CN101797312B - Method for preparing Alisma extract capable of inhibiting alpha-glucuroide activity - Google Patents
Method for preparing Alisma extract capable of inhibiting alpha-glucuroide activity Download PDFInfo
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- CN101797312B CN101797312B CN2010100458562A CN201010045856A CN101797312B CN 101797312 B CN101797312 B CN 101797312B CN 2010100458562 A CN2010100458562 A CN 2010100458562A CN 201010045856 A CN201010045856 A CN 201010045856A CN 101797312 B CN101797312 B CN 101797312B
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- rhizoma alismatis
- glucuroide
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Abstract
The invention discloses a method for preparing Alisma extract capable of inhibiting alpha-glucuroide activity, comprising the following steps: crushing Alisma, leaching, filtering, concentrating, standing and refrigerating, splitting clear liquid, and concentrating to obtain crude Alisma extract; and successively extracting by organic solvent, merging n-butanol extraction part partially, distilling by reduced pressure and concentrating, and drying to obtain the Alisma extract. The Alisma extract has the function of inhibiting the activity of alpha-glucuroide, the enzyme inhibition ratio can be 92.30%, the Alisma extract can be added in drug carriers to prepare granules, capsules, tablets, oral liquid or injection; or carriers are added into the Alisma extract to prepare heath-care beverage or heath-care food according to the conventional method. The invention is simple and feasible, and is suitable for promotion.
Description
Technical field
The invention belongs to the preparing technical field of alpha-glucosidase inhibitor.
Background technology
According to IDF's report, present global diabetes patient has exceeded 100,000,000, predicts 2025 and will break through 300,000,000, and main body is a type 2 diabetes mellitus.Prediabetes is Fructus Vitis viniferae IGR (impaired glucose regulation, IGR), interim comprise impaired fasting glucose (IFG) (impaired fasting glucose, IFG) and impaired glucose tolerance (impaired glucose tolerance, IGT).For IGT and type 2 diabetes mellitus early stage patient, all show as postprandial hyperglycemia, and postprandial hyperglycemia to the harm of body considerably beyond the empty stomach hyperglycemia.Postprandial hyperglycemia not only very easily brings out the pathological changes of tissues such as trunk, blood capillary, peripheral nerve, cause various complication, and the dependency between the general mortality rate of itself and coronary heart disease and diabetes is greater than the empty stomach hyperglycemia, so reduce post-prandial glycemia is the generation of prevention type 2 diabetes mellitus, reduces its complication and reduces one of important measures of mortality rate.
Alpha-glucosidase is a kind of enzyme that can make non-absorbent complex carbohydrate resolve into the absorbable monosaccharide of small intestinal, alpha-glucosidase activity inhibitor is then done to raise in order to reduce post-prandial glycemia by suppressing this enzyme, mainly represent medicine that acarbose (Acarbose) and voglibose (Voglibose) are arranged, the former suppresses mucous membrane of small intestine brush border alpha-glucosidase by competitive and reversibility, the cracking of blocking-up starch and sucrose, thereby prolong glucose and fructose in gastral infiltration rate, reduce post-prandial glycemia; The latter is a selectivity disaccharidase hydrolase inhibitor, suitably delays the absorption of sugar, improves post-prandial glycemia, and is stronger than glucosidase inhibitor effect to the maltase and the saccharase inhibitory action of intestinal.They have untoward reaction such as abdominal discomfort, flatulence, aerofluxus.Therefore, other wards off new footpath, seeks the inhibitor safely and effectively of alpha-glucosidase, has important clinical application value for treatment, prevention non-insulin-dependent diabetes mellitus, obesity, hyperlipemia and hyperlipoproteinemia.
Summary of the invention
The objective of the invention is to the Chinese medicine Rhizoma Alismatis is raw material, by simple and practical PROCESS FOR TREATMENT, obtains suppressing the extract of alpha-glucosidase activity, utilizes this extract to add pharmaceutical carrier again and prepares the alpha-glucosidase inhibitor dosage form.
Technical scheme of the present invention is:
Adopt extraction from the Chinese medicine Rhizoma Alismatis, to extract and obtain crude extract, again the Rhizoma Alismatis crude extract is used petroleum ether, ethyl acetate, n-butanol extraction successively, obtain petroleum ether part, ethyl acetate part and n-butyl alcohol part respectively, n-butyl alcohol partly merges, distilling under reduced pressure concentrates, and promptly obtains Rhizoma Alismatis extract after the drying.
