CN103110890B - Effective part of Xierigasiwei (Mongolian medicine) as well as preparation method, quality detection method and application thereof - Google Patents
Effective part of Xierigasiwei (Mongolian medicine) as well as preparation method, quality detection method and application thereof Download PDFInfo
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Abstract
The invention discloses an effective part of Xierigasiwei (a Mongolian medicine) and a preparation method thereof. According to the effective part of the Xierigasiwei and the preparation method thereof, the preparation method comprises one or more of a solvent extraction method, a macroreticular resin method, a supercritical C02 fluid extraction method and a liquid-liquid counter-current extraction method, the sum of percentage compositions of components of total terpenoids, total phenols and total alkaloids in the prepared effective part of the Xierigasiwei is 5-99% (w/w), the contents of effective constituents are greatly increased, and the defects that decoctions and capsules are high in dosage are overcome. Meanwhile, the invention further provides a content measuring method for the total phenols, the total terpenoids, the total alkaloids, geniposide, curcumin and berberine hydrochloride, thus being beneficial for improving the controllability of the product quality and having a practical significance on the development of the Xierigasiwei.
Description
Technical field
The present invention relates to a kind of effective site of mongolian medicine, particularly assist day loud, high-pitched sound four taste effective site and preparation method thereof, quality determining method and the application in the field such as medicine and health food, belong to medical art.
Background technology
Association's day loud, high-pitched sound four taste is made up of Rhizoma Curcumae Longae, Cortex Phellodendri, Fructus Gardeniae, Fructus Tribuli 4 taste Chinese medicine, and Mongolian medicine is clinical more conventional, has diuresis, rushes down damp and hot function clearly; Being mainly used in urine apolipsis, frequent micturition, urgent micturition, being with blood in urine, bladder twinge, is the first-selected prescription that Mongolian medicine treats bladder heat syndrome.Main containing compositions such as phenolic acids, alkaloids, terpenoid in association's day loud, high-pitched sound four taste, but have no the patent of the effective site relating to association's day loud, high-pitched sound four taste and its production and use so far.
Summary of the invention
The object of the present invention is to provide a kind of association day loud, high-pitched sound four taste effective site and preparation method thereof, also be to provide a kind of quality determining method of assisting day loud, high-pitched sound four taste effective site, be also to provide a kind of to assist day loud, high-pitched sound four taste effective site be active component medicine or functional food.
The crude drug of day loud, high-pitched sound four taste effective site is assisted to consist of in the present invention:
Rhizoma Curcumae Longae 100-200 weight portion, Cortex Phellodendri 50-130 weight portion, Fructus Gardeniae 60-180 weight portion, Fructus Tribuli 100-200 weight portion.
The crude drug composition of day loud, high-pitched sound four taste flavone effective part is assisted to be preferably in the present invention:
Rhizoma Curcumae Longae 150 weight portion, Cortex Phellodendri 90 weight portion, Fructus Gardeniae 120 weight portion, Fructus Tribuli 150 weight portion.
Described Fructus Tribuli is that the dry mature fruit of zygophyllaceae plant Fructus Tribuli TribulusterrestrisL. obtains through micro-parch.
The present invention assists the crude drug of day loud, high-pitched sound four taste effective site, it can be the decoction pieces of ingredients in commercially available association day loud, high-pitched sound four taste, also can be arbitrary positions such as the stem of these plants, leaf, spica, fruit, underground rhizome and root or whole plant, preferred crude drug source be---Rhizoma Curcumae Longae: the dry rhizome of zingiberaceous plant Rhizoma Curcumae Longae (Curcuma longa L.).Cortex Phellodendri: the bark of rutaceae Cortex Phellodendri (Phellodendron amurense Rupr.) or wampee.Fructus Gardeniae: in the fruit of dicotyledon medicine Maguireothamnus speciosus Fructus Gardeniae (Gardenia jasminoides Ellis).Fructus Tribuli (micro-stir-fry): the dry mature fruit of zygophyllaceae plant Fructus Tribuli TribulusterrestrisL. obtains through micro-parch.Wherein said association's day loud, high-pitched sound four taste ingredients comprises the crude drug, decoction pieces and the processed product that process without any process of preparing Chinese medicine.
The present invention assists the preparation method of day loud, high-pitched sound four taste effective site can adopt any one method following, or the combination in any of these methods is prepared:
1, solvent extraction: get association day loud, high-pitched sound four taste compound recipe pharmaceutical decocting piece 0.5-1.5 weight portion, 50%-95% alcohol reflux 2-3 time, solvent load is 5 ~ 15 parts by volume, each extraction 1-2 hour, decompression and solvent recovery, obtain extract, be first suspended in water by extract, after making medical material and dispersion, liquor capacity is than being 1:3-1:10(w/v); Then with the solvent of low polarity, solvent load is the aqueous solution of 1-3 times of extract, extracts 2-4 time, obtains liposoluble constituent; Use the solvent of middle polarity again, solvent load is the aqueous solution of 1-3 times of extract, through 2-5 extraction composition wherein; The liposoluble constituent that forward extraction obtains with it is mixed to get association's day loud, high-pitched sound four taste effective site.
