CN1173908C - Method for converting derivative of internal inositol of buckwheat seed into its monomer and its seed - Google Patents
Method for converting derivative of internal inositol of buckwheat seed into its monomer and its seed Download PDFInfo
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- CN1173908C CN1173908C CNB011104724A CN01110472A CN1173908C CN 1173908 C CN1173908 C CN 1173908C CN B011104724 A CNB011104724 A CN B011104724A CN 01110472 A CN01110472 A CN 01110472A CN 1173908 C CN1173908 C CN 1173908C
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- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 title claims abstract description 61
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 title claims abstract description 57
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Abstract
The present invention relates to a biological method which enables an inner D-chiro-Inositol derivative and a Myo-Inositol derivative of a mature buckwheat seed to convert into a D-chiro-Inositol monomer and an inositol monomer. The method comprises the following steps that mature buckwheat seeds are germinated for 7 to 22 hours under the condition of a temperature between 22 and 30 DEG C and 0 to 30mM of NaCl salt concentration, and a method for obtaining an extract object from the seeds and an extract object are provided. The present invention also relates to the purpose when the extract object is in the preparation of anti-diabetes medicine and health products.
Description
The present invention relates to a kind of interior inositol derivative of buckwheat mature seed that makes and be converted into the method for monomer whose and the seed that obtains thereof, and the method and the extract that from this seed, obtain extract; The invention still further relates to the purposes of the extract that obtains through the inventive method.
Regular Insulin is the hormone that is produced by the pancreas islet organ, acts on human body vitals and perienchyma's regulation and control sugar, fat and other metabolic process after entering blood circulation.Regular Insulin is incorporated into the human cell surface receptor protein, activates a series of signal pipeline, causes the cell surface channel to be opened, thereby will reach blood sugar reducing function in the glucose introducing cell in the blood.Any step in this signal pipeline is obstructed, or efficient reduces and all can cause Regular Insulin ineffective, is called insulin resistance.For compensation insulin action efficient reduces, human body produces more Regular Insulin, is called hyperinsulinemia.Continue hyperinsulinism concentration in the blood and cause metabolism disorder, blood sugar increasing, the blood triglyceride level raises, and high density lipoprotein cholesterol reduces, and plasma fibrinogen raises, and hyperuricemia etc.The disease that is caused by insulin resistance is referred to as the X syndromes, comprises diabetes, hypertension, obesity, coronary heart disease, polycystic ovary syndrome etc.
What U.S. scientist was nearly 20 years studies have shown that, (with D-chirality-inositol (D-Chiro-inositol) and inositol (myo-Inositol) is that representative has been played the part of the key player in the signal transduction process of insulin action to inositol (INOSITOL) quasi-molecule.Discover to reach in the urine in the normal human blood and contain certain density D-chirality-inositol, in II paradiabetes people, then almost detect less than, therefore, point out these patients possibly can't produce the D-chirality-inositol of q.s, cause the signal transmission of insulin action to be obstructed.A plurality of breadboard results all confirm, by promoting insulin function effectively, Regular Insulin, blood triglyceride level etc. in lowering blood glucose, the blood to experimental animal compensation D-chirality-inositol or inositol.Ongoing human clinical's check also shows that the D-chirality-inositol of purifying has significant curative effect to II paradiabetes, polycystic ovary syndrome.Therefore, if can extract the inositol quasi-molecule effectively from crude substance, the medicine for the treatment of II paradiabetes, polycystic ovary syndrome, hypertension, obesity and other cardiovascular disorder for Development and Production has great scientific value and economic worth.
The mankind make buckwheat seed various food for a long time, and have this food of report useful to diabetes.United States Patent (USP) the 6th, 162 contains abundant D-chirality-inositol derivative and a small amount of inositol (myo-inositol) derivative in No. 795 disclosure buckwheat seeds, and proposes to extract the method for these derivatives (being called " fagopyritols ").But find after deliberation, naturally occurring D-chirality-inositol derivative and inositol derivative can't resolve into effective constituent (that is the D-chirality-inositol of Individual existence and inositol molecule) in the buckwheat seed in human body, also can't absorb, because there is not (the Gitzelmann ﹠amp that exists of alpha-tilactase in people's the stomach and intestine; Auricchio, 1965).Therefore, if can find a kind of cost-effective method that naturally occurring D-chirality-inositol and inositol derivative in the buckwheat seed is converted into the effective monomer molecule, can directly apply to Development and Production treatment type ii diabetes, polycystic ovary syndrome, hypertension, the medicine of obesity and other cardiovascular disorder and relevant protective foods.
