CN100556892C - A kind of method of from mulberry leaf, extracting the 1-S-GI - Google Patents
A kind of method of from mulberry leaf, extracting the 1-S-GI Download PDFInfo
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- CN100556892C CN100556892C CNB2007100674983A CN200710067498A CN100556892C CN 100556892 C CN100556892 C CN 100556892C CN B2007100674983 A CNB2007100674983 A CN B2007100674983A CN 200710067498 A CN200710067498 A CN 200710067498A CN 100556892 C CN100556892 C CN 100556892C
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Abstract
The invention provides a kind of method of extracting 1 one S-GIs (DNJ) from mulberry leaf, is by adding the mineral acid acidification in the extracting solution of mulberry leaf, remove impurity through cooling, obtaining through column chromatography.Can make Folium Mori extract content be not less than 10%.The Folium Mori extract that obtains with the inventive method confirms that through experimentation on animals normal mouse is not had influence, and diabetic mice is had apparent in view hypoglycemic activity, can use in the healthcare products of preparation lowering blood glucose.The inventive method is not owing to need to add flocculation agent, having guaranteed when reducing cost, improving content does not have ectogenic material to bring in the last handling process, after acidizing fluid is concentrated, directly adopt homemade ion-exchange chromatography to separate, greatly reduce cost.The inventive method is reasonable in design, and is easy and simple to handle, need not specific installation, is fit to very much suitability for industrialized production.
Description
Technical field
The present invention relates to field of plant extraction, be specifically related to a kind of extract and preparation method of mulberry leaf effective constituent.
Background technology
Mulberry leaf " angle's leaf " of meaning in " Bencao Tujing " has the effect of nourishing YIN and supplementing blood, wind-dispelling heat-dissipating, the ventilation of beneficial liver, antihypertensive diuretic.The pharmacological action of mulberry leaf is all on the books in bibliographys such as Compendium of Material Medica, " book on Chinese herbal medicine is looked for the truth ", " Shiliao Bencao ", " medicinal herb tea and distilled medicinal water ", " science of prescription ", and Japanese ancient book " eating tea health note " record mulberry leaf have the effect that improves " drinking-water is sick " (being diabetes).This shows that its pharmaceutical use of mulberry leaf and nourishing function do not have and sink in China's origin that still has a long history abroad, for our development and use provide reliable foundation.
Mulberry leaf still are the medicine-food two-purpose article of national health department's approval, and its bitter, sweet, cold is gone into lung channel, fries in shallow oil juice and can only quench one's thirst for tea.At present, main although China's Morus alba Resource Development is very abundant still as the sericulture feed, after annual the sericulture, all there are many mulberry leaf to go out of use. from following development trend, exploitation health mulberry leaf product has a high potential.1 one S-GIs that contain in the mulberry leaf (1-deoxynojirimycin DNJ) are the multiple alpha-glucosidase inhibitors on human small intestine's fine hair, can stop amylolysis to become glucose, block disaccharide lytic enzymes such as interior maltin of small intestinal cell brush border and sucrase competitively, the absorption process that makes polysaccharide, oligosaccharides and the disaccharide of ingesting be digested to monose such as glucose, fructose is stoped, then lowering blood glucose concentration has certain therapeutic action to treatment diabetes and complication thereof.
Existing mulberry leaf 1 one S-GIs (DNJ) extracting method mainly contains two kinds, and the content of the Folium Mori extract that method obtained 1 one S-GIs of CN1579445A only is 1.0～3.0%, mainly obtains behind alcohol precipitation by adding flocculation agent again; The method of CN1430964A has obtained high-load mulberry leaf total alkali and DNJ extract, but technological process is more loaded down with trivial details, be concentrated into extracting solution earlier to proportion 1.12-1.20 after in the pre-treatment process, needing to extract, add 1-5 times of water gaging dissolving of primary dose again, add flocculation agent again behind the adjusting pH and leave standstill 12 hours, and follow-up sepn process is also after removing impurity through ion exchange resin, need resin is poured out with refluxing extraction again behind the vexed 4h of putting of alkali, step is many, operation inconvenience, the cost height of suitability for industrialized production.
Summary of the invention
The objective of the invention is to overcome the deficiency that prior art exists, a kind of more easy method of reasonably extracting 1 one S-GIs (DNJ) from mulberry leaf is provided, be to obtain with separation method, can make Folium Mori extract content be not less than 10%.
