CN108567955A

CN108567955A - A kind of pharmaceutical composition and preparation method thereof of prevention diabetic nephropathy

Info

Publication number: CN108567955A
Application number: CN201810188020.4A
Authority: CN
Inventors: 赖先荣; 曾勇; 范刚
Original assignee: Chengdu University of Traditional Chinese Medicine
Current assignee: Chengdu University of Traditional Chinese Medicine
Priority date: 2017-03-08
Filing date: 2018-03-07
Publication date: 2018-09-25

Abstract

The invention discloses a kind of pharmaceutical compositions of prevention diabetic nephropathy, it is characterized in that：It is that the bulk pharmaceutical chemicals matched by following weight are prepared：1~15 part of turmeric, 1~8 part of barberry skin, 1~15 part of emblic, 1~8 part of puncture vine.The present invention promotes its clinical application and new drug development by observing four turmeric soup difference compatibility dosage to the pharmacodynamic results of diabetic nephropathy rats and Principal Component Analysis excavation being combined to be capable of the best dose-effect compatibility of four turmeric soup of effective prevention early diabetic nephropathy.

Description

A kind of pharmaceutical composition and preparation method thereof of prevention diabetic nephropathy

Technical field

The present invention relates to a kind of pharmaceutical compositions and preparation method thereof of prevention diabetic nephropathy, belong to field of medicaments.

Background technology

Diabetic nephropathy (diabetic nephropathy, DN), is one of most common chronic complicating diseases of diabetes, It is the main reason for leading to end stage renal failure.Therefore, the prevention of DN early stages is particularly important.DN belongs to " the capital Nysa of Tibetanmedicine Library " disease (translate by the Chinese：Frequent micturition disease) scope, " capital Buddhist nun " means frequent micturition, " Sa Ku " mean consumption physical efficiency muddiness liquid, i.e., urine frequently Numerous, cloudy urine, consumption physical efficiency are the substantive characteristics of the disease.Specific therapy with " brave western soup " (barberry skin, turmeric, emblic, Four taste Tibetan medicine prescription of puncture vine, i.e. the prescription source of Modern preparations " SIWEI JIANGHUANG TANGSAN ") decoction is for oral administration, and four turmeric soup cures mainly it Card, belongs to frequent micturition disease scope in Tibetanmedicine.

But Tibetan medicine and pharmacology classical works《Four doctor's allusion quotations》In only describe the titles of each flavour of a drug of the prescription, " having medicine immeasurable ", The dosage of specific drug is not recorded, the prescribed dose recorded in the later age Tibetan medicine drug standards is also variant, it is therefore desirable into one Prescribed dose and its mechanism of action of the step research for diabetic nephropathy, in favor of instructing Tibetan medicine's clinical application.

Invention content

The purpose of the present invention is to provide a kind of pharmaceutical compositions and preparation method thereof of prevention diabetic nephropathy.

The present invention provides a kind of pharmaceutical compositions of prevention diabetic nephropathy, it is the bulk pharmaceutical chemicals matched by following weight It is prepared：1~15 part of turmeric, 1~8 part of barberry skin, 1~15 part of emblic, 1~8 part of puncture vine.

Preferably, it is that the bulk pharmaceutical chemicals matched by following weight are prepared：It is 1~2 part of turmeric, 3~7 parts of barberry skin, remaining Sweet sub 1~7 part, 2~8 parts of puncture vine.

Into preferred, it is that the bulk pharmaceutical chemicals matched by following weight are prepared：3 parts of turmeric, 10 parts of barberry skin, emblic 3 parts, 6 parts of puncture vine.

Further, it is the extract of primary medicinal powder by each weight proportion bulk pharmaceutical chemicals, water or organic solvent, and medicine is added The preparation that acceptable auxiliary material or complementary ingredient are prepared on.

Further, the preparation is oral preparation.

Further, the preparation is powder, granule, capsule, tablet, pill, decoction or mixture.

The present invention provides a kind of preparation methods of described pharmaceutical composition, include the following steps：Take each weight proportion Bulk pharmaceutical chemicals directly beat powder, either be added water or organic solvent extraction add pharmaceutically acceptable auxiliary material or it is complementary at Point to get.

Further, the water extracting method is：The bulk pharmaceutical chemicals of each weight proportion, extracting in water are taken three times, to add for the first time 12 times of amounts of water, impregnate 1 hour, decoct 1 hour, and filtration adds water to decoct again 2 times, and each plus 10 times of amounts of water 1 hour every time, merge Filtrate, concentration to get.

The present invention provides use of the described pharmaceutical composition in preparing the drug for the treatment of and/or diabetes and nephropathy preventing On the way.

The present invention provides a kind of method of quality control of described pharmaceutical composition, the method measures gallic acid, tan Spend acid, magnoflorine, Berberine hydrochloride, Jatrorrhizine chloride, the content of 7 kinds of ingredients of palmatin hydrochloride and curcumin.

Further, the content of the ingredient is：Gallic acid 30.64mgg^-1, ellagic acid 48.60mgg^-1, lily magnolia Flower alkali 49.32mgg^-1, Berberine hydrochloride 39.47mgg^-1, Jatrorrhizine chloride 42.24mgg^-1, palmatin hydrochloride 47.66mg·g^-1, curcumin 62.73mgg^-1。

The present invention provides a kind of efficient liquid phase detection method of described pharmaceutical composition, chromatographic condition is：

Stationary phase：Using octadecylsilane chemically bonded silica as filler；

Mobile phase：Mobile phase A is acetonitrile, and Mobile phase B is 0.1% phosphate aqueous solution.

Further, the stationary phase is II chromatographic columns of Capcell Pak C18-MG；Chromatographic column specification：Column length is 25cm, internal diameter 4.6mm, grain size are 5 μm.

Further, flow velocity 0.8mLmin-1.

Further, 10 μ L of sample size.

Further, 30 DEG C of column temperature.

Further, 0~60min Detection wavelengths are 270nm, and 60~70min Detection wavelengths are 430nm.

Further, eluent gradient elution requirement is：0 → 8min, 3% → 3%A；8 → 15min, 3% → 17%A； 15 → 20min, 17% → 17%A；20 → 33min, 17% → 20%A；33 → 38min, 20% → 25%A；38 → 48min, 25% → 25%A；48 → 70min, 25%A~80%A.

