CN103550265A - Extraction method of active ingredients of tuckahoe peels and tuckahoe peel extracts - Google Patents
Extraction method of active ingredients of tuckahoe peels and tuckahoe peel extracts Download PDFInfo
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Abstract
The invention discloses an extraction method of active ingredients of tuckahoe peels. The extraction method comprises the steps of flushing the tuckahoe peels by hydrochloric acid, washing the tuckahoe peels till the peels are neutral, and carrying out air-drying; carrying out reflux extraction on the tuckahoe peels by ethanol, and recycling ethanol from an extracting solution until the extracting solution is dried; dissolving dry matter in water, adding a mixture of normal butanol and ethyl acetate to carry out extraction, and recycling solvents from an organic phase until the organic phase is dried so as to obtain a crude product; dissolving the crude product into the ethanol to prepare a solution, enabling the solution to pass through a neutral alumina chromatographic column, eluting the solution by a mixture of ethanol and ethyl acetate, and collecting an eluent; adding active carbon into the eluent, filtering, concentrating the filter liquid, standing, filtering and drying so as to obtain the active ingredients. The extraction method has the advantages that the method is stable, and the extraction efficiency is high; the yield of the extracts is greatly improved, the content of the active ingredients is greatly increased, and the content of 3beta-hydroxy-lanostene 7,9 (11), 24-triene-21-acid and dehydroeburicoic acid in the extracts is large; the extracts have the functions of reducing the blood sugar and the blood fat as well as preferable application prospect.
Description
Technical field
The present invention relates to the extracting method of effective ingredient in a kind of Cortex Sclerotii Poriae and extract obtained, be specifically related to a kind of 3 beta-hydroxies-Pilus Caprae seu Ovis steroid 7,9(11), the acid of 24-triolefin-21-and dehydrogenation, according to the extracting method of the larger Cortex Sclerotii Poriae effective ingredient of background of cloth acid content and extract obtained, belong to Chinese medicine extraction technical field.
Background technology
Poria is the crust of the dry sclerotia of On Polyporaceae Poria cocos (Schw.) Wolf, different name " Siberian cocklebur skin " (< < Traditional Chinese Medicine in Sichuan will > >), nature and flavor " sweet light; flat ", function " diuretic; detumescence ", cures mainly " edema, abdominal distention ".Cortex Sclerotii Poriae main chemical compositions has pachyman, triterpene, natural gum, protein, sterol and fatty acid etc., and modern study result shows that in Poria, triterpenes chemical composition has antitumor, antiinflammatory, immunomodulating isoreactivity.
At present, the extraction of effective ingredient in Poria is had to certain research both at home and abroad, therefrom extracted the effective ingredient such as triterpenoid compound, total saponins, pachyman, flavone compound.Relative, from Cortex Sclerotii Poriae, the research of effective component extracting is less, in Cortex Sclerotii Poriae, contain than more rich triterpenoid compound, patent 201210168614.1 discloses a kind of Cortex Sclerotii Poriae extract and the application in preparing diuresis medicine thereof, and scheme is: ethanol percolation for Cortex Sclerotii Poriae, be then extracted with ethyl acetate, then go up petroleum ether-ethyl acetate eluting for silicagel column, the concentrated extract that to obtain.The extract obtained effect with guarantor's sodium row potassium, can be used for diuresis.Patent 201210235819.7 discloses a kind of Cortex Sclerotii Poriae extract and the application in treatment chronic renal failure medicine thereof, and scheme is: by ethanol extraction for Cortex Sclerotii Poriae, be then extracted with ethyl acetate, obtain extract.Extract has certain curative effect to chronic renal failure.
Because containing multiple active ingredient in Cortex Sclerotii Poriae, therefore adopt the effective ingredient of diverse ways gained and content thereof to be not quite similar, the therapeutical effect showing is also not quite similar.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of extracting method of Cortex Sclerotii Poriae effective ingredient is provided, 3 beta-hydroxies-Pilus Caprae seu Ovis steroid 7 in the method gained effective ingredient, 9(11), 24-triolefin-21-acid and dehydrogenation are larger according to background of cloth acid content, and yield is higher.
The present invention also provides the Cortex Sclerotii Poriae extract that adopts said method gained.
