CN104688933A - Composition of Pu'er tea effective component and application of composition in preparation of medicine or health food for reducing blood glucose - Google Patents

Abstract

The invention provides a composition containing Pu'er tea effective component, which comprises 0-5 weight part of tea polyphenol component, 6-12 weight part of theabrownin component and 0-1 weight part of caffeine component; the invention also provides an application of the composition in preparation of medicine or food for treating or preventing diabetes.

Description

A kind of compositions of Folium camelliae assamicae effective ingredient and the application in the medicine or health food of preparation treatment blood sugar lowering thereof

Technical field

The present invention relates to medicine and field of health care food, be specifically related to a kind of compositions of the effective ingredient proportioning containing Folium camelliae assamicae and the application in the medicine or health food of preparation treatment blood sugar lowering thereof.

Background technology

Folium camelliae assamicae original producton location is mainly in Simao Diqu and the Xishuangbanna in Yunnan.Along with the popularization of large leaf, Sichuan, Guangdong, Guangxi also become the province that Folium camelliae assamicae is produced.Along with expanding economy, the enhancing of people's health care consciousness, Folium camelliae assamicae because of mellow time of its flavour sweet, the fine quality of CHENXIANG uniqueness and its special health-care effect of human body is played to concern and the attention of the whole society, product is deeply by the favor of consumer.

Warm in nature and the storage tolerance of Folium camelliae assamicae, be suitable for cooking with or bubble drink.The many historical book of Ancient Times in China has much about the record of Folium camelliae assamicae effect: ZHAO Xue-Min supplementary Amplifications of the Compendium of Materia Medica is recorded, and Pu Hei is as paint, and relieving alcoholic intoxication the first, eliminates indigestion and phlegm, and clearing stomach is promoted the production of body fluid, and power is outstanding greatly also; Cloud again in " woody part ", Pu'er tea paste can control all kinds of diseases and ailments, as tripe catches cold, disperses with ginger decoction, and perspiration can be healed, and it is dry that mouth breaks larynx, pain of being heated, and chews namely heal night of making a slip of the tongue with five points; " Pu'er is helped digest and is dispersed cold, has Detoxication in record that Chen Zonghai writes " interview of the Room, Simao "." clear scholar's Song hero " occupying diet spectrum with breath " cloud " Pu'er product person, highly seasoned kind wind-phlegm of telling disappears meat, all filthy pathogen in summer string-shaped mass in the abdomen stomachache, and the diseases such as cholera dysentery are from the beginning of, often healing of drink ".Forefathers think to Pu'er tea health-care experience as can be seen here, and Folium camelliae assamicae has and helps digestion except poison, and regulate the flow of vital energy swollen, removing heat-phlegm, wind dispelling relieving alcoholic intoxication, controls the effects such as dysentery is antibacterial.

Diabetes are a kind of metabolism disorder diseases, and feature shows as hyperglycemia, glycosuria, negative nitrogen balance, occurs ketonemia sometimes.Diabetes can cause a series of complication, as downright bad in retinopathy, nervous system disease and peripheral vessels etc.Type 2 diabetes mellitus also claims non-insulin-dependent diabetes mellitus, and a kind of disease of multifactor initiation, shows as insulin resistant, not only relevant with Hyperinsulinism and hyperglycemia, also relevant with arteriosclerosis, hypertension, dyslipidemia and X syndrome.

Chinese scholars has done a large amount of previous research work, and Folium camelliae assamicae has blood sugar lowering blood fat effect really from clinical angularly preliminary proof.According to the various ingredients contained in literature search Folium camelliae assamicae, such as abrownin component, tea polyphenols component and caffeine component etc. have corresponding report in blood sugar lowering, such as, in bibliographical information, tea polyphenols can reduce sky and takes blood glucose and post-prandial glycemia under heavy dose, tea pigment (comprising the water colo(u)rs such as theaflavin, thearubigins and abrownin) has been developed to capsule and has been applied in the middle of the auxiliary treatment of diabetes, especially with the auxiliary treatment etc. of the type 2 diabetes mellitus patient of microcirculation disturbance.But what effect is these components in Folium camelliae assamicae play all respectively in effect of lowering blood sugar, which component contributions is maximum, and which type of compatibility proportioning can play maximum function of reducing blood sugar, there is no conclusion.

For probing into the biologic activity after the respective effect of this three classes component and their compatibility, invention has been deep research, through the extraction and isolation to abrownin component, tea polyphenols component and caffeine component, and they being carried out multiple proportioning screening.

Summary of the invention

The invention provides a kind of compositions containing Folium camelliae assamicae effective ingredient, comprise the tea polyphenols component of 0 ~ 5 weight portion, the abrownin component of 6 ~ 12 weight portions and the caffeine component of 0 ~ 1 weight portion, described tea polyphenols component, abrownin component, caffeine component are prepared as follows:

Abrownin method for preparing ingredients thereof:

Folium camelliae assamicae adds water intensification lixiviate 1-3 time, each 0.5-1 hour, 90-100% ethanol (v/v) is added to alcohol content 70-80%(v/v) after extracting solution is concentrated, quiet to more than 12 hours, the drying of alcohol hypostasis obtains theabrownin crude product, theabrownin crude product use water redissolves, and filters, and filtrate is concentrated into and dryly obtains abrownin component;

Tea polyphenols method for preparing ingredients thereof:

Folium camelliae assamicae adds water intensification lixiviate 1-3 time, each 0.5-1 hour, adds 90-100% ethanol (v/v) to alcohol content 70-80%(v/v after extracting solution is concentrated), quietly obtain supernatant to more than 12 hours, obtain crude product through concentrate drying, crude product is soluble in water, add aluminum chloride, regulate pH value to 4-6 with sodium bicarbonate solution, leave standstill, centrifugal, sediment separate out sulfuric acid solution dissolves, and solution with ethyl acetate extracts, and extract removes desolventizing and obtains tea polyphenols component;

Caffeine method for preparing ingredients thereof:

Folium camelliae assamicae adds water intensification lixiviate 1-3 time, each 0.5-1 hour, 90-100% ethanol (v/v) is added to alcohol content 70-80%(v/v) after extracting solution is concentrated, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, add aluminum chloride, regulate pH value to 4-6 with sodium bicarbonate solution, leave standstill, centrifugal, obtain supernatant, supernatant obtains solid through concentrate drying, redissolve with water, filter, cross macroporous adsorptive resins, and wash with water and obtain effluent, loading effluent and water lotion concentrate, with chloroform extraction, extract obtains caffeine component through separation.

Further, tea polyphenols component of the present invention, abrownin component, caffeine component are preferably prepared as follows:

Abrownin method for preparing ingredients thereof:

Folium camelliae assamicae adds water 80 DEG C of high-temp extracting three times of 10 times amount, each 0.5-1 hour, merge extractive liquid, less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 95% ethanol alcohol and be sink to 80%, quiet to more than 12 hours, the drying of alcohol hypostasis vacuum decompression obtains theabrownin crude product, redissolves filter with water, filtrate less than 70 DEG C is evaporated to dry, obtains abrownin component;

Tea polyphenols method for preparing ingredients thereof:

Folium camelliae assamicae adds water 80 DEG C of high-temp extracting three times of 10 times amount, each 0.5-1 hour, and merge extractive liquid, less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 95% ethanol alcohol and be sink to 80%, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 5.4 with sodium bicarbonate solution, leave standstill, centrifugal, precipitation sulfuric acid solution turns molten, be extracted with ethyl acetate, extract removes desolventizing and obtains tea polyphenols component;

Caffeine method for preparing ingredients thereof:

Folium camelliae assamicae adds water 80 DEG C of high-temp extracting three times of 10 times amount, each 0.5-1 hour, merge extractive liquid, less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 95% ethanol alcohol and be sink to 80%, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 5.4 with sodium bicarbonate solution, leave standstill, centrifugal, supernatant obtains solid through concentrate drying, add water redissolution, filter, filtrate crosses macroporous adsorptive resins, and wash with water and obtain effluent and water lotion, reduced vacuum is concentrated into proportion 1.10 ~ 1.15, with chloroform extraction three times, extract obtains caffeine component through separation.

Compositions containing Folium camelliae assamicae effective ingredient of the present invention, preferably includes 5 weight portion tea polyphenols components, the abrownin component of 12 weight portions and the caffeine component of 0 weight portion.

