CN102670698B - The application of Radix Flemingiae Philippinensis extract in preparation control diabetes medicament - Google Patents
The application of Radix Flemingiae Philippinensis extract in preparation control diabetes medicament Download PDFInfo
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Abstract
The present invention discloses the application in a kind of Radix Flemingiae Philippinensis extract preparation control diabetes medicament, the refining Radix Flemingiae Philippinensis extract obtained after the enrichment Radix Flemingiae Philippinensis extract that the Radix Flemingiae Philippinensis alcohol extraction acid hypostasis that described Radix Flemingiae Philippinensis extract is the former extract of Radix Flemingiae Philippinensis obtained after Radix Flemingiae Philippinensis lower alcohol extraction, described former extract obtains after Acid precipitation, described Radix Flemingiae Philippinensis alcohol extraction acid hypostasis obtain after polyamide enrichment or described enrichment Radix Flemingiae Philippinensis extract go up polyamide purification again.Radix Flemingiae Philippinensis extract has the activity reducing blood glucose, improve diabetic animal life quality, resist response to oxidative stress, improve carbohydrate tolerance.Compared with metformin, have and can improve diabetic animal body weight, minimizing amount of drinking water and food ration, and the advantage of response to oxidative stress can be resisted.Therefore its activity improving diabetic animal carbohydrate tolerance is better than acarbose, and Radix Flemingiae Philippinensis extract has good research and development and is worth in preparation control diabetes medicament.
Description
Technical field
The invention belongs to the field of Chinese medicines, be specifically related to the application of Radix Flemingiae Philippinensis extract in preparation control diabetes medicament.
Background technology
Diabetes are diseases of the easy overheap of glucose in a kind of blood.External its another name of giving is " reticent killer " (Silent Killer), particularly " adult diabetes mellitus ".It is high especially that the middle age of more than 40 years old catches rate, in Japan, accounts for 10% in the population of more than 40 years old, namely just have " diabetes " patient in the middle of ten people.Once suffer from " diabetes ", will reduce 10 years more than life-span, contingent complication is throughout whole body.For many years, medical circle is devoted to the research to diabetes always.This is because once suffer from " diabetes ", the trouble of human body arrives with it, because immunologic function weakens, easily to infect caused by flu, pneumonia, pulmonary tuberculosis various catches, and not easily cures.And optionally destroy cell, phagocyte.The defensive enginery of inhibiting tumor cell can weaken greatly, and to making, cancerous cell is active, gathering.Therefore many people to diabetes what is said or talked about and complexion changed, treatment by every possible means.The Remedies for diabetes of chemical classes has Drugs Promoting Insulin Secretion, biguanides, AGI etc., but chemical classes drug side effect is large, and some diabetics is not easily taken.
Radix Flemingiae Philippinensis, have another name called colour of loess chicken, golden pheasant lands (" Zhiwu Mingshi Tukao "), rat tail, thoroughly Pheretima, Millettia Speciosa, winged steed, newton's head (" south of the Five Ridges gather medicinal herbs record "), hang hitching post, very heavyly to hang, follow closely root Herba chenopodii, arrow root, Glycine tomentella, colour of loess over sixty years of age (" Jiangxi Chinese medicine " (10): 64,1957), energetically yellow (" Guangxi wild resource plant "), oxtail swings (" Nanning City's medicine will ").Be grown in the thick grass of hillside.The ground such as distribution Fujian, Taiwan, Guangxi, Guangdong, Hubei, Guizhou, Jiangxi..Conically, long 15 ~ 30 centimetres, root head comparatively expands dry root.Appearance brownish red, has obvious hole skin.Skin zone easily peels off.Commodity are cut into the inclined tab shape being about 3 ~ 7 centimetres more.Matter is hard, and section white, mealiness, in radial striations.Nature and flavor: sweet, acrid, warm.Function cures mainly: expelling wind and removing dampness, and repercussive detoxifies.Dispelling the wind and dampness pathogens arthralgia pain, chronic nephritis, traumatic injury, carbuncle, throat moth.
Summary of the invention
The object of the present invention is to provide the application of Radix Flemingiae Philippinensis extract in preparation control diabetes medicament.
Object of the present invention can be achieved through the following technical solutions:
A kind of Radix Flemingiae Philippinensis extract, the refining Radix Flemingiae Philippinensis extract obtained after the enrichment Radix Flemingiae Philippinensis extract that it is the Radix Flemingiae Philippinensis alcohol extraction acid hypostasis that described Radix Flemingiae Philippinensis extract is the former extract of Radix Flemingiae Philippinensis obtained after Radix Flemingiae Philippinensis lower alcohol extraction, described former extract obtains after Acid precipitation, the sour hypostasis of described Radix Flemingiae Philippinensis alcohol extraction obtains after polyamide enrichment or described enrichment Radix Flemingiae Philippinensis extract go up polyamide purification again, the main active in described Radix Flemingiae Philippinensis extract is genistin and genistein.
Above-mentioned Radix Flemingiae Philippinensis extract, the main active genistin described in it and the content of genistein are 0.5% ~ 60%.Be preferably 25% ~ 60%.
Described Radix Flemingiae Philippinensis extract adopts following methods preparation: after taking Radix Flemingiae Philippinensis pulverizing medicinal materials, 10 ~ 20 times amount 30% ~ 90% ethanol extractions soak 1 ~ 3h, reflux, extract, 2 ~ 4h, extract 2 ~ 4 times, merging filtrate, reclaims ethanol and filtrate concentrate drying is obtained the former extract of Radix Flemingiae Philippinensis.
Described Radix Flemingiae Philippinensis extract adopts following methods preparation: after taking Radix Flemingiae Philippinensis pulverizing medicinal materials, 10 ~ 20 times amount 30% ~ 90% ethanol extractions soak 1 ~ 3h, reflux, extract, 2 ~ 4h, extract 2 ~ 4 times, merging filtrate, reclaim ethanol and filtrate concentrate drying is obtained the former extract of Radix Flemingiae Philippinensis, medicinal liquid pH value is made to be 1 ~ 5 with dripping concentrated hydrochloric acid after being concentrated into 0.25 ~ 2g crude drug/mL after 70% dissolve with ethanol former for Radix Flemingiae Philippinensis extract, 4 DEG C of standing 2 ~ 12h, decompress filter is dry after washing filter cake to neutrality obtains the sour hypostasis of Radix Flemingiae Philippinensis alcohol extraction.
Described Radix Flemingiae Philippinensis extract adopts following methods preparation: after taking Radix Flemingiae Philippinensis pulverizing medicinal materials, 10 ~ 20 times amount 30% ~ 90% ethanol extractions soak 1 ~ 3h, reflux, extract, 2 ~ 4h, extract 2 ~ 4 times, merging filtrate, reclaim ethanol and filtrate concentrate drying is obtained the former extract of Radix Flemingiae Philippinensis, former for Radix Flemingiae Philippinensis extract is dripped concentrated hydrochloric acid make medicinal liquid pH value be 1 ~ 5 with 70% dissolve with ethanol after being concentrated into 0.25 ~ 2g crude drug/mL, 4 DEG C leave standstill 2 ~ 12h, decompress filter washing filter cake is to neutral, it is the sample solution of 3 ~ 7.5mg/mL that Radix Flemingiae Philippinensis alcohol extraction acid hypostasis drying obtained is dissolved in obtained concentration in the alcoholic solution of 10%, by good polyamide resin column processed on sample solution, pillar blade diameter length ratio is 1:4 ~ 7, loading flow velocity is 1 ~ 4BV/h, applied sample amount is no more than 16BV, after loading, colourless to eluent with 10% alcoholic solution eluting impurity, first use 6BV 20% ~ 40% ethanol to carry out eluting to target compound genistin, elution flow rate 2BV/h, obtain genistin position, 35% ethanol elution remove impurity, 6BV 50% ~ 60% ethanol carries out eluting to target compound genistein, elution flow rate 2BV/h, obtains genistein position, merge genistin and genistein position, obtain enrichment Radix Flemingiae Philippinensis extract.Preferred: the concentration of described sample solution is 3mg/mL, and pillar blade diameter length ratio is 1:5.9, and loading flow velocity is 2BV/h, and the concentration of the eluant ethanol of genistin and genistein is respectively 20% and 50%, order number 60 ~ 100 order of polyamide.
Described refining Radix Flemingiae Philippinensis extract, after enrichment Radix Flemingiae Philippinensis extract is gone up polyamide purification again, obtains refining Radix Flemingiae Philippinensis extract.
The application of above-mentioned Radix Flemingiae Philippinensis extract in preparation control diabetes medicament.
