabolic disease of characteristic with the hyperglycemia.It shows as, and concentration of glucose raises unusually in blood and the urine, blood glucose, can occur typical " three-many-one-little " symptom when glucose in urine is too high, i.e. polydipsia, polyuria, polyphagia and lose weight, and with fatigue and weak.Diabetes generally are divided into several types such as type i diabetes, type ii diabetes and gestational diabetes.Diabetes not only can cause microvascular complication, like peripheral neuropathy, eye pathological changes, diabetic nephropathy etc., and can cause serious trunk complication, like myocardium infarction, apoplexy, lower limb vascular obstructive pulmonary disease etc.These complication are very harmful, reduced people's quality of life, brought heavy financial burden to people.The generation and the development of therefore treating diabetes, control of diabetes complication have seemed more and more important.
EPDML research shows that in this two big blood glucose target of fasting blood glucose level and level of postprandial blood sugar, level of postprandial blood sugar is particularly important, and it is the key factor that the patient of glucose tolerance impaired (IGT) develops into type ii diabetes.And level of postprandial blood sugar is the key factor that causes diabetes patient's trunk complication and microvascular complication, and these complication are main causes that cause that distribution of diabetes increases.So strict control post-prandial glycemia has very important significance to the control of diabetes, and the antidiabetic medicine that can significantly reduce level of postprandial blood sugar has very big using value.
Carbohydrate major part in the food is starch, polysaccharide or oligosaccharide.After meals were taken food, alpha-glucosidase inhibitor can be with disaccharidase, polysaccharide or starch competition and combine with alpha-glucosidase in saliva and the pancreatic juice, occupies the binding site on the enzyme, and the digestion of disaccharidase, polysaccharide or starch is obstructed.The carbohydrate that is not digested is transported to hypomere and colon in the small intestinal, thereby digesting and assimilating of carbohydrate occurred in the whole section small intestinal, delays and has prolonged the absorption of monosaccharide (glucose) after the meal, and increasing of post-prandial glycemia significantly reduced.
The root of moraceae plants, stem, leaf all contain alpha-glucosidase and suppress composition.Patent through retrieval one Chinese patent application numbers 20061011164.3 discloses a kind of from Ramulus Mori, extract preparation alpha-glucosidase inhibitor and application thereof, and this patent is to be that raw material extracts and suppresses alpha-glucosidase effective site with the Ramulus Mori.We suppress composition through the alpha-glucosidase in the experiment confirm Ramulus Mori and mainly are distributed in the cortex part, and the wooden part alpha-glucosidase inhibition of Ramulus Mori component content only is 1/10th of a cortex part.It is one of China's conventional industries that Bombyxmori Linnaeus is cultured, and cultures the area Bombyxmori Linnaeus and all will carry out a pruning to the secondary Ramulus Mori every year, and a large amount of Ramulus Moris are used for Wood Processing, and Wood Processing is only used the wooden part of Ramulus Mori, and the cortex part must be removed.Therefore, be medical material with the Ramulus Mori, wide material sources, low price.Because wooden part is removed, it is over half that the raw material volume is reduced, and saved the extraction solvent consumption greatly, and the labor hour and the energy when extracting; Its wooden part can be used for the raw material of Wood Processing; The cortex part also can be used as the raw material of natural textile fiber behind the water-soluble alkaloid that extracts hypoglycemic activity, have good comprehensive utilization prospect.
In addition; The percentage by weight that the resulting alkaloid valid target content with hypoglycemic activity of this patent accounts for extract is more than 50%; And its to represent the high-load of composition 1-deoxynojirmycin (1-deoxynojirimycin DNJ) be 50% of total alkaloids, that is to say that DNJ accounts for 25% of total extract.
Number of patent application is to have described among the 200810021592.X from Cortex Mori, to obtain a kind of extract with hypoglycemic activity, but not mentioned effective site, more not mentioned content of effective.
Summary of the invention
The object of the invention is to provide a kind of process for separation and purification and application thereof of from Ramulus Mori, extracting the water-soluble alkaloid with hypoglycemic activity.
