CN101073596B - Alpha-glycosidase inhibitor, its extraction and use - Google Patents
Alpha-glycosidase inhibitor, its extraction and use Download PDFInfo
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- CN101073596B CN101073596B CN2006100208151A CN200610020815A CN101073596B CN 101073596 B CN101073596 B CN 101073596B CN 2006100208151 A CN2006100208151 A CN 2006100208151A CN 200610020815 A CN200610020815 A CN 200610020815A CN 101073596 B CN101073596 B CN 101073596B
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Abstract
The invention is concerned with the method of preparation of the alpha-glycosidase inhibitor extracts from walnut. It contents the active ingradient of the shell and kernel of the walnut (also includes Juglans regia L., Juglans cathayensis Dode, and Juglans mandshurica Maxim). Thealpha-glycosidase inhibitor is extracted from the draff after oil fired by water, alcohol, or water-alcohol solution, through filtration, concentration, and drying at last. It can use for prepareing health products for diabetes therapy or blood suger controlling.
Description
Technical field
The invention belongs to biomedicine field, relate to a kind of alpha-glucosidase inhibitor that from Semen Juglandis, extracts and extracting method and purposes.
Background technology
Diabetes have become serious public health problem in the world wide, and the whole world has 1.32 hundred million people to suffer from diabetes approximately, will reach 2.4 hundred million in 2010.In China, show according to nearest Epidemiological study, the onset diabetes rate of China by 0.67% before 15 years rise in recent years 3.21%, wherein the type ii diabetes patient accounts for more than 95% of sum, and the men and women does not have significant difference.According to statistics, China's diabetes number was 6,500,000 in 1978, and the mid-80 is about 7,500,000, to nineteen ninety-five diabetics reach 1,500 ten thousand people, annually in recent years increasing progressively with the numeral more than 1,000,000 people, reached more than 2,000 ten thousand people to the end of the year 1998.Recently the diabetes survey of in 11 provinces and cities in the whole nation more than 40,000 20 years old to 74 years old permanent resident populations being carried out shows that the onset diabetes rate is 3.21%, and the sickness rate of " reserves " (impaired glucose tolerance) of diabetes is 4.76%.With 1,200,000,000 population estimations, the diabetics of 20～74 years old age bracket of China is more than 2,000 ten thousand, and " reserves " of diabetics is not less than 3,000 ten thousand, and both add up to 50,000,000.Brainstrust estimates that if diabetes can not get effective control, to 2010, China's number of the infected will reach 6,000 ten thousand.In addition, in China, the increase of onset diabetes rate is also relevant with China aged tendency of population.In the age was higher than 60 years old crowd, the sickness rate of diabetes had reached 11.62%, and what be worth proposing is, the trend that type ii diabetes increases in child and teenager with ining addition, and this is mainly relevant with the increase of obese people in child and the teenager.
Diabetes are because defect of insulin secretion or/and the insulin action obstacle causes, is to be one group of metabolic disease of characteristics with chronic blood sugar increasing and carbohydrate metabolism disturbance.Mainly be divided into two types: type i diabetes (being insulin dependent diabetes mellitus (IDDM)) and type ii diabetes (being non-insulin-dependent diabetes mellitus), the latter accounts for the 90%-95% of patient's sum.The clinical manifestation of diabetes is a feature with hyperglycemia, glycosuria, polydipsia, polyuria and polyphagia.The complication that diabetes cause (as acute complicationses such as cardiovascular, kidney, retina and neural chronic complicating diseases, diabetic ketoacidosis, hyperosmolar nonketotic diabetic comas) is the main cause that causes death.All diabetic complications can be by strict glycemic control, from preventing to the full extent.If diabetics can be controlled at the blood sugar level of oneself near in the normal range of blood sugar, then diabetic complication just can not occur, and perhaps occurs than later and degree is also lighter.
Diabetes are chronic diseases, and its harm and influence to body relates to many aspects, and present medical level also is difficult to radical cure.But pharmacotherapy to a great extent to the correction of the control of blood sugar level, metabolism disorder, complication prevent and conclusive effect has been played in minimizing.The drug main that is used for diabetes at present will comprise following type:
1. insulin: insulin is the good medicine that is widely used in the treatment diabetes always.Outside traditional drug administration by injection, main at present from route of administration and the novel insulin medicament of dosage form aspect developing, as protamine zine insulin, Semilente Insulin, oral insulin, suction insulin etc.