Above-mentioned application, it is that described medicine comprises the Radix Flemingiae Philippinensis extract and pharmaceutically acceptable carrier or the conventional adjuvant used for the treatment of effective dose.The dosage form of described medicine comprises pill, powder, capsule, tablet, granule, injection or compound preparation.
Beneficial effect of the present invention:
Radix Flemingiae Philippinensis extract has the activity reducing blood glucose, improve diabetic animal life quality, resist response to oxidative stress, improve carbohydrate tolerance.Compared with metformin, have and can improve diabetic animal body weight, minimizing amount of drinking water and food ration, and the advantage of response to oxidative stress can be resisted.Therefore its activity improving diabetic animal carbohydrate tolerance is better than acarbose, and Radix Flemingiae Philippinensis extract has good research and development and is worth in preparation control diabetes medicament.
Detailed description of the invention
The preparation of genistin and the former extract of genistein in embodiment 1 Radix Flemingiae Philippinensis
1. experiment material
Instrument: LC-20AT high performance liquid chromatograph and SPD-20A UV-detector (Japanese Shimadzu Corporation), SinoChrom ODS-BP 5 μm of chromatographic columns (Dalian Yilite Analytical Instrument Co., Ltd), HH-6 digital display thermostat water bath (Guo Hua Electrical Appliances Co., Ltd).
Reagent: trifluoroacetic acid aqueous solution (world Reagent Company of the U.S., lot number: 1103160), Chromatographic Pure Methanol (Hanbon Sci. & Tech. Co., Ltd., lot number: 112866), analytical pure formic acid (Nanjing Chemistry Reagent Co., Ltd., lot number: 10120111319), commercially available pure water (Hangzhou Wahaha Group Co., Ltd, lot number: 210636), analytical pure hydrochloric acid (Nanjing Chemistry Reagent Co., Ltd., lot number: 080230147), 95% industrial alcohol.
Medicine: genistin chemical reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 111709-200501), genistein chemical reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 111704-200501), Radix Flemingiae Philippinensis medical material (originating from Yang Shuo, Guangxi).
2. the experimental technique of Study on extraction
Liquid phase chromatogram condition: chromatographic column is Yi Lite SinoChrom ODS-BP 5 μm; Mobile phase is acetonitrile-0.2% formic acid water; Determined wavelength is 261nm; Column temperature is 30 DEG C; Flow velocity is 1mL/min; Gradient elution program is in table 1.
Table 1 Radix Flemingiae Philippinensis extract HPLC eluent gradient elution program
The preparation of reference substance solution: precision takes genistin reference substance 1mg, genistein reference substance 1mg, be placed in 25mL volumetric flask respectively, scale is settled to dissolve with methanol, accurate 15mL genistin reference substance solution, the 5mL genistein reference substance solution drawn is in 50mL volumetric flask, add methanol constant volume to scale, mix homogeneously, obtain the mixing reference substance solution containing 12 μ g/mL genistins, 4 μ g/mL genisteins, cross 0.45 μm of microporous filter membrane, measure by above-mentioned liquid phase chromatogram condition.
The preparation of need testing solution: prepare extracting solution according to Different Extraction Method under each investigation factor, extracting solution is diluted to crude drug content 0.02g/mL, crosses 0.45 μm of microporous filter membrane, obtains need testing solution.
(1). concentration of alcohol is investigated
Take Radix Flemingiae Philippinensis medical material (being ground into velvet-like) 10g, with 10 times amount 30% ethanol, 50% ethanol, 70% ethanol, 90% soak with ethanol 2h, reflux, extract, 2h, extract 3 times, merge extractive liquid, is settled to 500mL with Extraction solvent, obtains the Radix Flemingiae Philippinensis extracting solution of crude drug content 0.02g/mL.Cross 0.45 μm of microporous filter membrane, measure by above-mentioned chromatographic condition, calculate genistin and genistein content, obtain best concentration of alcohol.When concentration of alcohol is 70% in extracting solution genistin and genistein content the highest, so best concentration of alcohol is 70%.
(2). pulverizing medicinal materials degree is investigated
Radix Flemingiae Philippinensis medical material is made Φ 1cm*1cm cylindrical block, 1cm thin segment, velvet-like 3 kinds of specifications.Respectively take 10g, with 10 times amount 70% soak with ethanol 2h, reflux, extract, 2h, extract 3 times, merge extractive liquid, is settled to 500mL with Extraction solvent, obtains the Radix Flemingiae Philippinensis extracting solution of crude drug content 0.02g/mL.Cross 0.45 μm of microporous filter membrane, measure by above-mentioned chromatographic condition, calculate genistin and genistein content, obtain best pulverizing medicinal materials degree.The highest when pulverizing medicinal materials becomes the fine strip shape of 1cm length, in extracting solution genistin and genistein content the highest, therefore, select this to be best pulverizing medicinal materials degree.
(3). soak time is investigated
Take Radix Flemingiae Philippinensis medical material (1cm thin segment) 10g, with 10 times amount 70% soak with ethanol 0h, 1h, 2h, 3h, reflux, extract, 2h, extract 3 times, merge extractive liquid, is settled to 500mL with Extraction solvent, obtains the Radix Flemingiae Philippinensis extracting solution of crude drug content 0.02g/mL.Cross 0.45 μm of microporous filter membrane, measure by above-mentioned chromatographic condition, calculate genistin and genistein content, obtain best soak time.When soak time is 2h, in extracting solution genistin and genistein content the highest, select best soak time to be 2h.
(4). adopt the optimised process of orthogonal design screening Radix Flemingiae Philippinensis alcohol reflux
Design the factor level table (see table 2) of 3 factor 3 levels, solid-liquid ratio, extraction time, extraction time are investigated, select L
9(3
4) orthogonal table, with genistin and genistein content for index, the optimum extraction process of preferred Radix Flemingiae Philippinensis.
The extraction process factor level of genistin and genistein in table 2 Radix Flemingiae Philippinensis
In table 3 Radix Flemingiae Philippinensis, the extraction process orthogonal test of genistin and genistein arranges
Take Radix Flemingiae Philippinensis (1cm thin segment) 10g, 70% soak with ethanol 2h.Carry out orthogonal test by table 3, after extracting solution is settled to 500mL, cross 0.45 μm of microporous filter membrane, measure by liquid phase chromatogram condition, with genistin and genistein content for index carries out intuitive analysis and variance analysis.Preferred Radix Flemingiae Philippinensis optimum extraction process.
Table 4 with genistin content for Index Orthogonal Test intuitive analysis (R1 is for genistin)
Table 5 with genistein content for Index Orthogonal Test intuitive analysis (R2 is for genistein)
Table 6 with genistin content for Index Orthogonal Test variance analysis
Note: F
0.05 (2,2)=19.000
Table 7 with genistein content for Index Orthogonal Test variance analysis
Note: F
0.05 (2,2)=19.000
As shown in Table 4, with genistin content for index, it is C>B>A that each factor affects size, and optimised process is A
2b
3c
3; As shown in Table 5, with genistein content for index, it is B>C>A that each factor affects size, and optimised process is A
2b
3c
3.As shown in Table 6, with genistin content for index, factor A, B do not have statistical significance, and factor C has statistical significance; As shown in Table 7, with genistein content for index, factor A does not have statistical significance, and factor B, C have statistical significance.Because factor B and factor C drops on edge, and these two factors all have statistical significance, therefore carry out supplementary test to these two factors.
(5). the supplementary test of orthogonal experiment
Take Radix Flemingiae Philippinensis medical material (1cm thin segment) 10g, totally 3 parts, with 15 times amount 70% soak with ethanol 2h.1st part: reflux, extract, 3h, extract 3 times; 2nd part: reflux, extract, 4h, extract 3 times; 3rd part: reflux, extract, 3h, extract 4 times, merge extractive liquid, is settled to 500mL with Extraction solvent, obtains the Radix Flemingiae Philippinensis extracting solution of crude drug content 0.02g/mL respectively.Cross 0.45 μm of microporous filter membrane, measure by liquid phase chromatogram condition, calculating genistin and genistein content are in table 8.
Genistin and genistein extraction process supplementary test in the preferred Radix Flemingiae Philippinensis of table 8 orthogonal test
From supplementary test result, increase extraction time or extraction time can not improve the content of R1 or R2.Therefore optimum extraction process is A
2b
3c
3, in conjunction with experiment of single factor result, i.e. 1cm thin segment shape medical material 15 times amount 70% soak with ethanol 2h, reflux, extract, 3h, extracts 3 times.
(6). the demonstration test of optimum extraction process
Take Radix Flemingiae Philippinensis (1cm thin segment) 10g, totally 3 parts, with 15 times amount 70% soak with ethanol 2h, reflux, extract, 3h, extract 3 times, merge extractive liquid, is settled to 500mL with Extraction solvent, obtains the Radix Flemingiae Philippinensis extracting solution of crude drug content 0.02g/mL.Cross 0.45 μm of microporous filter membrane, measure by liquid phase chromatogram condition, calculating genistin and genistein content are in table 9.