According to another embodiment of the present invention, described compositions, preferably includes 0 weight portion tea polyphenols component, the abrownin component of 6 weight portions and the caffeine component of 0.5 weight portion

According to another embodiment of the present invention, described compositions, preferably includes 0 weight portion tea polyphenols component, the abrownin component of 12 weight portions and the caffeine component of 1 weight portion.

According to another embodiment of the present invention, described compositions, preferably includes 2.5 weight portion tea polyphenols components, the abrownin component of 12 weight portions and the caffeine component of 0 weight portion.

According to another embodiment of the present invention, described compositions, preferably includes 5 weight portion tea polyphenols components, the abrownin component of 6 weight portions and the caffeine component of 0 weight portion.

According to another embodiment of the present invention, described compositions, preferably includes 0 weight portion tea polyphenols component, the abrownin component of 12 weight portions and the caffeine component of 0 weight portion.

The present invention also provides the described compositions containing Folium camelliae assamicae effective ingredient, and it is preparing the application in the medicine or food for the treatment of or preventing diabetes.

Treatment of the present invention or the purposes prevented diabetes are realized by the activity of Inhibiting α-glucosidase.

Treatment of the present invention or the purposes prevented diabetes are by suppressing the activity of aldose reductase to realize.

Treatment of the present invention or the purposes prevented diabetes are by Profilin tyrosine phosphatase-1B(protein tyrosine phosphatase1B, PTP-1B) activity realize.

The present invention also comprises, the medicine containing described Folium camelliae assamicae effective ingredient or food compositions, and food of the present invention comprises health food, includes but not limited to following food form: beverage, milk product, Folium Camelliae sinensis, cake, confection etc.

Active constituents of medicine in pharmaceutical composition of the present invention, its in the composition shared percentage by weight can be 0.01-99.99%, all the other are medicine acceptable carrier.Pharmaceutical composition of the present invention, exists in a unit, and described unit dosage form refers to the unit of preparation, as every sheet of tablet, and every capsules of capsule, every bottle of oral liquid, granule every bag etc.

Pharmaceutical composition of the present invention can be any pharmaceutically useful dosage form, and these dosage forms comprise: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, injection, suppository, ointment, plaster, cream, spray, drop, patch.Preparation of the present invention, preferably peroral dosage form, as: capsule, tablet, oral liquid, granule, pill, powder, sublimed preparation, unguentum etc.

Pharmaceutical composition of the present invention, the preparation of its oral administration can containing conventional excipient, and such as binding agent, filler, diluent, tablet agent, lubricant, disintegrating agent, coloring agent, flavoring agent and wetting agent, can carry out coating to tablet if desired.

The filler be suitable for comprises cellulose, mannitol, lactose and other similar filler.Suitable disintegrating agent comprises starch, polyvinylpyrrolidone and starch derivatives, such as sodium starch glycollate.Suitable lubricant comprises, such as magnesium stearate.The suitable acceptable wetting agent of medicine comprises sodium lauryl sulphate.

By mixing, fill, the method that tabletting etc. are conventional prepares solid oral composition.Repeatedly mix and active substance can be made to be distributed in those compositionss of a large amount of filler of whole use.

The form of oral liquid can be such as aqueous or oily suspensions, solution, Emulsion, syrup or elixir, or can be the composite dry products of a kind of available water before use or other suitable carrier.This liquid preparation can containing conventional additive, such as suspending agent, such as sorbitol, syrup, methylcellulose, gelatin, hydroxyethyl-cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agent, such as lecithin, anhydro sorbitol monooleate or arabic gum; Non-aqueous carrier (they can comprise edible oil), the oily ester of the such as ester of almond oil, fractionated coconut oil, such as glycerol, propylene glycol or ethanol; Antiseptic, such as para hydroxybenzene methyl ester or propyl p-hydroxybenzoate or sorbic acid, and if need, can containing conventional flavouring agent or coloring agent.

For injection, the fluid unit dosage form of preparation contains active substance of the present invention and sterile carrier.According to carrier and concentration, this compound can be suspended or dissolve.The preparation of solution is normally by being dissolved in a kind of carrier by active substance, filter-sterilized before being loaded a kind of suitable bottle or ampoule, then seals.Adjuvant such as a kind of local anesthetic, antiseptic and buffer agent also can be dissolved in this carrier.In order to improve its stability, by freezing for this compositions after loading bottle, and under vacuo water can be removed.

Pharmaceutical composition of the present invention, applicable medicine acceptable carrier is optionally added when being prepared into medicament, described medicine acceptable carrier is selected from: mannitol, sorbitol, sodium pyrosulfite, sodium sulfite, sodium thiosulfate, cysteine hydrochloride, TGA, methionine, vitamin C, EDETATE SODIUM, Ethylenediaminetetraacetic Acid Calcium Salt, the alkali-metal carbonate of monovalence, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulphuric acid, phosphoric acid, aminoacid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and its derivates, alginate, gelatin, polyvinylpyrrolidone, glycerol, POLYSORBATE 80, agar, calcium carbonate, calcium bicarbonate, surfactant, Polyethylene Glycol, cyclodextrin, beta-schardinger dextrin-, phospholipid material, Kaolin, Pulvis Talci, calcium stearate, magnesium stearate etc.

Compositions of the present invention according to the situation determination usage and dosage of patient, can take three every day in use, each 1-20 agent, as: 1-20 bag or grain or sheet.

Be below the detailed content of this research:

Test the best compatibility screening experiment of 1 Folium camelliae assamicae active component blood sugar lowering

The present invention is the compatibility proportioning adopting orthogonal experimental method design tea polyphenols component, caffeine component and abrownin component, by orthogonal table L9(34) design arrangement 9 experimental grouies, with C57BL/6J mice as a control group, KKAy mice random packet, successive administration 4 weeks, using fasting glucose, carbohydrate tolerance and external target enzyme inhibition as observation index, the best prescription of screening compatibility proportioning.

1. experiment material

1.1 laboratory animal

KKAy mice (SPF level), female 180,5-6 week age.Purchased from Beijing HFK Bio-Technology Co., Ltd., credit number: SCXK (capital) 2009-0004.

1.2 main agents and medicine

1. Rosiglitazone Maleate Tablets, GlaxoSmithKline PLC (Tianjin) company limited, specification: 4mg/ sheet, lot number: 09090110.

2. tea polyphenols component, lot number 20111216, khaki powder.Thered is provided by Tian Shi power group academy Chinese medicine.Deposit in the sample cabinet of pharmacology institute's test sample room, preserve under lucifuge room temperature.

3. abrownin component, lot number 20111209, the little block of dark brown.Thered is provided by Tian Shi power group academy Chinese medicine.Deposit in the sample cabinet of pharmacology institute's test sample room, preserve under lucifuge room temperature.

4. caffeine component, lot number 20111115, pale powder.Thered is provided by Tian Shi power group academy Chinese medicine.Deposit in the sample cabinet of pharmacology institute's test sample room, preserve under lucifuge room temperature.

5. blood sugar test reagent (BIOSEN5030 type blood glucose/lactic acid analysis instrument matched reagent).

1.3 constituent contents measure

It should be noted that, because tea polyphenols component, abrownin component and caffeine component are extract, it itself not all one matter, it is not sterling, this several component can be purified without any a kind of extracting method at present, such as, in the tea polyphenols component adopting method of the present invention to prepare, except containing except tea polyphenols material, also can be mixed with some tea polysaccharide, abrownin and caffeine constituents, same, in abrownin component, also some tea polysaccharide, tea polyphenols and caffeine constituents etc. can be contained.Therefore, needs calculate in tea polyphenols component, abrownin component and the caffeine component prepared according to preparation method of the present invention, the content of definite tea polyphenols, abrownin, caffeine and tea polysaccharide.The computational methods of various composition are as follows:

The assay of accurate abrownin in each component: when measuring the sample of form of extract, the paste-forming rate according to institute's test sample product is converted abrownin computing formula in document, conversion the results are shown in following table.

Sampling amount=3 × paste-forming rate %

In formula: E _b---abrownin absorbance

The assay of accurate tea polyphenols in each component: the same National Standard of the People's Republic of China of tea polyphenols composition Precise levels assay method " GB/T8313-2008 "-Tea Polyphenols in Tea and catechin content assaying method.