Technical solution of the present invention is as follows:
An extracting method for Cortex Sclerotii Poriae effective ingredient, is characterized in that, comprises the following steps:
(1) Cortex Sclerotii Poriae is rinsed with the hydrochloric acid of 1.0-2.0wt%, then water rinses to neutral, dries;
(2) the alcoholic solution reflux, extract, of Cortex Sclerotii Poriae use 70-95 volume % step (1) being processed 2 times, merge extractive liquid,, extracting solution reclaims ethanol to dry;
(3) by the dry water dissolution of step (2), add the mixture extraction of n-butyl alcohol and ethyl acetate, organic facies reclaims solvent to dry, obtains crude product;
(4) crude product dissolve with ethanol, wiring solution-forming, neutral alumina chromatographic column on gained solution, then carries out eluting with the mixture of ethanol and ethyl acetate, collects eluent;
(5) in eluent, add active carbon, be heated to seethe with excitement and keep 30min, filtered while hot, uses washing with alcohol active carbon, and merging filtrate and washing liquid are concentrated into 1/3 of original volume, staticly settles, filters, is dried, and obtains effective ingredient.
In above-mentioned steps (2), Cortex Sclerotii Poriae and alcoholic solution with magnitude relation, be 1:5-20(g/ml).
In above-mentioned steps (2), each extraction time is 0.5-3.5h.
In above-mentioned steps (2), preferably use the ethanol extraction Cortex Sclerotii Poriae of 70 volume %, Cortex Sclerotii Poriae and alcoholic solution with magnitude relation, be 1:10(g/ml), each extraction time is 0.5 h.
In above-mentioned steps (3), n-butyl alcohol: the volume ratio of ethyl acetate is 5-10:1.5, preferably 7:1.5.
In above-mentioned steps (3), water: the volume ratio of the mixture of n-butyl alcohol and ethyl acetate is 1:1.5-2.
In above-mentioned steps (4), its 100-120 quality dissolve with ethanol doubly for crude product, wiring solution-forming.
In above-mentioned steps (4), the 15-20 that the quality of neutral alumina is crude product doubly.
In above-mentioned steps (4), the volume ratio of ethanol and ethyl acetate is 2:3-7, is preferably 2:5.
In above-mentioned steps (4), the 1.5-3 that the consumption of the mixture of ethanol and ethyl acetate is aluminium oxide doubly, is preferably 2 times.
In above-mentioned steps (5), activated carbon dosage is the 2-3% of crude product quality.
The Cortex Sclerotii Poriae extract that adopts said method to obtain also should be protected, and in extract, effective ingredient is mainly 3 beta-hydroxies-Pilus Caprae seu Ovis steroid 7, and 9(11), 24-triolefin-21-acid and dehydrogenation are according to background of cloth acid, and both content are more than 50%.
Extracting method of the present invention is stable, extraction efficiency is high, the yield of extract and the content of effective ingredient have greatly been improved, 3 beta-hydroxies in extract obtained-Pilus Caprae seu Ovis steroid 7,9(11), 24-triolefin-21-acid and dehydrogenation are larger according to background of cloth acid content, extract has the effect of blood sugar lowering, blood fat reducing, has good application prospect.
Accompanying drawing explanation
Fig. 1 is the chromatogram of embodiment 1 Cortex Sclerotii Poriae extract.
Fig. 2 is 3 beta-hydroxies-Pilus Caprae seu Ovis steroid 7,9(11), and the chromatogram of 24-triolefin-21-acid.
Fig. 3 is that dehydrogenation is according to the chromatogram of background of cloth acid.
