CN101959899A

CN101959899A - The new compound in sprout brown rice source and with it as the prevention of effective constituent or improve the preparation of DPN

Info

Publication number: CN101959899A
Application number: CN2009801078320A
Authority: CN
Inventors: 臼杵靖刚; 于宽仁; 森川桂子; 喜濑光男
Original assignee: Fancl Corp
Current assignee: Fancl Corp
Priority date: 2008-03-06
Filing date: 2009-03-06
Publication date: 2011-01-26
Also published as: KR20100120714A; WO2009110612A1; JPWO2009110612A1

Abstract

Problem of the present invention is to find the new function of sprout brown rice ingredient, and effective prevention is provided or improves DPN or the preparation of diabetic neuropathy or functional foodstuff, it is safe, can persistence absorb, can make in a large number, and add to easily in food etc.The steroline (Acylated steryl-β-glucoside (ASG) (acidylate-sterol-β-glucosides)) that the invention provides with the sprout brown rice source is the prevention of effective constituent or improves DPN or the preparation of diabetic neuropathy or functional foodstuff.

Description

The new compound in sprout brown rice source and with it as the prevention of effective constituent or improve the preparation of DPN

Technical field

The present invention relates to new compound contained in the sprout brown rice, relate to the utilization of this new compound.More specifically, relate to the prevention of this new compound or improve the purposes of diabetic neuropathy.

Background technology

According to statistics in 2000, diabetic subject's number in Asia was 8,450 ten thousand people, and global diabetic subject's number is 100,000,000 5,1,000,000 people.According to Japanese MHLW diabetes fact finding in 2002, the diabetic subject of Japan and the people that may suffer from diabetes reached 1,620 ten thousand people, just have 1 among in fact per 6.3 adults.In addition, it is predicted that the patient's number in Asia in 2015 will reach 100,000,000 3,2,300,000 people, the whole world will reach 200,000,000 2,1,000,000 people (with reference to non-patent literature 1).

Diabetes (Diabetes Mellitus:DM) cause unusually because of glycometabolic, because the glucose concn pathosis in the blood raises, thereby this disease can cause the complication of various features, perhaps has the danger that causes complication.Here, complication is meant disease or the symptom that produces based on this disease.Diabetes self there is no serious subjective symptoms, thereby all are all not receive treatment till causing complication and make that sb.'s illness took a turn for the worse under a lot of situation.

Complication as diabetes, cerebral infarction, cerebral apoplexy, myocardial infarction, diabetic nephropathy, artery of lower extremity occlusive sclerosis, diabetic retinopathy, tetter, infection disease, diabetic neuropathy, hyperlipidaemia, vascular dementia disease etc. are arranged, and wherein diabetic nephropathy, diabetic retinopathy, diabetic neuropathy are called as three big complication.

As the reason of diabetes, have plenty of because inherited genetic factors causes, but because the situation that living habit caused of diet etc. accounts for a greater part ofly, therefore the expectation for heath food and functional foodstuff improves gradually.In recent years, sprout brown rice gets most of the attention for the availability of the complication of diabetes, it is reported that it has the prevention that improves effect, cardiovascular system disease (inhibition thrombosis) of hyperlipidaemia, the preventive effect of diabetic nephropathy etc.

Here, sprout brown rice is the material of instigating brown rice germination to form, and the state of germination is less than about 1mm.It is characterized in that, in the process of germinateing, can produce known γ-An Jidingsuan (γ-aminobutyric acid (GABA)) with hypotensive effect or anti-stress effect.In addition, sprout brown rice contains abundant foodstuff fibre, VITAMIN, mineral substance and unknown lipid in the layer of rice bran or bud, and it is very general as new kernel grain and then as the research object that is used to constitute staple food in Japan.About sprout brown rice health being had various availabilities studies, it is reported, in experimentation on animals, it has the effect (with reference to non-patent literature 2) of reduction by glucose concn in the blood of streptozotocin (Streptozotocin (STZ)) inductive diabetes rat.Known in addition, compare with rice, the diet of sprout brown rice is for healthy person (with reference to non-patent literature 3) and hyperglycemic patients (with reference to non-patent literature 4), can reduce glucose concn and Regular Insulin in the blood after the meal, as the staple food that is used for prevent diabetes, its meaning has obtained very high evaluation.

Therefore all the time, sprout brown rice is utilized as heath food, and having to provide safe, the preparation that can life-time service or the possibility of food.In recent years, the prevention that improves effect, cardiovascular system disease (inhibition thrombosis) for hyperlipidaemia of sprout brown rice, the preventive effect of diabetic nephropathy etc. get most of the attention.

In addition, carried out studying (non-patent literature 5) for the effect of improving to diabetic neuropathy of sprout brown rice, but also unknown completely about its effective constituent.Therefore, the inventor etc. have the effective constituent of improving effect to diabetic neuropathy and identify for contained in the sprout brown rice, have found new compound thus, thereby have finished the present invention.

Patent documentation 1: TOHKEMY 2001-352916 communique

Patent documentation 2: TOHKEMY 2002-136263 communique

Patent documentation 3: TOHKEMY 2002-360192 communique

Non-patent literature 1:Zimmet P, AlbertiK G, Shaw J.Global andsocietal implications of the diabetes epidemic.Nature.2001 Dec13; 414 (6865): 782-7.Review

Non-patent literature 2:Hagiwara H, Seki T, Ariga T.The effect ofpre-germinated brown rice intake on blood glucose and PAI-1levels in streptozotocin-induced diabetic rats.Biosci BiotechnolBiochem.2004Feb; 68 (2): 444-7.

Non-patent literature 3:Ito Y, Mizukuchi A, Kise M, Aoto H, Yamamoto S, Yoshihara R, Yokoyama J.Postprandial bloodglucose and insulin responses to pre-germinated brown rice inhealthy subjects.J Med Invest.2005Aug; 52 (3-4): 159-64.

Non-patent literature 4:Ito Y, Shen M, Kise M, Hayamizu K, YoshinoG, Yoshihara R, Yokoyama J.Effect of pre-germinated brown riceon postprandial blood glucose and insulin level insubjects withhyperglycemia.Jpn J Food Chem 2005; 12 (2): 80-4.

Non-patent literature 5:Usuki S, Ito Y, Morikawa K, Kise M, Ariga T, Rivner M, Yu RK.Effect of pre-germinated brown rice intake ondiabetic neuropathy instreptozotocin-induced diabetic rats.NutrMetab (Lond) .2007Nov23; 4 (1): 25

Non-patent literature 6:Abbott CA, Mackness MI, Kumar S, BoultonAJ, Durrington PN.Serumparaoxonase activity, concentration, and phenotype distribution in diabetesmellitus and its relationshipto serum lipids and lipoproteins.Arterioscler Thromb Vasc Biol.1995Nov; 15 (11): 1812-8.

Non-patent literature 7:Silva IV, Caruso-Neves C, Azeredo IM, Carvalho TL, Lara LS, de Mello MC, LopesAG.Urea inhibition ofrenal (NA++K+) ATPase activity is reversed by cAMP.ArchBiochem Biophys.2002Oct 15; 406 (2): 183-9.