Diabetic nephropathy is one of diabetes chronic microvascular complication, refers to Glomerular lesions caused by diabetes：Kidney Bead capillary basement membrane thickens, mesentery is expanded, and extracellular matrix increases, and progressive stage may occur in which diffusivity glomerulosclerosis etc. Pathological change causes clinic albuminuria occur, gradual kidney function damage, hypertension, oedema, and late period serious renal function occurs and declines It exhausts.DN belong to Tibetanmedicine " Jing Nisaku " disease (Chinese is translated：Frequent micturition disease) scope, and the card that four turmeric soup cures mainly belongs to Tibetan Frequent micturition disease (diabetes, Jing Nisaku diseases) scope in medicine, full side is made of turmeric, barberry skin, emblic and puncture vine four traditional Chinese medicine. Turmeric in side, acrid flavour, hardship are warm-natured, control viral disease, frequent micturition, the heat evil of red bar of increasing Sheng are made, as monarch drug in a prescription；Barberry skin, bitter, puckery, property It is cool, removing toxic substances, clear kidney heat, the heat that cooperation turmeric increases Sheng except red bar, as ministerial drug；Puncture vine, it is sweet in flavor, puckery, it is warm-natured, kidney is supported, nephrosis is controlled, Monarch drug in a prescription inducing diuresis for treating strangurtia is assisted, as by medicine；Emblic, it is sweet in flavor, sour, puckery, it is cool in nature, and three are closed because of coordinating the drug actions of a prescription, as horse medicine (efficacy-enhancing ingredient).

The present invention is by observing pharmacodynamic results and combination of the four turmeric soup difference compatibility dosage to diabetic nephropathy rats Principal component analysis (principal components analysis, PCA) method and stepwise regression analysis showed, excavation can have Effect intervenes the four turmeric soup optimum medicine efficacy compatibility of early diabetic nephropathy, promotes its clinical application and new drug development.

The present invention is by observing pharmacodynamic results and combination of the four turmeric soup difference compatibility dosage to diabetic nephropathy rats Principal Component Analysis excavates the best dose-effect compatibility of four turmeric soup for capableing of effective prevention early diabetic nephropathy, promotes its clinic Using and new drug development.Particularly, metering proportion of the present invention is turmeric：Barberry skin：Emblic：Puncture vine=3：10：3：6 items Index illustrates turmeric in the present invention more close to blank group, and more far from model group：Barberry skin：Emblic：Puncture vine=3： 10：3：6 metering proportion is most notable to the improvement of the indices of DN, close to blank group.

Obviously, the above according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.

The specific implementation mode of form by the following examples remakes further specifically the above of the present invention It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on the above of the present invention The technology realized all belongs to the scope of the present invention.

Description of the drawings

Fig. 1 is SIWEI JIANGHUANG TANGSAN test sample (A) high-efficient liquid phase chromatogram, 1. gallic acids；2. magnoflorine；3. tan is spent Acid；4. Jatrorrhizine chloride；5. palmatin hydrochloride；6. Berberine hydrochloride；7. curcumin.

Fig. 2 is mixing reference substance (B) high-efficient liquid phase chromatogram, 1. gallic acids；2. magnoflorine；3. ellagic acid；4. salt Sour jateorrhizine；5. palmatin hydrochloride；6. Berberine hydrochloride；7. curcumin.

Fig. 3 is influence (× 15000) of the transmission electron microscope observing four turmeric side to DN rat kidney Pathomorphology.

Fig. 4 is principal component analysis-shot chart of the different compatibility dosage of four turmeric soup 8 to DN rat pharmacodynamic indexs；

Fig. 5 is principal component analysis-load diagram of the different compatibility dosage of four turmeric soup 8 to DN rat pharmacodynamic indexs.

Fig. 6 is principal component analysis-dendrogram of the different compatibility dosage of four turmeric soup 8 to DN rat pharmacodynamic indexs.

Fig. 7 is principal component analysis-shot chart of the four turmeric soup proportion optimizing to db/db pharmacodynamics in Mice indexs.

Fig. 8 is partial least squares analysis figure of the four turmeric soup proportion optimizing to db/db pharmacodynamics in Mice indexs.

Fig. 9 is hierarchical cluster analysis figure of the four turmeric soup proportion optimizing to db/db pharmacodynamics in Mice indexs.

Specific implementation mode

The raw material that is used in the specific embodiment of the invention, equipment are known product, pass through and buy commercial product and obtain.

The preparation of 1 pharmaceutical composition of the present invention of embodiment

1, extraction process

Prescription：3 parts of turmeric, 10 parts of barberry skin, 3 parts of emblic, 6 parts of puncture vine.

Above four taste, three times, for the first time plus 12 times of amounts of water, immersion 1 hour decoct 1 hour extracting in water, filtration, the dregs of a decoction Water is added to decoct again 2 times, every time plus 10 times of water is measured, and 1 hour every time, merging filtrate was concentrated into required concentration.

2, quality control

HPLC measures gallic acid in Tibetan medicine SIWEI JIANGHUANG TANGSAN, ellagic acid, magnoflorine, Berberine hydrochloride, hydrochloric acid medicine Root alkali, the method for 7 kinds of ingredients of palmatin hydrochloride and curcumin：

Using Capcell Pak C18-MG II (4.6mmI.D. × 250mm, 5 μm), mobile phase A is acetonitrile, Mobile phase B For 0.1% phosphate aqueous solution；Flow velocity 0.8mLmin-1；10 μ L of sample size；30 DEG C of column temperature；Detection wavelength be 270nm (0~ 60min) and 430nm (60~70min, curcumin)；Gradient elution.The content of 7 kinds of ingredients is respectively 30.64mgg^-1、 48.60mg·g^-1、49.32mg·g^-1、39.47mg·g^-1、42.24mg·g^-1、47.66mg·g^-1And 62.73mgg^-1。

This method specificity is strong, as a result accurately and reliably, can be used as the method for quality control of SIWEI JIANGHUANG TANGSAN.

The preparation of 2 pharmaceutical composition of the present invention of embodiment

1, extraction process

Prescription：3 parts of turmeric, 6 parts of barberry skin, 3 parts of emblic, 6 parts of puncture vine.

2, quality control

With embodiment 1.

Embodiment 3HPLC methods measure the content of 7 kinds of ingredients in Tibetan medicine SIWEI JIANGHUANG TANGSAN

1, material and instrument

1.1 instrument

HPLC chromatogram instrument (1260 types of Agilent, be equipped with on-line degassing machine, autosampler, diode array detector, Column oven, Agilent company of the U.S.)；

Chromatographic column：Capcell Pak C₁₈- MG II (4.6mm × 250mm, 5 μm, Japanese Shiseido company)；Excellent general pure water Manufacture system (model：UPH-I-10T, Chengdu Ultra Pure Science ＆ Technology Co., Ltd).

Blood glucose meter (ACCCU-CHEK Performa) (Roche brilliance type, mating blood sugar test paper and disposable blood taking needle, sieve Family name's diagnostic products (Shanghai) Co., Ltd. produces)；

The multi-functional readout instrument of all-wave length (VARIOSKAN FLASH 2.4.3, Thermo companies of the U.S.)；

Constant temperature culture oscillator (ZHWY-100H, Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd.)；Thoroughly

Radio mirror (H-600IV, Hitachi, Japan).

1.2 drugs and the dry rhizome that reagent turmeric is zingiberaceous plant turmeric Curcuma longa L., emblic is big The dry mature fruit of halberd section plant emblic Phyllanthus emblica L., puncture vine are zygophyllaceae plant puncture vine The dry mature fruit of Tribulus terrestris L., the above 3 taste medicinal material are purchased from Chengdu International trade city medicinal material city , it examines, meets through Chengdu University of Traditional Chinese Medicine Lai Xianrong assistant researcher《Chinese Pharmacopoeia》Have under version (one) text items in 2015 Close regulation.