The optimum extraction process demonstration test of table 9
From demonstration test result, the average content that optimum extraction process extracts genistin and genistein in Radix Flemingiae Philippinensis reaches 0.768mg/g and 0.280mg/g respectively, be better than any one experimental result in orthogonal test, RSD value is 0.52% and 1.05%, and process stabilizing is reliable.
Optimum extraction process: take Radix Flemingiae Philippinensis medical material (the long thin segment of 1cm) 150g, 15 times amount 70% ethanol extractions soak 2h, reflux, extract, 3h, extract 3 times.Merging filtrate, reclaim ethanol, water bath method medicinal liquid, vacuum drying, obtains former extract 21.2005g, preserves in exsiccator.In former extract, genistin and genistein content are 0.741%.
Embodiment 2. extracting solution acid precipitation method purifying dye wood glycosides and genistein technical study (preparation of Radix Flemingiae Philippinensis alcohol extraction acid hypostasis)
Genistin and genistein are all flavones ingredient, are soluble in hot water and are insoluble in cold water, are soluble in the character that alkali is insoluble in acid, carry out water precipitation or the composition genistin of Acid precipitation purification of target and genistein to Radix Flemingiae Philippinensis alcohol extract according to flavones ingredient.Measure genistin and genistein content with HPLC method, with its rate of transform and purity for index, obtain the best deposition and purification technique of genistin and genistein in Radix Flemingiae Philippinensis.
Experiment material
Instrument: LC-20AT high performance liquid chromatograph and SPD-20A UV-detector (Japanese Shimadzu Corporation), SinoChrom ODS-BP 5 μm of chromatographic columns (Dalian Yilite Analytical Instrument Co., Ltd), BSA124S analytical balance (Sai Duolisi scientific instrument company limited), DZF-6020 type vacuum drying oven (the permanent Science and Technology Ltd. in Shanghai one), RE-5203 rotary evaporator (Shanghai Yarong Biochemical Instrument Plant), HH-6 digital display thermostat water bath (Guo Hua Electrical Appliances Co., Ltd).
Reagent: trifluoroacetic acid aqueous solution (world Reagent Company of the U.S., lot number: 1103160), Chromatographic Pure Methanol (Hanbon Sci. & Tech. Co., Ltd., lot number: 112866), analytical pure formic acid (Nanjing Chemistry Reagent Co., Ltd., lot number: 10120111319), commercially available pure water (Hangzhou Wahaha Group Co., Ltd, lot number: 210636), analytical pure hydrochloric acid (Nanjing Chemistry Reagent Co., Ltd., lot number: 080230147), 95% industrial alcohol.
Medicine: genistin chemical reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 111709-200501), genistein chemical reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 111704-200501), Radix Flemingiae Philippinensis medical material (originating from Yang Shuo, Guangxi).
(1). the preparation of the former extract of Radix Flemingiae Philippinensis ethanol
Take Radix Flemingiae Philippinensis medical material (1cm thin segment) 150g, 15 times amount 70% ethanol extractions soak 2h, reflux, extract, 3h, extract 3 times.Merging filtrate, reclaim ethanol, water bath method medicinal liquid, vacuum drying, obtains former extract 21.2005g, preserves in exsiccator.In former extract, genistin and genistein content are 0.741%.
(2). medicinal liquid pH value is investigated
Precision takes the former extract of 1.4g Radix Flemingiae Philippinensis ethanol (being equivalent to 10g crude drug) 4 parts, appropriate 70% dissolve with ethanol, be concentrated into 10mL, guarantee without alcohol taste, drip concentrated hydrochloric acid and make medicinal liquid pH value be respectively 1,3,5,7,4 DEG C of standing 12h, decompress filter, washing filter cake, to neutral, by filtering residue vacuum drying, is weighed.Take the precipitate of 1/10 weight (being equivalent to 1g crude drug), 70% dissolve with ethanol is also settled to 50mL, crosses 0.45 μm of microporous filter membrane, measure by liquid phase chromatogram condition, calculate genistin and genistein content, calculate the rate of transform and purity, select optimal pH.Best medicinal liquid pH value is selected to be 3.
(3). liquor strength is investigated
Precision takes 1.4g Radix Flemingiae Philippinensis ethanol extraction (being equivalent to 10g crude drug) 4 parts, appropriate 70% dissolve with ethanol, be concentrated into 5mL, 10mL, 20mL, 40mL (being respectively 2g crude drug/mL, 1g crude drug/mL, 0.5g crude drug/mL, 0.25g crude drug/mL) respectively, guarantee without alcohol taste, drip concentrated hydrochloric acid and make medicinal liquid pH value be 3,4 DEG C of standing 12h, decompress filter, washing filter cake, to neutral, by filtering residue vacuum drying, is weighed.Take the precipitate of 1/10 weight (being equivalent to 1g crude drug), 70% dissolve with ethanol is also settled to 50mL, crosses 0.45 μm of microporous filter membrane, measure by liquid phase chromatogram condition, calculate genistin and genistein content, calculate the rate of transform and purity, select best liquor strength.Best liquor strength is selected to be 1g crude drug/mL.
(4). the sedimentation time is investigated
Precision takes 1.4g Radix Flemingiae Philippinensis ethanol extraction (being equivalent to 10g crude drug) 4 parts, appropriate 70% dissolve with ethanol, be concentrated into 10mL (1g crude drug/mL), guarantee without alcohol taste, drip concentrated hydrochloric acid and make medicinal liquid pH value be 3,4 DEG C of standing 2h, 4h, 8h, 12h, decompress filter, washing filter cake, to neutral, by filtering residue vacuum drying, is weighed.Take the precipitate of 1/10 weight (being equivalent to 1g crude drug), 70% dissolve with ethanol is also settled to 50mL, crosses 0.45 μm of microporous filter membrane, measure by liquid phase chromatogram condition, calculate genistin and genistein content, calculate the rate of transform and purity, select the best sedimentation time.The best sedimentation time is selected to be 4h.
(4). the checking research of the heavy optimised process of acid
Precision takes the former extract of 1.4g Radix Flemingiae Philippinensis ethanol (being equivalent to 10g crude drug) 3 parts, appropriate 70% dissolve with ethanol, be concentrated into 10mL (1g crude drug/mL), guarantee without alcohol taste, drip concentrated hydrochloric acid and make medicinal liquid pH value be 3,4 DEG C of standing 4h, decompress filter, washing filter cake, to neutral, by filtering residue vacuum drying, is weighed.Take the precipitate of 1/10 weight (being equivalent to 1g crude drug), 70% dissolve with ethanol is also settled to 50mL, crosses 0.45 μm of microporous filter membrane, measures by liquid phase chromatogram condition, calculate genistin and genistein content in 3 parts of parallel sample, calculate the rate of transform and purity.
The optimum Acid precipitation process certification test of table 10
From demonstration test result, genistin and genistein in the former extract of best Acid precipitation purifying process purification Radix Flemingiae Philippinensis ethanol, both can be made mean transferred rate to reach 86.88% and 93.00% respectively, and purity reaches 1.75% and 0.54% respectively, and gross mass mark reaches 2.29%.Genistin and genistein rate of transform RSD value are respectively 1.15% and 1.56%; Purity RSD value is respectively 2.31% and 2.65%.This purifying process repeatability is good, reliable and stable.
The polyamide process of enriching research of genistin and genistein alcohol extraction acid hypostasis in embodiment 3 Radix Flemingiae Philippinensis
Experiment material
Instrument: Agilent 1100 high performance liquid chromatograph (Agilent Technologies of the U.S.), SinoChrom ODS-BP 5 μm of chromatographic columns (Dalian Yilite Analytical Instrument Co., Ltd), THZ-D Desk type constant-temperatureoscillator oscillator (Taicang experimental facilities factory), BSA124S analytical balance (Sai Duolisi scientific instrument company limited), DZF-6020 type vacuum drying oven (the permanent Science and Technology Ltd. in Shanghai one), KQ-250E type ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).
Reagent: 14-30 order, 30-60 order, 60-100 order, 100-200 order column chromatography polyamide (the biochemical plastic molding and processing plant of City of Taizhou road and bridge tetramethyl, lot number: 20110722), trifluoroacetic acid aqueous solution (world Reagent Company of the U.S., lot number: 1012904), Chromatographic Pure Methanol (Hanbon Sci. & Tech. Co., Ltd., lot number: 112866), analytical pure formic acid (Nanjing Chemistry Reagent Co., Ltd., lot number: 10120111319), commercially available pure water (Hangzhou Wahaha Group Co., Ltd, lot number: 1123CH), 95% industrial alcohol.