The object of the present invention is to provide a kind of buckwheat seed that makes D-chirality-inositol derivative and inositol derivative in the buckwheat mature seed significantly be converted into D-chirality-inositol and inositol monomer methods and obtain.
Another object of the present invention is to provide a kind of method that contains inositol monomer extract of from buckwheat seed, extracting.
A further object of the present invention is to provide a kind of purposes that contains the monomeric extract of inositol that extracts through extracting method of the present invention from buckwheat seed.
The object of the present invention is achieved like this:
The present invention relates to a kind of interior D-chirality-inositol derivative of buckwheat mature seed and inositol derivative of making and significantly be converted into D-chirality-inositol and the monomeric biological method of inositol, this method comprises: the buckwheat mature seed is at 22 ℃～30 ℃, sprouted 7～22 hours under 0～30mM NaCl salt concentration conditions, preferably at 22 ℃～25 ℃, sprouted under 10～20mMNaCl salt concentration conditions 13～19 hours, be preferably in 25 ℃, sprouted 13 hours under the 20mM NaCl salt concentration conditions.
D-chirality-inositol derivative and inositol derivative in the seed that process the invention described above method was handled reduce significantly, and D-chirality-inositol and inositol monomer increase considerably.Quick in germination process, the sharply increase of alpha-galactosidase activity, prompting D-chirality-inositol derivative and inositol derivative are to produce D-chirality-inositol and inositol monomer by hydrolysis.
Buckwheat can obtain up to the D-chirality-inositol of 118ug/ grain seed and the inositol of 10ug/ grain seed being grown under 17 ℃ of conditions ripe then the sprouting in 13 hours the seed after the pollination under 25 ℃, 20mM NaCl salt concentration conditions.
The invention still further relates to the buckwheat seed after process the present invention transforms D-chirality-inositol and the monomeric biological method processing of inositol, and extract the extracting method that is rich in D-chirality-inositol and inositol a kind of buckwheat seed after handling.The step that this method comprises is:
(1) with the buckwheat mature seed at 22 ℃～30 ℃, sprouted 7～22 hours under 0～30mM NaCl salt concentration conditions;
(2) buckwheat seed of the sprouting after the collection and treatment is pressed the seed wet weight and is added 10～20 times of volumes (the 1g seed that wet: 30～70% alcohol 10～20ml alcohol) are milled into homogenate, filtration;
(3) collect filtrate, lyophilize, acquisition contains the extract based on soluble-carbohydrate of D-chirality-inositol and inositol monomer and derivative thereof.
In addition, the invention still further relates to the purposes of buckwheat seed extract in the preparation antidiabetic medicine through extracting method of the present invention obtains.Experiment shows that extract of the present invention has tangible hypoglycemic effect to diabetes rat.D-chirality-inositol and inositol monomer content are directly proportional in this hypoglycemic effect and the extract.In addition, the extract that the experimentation on animals result shows the buckwheat seed that extracts through the inventive method has better function of polysaccharide than the D-chirality-inositol and the inositol monomer of the purifying of isodose, points out this extract may contain it and plants effective sugar reducing substance.Rutin and manganese (Mn) are two kinds of wherein possible active substances.
Therefore, extract of the present invention can be mixed and made into tablet, granule, capsule, suspension, drug form compositions such as oral liquid with various pharmaceutically acceptable carriers.Common people's consumption is: 600-6000mg extract/sky.
This extract can directly add the protective foods for preparing prevention and treatment diabetes in the food.General add-on is: with per day for adults picked-up 300-3000mg extract is the appropriate dosage scope.
Description of drawings:
Fig. 1 is the hypoglycemic agent quantitative response figure of extract of the present invention.
In order further to understand the present invention, below with reference to embodiment and accompanying drawing the present invention is made detailed, non-limiting description.
Embodiment one
D-chirality-inositol derivative and inositol derivative in the buckwheat mature seed significantly are converted into the biological method of D-chirality-inositol and inositol.
With 10 gram buckwheat seeds, under the condition of following table 1 and 2, in the salt solution of respective concentration, sprout corresponding time observations.