The solvent of described low polarity is the mixture of any one solvent in petroleum ether, ether, hexane, gasoline or any several solvent; The solvent of described middle polarity is the mixture of any one solvent in chloroform, ethyl acetate, acetone, n-butyl alcohol or any several solvent.
2 Flavonoids by Macroporous Adsorption Resins: get association day loud, high-pitched sound four taste compound recipe pharmaceutical decocting piece 0.5-1.5 weight portion, 50%-95% alcohol reflux 2-3 time, solvent load is 5 ~ 15 parts by volume, each extraction 1-2 hour, decompression and solvent recovery, obtain extract, be first suspended in water by extract, after making medical material amount and dispersion, liquor capacity is than being 1:10-1:2.5(w/v); By the 6-10 volume macroporous adsorbent resin doubly of suitable crude drug amount, adsorption flow rate 2BV/h, resin column blade diameter length ratio is 1:4 ~ 1:13, carries out remove impurity with the water elution of 1-5 times of resin volume, and remove impurity flow velocity is 2 ~ 6BV/h; With the 50%-70% ethanol elution of 5-12 times of resin volume, elution flow rate is 1 ~ 3BV/h, collect 50%-70% ethanol elution, recycling design, drying under reduced pressure, again with the 71%-90% ethanol elution of 5-12 times of resin volume, elution flow rate is 4 ~ 9BV/h, collects 71%-90% ethanol elution, recycling design, drying under reduced pressure, mixes 50%-70% ethanol elution thing with 71%-90% ethanol elution thing to be and assists day loud, high-pitched sound four taste effective site.
Macroporous resin wherein used is nonpolar, any one type of low pole, middle polarity, alkalescence or faintly acid, as AB-8, D3520, D101, D4020, HPD400, S-8, HZ-806 etc., wherein be preferably the resin of low pole or middle polarity, as AB-8, D4020, D101 etc.Eluant wherein used can also be water or moisture ethanol, methanol, acetone etc., is preferably the ethanol of 0 ~ 100%.
Wherein 50%-70% ethanol elution thing mixes with 71%-90% ethanol elution thing can be that eluate directly mixes, and also comprises and mixing with 50%-70% ethanol elution thing by 71%-90% ethanol elution thing beta-cyclodextrin inclusion compound or after absorbing with calcium hydrogen phosphate again.Beta-schardinger dextrin-and 71%-90% ethanol elution thing amount are 4:1 ~ 6:1, and enclose temperature is 65 ~ 75 DEG C, and the enclose time is 1.5-3 hour.
3, supercritical C0
2fluids extraction: get association day loud, high-pitched sound four taste compound recipe pharmaceutical decocting piece 0.5-1.5 weight portion, add 30-90% methanol, supercritical fluid extraction 2-3 time, each extraction 1-3 hour, solvent load is 8L, is suspended in water by the extract of gained, then by the ethyl acetate of low polarity, extraction composition wherein, must assist day loud, high-pitched sound four taste effective site;
Directly can extract association's day loud, high-pitched sound four taste raw material, also can the product that above-mentioned either method and step obtain be extracted.Can use during extraction or not use any one kind solvent following and solvent mixture: water, alcohols, ketone and esters solvent.
4, liquid-liquid counter-current extraction: get association day loud, high-pitched sound four taste compound recipe pharmaceutical decocting piece 0.5-1.5 weight portion, 50%-80% alcohol reflux 2-4 time, solvent load is 5-15 parts by volume, each extraction 1-3 hour, decompression and solvent recovery, obtains extract; First extract is suspended in water, then liposoluble constituent is obtained with low polar solvent extraction, then with solvent extraction acquisition terpenoid, phenols, the alkaloids composition wherein of middle polarity, liposoluble constituent and terpenoid, phenols, alkaloids composition are mixed to get and assist day loud, high-pitched sound four taste extract.
Liposoluble constituent can be directly mix with terpenoid, phenols, alkaloids composition, also can mix with terpenoid, phenols, alkaloids composition by liposoluble constituent beta-cyclodextrin inclusion compound or after absorbing with calcium hydrogen phosphate again.Beta-schardinger dextrin-and liposoluble constituent amount are 4:1 ~ 6:1, and enclose temperature is 65 ~ 75 DEG C, and the enclose time is 1.5-3 hour.
The pass of weight portion of the present invention and parts by volume is the relation of g/mL.
Association of the present invention day loud, high-pitched sound four taste effective site, wherein the summation of total terpenoid, total phenols, total alkaloids constituents percentage composition is 5 ~ 99% (w/w), is wherein preferably 50 ~ 99%(w/w).
Phenolic active components most importantly curcumin, demethoxycurcumin, bisdemethoxycurcumin, 1 in association of the present invention day loud, high-pitched sound four taste extract, 7-bis(4-hydroxy-3-methoxyphenyl)-1,4,6-heptarien-3-one, 1,7-bis(4-hydroxyphenyl)-1, the compositions such as 4,6-heptatrien-3-one.The terpenoid active component most importantly composition such as geniposide, Gardenoside, shanzhiside, jasminoidin, geniposidic acid, geniposide O-gentibioside, Fructus Gardeniae ketoside, 10-acetyl geniposide, gardenia acid, ursolic acid in association of the present invention day loud, high-pitched sound four taste extract.The alkaloids active component most importantly composition such as berberine, palmatine, jateorhizine, obacunone, obakulactone, Cortex Phellodendri lactone A, Cortex Phellodendri lactone B, Cortex Phellodendri amide A in association of the present invention day loud, high-pitched sound four taste extract.