Medicine: genistin chemical reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 111709-200501), genistein chemical reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 111704-200501), Radix Flemingiae Philippinensis alcohol extraction acid hypostasis (being prepared by the heavy optimised process of acid in embodiment 2).
Liquid phase condition determination is the same.
(1). static adsorption and desorbing Effect study
Precision takes 0.3g Radix Flemingiae Philippinensis alcohol extraction acid hypostasis 4 parts, and be dissolved in 500mL 10% ethanol, obtaining concentration is 0.6mg/mL Radix Flemingiae Philippinensis alcohol extracting-water precipitating thing solution.Add the polyamide 0.5g of 14-30 order, 30-60 order, 60-100 order, 100-200 order 4 kinds of particle diameters respectively, be placed in constant temperature oscillator jolting, 1mL supernatant is drawn respectively at 1h, 2h, 4h, 8h, 12h, 24h, water bath method, with 0.5mL 70% dissolve with ethanol, measure wherein genistin and genistein content, obtain non-adsorption liquid concentration, calculate adsorption rate=(sample liquid concentration-non-adsorption liquid concentration)/sample liquid concentration * 100%, and be abscissa with time, adsorption rate is that vertical coordinate draws polyamide static adsorption kinetic curve.
The resin filter of absorption 24h is obtained, adds 500mL 80% ethanol and carry out desorption.Be placed in constant temperature oscillator jolting, 1mL supernatant is drawn respectively at 1h, 2h, 4h, 8h, 12h, 24h, water bath method, with 0.5mL 70% dissolve with ethanol, middle method measures wherein genistin and genistein content, obtains eluate concentration, calculates desorption efficiency=eluate concentration/(sample liquid concentration-non-adsorption liquid concentration) * 100%, and be abscissa with time, desorption efficiency is that vertical coordinate draws desorption kinetic curve.
Experimental result shows: 60-100 order is the suitableeest polyamide particle diameter.
(2). dynamic adsorption condition is investigated
The filling of polyamide resin column: take 60-100 order polyamide 5g, 95% industrial alcohol boils 30min, repeats 3 times, filtration obtains resin, be suspended in appropriate 95% industrial alcohol, load in glass column, measurement blade diameter length ratio is 1:5.9, resin bed volume (BV) is 37.1mL.Without after residue after 95% ethanol purge polyamide resin column to eluent evaporate to dryness, pure water is eluted to eluent without alcohol taste.
Sample concentration is investigated: precision takes 3g Radix Flemingiae Philippinensis alcohol extraction acid hypostasis 4 parts, be dissolved in 400mL, 600mL, 800mL, 1000mL 10% respectively in ethanol, obtain the Radix Flemingiae Philippinensis alcohol extraction acid hypostasis solution of 7.50mg/mL, 5.00mg/mL, 3.75mg/mL, 3.00mg/mL, in this, as sample solution loading, loading flow velocity 2BV/h.Collect loading effluent, accurate absorption 4mL, 6mL, 8mL, 10mL respectively, water bath method, 25mL is settled to 70% ethanol, measure wherein genistin and genistein content, obtain the genistin of loading effluent and genistein concentration, calculate adsorption rate=(sample solution concentration-loading outflow concentration)/sample solution concentration * 100%.Best sample concentration is selected to be 3mg/mL (in Radix Flemingiae Philippinensis alcohol extraction acid hypostasis).
Loading flow velocity is investigated: precision takes 3g Radix Flemingiae Philippinensis alcohol extraction acid hypostasis 4 parts, be dissolved in 1000mL 10% ethanol respectively, obtain the Radix Flemingiae Philippinensis alcohol extraction acid hypostasis solution 4 parts of 3.00mg/mL, in this, as sample solution loading, loading flow velocity is respectively 1BV/h, 2BV/h, 3BV/h, 4BV/h.Collect loading effluent, accurate absorption 10mL respectively, water bath method, 25mL is settled to 70% ethanol, measure wherein genistin and genistein content, obtain the genistin of loading effluent and genistein concentration, calculate adsorption rate=(sample solution concentration-loading outflow concentration)/sample solution concentration * 100%.Best loading flow velocity is selected to be 2BV/h.
Maximum applied sample amount is investigated: precision takes 3g Radix Flemingiae Philippinensis alcohol extraction acid hypostasis, is dissolved in 1000mL 10% ethanol, and obtain the Radix Flemingiae Philippinensis alcohol extraction acid hypostasis solution of 3.00mg/mL, in this, as sample solution loading, loading flow velocity is respectively 2BV/h.Every 1BV collects a effluent, every part of effluent precision draws 10mL, water bath method, be settled to 25mL with 70% ethanol, measure wherein genistin and genistein content, obtain every part of effluent genistin and genistein concentration, calculate slip, take loading volume as abscissa, slip is vertical coordinate, draws leakage plot.Maximum applied sample amount is defined as 16BV.
Dynamic desorption condition investigation-genistin elution flow rate is investigated:
Dress is loading also.10% ethanol elution remove impurity is colourless to eluent, 10BV 20% ethanol carries out eluting to genistin, elution flow rate is respectively 1BV/h, 2BV/h, 3BV/h, 4BV/h, collect eluent, accurate absorption 2mL, water bath method, 70% dissolve with ethanol is also settled to 5mL, HPLC detects eluent genistin content, calculates genistein elution amount, obtains eluting rate=elution amount/adsorbance * 100%.By eluent evaporate to dryness, weigh dry cream weight, obtain purity=elution amount/dry cream weight * 100%.2BV/h is selected to be best elution flow rate.
Dynamic desorption condition investigation-genistin eluting agent is investigated
Fill post and loading.10% ethanol elution remove impurity is colourless to eluent, 20% ethanol carries out eluting to genistin, elution flow rate is 2BV/h, every BV collects 1 part of eluent, accurate absorption 2mL, water bath method, 70% dissolve with ethanol is also settled to 5mL, HPLC detects eluent genistin content, calculates genistin elution amount, obtains accumulative eluting rate=accumulative elution amount/adsorbance * 100%.By eluent evaporate to dryness, weigh dry cream weight, obtain accumulative purity=accumulative elution amount/dry cream weight * 100%.Best eluting agent is 6BV.
Dynamic desorption condition investigation-genistein elution flow rate is investigated
With dress also loading.10% ethanol elution remove impurity is colourless to eluent, after 6BV 20% ethanol carries out eluting to genistin, and the remove impurity of 10BV 35% ethanol elution.10BV 50% ethanol carries out eluting to genistein, elution flow rate is respectively 1BV/h, 2BV/h, 3BV/h, 4BV/h, collect eluent, accurate absorption 2mL, water bath method, 70% dissolve with ethanol is also settled to 5mL, and HPLC detects eluent genistein content, calculate genistein elution amount, obtain eluting rate=elution amount/adsorbance * 100%.By eluent evaporate to dryness, weigh dry cream weight, obtain purity=elution amount/dry cream weight * 100%.2BV/h is selected to be best elution flow rate.
Dynamic desorption condition investigation-genistein eluting agent is investigated
To fill post and loading.10% ethanol elution remove impurity is colourless to eluent, and 6BV 20% ethanol is to R1(genistin) carry out eluting after, the remove impurity of 10BV 35% ethanol elution.50% ethanol carries out eluting to genistein, elution flow rate is 2BV/h, every BV collects 1 part of eluent, accurate absorption 2mL, water bath method, 70% dissolve with ethanol is also settled to 5mL, and HPLC detects eluent genistein content, calculate genistein elution amount, obtain accumulative eluting rate=accumulative elution amount/adsorbance * 100%.By eluent evaporate to dryness, weigh dry cream weight, obtain accumulative purity=accumulative elution amount/dry cream weight * 100%.Best eluting agent is 6BV.
(3). the best purifying process demonstration test of enrichment Radix Flemingiae Philippinensis extract
According to above research, obtaining genistin and genistein optimised process in polyamide purification Radix Flemingiae Philippinensis is: select 60-100 order polyamide to fill post, using 10% dissolve with ethanol sample as sample solution, sample concentration is 3mg/mL (in Radix Flemingiae Philippinensis alcohol extraction acid hypostasis), loading flow velocity is 2BV/h, and maximum applied sample amount is 16BV.Colourless to eluent with 10% ethanol elution remove impurity, 6BV 20% ethanol carries out eluting to target compound genistin, elution flow rate 2BV/h, obtains genistin position.The remove impurity of 10BV 35% ethanol elution, 6BV 50% ethanol carries out eluting to target compound genistein, elution flow rate 2BV/h, obtains genistein position.Merge genistin and genistein position, obtain purification of samples.
With this optimised process parallel preparation 3 increment product, detect genistin and the total purity of genistein, and calculate RSD value in table 11.