2. non-insulin class medicine:, only depend on insulin still to be difficult to satisfy the needs of numerous class diabetes mellitus types though the novel form of insulin, new route of administration research have been obtained many progress.The non-insulin paradiabetes drug main of at present developing and succeeding in developing will comprise several classes:
(1) reduce carbohydrate absorption medicine---the initial inducement of glycosidase inhibitor diabetes and complication thereof is the rising of blood sugar concentration, and blood glucose is from digestion, the absorption of saccharic group food.The digestion of saccharic food, absorption process be as shown in drawings: polysaccharide and oligosaccharide all are to be by little intestinal absorption and be transported to blood and tissue after the monosaccharide through amylase, alpha-glucosidase different enzymic digestions such as (comprising maltase, saccharase etc.).Inhibition to these enzymes is the important channel that blood sugar control raises.
Such mechanism of drug action is the alpha-glucosidase that suppresses the small intestinal epimere, hinders carbohydrate breakdown and becomes single glucose, delays its absorption, reduces postprandial hyperglycemia.Clinical practice is comparatively successful acarbose (acarbose), voglibose (voglibose, the amino sugar analog of finding), miglitol (miglitol) from the actinomycetes culture fluid.
(2) insulin secretion stimulators has the effect that promotes insulin secretion, comprises that mainly sulfonylurea is (as glimepiride, glimepiride) and non-sulfonylurea (as repaglinide, repaglinid, Nateglinide nateglinid) etc.
(3) euglycemic agent this be the medicine that a class can improve insulin sensitivity, it can not promote the generation of insulin, but can improve the sensitivity of insulin and receptor target tissue, and insulin is played a role, and improves glycometabolic disorder.As Thiazolidine ketone (troglitazone, thiophene lattice row are on an equal basis), two croak class (as metformin, buformin) etc.
(4) the other medicines research and development are above-mentioned all is the diabetes medicament of having succeeded in developing and having gone on the market, and in addition also has many medicines also in R﹠D process.As glucagon receptor antagonist class medicine, fatty acid metabolism agent interfering etc.In addition, the numerous complication that cause at diabetes also have a large amount of medicament research and development reports, as aldose reductase (aldosereductase, AR) inhibitor class medicine (as tolrestat, tolrestat, epalrestat, epalresta) etc.
Diabetes are chronic disease even lifelong disease, positive health preserving in Drug therapy, health care are particularly important to the raising of the control of conditions of patients and quality of life, thereby the health food of developing medicine and having a specific function is the important channel of diabetes mellitus prevention and auxiliary treatment.Because it is the first line of defence of prevention and treatment diabetes that blood sugar control raises, the health food that exploitation has the blood sugar lowering effect is the important measures of diabetes mellitus prevention and auxiliary treatment.Our experiment shows that the Semen Juglandis extract has α-Dian Fenmei and alpha-glucosidase suppresses active, and the activity intensity of its total extract and present widely used hypoglycemic drug acarbose are equal or higher.Preliminary data-searching shows, yet there are no Semen Juglandis (comprising complex peach and J) is used to suppress α-Dian Fenmei and alpha-glucosidase activity in field of medicaments report at present both at home and abroad.
Summary of the invention
Based on above-mentioned, in order to enlarge the screening scope of blood sugar lowering class medicine, provide new thinking for developing plant class blood sugar reducing health product and medicine, the object of the invention is that open Semen Juglandis extract is used to suppress the active this new purposes of alpha-glucosidase.
The extract that the inventive method obtains has vitro inhibition alpha-glucosidase (comprising α-Dian Fenmei and alpha-glucosidase) activity.Compare with clinical antidiabetic drug acarbose commonly used under the same conditions, the residue extract that wherein extracts oil suppresses alpha-amylase activity and is better than acarbose, and it is active more than 70% that the inhibition alpha-glucosidase activity reaches acarbose; Though Semen Juglandis extract suppresses alpha-amylase activity not as good as acarbose, its activity that suppresses alpha-glucosidase can be used as diabetes adjuvant therapy medicaments or health product and further researchs and develops apparently higher than acarbose.