(1) seed germination time, salt concn and sprouting temperature are to the influence of alpha-galactosidase activity in the seed.
In order to determine different influences of sprouting temperature, different salt concn, different sprout time to alpha-galactosidase activity in the buckwheat seed, the inventor has carried out the multiplefactor cross-over experiment.Sophisticated general buck wheat seed was soaked several minutes in clear water, place that (20mM 30mM) on the wetted 4-5 layer thieving paper, places in the dish for 0mM, 10mM, is placed under the differing temps (22 ℃, 25 ℃, 30 ℃) and sprouts with different concns NaCl solution.After sprouting 7 hours, 10 hours, 13 hours, 16 hours, 19 hours, 22 hours, detect the alpha-galactosidase activity respectively.25 ℃ the results are shown in table 1, and the result of other temperature is similar.
Table 125 ℃ following sprout time and salt concn are to the influence of alpha--galactosidase activity in the seed
| 0hr | 7hr | 10hr | 13hr | 16hr | 19hr | 22hr | |
| 0mM NaCl | 2.12 | 34.68 | 47.28 | 53.87 | 60.65 | 46.19 | 30.40 |
| 10mM NaCl | 2.12 | 51.84 | 60.16 | 71.97 | 63.58 | 51.02 | 32.39 |
| 20mM NaCl | 2.12 | 58.28 | 74.66 | 71.37 | 65.29 | 47.89 | 27.99 |
| 30mM NaCl | 2.12 | 39.54 | 55.37 | 64.39 | 54.27 | 42.03 | 24.19 |
Unit: contained alpha--tilactase units in every milligram of extract (is 1 unit with the required enzyme amount of per minute hydrolysis 1umolpnp-alpha-galactoside).
As can be seen from Table 1, the activity of sprouting alpha-tilactase in the seed of beginning back sharply increases, and peaks in 10 to 16 hours.The NaCl of lower concentration quickens the activation of alpha--tilactase and increases peak value.It is the highest to sprout the enzyme relative reactivity that recorded in 10 hours under the 20mM NaCl condition, is 35 times before handling.
Plant seed excites a series of biochemical reactions in germination process, so that seed germination, required various nutritive substances and the energy of growth to be provided.The inventor finds that after deliberation buckwheat seed can activate the expression of alpha-tilactase in germination process, and this kind of enzyme acts on the alpha-semi-lactosi glycosidic bond just.Therefore, a kind of biological method and chemical process that activates the galactoside expression of enzymes handled, and can transform the derivative of D-chirality-inositol and inositol effectively, discharges free D-chirality-inositol and inositol molecule.
In the research, find buckwheat seed under suitable temperature, salt concn and sprout time, activate the alpha-galactosidase gene and efficiently express, thereby significantly D-chirality-inositol and inositol derivative are converted into D-chirality-inositol and inositol molecule.
(2) seed germination temperature, salt concn, sprout time are converted into the influence of monomer molecule to INOSITOL derivative in the buckwheat seed
D-chirality-inositol and inositol derivative and monomeric content are shown in following table 2 under the above-mentioned multiplefactor cross-over experiment.