Table 11 process certification is tested
It is reliable that demonstration test shows this process stabilizing, and repeatability is good, can prepare the enrichment Radix Flemingiae Philippinensis extract sample that genistin and the total purity of genistein are 25%.
Embodiment 4 refines the preparation of Radix Flemingiae Philippinensis extract
Enrichment Radix Flemingiae Philippinensis extract prepared by Example 3 optimised process, again goes up polyamide chromatography post according to the optimised process operational approach of embodiment 3, obtains refining Radix Flemingiae Philippinensis extract, and the purity of this extract is 60%.
Embodiment 5 Radix Flemingiae Philippinensis extract Inhibiting α-glucosidase activity research
Alpha-glucosidase participates in glycometabolic important enzyme in intestinal, can generate glucose, thus absorbed into serum forms blood glucose through its catalyzing hydrolysis.Alpha-glucosidase inhibitor can suppress the activity of this kind of enzyme, thus reaches the object reducing post-prandial glycemia.
The activity of Radix Flemingiae Philippinensis extract Inhibiting α-glucosidase is verified in this experiment in vitro.With 4-nitrophenols-α-D-pyranglucoside (PNPG) for substrate, alpha-glucosidase is hydrolyzed to it, measures the activity of enzyme.PNPG is a kind of maltose analog, 37 DEG C, 4-nitrophenols (PNP) can be hydrolyzed to by alpha-glucosidase under the condition of pH=6.8.Alpha-glucosidase inhibitor can suppress this generation be hydrolyzed, and the PNP that hydrolysis is discharged reduces.And by measuring the absorbance of PNP under 405nm, the degree of this suppression can be calculated.
Experiment material
Instrument: TDL80-2B desk centrifuge (Anting Scientific Instrument Factory, Shanghai), RT-6000 enzyme micro-plate reader (Rayto Life and Analytical Sciences Co., Ltd.), liquid-transfering gun (Dragon Medical(Shanghai) Co., Ltd., 20-200 μ L), TP-214 ten thousand/analytical balance (Denver's instrument (Beijing) company limited), B-260 thermostat water bath (Shanghai Yarong Biochemical Instrument Plant).
Reagent: alpha-glucosidase (TCI), PNPG (Alfa Aesar, lot number: 10115970), potassium dihydrogen phosphate (Nanjing chemical reagent factory), sodium hydroxide (Shantou Xilong Chemical Factory Co., Ltd, lot number: 080630 l), sodium chloride (Nanjing Chemistry Reagent Co., Ltd., lot number: 09060310494), natrium carbonicum calcinatum (Nanjing Chemistry Reagent Co., Ltd., lot number: 060960237), dimethyl sulfoxine, 95% ethanol.
Medicine: acarbose sheet (Bayer HealthCare Co, lot number: 117893), the former extract of Radix Flemingiae Philippinensis (being prepared by the optimised process of embodiment 1).Radix Flemingiae Philippinensis alcohol extraction acid hypostasis (being prepared by embodiment 2 optimised process).The former extract of enrichment Radix Flemingiae Philippinensis (being prepared by the optimised process of embodiment 3).The refining former extract of Radix Flemingiae Philippinensis (being prepared by embodiment 4).
Related reagent is prepared:
Preparation (the pH=6.8 of phosphate buffered solution (PBS), 0.1M): prepare PBS according to 2005 editions Chinese Pharmacopoeias annex XV, precision takes potassium dihydrogen phosphate 0.68g, is placed in 25mL volumetric flask, distilled water is settled to scale, obtains 0.2mol/L potassium dihydrogen phosphate.Accurate weighing sodium hydroxide 0.2g, be placed in 25mL volumetric flask, distilled water is settled to scale, obtains 0.2mol/L sodium hydroxide solution.Precision pipettes 0.2mol/L potassium dihydrogen phosphate 25mL, and 0.2mol/L sodium hydroxide solution 11.8mL, in 50mL volumetric flask, add 0.425g sodium chloride, adding distil water is settled to scale, obtains pH=6.8,0.1M, the phosphate buffered solution of sodium chloride concentration 0.85%.
The preparation (0.2U/mL) of alpha-glucosaccharase enzymatic solution: precision takes alpha-glucosidase 0.01g, be placed in 5mL volumetric flask, add PBS and be settled to scale, stir 2h, the centrifugal 10min of 3500rpm, namely Aspirate supernatant obtains 2mg/mL alpha-glucosaccharase enzymatic solution.According in 2.2 to the mensuration of alpha-glucosaccharase enzyme activity unit, this concentration and 0.2U/mL.(define that 1 enzyme activity unit (U) is 37 DEG C, under pH=6.8 condition, hydrolysis PNPG per minute generates the enzyme amount required for 1 μm of ol PNP.）
The preparation (2.5mmol/L) of PNPG solution: precision takes PNPG 0.0038g, is placed in 5mL volumetric flask, adds PBS and is settled to scale, be stirred to dissolving, obtains 2.5mmol/mL PNPG solution.
The preparation (0.2mol/L) of sodium carbonate liquor: take natrium carbonicum calcinatum 0.212g, be placed in 10mL volumetric flask, adding distil water is settled to scale, obtains 0.3mol/L sodium carbonate liquor.
(1). the mensuration of alpha-glucosaccharase enzyme activity
The making of standard curve: precision takes paranitrophenol (PNP) 0.014g, is placed in 10mL volumetric flask, adds PBS (pH=6.8,0.1M) and is settled to scale, obtains 10mmol/L PNP solution.Respectively dilution become 8,4,2,1,0.5mmol/L PNP solution.According to reaction system, mixed by 160 μ L PNP solution, add 96 orifice plates with 80 μ L 0.2mol/L sodium carbonate liquors, if 4 multiple holes, measure light absorption value A under 405nm, with PNP concentration for abscissa, light absorption value is vertical coordinate, draws PNP standard curve.
The determination of enzyme activity unit: in 96 orifice plates, every hole adds 112 μ L PBS, 8 μ L DMSO, 20 μ L alpha-glucosaccharase enzymatic solution, hatch 15min for common 37 DEG C, add PNPG solution 20 μ L again, 37 DEG C of reaction 15min, add 0.2mol/L sodium carbonate liquor 80 μ L cessation reaction, light absorption value A is measured under 405nm, if 4 multiple holes, average, determine enzyme activity unit.Define that 1 enzyme activity unit (U) is 37 DEG C, under pH=6.8 condition, hydrolysis PNPG per minute generates the enzyme amount required for 1 μm of olPNP.It is 10.1mg alpha-glucosidase that mensuration obtains 1U.
(2). the mensuration of Radix Flemingiae Philippinensis extract to alpha-glucosaccharase enzyme inhibition rate and the calculating of IC50
The preparation of enrichment Radix Flemingiae Philippinensis extract solution: precision takes enrichment Radix Flemingiae Philippinensis extract 20mg, and be placed in 5mL volumetric flask, DMSO is settled to scale, obtains 4mg/mL enrichment Radix Flemingiae Philippinensis extract solution.
Accurate absorption 2.5mL 4mg/mL enrichment Radix Flemingiae Philippinensis extract solution, be placed in 5mL volumetric flask, DMSO is settled to scale, obtains 2mg/mL enrichment Radix Flemingiae Philippinensis extract solution.
Accurate absorption 2.5mL 2mg/mL enrichment Radix Flemingiae Philippinensis extract solution, be placed in 5mL volumetric flask, DMSO is settled to scale, obtains 1mg/mL enrichment Radix Flemingiae Philippinensis extract solution.
Accurate absorption 2.5mL 1mg/mL enrichment Radix Flemingiae Philippinensis extract solution, be placed in 5mL volumetric flask, DMSO is settled to scale, obtains 0.5mg/mL enrichment Radix Flemingiae Philippinensis extract solution.
Accurate absorption 2.5mL 0.5mg/mL enrichment Radix Flemingiae Philippinensis extract solution, be placed in 5mL volumetric flask, DMSO is settled to scale, obtains 0.25mg/mL enrichment Radix Flemingiae Philippinensis extract solution.
The preparation of Radix Flemingiae Philippinensis acid hypostasis solution: be respectively 2.5 by upper method compound concentration, 5,10,20, the Radix Flemingiae Philippinensis acid hypostasis solution series of 40mg Radix Flemingiae Philippinensis acid hypostasis/mL.
The preparation of refining Radix Flemingiae Philippinensis extract solution: be respectively 0.1 by upper method compound concentration, 0.21,0.42,0.835,1.67mg refines the refining Radix Flemingiae Philippinensis extract solution series of Radix Flemingiae Philippinensis extract/mL.