Phenolic compound structure main in association of the present invention day loud, high-pitched sound four taste extract is in table 1:
The phenolic compound structure that table 1 is main
Association of the present invention day loud, high-pitched sound four taste effective site can separately, combination or with other Chinese and western drugs, food compatibility, make medicine or the functional foods such as capsule, tablet, pill, granule, oral liquid, syrup, electuary, medicated wine, injection, unguentum, powder, beverage.
The quality determining method of association of the present invention day loud, high-pitched sound four taste effective site comprise in following content assaying method any one or a few:
1, the assay of total phenols in day loud, high-pitched sound four taste effective site is assisted
Accurate absorption concentration is the curcumin reference substance solution 0.5,1.0,1.5 of 23.68 μ g/ml, 2.0,2.5,3.0 and 3.5ml, put in 25ml measuring bottle, add dehydrated alcohol to 5ml, add 0.6% ferric chloride-0.9% potassium ferricyanide mixed solution 1.0ml of 0.3% sodium lauryl sulphate 2.0ml and 1: 0.9, mixing, in the dark place 5min, add 0.1mol/L hydrochloric acid solution to scale, in the dark place 20min, take developer as blank, measure trap at 720nm wavelength place; With curcumin reference substance sampling amount for abscissa, absorbance is vertical coordinate, drawing standard curve;
Precision takes each 3 parts of association's day loud, high-pitched sound four taste effective site sample, and every part of 30mg, puts in 50mL measuring bottle, add 70% appropriate amount of ethanol, supersound process 5 minutes, is diluted to scale with 70% dissolve with ethanol, shakes up, filter, discard just filtrate, precision measures subsequent filtrate 5.0mL in 10mL measuring bottle, adds 70% ethanol to scale, shake up, as sample solution;
The above-mentioned sample solution 1.0mL of accurate absorption puts in 25ml measuring bottle respectively, add dehydrated alcohol to 5ml, add 0.6% ferric chloride-0.9% potassium ferricyanide mixed solution 1.0ml of 0.3% sodium lauryl sulphate 2.0ml and 1: 0.9, mixing, in the dark place 5min, add 0.1mol/L hydrochloric acid solution to scale, in the dark place 20min, take developer as blank, measure trap at 720nm wavelength place; External standard two-point method calculates content, obtains the present invention and assists total phenols in day loud, high-pitched sound four taste effective site to count 15% ~ 26% with curcumin.
2. assist the assay of total terpene in day loud, high-pitched sound four taste effective site
Accurate jasminoidin reference substance solution 0.1,0.2,0.4,0.6,0.8 and the 1.0mL drawing 0.0948mg/mL, puts in 10mL tool plug scale test tube; Vanillin-concentrated sulfuric acid solution that concentration is 1mg/mL is slowly dripped in test tube, and jolting test tube frequently; Finally be settled to 5mL, shake up, room temperature leaves standstill 10min, measures absorbance in 438nm place; With jasminoidin reference substance sampling amount (mg) for abscissa, absorbance is vertical coordinate, drawing standard curve;
Precision takes each 3 parts of association's day loud, high-pitched sound four taste effective site sample, and every part of 25mg, puts in 50mL measuring bottle, add 70% appropriate amount of ethanol, supersound process 5 minutes, is diluted to scale with 70% dissolve with ethanol, shakes up, filter, discard just filtrate, precision measures subsequent filtrate 5.0mL in 10mL measuring bottle, adds 70% ethanol to scale, shake up, as sample solution;
The above-mentioned sample solution 1.0mL of accurate absorption puts in 10mL tool plug scale test tube respectively; Vanillin-concentrated sulfuric acid solution that concentration is 1mg/mL is slowly dripped in test tube, and jolting test tube frequently; Finally be settled to 5mL, shake up, room temperature leaves standstill 10min, measures absorbance in 438nm place; External standard two-point method calculates content, obtains the present invention and assists total terpene in day loud, high-pitched sound four taste effective site to count 25% ~ 36% with jasminoidin.
3. assist the assay of total alkaloids in day loud, high-pitched sound four taste effective site
The accurate berberine hydrochloride reference substance solution 0.5ml drawing 0.0592mg/mL, 1.5ml, 2.5ml, 3.5ml, 4ml, 5.5ml put in 10ml tool plug scale test tube, volatilize solvent, add 6ml bromothymol blue test solution sample dissolution respectively, then add 6ml chloroform, firmly jolting 2min, in impouring separatory funnel, leaves standstill 30min, get chloroform layer, extract three times with method, get in chloroform stratification 25ml volumetric flask, and complement to scale with chloroform; Take corresponding reagent as blank, measure absorbance in 415nm, with berberine hydrochloride reference substance sampling amount (mg) for abscissa, absorbance is vertical coordinate, drawing standard curve;
Precision takes association's day each 3 parts of loud, high-pitched sound four taste effective site sample, every part of 30mg, puts in 25mL measuring bottle, adds 70% appropriate amount of ethanol, supersound process 5 minutes, is diluted to scale, shakes up, as sample solution with 70% dissolve with ethanol;
The above-mentioned sample solution 1.0mL of accurate absorption puts in 10ml tool plug scale test tube respectively, volatilize solvent, add 6ml bromothymol blue test solution sample dissolution respectively, then add 6ml chloroform, firmly jolting 2min, in impouring separatory funnel, leave standstill 30min, get chloroform layer, extract three times with method, get in chloroform stratification 25ml volumetric flask, and complement to scale with chloroform; Take corresponding reagent as blank, measure absorbance in 415nm; External standard two-point method calculates content, obtains the present invention and assists total alkaloids in day loud, high-pitched sound four taste effective site to count 12% ~ 28% with berberine hydrochloride.