Barberry skin, place of production Tibet are provided by Tibet sweet dew Tibetan Pharmaceutical Factory, through Chengdu University of Traditional Chinese Medicine Fan Gang assistant researcher DNA Bar code identifies its kind, is that the Berberidaceae plant of Tibetan medicine's Clinical practice pierces red pearl Berberis dictyophylla Franch. cortex in drying stem meets the pertinent regulations under six provinces and regions Tibetan medicine standard barberry skin medicinal material items.

Assay SIWEI JIANGHUANG TANGSAN sample is provided by national medicine institute of Chengdu University of Traditional Chinese Medicine, by middle Chinese Preparation method is prepared under people republic the Sanitation Ministry medicine standard (Tibetan medicine first, nineteen ninety-five version) the kind item, turmeric 15g, barberry skin 12.5g, emblic 25g, puncture vine 25g.

Gallic acid reference substance (lot number 110831-201204), Jatrorrhizine chloride reference substance (lot number 110733- 201108), palmatin hydrochloride reference substance (lot number 110732-201309), Berberine hydrochloride reference substance (lot number 110713- 201212) it is provided by National Institute for Food and Drugs Control with curcumin reference substance (lot number 110823-201004), is measured for containing It is fixed to use；

Magnoflorine reference substance (CAS:2141-09-5,99.77%) and ellagic acid reference substance (CAS:476-66-4, MF: C14H608,99.65%) it is provided by Sichuan science spy bio tech ltd；

Phosphoric acid, acetonitrile are chromatographically pure, and water is ultra-pure water, remaining reagent is that analysis is pure.

Metformin hydrochloride (lot number 20160120, Guizhou Shengjitang Pharmaceutical Co., Ltd.), is matched with distilled water when in use At the solution of 20mg/mL；

STZ (lot number S-0130, Sigma companies)；

Blood urea nitrogen kit (lot number 20161111), serum creatinine kit (lot number 20160803), blood uric acid kit (lot number 20161118) and total serum protein quantification kit (lot number 20161115) are purchased from Nanjing, and to build up biotechnology limited Company；

1 kit (lot number 21F305) of TGF-β and VEGF kits (lot number 21F322) are purchased from Yi Kesai biotechnologies (too Storehouse) Co., Ltd.

2, method and result

2.1HPLC methods measure the content of 7 kinds of ingredients in Tibetan medicine SIWEI JIANGHUANG TANGSAN

2.1.1 chromatographic condition

Capcell Pak C₁₈- MG II (4.6mm × 250mm, 5 μm), mobile phase A are acetonitrile, and Mobile phase B is 0.1% phosphorus Aqueous acid, gradient elution (are shown in Table 1)；Detection wavelength is 270nm (0~60min) and 430nm (60~70min, curcumin)； Flow velocity 0.8mL/min；10 μ L of sample size；30 DEG C of column temperature.Wherein theoretical cam curve presses gallic acid, Berberine hydrochloride and curcumin Peak, which calculates, should be not less than 2000,5000 and 4000.

1 SIWEI JIANGHUANG TANGSAN HPLC gradient elution condition of table

T/min	A acetonitriles (%)	0.1% phosphoric acid solutions of B (%)
			0	3	97
8	3	97
			15	17	83
20	17	83
			33	19	81
38	25	75
			48	25	75
70	80	20

2.1.2 the preparation of reference substance solution

Take each reference substance appropriate, it is accurately weighed, in volumetric flask, add 90% methanol to dissolve and be diluted to scale, shake up, makes Obtain gallic acid 1.2480mg/mL, magnoflorine 1.4200mg/mL, ellagic acid 0.3780mg/mL, Jatrorrhizine chloride 0.3020mg/mL pairs of 0.1180mg/mL, palmatin hydrochloride 0.0799mg/mL, Berberine hydrochloride 0.4908mg/mL and curcumin According to product storing solution.It is accurate respectively to draw above-mentioned reference substance storing solution 1mL, it is put into same 10mL volumetric flasks, adds 80% methanol dilute It releases to scale, shakes up to get 124.80 μ g/mL of gallic acid, 142.00 μ g/mL of magnoflorine, 37.80 μ g/mL of ellagic acid, salt 11.80 μ g/mL of sour jateorrhizine, 7.99 μ g/mL of palmatin hydrochloride, 49.08 μ g/mL of Berberine hydrochloride, 0.20 μ g/mL of bisdemethoxycurcumin Mixed reference substance solution, be protected from light refrigeration it is spare.

2.1.3 prepared by test solution

It in the accurately weighed SIWEI JIANGHUANG TANGSAN test sample 0.5g of prescription ratio, sets in conical flask with cover, hydrochloric acid 90% is added Methanol solution (2:100, v/v) 50mL shakes up, and weighs, ultrasonic 20min, and cooling is re-weighed, and less loss weight is supplied with same solvent Amount, shakes up, and filters, and subsequent filtrate is with 0.45 μm of miillpore filter filtration to get test solution.

By test solution, mixed reference substance solution presses chromatographic condition under embodiment 3 " 2.1.1 " item, 10 μ L of sample introduction respectively. The results show that chromatographic peak corresponding with (mixing) reference substance chromatographic retention is presented in test sample chromatography.As depicted in figs. 1 and 2 It is shown.

2.1.4 assay

Laboratory self-control SIWEI JIANGHUANG TANGSAN test sample 0.5g is taken in prescription ratio, it is each 3 parts, accurately weighed, by embodiment 3 Under " 2.1.2 " item prepared by test solution preparation method, is measured according to chromatographic condition under embodiment 3 " 2.1.1 " item, calculates content, It is shown in Table 2.

2 SIWEI JIANGHUANG TANGSAN assay result (n=3) of table

	Sample 1	Sample 2	Sample 3	Average value
					Ingredient	/(mg.g-1)	/(mg.g-1)	/(mg.g-1)	/(mg.g-1)
Gallic acid	7.6496	7.6263	7.6687	7.6482
					Magnoflorine	13.7959	13.8347	13.7767	13.8024
Ellagic acid	3.7465	3.7116	3.7289	3.7290
					Jatrorrhizine chloride	0.9291	0.9442	0.9215	0.9316
Palmatin hydrochloride	0.6750	0.6848	0.6769	0.6789
					Berberine hydrochloride	4.6855	4.6797	4.6688	4.6780
Curcumin	3.7720	3.7939	3.8010	3.7890

As a result measure gallic acid in SIWEI JIANGHUANG TANGSAN, magnoflorine, ellagic acid, Jatrorrhizine chloride, palmatin hydrochloride, The content of Berberine hydrochloride and curcumin be respectively 7.6482mg/g, 13.8024mg/g, 3.7290mg/g, 0.9316mg/g, 0.6789mg/g, 4.6780mg/g and 3.7890mg/g.

Beneficial effects of the present invention are proved below by way of experimental example.