Non-patent literature 8:Chung BH, Wilkinson T, Geer JC, S egrest JP.Preparative and quantitativeisolation of plasma lipoproteins:rapid, single discontinuous density gradientultracentrifugation ina vertical rotor.J Lipid Res.1980Mar; 21 (3): 284-91.

Non-patent literature 9:Coste T, Pierlovisi M, Leonardi J, Dufayet D, Gerbi A, Lafont H, Vague P, Raccah D.Beneficial effects ofgamma linolenic acid supplementation on nerveconductionvelocity, Na+, K+ATPase activity, and membrane fatty acidcomposition in sciatic nerve of diabetic rats.J Nutr Biochem.1999Jul; 10 (7): 411-20.

Non-patent literature 10:Shaheen Faizi, Muhammad Ali, RubeenaSaleem, Irfanullah, Sarah Bibi, Complete1H and 13C NMRassignments of stigma-5-en-3-O-β-glucoside and itsacetylderivative.Magnetic Resonance in Chemistry, 39 (7): 399-405 (2001)

Summary of the invention

The problem that invention will solve

Therefore all the time, sprout brown rice is utilized as heath food, and having to provide safe, the preparation that can life-time service or the possibility of food.That is,, will isolating effective constituent do not absorb but, can obtain certain effect by with the whole directly picked-up of sprout brown rice in order to obtain the effect of improving of diabetic neuropathy of the present invention.In addition, this improve effect also may be by producing with multiple factor synergy.

On the other hand, if the evaluation of the effective constituent of having that effect succeed, then irrealizable in a large number or the throwing of high density and become possibility with the state institute of sprout brown rice itself, and can expect diabetic neuropathy pathogenetic separate bright.

Therefore, problem of the present invention is that contained having in the sprout brown rice improves the evaluation of the effective constituent of diabetic neuropathy function.

The scheme that is used to deal with problems

The inventor etc. further investigate in order to solve above-mentioned problem, found that contained steroline (Acylated steryl-β-glucoside (ASG, acidylate-sterol-β-glucosides) in the layer of the rice bran of sprout brown rice or sprout brown rice (rice bran).Among the application, term steroline, acidylate-sterol-β-glucosides, ASG use with same meaning.) can improve diabetic neuropathy, thus the present invention finished.

That is, the present invention includes following scheme.

(1) a kind of composition that prevents and improve DPN, it is an effective constituent with the steroline that contains 2-hydroxyl-octadecanoic acid.

(2), it is characterized in that the sterol skeleton of steroline is 5 α-courage steroid-8 (14)-alkene-3 β-alcohol according to (1) described prevention with improve the composition of DPN.

(3) a kind of preparation that prevents or improve DPN, it is effective constituent to contain 2-hydroxyl-octadecanoic acid as the steroline of fatty acid part.

(4) a kind of preparation that prevents or improve DPN, it is effective constituent to contain 5 α-courage steroid-8 (14)-alkene-3 β-alcohol as the steroline of sterol skeleton.

(5) a kind of preparation that prevents or improve DPN, it is to contain 2-hydroxyl-octadecanoic acid as fatty acid part and to contain 5 α-courage steroid-8 (14)-alkene-3 β-alcohol be effective constituent as the steroline of sterol skeleton.

(6) a kind of homocysteine thiolactone is separated enzyme (homocysteinethiolactonase) activated compositions, and it is effective constituent to contain 2-hydroxyl-octadecanoic acid as the steroline of fatty acid part.

(7) separate the enzyme activation composition according to (6) described homocysteine thiolactone, it is characterized in that the sterol skeleton of steroline is 5 α-courage steroid-8 (14)-alkene-3 β-alcohol.

(8) a kind of steroline, it is by general formula (A) expression, and satisfy following (i) or (ii) in arbitrary condition,

(i) X in the general formula (A) is selected from following group, and Y is 5 α-courage steroid-8 (14)-alkene-3 β-alcohol:

Palmitinic acid (16:0),

Stearic acid (18:0),

2-hydroxyl-octadecanoic acid (18:0 (2h)),

Oleic acid (18:1),

Linolic acid (18:2) or

Lignoceric acid (24:0);

(ii) the X in the general formula (A) is 2-hydroxyl-octadecanoic acid (18:0 (2h)), and Y is selected from following group:

Campesterol,

Stigmasterol,

5 α-courage steroid-8 (14)-alkene-3 β-alcohol or

β-Gu Zaichun.

The effect of invention

The layer of the rice bran with sprout brown rice or sprout brown rice of the present invention (rice bran) is that the acidylate-sterol-β-glucosides (ASG) of raw material has diabetic neuropathy and improves effect.

Therefore, can be according to the present invention and utilize as the preventive of diabetic neuropathy or activator.

And it is safe, can persistence picked-up, and can purifying or synthetic and a large amount of the manufacturing, can add in the food etc., therefore for the human or animal improve health or possibility that preventing disease etc. can be made contributions bigger.

Description of drawings

Fig. 1-1 is the structure of acidylate-sterol-β-glucosides (ASG).

Fig. 1-2 is the structure of campesterol and Stigmasterol.

Fig. 1-3 is the structure of 5 α-courage steroid-8 (14)-alkene-3 β-pure and mild β-Gu Zaichun.

Fig. 2 is in the presence of homocysteine thiolactone modified LDL (HT-modifiedLDL), and the ASG in sprout brown rice source is for the active influence of the membrane derived sodium/potassium ATP enzyme of neural axon (Na/K ATPase).

Fig. 3 further uses tlc (thinlayer chromatography, TLC) each fraction of classification sprout brown rice lipid composition, and carry out the painted result of orcinol (Orcinol).

Fig. 4 is lipid H4 (acidylate-sterol-β-glucosides) result relatively with A2 and helicobacter pylori (Helicobacterpylori) source.

The spectrogram (1) that Fig. 5-1 analyzes with nuclear magnetic resonance spectroscopy method (NMR) for the ASG (A2 component) to the sprout brown rice source.

The spectrogram (2) that Fig. 5-2 analyzes with nuclear magnetic resonance spectroscopy method (NMR) for the ASG (A2 component) to the sprout brown rice source.

The result that Fig. 6 analyzes with nuclear magnetic resonance spectroscopy method (NMR) for the ASG (A2 component) to the sprout brown rice source.

The result of Fig. 7 for utilizing gas chromatography-mass spectrometry (GCMS) to analyze.

Fig. 8 is that ASG is to the active influence of HTase.

Fig. 9 is the HPTLC stretch-out view-1 of each fraction.

Figure 10 is the HPTLC stretch-out view-2 of each fraction.

Description of reference numerals

Glycolipid composition contained among the extraction separation component Fr.2 of the rice bran of A2 sprout brown rice, that arrive with TLC-orcinol color developing detection.

Glycolipid composition contained among the extraction separation component Fr.2 of A3 rice bran, that arrive with TLC-orcinol color developing detection.

Glycolipid composition contained among the extraction separation component Fr.2 of the rice bran of A4 sprout brown rice, that arrive with TLC-orcinol color developing detection.