Technical solution of the present invention realizes by following processing step:
(1) pulverizes: dry Rhizoma Alismatis granule is crushed to 5～80 orders;
(2) the thick extraction: water or water-miscible organic solvent lixiviate Rhizoma Alismatis, obtain extracting solution, filter, be concentrated into small size, cooling adds ethanol in concentrated solution, leaves standstill cold preservation, divides and gets the supernatant, following layered material slag is centrifugal, and the gained clear liquid and the supernatant merge, and concentrates, and obtains the Rhizoma Alismatis crude extract;
(3) the extraction essence is carried: the Rhizoma Alismatis crude extract is placed separatory funnel, add isopyknic extractant then, used extractant is followed successively by: petroleum ether, ethyl acetate, n-butyl alcohol are the organic layer merging of n-butanol extraction with extractant, distilling under reduced pressure concentrates, and promptly obtains Rhizoma Alismatis extract after the drying.
In above-mentioned steps (2), the Rhizoma Alismatis lixiviate comprises lixiviate of concussion method or circumfluence method lixiviate, and extraction time is 0.5～7 hour, can extract 3 times; Used water-miscible organic solvent comprises: concentration is the aqueous solution of the alcohol that contains 1～6 carbon atom, alkane or ester below 100%, and its addition is particulate 2～10 times an of Rhizoma Alismatis; Adding the ethanol final concentration in the concentrated solution is 30～90%; Leaving standstill refrigerated storage temperature is 3～10 ℃, and cold preservation time is 12～48 hours.
Rhizoma Alismatis extract suppresses the mensuration of alpha-glucosidase activity effect:
(1) preparation of enzyme activity determination reagent
67mmol/L phosphate buffered solution (PH=6.8): with dipotassium hydrogen phosphate 7.646g, potassium dihydrogen phosphate 4.559g mixed dissolution is settled to 500mL in distilled water.
4-Nitrobenzol-α-D-pyranglucoside (PNPG) solution: 0.091g PNPG is dissolved in the 1mL dimethyl sulfoxide, adds the 1.6mL kaliumphosphate buffer, be made into the 0.116mol/L substrate solution.
Alpha-glucosaccharase enzymatic solution: claim 3.5mg solid enzyme, add 67mmol/L kaliumphosphate buffer 200 μ L, be made into 0.1U/ μ L enzymatic solution.
Na
2CO
3Solution: take by weighing 0.53g Na
2CO
3Be settled to 50mL with distilled water, be made into the solution of 0.1mol/L.
Reduced glutathion: the 10mg reduced glutathion is dissolved in the 10mL buffer solution of potassium phosphate.
(2) mensuration of extract inhibitory enzyme activity
In 2mL phosphate buffer (pH6.8), add 1mg/mL glutathione solution 50 μ L, add alpha-glucosidase 5 μ L, behind 37 ℃ of insulation 10min, add testing sample solution 100 μ L and PNPG 50 μ L (final concentration), 37 ℃ of reaction 10min.
Add Na
2CO
3Solution 10mL cessation reaction is measured absorption value at wavelength 400nm place.
Wherein enzyme activity unit is defined as: at 37 ℃, under the condition of pH 6.8, it is an enzyme activity unit (U) that the interior hydrolysis PNPG of 1min discharges the required enzyme amount of 1 μ mol PNP.The inhibitor unit of activity is defined as: reduce by 1 the inhibition dosage that enzyme activity unit is required under identical condition.With the positive contrast of acarbose.The results are shown in Table.
The enzyme inhibition rate formula:
Enzymatic activity suppression ratio=[A blank-(A sample-A background)]/A blank * 100%
A blank: do not add the reacted absorption value of sample
A sample: the absorption value behind the adding example reaction
A background: the absorption value that only adds sample.
The situation of each extract inhibitory enzyme activity of table 1 Rhizoma Alismatis
By above table 1 as can be known, alpha-glucosidase suppresses active strong composition and is present in the n-butyl alcohol part in the Rhizoma Alismatis.
The invention has the beneficial effects as follows: preparation is simple for Rhizoma Alismatis extract, is fit to very much apply.It is suitable with background technology that this extract suppresses the alpha-glucosidase activity effect, and its suppression ratio is not less than the positive control acarbose.Rhizoma Alismatis extract can join pharmaceutical carrier, mixes, and granulates, and drying is made granule or capsule or tablet or oral liquid or injection according to the medicament preparation method of routine then; Perhaps add carrier, make health beverage or health food according to conventional method.
