CN108310226A - It is a kind of to have effects that prevent the composition and the preparation method and application thereof of diabetes - Google Patents
It is a kind of to have effects that prevent the composition and the preparation method and application thereof of diabetes Download PDFInfo
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- CN108310226A CN108310226A CN201810245505.2A CN201810245505A CN108310226A CN 108310226 A CN108310226 A CN 108310226A CN 201810245505 A CN201810245505 A CN 201810245505A CN 108310226 A CN108310226 A CN 108310226A
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- OXGUCUVFOIWWQJ-XIMSSLRFSA-N acanthophorin B Natural products O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OXGUCUVFOIWWQJ-XIMSSLRFSA-N 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 150000004283 biguanides Chemical group 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
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- 239000004026 insulin derivative Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 238000004900 laundering Methods 0.000 description 1
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- 239000008188 pellet Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
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- OEKUVLQNKPXSOY-UHFFFAOYSA-N quercetin 3-O-beta-D-glucopyranosyl(1->3)-alpha-L-rhamnopyranosyl(1->6)-beta-d-galactopyranoside Natural products OC1C(O)C(C(O)C)OC1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OEKUVLQNKPXSOY-UHFFFAOYSA-N 0.000 description 1
- QPHXPNUXTNHJOF-UHFFFAOYSA-N quercetin-7-O-beta-L-rhamnopyranoside Natural products OC1C(O)C(O)C(C)OC1OC1=CC(O)=C2C(=O)C(O)=C(C=3C=C(O)C(O)=CC=3)OC2=C1 QPHXPNUXTNHJOF-UHFFFAOYSA-N 0.000 description 1
- OXGUCUVFOIWWQJ-HQBVPOQASA-N quercitrin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OXGUCUVFOIWWQJ-HQBVPOQASA-N 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
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- 229910021642 ultra pure water Inorganic materials 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
- A61K36/605—Morus (mulberry)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
- A61K36/074—Ganoderma
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/898—Orchidaceae (Orchid family)
- A61K36/8984—Dendrobium
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
Abstract
Have effects that prevent the composition and the preparation method and application thereof of diabetes the invention discloses a kind of, it is made of mulberry leaf, duck wheat, dendrobium candidum and lucidum spore powder.The present invention is according to traditional Chinese medical theory, raw material composition and its weight proportion are screened by many experiments, the experimental results showed that, the present invention is using mulberry-leaf extract, duck wheat extract, dendrobium candidum, lucidum spore powder as best composition, the content of active polysaccharide, rutin, Quercetin and naringenin isoreactivity ingredient can be improved, with good reducing blood sugar and blood fat and other effects, and it is green safe, long-term use has no toxic side effect.The present invention provides preparation method, and technological design is rationally, it can be achieved that industrialized production.
Description
Technical field
The present invention relates to a kind of compositions, and in particular to a kind of to have effects that prevent the composition of diabetes and its preparation side
Method and application.
Background technology
Diabetes are a kind of global common diseases and frequently-occurring disease, it has also become the third after tumour and angiocardiopathy
Big disease.It is the main ancillary pathology of diabetes that diabetes, which merge stagnant heat illness, clinically common are diabetic cardiomyopathy,
Diabetic cardiomyopathy includes glycosuria disease-specific Myocardial damage, microvascular lesion related with diabetes, macroangiopathic
Change, cardiac autonomic neuropathy etc., while being often accompanied by nonspecific coronary atherosclerotic heart disease and (or) high blood
Pressure heart disease may be collectively referred to as diabetic cardiomyopathy on the basis of with diabetes.Diabetic cardiomyopathy and non-glycosuria
The coronary atherosclerotic heart disease that patient occurs is compared with cardiomyopathy, there is that state of an illness weight, contradiction are more, treatment difficulty is big, in advance
Poor afterwards and high case fatality rate feature.
The drug for the treatment of diabetes and its complication is more at present, the Western medicine clinically used, though there is certain curative effect,
But most there are larger adverse reactions, and as occurred rebounding after being discontinued, patient's hepatorenal damage, striated muscle melt the side effects such as disease,
It is palliative, and traditional Chinese medicine has its unique advantages in terms for the treatment of diabetes, has curative effect lasting, makees without apparent poison pair
With the advantages of, but at present for diabetes Chinese medicine mostly relatively folk prescription, proved recipe, lack the clinical drug efficacy study of system perfecting, and
Chinese prescription foundation is had nothing in common with each other, rules for the treatment of disunity.
Therefore, it actively finds and makes great efforts exploratory development and provided that safety is good, and adverse reaction is low, there is prevention sugar well
The composition of the sick effect of urine is of great significance.
Invention content
Goal of the invention：Present invention aims to solve the deficiencies of the prior art, and provides a kind of a kind of scientific and reasonable, the safeties of proportioning
It is good, the composition with fine auxiliary hyperglycemic effect.Another object of the present invention be to provide the preparation method of the composition with
Its identification method is applied with it.
Technical solution：In order to achieve the goal above, the technical solution that the present invention takes is：
It is a kind of to have effects that prevent the composition of diabetes, which is characterized in that it includes following raw materials according：
Mulberry leaf, duck wheat, dendrobium candidum, lucidum spore powder.
Preferably, above-described to have effects that prevent the composition of diabetes, it is by following parts by weight
Raw material is made：
125~250 parts of mulberry-leaf extract, 125~250 parts of duck wheat extract, 250~500 parts of dendrobium candidum, ganoderma lucidum spore
125~250 parts of sub- powder.
Preferably, above-described to have effects that prevent the composition of diabetes, it is by following parts by weight
Raw material is made：
125 parts of mulberry-leaf extract, 125 parts of duck wheat extract, 250 parts of dendrobium candidum, 125 parts of lucidum spore powder.
The preparation method of the composition prevent with diabetes of the present invention, includes the following steps：
The mulberry-leaf extract is prepared by the following method to obtain：Mulberry leaf are taken, 10 times of amount volumetric concentrations 80% are added
Alcohol reflux extracts 2 times, and 1.5 hours every time, filtration obtained ethanol extract；The dregs of a decoction add 10 times of amount water to extract 1 time, each 1.5h,
Filtration, obtains water extract, and ethanol extract and aqueous extract are merged, 60 DEG C of relative densities 1.20 are concentrated under reduced pressure into, spare；It takes
Concentrate, decompression or spray drying, crush, and sieving obtains mulberry-leaf extract；
The duck wheat extract is prepared by the following method to obtain：Bitter buckwheat is taken, 10 times of amount volumetric concentrations are added
60% alcohol reflux extracts 3 times, and 1.5 hours every time, filtration, merging filtrate was recovered under reduced pressure ethyl alcohol, is concentrated into 60 DEG C of relative densities
1.20 spare；It takes concentrate, normal pressure or is dried under reduced pressure, low-temperature grinding, be sieved, obtain duck wheat extract, it is spare；
The dendrobium candidum is prepared by the following method to obtain：100 DEG C of dendrobium candidum is taken to dry 30 minutes, powder
It is broken, it sieves with 100 mesh sieve, obtains dendrobium officinale powder.