Influence of the 1 bulk pharmaceutical chemicals consumption proportion of experimental example to treatment diabetic nephropathy effect

1 material

1.1 animal

Cleaning grade male SD rat, 220~250g of weight test Company of Animals Ltd. center up to rich fruit by Chengdu and provide, Production licence number：SCXK (river) 2015~030.

1.2 drugs and reagent

Four turmeric soup is made of turmeric, barberry skin, emblic, puncture vine four traditional Chinese medicine, turmeric, emblic, 3 taste medicinal material of puncture vine It is purchased from Chengdu International trade city medicinal material market, identifies, meets through Chengdu University of Traditional Chinese Medicine Lai Xianrong assistant researcher《Middle traditional Chinese medicines Allusion quotation》Pertinent regulations under version (one) text items in 2015.Barberry skin medicinal material, place of production Tibet are provided by Tibet sweet dew Tibetan Pharmaceutical Factory, warp Chengdu University of Traditional Chinese Medicine relies first flourish, Fan Gang assistant researcher's identification, meets related under six provinces and regions Tibetan medicine standard barberry skin medicinal material items Regulation.Each flavour of a drug clinical medicine dose range recorded according to the drug standards with the 1/3 of minimum amount is lower bound, highest dosage 2 times be high limit, rat is used as equivalent dose (Rat Standard weight 0.2kg, adult's standard weight by 10 times of amounts of people's dosage 70kg), 8 compatibilities, compatibility amount and concentration such as table 1,2,2-1 are set as using uniform design：

1 four turmeric soup difference compatibility dosage of table and concentration (rat model dosage)

2 four turmeric soup difference compatibility dosage of table (by people's dosage of Shape Coefficient formula conversion)

Table 2-1 four turmeric soup difference compatibilities dosage is (by the people of 10 times of conversions of rat equivalent dose behaviour dosage

With dosage)

1. Metformin hydrochloride control group, by people's dosage 1.5g/70kg/ days, about 20mg/kg, rat pressed the 10 of people's dosage Amount is used as equivalent dose again, i.e. rat dosage is 200mg/kg/ days.

2. Berberine hydrochloride control group, treatment diabetic nephropathy clinical effectively people's dosage 1.2g/70kg/ days, 17.1mg/ Kg, rat are used as equivalent dose by 10 times of amounts of people's dosage, i.e. rat dosage is 171mg/kg/ days.

3. Berberine hydrochloride+Berbamine hydrochloride control group, by Berbamine hydrochloride people's dosage 336mg/70kg/ days, 4.8mg/ Kg, rat are used as equivalent dose by 10 times of amounts of people's dosage, i.e. rat dosage is 48mg/kg/ days；It is controlled by Berberine hydrochloride Clinical effectively people's dosage 1.2g/70kg/ days, 17.1mg/kg of diabetic nephropathy is treated, rat is by 10 times of amounts of people's dosage as equivalent Dosage, i.e. rat dosage are 171mg/kg/ days；It is mixed with by the above dosage.

4. the quantity of barberry skin control group, barberry skin is 3-5g/ days, the height of dosage is limited to 1.5-10g/ days, folding Calculation is that the dosage of rat is 0.428-0.714g/kg, based on 0.7g/kg.

Said preparation extraction process is：Each bulk pharmaceutical chemicals are weighed in the ratio of bulk pharmaceutical chemicals in the tested number 1~8 of table 1, divides 3 times and carries It takes, the 1st time plus 12 times amount water impregnates 1.0h, decocts 1.0h, gauze filtration；The filter residue that 1st time is obtained by filtration is decocted 2 times again, Every time plus 10 times are measured water, and 1h/ times, gauze filters.3 filtrate is collected together to the concentration for being concentrated into table 1.

Metformin hydrochloride (Guiyang City, Guizhou Province Qingzhen medical industry garden, Chinese medicines quasi-word H52020955), when in use It is made into 20mg.ml with distilled water^-1Solution；STZ (Sigma companies, lot number：S-0130)；Urine proteins, blood urea nitrogen (BUN), blood Creatinine (Scr), blood uric acid and total serum protein quantification kit (the above kit is purchased from Nanjing biology and builds up)；TGF-β1、 VEGF ELISA kits are purchased from Yi Kesai biotechnologies (granary) Co., Ltd.

1.3 instrument

Roche brilliance type (ACCCU-CHEK Performa) blood glucose meter (mating blood sugar test paper and disposable blood taking needle), sieve Family name's diagnostic products (Shanghai) Co., Ltd. produces；

The multi-functional Thermo companies of the readout instrument U.S. of VARIOSKAN FLASH 2.4.3 all-wave lengths；

Constant temperature culture oscillator (ZHWY-100H), Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd..

2 methods

2.1 animal packets, modeling and administration

It after rat adaptability is raised 1 week, is grouped at random after being layered by weight, chooses 7 rats as blank pair According to group, remaining is diabetic model group.

It is deprived of food but not water 12h, according to preliminary experiment, model group is with 60mg.kg^-1The STZ of weight single intraperitoneal injection 1% (it is dissolved in 0.1mol.L before use^-1, 4 DEG C, the sterile sodium citrate buffer solution of PH 4.5), blank group injects same dosage lemon Sour sodium buffer solution presses 2g.kg to model group after 4 hours with 50% glucose^-1Weight ig (gastric infusion), prevents rat hypoglycemia Death, after 72h tail point blood sampling survey fasting blood-glucose, with fasting blood-glucose ﹥ 16.7mmol.L^-1For Glycemia Decline success, non-Cheng Mo is picked It removes.

Model group and blank group ig physiological saline after early diabetic nephropathy is formed, remaining each group ig give corresponding medicine Object gives the drug of tested number 1~8 and each drug control group in table 1 respectively, all groups are pressed 1ml.100g^-1Weight Administration, 1 time a day, successive administration 30d.

2.2 samples are collected

After 30d is administered, animal is deprived of food but not water 12h, surveys last blood glucose, weighs, by 0.7ml/100g weight with 20% Urethane anesthetized rat opens abdominal cavity, abdominal aortic blood, 3 000r.min^-1Centrifugation, takes supernatant；It is quickly taken after taking serum Go out left kidney, remove coating, weigh, calculates renal index (kidney quality/weight).

The part nephridial tissue of 2~3 rats of another every group of selection is fixed with 2.5% glutaraldehyde solution, carries out ultra microstructure sight Examine and measure renal glomerular basement membrane thickness, Electronic Speculum result figure 3.

Fig. 3 shows that rats in normal control group glomerular basement membrane thickness is uniform, sertoli cell marshalling, the uniform nothing of matrix It thickens, deposit is had no between podocytic process；Model group basilar memebrane obviously thickens, and visible electron dense object deposits, sertoli cell podocytic process number Amount is reduced, is merged, and partly disappeared dissolving.Melbine group, each compatibility group intervention in Tibetan medicine four turmeric side compared with model group After can be effectively improved pathology damage, renal glomerular basement membrane thickness is reduced.Wherein melbine group, the 3rd group and 5th group pair The protective effect of DN Renal Glomeruli In Rats structural damages becomes apparent.