4. assist the assay of jasminoidin, curcumin, berberine hydrochloride in day loud, high-pitched sound four taste effective site
Chromatographic condition: chromatographic column: Elite C18 chromatographic column, 4.6mm × 250mm, 5 μm; Mobile phase: acetonitrile (A) – 0.4% potassium dihydrogen phosphate aqueous solution (B), condition of gradient elution: 0 ~ 15min, 8%A → 18%A; 15 ~ 18min, 18%A → 45%A; 18 ~ 22min, 45%A → 27%A; 22 ~ 35min, 27%A → 12%A; 35 ~ 40min, 12%A → 45%A; 40 ~ 55min, 48%A → 52%A; Flow velocity 1.0mLmin
-1; Determined wavelength is respectively 238nm, 346nm, 346nm; Column temperature 30 DEG C; Sample size 20 μ L;
Specification Curve of Increasing: precision takes in drying under reduced pressure to the jasminoidin reference substance 0.89mg and curcumin reference substance 0.98mg to 5ml measuring bottle of constant weight, precision takes berberine hydrochloride 0.81mg in 10ml volumetric flask, draw 2ml in the 5ml volumetric flask being mixed with above-mentioned two reference substances, being mixed with concentration is respectively 0.178,0.0392,0.0324mgmL
-1mixing reference substance storing solution, be settled to scale with 50 ethanol, shake up, obtain mixing reference substance solution; Get mixing reference substance solution sample introduction 2.0 respectively respectively, 4.0,6.0,8.0,10.0,20.0,25.0 μ l carry out HPLC analysis under above-mentioned chromatographic condition, with peak area Y for vertical coordinate, the quality X (μ g) of solution is abscissa drawing standard curve, obtains the regression equation of jasminoidin, curcumin, berberine;
Assay: precision takes each 3 parts of 3 batches of association's day loud, high-pitched sound four taste effective site samples, every part of 25mg, in 25ml volumetric flask, 70% ethanol standardize solution, shakes up rear use 0.45 μm of microporous filter membrane and filters, get subsequent filtrate, obtain need testing solution;
The above-mentioned need testing solution 20 μ L of accurate absorption, injection liquid chromatography, measures each chromatograph peak-to-peak area, calculates content, obtain the present invention to assist in day loud, high-pitched sound four taste effective site and be respectively 1.30-2.50%, 9.25-19.50%, 0.80-2.10% containing curcumin, jasminoidin, berberine hydrochloride.
The crude drug extract of the present invention to association's day loud, high-pitched sound four taste carries out purification and obtains association's day loud, high-pitched sound four taste effective site, the content of its effective ingredient is improved greatly, overcomes decoction, defect that capsule taking dose is large; Simultaneously, association's day loud, high-pitched sound four taste in the past lacks concrete quality standard Con trolling index, do not form the quality standard system of system, the present invention obtains association's day loud, high-pitched sound four taste effective site by purification, carry out the assay of assisting total phenols in day loud, high-pitched sound four taste effective site, total terpene, total alkaloids, jasminoidin, curcumin, berberine hydrochloride, be conducive to the controllability of improving the quality of products, to the exploitation of mongolian medicine association day loud, high-pitched sound four taste, there is realistic meaning.
Experimental example and embodiment further illustrate but are not limited to the present invention below.
Experimental example 1: the screening of association's day loud, high-pitched sound four taste effective site enrichment resin process used
Through By consulting literatures data and actual combination, adopt 4 factor 3 horizontal quadratures experiments, investigate factor: the volume ratio of (50%-70% ethanol) elution flow rate, blade diameter length ratio, sample concentration, crude drug and resin.With total terpenoid, total phenols, total alkaloids extracted amount for inspection target.Specifically see the following form.
Table 3 factor level table
The preparation of sample: take compound recipe association day loud, high-pitched sound four Chinese medicine material totally 18 parts, by L9(3)
4orthogonal design table, prepare each sample (repeating a, totally 18 times) respectively, filter, recycling design, vacuum drying, weighs, and sees the following form.
Table 4 sample preparation table
The preparation of sample solution: above-mentioned 18 increment product are placed in 25mL measuring bottle respectively, with 70% dissolve with ethanol, ultrasonic, draw a certain amount of liquid to be measured respectively, operation repetitive two parts, with curcumin, jasminoidin, berberine hydrochloride for reference substance, according to the content assaying method of assisting total phenols in day loud, high-pitched sound four taste effective site, total terpene, total alkaloids in the present invention, measure the amount of total phenols, total terpene, total alkaloids.