The method that the present invention relates to from Semen Juglandis (comprising Semen Juglandis Juglans regia L., Chinese walnut Juglans cathayensis Dode, Semen Caryae Cathayensis Juglans mandshurica Maxim), extract with the active material of inhibition alpha-glucosidase, this method be Semen Juglandis shell or core or its oil expression residue water, alcohol or alcohol-water mixture extract, extract after filtration, concentrate, the product that oven dry obtains, used alcohol is methanol or ethanol or propanol or butanols, is preferably ethanol or methanol.
The characteristics of the inventive method are: raw material sources are abundant, and extracting method is simple.
The specific embodiment
Embodiment one:
1. extracting method: complex peach and J strike shell and get core, and 45 ℃ of bakings are spent the night, and respectively take by weighing 100g, add each 300ml of 95% ethanol, and 45 ℃ of shaking table lixiviates 5 hours are filtered, concentrate, 45 ℃ of oven dry, complex Semen Persicae crude extract 2.36g, J core crude extract 2.07g.This crude extract is used for determination of activity.
2. determination of activity:
(1) suppresses alpha-amylase activity
1. solution allocation:
Starch solution: soluble starch 0.2M, the citric acid of pH=6.4-sodium hydrogen phosphate buffer dissolving places boiling water bath heating 5min, is chilled to room temperature, and adding distil water is to final concentration 1% (W/V).
Iodine solution: 0.5g I2 and 5.0g KI are dissolved in the 50ml distilled water, place brown bottle to preserve, and use behind the usefulness distilled water diluting by 1: 200.
α-Dian Fenmei: the extensive and profound in meaning star biotechnology in Beijing Co., Ltd, derive from bacillus cereus, be made into 2-4U/ml with preceding with distilled water.
Acarbose (positive control medicine): Bayer HealthCare Co.Tablet, a slice contain acarbose 50mg.Every adds the 20ml dissolved in distilled water, is made into experiment concentration 2.5mg/ml.
Sample solution: get complex peach and each 50mg of J core extract, adding distil water 5ml is made into the 10mg/ml solution for standby.
2. assay method:
Measuring principle:
Starch can with iodine generation color reaction, starch loses the color reaction ability behind α-Dian Fenmeishuixie.In starch and α-Dian Fenmei reaction system, add testing sample, with colorimetric method for determining color reaction degree, by with do not add comparing of sample, but quantitative assay sample alphalise starch enzyme inhibition activity.
List of references [1.2] design determination of activity is undertaken by following flow process:
Contrast is provided with:
Blank: replace enzyme liquid and sample solution with distilled water;
Positive control: replace sample with the 2.5mg/ml acarbose;
Negative control: replace sample with distilled water.
3. experimental result:
Annotate: because of enzyme in the experiment excessive relatively, so starch percent hydrolysis %=(OD
Blank-OD
Sample)/OD
Blank* 100%
Suppression ratio=100%-starch percent hydrolysis
(2) activity of inhibition alpha-glucosidase
1. solution allocation:
Maltose solution: 2% maltose solution (using 0.2M, the preparation of pH=6.8 phosphate buffer).
Alpha-glucosidase: available from Sigma (Fluka), derive from yeast, be made into 16.5U/ml with distilled water with preceding.
Glu measures test kit: available from Changchun remittance power Bioisystech Co., Ltd.
Sample solution: get complex peach and J core extract, be made into the 0.5mg/ml solution for standby.
2. assay method:
Measuring principle: alpha-glucosidase can the hydrolysis of catalysis maltose produce glucose, by the difference of assaying reaction system glucose growing amount when adding and do not add sample, can determine the inhibition activity of sample to alpha-glucosidase.
Contrived experiment is measured by following flow process:
Contrast is provided with: blank group replaces enzyme and sample with distilled water; Positive group replaces sample with acarbose; Negative group replaces sample with distilled water.
3. experimental result:
Annotate: glucose content C
Sample=OD
Sample/ OD
Mark(0.232) * C
Mark(5.5mmol)
Discharge the amount=(OD of glucose
Sample-OD
Empty)/OD
Mark* C
Mark
Alpha-glucosaccharase enzyme inhibition rate=(discharge the amount of glucose
NegativeThe amount of-release glucose
Sample)/discharge glucose amount
Negative
Embodiment two:
1. extracting method: J oil expression residue is beaten powder through drying, and sieves, and takes by weighing 100g, adds 95% ethanol 300ml, and 45 ℃ of shaking table lixiviates 5 hours are filtered, and concentrates, and 45 ℃ of oven dry must crude extract 5.01g, and this crude extract is used for determination of activity.