Method for preparing extractive of the present invention is realized by following steps:
(1) mulberry leaf powder is broken into meal, extracts 2～3 times with 8 times of amounts or above water or the heating of aqueous hydrophilic solvent, hydrophilic solvent can be methyl alcohol, ethanol, acetonitrile and acetone, and the temperature that heating is extracted is between 50～100 ℃, with the refluxing extraction best results;
(2) with a kind of pH1～3 that are acidified in extracting solution merging back adding hydrochloric acid, sulfuric acid, nitric acid or the phosphoric acid, between 10～-30 ℃, be cooled to precipitation fully, filtration or the centrifugal precipitation of removing;
(3) filtrate is passed through Zeo-karb after suitably concentrating, water or aqueous hydrophilic solvent be washed till colourless after, extremely closely colourless with alkaline methanol or aqueous ethanolic solution wash-out again, alkali wherein can be ammoniacal liquor, diethylamino, triethylamine, the concentration of alkali is 10%～50%, and methyl alcohol or concentration of ethanol are 10%～95% in the aqueous solution; The model of Zeo-karb is 001 * 1.1; 001 * 7; 001 * 14.5 or the D151 type.
(4) elutriant of collection alkaline methanol or aqueous ethanolic solution is condensed into medicinal extract or spraying drying, and the content that can obtain the 1-S-GI is not less than 10% Folium Mori extract.
With this Folium Mori extract that the inventive method obtains, the vehicle that can add the pharmaceutics permission is prepared into pharmaceutical preparation, and dosage form comprises tablet, capsule, pill, granule, aerosol, sprays.
With the Folium Mori extract 1-S-GI (DNJ) that the inventive method obtains, can in the healthcare products of preparation lowering blood glucose, use, confirm normal mouse not to be had influence, and diabetic mice is had apparent in view hypoglycemic activity through experimentation on animals.
Usefulness of the present invention is: proposed to carry out in leaching process acidification, the step that postcooling is removed impurity has vital role, owing to do not need to add flocculation agent, having guaranteed when reducing cost, improving content does not have ectogenic material to bring in the last handling process, after acidizing fluid is concentrated, directly adopt homemade ion-exchange chromatography to separate, greatly reduce cost.The inventive method is reasonable in design, and is easy and simple to handle, need not specific installation, is fit to very much suitability for industrialized production.
Embodiment
The present invention is described further in conjunction with specific embodiments.
The preparation of embodiment 1 Folium Mori extract
Get about mulberry leaf medicinal material 200g and pulverize, add 3 premium on currency, refluxing extraction 2.5 hours, filtered filtration residue add 3 premium on currency again and refluxed 2 hours, merging filtrate, 5.2L altogether.This extracting solution is used hcl acidifying to pH1～3 backs of 6N refrigerate 2h at-8 ℃, remove by filter precipitation, filtrate is passed through Zeo-karb 001 * 1.1 after being concentrated into 2L, wash with water after colourless, add again and make ammoniacal liquor content reach 50% wash-out in ammoniacal liquor to 50% aqueous ethanolic solution, collect this part elutriant, be condensed into medicinal extract and get 1.8g, the content of measuring the 1-S-GI with the HPLC method is 11.8%.
The preparation of embodiment 2 Folium Mori extracts
Get 500 gram left and right sides pulverizing medicinal materials, the methanol eddy that adds 5 liter 30% extracted 2 hours, and filtered filtration residue adds 4 liter 30% alcohol reflux 2 hours, merging filtrate, 8.2L altogether again.This extracting solution is used sulfuric acid acidation to pH1～3 backs of 5N refrigerate 2h at-15 ℃, remove by filter precipitation, filtrate is passed through Zeo-karb 001 * 7 after being concentrated into 5L, wash with water and to neutrality, be washed till colourless with 30% methyl alcohol again, then adding diethylamino to 70% methanol aqueous solution makes its content reach 20% wash-out, collect this part elutriant, be condensed into medicinal extract and get 4.1g, the content of measuring the 1-S-GI with the HPLC method is 13.1%.