The specific embodiment
The present invention will be further elaborated below.
embodiment 1
Cortex Sclerotii Poriae extracting method is as follows:
1, get 5kg Cortex Sclerotii Poriae, with the hydrochloric acid of 1.5wt%, rinse once, then water is rinsed acid well, to neutrality, dries;
2, by the alcoholic solution reflux, extract, of 70 volume % 2 times for the 5kg Cortex Sclerotii Poriae after drying, the consumption of alcoholic solution is 50L, and each extraction time is 0.5 h, merge extractive liquid,, and decompression recycling ethanol, to dry, obtains dry;
3, the dry of step 2 is fallen apart with the water-soluble of 500ml, then add the mixture (n-butyl alcohol: ethyl acetate volume ratio=7:1.5) extract, get organic facies, reclaim solvent to dry, obtain crude product 203g of 750ml n-butyl alcohol and ethyl acetate;
4, crude product 20.5L dissolve with ethanol, wiring solution-forming, get crude product 18 quality neutral alumina doubly, dress chromatographic column, above-mentioned crude product solution is added in chromatographic column, make solution pass through chromatographic column, then use ethanol and ethyl acetate (ethanol: mixture ethyl acetate volume ratio=2:5) carries out eluting, the collection eluent of two volumes times aluminium oxide;
5, to the active carbon that adds 6.1g in eluent, be heated to boiling, keep micro-and boil 30 minutes, filtered while hot, uses washing with alcohol active carbon, merging filtrate and washing liquid, be concentrated into 1/3 of original volume, staticly settle, filter, 60 ℃ dry, obtain Cortex Sclerotii Poriae extract 150g.Calculate extract yield=extract quality/Cortex Sclerotii Poriae quality=150/5000=3%.
To hydroxyl-Pilus Caprae seu Ovis steroid 7 in extract, 9(11), 24-triolefin-21-acid and dehydrogenation are measured according to the content of background of cloth acid, and method is as follows:
1, instrument and reagent: Waters highly effective liquid phase chromatographic system, Water600 pump, 996 diode array detector, M
32chromatographic work station, Lichrospher-C
18post, 200 * 4.6mm, 5 μ (Hanbon Sci. & Tech. Co., Ltd.'s product).
2, medicine: acetonitrile (chromatographically pure level), 3 beta-hydroxies-Pilus Caprae seu Ovis steroid 7,9(11), 24-triolefin-21-acid and dehydrogenation are according to background of cloth acid (Chinese medicine new formulation center, Shandong Province preparation, content all reaches more than 98.0%).
3, testing conditions: mobile phase: acetonitrile: 0.5% acetic acid water (80:20), detects wavelength 242nm.
4, the preparation of reference substance solution
Precision takes 3 beta-hydroxy-7, and 9(11), 24-triolefin-21-acid reference substance, puts in 10 mL volumetric flasks, and methanol is diluted to scale, makes 228 μ gmL
-1reference substance solution; Precision takes dehydrogenation according to background of cloth acid reference substance, puts in 10 mL volumetric flasks, and methanol is diluted to scale, makes 130 μ gmL
-1reference substance solution.
5, the preparation of need testing solution
Get Cortex Sclerotii Poriae extract approximately 40 mg, accurately weighed, to put in 100 mL volumetric flasks, dissolve with methanol is also diluted to scale, shakes up, and with the filtering with microporous membrane of 0.45 μ m, gets subsequent filtrate, obtains.
6, assay method
Accurate above-mentioned reference substance solution and each 10 μ L of need testing solution of drawing, in injection liquid chromatography, obtain the chromatogram 1-3 of extract and reference substance respectively.
According to calculated by peak area, 3 beta-hydroxies-Pilus Caprae seu Ovis steroid 7,9(11), 24-triolefin-21-acid content is 30.61%, dehydrogenation is that 32.52%, two kind of material amounts to 63.23% according to background of cloth acid content.
embodiment 2
Cortex Sclerotii Poriae extracting method is as follows:
1, get 5kg Cortex Sclerotii Poriae, with the hydrochloric acid of 1.0wt%, rinse once, then water is rinsed acid well, to neutrality, dries;
2, by the alcoholic solution reflux, extract, of 80 volume % 2 times for the 5kg Cortex Sclerotii Poriae after drying, the consumption of alcoholic solution is 75L, and each extraction time is 2h, merge extractive liquid,, and decompression recycling ethanol, to dry, obtains dry;
3, the dry of step 2 is fallen apart with the water-soluble of 500ml, then add the mixture (n-butyl alcohol: ethyl acetate volume ratio=5:1.5) extract, get organic facies, reclaim solvent to dry, obtain crude product 186g of 1000ml n-butyl alcohol and ethyl acetate;
4, crude product 19L dissolve with ethanol, wiring solution-forming, get crude product 15 quality neutral alumina doubly, dress chromatographic column, above-mentioned crude product solution is added in chromatographic column, make solution pass through chromatographic column, then use ethanol and ethyl acetate (ethanol: mixture ethyl acetate volume ratio=2:3) carries out eluting, the collection eluent of 1.5 volumes times aluminium oxide;
5, to the active carbon that adds 3.8g in eluent, be heated to boiling, keep micro-and boil 30 minutes, filtered while hot, uses washing with alcohol active carbon, merging filtrate and washing liquid, be concentrated into 1/3 of original volume, staticly settle, filter, 60 ℃ dry, obtain Cortex Sclerotii Poriae extract 123g.