The assay of accurate caffeine in each component: adopt high performance liquid chromatography to measure:

Chromatographic condition

Chromatographic column: Kromasil C18 (250mm × 4.6mm, 5 μm);

Mobile phase: methanol (B)-3% aqueous acetic acid (A) carries out linear gradient elution, as shown in the table:

Flow velocity: 1.0ml/min;

Determined wavelength: 280nm;

Column temperature: 30 DEG C;

Test sample sample size: 10 μ l;

Detect: writing time 50min.

The assay of accurate tea polysaccharide in each component: adopt sulphuric acid anthrone method to measure, concrete grammar is:

(1) preparation of need testing solution: get this product powder and be about 0.5-1g, put in 250mL flask, adds 80% ethanol 50mL, 95 DEG C of water-bath backflow 30min, filter, while hot by hot 80% washing with alcohol 3 times (30mL/ time), volatilize solvent, filter paper is put in flask together with filtering residue, adds 50mL water, reflux 1h, filtered while hot, with hot wash, merging filtrate, be settled to 100mL.The accurate 5mL of absorption extracts solution, puts in polyamide chromatography post (15cm × 2cm), with hot water elution, collects filtrate, is settled to 100mL, as need testing solution.

(2) preparation of reference substance solution

Get D-anhydrous glucose reference substance appropriate, accurately weighed (being accurate to 0.001g), be dissolved in water and dilute the solution made containing 0.2mg in every 1ml, product solution in contrast.

(3) drafting of standard curve

Respectively accurate draw reference substance solution 0.0,0.1,0.2,0.3,0.4,0.5ml puts in 10ml tool plug test tube respectively, add two pure water respectively to 1.0ml, the phenol solution that each precision adds 5% (takes 2.5g re-distilled phenol, is dissolved in water and is diluted to 50ml, shake up, to obtain final product.) 1.0ml, jolting mixes, add 5.0ml sulphuric acid, with the rapid jolting mixing of miniature whirlpool mixed instrument, ambient temperatare puts 30 minutes, with the 1st pipe for blank, according to ultraviolet visible spectrophotometry (China's coastal port annex VB), measure absorbance (measuring complete in 1 hour) at 487nm wavelength place.Make standard curve with absorbance (Y) and quality (X), regression equation is:

Y＝a·X+b

In formula:

Y is the absorbance of reference substance solution;

X is the quality (mg) of D-anhydrous glucose;

A, b are constant.

(4) mensuration of need testing solution

Accurate absorption 1ml need testing solution, according under the drafting item of standard curve, from " precision adds the phenol solution 1.0ml of 5% ", with method operation, measures absorbance at 487nm place respectively.

(5) date processing

Be calculated as follows content:

$C=\frac{(A-b)\×V}{a\×W\×1000}\×f\×100\%$

In formula:

C is the tea polysaccharide percentage composition in sample;

A is the absorbance of sample;

W is sample weighting amount (g);

V is constant volume (mL);

F is extension rate;

B is constant.

According to the Method for Accurate Calculation of above-mentioned various component content, in the tea polyphenols component can prepared according to the inventive method, abrownin component and caffeine component three kinds of components, the Precise levels of each composition is:

1.4 experimental apparatus

BIOSEN5030 type blood glucose/lactic acid analysis instrument, German EKF manufactures;

PL303 electronic balance: Mettler-Toledo Instrument (Shanghai) Co., Ltd..

T2000 electronic balance: two outstanding brother (group) company limited of the U.S..

2. experimental technique

2.1 orthogonal experiment method screening optimum proportioning

2.1.1 the determination of factor level

Tea polyphenols component, abrownin component, caffeine component, often kind of component drafts 3 dosage levels.Select the L of a Three factors-levels ₉(3 ⁴) orthogonal table experiment arrangement, material elements level arranges, in table 1.

Table 1 factor level table (g/kg)

2.1.2 orthogonal experiment arrangement

By L9(3 ⁴) formula that provides of orthogonal table, form 9 different prescriptions and test.Concrete grammar selects KKAy mice (SPF level), is divided into 9 groups at random.Press L9(3 respectively for each group ⁴) experiment group table (see table 2) designed by orthogonal table tests, with fasting blood sugar, carbohydrate tolerance for observation index.Last with the comprehensive grading of two indices for curative effect index, and analyze the best medication proportioning of 3 kinds of components and the primary-slave relation in prescription thereof in prescription.

Table 2L9(3 ⁴) orthogonal experiment calendar

2.1.3 prepared by orthogonal experiment component

By L9(3 ⁴) 9 of orthogonal trial experiment prescriptions, the laboratory sample before use in each prescription is respectively by the mixing of its dosage level ratio, and pulverizer is pulverized and mixed.

3.1.4 the actual dosage of Multiple components in each component of orthogonal experiment

Measure according to 1.3 joint constituent contents, calculate component content in each component, in table 3.

In each component of table 3, the actual dosage of Multiple components calculates

2.2 grouping and administrations:

Animal high lipid food measures fasting glucose according to fasting plasma glucose method after feeding surrounding, by the grouping of actual measurement fasting blood sugar stratified random.

That chooses fasting glucose >18.1mmol/L in 180 KKAy enters group, is divided into 10 groups at random: positive drug group (1.33mgkg-1d-1, n=15); Component 1 ~ 9 group (n=15).Normal C57BL/6J mice

(n=11) matched group is done.

Matched group: gavage distilled water (10ml/kg BW);

Positive drug group: with distilled water preparation positive drug solution, gavage (10ml/kg BW), dosage 1.33mg/kg/ day;

Each component group according to orthogonal table dosage distilled water obtain solution, gavage (10ml/kg BW).

2.3 observation index:

2.3.1 daily observation index

Experimental session observes animal spirit, activity, hair color and urine volume feces situation of change; Quantitative assay food-intake weekly and body weight.

2.3.2 fasting plasma glucose

Before grouping and after administration 7d, 14d, 21d, 28d respectively tail venous blood sampling survey fasting blood sugar.Get blood after the equal fasting of all animals (freely drinking water) 5h, each treated animal gastric infusion of 1h before getting blood, dock when getting blood and get peripheral blood 10 μ l, measure fasting glucose with BIOSEN5030 type blood glucose/lactic acid analysis instrument.

2.3.3 carbohydrate tolerance laboratory observation index and detection method

Administration 28d animal fasting 5h(freely drinks water), gavage group is 1h administration before getting blood, oral administration of glucose 2.0gkg-1 at the end of fasting, measure to the blood glucose value of 0h, 0.5h, 1h, 2h after glucose, observe each group give glucose after the change of each time point blood glucose value, and calculate Area under the curve of blood glucose.

Area under the curve of blood glucose (AUC)=1/2 × (0h blood glucose value+0.5h blood glucose value) × 0.5+1/2 × (0.5h blood glucose value+1h blood glucose value) × 0.5+1/2 × (1h blood glucose value+2h blood glucose value) × 1.

2.3.4 to the inhibitory action of alpha-glucosidase activity

Take sucrose as substrate, the alpha-glucosaccharase enzyme reaction that given the test agent and rat small intestine epimere extract, the burst size according to glucose judges the inhibitory action of sample to phlorose glycosidase activity.

The process of small intestinal: the fasting rat sacrificed by decapitation of 7 hours is dissected, get mid small bowel and be about 10cm length as experimental subject, then the content on removing intestinal wall is rinsed gently with 0.9% sodium-chloride water solution, and cut small intestinal open with operating scissors, wrap with tinfoil and put into liquid nitrogen quick-freezing immediately, then taking-up is put into-20 DEG C of refrigerator freezings and is stored for future use.

Enzyme liquid preparation to be measured: blade scrapes small intestinal capsula interna, and is dissolved to finite concentration with the PBS that concentration is 0.05mol/L, pH7.4, and refiner stirs evenly, and is sub-packed in (often pipe is about 1.5ml) in little centrifuge tube, freezing for subsequent use.

The mensuration of the inhibit activities of alpha-glucosidase is adopted with the following method: survey absorbance (A) 400nm and 503nhibiting concentration (IC ₅₀) value.IC ₅₀inhibitor concentration required when being defined as inhibitory enzyme activity 50%.