Glycolipid composition contained among the extraction separation component Fr.2 of A5 rice bran, that arrive with TLC-orcinol color developing detection.

Glycolipid composition contained among the extraction separation component Fr.3 of B1 rice bran, that arrive with TLC-orcinol color developing detection.

Glycolipid composition contained among the extraction separation component Fr.3 of B2 rice bran, that arrive with TLC-orcinol color developing detection.

Glycolipid composition contained among the extraction separation component Fr.3 of B3 rice bran, that arrive with TLC-orcinol color developing detection.

Glycolipid composition contained among the extraction separation component Fr.3 of B4 rice bran, that arrive with TLC-orcinol color developing detection.

Glycolipid composition contained among the extraction separation component Fr.3 of B5 rice bran, that arrive with TLC-orcinol color developing detection.

A2 (alkalescence) carries out the material that alkaline purification forms to the A2 of purifying.

Fr forms the lipid crude extract of the rice bran of sprout brown rice or brown rice with the further classification of silica gel column chromatography material.

GSL St Standard glycosphingolipid (standard sphingoglycolipid)

Cer Ceramide (ceramide)

GlcCer Glucosylceramide (glucosylceramide)

GalCer Galactosylceramide (galactosyl ceramide)

LacCer Lactosylceramide (lactose ceramide)

Gb3 Globotriaosylceramide (ceramide three hexosides)

Gb4 Golbotetraosylceramide (ceramide tetrahexose glycosides)

C/M/W chloroform/methanol/water

C: M: W chloroform: methyl alcohol: water

Glc Glucose (glucose)

Lac Lactose (lactose)

Suc Sucrose (sucrose)

BBG Bovine brain ganglioside mixture (ox cranial nerve joint glycosides lipoprotein mixture)

ASG Acylated steryl-β-glucoside (acidylate-sterol-β-glucosides), steroline

Acidylate-sterol-β-the glucosides in H4 (Helicobacter pylori genus) helicobacter pylori source

Sterol-β-the glucosides in H6 (Helicobacter pylori genus) helicobacter pylori source

The commercial criterion ASG in ASG-matreya soybean source

16:0 palmitinic acid (palmitic acid)

18:0 stearic acid (stearic acid)

18:0 (2h) 2-hydroxyl-octadecanoic acid (2-hydroxy-octadecanoicacid)

18:1 oleic acid (oleic acid)

18:2 linolic acid (linoleic acid)

22:0 docosoic (behenic acid)

24:0 Lignoceric acid (lignoceric acid)

The lytic enzyme of HTase homocysteine thiolactone (homocysteine thiolactone)

HT homocysteine thiolactone (homocysteine thiolactone)

The LDL low-density lipoprotein

HT-modifiedLDL homocysteine thiolactone modified LDL

HT-LDL homocysteine thiolactone modified LDL

Na/K ATPase sodium/potassium ATP enzyme

SG removes the material that lipid acid forms from the ASG in sprout brown rice source

S-ASG is from the ASG of soybean purifying

Embodiment

[definition]

Diabetic neuropathy is peripheral nerve pathology and autonomic neuropathy for one of complication of producing based on diabetes.Clinically, this pathology can cause the numb or pain of trick etc. in the early stage, can cause sensory paralyses, motorius imbalance in chronic phase.On the pathology, can list the sex change and the damage of the myelin and the neural axon of nervous tissue.These DPNs can be as the membrane derived sodium/potassium ATP enzyme of reduction, the neural axon of peripheral nerve conduction of velocity (Na, K-ATPase) active reduction and observe, quantitatively.

The effect of improving of diabetic neuropathy is meant, the effect that effective constituent had of the ASG in the ASG in sprout brown rice source or sprout brown rice source is meant in human or animal's individuality, tissue or cell: make by diabetic neuropathy to cause sodium/potassium ATP enzyme activity of reducing or active rising of HTase and near the effect of normal value or prevent the effect of the reduction of motor nerve conduction velocity.

DPN described in the present invention is meant, shows the morbid state of identical with diabetic neuropathy or similar physiology, cytohistology or biochemical observations, is not limited to owing to diabetes cause.Promptly, this DPN is meant, in maincenter and peripheral nerve, can observe as the contained active reduction of HTase in the active reduction of sodium/potassium ATP enzyme in neu source in the reduction of motor nerve conduction velocity on the especially damage of neural axon, myelin demyelination, the neurophysiology on the histopathology, the biological chemistry or high-density lipoprotein (HDL) (HDL) component in the serum, quantitative all DPNs.

HTase is meant the lytic enzyme of the homocysteine thiolactone (homocysteine thiolactone) as arteriosclerotic risk factor, can range paraoxonase (paraoxonase) family.According to report, therefore active reduce (with reference to the non-patent literature 6) of paraoxonase think that the active reduction of HTase not only can become arteriosclerotic risk factor in following the patient of DPN, but also with DPN to carry out degree relevant.

[experiment material and method]

＜sprout brown rice 〉

Modulate according to known method (with reference to patent documentation 1-3).

The extraction of＜classification-lipid composition 〉

About lipid composition, the chloroform-methanol (1: 1 and 2: 1, volume ratio) that uses 30ml and 20ml carries out 2 times and extracts from the rice bran of the sprout brown rice of 5g or brown rice, as lipid composition.

＜classification-silica gel chromatography 〉

The compositional analysis of glycolipid and the purifying of each composition

Add the lipid composition of sprout brown rice and brown rice to silica gel chromatographic column (1x15cm), feed solvent chloroform: hexane (1: 1) 40ml, chloroform: hexane (9: 1) 70ml, with resulting component as Fr.1.Then, feed solvent chloroform: methyl alcohol (9: 1) 80ml, with resulting component as Fr.2.Feed solvent chloroform in addition: methyl alcohol: water (7: 3: 0.1) 80ml, solvent chloroform: methyl alcohol: water (3: 6: 0.8) 120ml, with resulting component respectively as Fr.3 and Fr.4.Utilize rotatory evaporator to concentrate and do solid each component.Each component 1 μ g point is added on silica gel thin-layer (TLC) plate, utilizes solvent chloroform: methyl alcohol: water (70: 30: 0.1) launches.After the expansion, the orcinol reagent of on the TLC plate, spraying.The TLC plate is positioned on the hot plate, heated 5 minutes down at 110 ℃.Utilize densometer (ensitometer) to detected each glycolipid composition carries out quantitatively by the orcinol colour developing, obtain composition.

The purifying of A2 component

Further with each contained glycolipid composition (A2 among the extraction separation component Fr.2 of the rice bran of sprout brown rice and brown rice and the Fr.3, A4, B1, B2, B3, B4 B5) adds silica gel chromatographic column (1x15cm) to, use solvent (chloroform: methyl alcohol: water (70: 30: 0.1), flow velocity 0.2ml/min) by the separation and purification of fraction collector.

＜alkali decomposes 〉

The 0.5M NaOH/ methyl alcohol that in A2 (0.1mg), adds 0.5ml: water (4: 1) and dissolving, at room temperature stirred 20 hours.After the stirring, add the chloroform of 2.5ml: methyl alcohol (2: 1), carry out Folch and distribute.