Adopt high performance liquid chromatography to record 3 beta-hydroxies in extract-Pilus Caprae seu Ovis steroid 7,9(11) amount to 55.24% with 24-triolefin-21-acid content.
embodiment 3
Cortex Sclerotii Poriae extracting method is as follows:
1, get 5kg Cortex Sclerotii Poriae, with the hydrochloric acid of 2.0wt%, rinse once, then water is rinsed acid well, to neutrality, dries;
2, by the alcoholic solution reflux, extract, of 95 volume % 2 times for the 5kg Cortex Sclerotii Poriae after drying, the consumption of alcoholic solution is 25L, and each extraction time is 3h, merge extractive liquid,, and decompression recycling ethanol, to dry, obtains dry;
3, the dry of step 2 is fallen apart with the water-soluble of 500ml, then add the mixture (n-butyl alcohol: ethyl acetate volume ratio=10:1.5) extract, get organic facies, reclaim solvent to dry, obtain crude product 183g of 800ml n-butyl alcohol and ethyl acetate;
4, crude product 22L dissolve with ethanol, wiring solution-forming, get crude product 20 quality neutral alumina doubly, dress chromatographic column, above-mentioned crude product solution is added in chromatographic column, make solution pass through chromatographic column, then use ethanol and ethyl acetate (ethanol: mixture ethyl acetate volume ratio=7:5) carries out eluting, the collection eluent of 3 volumes times aluminium oxide;
5, to the active carbon that adds 5.5g in eluent, be heated to boiling, keep micro-and boil 30 minutes, filtered while hot, uses washing with alcohol active carbon, merging filtrate and washing liquid, be concentrated into 1/3 of original volume, staticly settle, filter, 60 ℃ dry, obtain Cortex Sclerotii Poriae extract 122g.
Adopt high performance liquid chromatography to record 3 beta-hydroxies in extract-Pilus Caprae seu Ovis steroid 7,9(11) amount to 53.57% with 24-triolefin-21-acid content.
the embodiment 1 of take is below extract obtained is example, and extract blood sugar lowering, effect for reducing blood fat are studied.
, laboratory animal
Healthy male mice in kunming, body weight 24～26g; Clean level Wistar rat, body weight 180～220g.
, instrument and reagent
T-CHOL (TC), triglyceride (TG), HDL-C (HDL-C) test kit: clinical reagent subsidiary factory of Beijing North fine chemistry Co., Ltd produces.
Alloxan: Hong Kong FARCO company produces.
Instrument for Measuring Blood Sugar: Britain Bao Ling Man produces.
Semi-automatic biochemical analyzer: Shanghai San analytical tool factory produces
3, experimental technique
3.1 impacts on blood glucose in diabetic mice
3.1.1 diabetic mice: adopt the method for tail vein injection alloxan (administration after animal fasting 24h, dosage is 45mg/kgbw) to set up diabetes animal model.Empty stomach (fasting 3h) blood glucose value after tail vein injection alloxan 7d is greater than to the mice of 10mmol/L as the diabetic mice of this test.
3.1.2 test dose and animal grouping: according to the blood glucose value of diabetic mice, it is divided into four groups at random, 1 model control group wherein, 3 extract doses groups (dosage be respectively 1.5,2.5,3.5g/kgbw).Every group of equal 12 mices.