Extraction from Ramulus Mori provided by the invention, separation, purification obtain to have the method for preparing that suppresses the active water solublity total alkaloids of alpha-glucosidase, comprise the steps:
1st, with the Ramulus Mori being raw material, serves as to extract solvent with water: alcohol=100%-20%: 0%-80%, and the volume that extracts solvent is 6-10 a times of Ramulus Mori weight; Regulate extracting solution PH2-7, reflux, extracting liquid filtering repeats 2 times and extracts, and removes medicinal residues, merges 2 times filtrating;
2nd, the extracting solution of heating concentrating under reduced pressure the 1st step acquisition, extremely 1/2～1 times of former Ramulus Mori weight;
3rd, adding volumn concentration is 75%～100% methanol or ethanol, and the amount of adding is 3～5 times of the concentrated extracting solution volume that obtained in the 2nd step, carries out alcohol at 4～30 ℃ and precipitates; Centrifugal or filter, remove residue and insoluble matter, obtain supernatant;
4th, get supernatant after alcohol deposition that the 3rd step obtained concentrates, carry out purification through cation exchange resin column, with the non-adsorbable impurity of deionized water flush away; Reuse 0.2～1.5mol ammonia eluting; Anion exchange resin on the eluent is collected not absorbed portion, transfers pH8;
5th, adopt membrane filtration, the filtrating lyophilization becomes dry powder, maybe will collect the direct spray drying forming of liquid and process dry powder, gets flaxen water solublity total alkaloids extract, and this extract has the inhibition of alpha-glucosidase active.
1-deoxynojirimycin content accounts for the 50-70% of total extract in the said flaxen water solublity total alkaloids extract.
The application of the Cortex Mori water solublity total alkaloids that the above method obtains combines described Cortex Mori water solublity total alkaloids with the excipient substance that pharmaceutical preparation is allowed, be used to prepare hypoglycemic drug, can be prepared into the various dosage forms of fit for service.
Advantage of the present invention and good effect:
1. the present invention is medical material with the Ramulus Mori, wide material sources, low price.Because wooden part is removed, it is over half that the raw material volume is reduced, and saved the extraction solvent consumption greatly, and the labor hour and the energy when extracting;
2. the present invention is medical material with the Ramulus Mori, through the improvement of purifying technique, makes that 1-deoxynojirimycin content reaches more than 50% in the active water solublity total alkaloids extract of the inhibition with alpha-glucosidase;
3. the present invention provides a new approach for the comprehensive utilization of Bombyxmori Linnaeus aquaculture: the wooden part of Ramulus Mori can be used for the raw material of Wood Processing; The cortex part also can be used as the raw material of natural textile fiber behind the water-soluble alkaloid that extracts hypoglycemic activity, have good comprehensive utilization prospect.
Description of drawings
Fig. 1 Ramulus Mori water-soluble alkaloid total extract (sample) is to the inhibition curve of alpha-glucosidase.
The specific embodiment
One. the preparation and the application of Cortex Mori water-soluble alkaloid total extract
At first Ramulus Mori is pulverized, added extraction solvent (water: alcohol=100%-20%: 0%-80% regulates extracting solution PH2-7 for extracting solvent), the volume that extracts solvent is 6-10 a times of Ramulus Mori weight (Kg); Reflux is filtered, and repeats to extract 2 times, merges 2 times filtrating;
Extracting solution is concentrated into 1/2～1 times of former Ramulus Mori weight;
Add volumn concentration and be 75%～100% methanol or ethanol, the amount of adding is 3～5 times of the concentrated extracting solution volume that obtained in the 2nd step, carries out alcohol at 4～30 ℃ and precipitates; Centrifugal or filter, remove residue and insoluble matter, obtain supernatant;
Get the supernatant after the alcohol deposition concentrates, carry out purification through cation exchange resin column, with the non-adsorbable impurity of deionized water flush away; Reuse 0.2～2mol ammonia eluting, eluent is used deionized water rinsing through anion exchange resin; Collect not absorbed portion, collect liquid and transfer pH8;
Adopt membrane filtration; The filtrating lyophilization becomes dry powder; The direct spray drying forming of maybe will filtrating is processed dry powder; Get flaxen water solublity total alkaloids extract, this extract has the inhibition of alpha-glucosidase active, and the content of 1-deoxynojirimycin is the 50-70% of water solublity total alkaloids extract.