Orthogonal experiments is analyzed:
Factor A, B, C, D all have significant difference, and namely the volume ratio of (50%-70% ethanol) elution flow rate, blade diameter length ratio, sample concentration, crude drug and resin all has significant difference.By the difference in SAS comparison factor A, B, C, D between each level, carry out horizontal correlation analysis, in factor A, level 1,2 is obviously better than 3; In factor B, 2,3 levels are obviously better than 1 level, and 2,3 levels do not have notable difference; In factor C, 2 levels are obviously better than 1,3 levels; In factor D, 1,2 levels are obviously better than 3 levels, and 1,2 levels do not have notable difference.
In sum: the best purifying process of mongolian medicine association day loud, high-pitched sound-4 effective site is: A1 B2 C2 D2.
Namely optimised process is: elution flow rate 3BV/h, blade diameter length ratio 1:10, sample concentration 2g/5ml, the volume ratio 1:7.5 of crude drug and resin.
Experimental example 2: the assay of total phenols in association's day loud, high-pitched sound four taste extract
1. instrument and condition determination:
TU-1901 type ultraviolet-uisible spectrophotometer (Beijing),
SARTORIUS-BS224S type electronic analytical balance (Beijing Sai Duolisi instrument system company),
KQ-250DE type numerical control supersonic cleaning apparatus (Kunshan Ultrasonic Instruments Co., Ltd.).
Measure wavelength: 720nm
2. reference substance and reagent:
Curcumin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 110823-201004),
Reagent: water is Wahaha Pure Water, and other reagent are analytical pure
3. the preparation of reference substance solution:
It is appropriate that precision takes curcumin reference substance, adds the reference substance solution that absolute methanol is prepared into 0.0592mg/mL.
4. the preparation of sample solution
Precision takes each 3 parts of association's day loud, high-pitched sound four taste effective site sample prepared by embodiment 3, and every part of 30mg, puts in 50mL measuring bottle, add 70% appropriate amount of ethanol, supersound process 5 minutes, is diluted to scale with 70% dissolve with ethanol, shakes up, filter, discard just filtrate, precision measures subsequent filtrate 5.0mL in 10mL measuring bottle, adds 70% ethanol to scale, shake up, as sample solution.
5. assist the assay of total phenols in day loud, high-pitched sound four taste effective site
The above-mentioned sample solution 1.0mL of accurate absorption puts in 25ml measuring bottle respectively, add dehydrated alcohol to 5ml, add 0.3% sodium lauryl sulphate 2.0ml and 0.6% ferric chloride-0.9% potassium ferricyanide (1: 0.9) mixed solution 1.0ml, mixing, in the dark place 5min, add 0.1mol/L hydrochloric acid solution to scale, in the dark place 20min, take developer as blank, measure trap at 720nm wavelength place; External standard two-point method calculates content, to obtain final product.
Table 5 effective site total phenol content
Experimental example 3: the assay of total terpene in association's day loud, high-pitched sound four taste extract
1. instrument and condition determination:
TU-1901 type ultraviolet-uisible spectrophotometer (Beijing),
SARTORIUS-BS224S type electronic analytical balance (Beijing Sai Duolisi instrument system company),
KQ-250DE type numerical control supersonic cleaning apparatus (Kunshan Ultrasonic Instruments Co., Ltd.).
Measure wavelength: 720nm
2. reference substance and reagent:
Jasminoidin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 110749-200714),
Reagent: water is Wahaha Pure Water, and other reagent are analytical pure
3. the preparation of reference substance solution:
It is appropriate that precision takes jasminoidin reference substance, adds the reference substance solution that absolute methanol is prepared into 0.0948mg/mL.
4. the preparation of sample solution
Precision takes each 3 parts of association's day loud, high-pitched sound four taste effective site sample prepared by enforcement 3, and every part of 25mg, puts in 50mL measuring bottle, add 70% appropriate amount of ethanol, supersound process 5 minutes, is diluted to scale with 70% dissolve with ethanol, shakes up, filter, discard just filtrate, precision measures subsequent filtrate 5.0mL in 10mL measuring bottle, adds 70% ethanol to scale, shake up, as sample solution.
5. assist the assay of total terpene in day loud, high-pitched sound four taste effective site
The above-mentioned sample solution 1.0mL of accurate absorption puts in 10mL tool plug scale test tube respectively.Vanillin-concentrated sulfuric acid solution that concentration is about 1mg/mL is slowly dripped in test tube, and jolting test tube frequently.Finally be settled to 5mL, shake up, room temperature leaves standstill 10min, measures absorbance in 438nm place; External standard two-point method calculates content, to obtain final product.
The total terpene content of table 6 effective site
Experimental example 4: the assay of total alkaloids in association's day loud, high-pitched sound four taste extract
1. instrument and condition determination:
TU-1901 type ultraviolet-uisible spectrophotometer (Beijing),
SARTORIUS-BS224S type electronic analytical balance (Beijing Sai Duolisi instrument system company),
KQ-250DE type numerical control supersonic cleaning apparatus (Kunshan Ultrasonic Instruments Co., Ltd.).
Measure wavelength: 720nm
2. reference substance and reagent:
Berberine hydrochloride reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 110713-200911),
Reagent: water is Wahaha Pure Water, and other reagent are analytical pure
3. the preparation of reference substance solution:
It is appropriate that precision takes berberine hydrochloride reference substance, adds the reference substance solution that absolute methanol is prepared into 0.0592mg/mL.