10 μ l α-amylase are dissolved in 4ml and contain 50mmol/L NaCl, 5mmol/L CaCl by the mensuration of alpha-amylase inhibition ₂, 0.5/L TritonX-100 pH6.9, as enzyme conserving liquid in 25mmol/L Piperazine-N, N '-bis2-ethanesulfonic acid buffer, during mensuration, dilute 40 times of uses with buffer.Iodine method measures A700nm and IC ₅₀value.

2.3.5 to the inhibitory action of aldose reductase activity

Utilize rat lens aldose reductase, with DL-glyceraldehyde for substrate, NADPH(has an absworption peak at 340nm) be coenzyme, by the change detection of OD340nm before and after detection reaction to the inhibit activities of aldose reductase.

2.3.6 to the inhibitory action of Protein Tyrosine Phosphatases activity

Product pNP after utilizing the polypeptide pNPP containing phosphoric acid to be fallen 1 phosphoric acid by PTP1B enzymolysis has the principle of absworption peak at wavelength 405nm place, represent the suppression situation of compound to enzymatic activity with the amount generating pNP after PTP1B effect.

The inhibitor of equal in quality is dissolved in the solution of same volume, adds in enzyme assay system as constituents for suppressing.Loading sequence is: inhibitor 200 μ l, 25mM pH5.0NaOAc-HAc, 1.4mM EDTA, and 1.4mM DTT, 10nM Δ PTP1B, finally adds the p-NPP that final concentration is 20mM in reaction system.At 37 DEG C of reaction 30min, measure the changing value of OD405, compare with the blank not adding inhibitor.

2.3.7 date processing and result judge

Measurement data is with mean ± standard deviation represent.Application Minitab statistical software analyzes, and comparing between two of multisample mean adopts one factor analysis of variance (one-way ANOVA).

3. experimental result

3.1 general daily observations

The experimental session Normal group C57BL/6J mice mental status is good, and be quick on the draw, move freely, fur is glossy, and food-intake, amount of drinking water are stable, and continued weight increases.Each group of diabetic animal presents significantly " three-many-one-little " symptom, the situation such as polydipsia occurs, polyphagia, polyuria, blood glucose increase, and each administration group mice hair color is normal, movable normal.The weight of animals and food-intake change are not quite.

The experimental result of 3.2 whole animal orthogonal designs

3.2.1 each component of compatibility affects result to the fasting blood sugar 4 weeks of KKAy mice

In 4 weeks that give compatibility component, positive drug group is all starkly lower than model group (p < 0.001) in administration 1-4 week fasting glucose; During component 2 groups the 2nd, 4 week, fasting blood sugar is starkly lower than model group (p < 0.001); During component 3 groups the 2nd, 3,4 week, fasting blood sugar is lower than model group (p < 0.05, p < 0.001); Fasting blood sugar during component 6 groups the 2nd, 3,4 week is lower than model group (p < 0.05, p < 0.001); Fasting blood sugar during component 8 groups the 4th week is lower than model group (p < 0.01).Experimental result and 5 weeks fasting glucose situations of change are in table 4.Component 5 groups, component 7 groups at administration the 1st week fasting blood sugar higher than model group (p < 0.001); Component 9 groups at administration the 1st, 3 weeks fasting blood sugars higher than model group (p < 0.001).

Experimental result shows, and with the prolongation of dosage period, have the component reducing fasting glucose effect, effect manifests gradually and tends towards stability.Individual components does not reduce the effect of fasting glucose.The each component of compatibility affects result of variations in table 4 to the fasting blood sugar 4 weeks of KKAy mice.

3.2.2 the fasting blood sugar the results of analysis of variance of orthogonal design

4th week fasting plasma glucose result is selected to carry out the variance analysis of orthogonal design according to change of blood sugar situation.In this experiment, each factor is abrownin component > caffeine component > tea polyphenols component on fasting glucose impact order.According to the result of variance analysis, optimum proportioning is: tea polyphenols component: abrownin component: caffeine component is 0g/kg:1.2g/kg:0g/kg, carries out calculating and caffeine 1.5%: abrownin 18.3%: tea polysaccharide 23.4% by actual constituent content in often kind of component: the proportioning of tea polyphenols 9.7% reduces at most the fasting glucose of KKAy mice.

What 3.2.3 each component of compatibility was tested KKAy glucose tolerance in mice affects result

In each compatibility component, component 2, component 3, component 6 0.5h, 1h blood glucose elevated-levels after gavage exogenous glucose is obvious, and comparatively model group is low, and AUC compares with model group obvious statistical significance (p < 0.001).The results are shown in Table 5.

The each component of table 5 affects (mmol/L) to the carbohydrate tolerance of KKAy mice

Note: compare with model group ^{* *}p < 0.001;

3.2.4 the carbohydrate tolerance AUC the results of analysis of variance of orthogonal design

The variance analysis of orthogonal design is carried out by carbohydrate tolerance AUC measurement result.In this experiment, each factor is tea polyphenols component > abrownin component > caffeine component on carbohydrate tolerance impact order.According to the result of variance analysis, optimum proportioning is: tea polyphenols component: abrownin component: caffeine component is 0g/kg:1.2g/kg:0g/kg, carries out calculating and caffeine 1.5%: abrownin 18.3%: tea polysaccharide 23.4%: tea polyphenols 9.7% by actual constituent content in often kind of component.

The experimental result of 3.3 external diabetes target enzyme orthogonal designs

3.3.1 each component of compatibility is to the inhibitory action result of alpha-glucosidase activity

Experimental result shows each component of compatibility under 100 μ g/ml concentration to the equal unrestraint effect of amylase.To the mensuration of saccharase and maltase activity under the multiple concentration of each component of compatibility, calculate the suppression IC of each sample ₅₀, the results are shown in Table 6.In each compatibility component, component 2, component 3 pairs of saccharases and maltase all do not detect and suppress IC ₅₀, therefore can not use Minitab statistical software statistical analysis, suppress IC ₅₀the inhibitory action of Notes of Key Data component 4, component 7, component 8 pairs of alpha-glucosidase activities is stronger.

The each component of table 6 compatibility is to the inhibitory action of saccharase and maltase activity

3.3.2 each component of compatibility is to the inhibitory action result of aldose reductase activity

Experimental result shows component 4 aldose reductase activity in each compatibility component and suppresses comparatively strong, suppresses IC50 to be 4.87 μ g/ml.The results are shown in Table 7.

The each component of table 7 compatibility is to the inhibitory action of aldose reductase activity

3.3.3 the results of analysis of variance of the AR enzyme inhibition rate of orthogonal design

The variance analysis of orthogonal design is carried out by aldose reductase activity suppression ratio measurement result.In this experiment, each factor is tea polyphenols component > abrownin component > caffeine component on aldose reductase activity suppression ratio impact order.According to the result of variance analysis, optimum proportioning is: tea polyphenols component: abrownin component: caffeine component is 0.5g/kg:0g/kg:0.05g/kg, carries out calculating and caffeine 18.7%: abrownin 0.82%: tea polysaccharide 0.53% by actual constituent content in often kind of component: the proportioning of tea polyphenols 64.1% is maximum to aldose reductase activity suppression ratio.

3.3.4 each component of compatibility is to the inhibitory action result of Protein Tyrosine Phosphatases activity

Experimental result shows in each compatibility component, except component 4, component 7, all has comparatively high inhibition effect to Protein Tyrosine Phosphatases activity.The results are shown in Table 8.

The each component of table 8 compatibility is to the inhibitory action of Protein Tyrosine Phosphatases activity

3.3.5 the results of analysis of variance of the Protein Tyrosine Phosphatases suppression ratio of orthogonal design

The variance analysis of orthogonal design is carried out by Protein Tyrosine Phosphatases maximum inhibition measurement result.In this experiment, each factor is abrownin component > tea polyphenols component > caffeine component on Protein Tyrosine Phosphatases maximum inhibition impact order.According to the result of variance analysis, optimum proportioning is: tea polyphenols component: abrownin component: caffeine component is 0g/kg:1.2g/kg:0g/kg, carries out calculating and caffeine 1.5%: abrownin 18.3%: tea polysaccharide 23.4% by actual constituent content in often kind of component: the proportioning of tea polyphenols 9.7% is maximum to Protein Tyrosine Phosphatases maximum inhibition.