The preparation of the former extract solution of Radix Flemingiae Philippinensis: the same method compound concentration is respectively 7.5,15,30,60, the former extract solution series of Radix Flemingiae Philippinensis of the former extract/mL of 120mg Radix Flemingiae Philippinensis.
The preparation of acarbose solution: get acarbose sheet 1 (including acarbose 50mg), be weighed as 0.1374g, grind, take powder 0.0550g (including acarbose 20mg), be placed in 5mL volumetric flask, add PBS and be settled to scale, the centrifugal 10min of 3500rpm, Aspirate supernatant, obtaining concentration is 4mg/mL acarbose solution.
Accurate absorption 2.5mL 4mg/mL acarbose solution, be placed in 5mL volumetric flask, PBS is settled to scale, obtains 2mg/mL acarbose solution.
Accurate absorption 2.5mL 2mg/mL acarbose solution, be placed in 5mL volumetric flask, PBS is settled to scale, obtains 1mg/mL acarbose solution.
Accurate absorption 2.5mL 1mg/mL acarbose solution, be placed in 5mL volumetric flask, PBS is settled to scale, obtains 0.5mg/mL acarbose solution.
Accurate absorption 2.5mL 0.5mg/mL acarbose solution, be placed in 5mL volumetric flask, PBS is settled to scale, obtains 0.25mg/mL acarbose solution.
Mensuration to alpha-glucosaccharase enzyme inhibition rate:
Set up negative group, positive drug (acarbose) 5 dosage group (4mg/mL, 2mg/mL, 1mg/mL, 0.5mg/mL, 0.25mg/mL), the former extract of Radix Flemingiae Philippinensis 5 dosage group (7.5 mg/mL, 15 mg/mL, 30 mg/mL, 60 mg/mL, 120 mg/mL), Radix Flemingiae Philippinensis acid hypostasis 5 dosage group (2.5 mg/mL, 5 mg/mL, 10 mg/mL, 20 mg/mL, 40mg/mL), enrichment Radix Flemingiae Philippinensis extract 5 dosage group (4mg/mL, 2mg/mL, 1mg/mL, 0.5mg/mL, 0.25mg/mL), refining Radix Flemingiae Philippinensis extract 5 dosage group (0.1 mg/mL, 0.21 mg/mL, 0.42 mg/mL, 0.835 mg/mL, 1.67mg/mL), and set up blank group to eliminate the impact of medicinal liquid Color pair experiment respectively.
Reaction is carried out, according to Zhang Li etc. on 96 orifice plates
[1]the method set up slightly is made an amendment, and often group establishes 4 multiple holes, and each group liquid feeding method is in table 12:
Table 12 respectively organizes liquid feeding method
Alpha-glucosaccharase enzymatic solution 20 μ L and PBS, DMSO, inhibitor solution 8 μ L are mixed, hatches 15min in 37 DEG C of water-baths, add substrate PNPG solution 20 μ L, 37 DEG C of water-bath 15min, add 0.2mol/L sodium carbonate liquor 80 μ L cessation reaction.The light absorption value A of hydrolyzate PNP is measured under microplate reader 405nm.With the suppression ratio of following formula calculation sample to alpha-glucosidase:
Wherein: sample light absorption value=sample sets light absorption value-sample blank group light absorption value
Negative light absorption value=feminine gender group light absorption value-feminine gender blank group light absorption value
(3). the Inhibition test result of alpha-glucosidase
Radix Flemingiae Philippinensis extract to the inhibitory action of alpha-glucosidase in table 13
Table 13 Radix Flemingiae Philippinensis extract and acarbose are to the suppression ratio of alpha-glucosidase
Data according to table 13 show, and this experiment, using different Radix Flemingiae Philippinensis extract as sample, all has very strong alpha-glucosaccharase enzyme inhibition activity.
Embodiment 5 Radix Flemingiae Philippinensis extract is on the impact of alloxan diabetes mouse blood sugar and oxidative stress
Experiment material
Instrument: TDL80-2B desk centrifuge (Anting Scientific Instrument Factory, Shanghai); RT-6000 enzyme micro-plate reader (Rayto Life and Analytical Sciences Co., Ltd.); BSA124S analytical balance (Sai Duolisi scientific instrument company limited); the miniature vortex mixed instrument of WH-2 (Shanghai Hu Xi analytical tool factory); pipettor (0.5-10 μ L; 20-200 μ L, 100-1000 μ L, Dragon Medical(Shanghai) Co., Ltd.).
Reagent: alloxan (Shanghai Ru Ji biotechnology Development Co., Ltd, lot number: 080825), Glucose estimation kit (ShangHai RongSheng Biology Pharmacy Co., Ltd, lot number: 20111201), (Bioengineering Research Institute is built up in Nanjing to SOD test kit (WST-1 method), lot number: 20120420), (Bioengineering Research Institute is built up in Nanjing to malonaldehyde (MDA) testing cassete, lot number: 20120423), (Bioengineering Research Institute is built up in Nanjing to glutathione-peroxidase (GSH-Px) testing cassete, lot number: 20111202), glucose (Nanjing Chemistry Reagent Co., Ltd., lot number: 081160760), sodium chloride (Nanjing Chemistry Reagent Co., Ltd., lot number: 061110999), 95% ethanol.
Medicine: the former extract of enrichment Radix Flemingiae Philippinensis (prepared by the optimised process of embodiment 3, HPLC measures genistin and genistein total content is 25%).The refining former extract of Radix Flemingiae Philippinensis (prepared by embodiment 4, HPLC measures genistin and genistein total content is 60%).Metformin hydrochloride tablet (Zhonghui Pharmaceutical Co., Ltd., Beijing, lot number: 20100206), acarbose tablet (Bayer HealthCare Co, lot number: BJ05904).
Animal: ICR mice (♂, 20-25g, Yangzhou University's comparative medicine center, credit number: SCXK (Soviet Union) 2007-0001).
(1) to the preparation of drug solns
The preparation of enrichment Radix Flemingiae Philippinensis extract medicinal liquid:
The preparation of high dose enrichment Radix Flemingiae Philippinensis extract: take enrichment Radix Flemingiae Philippinensis extract 0.5g, be placed in 50mL volumetric flask, 0.4%CMC-Na is settled to scale, and jolting makes precipitation suspendible, obtains 10mg/mL Radix Flemingiae Philippinensis extract suspension.
The preparation of middle dosage enrichment Radix Flemingiae Philippinensis extract: take enrichment Radix Flemingiae Philippinensis extract 0.25g, be placed in 50mL volumetric flask, 0.4%CMC-Na is settled to scale, and jolting makes precipitation suspendible, obtains 5mg/mL Radix Flemingiae Philippinensis extract suspension.
The preparation of low dosage enrichment Radix Flemingiae Philippinensis extract: take enrichment Radix Flemingiae Philippinensis extract 0.125g, be placed in 50mL volumetric flask, 0.4%CMC-Na is settled to scale, and jolting makes precipitation suspendible, obtains 2.5mg/mL Radix Flemingiae Philippinensis extract suspension.
The preparation (compound method is the same) of refining Radix Flemingiae Philippinensis extract medicinal liquid: the middle dosage that high dose refines Radix Flemingiae Philippinensis extract suspension, concentration is 2.1mg/mL of compound concentration to be concentration be 4.2mg/mL refines Radix Flemingiae Philippinensis extract suspension and concentration is respectively that the low dosage of 1.05mg/mL refines Radix Flemingiae Philippinensis extract suspension.
The preparation of metformin hydrochloride solution: every hydrochloric metformin 0.25g of sheet metformin hydrochloride tablet.Metformin hydrochloride tablet sheet remeasurement: get 10 metformin hydrochloride tablets at random, analytical balance is measured its every blade and is heavily respectively, 0.3083g, 0.3254g, 0.3138g, 0.3158g, 0.3130g, 0.3143g, 0.3250g, 0.3218g, 0.3292g, 0.3301g.Asking 10 average sheets is heavily 0.31967g, thinks that metformin hydrochloride tablet sheet is heavily 0.31967g.Get metformin hydrochloride tablet 1, grind, analytical balance precision takes powder 0.4799g, adds in 50mL volumetric flask, adds water and be settled to scale, and obtaining Determination of metformin hydrochloride is 7.5mg/mL solution.
The preparation of acarbose solution: every sheet acarbose tablet is containing acarbose 50mg.Get acarbose tablet 1, grind, be placed in 50mL volumetric flask, add water and be settled to scale, obtaining acarbose concentration is 1mg/mL solution.
(2). the foundation of alloxan diabetes mouse model and grouping
144 ICR mices (♂, 20-25g), adaptability raises 3d, randomly draws 16 as blank group.Remain 128 mices and carry out modeling.