Collect lower floor, and concentrate.The lower floor that is distributed is added on the chloroform TLC plate with a small amount of point, utilizes solvent chloroform: methyl alcohol: water (70: 30: 0.1) launches.After the expansion, the orcinol reagent of on the TLC plate, spraying.The TLC plate is positioned on the hot plate, heated 5 minutes down at 110 ℃.Carry out the orcinol colour developing, relatively the TLC of A2 before and after the alkaline purification and A4 launches degree of excursion.

＜classification-tlc 〉

(Silica gel 60, the position apart from below 1.5cm Merck) utilizes the microsyringe coated sample at the commercially available vertical 10cmTLC plate that has coated silica gel.Utilize and launch solvent chloroform: methyl alcohol: water (70: 30: 0.1) launches.Carry out air-dry after the expansion.

＜orcinol (orcinol) dyeing 〉

By spray pattern, the 0.2% orcinol/2N sulfuric acid of on the TLC plate, spraying equably.The TLC plate of being sprayed is heated being heated on 110 ℃ the hot plate.Pass through present special red bean look colour developing isolating sacchariferous each composition on the TLC plate is identified.

＜gas chromatography-mass spectrometry (GCMS) 〉

A2 (50 μ g) is dissolved in 1N hydrochloric acid/methyl alcohol of 1.0ml, 86 ℃ of following hydrolysis 16 to 24 hours.After the reaction, be cooled to room temperature, add the hexane of 1.0ml then, stirring, centrifugal is separated into 2 layers.The hexane that adds 1.0ml in lower floor once more carries out 2 layers of separation.In addition, carry out 2 layers of separation again for lower floor.Only collect the upper strata of containing a large amount of hexanes, dry in nitrogen, as fatty acid component.The GCMS that hexane layer is used for lipid acid analyzes.On the other hand, in remaining hydrochloric acid methanol layer, add the ether of equivalent, carry out 2 layers of distribution.The ether layer is dry in nitrogen, and the GCMS that is used for sterol as the sterol component analyzes.GCMS analyzes by being installed in Hewlett-Packard GC-MS (5972MS﹠amp; Capillary column DB-1 (50mX0.25mm) 5890GC) and implementing.The analysis of lipid acid is according to 10 ℃/minute gradient increased temperature program of 70 ℃ of initial stage temperature (5 minutes), 10 ℃/minute (18 minutes), finishing temperature 250 ℃ (15 minutes) and implement.The analysis of sterol is according to the gradient increased temperature program of 70 ℃ of initial stage temperature (1 minute), 10 ℃/minute (18 minutes), finishing temperature 250 ℃ (21 minutes) and implement.

＜nuclear magnetic resonance spectroscopy method (NMR) 〉

A2 (1.0mg) is dissolved in the CD of 0.5ml ₂Cl ₂In, after the acquisition preliminary NMR data, with CD ₂Cl ₂Drying is removed under nitrogen.Be dissolved in the CDCl of 0.5ml once more ₃In, carry out formal mensuration.Data get for measuring with 25 ℃ of following 800MHz and 900MHz.About the collection of data, except ¹Beyond the spectrogram of HNMR, also collect 2 dimension spectrogram [COSY, HSQC (carbon-proton one-bond correlated data, carbon-proton singly-bound related data), HMBC (carbon-proton multiple bondcorrelated data, carbon-proton Multiple Bonds related data)].

The separation of＜lipoprotein 〉

Lipoprotein is prepared according to the step (with reference to non-patent literature 8) of former report.Briefly, collect the fresh serum of gathering from normal rat, use solid K Br and make density be 1.3g/ml.Add the common physiological saline of one deck (3.5ml, 1.006g/ml) in above-mentioned synthetic serum (1.5ml, 1.3g/ml) top, utilize the super centrifugal discontinuous density gradient of in centrifuge tube, making.Utilize the TV865 rotor, by 369548g, 4 ℃, 45 minutes the super centrifugal lipoprotein that separates.Reclaim 3 kinds of main lipoprotein components (VLDL, LDL, HDL), under 4 ℃, carry out the dialysis in an evening for PBS.In this specification sheets, LDL is meant the component that obtains by this method.

＜HTase determination of activity 〉

HTase activity among the rat blood serum HDL is used commercially available mensuration test kit and is measured (AlfresaAutoHTLase; Alfresa Pharma Corp., Osaka, Japan).This test kit utilizes γ-sulfo-butyrolactone (thiobutyrolactone) as substrate.HTase hydrolysis of lactone ring generates free sulphur alcohol radical (thiol).Thiol group is by with 5,5 '-two (the 2-nitrobenzoic acids) (5 of dithio, 5 '-dithiobis (2-nitrobenzoic acid)) reaction, 5-sulfo--2-nitrobenzoic acid (5-thio-2-nitrobenzoic acid) that generation can be measured under the absorption of 450nm.Measure the absorption of 450nm, calculate enzymic activity.

＜be used for the modulation of the active thick purifying sciatic nerve film of sodium/potassium ATP enzyme 〉

Thick purification membrane is according to step (with reference to the non-patent literature 7) modulation of report in the past.Briefly, the sciatic nerve with rat is cooling off the middle fragmentation of isotonic solution (250mM sucrose, 10mM HEPES-Tris damping fluid (pH7.6), 2mM EDTA, 1mM PMSF) and is homogenizing.This liquid that homogenizes with 10 minutes, 4 ℃, the condition centrifugation of 3000rpm, is reclaimed supernatant liquor, and then with 45 minutes, 45000rpm centrifugation.After giving up supernatant liquor, precipitation is suspended in the 250mM sucrose solution (being dissolved in the 10mM HEPES-Tris damping fluid (pH7.6)) of 100 μ l.

The adjustment of＜homocysteine thiolactone modified LDL 〉

Carry out under the experiment condition of the former report of the homocysteine thiolactoneization of invisible spectro LDL by (non-patent literature 5).Briefly, with an amount of LDL solution (LDL protein, 100 μ g) be suspended among the 10mM PBS (pH8.2), integral body 37 ℃ of down soft mixing, is hatched the full-cream matter component (TLp) or the ASG (0.01 to 10.0 μ g) of homocysteine thiolactone (100 μ mol/L) and represented amount 2 hours simultaneously.After hatching,, make this mixed solution by using in 10mM PBS (pH8.2) the equilibrated Bio-gelP-2 post in order to remove unreacted homocysteine thiolactone.

＜Na/K atpase activity is measured 〉

Sodium/potassium ATP enzyme activity is measured according to former report (with reference to non-patent literature 7).Briefly, be used to measure consisting of of sodium/potassium dependency active sodium/potassium ATP enzyme determination of activity solution (0.2ml): 10mM MgCl ₂, the thick purification membrane protein of 20mM HEPES-Tris (pH7.0), 120mM NaCl, 30mM KCl, 0.5mg/ml and 25mM[γ- ³²P] and ATP (10,000cpm).To measure solution after 37 degree are hatched 15 minutes, add the gac of 0.1mg/ml, with 15,000rpm centrifugation 15 minutes.Reclaim supernatant liquor, measure inorganic by scintillometer ³²The P radioactivity.