3.1.3 the mensuration of fasting glucose: above-mentioned each dosage treated animal gives after extract 30d (model control group is to distilled water) continuously, the ophthalmic corner of the eyes is got blood and is surveyed whole blood fasting blood sugar (fasting 3h).
impact on Experimental Hyperlipemia rat fat
3.2.1 adopt rat hyperlipoidemia method to test.With normal feedstuff feed rat, observe after 5d, get on an empty stomach tail blood, enzymatic assays serum total cholesterol (TC), triglyceride (TG), HDL-C (HDL-C), according to TC level, animal is divided into 4 groups at random: high fat matched group, three extract groups (dosage be respectively 1.5,2.5,3.5g/kgbw).Extract is pressed desired concn preparation with distilled water.From formal test, each treated animal is used high lipid food instead, extract group gavage gives the extract of various dose, high fat matched group gavage gives the distilled water of same volume, gavage amount is pressed 1.0ml/100gbw and is calculated, once a day, after 40d, get on an empty stomach the above-mentioned every blood lipids index of tail hematometry continuously.
3.2.2 animal feed
Normal feedstuff: normal granules feedstuff.
High lipid food (wt%): normal feedstuff 79%, cholesterol 1%, Adeps Sus domestica 10%, fresh-laid egg yellow liquor 10%.
, date processing
Adopt t check and variance analysis to carry out statistical procedures.
, results and analysis
the impact of 5.1 extracts on diabetic mice fasting glucose
Each organize diabetic mice before contact extract the average fasting blood sugar of (i.e. test before) all without significant difference (p>0.05); And give (after test) after extract 30d (model control group is to distilled water) at continuous per os, all there is certain variation in its average fasting blood sugar and average fasting glucose reduction value.The results are shown in Table 1.
As can be seen from Table 1, the fasting blood sugar after the basic, normal, high dosage group of the present invention mouse test is all lower than model control group, and middle agent to organize performance difference most extremely remarkable; Before and after test, blood glucose reduction value is that middle dosage group raises obviously equally.This result of the test explanation extract of the present invention has some improvement to the fasting glucose tool of diabetic mice.
the impact of extract on rat fat
Extract of the present invention the results are shown in Table 2 to the impact of rat fat.
As can be seen from Table 2, each organizes rat (before test) before contact extract, equal no significant difference between every blood lipids index group.And after giving extract of the present invention (model control group is to the distilled water) 40d of the above-mentioned various dose of rat oral gavage (after test), the serum TC of model control group, TG all show obvious rising, the success of hyperlipemia animal model copy is described; Meanwhile, the rising of the serum TC of basic, normal, high dosage group has been subject to obvious inhibition, and the rising of HDL-C is remarkable compared with model control group comparing difference.This result of the test shows, extract of the present invention can be resisted the rising effect of high lipid food to Serum TC significantly, and this effect is likely that the part TC that the HDL-C by rising animal disposes in body completes.This explanation extract of the present invention preventive effect certain to being formed with of serum lipids in rats.
Claims (10)
1. an extracting method for Cortex Sclerotii Poriae effective ingredient, is characterized in that, comprises the following steps:
(1) Cortex Sclerotii Poriae is rinsed with the hydrochloric acid of 1.0-2.0wt%, then water rinses to neutral, dries;
(2) the alcoholic solution reflux, extract, of Cortex Sclerotii Poriae use 70-95 volume % step (1) being processed 2 times, merge extractive liquid,, extracting solution reclaims ethanol to dry;
(3) by the dry water dissolution of step (2), add the mixture extraction of n-butyl alcohol and ethyl acetate, organic facies reclaims solvent to dry, obtains crude product;
(4) crude product dissolve with ethanol, wiring solution-forming, neutral alumina chromatographic column on gained solution, then carries out eluting with the mixture of ethanol and ethyl acetate, collects eluent;
(5) in eluent, add active carbon, be heated to seethe with excitement and keep 30min, filtered while hot, uses washing with alcohol active carbon, and merging filtrate and washing liquid are concentrated into 1/3 of original volume, staticly settles, filters, is dried, and obtains effective ingredient.
2. extracting method according to claim 1, is characterized in that: in step (2), Cortex Sclerotii Poriae and alcoholic solution with magnitude relation, be 1:5-20(g/ml).
3. extracting method according to claim 1, is characterized in that: in step (2), each extraction time is 0.5-3.5h.