3 kinds in table 2 is different sprouts D-chirality-inositol and inositol derivative and monomeric content in temperature, 3 kinds of different salt concn, the 6 kinds of different sprout time buckwheats
Unit: ug/ grain seed
| 22 ℃ of sproutings | |||||||
| 0hr | 7hr | 10hr | 13hr | 16hr | 19hr | 22hr | |
| 0mM NaCl | |||||||
| D-chirality-inositol derivative | 320.50 | 269.68 | 184.71 | 121.69 | 78.47 | 50.37 | 31.78 |
| D-chirality-inositol | 14.72 | 17.54 | 21.73 | 26.22 | 34.66 | 41.29 | 37.91 |
| Inositol derivative | 31.85 | 20.01 | 16.54 | 10.33 | 6.05 | 3.21 | 3.22 |
| Inositol | 1.85 | 2.58 | 3.16 | 2.99 | 5.47 | 6.79 | 5.33 |
| 10mM NaCl | |||||||
| D-chirality-inositol derivative | 320.50 | 245.29 | 152.52 | 93.16 | 50.32 | 28.92 | 16.02 |
| D-chirality-inositol | 14.72 | 26.88 | 42.21 | 63.89 | 75.14 | 68.99 | 61.82 |
| Inositol derivative | 31.85 | 19.04 | 14.39 | 8.97 | 3.92 | 2.16 | 2.35 |
| Inositol | 1.85 | 4.65 | 5.38 | 7.64 | 8.35 | 7.77 | 5.36 |
| 20mM NaCl | |||||||
| D-chirality-inositol derivative | 320.50 | 218.06 | 117.90 | 63.59 | 23.91 | 14.60 | 8.77 |
| D-chirality-inositol | 14.72 | 35.18 | 60.19 | 87.47 | 104.52 | 98.78 | 62.44 |
| Inositol derivative | 31.85 | 17.65 | 12.40 | 9.52 | 4.47 | 3.07 | 3.29 |
| Inositol | 1.85 | 4.98 | 5.46 | 8.25 | 9.12 | 9.00 | 7.29 |
| 30mM NaCl | |||||||
| D-chirality-inositol derivative | 320.50 | 238.47 | 169.54 | 86.75 | 43.43 | 34.88 | 21.47 |
| D-chirality-inositol | 14.72 | 30.15 | 44.19 | 56.29 | 73.47 | 94.55 | 85.39 |
| Inositol derivative | 31.85 | 19.02 | 18.45 | 9.48 | 6.42 | 3.11 | 2.75 |
| Inositol | 1.85 | 4.42 | 4.97 | 6.40 | 6.99 | 8.77 | 7.20 |
| 25 ℃ of sproutings | |||||||
| 0hr | 7hr | 10hr | 13hr | 16hr | 19hr | 22hr | |
| 0mM NaCl | |||||||
| D-chirality-inositol derivative | 320.50 | 243.49 | 157.36 | 99.48 | 56.27 | 35.49 | 18.90 |
| D-chirality-inositol | 14.72 | 23.53 | 29.55 | 41.86 | 50.62 | 39.88 | 27.21 |
| Inositol derivative | 31.85 | 18.18 | 13.33 | 8.44 | 4.27 | 3.01 | 3.18 |
| Inositol | 1.85 | 3.20 | 5.44 | 6.39 | 7.88 | 7.60 | 5.99 |
| 10mM NaCl | |||||||
| D-chirality-inositol derivative | 320.50 | 211.80 | 116.25 | 55.43 | 26.26 | 15.25 | 8.34 |
| D-chirality-inositol | 14.72 | 44.63 | 76.72 | 105.46 | 98.38 | 102.79 | 91.88 |
| Inositol derivative | 31.85 | 17.35 | 15.42 | 10.05 | 3.48 | 2.08 | 2.12 |
| Inositol | 1.85 | 6.33 | 7.24 | 11.74 | 13.86 | 12.78 | 10.53 |
| 20 mM NaCl | |||||||
| D-chirality-inositol derivative | 320.50 | 179.33 | 73.25 | 32.11 | 12.50 | 7.88 | 8.91 |
| D-chirality-inositol | 14.72 | 64.76 | 90.82 | 117.76 | 111.38 | 94.29 | 81.20 |
| Inositol derivative | 31.85 | 15.37 | 11.45 | 7.23 | 2.45 | 1.02 | 1.88 |
| Inositol | 1.85 | 5.39 | 8.40 | 9.88 | 9.13 | 7.59 | 8.32 |
| 30mM NaCl | |||||||
| D-chirality-inositol derivative | 320.50 | 221.48 | 164.53 | 59.00 | 37.14 | 15.28 | 15.76 |
| D-chirality-inositol | 14.72 | 29.82 | 47.17 | 67.12 | 83.15 | 75.51 | 68.02 |
| Inositol derivative | 31.85 | 21.00 | 18.47 | 7.69 | 4.54 | 3.25 | 3.46 |
| Inositol | 1.85 | 4.43 | 6.85 | 8.12 | 8.51 | 7.97 | 6.33 |
| 30 ℃ of sproutings | |||||||
| 0hr | 7hr | 10hr | 13hr | 16hr | 19hr | 22hr | |
| 0mM NaCl | |||||||
| D-chirality-inositol derivative | 320.50 | 102.78 | 67.83 | 21.79 | 27.57 | 16.99 | 15.60 |
| D-chirality-inositol | 14.72 | 26.39 | 27.57 | 36.66 | 32.76 | 38.39 | 28.35 |
| Inositol derivative | 31.85 | 13.26 | 12.83 | 3.86 | 5.77 | 1.68 | 3.27 |
| Inositol | 1.85 | 6.11 | 6.33 | 7.86 | 5.79 | 6.35 | 4.93 |
| 10mM NaCl | |||||||