The preparation of embodiment 3 Folium Mori extracts
Get about mulberry leaf medicinal material 1kg and pulverize, add 8 liter 20% acetone, refluxing extraction 2.5 hours, filtered filtration residue add 8 liter 20% acetone again and refluxed merging filtrate, 12.6L altogether 2 hours; This extracting solution is acidified to pH1～3 backs at-30 ℃ of refrigeration 2h with the phosphoric acid of 5N, remove by filter precipitation, filtrate is passed through Zeo-karb 001 * 14.5 after being concentrated into 6L, wash with water and to neutrality, be washed till colourless with 60% acetone again, then adding diethylamino to 60% methanol aqueous solution makes its content reach 30% wash-out, collect this part elutriant, be condensed into medicinal extract and get 9.6g, the content of measuring the 1-S-GI with the HPLC method is 12.6%.
The preparation of embodiment 4 Folium Mori extracts
Get about mulberry leaf medicinal material 1kg and pulverize, add 10 liter 10% acetonitrile, refluxing extraction 2 hours, filtered filtration residue add 8 liter 10% acetonitrile again and refluxed merging filtrate, 15.6L altogether 2 hours; This extracting solution is acidified to pH1～3 backs at-30 ℃ of refrigeration 2h with the phosphoric acid of 5N, remove by filter precipitation, filtrate is passed through Zeo-karb D151 after being concentrated into 6L, wash with water and to neutrality, be washed till colourless with 60% acetonitrile again, then add triethylamine to 70% methanol aqueous solution and make its content reach 10% wash-out,, collect this part elutriant, be condensed into medicinal extract and get 11.2g, the content of measuring the 1-S-GI with the HPLC method is 10.9%.
The preparation of embodiment 5 Folium Mori extracts
Get about mulberry leaf medicinal material 1kg and pulverize, add 8 liter 50% ethanol, refluxing extraction 2 hours, filtered filtration residue add 6 liter 10% acetonitrile again and refluxed merging filtrate, 11L altogether 2 hours; This extracting solution is acidified to pH1～3 backs at-30 ℃ of refrigeration 2h with the nitric acid of 6N, remove by filter precipitation, filtrate is passed through Zeo-karb 001 * 7 after being concentrated into 6L, wash with water and to neutrality, be washed till colourless with 60% ethanol again, continue with 50% ammoniacal liquor ethanolic soln wash-out, collect this part elutriant, be condensed into medicinal extract and get 12.3g, the content of measuring the 1-S-GI with the HPLC method is 12.4%.
Embodiment 6
Content assaying method of the present invention adopts high-efficient liquid phase technique, comprises following steps: the HPLC condition: the C18 post, and 30 ℃ of column temperatures, moving phase is acetonitrile: 0.1% aqueous acetic acid (35: 65), flow velocity 0.8ml/min detects wavelength 254nm;
Pre-column derivatization: be reflected in the centrifuge tube of 1.5ml and carry out.Precision takes by weighing an amount of sample and is settled to 100ml with distilled water, get 20 μ l sample solutions, add borate buffer solution (pH=8.5) 20 μ l, the acetonitrile solution that adds the FMOC-Cl (fluorenes methoxy acyl chlorides) of 40 μ l 5mmol/L again, jolting, reaction is 20 minutes under the room temperature, and the glycine solution that adds 20 μ l 0.1mol/L again reacts completely remaining derivatization reagent, adds 400 μ l, 0.1% aqueous acetic acid after 10 minutes.
The preparation of typical curve: it is an amount of that precision takes by weighing 1-S-GI standard substance, be settled to 100ml with distilled water and promptly get standardized solution, mother liquor is diluted to the standardized solution series of different concns with distilled water, get 10 μ l standardized solution respectively and measure peak area with the derive liquid phase of going forward side by side of above-mentioned derivatize step, with the standard substance quality is X-coordinate, and peak area is an ordinate zou base of calculation curve.
Embodiment 7 tablets
Get the Folium Mori extract 30g that above-mentioned 1～5 arbitrary embodiment method obtains, pulverize the back and cross 80 mesh sieves,, add 10% starch slurry 10g and make softwood adding Magnesium Stearate 1g, be pressed into 1000 behind the dry starch 10g mixing, promptly with starch 60g mixing.Every contains Folium Mori extract 30mg.
Embodiment 8 capsules
Get the Folium Mori extract 30g that above-mentioned 1～5 arbitrary embodiment method obtains, pulverize the back and cross 80 mesh sieves, add 10% starch slurry and make softwood, after granulating with 14 mesh sieves, put 60 ℃～70 ℃ dry backs, be inserted in No. 1 Capsules in whole of 12 mesh sieves.Adorn 1000 altogether, every capsules contains Folium Mori extract 30mg.