The Quality evaluation of the composition prevent with diabetes of the present invention：Include the following steps：
(1) qualitative research
(1.1) microscopical characters of glossy ganoderma spore powder with crushed sporoderm
Microscopical characters：Glossy ganoderma spore powder with crushed sporoderm is sepia powder, and appearance is extremely fine and smooth, without luming at top, is referred to mother
Gently consult with index finger and touch, there is smooth fine and smooth sense, no adhesion, the feeling without foreign matters such as grit；It is seen under 1500 times of amplification factor
It examines, exosporium-broken spore is not complete, plentiful, double-walled construction is clear, and outer wall is colourless, and inner wall has spinule, filbert；Individual morphology is consistent,
It is oval；Top is truncate, and size is about 8.5~11 × 5.2~6.9 μm；And exosporium-broken spore then has several forms, according to broken wall journey
Sequence difference can be divided into slight broken wall --- spore shape completely, on wall has aperture, moderate broken wall ---, and spore shape is imperfect, there have to be scarce
Damage, severe broken wall --- broken spore coat and spore content；
(1.2) sitosterol thin layer differentiates in duck wheat：
Duck wheat control medicinal material and each 3g of composition powder are weighed respectively, and 25mL chloroformic solutions, ultrasonic extraction is added
30min, filtering, takes 10mL filtrates, is evaporated on evaporating dish, is dissolved with chloroform and be settled to 2mL；Draw composition test liquid, hardship
Buckwheat control medicinal material solution l0 μ L are put respectively to be contained in same on the silica G plate that 0.3% sodium carboxymethylcellulose is alite paste, with body
Product is than being 7：3 petroleum ether-ether is solvent, is unfolded, and takes out, dries, and is sprayed with 10% sulfuric acid solution, 105 DEG C are dried to spot
Point colour developing, daylight are inspected；
(2) quantitative study
(2.1) determination of polysaccharide in lucidum spore powder
Precision weighs anthrone 0.1g, is added 80% sulfuric acid solution 100mL, and stirring is allowed to dissolve, shake up to get；Anthrone-
Sulfuric acid chromogenic reaction is measured with colorimetric method at 625nm using DEXTROSE ANHYDROUS product as a contrast；
The preparation of reference substance solution：
Precision weighs the dry DEXTROSE ANHYDROUS to constant weight, accurately weighed, is placed in measuring bottle, adds distillation water dissolution and dilutes
It to scale, shakes up, is configured to reference substance solution；
The preparation of test solution
Lucidum spore powder powder is taken, it is accurately weighed, it sets in round-bottomed flask, water is added to stand, be heated to reflux, filter while hot, use
A small amount of hot water washing nozzle and filter residue, filter residue and filter paper are set in flask, water is added, is heated to reflux, is filtered while hot, merging filtrate,
It sets and is evaporated in water-bath, residue water dissolution is slowly added dropwise ethyl alcohol, shakes up while stirring, centrifugation, discards supernatant liquid, and sediment is used
Hot water dissolving is simultaneously transferred in measuring bottle, lets cool, adds water to scale, shakes up, and takes solution appropriate, and centrifugation, precision measures supernatant and sets
In measuring bottle, add water to scale, shake up to get；
Specification Curve of Increasing
Precision measures reference substance solution 0.4,0.6,0.8,1.0,1.2mL, sets in 10mL tool plug test tubes, respectively adds water to respectively
2.0mL, rapid accurate addition Liu Suan Onion ketone solution, shakes up, and after placement, sets cooling in ice bath immediately, is sky with corresponding reagent
In vain, according to UV-VIS spectrophotometry, absorbance, using absorbance as ordinate, a concentration of horizontal seat are measured at 625nm wavelength
Mark draws standard curve；
(2.2) naringenin assay in dendrobium candidum
Using dendrobium candidum active ingredient shaddock ped cellulose content in high effective liquid chromatography for measuring composition；Chromatographic condition is：
XB C18 chromatographic columns, specification be 4.6mm × 250mm, 5 μm；30 DEG C of column temperature；0.2% phosphoric acid of mobile phase is that A phases-methanol is B phases,
Gradient elution；Detection wavelength 290nm；Flow velocity 1.0mL/min；
Take naringenin reference substance appropriate, it is accurately weighed, it sets in 50mL brown measuring bottles, methanol is added to be dissolved to scale, shake up, make
It is spare at naringenin reference substance solution；
Dendrobium candidum is taken, is set in round-bottomed flask, the mixed solution of methanol and 20% hydrochloric acid is added in precision, weighs, sets water-bath
In be heated to reflux, let cool, be re-weighed, supply less loss weight with methanol, shake up, stand, take supernatant to be filtered through 0.22 μm of miillpore filter
It crosses, takes subsequent filtrate, 10 μ L of sample introduction that it is spare that test solution is made；
Precision draws reference substance solution and test solution injects liquid chromatograph, using one point external standard method, by above-mentioned color
Spectral condition is measured；
(2.3) in duck wheat extract rutin, quercetin content assay method foundation
The preparation of reference substance solution
Precision weighs control substance of Rutin, and Quercetin reference substance is respectively placed in volumetric flask, is dissolved with methanol and is settled to quarter
Degree, shakes up to get rutin and Quercetin reference substance solution；
The preparation of test solution
Duck wheat medicinal material is taken, alcohol reflux extraction is added, merging filtrate is concentrated under reduced pressure, and residue is dissolved with methanol, is transferred to
In measuring bottle, add methanol to scale, shake up, filter, take subsequent filtrate to get；
Precision draws control substance of Rutin and Quercetin reference substance solution, is half-and-half diluted 6 times with methanol, is implanted sequentially liquid phase color
In addition spectrometer, bioassay standard curve draw duck wheat test solution and inject liquid chromatograph；
Chromatographic condition is：Chromatographic column Thermo C18,4.6mm × 250mm, 5 μm；Mobile phase is -0.2% formic acid water of acetonitrile
Solution, gradient elution：0~18min, acetonitrile 17~40%, 18~19min, acetonitrile 40~17%；Volume flow is 1mL/min；
Detection wavelength：λ=256nm；Sample size：10μL；
With a concentration of abscissa C of each reference substance, standard curve is drawn by ordinate of the peak area A of each reference substance；
(2.4) in mulberry leaf the content of rutin assay：
The preparation of reference substance solution
Take control substance of Rutin appropriate, it is accurately weighed, solution of every 1mL containing 0.1mg is made with methanol；
The preparation of test solution
Mulberry Leaf is taken, it is accurately weighed, it sets in round-bottomed flask, adds methanol, be heated to reflux, filter, filter residue again with methanol is same
Method is extracted 2 times, and solvent is recovered under reduced pressure in merging filtrate, and residue is dissolved with methanol, is transferred in 25mL measuring bottles, adds methanol to scale,
Shake up, filter, take subsequent filtrate to get；
The preparation of standard curve
Take 500 μ L of control substance of Rutin solution, with methanol half-and-half dilute 6 times, obtain concentration be respectively 0.0095mg/mL,
The reference substance solution of 0.019mg/mL, 0.0379mg/mL, 0.0758mg/mL, 0.1517mg/mL, 0.3034mg/mL inject liquid
Chromatography, sample introduction measure；
Chromatographic condition is：Chromatographic column be C18 columns, 4.6mm × 250mm, 5 μm；Mobile phase is that -0.5% phosphoric acid of methanol is water-soluble
Liquid, gradient elution：0~5min, 35% methanol；8~16min, 40% methanol；19~24min, 51% methanol；26~30min,
70% methanol；30~35min, 35% methanol.Volume flow 1mL/min；Detection wavelength 358nm, 10 μ L of sample size；
With a concentration of abscissa, peak area is that ordinate draws standard curve.
Preferably, the Quality evaluation of the above-described composition prevent with diabetes：Including
Following steps：
(1) qualitative research
(1.1) microscopical characters of glossy ganoderma spore powder with crushed sporoderm
Microscopical characters：Glossy ganoderma spore powder with crushed sporoderm is sepia powder, and appearance is extremely fine and smooth, without luming at top, is referred to mother
Gently consult with index finger and touch, there is smooth fine and smooth sense, no adhesion, the feeling without foreign matters such as grit；It is seen under 1500 times of amplification factor
It examines, exosporium-broken spore is not complete, plentiful, double-walled construction is clear, and outer wall is colourless, and inner wall has spinule, filbert；Individual morphology is consistent,
It is oval；Top is truncate, and size is about 8.5~11 × 5.2~6.9 μm；And exosporium-broken spore then has several forms, according to broken wall journey
Sequence difference can be divided into slight broken wall --- spore shape completely, on wall has aperture, moderate broken wall ---, and spore shape is imperfect, there have to be scarce
Damage, severe broken wall --- broken spore coat and spore content；
(1.2) sitosterol thin layer differentiates in duck wheat：
Duck wheat control medicinal material and each 3g of composition powder are weighed respectively, and 25mL chloroformic solutions, ultrasonic extraction is added
30min, filtering, takes 10mL filtrates, is evaporated on evaporating dish, is dissolved with chloroform and be settled to 2mL；Draw composition test liquid, hardship
Buckwheat control medicinal material solution l0 μ L are put respectively to be contained in same on the silica G plate that 0.3% sodium carboxymethylcellulose is alite paste, with body
Product is than being 7：3 petroleum ether-ether is solvent, is unfolded, and takes out, dries, and is sprayed with 10% sulfuric acid solution, 105 DEG C are dried to spot
Point colour developing, daylight are inspected；
(2) quantitative study
(2.1) determination of polysaccharide in lucidum spore powder
Precision weighs anthrone 0.1g, is added 80% sulfuric acid solution 100mL, and stirring is allowed to dissolve, shake up to get；Anthrone-
Sulfuric acid chromogenic reaction is measured with colorimetric method at 625nm using DEXTROSE ANHYDROUS product as a contrast；
The preparation of reference substance solution：
Precision weighs 105 DEG C of dryings to the DEXTROSE ANHYDROUS 1.521mg of constant weight, accurately weighed, is placed in 10mL measuring bottles, adds
Distillation water dissolution is simultaneously diluted to scale, shakes up, is configured to a concentration of 0.1521mgmL-1Reference substance solution；
The preparation of test solution
Lucidum spore powder powder 2g is taken, it is accurately weighed, it sets in round-bottomed flask, adds water 60mL to stand 1 hour, be heated to reflux 4
Hour, it filters while hot, with a small amount of hot water washing nozzle and filter residue, filter residue and filter paper is set in flask, adds water 60mL, is heated to reflux