| D-chirality-inositol derivative | 320.50 | 113.75 | 64.87 | 32.81 | 14.73 | 9.38 | 10.53 |
| D-chirality-inositol | 14.72 | 39.88 | 53.31 | 64.88 | 53.91 | 54.79 | 47.29 |
| Inositol derivative | 31.85 | 14.20 | 10.72 | 11.38 | 7.69 | 5.66 | 5.11 |
| Inositol | 1.85 | 4.71 | 6.55 | 7.34 | 8.20 | 8.11 | 6.93 |
| 20mM NaCl | |||||||
| D-chirality-inositol derivative | 320.50 | 124.39 | 70.20 | 41.54 | 24.55 | 22.43 | 12.60 |
| D-chirality-inositol | 14.72 | 59.38 | 73.54 | 88.66 | 69.49 | 72.54 | 61.20 |
| Inositol derivative | 31.85 | 18.47 | 13.49 | 12.00 | 8.68 | 5.65 | 5.11 |
| Inositol | 1.85 | 3.82 | 8.04 | 8.26 | 9.34 | 7.75 | 7.32 |
| 30mM NaCl | |||||||
| D-chirality-inositol derivative | 320.50 | 163.82 | 139.83 | 78.99 | 46.52 | 33.00 | 32.57 |
| D-chirality-inositol | 14.72 | 33.15 | 42.82 | 73.19 | 90.65 | 76.18 | 73.48 |
| Inositol derivative | 31.85 | 20.54 | 18.84 | 13.45 | 8.67 | 5.99 | 6.40 |
| Inositol | 1.85 | 5.96 | 7.72 | 10.39 | 10.49 | 9.73 | 7.64 |
From the result of table 2, can draw several conclusions:
1) be accompanied by the minimizing of D-chirality-inositol and inositol derivative in the germination process, D-chirality-inositol and inositol monomer content increase, and reach peak value at 13 to 19 hours.
2) sprout temperature and rise and quicken the disappearance and the monomeric accumulation of D-chirality-inositol and inositol derivative, but monomer content reduces on the contrary when surpassing 25 ℃.Monomer content can be by multiple factor affecting, comprises that derivative degraded, monomer are used in its and plant that biosynthetic pathway, monomer are degraded, monomer is newly synthetic etc.
3) Low Concentration NaCl increases D-chirality-inositol inositol monomer content greatly, and this effect is not to realize by influencing the derivative degraded.NaCl may be by reducing monomeric utilization or degraded or increasing the monomeric synthetic effect that monomer content increases greatly that reaches.
4) monomer cumulative top condition is 25 ℃, and 20mM NaCl sprouted 13 hours, contained 118ug D-chirality-inositol and 10ug inositol in every seed.
Embodiment two
The method of D-chirality-inositol and inositol in the extraction buckwheat seed
The buckwheat mature seed at 25 ℃, was sprouted 13 hours under the 20mM NaCl salt concentration conditions;
Under this experiment condition, the every batch of extraction is a material with the germinating seed of 10g buckwheat seed or 10g buckwheat seed, adds 50% alcohol (the 1g seed weight: 15ml) be milled into homogenate of 15 times of volumes by wet weight.
Experimental result finds, with the liquid extracting of milling of the interior alcohol of 30%-70% concentration range, mill respectively that its extraction efficiency does not have significant difference between the liquid, but we finds that the alcohol of higher concentration can increase the stability of D-chirality-inositol and inositol and derivative thereof.Therefore, preferably with 50% alcohol as the extracting solution of milling.Mill is that (Proctor-Silex, Inc. USA) at room temperature carry out, and 10-20 was overheated to prevent refiner second at interval in every homogenate 1-2 minute with a small-sized refiner.With three layers of filtered through gauze homogenate, filtrate is stored under-4 ℃ to-20 ℃.In order to increase extraction yield, remaining filter residue extracts 1-2 time with 50% alcohol again.Merging filtrate, (Labcono, USA) middle dry a few hours are until obtaining the exsiccant extract at lyophilizer.