[result and investigation]

The classification of the effective constituent in＜sprout brown rice source 〉

Contained lipid composition in the layer of the rice bran of sprout brown rice is classified as

fraction

1,2,3 and 4; and then use the tlc classification, hence one can see that, and the effective constituent of improving function with diabetic neuropathy shown in the non-patent literature 5 is acidylate-sterol-β-glucosides (ASG).

It is emphasized that effective constituent only is included among the one-component A2 (below, be designated as A-2 sometimes, not special the differentiation).That is, have at the whole food that confirms sprout brown rice and so under the situation of specific pharmacy effect, the performance effect also is understandable even the multiple factor acts synergistically.In addition, though one-component A2 comprises multiple ASG, in the classification of using tlc, they can't be separated one by one.Its reason is considered to, and the structure of each ASG and chemical property are similar mutually.

When mentioning " ASG " in this specification sheets, expression is as the ASG of generic name.Mention " the sprout brown rice source () ASG " time, expression is equivalent to the set of the multiple ASG of A2 component.But, under the clear and definite situation of context, " ASG " sometimes also expression be equivalent to the set of the multiple ASG of A2 component.

The present invention relates to the ASG in this sprout brown rice source, in addition, relate to the improvement of the ASG that has used this sprout brown rice source or the preparation of prevent diabetes DPN.

The evaluation of the effective constituent in＜sprout brown rice source 〉

For the A2 component, use NMR to analyze, the result is identical with expection, confirms that the A2 component is made of ASG, and the sterol that constitutes the ASG of A2 as can be known is campesterol, Stigmasterol, 5 α-courage steroid-8 (14)-alkene-3 β-pure and mild β-Gu Zaichun.In addition, the lipid acid that constitutes the ASG of A2 as can be known is that (16:0 below represents with dummy suffix notation palmitinic acid sometimes.Other lipid acid are also identical.), stearic acid (18:0), 2-hydroxyl-octadecanoic acid (18:0 (2h)), oleic acid (18:1), linolic acid (18:2) and Lignoceric acid (24:0).The structure of the sterol of the ASG of the general structure of ASG and formation A2 is shown in Fig. 1.

As shown in Figure 1, the ASG of A2 component is combined with any of above-mentioned 4 kinds of sterols on the skeleton of sugar (Sugar), and is combined with above-mentioned 6 kinds of lipid acid any.24 kinds of combinations are arranged in theory, therefore, can comprise 24 kinds of ASG, but the ASG of the A2 component of reality may be defined as less kind.In addition, sugar has α to combine and the β combination with the combination of sterol as can be known, but under the situation of the ASG that sprout brown rice is originated, only is that β is in conjunction with (with reference to following content).

The ASG in＜sprout brown rice source is for the active effect of HTase 〉

HTase is meant the lytic enzyme of the homocysteine thiolactone (homocysteine thiolactone) as arteriosclerotic risk factor, can range paraoxonase (paraoxonase) family.According to report, therefore active reduce (with reference to the non-patent literature 5) of paraoxonase think that the active reduction of HTase not only can become arteriosclerotic risk factor in following the patient of DPN, but also with DPN to carry out degree relevant.The ASG of the present application can improve the HTase activity among the rat blood serum LDL, hints that thus it has as the function of improving or preventing the preparation of DPN.

The ASG in＜sprout brown rice source is for the active specificity effect of HTase 〉

Under the situation of the ASG (ASG) that adds the sprout brown rice source, the activity of having observed the HTase of dose-dependently rises, relative therewith, under the situation of the ASG (S-ASG) that adds SG (from the ASG in sprout brown rice source, remove lipid acid and material) and soybean source, do not observe the activity rising (table 6) of HTase.It shows that the active effect that dose-dependently ground improves HTase is the specific effect that ASG had in sprout brown rice source.

The DPN of the ASG in＜sprout brown rice source improve effect

Known DPN can be as the reduction or the membrane derived sodium/potassium ATP enzyme (Na of neural axon of peripheral nerve conduction of velocity, K-ATPase) active reduction and observe, quantitatively (non-patent literature 5 and 9), in this research, from being used to handle the viewpoints such as convenience of a large amount of samples, with the active index of sodium/potassium ATP enzyme as DPN.That is, the active junior of sodium/potassium ATP enzyme represents that the degree of DPN is more serious.

The reason of the active reduction of sodium/potassium ATP enzyme that neural axon is membrane derived is considered to: LDL is modified by homocysteine thiolactone, and the function of LDL is suppressed.In this research,, in the presence of homocysteine thiolactone modified LDL (HT-modified LDL), measure sodium/potassium ATP enzyme activity from the synthetic thick purifying sciatic nerve film of healthy rat in order to reproduce DPN.

In the presence of homocysteine thiolactone modified LDL (HT-modified LDL), the ASG in sprout brown rice source has improved membrane derived sodium/potassium ATP enzyme (Na/K ATPase) activity of neural axon, and this raising presents dose-dependently (Fig. 2).Its ASG that shows the sprout brown rice source has the function of improving DPN.

The DPN that the ASG in＜sprout brown rice source is special improve effect

Under the situation of the ASG (ASG) that adds the sprout brown rice source, the activity of having observed the sodium/potassium ATP enzyme of dose-dependently rises, relative therewith, under the situation of the ASG (S-ASG) that adds SG and soybean source, do not observe the activity rising (table 7) of sodium/potassium ATP enzyme.It shows that the active effect that dose-dependently ground improves sodium/potassium ATP enzyme is the specific effect that ASG had in sprout brown rice source.

The effective constituent (with the comparison of S-ASG) of the ASG in＜sprout brown rice source 〉

As previously mentioned, the ASG in sprout brown rice source is not single ASG, but the mixture of multiple ASG (table 4 and table 5).Comprise among the ASG in sprout brown rice source have campesterol, Stigmasterol, β-Gu Zaichun or 5 α-courage steroid-8 (14)-alkene-3 β-alcohol is as sterol ASG partly.On the other hand, only comprise among the ASG in soybean source and have campesterol, Stigmasterol or β-Gu Zaichun ASG as the sterol part.This has hinted that consumingly the real effective constituent of the ASG in sprout brown rice source may be to have the ASG of 5 α-courage steroid-8 (14)-alkene-3 β-alcohol as the sterol part.In theory, it also is possible having the multiple factor synergy of more weak effect and producing more potent fruit, but is difficult to the imagination in the reality.

In addition, comprise among the ASG in sprout brown rice source have 16:0,18:0,18:0 (2h), 18:1,18:2 or 24:0 be as the ASG of fatty acid part.On the other hand, only comprise among the ASG in soybean source have 16:0,18:0,18:1,18:2,22:0 (docosoic) or 24:0 as the ASG of fatty acid part.This has hinted that consumingly the real effective constituent of the ASG in sprout brown rice source may be to have the ASG of 18:0 (2h) as fatty acid part.Equally, in theory, it also is possible having the multiple factor synergy of more weak effect and producing more potent fruit, but is difficult to the imagination in the reality.