Embodiment 9 pills
Get the Folium Mori extract 30g that above-mentioned 1～5 arbitrary embodiment method obtains, pulverize the back and cross 80 mesh sieves, proper honey is made the general ball of making of tamanori.
Embodiment 10 granules
Get the Folium Mori extract 30g that above-mentioned 1～5 arbitrary embodiment method obtains, pulverize the back and cross 80 mesh sieves, add 10% starch slurry and make softwood, after granulating with 14 mesh sieves, put 60 ℃～70 ℃ dry backs in the whole grain of 12 mesh sieves, packing, sealing, packing is promptly.
Embodiment 11 sprayss
Get the Folium Mori extract 30g that above-mentioned 1～5 arbitrary embodiment method obtains, pulverize the back and cross 80 mesh sieves, suitable quantity of water dissolving, sterilization back can.
Embodiment 12DNJ extract is to the influence of normal mouse blood sugar level
Normal mouse is divided into 5 groups at random, every group 10, the basic, normal, high dosage group of DNJ extract gives 200,400 respectively, 800mg/kg, and negative control group gives 0.5%CMC-Na 20ml/kg, positive controls gives gliclazide 100mg/kg, every day 1 gastric infusion, for three days on end, fasting 12h before the last administration, before administration and after the administration 1,2h tail vein gets blood, 3000rpm * 10min, separation of serum is used the determination of glucose oxidase blood glucose value.
To normal mouse administration 3 days, after the last administration behind the 1h, compare with negative control group, DNJ extract 400,800mg/kg dosage group have obvious functions of blood sugar effect (P＜0.05 and P＜0.01), and positive control drug gliclazide 100mg/kg group also has obvious functions of blood sugar effect (P＜0.001); Behind the 2h, compare with negative control group after the last administration, positive controls still has significant hypoglycemic activity (P＜0.001), and the hypoglycemic activity of DNJ extract not obvious (P＞0.05).
Embodiment 13DNJ extract causes the influence of hyperglycemia to normal injected in mice glucose
Normal mouse is divided into 5 groups at random, and 10 every group, fasting is gastric infusion after 12 hours, the basic, normal, high dosage group of DNJ extract gives 200,400 respectively, 800mg/kg, negative control group gives 0.5%CMC-Na20ml/kg, and positive controls gives gliclazide 100mg/kg, behind the administration 1h, every mouse peritoneal injectable dextrose monohydrate 2g/kg, behind the injectable dextrose monohydrate 1h, mouse is plucked eyeball and gets blood, 3000rpm * 10min, separation of serum is used the determination of glucose oxidase blood glucose value.
Abdominal injection glucose 2g/kg again behind the normal mouse gastric infusion 1h measures blood glucose value behind the 1h.Compare with negative control group, DNJ extract 400,800mg/kg dosage group have obvious functions of blood sugar effect (P＜0.001 and P＜0.01), and positive control drug gliclazide 100mg/kg group also has obvious functions of blood sugar effect (P＜0.001).
Embodiment 14DNJ extract is to the influence of normal mouse starch tolerance
Normal mouse is divided into 5 groups at random, every group 10, fasting is gastric infusion after 12 hours, the basic, normal, high dosage group of DNJ extract gives 50,100 respectively, 200mg/kg, negative control group gives 0.5%CMC-Na 20ml/kg, positive controls gives acarbose 20mg/kg, behind the administration 0.5h, every mouse is irritated stomach with Zulkovsky starch 5g/kg, before administration, give behind the starch 0.5,1,2h tail vein gets blood, 3000rpm * 10min, separation of serum is used the determination of glucose oxidase blood glucose value.
Zulkovsky starch 5g/kg irritates stomach behind the normal mouse gastric infusion 0.5h, compare with negative control group, DNJ extract 100mg/kg dosage group 1h after giving starch has obvious functions of blood sugar effect (P＜0.05), and positive control drug acarbose 20mg/kg group is all having obvious functions of blood sugar effect (P＜0.05 and P＜0.01) to 0.5h behind the starch and 1h.