4. the preparation of sample solution
Precision takes each 3 parts of association's day loud, high-pitched sound four taste effective site sample prepared by embodiment 3, and every part of 30mg, puts in 25mL measuring bottle, adds 70% appropriate amount of ethanol, supersound process 5 minutes, is diluted to scale, shakes up, as sample solution with 70% dissolve with ethanol.
5. assist the assay of total alkaloids in day loud, high-pitched sound four taste effective site
The above-mentioned sample solution 1.0mL of accurate absorption puts in 10ml tool plug scale test tube respectively, volatilize solvent, add 6ml bromothymol blue test solution sample dissolution respectively, then add 6ml chloroform, firmly jolting 2min, in impouring separatory funnel, leave standstill 30min, get chloroform layer, extract three times with method, get in chloroform stratification 25ml volumetric flask, and complement to scale with chloroform.Take corresponding reagent as blank, measure absorbance in 415nm; External standard two-point method calculates content, to obtain final product.
Table 7 effective site total alkaloid content
Experimental example 5: the assay of jasminoidin, curcumin, berberine hydrochloride in association's day loud, high-pitched sound four taste effective site
1. instrument and condition determination:
The full-automatic sample introduction high performance liquid chromatograph of HIT LC-2000, L-2455 UV-detector, AgilentChemstation; AB135-S type ultrasonic cleaning instrument, AB135-S electronic balance (Mettler Toledo company) electronic balance.
Chromatographic condition: chromatographic column: Elite C18 (4.6mm × 250mm, 5 μm) chromatographic column; Mobile phase: acetonitrile (A) – 0.4% potassium dihydrogen phosphate aqueous solution (B), gradient elution (0 ~ 15min, 8%A → 18%A; 15 ~ 18min, 18%A → 45%A; 18 ~ 22min, 45%A → 27%A; 22 ~ 35min, 27%A → 12%A; 35 ~ 40min, 12%A → 45%A; 40 ~ 55min, 48%A → 52%A), flow velocity 1.0mLmin
-1; Determined wavelength is respectively 238nm, 346nm, 346nm; Column temperature 30 DEG C; Sample size 20 μ L.Under above-mentioned chromatographic condition, the separating degree of jasminoidin, curcumin, berberine hydrochloride and adjacent chromatographic peak is all greater than 1.5, and symmetrical factor is between 0.95 ~ 1.05.
2. reference substance and reagent:
Jasminoidin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 110749-200714); Berberine hydrochloride reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 110713-200911); Curcumin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 110823-201004); In mongolian medicine compound recipe association day loud, high-pitched sound four taste, ingredients provides by Anguo City Condar Chinese crude drug company limited, Inner Mongol Tian Li medical material company limited, and acetonitrile is Fisher chromatographically pure reagent, and water is Wahaha Pure Water, and other reagent is analytical pure.
3. the preparation of reference substance solution:
Precision takes puts in 5ml measuring bottle through drying under reduced pressure to the jasminoidin reference substance 0.89mg of constant weight and curcumin reference substance 0.98mg, precision takes berberine hydrochloride 0.81mg in 10ml volumetric flask, draw 2ml in the 5ml volumetric flask being mixed with above-mentioned two reference substances, being mixed with concentration is respectively 0.178,0.0392,0.0324mgmL
-1mixing reference substance storing solution, be settled to scale with 50 ethanol, shake up, reference substance storing solution must be mixed.
4. the preparation of sample solution
Precision takes each 3 parts of 3 batches of association's day loud, high-pitched sound four taste effective site samples prepared by embodiment 3, every part of about 25mg, and in 25ml volumetric flask, 70% ethanol standardize solution, shakes up rear use 0.45 μm of microporous filter membrane and filter, get subsequent filtrate, to obtain final product.
5. assist the assay of jasminoidin, curcumin, berberine hydrochloride in day loud, high-pitched sound four taste effective site
The above-mentioned need testing solution 20 μ L of accurate absorption, injection liquid chromatography, measures each chromatograph peak-to-peak area, calculates content.
Table 8 effective site jasminoidin, curcumin, content of berberine
Experimental example 6: mouse corrosion disease, analgesic experiment
1. experiment purpose and requirement
This experiment, by giving mice mongolian medicine compound recipe association day loud, high-pitched sound four taste, investigates the pharmacological action of its anti-inflammatory and antalgic.Require that experiment should be carried out under clean, clean and tidy, aseptic environment, see whether it has significant difference.
2. instrument and batching
Body weight 18g-22g mice, (former formula extraction dry powder is prepared from according to the disclosed method of Chinese people's the Sanitation Ministry medicine standard " mongolian medicine fascicle " former formula extraction dry powder, will: Rhizoma Curcumae Longae, Cortex Phellodendri, Fructus Gardeniae, Fructus Tribuli four Chinese medicine material are according to ratio described in this article, be ground into middle powder, sieve, mix, obtain lower with), effective site dry powder (by embodiment 3 method preparation), positive QIANLIEKANG PIAN (Kang Enbei Zhejiang Pharmaceutical Co), positive SANJIN PIAN (Sanjin Pharmaceutical Co., Ltd., Guilin); 0.5% carboxymethylcellulose sodium solution, 0.7% acetum, dimethylbenzene, irrigation stomach device, shears, the card punch of diameter 8mm, electronic balance, GB204 type precise electronic claims, thermostatic drying chamber (by Shanghai instrument plant system).