Therefore, contained real effective constituent very likely is to have 5 α-courage steroid-8 (14)-alkene-3 β-alcohol as sterol partly or have the ASG of 18:0 (2h) as fatty acid part among the ASG in sprout brown rice source.In theory, comprise the ASG of 4 * 6=24 kind among the ASG in sprout brown rice source, but according to this research, the candidate of contained real effective constituent is defined as the ASG of 6+4-1=9 kind among the ASG in sprout brown rice source.Promptly, have 5 α-courage steroid-8 (14)-alkene-3 β-alcohol as sterol part and have 16:0,18:0,18:0 (2h), 18:1,18:2 or 24:0 6 kinds of ASG, or have campesterol, Stigmasterol, β-Gu Zaichun or 5 α-courage steroid-8 (14)-alkene-3 β-alcohol as the sterol part and have the 4 kind ASGs (a kind be repeated calculating) of 18:0 (2h) as fatty acid part as fatty acid part.

The present application relates to the real effective constituent of the ASG in this sprout brown rice source, relates to the ASG in the sprout brown rice source that comprises real effective constituent.

The effective constituent (with the comparison of SG) of the ASG in＜sprout brown rice source 〉

In this research, fatty acid part is removed in the ASG hydrolysis in sprout brown rice source, modulated SG thus, compare with this SG.The SG that has lost fatty acid part does not as can be known have the improvement effect (table 6 and table 7) of DPN, and the existence of fatty acid part is necessary.

Application on the＜industry 〉

The ASG (single or multiple) in sprout brown rice of the present invention source can directly use the component that obtains by aforesaid method, but can be dissolved or dispersed in the suitable liquid usually, perhaps mix or adsorb thereon, according to circumstances further add emulsifying agent, dispersion agent, suspension agent, spreading agent (spreadingagent), permeate agent, wetting agent, stablizer etc. therein and use as preparations such as emulsion, finish, wettable powder, powder, tablet, capsule, liquors with suitable powder carrier.

The usage quantity of the component when using as preparation is according to the form of preparation and different, but so long as effectively throw with amount and get final product, no problem in the security, so the upper limit does not have special stipulation.

In addition, functional foodstuff described in the present invention is meant, with the ASG in sprout brown rice source as effective constituent and have prevention or improve the food of the function of DPN, perhaps can expect the food of the performance of these functions, comprise in heath food, specific food for health care, the trophic function food any by the picked-up of food.

As food, can list dessert such as chewing gum, candy, sheet dessert, elasticity jelly (Gummy Jellies), chocolate, biscuit or snack, cold drinks such as ice-creams, fruit syrup ice-creams or ice system dessert, beverage, pudding, jam, milk-product, food flavouring etc., these food can dailyly absorb.As the addition of the lipid composition of the present invention in these food, according to the form of food and different, but since no problem in the security, thereby its concentration there is no need to be provided with the upper limit.

[reference example 1]

[classification of the effective constituent in sprout brown rice source]

Utilize the method described in experiment material and the method that the sprout brown rice lipid composition is classified as

fraction

1,2,3 and 4 (Fr1, Fr2, Fr3, Fr4).Carry out the HTase determination of activity for each fraction, result Fr2 as can be known has activity (table 1), and the effective constituent with function of improving diabetic neuropathy is included among this Fr2.That is, have only the Fr.2 that adds 1.0 μ g to compare with the situation of not adding any additive (None), observe the active rising of HTase as the situation of additive (Additive).

Table 1

^aNumerical value is the average (n=4) in each experiment

^bNumerical value is represented with the active % of contrast HTase that does not contain additive for contain the active average of HTase (n=2) of 1.0 μ g additives (each fraction of Fr.1～4 of total TLp or TLp) in each experiment.

(thin layer chromatography TLC) carries out classification to each fraction, the results are shown among Fig. 3 A orcinol (Orcinol) is painted further to use tlc.Can observe, according to the order of Fr1, Fr2, Fr3, from TLC unfolded top downwards, the different composition of polarity according to the order of degree of excursion by wash-out.Use GSLSt to serve as a mark.The main band of Fr2 and Fr3 is distinguished called after A1-A5 and B1-B5 successively from the top.Carry out the HTase determination of activity for each band, result A2 as can be known has activity (table 2), and the effective constituent with function of improving diabetic neuropathy is included among this A2.That is, have only the A2 (deriving from the lipid (TLp) of sprout brown rice) that adds 0.1 μ g to compare with the situation of not adding any additive (None), observe the active remarkable rising of HTase as the situation of additive (Additive).In addition, when the lipid (TLb) to common brown rice carries out classification, do not observe the band suitable (ND, the abbreviation of not determined (not detecting)) with A2.

Table 2

^aNumerical value is the average (n=4) in each experiment, and represents % with total glycolipid with TLb or TLb

^bNumerical value is for containing 0.1 μ g additive (glycoconjugate in each experiment; The active average of HTase (n=2) of A2～B5) is represented with the active % of contrast HTase that does not contain additive.

For purified A2, A4 with through the A2 of alkaline purification, the TLC unfolded be the results are shown among Fig. 3 B.If A2 is carried out alkaline purification, then demonstrate the degree of excursion identical with A4.The variation of degree of excursion does not appear in A4 because of alkaline purification.The ASG that is originated by the soybean of Matreya company sale demonstrates the degree of excursion identical with A2, by carrying out alkaline purification, demonstrates the degree of excursion identical with A4.In addition, as shown in Figure 4, the lipid H4 (acidylate-sterol-β-glucosides) with helicobacter pylori (Helicobacterpylori) source demonstrates identical degree of excursion as can be known.H4 also demonstrates the identical degree of excursion of H6 of originating with helicobacter pylori by alkaline purification.When A4 and H6 were compared, degree of excursion was different slightly, but demonstrates roughly the same degree of excursion.Hence one can see that, and A2, H4, A4 and H6 are the structure of acidylate.Think not being both slightly of degree of excursion because sterol-β-glucosides combines with difference, the β of sterol-β-glucosides or α produces in conjunction with the characteristic of this steric isomer.That is, A2 is acidylate-sterol-β-glucosides as can be known.ASG is by shown in the general formula of Fig. 1, has as the sugar at center and sterol and lipid acid (among the figure by shown in the R) bonded structure.

Embodiment 1

[evaluation of the effective constituent in sprout brown rice source]

＜utilize the structural analysis of NMR 〉

Be the structure of the sterol of the ASG that understands bright formation sprout brown rice source and lipid acid and above-mentioned combination (α in conjunction with or β in conjunction with) that the inventor etc. have carried out nuclear magnetic resonance spectroscopy method (NMR).It the results are shown in Fig. 5 and table 3.