4. conclusion

Orthogonal design test is the organic conception from comprehensive test design, from bulk testing, select the representative strong testing site of part test, utilize orthogonal table equilibrium dispersion, neatly can ratio characteristic, it not the quality of direct comparative test result, but by the size of hydraulic test value, analysis is made to the quality of each factor primary and secondary and level.Orthogonal design can overcome medicament categories to be fixed, and the shortcomings such as dosage is single, can arrange multifactorial experiment by orthogonal table, level can be optional, finally finds out preferred plan (optimum formula) according to result of the test.This experiment designs different compatibility totally 9 groups according to orthogonal array.As 3 kinds of mutual compatibilities of component, though all compatible combination can not be reflected comprehensively, certain aspect can reflect the power of the compatibility relationship under effect condition between 3 taste components.

From experimental result, on whole animal model, dark brown component has the greatest impact on fasting blood sugar and carbohydrate tolerance AUC and affects significantly, and the content increasing abrownin component effectively can improve the symptom of diabetic animal.Caffeine component shows in orthogonal test to be affected significantly fasting blood sugar and carbohydrate tolerance AUC, and tea polyphenols component affects also not remarkable on fasting blood sugar and carbohydrate tolerance AUC in orthogonal test.

In external target enzyme inhibition test, tea polyphenols component has stronger inhibitory action to saccharase and maltase activity, in the external experiment of the inhibitory action to aldose reductase activity, the inhibitory action of tea polyphenols component to aldose reductase is maximum also the most remarkable, and abrownin component and caffeine component do not make significant difference to aldose reductase.In orthogonal test, the inhibitory action of abrownin component to PTP-1B is maximum also the most remarkable, and tea polyphenols component and caffeine component do not make significant difference.

This research comprehensive shows, the material base of Pu'er tea blood sugar lowering is abrownin component, tea polyphenols component.Best compatibility proportioning combination: tea polyphenols component: abrownin component: caffeine component is 0.5:1.2:0, carries out calculating and caffeine 4.5% by actual constituent content in often kind of component: abrownin 13.2%: tea polysaccharide 18.2%: the proportioning of tea polyphenols 27.6%

Test 2 Folium camelliae assamicae active component compatibility components to study STZ diabetes rat blood sugar reducing function

The animal model STZ that recommendation declared by the classical way selecting several compatibility components of Folium camelliae assamicae active component to adopt pharmacological evaluation hypoglycemic medicine drug effect the most often to use and new drug causes diabetes rat model, observe the precious impact that the fasting glucose of diabetic model rats, carbohydrate tolerance and insulin resistant are caused on STZ of Folium camelliae assamicae, confirm the best compatibility proportioning of Folium camelliae assamicae active component blood sugar lowering.

1. experiment material

1.1 laboratory animal

Sprague-Dawley rat 85, male, SPF level, body weight 220-250g, is provided by Beijing Vital River Experimental Animals Technology Co., Ltd., licence: SCXK(capital) 2012-0001.

1.2 main agents and medicine

(1) Rosiglitazone Maleate Tablets, lot number: 09090110, GlaxoSmithKline PLC (Tianjin) company limited, specification: 4mg/ sheet.

(2) tea polyphenols component, lot number 20111216, khaki powder.Thered is provided by Tian Shi power group academy Chinese medicine.Deposit in the sample cabinet of pharmacology institute's test sample room, preserve under lucifuge room temperature.

(3) abrownin component, lot number 20111209, the little block of dark brown.Thered is provided by Tian Shi power group academy Chinese medicine.Deposit in the sample cabinet of pharmacology institute's test sample room, preserve under lucifuge room temperature.

(4) caffeine component, lot number 20111115, pale powder.Thered is provided by Tian Shi power group academy Chinese medicine.Deposit in the sample cabinet of pharmacology institute's test sample room, preserve under lucifuge room temperature.

(5) streptozotocin (Streptozotocin, STZ), lot number: S0130-1G, sigma company of the U.S., uses front Fresh.

(6) Rat/mouse Insulin ELISA Kit, lot number: ST2347EN00, is purchased from MILLIPORE company of the U.S..

(7) blood sugar test reagent (BIOSEN5030 type blood glucose/lactic acid analysis instrument matched reagent).

(8) sodium citrate (Na3C6H5O72H2O): Tianjin chemical reagent one factory, lot number: 1108192.

(9) anhydrous citric acid (C6H8O7H2O): Tianjin chemical reagent one factory, lot number: 1108241.

(10) CHOL detectable, R1 lot number DK334, R2 lot number DK335, Japanese Wako Pure Chemical Industries, Ltd..

(11) TG detectable, R1 lot number EE607, R2 lot number DL018, Japanese Wako Pure Chemical Industries, Ltd..

(12) HDL-C detectable, R1 lot number DG373, R2 lot number DG374, Japanese Wako Pure Chemical Industries, Ltd..

(13) LDL-C detectable, R1 lot number DH017, R2 lot number DH018, Japanese Wako Pure Chemical Industries, Ltd..

(14) GLU detectable, R1 lot number DH004, R2 lot number DH005, Japanese Wako Pure Chemical Industries, Ltd..

(15) FRUC detectable, lot number 11102406, Fahrenheit Asia-Pacific, Shanghai Biology Pharmacy Co., Ltd.

1.3 experimental apparatus

BIOSEN5030 type blood glucose/lactic acid analysis instrument, German EKF company manufactures.

The multi-functional microplate reader of Infinite200, TECAN company of Sweden.

Hitachi 7020 type full automatic biochemical apparatus: FDAC Co., Ltd..

PL303 electronic balance: Mettler-Toledo Instrument (Shanghai) Co., Ltd..

T2000 electronic balance: two outstanding brother (group) company limited of the U.S..

2. experimental technique

The foundation of 2.1STZ diabetes model

After SD male rat 85 adaptabilities feed 1w, be divided into Normal group and Glycemia Decline group at random.Normal group 10, Glycemia Decline group 75.Modeling group 75 Rat Fast 16h, through abdominal cavity shot STZ55mg.kg ^-1(0.1mmol.L ^-1, Ph4.4 citric acid solution is mixed with 2% solution), normal control treated animal injects isodose citrate buffer solution in contrast.After injection 3d, after animal fasting 5h, tail vein aculeus blood-taking surveys blood glucose, and blood glucose value is greater than 10mmol.L ^-1person is defined as diabetes rat.

2.2 grouping and administrations:

With reference to early-stage Study result, select the object that three proportioning groups are investigated as function of reducing blood sugar altogether.Comprise the best compatibility proportioning combination with whole animal model orthogonal; Be difficult to remove completely in conjunction with actual production technique caffeine component, and the situation that in Pu'er tea, polyphenol content is not high, reduce caffeine amounts of components as far as possible, increase the proportioning combination of dark brown component; Proportioning group is investigated as last using the best compatibility proportioning that orthogonal is comprehensively analyzed.In component compatibility situation and each component, each composition actual content is in table 9, table 10.

The component compatibility situation of table 9 three proportioning groups

Each composition actual content in each component of table 10

The diabetes rat of Cheng Mo is divided into model group, positive drug group, component proportion 1, component proportion 2, component proportion 3 at random, totally 5 treated animals, often organizes 12.Normal group 1 group, 10 animals.Every day gavage, continuous gavage 4 weeks.

Normal group: gavage distilled water (10ml/kg BW);

Model control group: gavage distilled water (10ml/kg BW);

Positive drug group: with distilled water preparation positive drug solution, gavage (10ml/kg BW), dosage 1.33mg/kg/ day;

Each group of component proportion according to table 1 dosage distilled water obtain solution, gavage (10ml/kg BW).

2.3 observation index:

2.3.1 daily observation index

Experimental session observes animal spirit, activity, hair color and urine volume feces situation of change; Quantitative assay food-intake weekly and body weight.

2.3.2 fasting plasma glucose

Before grouping and after administration 7d, 14d, 21d, 28d respectively tail venous blood sampling survey fasting blood sugar.Get blood after the equal fasting of all animals (freely drinking water) 5h, each treated animal gastric infusion of 1h before getting blood, when getting blood, tail vein aculeus blood-taking, gets peripheral blood 10 μ l, measures fasting glucose with BIOSEN5030 type blood glucose/lactic acid analysis instrument.