With normal saline 6mg/mL alloxan solution.For subsequent use in brown bottle.After water 17h be can't help to mice fasting, tail vein injection alloxan solution, administration volume 10mL/kg.After injection 4.5h, gavage gives 50% D/W, prevents hypoglycemia from occurring.After 72h, under water 8h situation is can't help in mice fasting, eyeground vein clump gets blood, after leaving standstill 1h, and the centrifugal 10min of 3000rpm, separation of serum.Glucose in serum content is measured with Glucose estimation kit (glucose oxidase-peroxidase method) method, obtain mice fasting blood sugar, the mice of screening 112 fasting glucose >=11.1mmol/L, as diabetes model, and carry out random packet with fasting blood sugar, be divided into model control group, positive controls (metformin group), enrichment Radix Flemingiae Philippinensis extract high dose group, dosage group in enrichment Radix Flemingiae Philippinensis extract, enrichment Radix Flemingiae Philippinensis extract low dose group, refining Radix Flemingiae Philippinensis extract high dose group, dosage group and refining Radix Flemingiae Philippinensis extract low dose group 8 groups in refining Radix Flemingiae Philippinensis extract, modeling and grouping situation are as table 14.
The foundation of table 14 alloxan diabetes mouse model
Note: compare with blank group, * * P<0.01
Through SPSS11.5 statistics, each group modeling fasting blood sugar and blank group have pole significant difference (P<0.01), prove modeling success; Except blank group, between all the other 8 groups, modeling blood glucose value is thought from same sample population (P>0.7), can carry out parallel laboratory test research and compare.
(3). medication
Every 2d records a Mouse Weight, 20mL/kg gastric infusion, administration 14d.Each group of mice gives relative medicine solution or blank solution, blank group: 0.4%CMC-Na aqueous solution; Model control group: 0.4%CMC-Na aqueous solution; Positive controls (metformin group): 7.5mg/mL metformin hydrochloride solution (dosage 150mg/kg); Enrichment Radix Flemingiae Philippinensis extract high dose group: 10mg/mL enrichment Radix Flemingiae Philippinensis extract solution (dosage 200mg/kg); Dosage group in enrichment Radix Flemingiae Philippinensis extract: 5mg/mL enrichment Radix Flemingiae Philippinensis extract solution (dosage 100mg/kg); Enrichment Radix Flemingiae Philippinensis extract low dose group: 2.5mg/mL enrichment Radix Flemingiae Philippinensis extract solution (dosage 50mg/kg); Refining Radix Flemingiae Philippinensis extract high dose group: 4.2mg/mL refines Radix Flemingiae Philippinensis extract solution (dosage 84mg/kg); In refining Radix Flemingiae Philippinensis extract, dosage group: 2.1mg/mL refines Radix Flemingiae Philippinensis extract solution (dosage 42mg/kg); Refining Radix Flemingiae Philippinensis extract low dose group: 1.05mg/mL refines Radix Flemingiae Philippinensis extract solution (dosage 21mg/kg).
(4). the mensuration of body weight, index of drinking water, index of ingesting
Every 2d records a Mouse Weight; Every 2d record one time the same day amount of drinking water, drinking-water index (%)=amount of drinking water/body weight * 100%; Every 2d record one time the same day food ration, index of ingesting (%)=food ration/body weight * 100%.
(5). the mensuration of fasting glucose
After successive administration 7d, 14d, measure mice fasting blood sugar.Water 8h is can't help in mice fasting, and eyeground vein clump gets blood, the centrifugal 10min of 3000rpm after 1h, is separated upper serum, measures mice fasting blood sugar by Glucose estimation kit method.Investigate Radix Flemingiae Philippinensis extract hypoglycemic activity.
(6). the mensuration of oxidative stress index
After successive administration 14d, pluck eye and get blood, the centrifugal 10min of 3000rpm after 1h, be separated upper serum, slightly make an amendment by kit method and measure SOD in serum, MDA, GSH-Px respectively.Investigate Radix Flemingiae Philippinensis extract antioxidant activity.
(7). the mensuration of glucose tolerance
Water 8h is can't help in mice fasting, by i.g. medication.I.g. 5g/kg starch solution after 1h.When giving starch solution 0h, 0.5h, 1h, 2h, to the blood sampling of mice eyeground vein clump, the centrifugal 10min of 3000rpm after 1h, is separated upper serum, measures postprandial plasma glucose level by Glucose estimation kit method.And calculate AUC area=(A0+A0.5) * 0.5/2+ (A0.5+A1) * 0.5/2+ (A1+A2) * 1/2, wherein A0, A0.5, A1, A2 represent the blood glucose value after giving starch solution 0h, 0.5h, 1h, 2h respectively.
In experimentation, because mice is in high sugared state, often have dead generation, thus animal number of elements during individual experimental group statistics and uneven with, blank group is 16, model group is 12, and enrichment Radix Flemingiae Philippinensis high dose group is 14, and in enrichment Radix Flemingiae Philippinensis, dosage group is 14, enrichment Radix Flemingiae Philippinensis low dose group is 13, refining Radix Flemingiae Philippinensis high dose group is 14, and in refining Radix Flemingiae Philippinensis, dosage group is 14, and refining Radix Flemingiae Philippinensis low dose group is 13.
(8). experimental result
Enrichment Radix Flemingiae Philippinensis extract and refining Radix Flemingiae Philippinensis extract on the impact of alloxan diabetes Mouse Weight, index of drinking water, index of ingesting in table 15.Show diabetic mice body weight change situation during administration
Mouse Weight table (n=14) during table 15 administration
Note: compare with blank group, * P<0.05, * * P<0.01; Compare with model control group,
#p<0.05,
##p<0.01(is empty: blank group, mould: model control group, two: positive controls (metformin group), Fu Gao: enrichment Radix Flemingiae Philippinensis extract high dose group, Fu Zhong: dosage group in enrichment Radix Flemingiae Philippinensis extract, rich low: enrichment Radix Flemingiae Philippinensis extract low dose group, essence is high: refining Radix Flemingiae Philippinensis extract high dose group, in essence: dosage group in refining Radix Flemingiae Philippinensis extract, essence is low: refining Radix Flemingiae Philippinensis extract low dose group.）
Blank group body weight increases day by day as shown in Table 15, and model control group body weight alleviates day by day, and from the 2nd day, the body weight that every 2d measures all produced pole significant difference (P<0.01) with blank group.Metformin group and model group there was no significant difference, metformin can not improve the symptom that diabetic mice body weight alleviates.And enrichment Radix Flemingiae Philippinensis extract high, medium and low dosage group and the high, medium and low dosage group of refining Radix Flemingiae Philippinensis extract were from the 4th day, the body weight that every 2d measures all produces pole significant difference (P<0.01) with model control group, proves that enrichment Radix Flemingiae Philippinensis extract and refining Radix Flemingiae Philippinensis extract all have the effect improving diabetic mice body weight and alleviate.
Table 16 shows each group of mice drinking-water index variation situation.
Mice drinking-water index table (n=14) during table 16 administration
Model control group drinking-water index is apparently higher than blank group as shown in Table 16, proves that " polydipsia " symptom appears in diabetic mice.Metformin and enrichment Radix Flemingiae Philippinensis extract high, medium and low dosage group and refining Radix Flemingiae Philippinensis extract high, medium and low dosage group all show the effect of suitable reduction drinking water for animals index.
Table 17 shows each group of mice and to ingest index variation situation.
During table 17 administration, mice ingests index table (n=14)
Model control group ingests index apparently higher than blank group as shown in Table 17, proves that " polyphagia " symptom appears in diabetic mice.Metformin and enrichment Radix Flemingiae Philippinensis extract high, medium and low dosage group and the high, medium and low dosage group of refining Radix Flemingiae Philippinensis extract all show the effect reducing animal ingestion index.During the effect that wherein enrichment Radix Flemingiae Philippinensis high dose group and refining Radix Flemingiae Philippinensis high dose group reduce animal ingestion index is better than, low dose group and metformin group.
Radix Flemingiae Philippinensis extract is on the impact of alloxan diabetes mice fasting glucose
Mice fasting blood sugar is detected in table 18 after successive administration 7d and 14d.
Table 18 mouse blood sugar change list (n=14)
Note: compare with blank group, * P<0.05, * * P<0.01; Compare with model control group,
#p<0.05,
##p<0.01
As shown in Table 18, each group modeling blood glucose and blank group all have pole significant difference (P<0.01), prove modeling success.The change of 14d inner model matched group fasting blood sugar is little, proves that the diabetes model set up is stablized.Metformin, when 7d and 14d, all shows obvious hypoglycemic activity, has pole significant difference (P<0.01) with model group.Enrichment Radix Flemingiae Philippinensis extract high, medium and low dosage group and the high, medium and low dosage group of refining Radix Flemingiae Philippinensis extract all show the activity reducing fasting glucose, and show certain dose dependent.Wherein high dose group all produces pole significant difference (P<0.01) with model control group when 7d, 14d; Middle dosage group when 7d and model control group produce significant difference (P<0.05), during 14d produce pole significant difference (P<0.01); Low dose group all produces significant difference (P<0.05) with model control group when 7d, 14d.