Ramulus Mori 1Kg with pulverizing adds water 10L, transfers pH2, and reflux 2 hours is filtered; Repeat to extract 2 times, merge filtrating 2 times, be concentrated into 500mL, add 95% ethanol 1500mL, the room temperature hold over night; Filter, remove insoluble matter, Zerolit 225 cationic resin on the filtrating are with the non-adsorbable impurity of deionized water flush away; Reuse 1mol ammonia eluting, D380 resin anion (R.A.) on the eluent is collected not absorbed portion, transfers pH8; Cross film, filtrating is dry, promptly gets faint yellow water solublity total alkaloids extract 1.3g, DNJ-content 62%.
Embodiment 2
Ramulus Mori 1Kg with pulverizing adds 80% methanol 8L, transfers pH7, and reflux 2 hours is filtered; Repeat to extract 2 times, merge filtrating 2 times, be concentrated into 500mL, add 95% ethanol 1500mL, the room temperature hold over night; Filter, remove insoluble matter, last 0017 cationic resin of filtrating is with the non-adsorbable impurity of deionized water flush away; Reuse 0.5mol ammonia eluting, D261 resin anion (R.A.) on the eluent is collected not absorbed portion, transfers pH8; Use membrane filtration, filtrating is dry, promptly gets faint yellow water solublity total alkaloids extract 11g, DNJ-content 69%.
Ramulus Mori 1Kg with pulverizing adds 60% ethanol 6L, transfers pH4, and reflux 2 hours is filtered; Repeat to extract 2 times, merge filtrating 2 times, be concentrated into 500mL, add 95% ethanol 1500mL, the room temperature hold over night; Filter, remove insoluble matter, Amberlite IR120 cationic resin on the filtrating is with the non-adsorbable impurity of deionized water flush away; Reuse 0.2mol ammonia eluting, D371 resin anion (R.A.) on the eluent is collected not absorbed portion, transfers pH8; Use membrane filtration, filtrating is dry, promptly gets faint yellow water solublity total alkaloids extract 1.6g, DNJ-content 57%
Embodiment 4, (the water solublity total alkaloids extract is used to prepare hypoglycemic drug)
Take by weighing Ramulus Mori water solublity total alkaloids extract 30g, add sodium carboxymethyl cellulose (in add) 5.0g, cross-linking sodium carboxymethyl cellulose (in add) 4.5g; Starch 88g, mixing is prepared soft material with 50% ethanol; Cross 16 mesh sieves and granulate, drying adds micropowder silica gel 1.5g; Magnesium stearate 1.0g crosses 16 mesh sieve granulate tablettings, makes 1000.
Two, the distribution experiment of DNJ composition in the Ramulus Mori
1. take by weighing the dry Ramulus Mori 100g of the different cultivars of pulverizing 2 mesh sieves, 3 parts of each kinds, the aqueous solution of adding pH1, reflux is filtered, and repeats to extract 2 times, merges 2 times filtrating, is concentrated into certain volume, measures DNJ content.The result sees table 1;
2. take by weighing the dry Ramulus Mori reel 100g of the different cultivars of pulverizing 2 mesh sieves, 3 parts of each kinds, the aqueous solution of adding pH1, reflux is filtered, and repeats to extract 2 times, merges 2 times filtrating, is concentrated into certain volume, measures DNJ content.The result sees table 1;
3. take by weighing the dry Ramulus Mori 100g of the different cultivars of pulverizing 2 mesh sieves, 3 parts of each kinds, the aqueous solution of adding pH1, reflux is filtered, and repeats to extract 2 times, merges 2 times filtrating, is concentrated into certain volume, measures DNJ content.The result sees table 1;
Each position DNJ percentage composition of the Ramulus Mori of table 1, different cultivars
Experimental result shows, though the content of DNJ is variant in the different cultivars Ramulus Mori, DNJ content does not have significant difference; And the distribution of DNJ in Ramulus Mori, Ramulus Mori reel has notable difference: the highest at cortex part content, and Ramulus Mori reel part DNJ component content only is 1/10th of a cortex part, in addition lower.