2.3 observation index and detection method

2.3.1 rat general state

Situations such as including rat spirit, hair color, weight, diet drinking-water, urine volume.

2.3.2 Biochemical Indexes

Serum FBG, BUN, Scr, UA, total serum protein are measured using 7600 type automatic clinical chemistry analyzers.

2.3.3ELISA method detection serum TGF-β 1, VEGF.

3 results

3.1 general status are observed

Compared with normal group, there is much more apparent drinks, diuresis, more foods in model group, and gradually appears apathetic, and reaction is late The symptoms such as blunt, hair color yellowing is matt, and depilation, weight decline；Compared with model group, administration group animal state is preferable, weight Increase higher than model group.

The influence of 3.2 pairs of Histopathology

Four rats are extracted in blank group, model group and administration group respectively and do preliminary experiment, tissues observed pathomorphism is seen It examines.

Normal rats nephridial tissue has no pathological change；Model group rats nephridial tissue visible part glomerulus falls off, and kidney is small Ball mesangial matrix *, capillary loops are closed, and renal tubule brush border falls off, a small amount of fibrosis around blood vessel, each treatment group's nephridial tissue Pathological change has different degrees of improvement compared with model group.

3.3 PCA analysis results

3.3.1 four turmeric soup difference compatibility dosage-Pharmacodynamic Data Analysis

3 four turmeric soup difference compatibility dosage of table to the Pharmacodynamic Data of diabetic nephropathy (N=7)

Note：The * P compared with model group<0.05,**P<0.01.

The present invention has carried out acting on relevant drug effect with " controlling diabetic nephropathy " to 8 different compatibility dosage of four turmeric soup Research is learned, the results are shown in Table 3.

The experimental results showed that：Using STZ induced diabetes nephrotic rats models, after modeling 10 weeks, it is administered 4 weeks, control Group, four turmeric soup difference compatibility group indices compared with model group have different degrees of improvement, specially：

1. compared with blank group, the renal index of model group increases significant meaning (P ＜ 0.01)；Compared with model group, The significant meaning (P ＜ 0.05) of renal index reduction of melbine group, 1,3 group of compatibility dosage group.

2. compared with blank group, the blood urea nitrogen level of model group increases significant meaning (P ＜ 0.01)；With model group Compare, melbine group, Berberine hydrochloride+Berbamine hydrochloride group, barberry skin group, the blood urea nitrogen of compatibility dosage group 1~6 are horizontal Reduce significant meaning (P ＜ 0.05).

3. compared with blank group, the serum creatinine level of model group increases significant meaning (P ＜ 0.01)；With model group ratio Compared with the blood flesh of, melbine group, jamaicin group, Berberine hydrochloride+Berbamine hydrochloride group, barberry skin group, compatibility dosage group 2~7 Acid anhydride level reduces significant meaning (P ＜ 0.05).

4. compared with blank group, the serum uric acid level of model group increases significant meaning (P ＜ 0.01)；With model group ratio Compared with the blood urine sour water of, melbine group, Berberine hydrochloride+Berbamine hydrochloride group, barberry skin group, compatibility dosage group 1,3 and 5~8 The low significant meaning (P ＜ 0.05) of pancake.

5. compared with blank group, the total serum protein level of model group reduces significant meaning (P ＜ 0.01)；With model Group compares, and melbine group, Berberine hydrochloride+Berbamine hydrochloride group, the total serum protein level of compatibility dosage group 1~7 increase Significant meaning (P ＜ 0.05).

6. compared with blank group, the fasting blood glucose level of model group increases significant meaning (P ＜ 0.01)；With model group Compare, melbine group, jamaicin group, Berberine hydrochloride+Berbamine hydrochloride group, the empty stomach of barberry skin group, each compatibility dosage group Blood glucose level reduces significant meaning (P ＜ 0.05), and is superior to melbine group.

7. compared with blank group, the TGF-β 1 of model group is horizontal to increase significant meaning (P ＜ 0.01)；With model group ratio Compared with the TGF- of, melbine group, jamaicin group, Berberine hydrochloride+Berbamine hydrochloride group, barberry skin group, compatibility dosage group 1,3,5 β 1 is horizontal to reduce significant meaning (P ＜ 0.05).

8. compared with blank group, the VEGF levels of model group increase significant meaning (P ＜ 0.01)；With model group ratio Compared with the VEGF levels of compatibility dosage group 1~7 reduce significant meaning (P ＜ 0.05).

This experimental result shows that, with the progress of diabetic nephropathy, serum TGF-β 1, VEGF levels gradually rise, and serum TGF-β 1 is horizontal to be proportionate with VEGF.Speculate that kidney local T GF- β 1, vegf expression increase, the two collaboration promotes new vessels Generation, change the microvessel structure of kidney, take part in the generation of diabetic nephropathy.Each dosage ratio as can be seen from Figure 4 Group has preferable degree of being spatially separating with blank group, model group, illustrates that each dosage ratio group has the spy different from model group, blank group Sign, but it is closer to model group, and further from blank group, illustrate that each proportioning group has certain change to the indices of DN It is kind, it can be difficult to being improved to the degree of blank group；1,2,3,4,5,6 group of proportioning is preferable, wherein the influence of the 5th group of proportioning is more bright It is aobvious.Fig. 5 indicates the correlation of each data variable and principal component, represents the influence degree of each data variable, it can be seen that renal index, BUN levels, FBG levels, TGF-β 1, VEGF levels are maximum to the load of principal component 1 (PC1), show they be it is main influence because Element；This may be related with VEGF with the drug hypoglycemic effect, inhibition extracellular matrix composition-factor TGF-β 1.However diabetes The development of nephrosis and the increase of extracellular mechanism and accumulation are closely related, research shows that conversion growth factor (Transforming Growth Factor, TGF-β 1) and VEGF can all increase the synthesis of extracellular matrix.TGF-β 1 is that diabetic nephropathy was fallen ill Core factor in journey in complicated cytokine network, there are five types of isomers, only TGF-β 1 is being fed in this five kinds of isomers The expression of newborn animal kidney is most.TGF-β 1, which has, to be promoted cellular mast, increases extracellular matrix generation, by inhibiting substrate degradation Enzymatic synthesis and the effect for reducing extracellular matrix degradation.VEGF is a kind of vascular endothelial growth factor of high degree of specificity, is passed through Change blood vessel inner skin cell function and structure, the permeability for increasing glomerular capillary, promote extracellular matrix synthesis and Participate in the disease progression of DN.Clinical research shows that diabetic urinates VEGF levels in DN early stages and raises, and Urinary VEGF levels with DN lesion growths there is clinical correlation, the high expression level that diabetic urinates VEGF to lead to glomerular filtration The pathology of membrane structure sexually revises, and the macromoleculars such as albumin is caused to pass through.