Under this experiment condition, the germinating seed of 10g buckwheat seed or 10g buckwheat seed on average can obtain the dry extract of 0.5g.
The technology of scale operation extract is slightly modified on this basis, and acquires main equipment such as runner milling, homogenate stirrer, steam raising stove, industrial centrifugal machine, flame filter press etc.
The extract of the ripe buckwheat seed preparation of never sprouting contains about 28% the D-chirality-inositol and the monomer of inositol derivative and minute quantity (＜1%), and at 25 ℃, 20mM NaCl,, sprout the extract for preparing in 13 hours the seed and contain D-chirality-inositol monomer of about 10%, 1% inositol monomer, and the derivative of a small amount of (＜3%).
Embodiment three
Repeat with embodiment two identical method stepss, different is that buckwheat mature seed 10 is restrained at 22 ℃, sprouts 7 hours under the 10mM NaCl salt concentration conditions; The alcohol that extracts usefulness is 30% alcohol.Get dried extract 0.45 gram.
Embodiment four
Repeat with embodiment two identical method stepss, different is with the buckwheat mature seed at 30 ℃, sprouts 22 hours under the 30mMNaCl salt concentration conditions; The alcohol that extracts usefulness is 70% alcohol.Get dried extract 0.51 gram.
Embodiment five
Present embodiment is the purifying and the mensuration of D-chirality-inositol, inositol and derivative
In order to be further purified the inositol quasi-molecule, the extract that embodiment two obtains is milled into homogenate by 75% propionitrile (acetonitrile) of dry weight 0.5 times of volume of adding (0.5ml/g).The preparation separator column, this separator column is with having silica gel (9%) filling (Aldrich Chemical Company Inc. product) that 3-aminopropyl (aminopropyl) is modified, and homogenate obtains separating on this post.With 60-75% propionitrile wash-out, 0.4ml/Min.With 5ml is that the different components elutriant is collected by unit, and 1ml evaporation drying wherein is used for gas-chromatography and decomposes.Each component is analyzed on Hewlett-Packard 5890 Series II gas chromatographs, and separator column is DB-1701 (30-mlength, 0.25-mm i.d., 0.25um film thickness, J﹠amp; W Scientific product).Column temperature is 275 ℃ and 325 ℃.Carrying gas (Carrier gas) is helium (1.0ml/min), splitl: 100; Detecting gas is hydrogen (30ml/min), air (300ml/min) and nitrogen (30ml/min).Inositol quasi-molecule content is according to typical curve, and calculate with the retention time of standard specimen and peak area contrast.D-chirality-inositol, the standard specimen of inositol and derivative thereof are all available from SERVA company.
The mensuration of embodiment six alpha-galactosidase activities
Collect buckwheat seed or germinating seed 10 grams, containing 40mM NaCl, mill centrifugal 5 minutes of 13200g in the 30mM potassium phosphate buffer (pH7.5) of 3mM DTT.In supernatant liquor, add ammonium sulfate to 60% saturation ratio, spend the night centrifugal 15 minutes of 18000g.Precipitation is dissolved in 0.01M sodium phosphate buffer (pH7.0), dialysis, and centrifugal again 15 minutes of 18000g is to remove insolubles.Be splined on DEAE-Sephadex A-50 post (use in advance 0.01M pH7.0 sodium phosphate buffer saturated), use the 0.01M sodium phosphate buffer respectively, 0.05M damping fluid, 0.1M buffer solution elution.The alpha-galactoside mainly is present in the 0.05M sodium phosphate elutriant.The dialysis (pH4.0) in the 0.05M sodium acetate soln again of thick enzyme solution, be further purified with CM-Sephadex C-50 post (use in advance 0.05M sodium-acetate pH4.0 saturated), use 0.05M sodium-acetate buffer (pH4.0) and linear gradient NaCl eluant solution respectively, collect the part that is rich in the alpha-tilactase.Be further purified with Sephadex G-100 post at last, and concentrate, obtain the alpha-tilactase of purifying.Its purity of electrophoretic examinations on 10% SDS-polyacrylamide glue, its molecular weight is between 45-47KD.