The variation of the degree of excursion on the TLC plate before and after decomposing from the alkali of A2, with the TLC plate of other samples (A4, H6, A4, S2 etc.) on the comparison of degree of excursion (movement), measurable A2 is ASG, but understands that by the NMR analytical solution which kind of ASG A2 is.The analytical procedure of NMR data is with reference to non-patent literature 10.Separating bright content by NMR is 4 following points: comprise glucose, lipid acid and the sterol of each 1 molecule among (1) A2, (2) become ester at 6 conjugated fatty acids of glucose, and (3) glucose forms β with sterol and combines, and (4) main sterol is a Sitosterol.

¹H- ¹³The signals of the 2 dimension spectrograms of C NMR can belong in the signal of each contained in glucose, Sitosterol, lipid acid proton (table 3).

With signal value as the proton (glucose 1～glucose 6) of glucose, Sitosterol (Sitosterol C, Sitosterol CH, Sitosterol CH2, Sitosterol CH3), lipid acid (acyl group CH2, acyl group H1) and be recorded in the table 3.Coding as shown in Figure 6.

Table 3

The H1 of glucose 1 is 4.359ppm, takes place to be coupled (7.7Hz) with the scalar of H2, and hence one can see that, and glucose is the β combination.About glucose, the H6 of glucose 6 and H6 ' be to the downfield displacement, hence one can see that 6 by acidylate.About 6 acidylate, 2.32 with the signal of 1.59ppm are and identical carbonyl bonded CH2 proton, hence one can see that 6 lipid acid that are combined with long-chain.Can find out also that from table 3 the distinctive signal indicating of sterol skeleton confirms the result of Sitosterol as main component basically.

＜utilize the structural analysis of GCMS 〉

Because the NMR structural analysis can't all identify composition contained in sterol and the lipid acid, therefore obtain the sterol composition that constitutes A2 and as the ratio of components of fatty acid component by the GCMS structural analysis.

The composition of sterol that constitutes the ASG in the composition of sterol of ASG of A2 and soybean source is shown in table 4.

Table 4

Campesterol, Stigmasterol and β-Gu Zaichun are also contained among the ASG in soybean source, and this is well-known.It should be noted that among the A2 and contain new sterol i.e. " 5 α-courage steroid-8 (14)-alkene-3 β-alcohol " with higher ratio.To identify that the data of GCMS of the structure of 5 α-courage steroid-8 (14)-alkene-3 β-alcohol are shown in the A of Fig. 7, its structure is shown in Fig. 1.

Then, the composition of lipid acid of the analysis that utilizes NMR being separated the ASG in the composition of lipid acid of bright, as to constitute A2 ASG and soybean source is shown in table 5.

Table 5

16:0,18:0,18:1,18:2,22:0 and 24:0 are also contained among the ASG in soybean source, and this is well-known.It should be noted that and contain new lipid acid i.e. " 18:0 (2h) " among the A2.The GCMS spectrogram that to identify 18:0 (2h) is shown among the B of Fig. 7.

Embodiment 2

[ASG in sprout brown rice source is to the active effect of HTase in serum hdl source]

ASG for the sprout brown rice source investigates the active effect of HTase.The simple declaration experimental technique.Modulation HDL from the pooled serum (pooled serum) of normal rat, with its enzyme source as HTase, behind the interpolation ASG, the activation of investigation HTase.

It is the results are shown in Fig. 8.The amount of the ASG that is added is between 0 to the 0.1 μ g, has observed the active rising of HTase of dose-dependently.The activity of this HTase rises saturated when the amount of the ASG that is added is 0.5 μ g, does not also have bigger variation when further adding.

Embodiment 3

[ASG in sprout brown rice source is for the active specificity effect of HTase]

For whether the ASG that understands bright above-mentioned sprout brown rice source is the specific effect that ASG had in sprout brown rice source for the active effect of HTase, the inventor etc. have repeated the test identical with Fig. 8.For the ASG (ASG) in sprout brown rice source, from the ASG in sprout brown rice source, remove lipid acid and material (SG) and the ASG (S-ASG) that originates of soybean, investigated the active variation of HTase, the results are shown in the table 6.HTase activity under any one the situation of not adding among ASG, SG or the S-ASG as 100, is represented with relative value.

Table 6

Under the situation of the ASG (ASG) that has added the sprout brown rice source, obtained the result identical with Fig. 8, relative therewith, under the situation of the ASG (S-ASG) that has added SG and soybean source, the activity of not observing the HTase of dose-dependently rises.

Embodiment 4

[DPN of the ASG in sprout brown rice source improve effect]

For the ASG that confirms sprout brown rice source whether have with the same diabetic neuropathy of sprout brown rice improve effect (non-patent literature 5), in the presence of homocysteine thiolactone modified LDL (HT-modified LDL), for the ASG in sprout brown rice source to sodium/potassium ATP enzyme (Na/K ATPase) active influence investigate.The simple declaration experimental technique.The thick component of modulation Na/KATP enzyme from the sciatic nerve of normal rat.Synthetic LDL from normal rat serum is carried out homocysteineization and ASG adds together, and investigation is for the active influence of Na/K ATP enzyme.

It is the results are shown in Fig. 2.The amount of the ASG that is added has been observed the rising of the Na/K atpase activity of dose-dependently between 0 to 1.0 μ g.The activity of this Na/K ATP enzyme rises saturated when the amount of the ASG that is added is 1.0 μ g, does not also have bigger variation when further adding.

Embodiment 5

[the specific DPN of ASG in sprout brown rice source improve effect]

For whether the ASG that understands bright above-mentioned sprout brown rice source is that the ASG in sprout brown rice source is peculiar for the active effect of sodium/potassium ATP enzyme, the inventor etc. have repeated the test identical with Fig. 2.For the ASG (ASG) in sprout brown rice source, from the ASG in sprout brown rice source, remove lipid acid and material (SG) and the ASG (S-ASG) that originates of soybean, investigated the active variation of sodium/potassium ATP enzyme, the results are shown in the table 7.With not adding sodium/potassium ATP enzyme activity under any one the situation of ASG, SG or S-ASG, represent with relative value as 100.

Table 7

Under the situation of the ASG (ASG) that has added the sprout brown rice source, obtained the result identical with Fig. 2, relative therewith, under the situation of the ASG (S-ASG) that has added SG and soybean source, the activity of not observing the sodium/potassium ATP enzyme of dose-dependently rises.

Embodiment 6

[utilizing the extracting method of the ASG of solvent-extraction process]

The rice bran 5g of weighing sprout brown rice uses the hexane of 30mL to stir, washs after 15 minutes, filters with B (Buechner funnel), obtains residue.Filtrate is discarded.After under these conditions residue further being washed 2 times, in residue, add the mixed solution (ethanol: water=2: 1 or 1: 1 of ethanol, ethanol and distilled water.Below abbreviate ethanol water (2: 1), ethanol water (1: 1) sometimes respectively as.) or the mixed solution (chloroform: methyl alcohol=2: 1 of chloroform and methyl alcohol.Below, abbreviate chloroform methanol (2: 1) sometimes as.) 30mL, stir, extracted 30 minutes, filter with B, obtain filtrate.Residue is discarded.Whole filtrate of ethanol, ethanol water (2: 1), ethanol water (1: 1) or chloroform methanol (2: 1) of amount is moved in the eggplant type flask, use vaporizer to concentrate and do admittedly, obtain AS G enriched material E (extraction using alcohol), F (ethanol water (2: 1) extraction), G (ethanol water (1: 1) extraction), the H (chloroform methanol (2: 1) extraction) in sprout brown rice source.