It 3 hours, filters while hot, merging filtrate, sets and be evaporated in water-bath, residue water 5mL dissolves, and ethyl alcohol is slowly added dropwise while stirring
75mL shakes up, and is placed 12 hours at 4 DEG C, and centrifugation discards supernatant liquid, and sediment is with hot water dissolving and is transferred in 50mL measuring bottles,
It lets cool, adds water to scale, shake up, take solution appropriate, centrifuge, precision measures supernatant 3mL and sets in 25mL measuring bottles, adds water to quarter
Degree, shake up to get；
Specification Curve of Increasing
Precision measures reference substance solution 0.4,0.6,0.8,1.0,1.2mL, sets in 10mL tool plug test tubes, respectively adds water to respectively
2.0mL, rapid accurate addition Liu Suan Onion ketone solution (accurate Cheng Qu Onion ketone 0.lg, add sulfuric acid 100mL to make dissolving, shake up) 6mL, stands
It shakes up, after placing 15 minutes, sets cooling 15 minutes in ice bath and take out immediately, using corresponding reagent as blank, according to ultraviolet-visible
Spectrophotometry measures absorbance at 625nm wavelength, and using absorbance as ordinate, it is bent to draw standard for a concentration of abscissa
Line, y=0.0051x-0.0036；
(2.2) naringenin assay in dendrobium candidum
Using dendrobium candidum active ingredient shaddock ped cellulose content in high effective liquid chromatography for measuring composition；Chromatographic condition is：
XB C18 chromatographic columns, specification be 4.6mm × 250mm, 5 μm；30 DEG C of column temperature；0.2% phosphoric acid of mobile phase is that A phases-methanol is B phases,
Gradient elution；Detection wavelength 290nm；Flow velocity 1.0mL/min；
Take naringenin reference substance appropriate, it is accurately weighed, it sets in 50mL brown measuring bottles, methanol is added to be dissolved to scale, shake up, make
Contain the reference substance solution of 26 μ g at every 1mL, it is spare；
Take composition 0.5g, it is accurately weighed, set in the round-bottomed flask of 50mL, precision be added the methanol that volume ratio is 4: 1 and
The mixed solution 25mL of 20% hydrochloric acid, weighs, sets in water-bath and be heated to reflux, let cool, be re-weighed, and supplies less loss weight with methanol, shakes
It is even, it stands, takes supernatant to be filtered through 0.22 μm of miillpore filter, take subsequent filtrate, 10 μ L of sample introduction that it is spare that test solution is made；
Precision draws reference substance solution and test solution injects liquid chromatograph, and naringenin is measured using one point external standard method
Content；
(2.3) in duck wheat extract rutin, quercetin content assay method foundation
The preparation of reference substance solution
Precision weighs control substance of Rutin 5.952mg, and Quercetin reference substance 3.075mg is respectively placed in 10mL volumetric flasks, uses
Methanol dissolves and is settled to scale, shake up to get rutin and Quercetin reference substance solution concentration be respectively 0.5952mg/mL and
0.3075mg/mL；
The preparation of test solution
Duck wheat medicinal material is taken, 10 times of amounts are added, 60% alcohol reflux of volumetric concentration extracts 3 times, 1 hour every time, merges filter
Liquid is concentrated under reduced pressure, and residue is dissolved with methanol, is transferred in 25mL measuring bottles, added methanol to scale, shake up, and filters, takes subsequent filtrate,
To obtain the final product；
Precision draws control substance of Rutin and Quercetin reference substance solution, and constant gradient dilutes 6 parts successively respectively, is implanted sequentially liquid
In addition chromatography, bioassay standard curve draw duck wheat test solution and inject liquid chromatograph；
Chromatographic condition is：Chromatographic column Thermo C18,4.6mm × 250mm, 5 μm；Mobile phase is -0.2% formic acid water of acetonitrile
Solution, gradient elution：0~18min, acetonitrile 17~40%, 18~19min, acetonitrile 40~17%；Volume flow is 1mL/min；
Detection wavelength：λ=256nm；Sample size：10μL；
With a concentration of abscissa of each reference substance, (C draws standard curve, reed by ordinate of the peak area A of each reference substance
Fourth equation of linear regression is Y=2 × 107+2506.8；The equation of linear regression of Quercetin is Y=4 × 107- 11208；
(2.4) in mulberry leaf the content of rutin assay：
The preparation of reference substance solution
Take control substance of Rutin appropriate, it is accurately weighed, solution of every 1mL containing 0.1mg is made with methanol；
The preparation of test solution
Mulberry Leaf lg is taken, it is accurately weighed, it sets in round-bottomed flask, adds methanol 50mL, be heated to reflux 30 minutes, filter, filter
Slag again with methanol 50mL is extracted 2 times with method, and solvent is recovered under reduced pressure in merging filtrate, and residue is dissolved with methanol, is transferred to 25mL amounts
Bottle in, add methanol to scale, shake up, filter, take subsequent filtrate to get；
The preparation of standard curve
Take 500 μ L of control substance of Rutin solution, with methanol half-and-half dilute 6 times, obtain concentration be respectively 0.0095mg/mL,
The reference substance solution of 0.019mg/mL, 0.0379mg/mL, 0.0758mg/mL, 0.1517mg/mL, 0.3034mg/mL inject liquid
Chromatography, sample introduction measure；
Chromatographic condition is：Chromatographic column be C18 columns, 4.6mm × 250mm, 5 μm；Mobile phase is that -0.5% phosphoric acid of methanol is water-soluble
Liquid, gradient elution：0~5min, 35% methanol；8~16min, 40% methanol；19~24min, 51% methanol；26~30min,
70% methanol；30~35min, 35% methanol.Volume flow 1mL/min；Detection wavelength 358nm, 10 μ L of sample size；
It is mapped with a concentration of abscissa, it is y=2 × 10 to obtain standard curve7X-10419, the range of linearity be 0.0095~
0.3034mg/mL, correlation coefficient r 1.
It is of the present invention to have effects that prevent composition the answering in preparing hypoglycemic drug or health products of diabetes
With.
It is of the present invention to have effects that prevent composition the answering in preparing blood lipid-lowering medicine or health products of diabetes
With.
It is of the present invention to have effects that the composition for preventing diabetes is preparing hypoglycemic and blood lipid-lowering medicine or health care
Application in product.
Advantageous effect：It is provided by the invention to have effects that the composition for preventing diabetes has the following advantages：
1, the present invention screens raw material composition and its weight proportion, experimental result according to traditional Chinese medical theory by many experiments
Show the present invention with weight ratio for 3:1:1 duck wheat, dendrobium candidum, lucidum spore powder have very as best composition
Good effect of lowering blood sugar, green safe property, long-term use have no toxic side effect.
2, results of animal shows：Composition provided by the invention has extraordinary hypoglycemic, reducing blood lipid, improves sugar
Tolerance increases insulin generation and other effects.
Specific implementation mode
With reference to specific embodiment, the present invention is furture elucidated, it should be understood that these embodiments be merely to illustrate the present invention and
It is not used in and limits the scope of the invention, after having read the present invention, various of equal value shapes of the those skilled in the art to the present invention
The modification of formula falls within the application range as defined in the appended claims.
Embodiment 1
1, mulberry leaf alcohol reflux extracting process optimization test
Factor level designs
Using L9(34) orthogonal array, using rutin in mulberry leaf and general flavone content as index, investigate determining alcohol, solid-liquid ratio,
Influence of the extraction time to index constituents extraction.
1 orthogonal test factor level table of table
| Factor | A determining alcohols (%) | B solid-liquid ratios | C extraction times | D blank |
| 1 | 60 | 1:8 | 1 | 1 |
| 2 | 70 | 1:10 | 2 | 2 |
| 3 | 80 | 1:12 | 3 | 3 |
2, interpretation of result
According to L9(34) orthogonal arrage progress orthogonal test, mulberry leaf medicinal material 10g is weighed, alcohol reflux is added, is filtered, liquid is merged.
Every group of experiment is 3 times parallel.
2 orthogonal experiments of table
3 rutin variance analysis of table
| Factor | Sum of square of deviations | Degree of freedom | F ratios | F critical values | Conspicuousness |
| Determining alcohol | 0.041 | 2 | 20.500 | 19.000 | * |
| Solid-liquid ratio | 0.011 | 2 | 5.500 | 19.000 | |
| Extraction time | 0.147 | 2 | 73.500 | 19.000 | * |
| Error | 0.00 | 2 |
4 general flavone variance analysis of table
| Factor | Sum of square of deviations | Degree of freedom | F ratios | F critical values | Conspicuousness |
| Determining alcohol | 0.273 | 2 | 9.750 | 19.000 | |
| Solid-liquid ratio | 0.175 | 2 | 6.250 | 19.000 | |
| Extraction time | 0.469 | 2 | 16.750 | 19.000 | |
| Error | 0.03 | 2 |
The result shows that when using rutin as Testing index, two significant differences of factor of determining alcohol and extraction time, and it is main
Secondary factor sequence is：Extraction time>Determining alcohol>Solid-liquid ratio, therefore it is solid-liquid ratio 1 to select the optimised process of mulberry leaf alcohol extracting：12, alcohol
A concentration of 80%, extraction time is 3 times, but in view of cost-effective, therefore selects solid-liquid ratio for 1：10.
When using general flavone as index, each factor there are no significant difference, therefore select best alcohol extraction process for solid-liquid ratio 1:
10, determining alcohol 80%, extraction time is 3 times.
10 times are added to measure 80% edible ethanol refluxing extraction 2 times, 1.5 hours every time, filtration, the dregs of a decoction added 10 times of amount water extractions 1
Secondary, each 1.5h, filtration merges with ethanol extract.Ethyl alcohol is recovered under reduced pressure, is concentrated into relative density 1.20 (60 DEG C) duck wheat
Extraction process optimization test
The determination of factor level
By preliminary experiment, influencing alcohol extraction process principal element has determining alcohol, extraction time, solid-liquid ratio.This experiment uses L9
(34) orthogonal array, using the content of rutin and Quercetin as evaluation index, investigation determining alcohol, extraction time, solid-liquid ratio three
Influence of the factor to index constituents extraction.
5 factor level table of table
| Factor | A determining alcohols (%) | B extraction times | C solid-liquid ratios (g:mL) | Blank D |
| 1 | 60 | 1 | 1:6 | 1 |
| 2 | 70 | 2 | 1:8 | 2 |
| 3 | 80 | 3 | 1:10 | 3 |
Orthogonal test
Duck wheat medicinal material 10g is weighed, is that evaluation refers to the content of rutin and Quercetin according to orthogonal array experiment arrangement
Mark carries out preferably alcohol extraction process, and orthogonal experiments see the table below.