Radix Flemingiae Philippinensis extract is on the impact of alloxan diabetes mice serum SOD, MDA, GSH-Px
After successive administration 14d, measure SOD, MDA, GSH-Px (table 19) in serum, checking Radix Flemingiae Philippinensis extract antioxidant activity.
Table 19 mice serum MDA, SOD, GSH-Px content
Note: compare with blank group, * P<0.05, * * P<0.01; Compare with model control group,
#p<0.05,
##p<0.01
As shown in Table 19, model control group serum MDA, SOD, GSH-Px all pole are significantly higher than blank group (P<0.01), and diabetic animal models creates very strong response to oxidative stress.Metformin can not eliminate this reaction.And enrichment Radix Flemingiae Philippinensis extract high, medium and low dosage group and the high, medium and low dosage group of refining Radix Flemingiae Philippinensis extract all can improve the response to oxidative stress of animal, improve SOD in serum, GSH-Px enzyme activity, reduce Content of MDA.A good appetite suddenly appearing in a serious disease, low dose group serum MDA indicator and model matched group compare generation significant difference outer (P<0.05), and all the other all create pole significant difference (P<0.01).
Radix Flemingiae Philippinensis extract is on the impact of alloxan diabetes glucose tolerance in mice
After giving starch solution to animal pattern, 0,0.5,1, after 2h, blood glucose value is in table 20.
Table 20 glucose tolerance in mice
Note: compare with blank group, * P<0.05, * * P<0.01; Compare with model control group,
#p<0.05,
##p<0.01(is empty: blank group, mould: model control group, Ah: positive controls (acarbose), Fu Gao: enrichment Radix Flemingiae Philippinensis extract high dose group, Fu Zhong: dosage group in enrichment Radix Flemingiae Philippinensis extract, rich low: enrichment Radix Flemingiae Philippinensis extract low dose group, essence is high: refining Radix Flemingiae Philippinensis extract high dose group, in essence: dosage group in refining Radix Flemingiae Philippinensis extract, essence is low: refining Radix Flemingiae Philippinensis extract low dose group.）
As shown in Table 20, alloxan diabetes glucose tolerance in mice degradation (comparing P<0.01 with blank group), and positive drug acarbose can improve this symptom, make animal pattern post-prandial glycemia unlikely too high, compare with model control group and have pole significant difference (P<0.01), enrichment Radix Flemingiae Philippinensis extract middle and high dosage group has similar to positive drug acarbose or the better activity improving carbohydrate tolerance with refining Radix Flemingiae Philippinensis extract middle and high dosage group.Low dose group also creates significant difference (P<0.05) with model group.
Conclusion:
Experiment, with the method for tail vein injection alloxan, successfully establishes diabetic mouse model.Experiment demonstrates Radix Flemingiae Philippinensis extract first and has the activity reducing blood glucose, improve diabetic animal life quality, resist response to oxidative stress, improve carbohydrate tolerance.Compared with metformin, have and can improve diabetic animal body weight, minimizing amount of drinking water and food ration, and the advantage of response to oxidative stress can be resisted.Therefore its activity improving diabetic animal carbohydrate tolerance is better than acarbose, and Radix Flemingiae Philippinensis extract has good research and development and is worth.
Embodiment 6 prepares granule
Take enrichment Radix Flemingiae Philippinensis extract prepared by the optimised process in embodiment 3, add the adjuvant dextrin of extract 2 times of quality, mix homogeneously, cross 65 mesh sieves, be binding agent with appropriate 95% ethanol, make moist wood, granulate through 20 mesh sieve extruding, after 65 DEG C of dryings, obtain granule.
Embodiment 5 prepares tablet
Refining Radix Flemingiae Philippinensis extract prepared by the optimised process taken in embodiment 4, adds appropriate supplementary product starch and magnesium stearate, and mixing, beats sheet, obtain tablet.
Claims (7)
1. a Radix Flemingiae Philippinensis extract, it is characterized in that described Radix Flemingiae Philippinensis extract is the refining Radix Flemingiae Philippinensis extract obtained after Radix Flemingiae Philippinensis alcohol extraction acid hypostasis, the described Radix Flemingiae Philippinensis alcohol extraction acid enrichment Radix Flemingiae Philippinensis extract that obtains after polyamide enrichment of hypostasis or described enrichment Radix Flemingiae Philippinensis extract that former extract obtains after Acid precipitation go up polyamide purification again, the main active in described Radix Flemingiae Philippinensis extract is genistin and genistein;
Described Radix Flemingiae Philippinensis extract adopts following methods preparation: after taking Radix Flemingiae Philippinensis pulverizing medicinal materials, 10 ~ 20 times amount 30% ~ 90% ethanol extractions soak 1 ~ 3h, reflux, extract, 2 ~ 4h, extract 2 ~ 4 times, merging filtrate, reclaim ethanol and filtrate concentrate drying is obtained the former extract of Radix Flemingiae Philippinensis, medicinal liquid pH value is made to be 1 ~ 5 with dripping concentrated hydrochloric acid after being concentrated into 0.25 ~ 2g crude drug/mL after 70% dissolve with ethanol former for Radix Flemingiae Philippinensis extract, 4 DEG C of standing 2 ~ 12h, decompress filter is dry after washing filter cake to neutrality obtains the sour hypostasis of Radix Flemingiae Philippinensis alcohol extraction;
described Radix Flemingiae Philippinensis extract adopts following methods preparation: after taking Radix Flemingiae Philippinensis pulverizing medicinal materials, 10 ~ 20 times amount 30% ~ 90% ethanol extractions soak 1 ~ 3h, reflux, extract, 2 ~ 4h, extract 2 ~ 4 times, merging filtrate, reclaim ethanol and filtrate concentrate drying is obtained the former extract of Radix Flemingiae Philippinensis, former for Radix Flemingiae Philippinensis extract is dripped concentrated hydrochloric acid make medicinal liquid pH value be 1 ~ 5 with 70% dissolve with ethanol after being concentrated into 0.25 ~ 2g crude drug/mL, 4 DEG C leave standstill 2 ~ 12h, decompress filter washing filter cake is to neutral, it is the sample solution of 3 ~ 7.5mg/mL that Radix Flemingiae Philippinensis alcohol extraction acid hypostasis drying obtained is dissolved in obtained concentration in the alcoholic solution of 10%, by good polyamide resin column processed on sample solution, pillar blade diameter length ratio is 1:4 ~ 7, loading flow velocity is 1 ~ 4BV/h, applied sample amount is no more than 16BV, after loading, colourless to eluent with 10% alcoholic solution eluting impurity, first use 6BV 20% ~ 40% ethanol to carry out eluting to target compound genistin, elution flow rate 2BV/h, obtain genistin position, 35% ethanol elution remove impurity, 6BV 50% ~ 60% ethanol carries out eluting to target compound genistein, elution flow rate 2BV/h, obtains genistein position, merge genistin and genistein position, obtain enrichment Radix Flemingiae Philippinensis extract,
described refining Radix Flemingiae Philippinensis extract, after enrichment Radix Flemingiae Philippinensis extract is gone up polyamide purification again, obtains refining Radix Flemingiae Philippinensis extract.
2. Radix Flemingiae Philippinensis extract according to claim 1, is characterized in that the content of described main active genistin and genistein is 0.5% ~ 60%.
3. Radix Flemingiae Philippinensis extract according to claim 2, is characterized in that the content of described main active genistin and genistein is 25% ~ 60%.
4. Radix Flemingiae Philippinensis extract according to claim 1, it is characterized in that the concentration of described sample solution is 3mg/mL, pillar blade diameter length ratio is 1:5.9, and loading flow velocity is 2BV/h, the concentration of the eluant ethanol of genistin and genistein is respectively 20% and 50%, order number 60 ~ 100 order of polyamide.
5. the application of the Radix Flemingiae Philippinensis extract in claim 1 ~ 4 described in any one in preparation control diabetes medicament.
6. application according to claim 5, is characterized in that described medicine comprises the Radix Flemingiae Philippinensis extract and pharmaceutically acceptable carrier or the conventional adjuvant used for the treatment of effective dose.
7. application according to claim 6, is characterized in that the dosage form of described medicine comprises pill, powder, capsule, tablet, granule, injection or compound preparation.
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| 王明煜.《蔓性千斤拔有效组分提取分离及其抑菌、防治血栓作用》.《蔓性千斤拔有效组分提取分离及其抑菌、防治血栓作用》.2008,第22-34页. * |
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