The purity of enzymic activity adopts following method.(p-Nitrophenyl (PNP) alpha-galactoside) (Sigma Chemical Co.) is substrate with p-oil of mirbane (PNP) alpha galactosides, contain 0.05M sodium phosphate (pH6.0) .2mM PNP-galactoside (PNP-galactoside) and enzyme in the reaction solution, reaction volume is 0.1ml.37 ℃ of insulations are after 12 hours, and reaction (pH9.8) stops with 0.7ml 0.2M Sodium Tetraborate (sodium borate).Under 400nm, measure the decomposition amount of p-nitrophenols (p-nitrophenol).Unit of enzyme activity is the required enzyme amount of per minute hydrolysis 1umol substrate under these conditions.
The rat hypoglycemic experiment of embodiment seven extracts of the present invention
(1) rat chemical induction diabetes experimental model: choosing weight in average is the male Sprague-Dawley mouse of 250-280 gram, press the dosage IP injection STZ (Streptozocin of 45mg/kg, Sigma ChemicalsCo. is dissolved in the citrate buffer solution) solution.From the caudal vein blood drawing, use glucose oxidase method (Beckman glucose analyser, Fullerton, CA) mensuration blood-sugar content wherein after 24 hours.Choose blood sugar 350 to 400mg/dl rat as diabetes experimental animal (glucose level of normal rat is 116mg/dl).
(2) the above-mentioned diabetes rat random packet of choosing through chemical induction, (extract is dissolved in the 0.5ml physiological saline, uses the dropper feeding, once a day) in every group of extract processing that obtains with the process embodiment of the invention two of doses.Positive control (the D-chirality-inositol and the inositol of purifying) also is dissolved in feeding in the physiological saline.Before handling and handle after 1 day, 2 days, 4 days and 8 days, extract the 0.5ml blood sample with caudal vein and be used to measure glucose content.
Give diabetes rat according to the dosage in the following table 3, its result is:
The hypoglycemic effect of table 3 extract in STZ inductive diabetes rat
| The extract consumption | | ||||
| 0 day | 1 day | 2 days | 4 days | 8 days | |
| 0mg/ | 100 | 102 | 104 | 109 | 106 |
| 25mg/ | 100 | 98 | 97 | 94 | 89* |
| 50mg/ | 100 | 88* | 73** | 61** | 60** |
| 100mg/ | 100 | 72** | 68** | 59** | 58** |
*：P＜0.05
**：P＜0.01
Above-mentioned numerical value is the mean value of 10 experimental mouse, and the time blood-sugar content was made as 100 in 0 day, and point data is adjusted accordingly At All Other Times.Above-mentioned four groups of 0 day time blood sugar concentrations handling are respectively: 0mg/kg (contrast), and 382.3+/-15.1mg/dl; 25mg/kg, 376.4+/-18.7mg/dl; 50mg/kg, 377.9+/-20.2mg/dl; 100mg/kg, 372.0+/-16.3mg/dl.
As can be seen, when this extract using dosage was 50mg/kg, the comparison of experimental mouse blood-sugar content was according to having descended 39% after 4 days from table 3 and Fig. 1.And under 25mg/kg dosage, in 4 days with not obviously difference of contrast, just first meeting effect after 8 days.When using dosage further increased to 100mg/kg, the blood-sugar content comparison reached 41% according to descending after having descended 28%, 4 day after 1 day.
Main active ingredient and content in the table 4 extract sample of the present invention
| Sample number | Sprouting condition | The DCI derivative | The MI derivative | DCI | MI | Rutin | Manganese |
| 1 | Before the sprouting | 13992 | 1364 | 572 | 88 | 2436 | 27 |
| 2 | 0mM NaCl，25℃，16hr | 2552 | 264 | 2156 | 308 | 2631 | 25 |
| 3 | 10mM NaCl，25℃，13hr | 2332 | 484 | 4356 | 396 | 2698 | 31 |
| 4 | 20mM NaCl，25℃，13hr | 1188 | 308 | 5236 | 484 | 2579 | 29 |
| 5 | Over against shining (DCI of purifying and MI) | 0 | 0 | 5236 | 484 | 0 | 0 |
| 6 | Negative contrast (weighting material) | 0 | 0 | 0 | 0 | 0 | 0 |
DCI and MI in the table represent respectively: D-chirality-inositol and inositol.