2 four turmeric soup of experimental example optimizes influence of the dosage to Spontaneous Diabetic nephrosis db/db mouse models

Fig. 7 is principal component analysis-shot chart of the four turmeric soup proportion optimizing to db/db pharmacodynamics in Mice indexs；Fig. 8 is Partial least squares analysis figure of the four turmeric soup proportion optimizing to db/db pharmacodynamics in Mice indexs；Fig. 9 optimizes for four turmeric soup With the hierarchical cluster analysis figure for comparing db/db pharmacodynamics in Mice indexs.Related Pharmacodynamic Data carries out PCA principal component analysis, HCA Hierarchical cluster analysis, the results showed that it is there is administration group to be obviously divided into two classes with model group (db/db groups) more, illustrate that administration group has DN It is apparent to improve.

1, animal

Using Nanjing University-db/db mouse (model group) of Nanjing biological medicine research institute offer, db/m mouse (blank Group).

Db/db mouse models behave like the diabetes B of the mankind, are common for researching and developing treatment diabetes B drug One of 5 kinds of animal models and one of the animal models recommended of FDA, the characteristics of can reflecting human diseases substantially, in having Etc. the features such as degree are fat, blood fat is high.

The result of study of diabetes B rat model, determining four turmeric soup is induced to optimize dose ratio and be according to STZ Turmeric：Barberry skin：Emblic：Puncture vine=3：10：3：6, it is turmeric to convert as people with daily dosage：Barberry skin：Emblic：Puncture vine =1.5g：5g：1.5g：3g calculates animal dosage according to Shape Coefficient formula and (because the obese degree of db/db mouse is higher, does not have There is the Shape Coefficient of standard, but it belongs to mouse scope, therefore it is 0.0898, db/db that the Shape Coefficient of db/db mouse, which is fixed tentatively, Standard weight 0.05kg, db/m mouse standard weight 0.02kg, the adult standard weight 70kg of mouse).Calculate people's use etc. Effect dosage is equivalent to about 9 times of db/db mouse.It is thus determined that four turmeric soup optimization dosage is grouped into low dose group (0.978g/ Kg is approximately equivalent to 4 times), middle dose group (1.957g/kg is approximately equivalent to 9 times), (3.914g/kg is approximately equivalent to 20 to high dose group Again), former dosage group (1.305g/kg is approximately equivalent to 9 times) is another to set jamaicin group (0.157g/kg is approximately equivalent to 9 times), two First biguanides group (0.196g/kg is approximately equivalent to 9 times), barberry skin group (0.652g/kg is approximately equivalent to 9 times).

The influence of 1.1 pairs of kidney organ's indexes and biochemical indicator

Table 9 is the shadow that SIWEI JIANGHUANG TANGSAN optimizes dosage to db/db diabetic mouse models general indicator and biochemical indicator It rings.

Compared with db/m groups, the kidney organ's index and weight pole conspicuousness of db/db groups increase (P ﹤ 0.01)；With db/db Group compares, and high dose group, jamaicin group, kidney organ's index conspicuousness of former dosage group decline (P ﹤ 0.05), middle dose group, small Bark of a cork tree alkali group, former dosage group, the weight conspicuousness of barberry skin group decline (P ﹤ 0.05).

Compared with db/m groups, the pole fasting blood-glucose index (FBG) conspicuousness of db/db groups increases (P ﹤ 0.01)；With db/db groups Compare, melbine group, high dose group, the FBG conspicuousnesses of jamaicin group decline (P ﹤ 0.05).

Compared with db/m groups, the horizontal pole (BUN) conspicuousness of blood urea nitrogen of db/db groups increases (P ﹤ 0.01)；With db/db groups Compare, melbine group, high dose group, middle dose group, low dose group, former dosage group, jamaicin group, the BUN of barberry skin group are aobvious Work property declines (P ﹤ 0.05).

Compared with db/m groups, the pole urinary albumin excretion ratio (UAE) conspicuousness of db/db groups increases (P ﹤ 0.01)；With db/db Group compares, melbine group, low dose group, middle dose group, high dose group, former dosage group, jamaicin group, barberry skin group UAE Pole conspicuousness declines (P ﹤ 0.01).

Compared with db/m groups, the pole Urinary Microalbumin Excretion rate (UAlb) conspicuousness of db/db groups increases (P ﹤ 0.01)；With Db/db groups compare, melbine group, low dose group, middle dose group, high dose group, former dosage group, jamaicin group, barberry skin group UAlb conspicuousnesses decline (P ﹤ 0.05).

Compared with db/m groups, the pole serum creatinine level (Scr) conspicuousness of db/db groups increases (P ﹤ 0.01)；With db/db groups Compare, melbine group, low dose group, middle dose group, high dose group, former dosage group, jamaicin group, the Scr of barberry skin group are aobvious Work property declines (P ﹤ 0.05).

Compared with db/m groups, the pole serum uric acid level (UA) conspicuousness of db/db groups increases (P ﹤ 0.01)；With db/db groups Compare, melbine group, low dose group, middle dose group, former dosage group, jamaicin group, the UA conspicuousnesses of barberry skin group decline (P ﹤ 0.05)。

Compared with db/m groups, horizontal (TGF-β 1) pole conspicuousnesses of transforming growth factor-β l of db/db groups increase (P ﹤ 0.01)；Compared with db/db groups, melbine group, low dose group, middle dose group, high dose group, former dosage group, jamaicin group, 1 conspicuousness of TGF-β of barberry skin group declines (P ﹤ 0.05).TGF-β 1 is the strongest cause kidney fibrosis cell factor of effect.

Compared with db/m groups, vascular endothelial growth factor (VEGF) conspicuousness of db/db groups increases (P ﹤ 0.05)；With Db/db groups compare, and the VEGF conspicuousnesses of jamaicin group decline.

9 SIWEI JIANGHUANG TANGSAN of table optimizes influence of the dosage to db/db diabetic mouse models general indicator and biochemical indicator

Data format is expressed as means standard deviation, n=6；*p<0.05,**p<0.01, the compared with db/db groups

In conclusion four turmeric soup optimization dosage is equal to the renal index of diabetic nephropathy (DN) and every biochemical indicator It improves significantly, mechanism of action may be related with improvement renal fibrosis and adjusting 1 expression of TGF-β.

1.2 on the histological influence of Pathological

HE coloration results show：Compared with db/m groups, the renal cells oedema of db/db groups, kidney peplos are narrow, on Chrotoplast falls off, steatosis is notable；Compared with db/db groups, melbine group, low dose group, middle dose group, high dose group, Former dosage group, jamaicin group, the above pathological manifestations of barberry skin group are improved.PASM coloration results show：With db/m groups Compare, the renal tubular basement membrane thickness of db/db groups dramatically increases (IOD) (P ﹤ 0.01)；Compared with db/db groups, melbine group, Low dose group, middle dose group, high dose group, former dosage group, jamaicin group, barberry skin group renal tubular basement membrane thickness significantly drop Low (IOD) (P ﹤ 0.01).