2. determination of activity:
1. solution allocation: with embodiment one.
2. activity determination method: with embodiment one.
3. experimental result:
(1) suppresses alpha-amylase activity
(2) activity of inhibition alpha-glucosidase
Annotate: OD
Mark=0.235
Embodiment three:
1. extracting method: J shell drying, beat powder, sieve, take by weighing 28g, add 95% ethanol 84ml, 45 ℃ of shaking table lixiviates 5 hours are filtered, concentrate, 45 ℃ of oven dry, extract 0.23g, this extract is used for determination of activity.
2. determination of activity:
1. solution allocation: with embodiment one.
2. activity determination method: with embodiment one.
3. experimental result:
(1) suppresses alpha-amylase activity
(2) activity of inhibition alpha-glucosidase
Annotate: OD
Mark=0.235
Embodiment four:
1. extracting method: the oven dry of J oil expression residue, beat powder, sieve, take by weighing 20g, add water 60ml, 45 ℃ of shaking table lixiviates 5 hours are filtered, and concentrate, and 45 ℃ of oven dry get extract 0.24g, and this extract is used for determination of activity.
2. determination of activity:
1. solution allocation: with embodiment one.
2. activity determination method: with embodiment one.
3. experimental result:
Suppress alpha-amylase activity
Embodiment five:
1. extracting method: J oil expression residue drying, beat powder, sieve, take by weighing 20g, add 50% ethanol 60ml, 50 ℃ of shaking table lixiviates 8 hours are filtered, and concentrate, and 45 ℃ of oven dry must extract 0.27g, and this extract is used for determination of activity.
2. determination of activity:
1. solution allocation: with embodiment one.
2. activity determination method: with embodiment one.
3. experimental result:
(1) suppresses alpha-amylase activity
(2) activity of inhibition alpha-glucosidase
Annotate: OD
Mark=0.239
List of references:
[1] B Shi Teer Mach. measure α-Dian Fenmei with extraction chemistry company law (modified model of Amano method). the assay method of enzyme. the .1992 of China Light Industry Press, 8:40
[2] Cheng Xiuli. α-Dian Fenmei. the determination of activity of inhibitor. Chinese new medicine .2004,3 (4): 71.
Claims (2)
1. alpha-glucosidase inhibitor is characterized in that the effective ingredient that contains in the shell of Semen Juglandis Juglans regia L. or Chinese walnut Juglans cathayensis Dode or Semen Caryae Cathayensis Juglans mandshurica Maxim or the core; Its extracting method is will be through shell or the shell core mixture or the Semen Juglandis oil expression residue of the Semen Juglandis of mechanical activation comminution, the alcohol or the alcohol-water mixture that add 3 times of amount w/vs, putting 45 ℃ of shaking table lixiviates 5 hours, filter, spends the night in 45 ℃ of baking ovens and obtains the extract alpha-glucosidase inhibitor in the concentrated back of filtrate decompression.
2. the described alpha-glucosidase inhibitor of claim 1 make to suppress alpha-amylase activity or is suppressing purposes in the product of alpha-glucosidase activity.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101347572B (en) * | 2008-09-02 | 2010-10-27 | 盛秀芬 | Medicament for treating diabetes and preparation method |
| CN101530464B (en) * | 2009-04-03 | 2013-03-20 | 司高 | Alcohol extract of walnut kernel and use thereof |
| CN103535708B (en) * | 2011-07-19 | 2015-07-01 | 江西江中制药(集团)有限责任公司 | Technology for reducing in-vivo decomposition and absorption speeds of starch |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1282539A (en) * | 1999-08-02 | 2001-02-07 | 张发义 | Walnut kernel juice and its preparing process |
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| CN1282539A (en) * | 1999-08-02 | 2001-02-07 | 张发义 | Walnut kernel juice and its preparing process |
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Owner name: GUANGYUAN TIANHUANGSHAN WALNUT FOOD CO., LTD. Free format text: FORMER OWNER: CHENDU BIOLOGY INST., CHINESE ACADEMY OF SCIENCES Effective date: 20140630 |
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