Unit: ug/50mg extract
Table 5 contrast, the buckwheat extract of not sprouting and the hypoglycemic effect of extract in STZ inductive diabetes rat of different sprouting conditions
| Sample number | Sprouting condition | Before the processing | Handle after 2 days | Handle after 4 days |
| 1 | Before the sprouting | 100 | 99 | 96 |
| 2 | 0mM NaCl，25℃，16hr | 100 | 92 | 87* |
| 3 | 10mM NaCl，25℃，13hr | 100 | 82* | 64** |
| 4 | 20mM NaCl，25℃，13hr | 100 | 69** | 58** |
| 5 | Over against shining (DCI of purifying and MI packing) | 100 | 88* | 76** |
| 6 | Negative contrast (weighting material) | 100 | 101 | 102 |
*：P＜0.05**：P＜0.01
Illustrate: extract of the present invention is by preparing before sprouting or in the seed of sprouting under the different condition.Consumption is 50mg/kg.Above-mentioned numerical value is the mean value of 10 experimental mouse, and the time blood-sugar content was made as 100 in 0 day, and point data is adjusted accordingly At All Other Times.
This shows that extract of the present invention shows as:
1. the hypoglycemic effect is directly proportional with wherein contained D-chirality-inositol and inositol amount of monomer, that is monomer content is high more, and hypoglycemic effect is obvious more.
2. the hypoglycemic effect can not be explained by D-chirality-inositol and inositol monomer fully, also has other material synergy.Rutin and manganese (Mn) are two kinds of wherein possible active substances.
Rutin (Rutin) is a kind of flavonoid glucosides that plant metabolism produces.U.S. Patent number 6,096,364 show that rutin has the hypoglycemic effect.There is report to point out that it can opposing kapillary fragility (Griffith etc., 1944 relevant with hypertension or hemorrhagic disease in addition; Schilcher etc., 1990).The rutin of extracting from buckwheat also shows oxidation resistant effect (Watanbe, 1998).Buckwheat seed contains a certain amount of Mn equally.Discover D-chirality-inositol and the shared of Mn of Fonteles etc. (1999) are used separately more fast and the more substantial blood-sugar content that has reduced in the STZ inductive diabetes rat than D-chirality-inositol.Studies show that Mn may be a kind of Signal Regulation molecule in the insulin metabolism, discharge, regulate in the molecule activation playing an important role at blood sugar absorption, Regular Insulin.
Claims (8)
1. one kind makes interior D-chirality-inositol derivative of buckwheat mature seed and inositol derivative be converted into D-chirality-inositol and the monomeric biological method of inositol, this method comprises: the buckwheat mature seed was sprouted under 0～30mMNaCl salt concentration conditions 7～22 hours at 22 ℃～30 ℃.
2. the method for claim 1 is characterized in that the buckwheat mature seed at 22 ℃～25 ℃, sprouted under 10～20mMNaCl salt concentration conditions 13～19 hours.
3. the method for claim 1 is characterized in that the buckwheat mature seed at 25 ℃, sprouts 13 hours under the 20mMNaCl salt concentration conditions.
4. an Accessory Right requires to extract in the 1 described buckwheat seed extracting method that is rich in D-chirality-inositol and inositol, described buckwheat seed is to obtain like this: make the buckwheat mature seed at 22 ℃～30 ℃, sprouted under 0～30mMNaCl salt concentration conditions 7～22 hours; Described extracting method is characterised in that it step that comprises is:
(1) buckwheat seed of the sprouting after the collection and treatment is pressed 30～70% alcohol that the wet seed of 1g adds 10～20ml with the seed wet weight, is milled into homogenate, filters;
(2) collect filtrate, lyophilize, acquisition contains the extract based on soluble-carbohydrate of D-chirality-inositol and inositol monomer and derivative thereof.
5. extract that obtains through the described method of claim 4.
6. the purposes of extract as claimed in claim 5 in the preparation antidiabetic medicine.
7. the purposes of extract as claimed in claim 5 in preparing prevention or anti-diabetic food, healthcare products.
8. pharmaceutical composition is characterized in that it contains the extract as claimed in claim 5 and the pharmaceutically acceptable carrier of effective dose.
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| CN101904907B (en) * | 2009-06-03 | 2012-04-18 | 上海诺金科生物科技有限公司 | Buckwheat extract abundant in inositol and general flavone and preparation method thereof |
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