2.3.3 carbohydrate tolerance laboratory observation index and detection method

Administration 28d animal fasting 5h(freely drinks water), gavage group is 1h administration before getting blood, oral administration of glucose 2.0gkg at the end of fasting ^-1, measure to the blood glucose value of 0h, 0.5h, 1h, 2h after glucose, observe each group give glucose after the change of each time point blood glucose value, and calculate Area under the curve of blood glucose.

Area under the curve of blood glucose (AUC)=1/2 × (0h blood glucose value+0.5h blood glucose value) × 0.5+1/2 × (0.5h blood glucose value+1h blood glucose value) × 0.5+1/2 × (1h blood glucose value+2h blood glucose value) × 1.

2.3.4 Diagnostic Value of Fasting Serum insulin assay

After administration 30d, water 12h is can't help in fasting, lumbar injection chloral hydrate anesthesia (0.04ml10g ^-1), socket of the eye venous blood sampling, room temperature leaves standstill 30min, and 2500g × 10min4 DEG C is centrifugal, separation of serum, and employing double antibodies sandwich enzyme exempts from ELISA method, according to test kit description step measurements.

2.3.5 blood biochemical detects

After administration 30d, water 12h is can't help in fasting, lumbar injection chloral hydrate anesthesia (0.04ml10g-1), socket of the eye venous blood sampling, and 3000 revs/min centrifugal, and separation of serum detects GLU, CHOl, TG, HDL-C, LDL-C, FRUC content with full automatic biochemical apparatus.

2.3.6 date processing and result judge

Measurement data is with mean ± standard deviation represent.Application SPSS11.0 statistical software analyzes, and comparing between two of multisample mean adopts one factor analysis of variance (one-way ANOVA).

3. experimental result

3.1 general daily observation and body weight change

Experimental session is respectively organized diabetic animal and is presented significantly " three-many-one-little " symptom, the situations such as generation polydipsia, polyphagia, polyuria, blood glucose increase, the glucose in urine positive, refusing to eat, dull, fur is loose.It is slow that diabetes each treated animal body weight increases compared with normal group.

3.2 impacts on fasting glucose

The rising more remarkable in negative control group of model group blood glucose value, proves animal model success.Positive drug group is 2-4 week upon administration, and fasting blood sugar is all starkly lower than model group (p < 0.05, p < 0.01).During component 1 group the 2nd, 3,4 week, fasting blood sugar is lower than model group (p < 0.01, p < 0.001); During component 2 groups the 2nd week, fasting blood sugar is starkly lower than model group (p < 0.01); During component 3 groups the 2nd, 3,4 week, fasting blood sugar is starkly lower than model group (p < 0.01); Each component affects result of variations in table 11 to the fasting blood sugar 4 weeks of diabetes rat.

The each compatibility component of table 11 affects (mmol/L) the fasting glucose of diabetes rat

Note: compare with model group ^*p < 0.05, ^*p < 0.01, ^{* *}p < 0.001;

3.3 impacts on carbohydrate tolerance

The obvious comparatively model group low (p < 0.05, p < 0.01) of component 1 group 0.5h, 2h mean blood glucose after gavage exogenous glucose, AUC compares with model group obvious significant difference (p < 0.01).After component 2 groups of gavage exogenous glucoses, significantly (p < 0.05), AUC more also has significant difference (p < 0.05) with model group simultaneously for 1h mean blood glucose and model group comparing difference.The obvious comparatively model group low (p < 0.01, p < 0.001) of component 3 groups 0.5h, 1h, 2h mean blood glucose after gavage exogenous glucose, AUC compares with model group simultaneously obvious significant difference (p < 0.001).The results are shown in Table 12.

The each compatibility component of table 12 is on the impact (mmol/L) of the carbohydrate tolerance of diabetes rat

Note: compare with model group ^*p < 0.05, ^*p < 0.01, ^{* *}p < 0.001;

3.4 impacts on animal Diagnostic Value of Fasting Serum insulin

As seen from Table 13, model group serum insulin significantly increases, and have highly significant (P<0.001) with matched group comparing difference, positive drug group, component 1 group, component 2 groups, component 3 groups all can significantly reduce rat blood serum insulin, compare with model group and have significant difference (P<0.05, P<0.001).

The each compatibility component of table 13 is on the impact of animal Diagnostic Value of Fasting Serum insulin

3.5 blood biochemical testing results

From table 14, table 15, serum GLU testing result is consistent with fasting plasma glucose, and component 1 group, component 2 groups compare significant difference (P<0.05) with model group.FRUC, TC, TG, HDL-C, LDL-C index is equal zero difference between respectively organizing.

The each compatibility component of table 14 affects I to biochemical indicator

Note: compare with model group ^*p < 0.05, ^*p < 0.01;

The each compatibility component of table 15 affects II to biochemical indicator

4 experiment conclusion and discussion

This experimental selection evaluates the classical way of hypoglycemic medicine drug effect, namely the animal model STZ that recommendation declared by new drug causes diabetes rat model, observe three Folium camelliae assamicae active component compatibilities cause the fasting glucose of diabetic model rats, carbohydrate tolerance, insulin and biochemical indicator impact on STZ, confirm the best compatibility proportioning of Folium camelliae assamicae active component blood sugar lowering.

Three proportioning groups of experimental selection are the best compatibility proportioning component 1 selected with whole animal model orthogonal result respectively; In conjunction with the proportioning component 2 that production technology is determined; The best compatibility proportioning component 3 of selection is comprehensively analyzed with vivo and vitro orthogonal.

Fasting glucose experimental result shows three compatibility components has obvious reducing effect to diabetic animal fasting glucose, and wherein the effect of component 1 and component 3 is stablized.Carbohydrate tolerance experiment is similar to fasting glucose result, and wherein component 3 improves the effect of carbohydrate tolerance the most obviously, does not have difference between each component.When assay interpretation of result in conjunction with three kinds of compatibility components can show that the content of abrownin component in compatibility component, tea polyphenols component is higher, blood sugar lowering is better with the effect improving carbohydrate tolerance, can have a negative impact when caffeine constituent content is too high to hypoglycemic effect.

Insulin resistant (or tissue insulin sensitivity) is the important pathogenic factors of diabetes, the important indicator being the research carbohydrate metabolism disturbance cause of disease and contacting with complication.The Diagnostic Value of Fasting Serum insulin of model group, apparently higher than negative control group, proves that this laboratory animal is the typical model of insulin resistant.Component 1 group, component 2 groups, component 3 groups all can significantly reduce rat blood serum insulin, and show that three compatibility components well can improve insulin resistant, Enhancement test animal is to the sensitivity of insulin.

By this efficacy validation test can determine that three kinds of component compatibilities all can reach more satisfactory hypoglycemic effect, comprehensive multinomial observation index wherein tea polyphenols component 0.5g/kg, dark brown component 1.2g/kg such proportioning combination can reach best hypoglycemic effect.

Detailed description of the invention

Further illustrate the present invention by the following examples

Embodiment 1

Abrownin method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting three times, each 0.5-1 hour, merge extractive liquid, less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 95% ethanol alcohol and be sink to 80%, quiet to more than 12 hours, the drying of alcohol hypostasis vacuum decompression obtains theabrownin crude product, and redissolve with water and filter, filtrate less than 70 DEG C is evaporated to and dryly obtains abrownin component;

Tea polyphenols method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting three times, each 0.5-1 hour, merge extractive liquid, less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 95% ethanol alcohol and be sink to 80%, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 5.4 with sodium bicarbonate solution, leave standstill, centrifugal, sediment separate out sulfuric acid solution turns molten, be extracted with ethyl acetate, extract removes desolventizing and obtains tea polyphenols component;

Caffeine method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting three times, each 0.5-1 hour, merge extractive liquid, less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 95% ethanol alcohol and be sink to 80%, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 5.4 with sodium bicarbonate solution, leave standstill, centrifugal, supernatant obtains solid through concentrate drying, redissolve with water, filter, cross macroporous adsorptive resins, and wash with water and obtain effluent, loading effluent and water lotion reduced vacuum are concentrated into proportion 1.10 ~ 1.15, with chloroform extraction three times, extract obtains caffeine component through separation.

Tea polyphenols component above-mentioned steps obtained, abrownin component and caffeine component carry out taking, mixing according to 5 weight portion tea polyphenols components, the abrownin component of 12 weight portions and the caffeine component of 0 weight portion, obtain the compositions of Folium camelliae assamicae effective ingredient of the present invention.Calculating and caffeine 4.47% is carried out, abrownin 13.18%, tea polysaccharide 18.16%, tea polyphenols 27.55% by actual constituent content in often kind of component.