6 intuitive analytical table of table
7 rutin variance analysis of table
| Factor | Sum of square of deviations | Degree of freedom | F ratios | F critical values | Conspicuousness |
| Determining alcohol | 12.905 | 2 | 3.654 | 19.000 | |
| Solid-liquid ratio | 12.114 | 2 | 3.430 | 19.000 | |
| Extraction time | 2.433 | 2 | 0.689 | 19.000 | |
| Error | 3.53 | 2 |
8 Quercetin variance analysis of table
| Factor | Sum of square of deviations | Degree of freedom | F ratios | F critical values | Conspicuousness |
| Determining alcohol | 0.015 | 2 | 7.500 | 19.000 | |
| Solid-liquid ratio | 0.045 | 2 | 22.500 | 19.000 | * |
| Extraction time | 0.003 | 2 | 1.500 | 19.000 | |
| Error | 0.00 | 2 |
Influence Extraction of rutin secondary factors be：Determining alcohol>Extraction time>Solid-liquid ratio, each factor there are no significant difference.
The secondary factors for influencing Quercetin extraction are extraction time>Determining alcohol>The significant difference of solid-liquid ratio, wherein solid-liquid ratio, therefore most
Good process combination is A1B3C3, i.e. 60% ethyl alcohol, extraction 3 times, solid-liquid ratio 1:10.
Process certification
On the basis of orthogonal test, A is selected1B3C3(60% ethyl alcohol extracts 3 times, solid-liquid ratio 1:10) verification examination is done
It tests, the results are shown in Table 9.
9 process certification result (n=3) of table
| Serial number | Rutin content (%) | Quercetin content (%) |
| 1 | 1.462 | 0.074 |
| 2 | 1.452 | 0.073 |
| 3 | 1.474 | 0.074 |
| Average content | 1.463 | 0.074 |
| RSD (%) | 0.72 | 1.12 |
The result shows that the process stabilizing is feasible.
Embodiment 2
Have effects that prevent the composition of diabetes, it is made of the raw material of following parts by weight：
Mulberry-leaf extract 125g, duck wheat extract 125g, dendrobium candidum 250g, lucidum spore powder 125g.
The mulberry-leaf extract is prepared by the following method to obtain：Mulberry leaf are taken, 10 times of amount volumetric concentrations 80% are added
Alcohol reflux extracts 2 times, and 1.5 hours every time, filtration obtained ethanol extract；The dregs of a decoction add 10 times of amount water to extract 1 time, each 1.5h,
Filtration, obtains water extract, and ethanol extract and aqueous extract are merged, 60 DEG C of relative densities 1.20 are concentrated under reduced pressure into, spare；It takes
Concentrate, decompression or spray drying, crush, and sieving obtains mulberry-leaf extract；
The duck wheat extract is prepared by the following method to obtain：Bitter buckwheat is taken, 10 times of amount volumetric concentrations are added
60% alcohol reflux extracts 3 times, and 1.5 hours every time, filtration, merging filtrate was recovered under reduced pressure ethyl alcohol, is concentrated into 60 DEG C of relative densities
1.20 spare；It takes concentrate, normal pressure or is dried under reduced pressure, low-temperature grinding, be sieved, obtain duck wheat extract, it is spare；
The dendrobium candidum is prepared by the following method to obtain：100 DEG C of dendrobium candidum is taken to dry 30 minutes, powder
It is broken, it sieves with 100 mesh sieve, obtains dendrobium officinale powder.
The lucidum spore powder is produced by the Jiangsu bio tech ltd Hong Shou and is provided.
Embodiment 3
The Quality evaluation of the composition with prevention diabetes described in embodiment 2：Include the following steps：
(1) qualitative research
(1.1) microscopical characters of glossy ganoderma spore powder with crushed sporoderm
Microscopical characters：Glossy ganoderma spore powder with crushed sporoderm is sepia powder, and appearance is extremely fine and smooth, without luming at top, is referred to mother
Gently consult with index finger and touch, there is smooth fine and smooth sense, no adhesion, the feeling without foreign matters such as grit；It is seen under 1500 times of amplification factor
It examines, exosporium-broken spore is not complete, plentiful, double-walled construction is clear, and outer wall is colourless, and inner wall has spinule, filbert；Individual morphology is consistent,
It is oval；Top is truncate, and size is about 8.5~11 × 5.2~6.9 μm；And exosporium-broken spore then has several forms, according to broken wall journey
Sequence difference can be divided into slight broken wall --- spore shape completely, on wall has aperture, moderate broken wall ---, and spore shape is imperfect, there have to be scarce
Damage, severe broken wall --- broken spore coat and spore content；
(1.2) sitosterol thin layer differentiates in duck wheat：
Duck wheat control medicinal material and each 3g of composition powder are weighed respectively, and 25mL chloroformic solutions, ultrasonic extraction is added
30min, filtering, takes 10mL filtrates, is evaporated on evaporating dish, is dissolved with chloroform and be settled to 2mL；Draw composition test liquid, hardship
Buckwheat control medicinal material solution l0 μ L are put respectively to be contained in same on the silica G plate that 0.3% sodium carboxymethylcellulose is alite paste, with body
Product is than being 7：3 petroleum ether-ether is solvent, is unfolded, and takes out, dries, and is sprayed with 10% sulfuric acid solution, 105 DEG C are dried to spot
Point colour developing, Rf values identical as reference substance have the spot of same color；
(2) quantitative study
(2.1) determination of polysaccharide in lucidum spore powder
Precision weighs anthrone 0.1g, is added 80% sulfuric acid solution 100mL, and stirring is allowed to dissolve, shake up to get；Anthrone-
Sulfuric acid chromogenic reaction is measured with colorimetric method at 625nm using DEXTROSE ANHYDROUS product as a contrast；
The preparation of reference substance solution：
Precision weighs 105 DEG C of dryings to the DEXTROSE ANHYDROUS 1.521mg of constant weight, accurately weighed, is placed in 10mL measuring bottles, adds
Distillation water dissolution is simultaneously diluted to scale, shakes up, is configured to a concentration of 0.1521mgmL-1Reference substance solution；
The preparation of test solution
Lucidum spore powder powder 2g is taken, it is accurately weighed, it sets in round-bottomed flask, adds water 60mL to stand 1 hour, be heated to reflux 4
Hour, it filters while hot, with a small amount of hot water washing nozzle and filter residue, filter residue and filter paper is set in flask, adds water 60mL, is heated to reflux
It 3 hours, filters while hot, merging filtrate, sets and be evaporated in water-bath, residue water 5mL dissolves, and ethyl alcohol is slowly added dropwise while stirring
75mL shakes up, and is placed 12 hours at 4 DEG C, and centrifugation discards supernatant liquid, and sediment is with hot water dissolving and is transferred in 50mL measuring bottles,
It lets cool, adds water to scale, shake up, take solution appropriate, centrifuge, precision measures supernatant 3mL and sets in 25mL measuring bottles, adds water to quarter
Degree, shake up to get；
Specification Curve of Increasing
Precision measures reference substance solution 0.4,0.6,0.8,1.0,1.2mL, sets in 10mL tool plug test tubes, respectively adds water to respectively
2.0mL, rapid accurate addition Liu Suan Onion ketone solution (accurate Cheng Qu Onion ketone 0.lg, add sulfuric acid 100mL to make dissolving, shake up) 6mL, stands
It shakes up, after placing 15 minutes, sets cooling 15 minutes in ice bath and take out immediately, using corresponding reagent as blank, according to ultraviolet-visible
Spectrophotometry measures absorbance at 625nm wavelength, and using absorbance as ordinate, it is bent to draw standard for a concentration of abscissa
Line, y=0.0051x-0.0036；
(2.2) naringenin assay in dendrobium candidum
Using dendrobium candidum active ingredient shaddock ped cellulose content in high effective liquid chromatography for measuring composition；Chromatographic condition is：
XB C18 chromatographic columns, specification be 4.6mm × 250mm, 5 μm；30 DEG C of column temperature；0.2% phosphoric acid of mobile phase is that A phases-methanol is B phases,
Gradient elution；Detection wavelength 290nm；Flow velocity 1.0mL/min；
Take naringenin reference substance appropriate, it is accurately weighed, it sets in 50mL brown measuring bottles, methanol is added to be dissolved to scale, shake up, make
Contain the reference substance solution of 26 μ g at every 1mL, it is spare；
Take composition 0.5g, it is accurately weighed, set in the round-bottomed flask of 50mL, precision be added the methanol that volume ratio is 4: 1 and
The mixed solution 25mL of 20% hydrochloric acid, weighs, sets in water-bath and be heated to reflux, let cool, be re-weighed, and supplies less loss weight with methanol, shakes
It is even, it stands, takes supernatant to be filtered through 0.22 μm of miillpore filter, take subsequent filtrate, 10 μ L of sample introduction that it is spare that test solution is made；
Precision draws reference substance solution and test solution injects liquid chromatograph, and naringenin is measured using one point external standard method
Content；
(2.3) in duck wheat extract rutin, quercetin content assay method foundation
The preparation of reference substance solution