Three, Cortex Mori water-soluble alkaloid total extract is to the alpha-glucosidase inhibitory action
With each 10 milligrams of DNJ standard substance, Cortex Mori water-soluble alkaloid total extract and acarbose standard substance, use 1mL0.2mol/L PBS (pH=6.8) with the sample ultrasonic dissolving respectively, and 10 times of serial dilutions are 1,0.1; 0.01,0.001,0.0001, the sample of 0.00001mg/mL; Sample thief 20 μ L add in 96 orifice plates respectively, add the thick enzymatic solution of 30 μ L intestinal, 37 ℃ hatch 5min after, add 100 μ L maltose and start reaction; Behind 37 ℃ of reaction 30min, add the hydrochloric acid cessation reaction of 10 μ L 0.1mol/L, get reactant liquor 50 μ L, add 37 ℃ of reactions of glucose working solution 15min of 150 μ L; 490nm measures the OD value, according to the experimental data mapping, sees Fig. 1.
Inhibition curve result according to alpha-glucosidase is as shown in the figure; Ramulus Mori water solublity total alkaloids extract (among the figure for sample), 1-deoxynojirimycin (DNJ) standard substance, acarbose demonstrate parallel alpha-glucosidase is suppressed effect, wherein Ramulus Mori water solublity total alkaloids extract (being sample among the figure) is than the inhibition alpha-glucosidase IC of acarbose
50The low nearly one magnitude of concentration.
Claims (3)
1. an extraction from Ramulus Mori, separation, purification obtain to have the method for preparing that suppresses the active water solublity total alkaloids of alpha-glucosidase, it is characterized in that comprising the steps:
1st, with the Ramulus Mori being raw material, serves as to extract solvent with water: alcohol=100%-20%: 0%-80%, and the volume that extracts solvent is 6-10 a times of Ramulus Mori weight; Regulate extracting solution PH2-7, reflux, extracting liquid filtering repeats 2 times and extracts, and removes medicinal residues;
2nd, the extracting solution of heating concentrating under reduced pressure the 1st step acquisition, extremely 1/2～1 times of former Ramulus Mori weight;
3rd, adding volumn concentration is 75%～100% methanol or ethanol, and the amount of adding is 3～5 times of the concentrated extracting solution volume that obtained in the 2nd step, carries out alcohol at 4～30 ℃ and precipitates; Centrifugal or filter, remove residue and insoluble matter, obtain supernatant;
4th, get supernatant after alcohol deposition that the 3rd step obtained concentrates, carry out purification through cation exchange resin column, with the non-adsorbable impurity of deionized water flush away; Reuse 0.2～1.5mol ammonia eluting; Anion exchange resin on the eluent is collected not absorbed portion, collects liquid and transfers pH8;
5th, adopt membrane filtration, the filtrating lyophilization becomes dry powder, maybe will collect the direct spray drying forming of liquid and process dry powder, gets flaxen water solublity total alkaloids extract, and this extract has the inhibition of alpha-glucosidase active.
2. method according to claim 1 is characterized in that 1-deoxynojirimycin content in the said flaxen water solublity total alkaloids extract accounts for the 50%-70% of total extract.
3. the application of the Cortex Mori water solublity total alkaloids of the said method acquisition of claim 1; It is characterized in that described Cortex Mori water solublity total alkaloids is combined with the excipient substance that pharmaceutical preparation is allowed; Be used to prepare hypoglycemic drug, be prepared into the various dosage forms of fit for service.
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Cited By (2)
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| CN103755623A (en) * | 2014-01-03 | 2014-04-30 | 广州军区广州总医院 | Response surface method optimized method for extracting 1-deoxynojirimycin in mulberry leaves with acidic ethanol solution |
| CN105535112A (en) * | 2015-12-29 | 2016-05-04 | 浙江省农业科学院 | Extraction technology of hypoglycemic medicinal active substances of mulberry leaves and mulberries and formula |
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Application publication date: 20120725 |