Claims (4)
1. a method of extracting the 1-S-GI from mulberry leaf is that mulberry leaf powder is broken into meal, and water or the heating of aqueous hydrophilic solvent are extracted, and it is characterized in that realizing by following steps:
(1) mulberry leaf powder is broken into meal, water or the heating of aqueous hydrophilic solvent are extracted 2～3 times, and the temperature that heating is extracted is between 50～100 ℃;
(2) extracting solution is merged back adding mineral acid and be acidified to pH1～3, between 10～-30 ℃, be cooled to precipitation fully, filter or the centrifugal precipitation of removing;
(3) filtrate concentrates the back by Zeo-karb, water or aqueous hydrophilic solvent be washed till colourless after, again with alkaline methanol or aqueous ethanolic solution wash-out to closely colourless;
(4) collect elutriant, be condensed into medicinal extract or spraying drying, the content that promptly obtains the 1-S-GI is not less than 10% Folium Mori extract.
2. a kind of method of extracting the 1-S-GI from mulberry leaf according to claim 1 is characterized in that: the mineral acid in the step (2) is selected a kind of in hydrochloric acid, sulfuric acid, nitric acid or the phosphoric acid for use.
3. a kind of method of from mulberry leaf, extracting the 1-S-GI according to claim 1, it is characterized in that: alkaline methanol described in the step (3) or the alkali in the aqueous ethanolic solution are selected a kind of in ammoniacal liquor, diethylamino, the triethylamine for use, the concentration of alkali is 10%～50%, and methyl alcohol or concentration of ethanol are 10%～95% in the aqueous solution.
4. a kind of method of extracting the 1-S-GI from mulberry leaf according to claim 1 is characterized in that: hydrophilic solvent is selected a kind of in methyl alcohol, ethanol, acetonitrile or the acetone for use.
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Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101654428B (en) * | 2009-09-11 | 2012-05-23 | 成都市金医生科技健康产业有限公司 | Method for extracting and separating 1-deoxynojirimycin with high purity from natural products |
| CN101671294B (en) * | 2009-09-22 | 2011-12-21 | 广东太阳神集团有限公司 | Method for continuously extracting and separating 1-deoxynojirimycin (DNJ) and flavone from folium mori |
| CN102190615B (en) * | 2011-04-08 | 2012-12-19 | 长沙华诚生物科技有限公司 | Method for extracting and separating 1-deoxynojirimycin from mulberry leaves |
| CN102276515B (en) * | 2011-09-13 | 2013-05-08 | 西南大学 | Method for extracting deoxynojirimycin |
| CN102675188B (en) * | 2012-05-21 | 2014-10-22 | 江苏科技大学 | Extraction method of 1-desoxynojirimycin in mulberry leaf |
| CN103204800B (en) * | 2013-05-14 | 2017-07-04 | 成都科源生物技术有限公司 | A kind of extracting method of 1 DNJ |
| CN103755623B (en) * | 2014-01-03 | 2015-12-30 | 广州军区广州总医院 | The acid ethanol solution that a kind of response phase method is optimized extracts the method for 1-Deoxynojirimycin in Mulberry Leaves |
| CN103961410A (en) * | 2014-05-07 | 2014-08-06 | 山东富而美生物科技有限公司 | Mulberry enzyme capsule and preparation method thereof |
| CN103965096B (en) * | 2014-05-09 | 2016-08-24 | 江西海富生物工程有限公司 | A kind of preparation method being applicable to industrial 1-DNJ |
| CN104007205B (en) * | 2014-06-13 | 2015-09-30 | 漳州片仔癀药业股份有限公司 | A kind of detection method for the treatment of the pharmaceutical preparation of diabete |
| CN105130878B (en) * | 2015-08-20 | 2017-09-29 | 安徽珂欣茧业有限公司 | A kind of method that DNJ is extracted from mulberry leaf |
| CN108828121B (en) * | 2018-06-14 | 2020-06-05 | 华润三九医药股份有限公司 | Method for detecting content of α -high nojirimycin in white tree medicinal material |
| JP2023522598A (en) * | 2020-04-24 | 2023-05-31 | ソシエテ・デ・プロデュイ・ネスレ・エス・アー | Use of mulberry extract to control postprandial glycemic response |
| CN112603941A (en) * | 2021-01-13 | 2021-04-06 | 湘潭市中心医院 | Preparation method of traditional Chinese medicine extract for regulating glycolipid metabolism |
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