Precision weighs control substance of Rutin 5.952mg, and Quercetin reference substance 3.075mg is respectively placed in 10mL volumetric flasks, uses
Methanol dissolves and is settled to scale, shake up to get rutin and Quercetin reference substance solution concentration be respectively 0.5952mg/mL and
0.3075mg/mL；
The preparation of test solution
Duck wheat medicinal material is taken, 10 times of amounts are added, 60% alcohol reflux of volumetric concentration extracts 3 times, 1 hour every time, merges filter
Liquid is concentrated under reduced pressure, and residue is dissolved with methanol, is transferred in 25mL measuring bottles, added methanol to scale, shake up, and filters, takes subsequent filtrate,
To obtain the final product；
Precision draws control substance of Rutin and Quercetin reference substance solution, and constant gradient dilutes 6 parts successively respectively, is implanted sequentially liquid
In addition chromatography, bioassay standard curve draw duck wheat test solution and inject liquid chromatograph；
Chromatographic condition is：Chromatographic column Thermo C18,4.6mm × 250mm, 5 μm；Mobile phase is -0.2% formic acid water of acetonitrile
Solution, gradient elution：0~18min, acetonitrile 17~40%, 18~19min, acetonitrile 40~17%；Volume flow is 1mL/min；
Detection wavelength：λ=256nm；Sample size：10μL；
With a concentration of abscissa of each reference substance, (C draws standard curve, reed by ordinate of the peak area A of each reference substance
Fourth equation of linear regression is Y=2 × 107+2506.8；The equation of linear regression of Quercetin is Y=4 × 107- 11208；
(2.4) in mulberry leaf the content of rutin assay：
The preparation of reference substance solution
Take control substance of Rutin appropriate, it is accurately weighed, solution of every 1mL containing 0.1mg is made with methanol；
The preparation of test solution
Mulberry Leaf lg is taken, it is accurately weighed, it sets in round-bottomed flask, adds methanol 50mL, be heated to reflux 30 minutes, filter, filter
Slag again with methanol 50mL is extracted 2 times with method, and solvent is recovered under reduced pressure in merging filtrate, and residue is dissolved with methanol, is transferred to 25mL amounts
Bottle in, add methanol to scale, shake up, filter, take subsequent filtrate to get；
The preparation of standard curve
Take 500 μ L of control substance of Rutin solution, with methanol half-and-half dilute 6 times, obtain concentration be respectively 0.0095mg/mL,
The reference substance solution of 0.019mg/mL, 0.0379mg/mL, 0.0758mg/mL, 0.1517mg/mL, 0.3034mg/mL inject liquid
Chromatography, sample introduction measure；
Chromatographic condition is：Chromatographic column be C18 columns, 4.6mm × 250mm, 5 μm；Mobile phase is that -0.5% phosphoric acid of methanol is water-soluble
Liquid, gradient elution：0~5min, 35% methanol；8~16min, 40% methanol；19~24min, 51% methanol；26~30min,
70% methanol；30~35min, 35% methanol.Volume flow 1mL/min；Detection wavelength 358nm, 10 μ L of sample size；
It is mapped with a concentration of abscissa, it is y=2 × 10 to obtain standard curve7X-10419, the range of linearity be 0.0095~
0.3034mg/mL, correlation coefficient r 1.
By the Qualitive test present invention with mulberry-leaf extract 125g, duck wheat extract 125g, dendrobium candidum 250g, ganoderma lucidum
The composition of conidia powder 125g, active polysaccharide, rutin, Quercetin and shaddock ped cellulose content are high in composition, are carried higher than single mulberry leaf
Take polysaccharide, rutin, quercitrin in the composition of object 125g, duck wheat extract 125g, dendrobium candidum 250g, lucidum spore powder 125g
The sum of the content of element and naringenin.Illustrate mulberry-leaf extract 125g, duck wheat extract 125g, dendrobium candidum 250g, ganoderma lucidum spore
The dissolution content of active component polysaccharides, rutin, Quercetin and naringenin can be significantly improved after the combination of sub- powder 125g.
The both effectiveness experiment of 4 reducing blood sugar and blood fat of embodiment
One, experiment material
1. drug, reagent and its preparation method
1.1 drugs and reagent
Test medicine：Composition (mulberry-leaf extract 125g, the duck wheat extract that the embodiment of the present invention 2 is prepared
125g, dendrobium candidum 250g, lucidum spore powder 125g) clinical quasi- dosage is 5g (amount of formulation)/60kg, mouse designs agent in experiment
Amount is 2.5g/kg (being respectively 30 times of quantity)；Rat design dosage be 0.21g/kg, 0.42g/kg, 0.84g/kg,
1.26g/kg (being respectively 2.5,5,10,15 times of quantity).
Melbine, Guizhou Shengjitang Pharmaceutical Co., Ltd., lot number：20160505.Rat design dosage is in experiment
0.125g/kg。
Alloxan, Sigma companies, lot number：Lot number：BCBW1234V.
Physiological saline, Shuanghe Pharmaceutical Ind Co., Ltd., Anhui, lot number：1603012D.
Citric acid, Sinopharm Chemical Reagent Co., Ltd., lot number：20130801.
Sodium citrate, Chengdu Ke Long chemical reagents factory, lot number：T20100913.
Glucose, Amresco companies of the U.S., lot number：0188.
Triglycerides (TG) assay kit (single reagent GPO-PAP methods), Bioengineering Research Institute, lot number are built up in Nanjing：
20161126。
Total cholesterol (TC) assay kit (single reagent GPO-PAP methods), Bioengineering Research Institute, lot number are built up in Nanjing：
20161126。
Rat blood serum insulin assay kit (Elisa methods), Bioengineering Research Institute, lot number are built up in Nanjing：
20161201。
Glycated serum protein (GSP) kit (NBT methods), Bioengineering Research Institute, lot number are built up in Nanjing：20161128.
Rat glycosylated hemoglobin assay kit (Elisa methods), Bioengineering Research Institute, lot number are built up in Nanjing：
20161201.1.2 drugs and preparation of reagents method
1.2.1 normal mouse blood glucose is tested
Present composition high dose group (2.5g amount of formulation/kg)：Extract powder 50g is weighed, 400ml is made into distilled water,
A concentration of 0.125g/ml, administration volume are 20ml/kg.Dosage is calculated by amount of formulation.
1.2.2 blood glucose in diabetic rats is tested
The present composition I (0.21g amount of formulation/kg)：Extract powder 4.2g is weighed, 400ml is made into distilled water, it is a concentration of
0.0105g/ml, administration volume are 20ml/kg.Dosage is calculated by amount of formulation.
The present composition II (0.42g amount of formulation/kg)：Extract powder 8.4g is weighed, 400ml, concentration are made into distilled water
For 0.021g/ml, administration volume is 20ml/kg.Dosage is calculated by amount of formulation.
The present composition III (0.84g amount of formulation/kg)：Extract powder 16.8g is weighed, 400ml, concentration are made into distilled water
For 0.042g/ml, administration volume is 20ml/kg.Dosage is calculated by amount of formulation.
The present composition IV (1.26g amount of formulation/kg)：Extract powder 25.2g is weighed, 400ml, concentration are made into distilled water
For 0.063g/ml, administration volume is 20ml/kg.Dosage is calculated by amount of formulation.
Melbine (0.125g/kg) takes 20 (0.25g/), 400ml is made into distilled water, a concentration of
0.0125g/ml, administration volume are 10ml/kg.Dosage is calculated by amount of formulation.
Alloxan (100mg/kg) weighs alloxan powder 2g and is dissolved in 200ml citric acid-sodium citrate buffer solutions,
A concentration of 0.01g/ml, intraperitoneal injection volume are 10ml/kg.
2. experimental animal and feed
Cleaning grade ICR male mices, weight 18-22g, are provided by Yangzhou University's comparative medicine center by 30, up-to-standard
Credit number：SCXK (Soviet Union) 2012-0004, for testing " influence to intact animal fasting blood-glucose ".
SPF grades of male SD rats, weight 150-170g, are provided by Zhejiang Province's Experimental Animal Center by 120, up-to-standard
Credit number：SCXK (Zhejiang) 2014-0001, for testing " influence to hyperglycemia model animal ".
Experimental mouse pellet, high thermal energy fodder are provided by Jiangning county Qinglongshan animal reproduction field.
3. laboratory apparatus
TD6001 electronic balances, Tianjin heavenly steed instrument plant.
Electronic analytical balance (FA2104), Shanghai precision instrument Co., Ltd.
Blood sugar test test paper (FAD glucose dehydrogenase methods), Bayer HealthCare Co.
Visit safe and comfortable contour TS blood glucose meters, Bayer HealthCare Co.
DHG-9140A type electric heating constant-temperature blowing drying boxes, the upper macro experimental facilities Co., Ltd of Nereid.
Direct-Q ultra-pure water instrument, MILLIPORE companies of the U.S..
Mix-100 mixing elfins, Hangzhou Ao Sheng Instrument Ltd..
Synergy HT microplate reader, Bio-Tek companies of the U.S..
Two, experiment condition and statistical method
Mouse uses plain particles forage feed, rat laundering period and formal experiment to be raised with plain particles feed for the 1st week,
Replace high thermal energy fodder within 3 weeks afterwards.Temperature：20~24 DEG C；Humidity：50~60%.Using 22.0 statistical softwares of SPSS, multigroup
Compare and use one-way analysis of variance, compare two-by-two between group and examined using t, data withIt indicates.