Embodiment 2

Abrownin method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting 1 time, 0.5 hour time, extracting solution less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 90% ethanol alcohol and be sink to alcohol content 70%, quiet to more than 12 hours, the drying of alcohol hypostasis vacuum decompression obtains theabrownin crude product, and redissolve with water and filter, filtrate less than 70 DEG C is evaporated to and dryly obtains abrownin component;

Tea polyphenols method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting 1 time, 0.5 hour time, extracting solution less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 90% ethanol alcohol and be sink to alcohol content 70%, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 4 with sodium bicarbonate solution, leave standstill, centrifugal, sediment separate out sulfuric acid solution turns molten, solution with ethyl acetate extracts, and extract removes desolventizing and obtains tea polyphenols component;

Caffeine method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting 1 time, 0.5 hour time, extracting solution less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 90% ethanol alcohol and be sink to alcohol content 70%, quiet to more than 12 hours, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 4 with sodium bicarbonate solution, leave standstill, centrifugal, obtain supernatant, supernatant obtains solid through concentrate drying, redissolve with water, filter, cross macroporous adsorptive resins, and wash with water and obtain effluent, loading effluent and water lotion reduced vacuum are concentrated into proportion 1.10 ~ 1.15, with chloroform extraction three times, extract obtains caffeine component through separation.

Abrownin component above-mentioned steps obtained, tea polyphenols component and caffeine component carry out taking, mixing according to 0 weight portion tea polyphenols component, the abrownin component of 6 weight portions and the caffeine component of 0.5 weight portion, obtain the compositions of Folium camelliae assamicae effective ingredient of the present invention.Calculating and caffeine 8.29%, abrownin 16.89%, tea polysaccharide 21.75%, tea polyphenols 9.03% is carried out by actual constituent content in often kind of component.

Embodiment 3

Abrownin method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting 2 times, each 1 hour, extracting solution less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 100% ethanol to alcohol content 80%, quiet to more than 12 hours, the drying of alcohol hypostasis vacuum decompression obtains theabrownin crude product, and redissolve with water and filter, filtrate less than 70 DEG C is evaporated to and dryly obtains abrownin component;

Tea polyphenols method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting 2 times, each 1 hour, and extracting solution less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 100% ethanol to alcohol content 80%, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 6 with sodium bicarbonate solution, leave standstill, centrifugal, sediment separate out sulfuric acid solution turns molten, solution with ethyl acetate extracts, and extract removes desolventizing and obtains tea polyphenols component;

Caffeine method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting 2 times, each 1 hour, extracting solution less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 100% ethanol to alcohol content 80%, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 6 with sodium bicarbonate solution, leave standstill, centrifugal, obtain supernatant, supernatant obtains solid through concentrate drying, redissolve with water, filter, cross macroporous adsorptive resins, and wash with water and obtain effluent, loading effluent and water lotion reduced vacuum are concentrated into proportion 1.10 ~ 1.15, with chloroform extraction three times, extract obtains caffeine component through separation.

Abrownin component above-mentioned steps obtained, tea polyphenols component and caffeine component carry out taking, mixing according to 0 weight portion tea polyphenols component, the abrownin component of 12 weight portions and the caffeine component of 1 weight portion, obtain the compositions of Folium camelliae assamicae effective ingredient of the present invention.Calculating and caffeine 8.29%, abrownin 16.89%, tea polysaccharide 21.75%, tea polyphenols 9.03% is carried out by actual constituent content in often kind of component.

Embodiment 4

Abrownin method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting 2 times, each 0.5 hour, extracting solution less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 95% ethanol to alcohol content 80%, quiet to more than 12 hours, the drying of alcohol hypostasis vacuum decompression obtains theabrownin crude product, and redissolve with water and filter, filtrate less than 70 DEG C is evaporated to and dryly obtains abrownin component;

Tea polyphenols method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting 2 times, each 0.5 hour, and extracting solution less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 95% ethanol to alcohol content 80%, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 5 with sodium bicarbonate solution, leave standstill, centrifugal, sediment separate out sulfuric acid solution turns molten, solution with ethyl acetate extracts, and extract removes desolventizing and obtains tea polyphenols component;

Caffeine method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting 2 times, each 0.5 hour, extracting solution less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 95% ethanol to alcohol content 80%, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 5 with sodium bicarbonate solution, leave standstill, centrifugal, obtain supernatant, supernatant obtains solid through concentrate drying, redissolve with water, filter, cross macroporous adsorptive resins, and wash with water and obtain effluent, loading effluent and water lotion reduced vacuum are concentrated into proportion 1.10 ~ 1.15, with chloroform extraction three times, extract obtains caffeine component through separation.

Abrownin component above-mentioned steps obtained, tea polyphenols component and caffeine component carry out taking, mixing according to 2.5 weight portion tea polyphenols components, the abrownin component of 12 weight portions and the caffeine component of 0 weight portion, obtain the compositions of Folium camelliae assamicae effective ingredient of the present invention.Calculating and caffeine 3.24%, abrownin 15.3%, tea polysaccharide 20.33%, tea polyphenols 20.17% is carried out by actual constituent content in often kind of component.

Embodiment 5

Abrownin method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting 3 times, each 1 hour, extracting solution less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 80% ethanol to alcohol content 70%, quiet to more than 12 hours, the drying of alcohol hypostasis vacuum decompression obtains theabrownin crude product, and redissolve with water and filter, filtrate less than 70 DEG C is evaporated to and dryly obtains abrownin component;

Tea polyphenols method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting 3 times, each 1 hour, and extracting solution less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 80% ethanol to alcohol content 70%, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 6 with sodium bicarbonate solution, leave standstill, centrifugal, sediment separate out sulfuric acid solution turns molten, solution with ethyl acetate extracts, and extract removes desolventizing and obtains tea polyphenols component;

Caffeine method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting 3 times, each 1 hour, extracting solution less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 80% ethanol to alcohol content 70%, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 6 with sodium bicarbonate solution, leave standstill, centrifugal, obtain supernatant, supernatant obtains solid through concentrate drying, redissolve with water, filter, cross macroporous adsorptive resins, and wash with water and obtain effluent, loading effluent and water lotion reduced vacuum are concentrated into proportion 1.10 ~ 1.15, with chloroform extraction three times, extract obtains caffeine component through separation.

Abrownin component above-mentioned steps obtained, tea polyphenols component and caffeine component carry out taking, mixing according to 5 weight portion tea polyphenols components, the abrownin component of 6 weight portions and the caffeine component of 0 weight portion, obtain the compositions of Folium camelliae assamicae effective ingredient of the present invention.Calculating and caffeine 6.09%, abrownin 10.39%, tea polysaccharide 15.31%, tea polyphenols 37.29% is carried out by actual constituent content in often kind of component.

Embodiment 6

Abrownin method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting 3 times, each 0.5 hour, extracting solution less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 95% ethanol to alcohol content 85%, quiet to more than 12 hours, the drying of alcohol hypostasis vacuum decompression obtains theabrownin crude product, and redissolve with water and filter, filtrate less than 70 DEG C is evaporated to and dryly obtains abrownin component;

Tea polyphenols method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting 3 times, each 0.5 hour, extracting solution less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 95% ethanol to alcohol content 85%, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 5.4 with sodium bicarbonate solution, leave standstill, centrifugal, sediment separate out sulfuric acid solution turns molten, solution with ethyl acetate extracts, and extract removes desolventizing and obtains tea polyphenols component;

Caffeine method for preparing ingredients thereof:

Folium camelliae assamicae adds 10 times of water gagings, 80 DEG C of high-temp extracting 3 times, each 0.5 hour, extracting solution less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 95% ethanol to alcohol content 85%, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 5.4 with sodium bicarbonate solution, leave standstill, centrifugal, obtain supernatant, supernatant obtains solid through concentrate drying, redissolve with water, filter, cross macroporous adsorptive resins, and wash with water and obtain effluent, loading effluent and water lotion reduced vacuum are concentrated into proportion 1.10 ~ 1.15, with chloroform extraction three times, extract obtains caffeine component through separation.