The specific embodiment
Embodiment 1: Rhizoma Alismatis is pulverized, cross 5 mesh sieves, take by weighing 100g, add 1000mL water at 60 ℃ of water-bath concussion lixiviate 7h.Add for the second time 800mL water at 60 ℃ of water-bath concussion lixiviate 5h.Add 600mL30% ethanol for the third time at 60 ℃ of water-bath concussion lixiviate 3h.Merge three times extracting solution, be concentrated into 250mL, filter.In concentrated solution, add ethanol, make that ethanol content reaches 30% in the concentrated solution.In 3 ℃, leave standstill 48h.Getting supernatant, centrifugal (4950r/min, 15min), getting precipitation, centrifugal (4950r/min 15min), merges centrifugal liquid.After centrifugal liquid is concentrated into no ethanol and flows out, extract respectively with isopyknic petroleum ether, ethyl acetate, n-butyl alcohol, each three times, merge corresponding organic layer, obtain respectively petroleum ether part, ethyl acetate partly, the n-butyl alcohol part.The distilling under reduced pressure of n-butyl alcohol part is concentrated, promptly obtain having the Rhizoma Alismatis extract that suppresses alpha-glucosidase activity after the drying.
The yield situation of each extract of table 2 Rhizoma Alismatis
Embodiment 2: Rhizoma Alismatis is pulverized, cross 10 mesh sieves, take by weighing 100g, add 700mL30% ethanol at 60 ℃ of water-bath reflux, extract, 4h.For the second time add 500mL30% ethanol at 60 ℃ of water-bath reflux, extract, 3h.Add 300mL30% ethanol for the third time at 60 ℃ of water-bath reflux, extract, 2h.Merge three times extracting solution, be concentrated into 250mL, filter.In concentrated solution, add ethanol, make that ethanol content reaches 60% in the concentrated solution.In 7 ℃, leave standstill 24h.Getting supernatant, centrifugal (4950r/min, 15min), getting precipitation, centrifugal (4950r/min 15min), merges centrifugal liquid.After centrifugal liquid is concentrated into no ethanol and flows out, extract respectively with isopyknic petroleum ether, ethyl acetate, n-butyl alcohol, each three times, merge corresponding organic layer, obtain respectively petroleum ether part, ethyl acetate partly, the n-butyl alcohol part.The distilling under reduced pressure of n-butyl alcohol part is concentrated, promptly obtain having the Rhizoma Alismatis extract that suppresses alpha-glucosidase activity after the drying.
The yield situation of each extract of table 3 Rhizoma Alismatis
Embodiment 3: Rhizoma Alismatis is pulverized, cross 20 mesh sieves, take by weighing 100g, add 800mL30% methanol at 60 ℃ of water-bath reflux, extract, 4h.For the second time add 600mL30% methanol at 60 ℃ of water-bath reflux, extract, 3h.Add 400mL30% methanol for the third time at 60 ℃ of water-bath reflux, extract, 2h.Merge three times extracting solution, be concentrated into 250mL, filter.In concentrated solution, add ethanol, make that ethanol content reaches 45% in the concentrated solution.In 5 ℃, leave standstill 24h.Getting supernatant, centrifugal (4950r/min, 15min), getting precipitation, centrifugal (4950r/min 15min), merges centrifugal liquid.After centrifugal liquid is concentrated into no ethanol and flows out, extract respectively with isopyknic petroleum ether, ethyl acetate, n-butyl alcohol, each three times, merge corresponding organic layer, obtain respectively petroleum ether part, ethyl acetate partly, the n-butyl alcohol part.The distilling under reduced pressure of n-butyl alcohol part is concentrated, promptly obtain having the Rhizoma Alismatis extract that suppresses alpha-glucosidase activity after the drying.
The yield situation of each extract of table 4 Rhizoma Alismatis
Embodiment 4: Rhizoma Alismatis is pulverized, cross 65 mesh sieves, take by weighing 100g, add 500mL75% ethanol at 60 ℃ of water-bath reflux, extract, 3h.For the second time add 300mL75% ethanol at 60 ℃ of water-bath reflux, extract, 2h.Add 200mL75% ethanol for the third time at 60 ℃ of water-bath reflux, extract, 1h.Merge three times extracting solution, be concentrated into 250mL, filter.In concentrated solution, add ethanol, make that ethanol content reaches 85% in the concentrated solution.In 10 ℃, leave standstill 12h.Getting supernatant, centrifugal (4950r/min, 15min), getting precipitation, centrifugal (4950r/min 15min), merges centrifugal liquid.After centrifugal liquid is concentrated into no ethanol and flows out, extract respectively with isopyknic petroleum ether, ethyl acetate, n-butyl alcohol, each three times, merge corresponding organic layer, obtain respectively petroleum ether part, ethyl acetate partly, the n-butyl alcohol part.The distilling under reduced pressure of n-butyl alcohol part is concentrated, promptly obtain having the Rhizoma Alismatis extract that suppresses alpha-glucosidase activity after the drying.