Three, experimental method and result
(1) intact animal hypoglycemic is tested
1. grouping, modeling and medication
Cleaning grade ICR male mices 30,18-22g are taken, after adapting to raising 3-5 days, it is preceding on an empty stomach to measure administration by fasting 5h
Blood glucose value.Then two groups are randomly divided into, every group 15, respectively (1) blank control group：Physiological saline 20ml/kg；(2) this hair
Bright composition high dose group：2.5g/kg.Daily gavage 1 time, gavage volume are 20ml/kg, fasting 5h after successive administration 35 days,
Measure fasting blood sugar after being administered.
2. observation index
2.1 ordinary circumstance
Observation mouse diet, drinking-water and urination daily.
2.2 weight
Measure administration before (0 week), administration mid-term (2 weeks), be administered after (5 weeks) weight.
2.3 blood glucose value
Fasting 5h after successive administration 35 days measures fasting blood sugar, and compares two groups of animal blood glucose values.
3. result
3.1 ordinary circumstances are observed
Blank control group and present composition high dose group mouse diet, drinking-water situation are normal, and urination is normal, gives
Without significant difference (being shown in Table 10) between the Mice Body recombination of blank control group, present composition high dose group before, during and after medicine.
10 present composition of table to normal mouse weight influence (N=15)
The influence of 3.2 pairs of normal mouse fasting blood-glucoses
Experimental result is shown：Present composition high dose group has no significant effect normal mouse fasting blood-glucose, with blank
Control group compares that there was no significant difference (being shown in Table 11).
Influence of 11 present composition of table to normal mouse fasting blood-glucose (mmol/L)
(2) to the influence of hyperglycemia model animal
1. basal plasma glucose
Cleaning grade male SD rat 120, weight 150-170g is taken, common that material is maintained to adapt to after raising 3-5 days, fasting 4
Hour, measure basal plasma glucose value (i.e.：It measures to (i.e. 0h) blood glucose value before glucose；After 2.5g/kg glucose, measurement 0.5,
2h blood glucose values).
2. grouping, administration and modeling
Fasting blood-glucose is chosen in the rat of 4-7mmol/L and carries out being grouped (without significant difference between fasting blood-glucose group) at random,
Every group 15, i.e. (1) blank control group, are not processed；(2) model control group：NS 10ml/kg；(3) melbine group：
0.125g/kg；(4) I group of the present composition：0.21g/kg；(5) II group of the present composition：0.42g/kg；(6) of the invention
III group of composition：0.84g/kg；(7) VII group of the present composition：1.26g/kg.In addition to (1) is organized, remaining each group successive administration 33
It.With material raising is maintained, high thermal energy fodder is replaced within latter 3 weeks within first 1 week.Fasting (can't help water) for 24 hours after being administered 4 weeks, and it is phonetic to give four oxygen
Pyridine 100mg/kg intraperitoneal injections continue to give high thermal energy fodder feeding 5 days after injection.3. observation index
3.1 ordinary circumstance
Observation rat diet, drinking-water and urination daily.
3.2 weight
Measure administration before (0 week), administration in (3 weeks), be administered after (5 weeks) weight.
3.3 fasting blood-glucoses and sugar tolerance
After administration 33 days, each group animal fasting 4h detects fasting blood-glucose and sugar tolerance (each group orally administration Portugal after 15min
Grape sugar 2.5g/kg is measured respectively to the blood glucose value of each group rat 0.5 after glucose, 2h), calculate Area under the curve of blood glucose (AUC)
Variation.
Area under the curve of blood glucose=0.25 × (0h blood glucose value+4 × 0.5h+3 × 2h of blood glucose value blood glucose values).
3.4 serum insulins, cholesterol, triglyceride and glycated serum protein, glycosylated hemoglobin
After blood sugar detection, rat eye socket takes blood 4ml, is stored at room temperature centrifugation (3000rpm, 10min) after 2h and takes serum,
Test serum insulin and cholesterol, triglyceride, glycated serum protein are horizontal, while anticoagulated whole blood 1ml being taken to survey HbAle
The level of albumen.
4. result
4.1 ordinary circumstances are observed
Model group rats dietary amount, amount of drinking water, weight increased, and urination is more；Melbine group with of the present invention group
It closes object group I, II, III, IV to be reduced compared with model group diet amount of drinking water, urine volume is reduced.After alloxan is injected intraperitoneally, model
Group rats death 6, dead 5 of melbine group, dead 3 of I group of the present composition, II group dead 2 of the present composition
Only, the present composition III dead 5, the present composition IV dead 5.
The influence of 4.2 pairs of diabetes rat body weights
(3 weeks) in (0 week) before administration, administration, be administered between the weight group for terminating (5 weeks) each group animal without significant difference (see
Table 12).
Influence of 12 present composition of table to rat body weight
Note：Before administration, administration in every group of number of rats n=15##P<0.01 compared with blank control group
The influence of 4.3 pairs of diabetes rat fasting blood-glucoses
Experimental result is shown：Model group rats fasting blood-glucose compared with blank control group rat is significantly raised, melbine
Group, II, III group of rat fasting blood-glucose of the present composition significantly reduce compared with model group and (are shown in Table 13).
Influence of 13 present composition of table to hyperglycemic rat fasting blood-glucose (mmol/L)
##P<0.01 compared with blank control group, * * P<0.01, * P<0.05 compared with model group
The influence of 4.4 pairs of diabetes rat sugar tolerances
Experimental result is shown：Model group rats Area under the curve of blood glucose compared with blank control group rat obviously increases, and two
First biguanides group, the present composition I, II, III, IV group of Area under the curve of blood glucose significantly reduce compared with model group and (are shown in Table 14).
Influence of 14 present composition of table to rat blood sugar area under the curve
##P<0.01 compared with blank control group, * * P<0.01 compared with model group
The influence of 4.5 pairs of diabetes rat glycated serum proteins and hemoglobin
Experimental result is shown：Without aobvious between blank control group, model group, each administration group glycated serum protein and hemoglobin group
Write difference (P>0.05) (15 are shown in Table).
Influence of 15 present composition of table to rat glycated serum protein, glycosylated hemoglobin
The influence of 4.6 pairs of diabetes rat Diagnostic Value of Fasting Serum insulin
Experimental result is shown：Model group rats Diagnostic Value of Fasting Serum insulin compared with blank control group significantly reduces, each to be administered
Group can increase the level of serum insulin, wherein melbine group, II, III group of the present composition and model to some extent
Group significantly increases compared to Diagnostic Value of Fasting Serum insulin level and (is shown in Table 16).
Influence of 16 present composition of table to rat limosis serum insulin
##P<0.01 compared with blank control group, * * P<0.01, * P<0.05 compared with model group
The influence of 4.7 pairs of diabetes rat blood fat
Experimental result is shown：Model group rats serum levels of triglyceride and total cholesterol level compared with blank control group is notable
It increases, II, III group of melbine group, present composition reduction serum levels of triglyceride are horizontal, melbine group, present invention combination
Object I, II, III group of reduction serum total cholesterol level, with significant difference (being shown in Table 17) compared with model group.
Influence of 17 present composition of table to rat blood serum triglyceride
##P<0.01 compared with blank control group, * * P<0.01, * P<0.05 compared with model group
Above the experimental results showed that：
1. present composition high dose group (2.5g/kg, 30 times) has no significant effect normal mouse fasting blood sugar.
2. the present composition I (0.21g/kg, 2.5 times) prevention administration can reduce blood glucose in diabetic rats area under the curve
(P<0.01), serum cholesterol (P<0.05)；
The present composition II (0.42g/kg, 5 times) prevention administration can significantly reduce diabetes rat fasting blood-glucose (P<
0.05), Area under the curve of blood glucose (P<0.01) serum insulin (P, is increased<0.05) serum levels of triglyceride (P, is reduced<0.01)
With cholesterol (P<0.01)；
The present composition III (0.84g/kg, 10 times) prevention administration can significantly reduce diabetes rat fasting blood-glucose (P<
0.01), Area under the curve of blood glucose (P<0.01) serum insulin (P, is increased<0.01) serum levels of triglyceride (P, is reduced<0.01)
With cholesterol (P<0.01)；
The present composition IV (1.26g/kg, 15 times) prevention administration can significantly reduce below blood glucose in diabetic rats curve
Product (P<0.01).
The present invention presses the above hypoglycemic experiment screening mulberry-leaf extract, duck wheat extract, dendrobium candidum, Reishi sporule
Other weight ratios of powder, including 1:1:1:1、1:2:1:1、2:1:1:1、1:1:1:2、3:1:1:1、1:3:1:1、1:1:3:1、1:
1:1:3 different weight than raw material composition, comparing result shows mulberry-leaf extract of the present invention, duck wheat extract, iron sheet stone
Dry measure used in former times, lucidum spore powder weight ratio be：1:1:2:When 1, have effects that best hypoglycemic, reducing blood lipid.Show in the weight
Under proportioning, 4 taste raw materials play synergistic effect, are matched for optimum amount of the present invention.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of have effects that prevent the composition of diabetes, which is characterized in that it includes following raw materials according：
Mulberry leaf, duck wheat, dendrobium candidum, lucidum spore powder.
2. according to claim 1 have effects that prevent the composition of diabetes, which is characterized in that it is by following parts by weight
Several raw materials are made：
125~250 parts of mulberry-leaf extract, 125~250 parts of duck wheat extract, 250~500 parts of dendrobium candidum, lucidum spore powder
125~250 parts.