3. principle, step and method
3.1 mice granuloma experiments
Experimental principle: because mouse back implants cotton balls, can cause local proliferation to produce granuloma, and through giving mongolian medicine compound recipe association day loud, high-pitched sound four taste, its administration group and the granuloma of matched group weight of rising in value has obviously different, thus provable compound recipe has antiinflammatory action.
Step and method: get body weight 18-22g70 mice, male and female half and half, be divided into 7 groups at random by body weight: (1) blank group; (2) former formula extraction group; (3-5) effective site (low dosage, middle dosage, high dose); (6) the positive SANJIN PIAN group of positive QIANLIEKANG group (7), often organizes 10 mices.
With ether by after mouse anesthesia, respectively at mouse back place parallel with forelimb subcutaneous implantation autoclaving cotton balls two (note: should split when two cotton balls are implanted and vertebra both sides, and each cotton balls is accurately weighed 10mg).Post operation starts gastric infusion on the 2nd day, and blank group gives distilled water, the drug solution of each group gavage equal-volume distilled water, by 1 time/d administration, and, successive administration 7d.The cervical vertebra that dislocates after last administration 1h puts to death mice, and granuloma induced by implantation of cotton pellets is taken out in operation, and stripping is fatty tissue to the greatest extent, and be then placed in 60 DEG C of drying bakers, be dried to constant weight, taking-up is weighed, and its weight deducts cotton balls weight and is granuloma dry weight.
3.2 mice ear experiments
Experimental principle: because dimethylbenzene can cause mice to have inflammation, therefore be applied to by dimethylbenzene on mouse ear and can cause inflammation, thus ear swelling, thus the weight of ear increases.After this medicine, if any the effect of antiinflammatory action, weight will reduce to some extent, and whether the weight that therefore just can calculate minimizing according to weight difference method weighs it has antiinflammatory action.Left and right auricle weight difference is swelling.Swelling suppression ratio=(blank group swelling-administration group swelling)/blank group swelling.The swelling of matched group and administration group is carried out statistical procedures (SPSS13.0 statistical software is t and checks).
Step and method: get body weight 18g-22g mice 60, male and female half and half, be divided into 6 groups at random, i.e. (l) blank group; (2) former formula extraction group; (3-5) effective site (low dosage, middle dosage, high dose); (6) the positive SANJIN PIAN group of positive QIANLIEKANG group (7), often organizes 10.Gastric infusion respectively, blank group gives equal-volume distilled water, once a day, altogether to 7 days.Last administration is after 30 minutes, get 0.lml dimethylbenzene be applied to each Mus auris dextra before and after two sides cause inflammation.After 1 hour, put to death by mice cervical dislocation, cut two ears along auricle baseline, lay round auricle at the two same positions of ear respectively with the card punch of diameter 8mm, use scales/electronic balance weighing rapidly, every Mus auris dextra sheet deducts left auricle weight and is auricle inflammation swelling.Each group is compared with blank group, carries out t inspection.
3.3 mice acetic acid twisting experiments
Experimental principle: 0.7% acetum can cause mice pain to occur writhing response, after this medicine, if any the effect of analgesic activity, writhing number of times will reduce to some extent, therefore just can weigh it and whether has analgesic activity according to observing writhing number of times with blank.Calculate analgesia percentage rate:
Analgesia percentage rate (%)=[(blank group writhing number of times one administration group writhing number of times)/blank group writhing number of times] * 100%
Step and method: get body weight 18g-20g mice 70, male and female half and half, be divided into 7 groups at random by body weight, (l) blank group; (2) former formula extraction group; (3-5) effective site (low dosage, middle dosage, high dose); (6) the positive SANJIN PIAN group of positive QIANLIEKANG group (7).The distilled water solution of the corresponding medicine of each administration group gavage, blank group gavage gives isopyknic distilled water, and administration volume is 0.2ml/l0g body weight.Administration 7 days.Last administration is after 30 minutes, to every Mus lumbar injection 0.7% acetum 0.2ml/ only, observes and records the writhing number of times of each mice in 45 minutes.Each group is compared from group with sky, carries out t inspection.
4. experimental data and result
(1) experimental data:
Table 9 mice granuloma experimental result
n=10
Note: * P < 0.05, * * P < 0.01
Table 10 mice dimethylbenzene experimental result
n=10
Note: * P < 0.05, * * P < 0.01
Table 11 mice acetic acid twisting experimental result
n=10
Note: * P < 0.05, * * P < 0.01
(2) experimental result:
This experimental selection antiinflammatory, analgesia 2 pharmacology indexs, compare the antiinflammatory of mongolian medicine compound recipe association day loud, high-pitched sound four taste effective site (basic, normal, high dosage) and former formula extraction, analgesia drug action.Experimental result shows, in antiinflammatory experiment, in granuloma experimental result, in mongolian medicine compound recipe association day loud, high-pitched sound four taste effective site, dosage group has significant differences; In mice ear experimental result, in mongolian medicine compound recipe association day loud, high-pitched sound four taste effective site, dosage group has significant differences; In analgesic experiment, in mongolian medicine compound recipe association day loud, high-pitched sound four taste effective site, dosage group has significant differences.