Abrownin component above-mentioned steps obtained, tea polyphenols component and caffeine component carry out taking, mixing according to 0 weight portion tea polyphenols component, the abrownin component of 12 weight portions and the caffeine component of 0 weight portion, obtain the compositions of Folium camelliae assamicae effective ingredient of the present invention.Calculating and caffeine 1.5%, abrownin 18.3%, tea polysaccharide 23.4%, tea polyphenols 9.7% is carried out by actual constituent content in often kind of component.

Embodiment 7

According to the compositions of the Folium camelliae assamicae effective ingredient that embodiment 1 obtains, two kinds of components carried out being mixed to get mixture, add customary adjuvant, the conventional method on application galenic pharmacy, makes tablet.

Embodiment 8

According to the compositions of the Folium camelliae assamicae effective ingredient that embodiment 2 obtains, two kinds of components carried out being mixed to get mixture, add customary adjuvant, the conventional method on application galenic pharmacy, makes capsule.

Embodiment 9

According to the compositions of the Folium camelliae assamicae effective ingredient that embodiment 3 obtains, two kinds of components carried out being mixed to get mixture, add customary adjuvant, the conventional method on application galenic pharmacy, makes granule.

Embodiment 10

According to the compositions of the Folium camelliae assamicae effective ingredient that embodiment 4 obtains, two kinds of components carried out being mixed to get mixture, add customary adjuvant, the conventional method on application galenic pharmacy, makes powder.

Embodiment 11

According to the compositions of the Folium camelliae assamicae effective ingredient that embodiment 5 obtains, two kinds of components carried out being mixed to get mixture, add customary adjuvant, the conventional method on application galenic pharmacy, makes drop pill.

Embodiment 12

According to the compositions of the Folium camelliae assamicae effective ingredient that embodiment 6 obtains, two kinds of components carried out being mixed to get mixture, add customary adjuvant, the conventional method on application galenic pharmacy, makes injection.

Embodiment 13

According to the compositions of the Folium camelliae assamicae effective ingredient that embodiment 1 ~ 6 either method obtains, join by a certain percentage in tea product with common process, make the health tea with weight losing function.

Embodiment 14

According to the compositions of the Folium camelliae assamicae effective ingredient that embodiment 1 ~ 6 either method obtains, join by a certain percentage in milk product with common process, make the health food with weight losing function.

Embodiment 15

According to the compositions of the Folium camelliae assamicae effective ingredient that embodiment 1 ~ 6 either method obtains, join by a certain percentage in beverage with common process, make the health beverage with weight losing function.

Embodiment 16

According to the compositions of the Folium camelliae assamicae effective ingredient that embodiment 1 ~ 6 either method obtains, join by a certain percentage in cake with common process, make the health care cake with weight losing function.

Claims (14)

1. the compositions containing Folium camelliae assamicae effective ingredient, comprise the tea polyphenols component of 0 ~ 5 weight portion, the abrownin component of 6 ~ 12 weight portions and the caffeine component of 0 ~ 1 weight portion, the preparation method of described tea polyphenols component, abrownin component, caffeine component is as follows:

Abrownin method for preparing ingredients thereof:

Folium camelliae assamicae adds water intensification lixiviate 1-3 time, each 0.5-1 hour, adds 90-100% ethanol to alcohol content 70-80% after extracting solution is concentrated, quiet to more than 12 hours, the drying of alcohol hypostasis obtains theabrownin crude product, and theabrownin crude product water redissolves, filter, filtrate is concentrated into and dryly obtains abrownin component;

Tea polyphenols method for preparing ingredients thereof:

Folium camelliae assamicae adds water intensification lixiviate 1-3 time, each 0.5-1 hour, adds 90-100% ethanol to alcohol content 70-80% after extracting solution is concentrated, quietly obtain supernatant to more than 12 hours, obtain crude product through concentrate drying, crude product is soluble in water, add aluminum chloride, regulate pH value to 4-6 with sodium bicarbonate solution, leave standstill, centrifugal, sediment separate out sulfuric acid solution dissolves, and solution with ethyl acetate extracts, and extract removes desolventizing and obtains tea polyphenols component;

Caffeine method for preparing ingredients thereof:

Folium camelliae assamicae adds water intensification lixiviate 1-3 time, each 0.5-1 hour, 90-100% ethanol is added to alcohol content 70-80% after extracting solution is concentrated, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, add aluminum chloride, regulate pH value to 4-6 with sodium bicarbonate solution, leave standstill, centrifugal, obtain supernatant, supernatant obtains solid through concentrate drying, redissolve with water, filter, cross macroporous adsorptive resins, and wash with water and obtain effluent, loading effluent and water lotion concentrate, with chloroform extraction, extract obtains caffeine component through separation.

2. compositions according to claim 1, comprises 5 weight portion tea polyphenols components, the abrownin component of 12 weight portions and the caffeine component of 0 weight portion.

3. compositions according to claim 1, comprises 0 weight portion tea polyphenols component, the abrownin component of 6 weight portions and the caffeine component of 0.5 weight portion.

4. compositions according to claim 1, comprises 0 weight portion tea polyphenols component, the abrownin component of 12 weight portions and the caffeine component of 1 weight portion.

5. compositions according to claim 1, comprises 2.5 weight portion tea polyphenols components, the abrownin component of 12 weight portions and the caffeine component of 0 weight portion.

6. compositions according to claim 1, comprises 5 weight portion tea polyphenols components, the abrownin component of 6 weight portions and the caffeine component of 0 weight portion.

7. compositions according to claim 1, comprises 0 weight portion tea polyphenols component, the abrownin component of 12 weight portions and the caffeine component of 0 weight portion.

8., according to the arbitrary described compositions of claim 1 ~ 7, wherein the preparation method of tea polyphenols component, abrownin component, caffeine component is as follows:

Abrownin method for preparing ingredients thereof:

Folium camelliae assamicae adds water 80 DEG C of high-temp extracting three times of 10 times amount, each 0.5-1 hour, merge extractive liquid, less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 95% ethanol alcohol and be sink to 80%, quiet to more than 12 hours, the drying of alcohol hypostasis vacuum decompression obtains theabrownin crude product, redissolves filter with water, filtrate less than 70 DEG C is evaporated to dry, obtains abrownin component;

Tea polyphenols method for preparing ingredients thereof:

Folium camelliae assamicae adds water 80 DEG C of high-temp extracting three times of 10 times amount, each 0.5-1 hour, and merge extractive liquid, less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 95% ethanol alcohol and be sink to 80%, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 5.4 with sodium bicarbonate solution, leave standstill, centrifugal, precipitation sulfuric acid solution turns molten, be extracted with ethyl acetate, extract removes desolventizing and obtains tea polyphenols component;

Caffeine method for preparing ingredients thereof:

Folium camelliae assamicae adds water 80 DEG C of high-temp extracting three times of 10 times amount, each 0.5-1 hour, merge extractive liquid, less than 70 DEG C is evaporated to relative density 1.05 ~ 1.10, add 95% ethanol alcohol and be sink to 80%, quiet to more than 12 hours, obtain supernatant, crude product is obtained through concentrate drying, crude product is soluble in water, the aluminum chloride of quality such as to add, regulate pH value to 5.4 with sodium bicarbonate solution, leave standstill, centrifugal, supernatant obtains solid through concentrate drying, add water redissolution, filter, filtrate crosses macroporous adsorptive resins, and wash with water and obtain effluent and water lotion, reduced vacuum is concentrated into proportion 1.10 ~ 1.15, with chloroform extraction three times, extract obtains caffeine component through separation.

9. the application of the arbitrary described compositions of claim 1 ~ 7 in preparation treatment or the medicine prevented diabetes or food.

10. applying as claimed in claim 9, is realized by the activity of Inhibiting α-glucosidase.

11. apply as claimed in claim 9, are by suppressing the activity of aldose reductase to realize.

12. apply as claimed in claim 9, are to be realized by the activity of Profilin tyrosine phosphatase-1B.

13. containing the medicine for medical application of the arbitrary described compositions of claim 1 ~ 7 or food compositions.

14. compositionss according to claim 12, food form is wherein selected from: beverage, milk product, Folium Camelliae sinensis, cake, confection; Medicament forms is wherein selected from: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, injection, suppository, ointment, plaster, cream, spray, drop, patch.