Above-mentioned ASG enriched material E, F, G add chloroform methanol (2: 1) 30mL, dissolve 15 minutes once more after, use chloroform methanol (2: 1) constant volume to be 500mL.Above-mentioned ASG enriched material H adds chloroform methanol (2: 1) 30mL, dissolve 15 minutes once more after, use chloroform methanol (2: 1) constant volume to be 1500mL.Each enriched material E, F behind the constant volume, G, H 2mL is centrifugal under the condition of 1500g * 10 minute, with supernatant liquor as working sample.In addition, the ASG in each regenerant uses high performance liquid chromatography (HPLC) to obtain the containing ratio of ASG.Being described in detail as follows of the analytical procedure of ASG is described.

[analytical procedure of ASG]

The analysis condition that the HPLC of ASG analyzes use table 8 carries out.

Table 8

Analysis condition

Moving phase and gradient condition are shown in table 9.

Table 9

Moving phase and gradient condition ^§

§ analyzes the solvent of A and B under 0,15,20,25,30 minute above-mentioned gradient condition.

* A=chloroform, B=methyl alcohol: water (95: 5, volume/volume)

[analytical results]

Each analytical results that extracts the ASG containing ratio in the component is shown in table 10.

Table 10

The ASG enriched material	Concentrate dry substance weight (ASG containing ratio)
		E	72.6mg(15.8％)
F	45.1mg(19.4％)
		G	147.5mg(2.01％)
H	75.0mg(1.26％)

Embodiment 7

[utilizing the purifying of the ASG that Iatrobeads (particulate porous property silica particles) chromatography carries out]

In order to obtain highly purified ASG, extract purifying by the Iatrobeads chromatography based on the analytical results of the foregoing description 6.

The rice bran 25g of weighing sprout brown rice reclaims component by Iatrobeads chromatography (Iatrobeads chromatography), obtains fraction 2.Further, fraction was opened in 8～12 minutes, they are done admittedly, obtain concentrated do I (I of letter) admittedly by the Iatrobeads chromatography with fraction 2 concentrated doing admittedly.

＜post 〉

φ 2cm * 30cm (filling gel (Iatrobeads 6RS-8060, the ヤトロ of Mitsubishi Chemical of Co., Ltd. Application system))

＜method 〉

1. behind the rice bran 25g with the hexane wash sprout brown rice, use chloroform: hexane=1: 1 extraction TL component (TL).

2. with rotatory evaporator that TL is dried fully solid.

3. will do the chloroform that TL after solid is dissolved in 30ml: hexane (C: H)=1: 1 in.

4. feed the C of 200ml: H=9 respectively: 1, reclaim and see through liquid (fraction 1).

5. feed the chloroform of 200ml: methyl alcohol=9: 1, reclaim and see through liquid (fraction 2).

6. feeding chloroform: methyl alcohol: water (C: M: W)=7: 3: 0.1, reclaim and see through liquid (fraction 3).

7. feed the C of 200ml: M: W=30: 60: 0.8, reclaim and see through liquid (fraction 4).

8. each fraction is supplied to confirm the ASG elutriated fraction in HPTLC (silica gel 60, メ Le Network corporate system).

HPTLC plate (Silicagel60 at the commercially available long 10cm that has been coated with silica gel, Merck) position apart from below 1.5cm, utilize microsyringe that each fraction 5 μ L is carried out the sample coating, by after launching solvent chloroform and launching, carry out air-dryly, utilize chloroform once more: methyl alcohol (95: 5) carries out launching for 2 times and is air-dry.

＜result 〉

Under these conditions each fraction is supplied to confirm the existence of ASG in tlc, contained ASG all is eluted in the fraction 2 among the TL as a result.It the results are shown in Fig. 9.

[utilizing purifying follow-up of the ASG that the Iatrobeads chromatography carries out]

＜post 〉

φ 1cm * 40cm (filling the Iatrobeads of 20cm)

＜method 〉

1. after utilizing vaporizer to do above-mentioned fraction 2 admittedly, be dissolved in the chloroform of 10ml: hexane=in 1: 1.

With the lysate application of sample to post.

3. feed the chloroform of 75ml: hexane (C: H)=1: 1.

4. feed the chloroform (C) of 75ml.

5. feed C: M=95: 5, collect according to the mode of 2ml/ fraction.

6. be collected into fraction No.30, with above-mentioned same condition under confirm the wash-out of ASG by HPTLC.

＜result 〉

Near fraction No.10～12, can confirm the band of ASG.In addition, the elution time of per 1 fraction is 5 minutes.It the results are shown in Figure 10.

[analytical procedure of ASG]

The analysis of ASG containing ratio is carried out similarly to Example 6.That is, the moving phase of the analysis condition of use table 8 and table 9 and gradient condition are carried out.

[analytical results]

The analytical results of ASG is shown in table 11.

Table 11

The ASG enriched material	Concentrate dry substance weight (ASG containing ratio)
		I	(10.3mg more than 98.0%)

Utilizability on the industry

The ASG in sprouted unpolished rice of the present invention source has prevention or improves the effect of diabetic neuropathy.

Therefore, according to the present invention, can utilize as prevention or the preparation that improves diabetic neuropathy.

And, all the time, sprouted unpolished rice is utilized as healthy food, therefore the ASG's in sprouted unpolished rice source is safe, can also continuation absorb, and can make in a large number, and add to easily in the food etc., therefore for the human or animal improve health or possibility that prevent disease etc. can be made contributions bigger.

Claims

1. composition that prevents and improve DPN, it is an effective constituent with the steroline that contains 2-hydroxyl-octadecanoic acid.

2. prevention according to claim 1 and improve the composition of DPN is characterized in that the sterol skeleton of steroline is 5 α-courage steroid-8 (14)-alkene-3 β-alcohol.

3. preparation that prevents or improve DPN, it is effective constituent to contain 2-hydroxyl-octadecanoic acid as the steroline of fatty acid part.

4. preparation that prevents or improve DPN, it is effective constituent to contain 5 α-courage steroid-8 (14)-alkene-3 β-alcohol as the steroline of sterol skeleton.

5. preparation that prevents or improve DPN, it is to contain 2-hydroxyl-octadecanoic acid as fatty acid part and to contain 5 α-courage steroid-8 (14)-alkene-3 β-alcohol be effective constituent as the steroline of sterol skeleton.

6. a homocysteine thiolactone is separated the enzyme activation composition, and it is effective constituent to contain 2-hydroxyl-octadecanoic acid as the steroline of fatty acid part.

7. homocysteine thiolactone according to claim 6 is separated the enzyme activation composition, it is characterized in that, the sterol skeleton of steroline is 5 α-courage steroid-8 (14)-alkene-3 β-alcohol.

8. steroline, it is by general formula (A) expression, and satisfy following (i) or (ii) in arbitrary condition,