The yield situation of each extract of table 5 Rhizoma Alismatis
Embodiment 5: Rhizoma Alismatis is pulverized, cross 80 mesh sieves, take by weighing 100g, add 600mL80% methanol at 60 ℃ of water-bath reflux, extract, 1h.For the second time add 300mL80% methanol at 60 ℃ of water-bath reflux, extract, 0.5h.Add 300mL80% methanol for the third time at 60 ℃ of water-bath reflux, extract, 0.5h.Merge three times extracting solution, be concentrated into 250mL, filter.In concentrated solution, add ethanol, make that ethanol content reaches 90% in the concentrated solution.In 10 ℃, leave standstill 12h.Getting supernatant, centrifugal (4950r/min, 15min), getting precipitation, centrifugal (4950r/min 15min), merges centrifugal liquid.After centrifugal liquid is concentrated into no ethanol and flows out, extract respectively with isopyknic petroleum ether, ethyl acetate, n-butyl alcohol, each three times, merge corresponding organic layer, obtain respectively petroleum ether part, ethyl acetate partly, the n-butyl alcohol part.The distilling under reduced pressure of n-butyl alcohol part is concentrated, promptly obtain having the Rhizoma Alismatis extract that suppresses alpha-glucosidase activity after the drying.
The yield situation of each extract of table 6 Rhizoma Alismatis
Claims (5)
1. the preparation method of an Alisma extract capable of inhibiting alpha-glucuroide activity, it is characterized in that: described preparation method may further comprise the steps:
(1) pulverizes: the Rhizoma Alismatis raw material pulverizing;
(2) the thick extraction: water or water-miscible organic solvent lixiviate Rhizoma Alismatis, obtain extracting solution, filter, be concentrated into small size, cooling adds ethanol in concentrated solution, leaves standstill cold preservation, divides and gets the supernatant, following layered material slag is centrifugal, and the gained clear liquid and the supernatant merge, and concentrates, and obtains the Rhizoma Alismatis crude extract;
(3) the extraction essence is carried: the Rhizoma Alismatis crude extract is placed separatory funnel, add isopyknic extractant then, used extractant is followed successively by: petroleum ether, ethyl acetate, n-butyl alcohol are the organic layer merging of n-butanol extraction with extractant, distilling under reduced pressure concentrates, and promptly obtains Rhizoma Alismatis extract after the drying.
2. the preparation method of Alisma extract capable of inhibiting alpha-glucuroide activity according to claim 1, it is characterized in that: Rhizoma Alismatis is exsiccant Rhizoma Alismatis granule, is crushed to 5～80 orders.
3. the preparation method of Alisma extract capable of inhibiting alpha-glucuroide activity according to claim 1 is characterized in that: lixiviate comprises lixiviate of concussion method or circumfluence method lixiviate in thick the extraction, and extraction time is 0.5～7 hour, can extract 3 times.
4. the preparation method of Alisma extract capable of inhibiting alpha-glucuroide activity according to claim 1, it is characterized in that: the used water-miscible organic solvent of lixiviate is that concentration is the aqueous solution of the alcohol that contains 1～6 carbon atom, alkane or ester below 100% in thick the extraction, and water or water-miscible organic solvent addition are particulate 2～10 times of Rhizoma Alismatis; Adding the ethanol final concentration in the concentrated solution in thick the extraction is 30～90%.
5. the preparation method of Alisma extract capable of inhibiting alpha-glucuroide activity according to claim 1, it is characterized in that: it is 3～10 ℃ that the Rhizoma Alismatis extracting solution leaves standstill refrigerated storage temperature, cold preservation time is 6～48 hours.
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| CN113663043B (en) * | 2021-08-24 | 2022-05-20 | 广东工业大学 | Composition with alpha-glucosidase inhibition effect and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101406576A (en) * | 2008-11-24 | 2009-04-15 | 浙江省中药研究所有限公司 | Chinese medicine for treating diabetes and preparation method |
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| CN101406576A (en) * | 2008-11-24 | 2009-04-15 | 浙江省中药研究所有限公司 | Chinese medicine for treating diabetes and preparation method |
Non-Patent Citations (2)
| Title |
|---|
| Xin-Bo Yang et al..Effects of rhizome alismatis extract on blood biochemical indices and insulin in hyperglycemic mice.《Chinese Journal of Clinical Rehabilitation》.2004,第8卷(第6期),1196-1197. * |
| 易醒 等.泽泻的研究现状与展望.《时珍国医国药》.2007,第18卷(第2期),331. * |
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