3. according to claim 2 have effects that prevent the composition of diabetes, which is characterized in that it is by following parts by weight
Several raw materials are made：
125 parts of mulberry-leaf extract, 125 parts of duck wheat extract, 250 parts of dendrobium candidum, 125 parts of lucidum spore powder.
4. the preparation method of the composition prevent with diabetes according to claim 2 or 3, which is characterized in that
The mulberry-leaf extract is prepared by the following method to obtain：Mulberry leaf are taken, 10 times of amount 80% ethyl alcohol of volumetric concentration are added
Refluxing extraction 2 times, 1.5 hours every time, filtration obtained ethanol extract；The dregs of a decoction add 10 times of amount water to extract 1 time, each 1.5h, filter
It crosses, obtains water extract, ethanol extract and aqueous extract are merged, 60 DEG C of relative densities 1.20 are concentrated under reduced pressure into, it is spare；It takes dense
Contracting liquid, decompression or spray drying, crush, and sieving obtains mulberry-leaf extract；
The duck wheat extract is prepared by the following method to obtain：Bitter buckwheat is taken, 10 times of amount 60% second of volumetric concentration are added
Alcohol reflux extracts 3 times, and 1.5 hours every time, filtration, merging filtrate was recovered under reduced pressure ethyl alcohol, is concentrated into 60 DEG C of relative densities 1.20,
It is spare；It takes concentrate, normal pressure or is dried under reduced pressure, low-temperature grinding, be sieved, obtain duck wheat extract, it is spare；
The dendrobium candidum is prepared by the following method to obtain：It takes 100 DEG C of dendrobium candidum to dry 30 minutes, crushes, mistake
100 mesh sieve, and obtain dendrobium officinale powder.
5. claims 1 to 3 any one of them has effects that prevent the Quality evaluation of the composition of diabetes：Its feature
It is to include the following steps：
(1) qualitative research
(1.1) microscopical characters of glossy ganoderma spore powder with crushed sporoderm
Microscopical characters：Glossy ganoderma spore powder with crushed sporoderm is sepia powder, and appearance is extremely fine and smooth, without luming at top, is referred to mother and is eaten
Finger, which is gently consulted, to be touched, and has smooth fine and smooth sense, no adhesion, the feeling without foreign matters such as grit；It is observed under 1500 times of amplification factor, no
Exosporium-broken spore is complete, plentiful, double-walled construction is clear, and outer wall is colourless, and inner wall has spinule, filbert；Individual morphology is consistent, in ellipse
Shape；Top is truncate, and size is about 8.5~11 × 5.2~6.9 μm；And exosporium-broken spore then has several forms, it is different according to broken wall program
Can be divided into slight broken wall --- spore shape is complete, has aperture, moderate broken wall on wall --- spore shape is imperfect, has defect, again
Spend broken wall --- broken spore coat and spore content；
(1.2) sitosterol thin layer differentiates in duck wheat：
Duck wheat control medicinal material and each 3g of composition powder are weighed respectively, and 25mL chloroformic solutions, ultrasonic extraction 30min, mistake is added
Filter, takes 10mL filtrates, is evaporated on evaporating dish, is dissolved with chloroform and be settled to 2mL；Draw composition test liquid, duck wheat control
Medicinal material solution l0 μ L are put respectively to be contained in same on the silica G plate that 0.3% sodium carboxymethylcellulose is alite paste, with volume ratio for 7：
3 petroleum ether-ether is solvent, is unfolded, and takes out, dries, and is sprayed with 10% sulfuric acid solution, 105 DEG C are dried to spot development, day
Light is inspected；
(2) quantitative study
(2.1) determination of polysaccharide in lucidum spore powder
Precision weighs anthrone 0.1g, is added 80% sulfuric acid solution 100mL, and stirring is allowed to dissolve, shake up to get；Anthrone Sulphuric acid
Chromogenic reaction is measured with colorimetric method at 625nm using DEXTROSE ANHYDROUS product as a contrast；
The preparation of reference substance solution：
Precision weighs the dry DEXTROSE ANHYDROUS to constant weight, accurately weighed, is placed in measuring bottle, adds distillation water dissolution and is diluted to quarter
Degree, shakes up, is configured to reference substance solution；
The preparation of test solution
Lucidum spore powder powder is taken, it is accurately weighed, it sets in round-bottomed flask, water is added to stand, be heated to reflux, filter while hot, with a small amount of
Hot water washing nozzle and filter residue, filter residue and filter paper are set in flask, add water, are heated to reflux, and are filtered while hot, and merging filtrate sets water
It is evaporated in bath, residue water dissolution is slowly added dropwise ethyl alcohol, shakes up while stirring, and centrifugation discards supernatant liquid, sediment hot water
It dissolves and is transferred in measuring bottle, let cool, add water to scale, shake up, take solution appropriate, centrifuge, precision measures supernatant and sets measuring bottle
In, add water to scale, shake up to get；
Specification Curve of Increasing
Precision measures reference substance solution 0.4,0.6,0.8,1.0,1.2mL, sets in 10mL tool plug test tubes, respectively adds water to respectively
2.0mL, rapid accurate addition Liu Suan Onion ketone solution, shakes up, and after placement, sets cooling in ice bath immediately, is sky with corresponding reagent
In vain, according to UV-VIS spectrophotometry, absorbance, using absorbance as ordinate, a concentration of horizontal seat are measured at 625nm wavelength
Mark draws standard curve；
(2.2) naringenin assay in dendrobium candidum
Using dendrobium candidum active ingredient shaddock ped cellulose content in high effective liquid chromatography for measuring composition；Chromatographic condition is：XB
C18 chromatographic columns, specification be 4.6mm × 250mm, 5 μm；30 DEG C of column temperature；0.2% phosphoric acid of mobile phase is that A phases-methanol is B phases, gradient
Elution；Detection wavelength 290nm；Flow velocity 1.0mL/min；
Take naringenin reference substance appropriate, it is accurately weighed, it sets in 50mL brown measuring bottles, methanol is added to be dissolved to scale, shake up, shaddock is made
Skin element reference substance solution, it is spare；
Dendrobium candidum is taken, is set in round-bottomed flask, the mixed solution of methanol and 20% hydrochloric acid is added in precision, weighs, and sets in water-bath and adds
Heat reflux, lets cool, is re-weighed, supply less loss weight with methanol, shake up, and stands, takes supernatant to be filtered through 0.22 μm of miillpore filter, take
Subsequent filtrate, 10 μ L of sample introduction, it is spare to be made test solution；
Precision draws reference substance solution and test solution injects liquid chromatograph, using one point external standard method, by above-mentioned chromatostrip
Part is measured；
(2.3) in duck wheat extract rutin, quercetin content assay method foundation
The preparation of reference substance solution
Precision weighs control substance of Rutin, and Quercetin reference substance is respectively placed in volumetric flask, and scale is dissolved and be settled to methanol,
It shakes up to get rutin and Quercetin reference substance solution；
The preparation of test solution
Duck wheat medicinal material is taken, alcohol reflux extraction is added, merging filtrate is concentrated under reduced pressure, and residue is dissolved with methanol, is transferred to measuring bottle
In, add methanol to scale, shake up, filter, take subsequent filtrate to get；
Precision draws control substance of Rutin and Quercetin reference substance solution, is half-and-half diluted 6 times with methanol, is implanted sequentially liquid chromatogram
In addition instrument, bioassay standard curve draw duck wheat test solution and inject liquid chromatograph；
Chromatographic condition is：Chromatographic column Thermo C18,4.6mm × 250mm, 5 μm；Mobile phase is that -0.2% formic acid of acetonitrile is water-soluble
Liquid, gradient elution：0~18min, acetonitrile 17~40%, 18~19min, acetonitrile 40~17%；Volume flow is 1mL/min；Inspection
Survey wavelength：λ=256nm；Sample size：10μL；
With a concentration of abscissa C of each reference substance, standard curve is drawn by ordinate of the peak area A of each reference substance；
(2.4) in mulberry leaf the content of rutin assay：
The preparation of reference substance solution
Take control substance of Rutin appropriate, it is accurately weighed, solution of every 1mL containing 0.1mg is made with methanol；
The preparation of test solution
Mulberry Leaf is taken, it is accurately weighed, it sets in round-bottomed flask, adds methanol, be heated to reflux, filter, filter residue again with methanol is carried with method
It takes 2 times, solvent is recovered under reduced pressure in merging filtrate, and residue is dissolved with methanol, is transferred in 25mL measuring bottles, adds methanol to scale, shakes
It is even, filtration, take subsequent filtrate to get；
The preparation of standard curve
500 μ L of control substance of Rutin solution are taken, are half-and-half diluted 6 times with methanol, it is respectively 0.0095mg/mL, 0.019mg/ to obtain concentration
The reference substance solution of mL, 0.0379mg/mL, 0.0758mg/mL, 0.1517mg/mL, 0.3034mg/mL inject liquid chromatogram
Instrument, sample introduction measure；
Chromatographic condition is：Chromatographic column be C18 columns, 4.6mm × 250mm, 5 μm；Mobile phase is -0.5% phosphate aqueous solution of methanol, ladder
Degree elution：0~5min, 35% methanol；8~16min, 40% methanol；19~24min, 51% methanol；26~30min, 70% first
Alcohol；30~35min, 35% methanol, volume flow 1mL/min；Detection wavelength 358nm, 10 μ L of sample size；
With a concentration of abscissa, peak area is that ordinate draws standard curve.