1.3 couples of kidney H I F-1a, TGF-β 1, VEGF gene expressions influence

HIF-1 α, TGF-β β 1 and VEGF have been confirmed as the crucial adjusting factor of DN kidney region fibrosis, using real-time The mRNA of quantitative PCR detection DN correlation factors is expressed.Compared with db/m groups, HIF-1a, TGF-β 1, the VEGF genes of db/db groups (mRNA) expression extremely significantly increases (P ＜ 0.01)；Compared with db/db groups, melbine group, low dose group, middle dose group, Jamaicin group, barberry skin group HIF-1 α genes (mRNA) expression significantly reduce (P ＜ 0.05), melbine group, low dose Amount group, middle dose group, high dose group, the TGF-β 1 of jamaicin group, VEGF genes (mRNA) expression significantly reduce (P ＜ 0.05)。

The influence that 1.4 couples of kidney H I F-1a, TGF-β 1, vegf protein are expressed

The protein expression of DN correlation factors is detected using western blots methods.Compared with db/m groups, db/db groups HIF-1a, TGF-β 1, vegf protein expression extremely significantly increase (P ＜ 0.01)；It is melbine group, low compared with db/db groups Dosage group, middle dose group, high dose group, former dosage group, jamaicin group, the HIF-1a of barberry skin group, TGF-β 1, vegf protein table (P ＜ 0.01) is significantly reduced up to level.

The in situ detection of 1.5 couples of kidney H I F-1a, TGF-β 1, VEGF Tissue distributions

The Tissue distribution feature of DN correlation factors is detected using ImmunohistochemistryMethods Methods.Compared with db/m groups, db/db groups HIF-1a, TGF-β 1, vegf expression level extremely significantly increase (P ＜ 0.01)；Compared with db/db groups, melbine group, low dosage 1 protein expression level pole of the TGF-β significant decrease of group, middle dose group, high dose group, former dosage group, jamaicin group, barberry skin group (P ＜ 0.01), melbine group, low dose group, middle dose group, high dose group, jamaicin group, barberry skin group vegf protein table (P ＜ 0.05) is significantly reduced up to level, melbine group, low dose group, middle dose group, high dose group, jamaicin group, barberry skin The HIF-1a protein expression levels pole of group significantly reduces (P ＜ 0.01),.

The influence of 1.6 pairs of intestinal floras

Compared with db/m groups, the group's composition and diversity of db/db groups have pole significant difference (P ＜ 0.01), firmicutes Door Lactobacillus ratio dramatically increases, it is known that Bacillus acidi lactici and lipid metaboli index LDL-C (low-density lipoprotein) notable negative It closes, the quantity of Bacillus acidi lactici increases in enteron aisle, and the LDL-C of excessive level is improved and reduces.It is small compared with db/db groups The group's composition and diversity of bark of a cork tree alkali group have significant difference (P ＜ 0.05), significantly reduce the ratio of Lactobacillus, still Ratio is also close with normal group after it is reduced and is increased slightly, hence it is evident that increases Proteobacteria Enterobacter, Escherichia coli will The ratio for congratulating Pseudomonas, these results suggest that Berberine hydrochloride has apparent influence to intestinal flora, newborn with the reduction of LDL-C Acidfast bacilli category ratio restores to prompt the barberry micromicro of hydrochloric barberry alkali composition with preferable to the degree close with Normal group The disorders of lipid metabolism of db/db groups is corrected on ground, is improved significantly to hyperlipidemia；Low dose group, high dose group, former dosage group pair The Microbial community composition and diversity of intestinal flora does not have a significant impact (P ＞ 0.05), low dose group, high dose group, original Dosage group increases Lactobacillus ratio, and [Liu Tingting, Cai De letter real-time quantitative PCR methods analyze diabetes B Intestinal Mucosal Injury in Patients Undergoing breast Change [J] the world Chinese of acidfast bacilli strain quantity digests magazine, 2012,20 (15):1359-1365.], this and db/db groups In the diabetes middle and later periods, the correction of disorders of lipid metabolism needs the therapeutic process of longer-term, therefore its Lactobacillus ratio for many places Increase to reduce excessively high LDL-C concentration, illustrates that SIWEI JIANGHUANG TANGSAN can improve LDL-C type hyperlipidemias.

2 four turmeric soups optimize influence of the dosage to STZ induced diabetic rats model serum biomarkers

STZ induced diabetic rats model serum biomarkers are analyzed using LC-MS metabolism group method.With Db/db groups compare, the common difference generation of 4 groups of db/m groups, 1 group of uniform design compatibility, 2 groups of uniform design compatibility, jamaicin group etc. It is Xanthosine (P ﹤ 0.05) to thank to marker；Compared with db/db groups, db/m groups, 1 group of uniform design compatibility, uniform design are matched 52 groups, barberry skin group, 5 groups of berbamine-jamaicin group etc. common difference metabolic markers be Ornithine (P ＜ 0.05).

Xanthosine (xanthosine)：Xanthosine is the product of body eubolism, generally may be used also in vivo It is further broken into xanthine, finally oxidation forms uric acid (uric acid, UA) and excreted with urine.Glycosuria is induced in STZ In the research of sick rat model, compared with blank group, the pole serum uric acid level (UA) conspicuousness of model group increases (P ＜ 0.01)；Compared with model group, the UA conspicuousnesses of 1 group of uniform design compatibility decline (P ＜ 0.05).In db/db diabetic mice moulds In the research of type, compared with db/m groups, the pole serum uric acid level (UA) conspicuousness of db/db groups increases (P ＜ 0.01)；With db/ Db groups compare, and low dose group, middle dose group, the UA conspicuousnesses of jamaicin group decline (P ＜ 0.05).Illustrate xanthosine conduct Significant difference metabolin has pharmacological significance and clinical value.

Ornithine (ornithine)：Ornithine recycles (urea cycle), and urea is row in most of land_atmosphere coupling model bodies Go out the mode of ammonia, any one step of urea cycle, which goes wrong, to be likely to generate disease.Most of ammonia in human body is in liver It is interior that urea synthesis is recycled by ornithine.A part is used for the amination of ketone acid, glutamine is synthesized, by kidney renal tubule Chrotoplast generates, and forms amine salt in kidney, is discharged from urine, and ammonia is mainly excluded with ammonium salts external in urinary ammonia urine, is to adjust One of critical function of electrolyte balance.Urea nitrogen accounts for important in the diseases such as diagnosis diabetic ketoacidosis, hepatosis Status.In the research of STZ induced diabetic rats models, compared with blank group, horizontal pole (BUN) of blood urea nitrogen of model group Conspicuousness increases (P ＜ 0.01)；Compared with model group, 1 group of uniform design compatibility, 2 groups of uniform design compatibility, barberry skin group BUN conspicuousnesses decline (P ＜ 0.05).In the research of db/db diabetic mouse models, compared with db/m groups, the blood of db/db groups The pole urea nitrogen levels (BUN) conspicuousness increases (P ＜ 0.01)；Compared with db/db groups, low dose group, middle dose group, high dose Group, the poles the BUN conspicuousness of jamaicin group decline (P ＜ 0.01).Illustrate that ornithine has pharmacology as significant difference metabolin Learn meaning and clinical value.