4. extracting method according to claim 1, is characterized in that: in step (2), with the ethanol extraction Cortex Sclerotii Poriae of 70 volume %, Cortex Sclerotii Poriae and alcoholic solution with magnitude relation, be 1:10(g/ml), each extraction time is 0.5 h.
5. extracting method according to claim 1, is characterized in that: in step (3), and n-butyl alcohol: the volume ratio of ethyl acetate is 5-10:1.5, preferably 7:1.5; Water: the volume ratio of the mixture of n-butyl alcohol and ethyl acetate is 1:1.5-2.
6. extracting method according to claim 1, is characterized in that: in step (4), and its 100-120 quality dissolve with ethanol doubly for crude product, wiring solution-forming; The quality of neutral alumina is 15-20 times of crude product.
7. extracting method according to claim 1, is characterized in that: in step (4), the volume ratio of ethanol and ethyl acetate is 2:3-7, is preferably 2:5; The consumption of the mixture of ethanol and ethyl acetate is 1.5-3 times of aluminium oxide, is preferably 2 times.
8. extracting method according to claim 1, is characterized in that: in step (5), activated carbon dosage is the 2-3% of crude product quality.
9. extracting method according to claim 1, is characterized in that: in gained effective ingredient, mainly comprise 3 beta-hydroxies-Pilus Caprae seu Ovis steroid 7,9(11), 24-triolefin-21-acid and dehydrogenation are according to background of cloth acid, and both content are more than 50%.
10. adopt the Cortex Sclerotii Poriae extract of the extracting method extraction gained of the Cortex Sclerotii Poriae effective ingredient described in any one in claim 1-9.
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| CN105211989A (en) * | 2015-10-15 | 2016-01-06 | 兰军胜 | A kind of preparation method of clearing heat and removing internal heat beverage |
| WO2017193900A1 (en) * | 2016-05-10 | 2017-11-16 | 杏辉天力（杭州）药业有限公司 | Poria cocos skin extract, poricoic acid a, and poricoic acid b and application thereof for blood sugar modulation |
| CN107412236A (en) * | 2016-05-24 | 2017-12-01 | 施纯青 | The purposes of Antrodia camphorata abstraction purification thing |
| JP2019529533A (en) * | 2016-09-06 | 2019-10-17 | シンファー ティアン−リー ファーマシューティカル カンパニー リミテッド（ハンツォウ）Ｓｉｎｐｈａｒ Ｔｉａｎ−Ｌｉ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｃｏ．， Ｌｔｄ． （Ｈａｎｇｚｈｏｕ） | Use of poly extract and its active ingredients in skin care and / or promoting wound healing |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105211989A (en) * | 2015-10-15 | 2016-01-06 | 兰军胜 | A kind of preparation method of clearing heat and removing internal heat beverage |
| WO2017193900A1 (en) * | 2016-05-10 | 2017-11-16 | 杏辉天力（杭州）药业有限公司 | Poria cocos skin extract, poricoic acid a, and poricoic acid b and application thereof for blood sugar modulation |
| TWI676473B (en) * | 2016-05-10 | 2019-11-11 | 杏輝藥品工業股份有限公司 | Uses of poria cocos epidermis extract, poricoic acid a, and poricoic acid b in regulating blood glucose level |
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| JP2019529533A (en) * | 2016-09-06 | 2019-10-17 | シンファー ティアン−リー ファーマシューティカル カンパニー リミテッド（ハンツォウ）Ｓｉｎｐｈａｒ Ｔｉａｎ−Ｌｉ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｃｏ．， Ｌｔｄ． （Ｈａｎｇｚｈｏｕ） | Use of poly extract and its active ingredients in skin care and / or promoting wound healing |
| WO2020155945A1 (en) * | 2019-01-30 | 2020-08-06 | 中国科学院微生物研究所 | Preparation method for indian bread peel triterpene composition and application thereof |
| CN110988191A (en) * | 2019-12-23 | 2020-04-10 | 哈尔滨市康隆药业有限责任公司 | HPLC content determination method of Shenzhu kang syrup |
| CN112326862A (en) * | 2020-09-29 | 2021-02-05 | 鸿翔中药科技有限责任公司 | Poria cocos formula granules, preparation method thereof and thin-layer identification method |
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