6. the Quality evaluation of the composition prevent with diabetes according to claim 5：It is characterized in that
Include the following steps：
(1) qualitative research
(1.1) microscopical characters of glossy ganoderma spore powder with crushed sporoderm
Microscopical characters：Glossy ganoderma spore powder with crushed sporoderm is sepia powder, and appearance is extremely fine and smooth, without luming at top, is referred to mother and is eaten
Finger, which is gently consulted, to be touched, and has smooth fine and smooth sense, no adhesion, the feeling without foreign matters such as grit；It is observed under 1500 times of amplification factor, no
Exosporium-broken spore is complete, plentiful, double-walled construction is clear, and outer wall is colourless, and inner wall has spinule, filbert；Individual morphology is consistent, in ellipse
Shape；Top is truncate, and size is about 8.5~11 × 5.2~6.9 μm；And exosporium-broken spore then has several forms, it is different according to broken wall program
Can be divided into slight broken wall --- spore shape is complete, has aperture, moderate broken wall on wall --- spore shape is imperfect, has defect, again
Spend broken wall --- broken spore coat and spore content；
(1.2) sitosterol thin layer differentiates in duck wheat：
Duck wheat control medicinal material and each 3g of composition powder are weighed respectively, and 25mL chloroformic solutions, ultrasonic extraction 30min, mistake is added
Filter, takes 10mL filtrates, is evaporated on evaporating dish, is dissolved with chloroform and be settled to 2mL；Draw composition test liquid, duck wheat control
Medicinal material solution l0 μ L are put respectively to be contained in same on the silica G plate that 0.3% sodium carboxymethylcellulose is alite paste, with volume ratio for 7：
3 petroleum ether-ether is solvent, is unfolded, and takes out, dries, and is sprayed with 10% sulfuric acid solution, 105 DEG C are dried to spot development, day
Light is inspected；
(2) quantitative study
(2.1) determination of polysaccharide in lucidum spore powder
Precision weighs anthrone 0.1g, is added 80% sulfuric acid solution 100mL, and stirring is allowed to dissolve, shake up to get；Anthrone Sulphuric acid
Chromogenic reaction is measured with colorimetric method at 625nm using DEXTROSE ANHYDROUS product as a contrast；
The preparation of reference substance solution：
Precision weighs 105 DEG C of dryings to the DEXTROSE ANHYDROUS 1.521mg of constant weight, accurately weighed, is placed in 10mL measuring bottles, adds distillation
Water dissolution is simultaneously diluted to scale, shakes up, is configured to a concentration of 0.1521mgmL-1Reference substance solution；
The preparation of test solution
Lucidum spore powder powder 2g is taken, it is accurately weighed, it sets in round-bottomed flask, adds water 60mL to stand 1 hour, be heated to reflux 4 hours,
It filters while hot, with a small amount of hot water washing nozzle and filter residue, filter residue and filter paper is set in flask, adds water 60mL, it is small to be heated to reflux 3
When, it filters while hot, merging filtrate, sets and be evaporated in water-bath, residue water 5mL dissolves, and ethyl alcohol 75mL is slowly added dropwise while stirring, shakes
It is even, it is placed 12 hours at 4 DEG C, centrifuges, discard supernatant liquid, sediment is with hot water dissolving and is transferred in 50mL measuring bottles, lets cool, adds
Water shakes up to scale, takes solution appropriate, and centrifugation, precision measures supernatant 3mL and sets in 25mL measuring bottles, adds water to scale, shakes up,
To obtain the final product；
Specification Curve of Increasing
Precision measures reference substance solution 0.4,0.6,0.8,1.0,1.2mL, sets in 10mL tool plug test tubes, respectively adds water to respectively
2.0mL, rapid accurate addition Liu Suan Onion ketone solution (accurate Cheng Qu Onion ketone 0.lg, add sulfuric acid 100mL to make dissolving, shake up) 6mL, stands
It shakes up, after placing 15 minutes, sets cooling 15 minutes in ice bath and take out immediately, using corresponding reagent as blank, according to ultraviolet-visible
Spectrophotometry measures absorbance at 625nm wavelength, and using absorbance as ordinate, it is bent to draw standard for a concentration of abscissa
Line, y=0.0051x-0.0036；
(2.2) naringenin assay in dendrobium candidum
Using dendrobium candidum active ingredient shaddock ped cellulose content in high effective liquid chromatography for measuring composition；Chromatographic condition is：XB
C18 chromatographic columns, specification be 4.6mm × 250mm, 5 μm；30 DEG C of column temperature；0.2% phosphoric acid of mobile phase is that A phases-methanol is B phases, gradient
Elution；Detection wavelength 290nm；Flow velocity 1.0mL/min；
Take naringenin reference substance appropriate, it is accurately weighed, it sets in 50mL brown measuring bottles, methanol is added to be dissolved to scale, shake up, be made every
1mL contains the reference substance solution of 26 μ g, spare；
Composition 0.5g is taken, it is accurately weighed, it sets in the round-bottomed flask of 50mL, the methanol and 20% that volume ratio is 4: 1 is added in precision
The mixed solution 25mL of hydrochloric acid, weighs, sets in water-bath and be heated to reflux, let cool, be re-weighed, and supplies less loss weight with methanol, shakes up, quiet
It sets, supernatant is taken to be filtered through 0.22 μm of miillpore filter, take subsequent filtrate, 10 μ L of sample introduction that it is spare that test solution is made；
Precision draws reference substance solution and test solution injects liquid chromatograph, and measuring naringenin using one point external standard method contains
Amount；
(2.3) in duck wheat extract rutin, quercetin content assay method foundation
The preparation of reference substance solution
Precision weighs control substance of Rutin 5.952mg, and Quercetin reference substance 3.075mg is respectively placed in 10mL volumetric flasks, uses methanol
Dissolve and be settled to scale, shake up to get rutin and Quercetin reference substance solution concentration be respectively 0.5952mg/mL and
0.3075mg/mL；
The preparation of test solution
Duck wheat medicinal material is taken, 10 times of amounts are added, 60% alcohol reflux of volumetric concentration extracts 3 times, and 1 hour every time, merging filtrate subtracted
Pressure concentration, residue dissolves with methanol, is transferred in 25mL measuring bottles, added methanol to scale, shake up, and filters, take subsequent filtrate to get；
Precision draws control substance of Rutin and Quercetin reference substance solution, and constant gradient dilutes 6 parts successively respectively, is implanted sequentially liquid phase color
In addition spectrometer, bioassay standard curve draw duck wheat test solution and inject liquid chromatograph；
Chromatographic condition is：Chromatographic column Thermo C18,4.6mm × 250mm, 5 μm；Mobile phase is that -0.2% formic acid of acetonitrile is water-soluble
Liquid, gradient elution：0~18min, acetonitrile 17~40%, 18~19min, acetonitrile 40~17%；Volume flow is 1mL/min；Inspection
Survey wavelength：λ=256nm；Sample size：10μL；
With a concentration of abscissa of each reference substance, (C draws standard curve, rutin line by ordinate of the peak area A of each reference substance
Property regression equation be Y=2 × 107+2506.8；The equation of linear regression of Quercetin is Y=4 × 107- 11208；
(2.4) in mulberry leaf the content of rutin assay：
The preparation of reference substance solution
Take control substance of Rutin appropriate, it is accurately weighed, solution of every 1mL containing 0.1mg is made with methanol；
The preparation of test solution
Mulberry Leaf lg is taken, it is accurately weighed, it sets in round-bottomed flask, adds methanol 50mL, be heated to reflux 30 minutes, filter, filter residue is again
With methanol 50mL, being extracted 2 times with method, solvent is recovered under reduced pressure in merging filtrate, and residue is dissolved with methanol, is transferred in 25mL measuring bottles,
Add methanol to scale, shake up, filter, take subsequent filtrate to get；
The preparation of standard curve
500 μ L of control substance of Rutin solution are taken, are half-and-half diluted 6 times with methanol, it is respectively 0.0095mg/mL, 0.019mg/ to obtain concentration
The reference substance solution of mL, 0.0379mg/mL, 0.0758mg/mL, 0.1517mg/mL, 0.3034mg/mL inject liquid chromatogram
Instrument, sample introduction measure；
Chromatographic condition is：Chromatographic column be C18 columns, 4.6mm × 250mm, 5 μm；Mobile phase is -0.5% phosphate aqueous solution of methanol, ladder
Degree elution：0~5min, 35% methanol；8~16min, 40% methanol；19~24min, 51% methanol；26~30min, 70% first
Alcohol；30~35min, 35% methanol, volume flow 1mL/min；Detection wavelength 358nm, 10 μ L of sample size；
It is mapped with a concentration of abscissa, it is y=2 × 10 to obtain standard curve7X-10419, the range of linearity be 0.0095~
0.3034mg/mL, correlation coefficient r 1.
7. claims 1 to 3 any one of them has effects that the composition for preventing diabetes is preparing hypoglycemic drug or guarantor
Application in strong product.
8. claims 1 to 3 any one of them has effects that the composition for preventing diabetes is preparing blood lipid-lowering medicine or guarantor
Application in strong product.
9. claims 1 to 3 any one of them has effects that the composition for preventing diabetes is preparing hypoglycemic and reducing blood lipid
Application in drug or health products.
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