By above-mentioned experimental data it is found that compared to 1 group in embodiment 1~8 groups, metering proportion of the present invention is turmeric：It is small Bark of a cork tree skin：Emblic：Puncture vine=3：10：3：6 indices illustrate this hair more close to blank group, and more far from model group Bright middle turmeric：Barberry skin：Emblic：Puncture vine=3：10：3：6 metering proportion is most notable to the improvement of the indices of DN, Effect for treating diabetic nephropathy is optimal.

The recipe parameters research of 3 pharmaceutical composition of the present invention of experimental example

According in the SIWEI JIANGHUANG TANGSAN drug standards and the Conventional wisdom of Tibetan medicine's medication, dosage form is decoction made from powder, using adding water The method of decoction extracts, and the recipe parameters such as amount of water, extraction time, extraction time are indefinite.The present invention is with nutgall Acid, jamaicin, curcumin, paste-forming rate are inspection target, optimize water boiling method extraction process using uniform design.

Four turmeric capsules preparation technique is：Four taste of this product, turmeric are ground into coarse powder, are made with 10 times of 70% ethyl alcohol of amount molten Agent impregnates 12 hours, and slowly diacolation, collects percolate, ethyl alcohol is recovered under reduced pressure and is concentrated into thick paste, and it is suitable that pregelatinized starch is added Amount, is dried under reduced pressure, is ground into fine powder；Barberry skin is broken into coarse powder, adds 14 times of 75% ethyl alcohol of amount, reflux respectively for the first time and for the second time Filtrate is collected in extraction 2 hours, third time plus 12 times of 75% ethyl alcohol of amount, refluxing extraction 1 hour, merging filtrate, filtration；Emblic It is ground into coarse powder with puncture vine, for the first time plus 14 times are measured 90% ethyl alcohol, and refluxing extraction 2 hours adds 12 times of 70% ethyl alcohol of amount for the second time, Refluxing extraction 1 hour, merging filtrate, filtration collect filtrate, above two extracting solution are merged, ethyl alcohol is recovered under reduced pressure and concentrates It to thick paste, is dried under reduced pressure, is ground into fine powder.Above-mentioned fine powder is mixed, (extract powder is pressed：Microcrystalline cellulose=13:2) ratio is mixed It is even, add 80% ethanol solution that particle is made, Magnesium Stearate proper quantity, mixing is added, 60 DEG C of dryings are packed into capsule, are made 1000, To obtain the final product.

To sum up, the present invention is by observing four turmeric soup difference compatibility dosage to the pharmacodynamic results of diabetic nephropathy rats simultaneously The best dose-effect compatibility of four turmeric soup for capableing of effective prevention early diabetic nephropathy is excavated in conjunction with Principal Component Analysis, promotes it Clinical application and new drug development.Particularly, metering proportion of the present invention is turmeric：Barberry skin：Emblic：Puncture vine=3：10：3：6 Indices illustrate turmeric in the present invention more close to blank group, and more far from model group：Barberry skin：Emblic：Puncture vine= 3：10：3：6 metering proportion is most notable to the improvement of the indices of DN, close to blank group.

Claims

1. a kind of pharmaceutical composition of prevention diabetic nephropathy, it is characterized in that：It is prepared by the bulk pharmaceutical chemicals that following weight matches It forms：1~15 part of turmeric, 1~8 part of barberry skin, 1~15 part of emblic, 1~8 part of puncture vine.

2. pharmaceutical composition as described in claim 1, it is characterized in that：It is the bulk pharmaceutical chemicals matched by following weight prepare and At：1~2 part of turmeric, 3~7 parts of barberry skin, 1~7 part of emblic, 2~8 parts of puncture vine.

3. pharmaceutical composition as claimed in claim 1 or 2, it is characterized in that：It is prepared by the bulk pharmaceutical chemicals that following weight matches It forms：3 parts of turmeric, 10 parts of barberry skin, 3 parts of emblic, 6 parts of puncture vine.

4. the pharmaceutical composition as described in claims 1 to 3 any one, it is characterized in that：It is by each weight proportion bulk pharmaceutical chemicals Primary medicinal powder, water or organic solvent extract, pharmaceutically acceptable auxiliary material is added or complementary ingredient is prepared Preparation.

5. pharmaceutical composition as claimed in claim 4, it is characterized in that：The preparation is oral preparation；Preferably, described Preparation is powder, granule, capsule, tablet, pill, decoction or mixture.

6. a kind of preparation method of Claims 1 to 5 any one described pharmaceutical composition, it is characterized in that：Include the following steps： The bulk pharmaceutical chemicals for taking each weight proportion directly beat powder, or water or organic solvent extraction is added, and add pharmaceutically acceptable auxiliary Material or complementary ingredient to get；

Preferably, the water extracting method is：The bulk pharmaceutical chemicals of each weight proportion, extracting in water are taken three times, to add 12 times of water for the first time Amount is impregnated 1 hour, is decocted 1 hour, and filtration adds water to decoct again 2 times, each plus 10 times of amounts of water, 1 hour every time, merging filtrate, Concentration to get.

7. Claims 1 to 5 any one described pharmaceutical composition is in preparing the drug for the treatment of and/or diabetes and nephropathy preventing Purposes.

8. a kind of method of quality control of Claims 1 to 5 any one described pharmaceutical composition, it is characterized in that：The method Measure gallic acid, ellagic acid, magnoflorine, Berberine hydrochloride, Jatrorrhizine chloride, 7 kinds of ingredients of palmatin hydrochloride and curcumin Content；

Preferably, the content of the ingredient is：Gallic acid 30.64mgg^-1, ellagic acid 48.60mgg^-1, magnoflorine 49.32mg·g^-1, Berberine hydrochloride 39.47mgg^-1, Jatrorrhizine chloride 42.24mgg^-1, palmatin hydrochloride 47.66mg g^-1, curcumin 62.73mgg^-1。

9. a kind of efficient liquid phase detection method of Claims 1 to 5 any one described pharmaceutical composition, it is characterized in that：Chromatography Condition is：

Stationary phase：Using octadecylsilane chemically bonded silica as filler；

10. efficient liquid phase detection method as claimed in claim 9, it is characterized in that：The stationary phase is Capcell Pak II chromatographic columns of C18-MG；Chromatographic column specification：Column length is 25cm, and internal diameter 4.6mm, grain size is 5 μm；

And/or flow velocity 0.8mLmin-1；

And/or 10 μ L of sample size；

And/or 30 DEG C of column temperature；

And/or 0~60min Detection wavelengths are 270nm, 60~70min Detection wavelengths are 430nm；

And/or eluent gradient elution requirement is：0 → 8min, 3% → 3%A；8 → 15min, 3% → 17%A；15→ 20min, 17% → 17%A；20 → 33min, 17% → 20%A；33 → 38min, 20% → 25%A；38 → 48min, 25% → 25%A；48 → 70min, 25%A~80%A.