Therefore, by above experimental design model, all prove that mongolian medicine compound recipe association day loud, high-pitched sound four taste effective site has obvious anti-inflammatory and analgesic effect.Considered by anti-inflammatory and analgesic effect, the mongolian medicine compound recipe association day loud, high-pitched sound four taste effective site drug action of gained of the present invention is obviously better than former formula extraction.
Experimental example 7: bacteriostatic test
Strain used: staphylococcus aureus, alpha streptococcus, escherichia coli, bacillus pyocyaneus, Candida albicans (above bacterial strain is reference culture).Staphylococcus aureus, escherichia coli, bacillus pyocyaneus ordinary culture medium are cultivated, and alpha streptococcus blood serum medium is cultivated, and Candida albicans sabouraud culture medium is cultivated.
Medicine used: former formula extraction dry powder (with experimental example 6), effective site dry powder (by the preparation of embodiment 3 method)
Extracorporeal bacteria inhibitor test
Minimal inhibitory concentration of drug (MIC) measures: by medicine 0.2g/mL, in test tube, continuous two-fold dilution is done by corresponding culture medium, 1:1,1:2,1:4,1:8,1:16,1:32,1:64, often adding bacteria containing amount in pipe is 1 × 106/mL bacterium liquid 0.1mL, put in 37 DEG C of incubators and cultivate 24h, observe minimum inhibitory concentration (MIC).
Medicine minimum bactericidal concentration (MBC) measures: adopt meat soup two-fold dilution viable plate count method to measure minimal bactericidal concentration (MBC), namely first MIC is measured, the each pipe culture of bacterial growth will be had no again successively draw 0.1mL respectively and be poured on plate, 37 DEG C, cultivate 18h again, the minimum dilution drug level that on plate, clump count is less than 5 is minimum bactericidal concentration (MBC), and observes minimum bactericidal concentration.
Table 12 result of the test
Experiment conclusion: association's day loud, high-pitched sound four taste effective site and former formula extraction all have bacteriostasis to staphylococcus aureus, alpha streptococcus, escherichia coli, bacillus pyocyaneus, Candida albicans, the bacteriostasis of the more former formula extraction of association's day loud, high-pitched sound four taste effective site is stronger.
Following embodiment all can realize the effect of above-mentioned experimental example.
Detailed description of the invention
Embodiment 1: the preparation-Flavonoids by Macroporous Adsorption Resin of association's day loud, high-pitched sound four taste effective site
Get and assist day loud, high-pitched sound four taste compound recipe pharmaceutical decocting piece 1kg, 50% ethanol 10L reflux, extract, 2 times, each extraction 2 hours, decompression and solvent recovery, obtains extract, and the aqueous dispersion adding certain volume is dissolved, and makes medical material amount and the rear liquor capacity ratio of dispersion be 1:
2.5(sample concentration 2g/5ml).By 7.5LAB-8 macroporous adsorbent resin, adsorption flow rate 2Bv/h, resin column blade diameter length ratio is 1:10, remove impurity is carried out with the water elution of 1.5 times of resin volumes, remove impurity flow velocity is 2Bv/h, with 70% ethanol elution of 8 times of resin volumes, elution flow rate is 2Bv/h, collect 70% ethanol elution, recycling design, drying under reduced pressure, again with 90% ethanol elution of 8 times of resin volumes, elution flow rate is 4 ~ 9BV/h, collect 90% ethanol elution, recycling design, drying under reduced pressure, mix with 70% ethanol elution thing to be again after 90% ethanol elution thing calcium hydrogen phosphate is absorbed and assist day loud, high-pitched sound four taste effective site.
Embodiment 2: the preparation-Flavonoids by Macroporous Adsorption Resin of association's day loud, high-pitched sound four taste effective site
Get association day loud, high-pitched sound four taste compound recipe pharmaceutical decocting piece 1kg, add 70% ethanol 15L supersound extraction 3 times, each extraction 1.5 hours, decompression and solvent recovery, obtain extract, the aqueous dispersion adding certain volume is dissolved, and after making medical material amount and dispersion, liquor capacity is than being 1:2.5(sample concentration 2g/5ml).By 10L AB-8 macroporous adsorbent resin, adsorption flow rate 2Bv/h, resin column blade diameter length ratio is 1:6, carry out remove impurity with the water elution of 2 times of resin volumes, remove impurity flow velocity is 4Bv/h, with 70% ethanol elution of 12 times of resin volumes, elution flow rate is 1Bv/h, collects 50% ethanol elution, recycling design, drying under reduced pressure, again with 90% ethanol elution of 10 times of resin volumes, elution flow rate is 4 ~ 9BV/h, collects 90% ethanol elution, recycling design, drying under reduced pressure; By 90% ethanol elution thing beta-cyclodextrin inclusion compound, beta-schardinger dextrin-and 90% ethanol elution thing amount are 5:1, and enclose temperature is 70 DEG C, and the enclose time is 2 hours, mixes after enclose with 70% ethanol elution thing again, are association's day loud, high-pitched sound four taste effective site
Embodiment 3: the preparation-Flavonoids by Macroporous Adsorption Resin of association's day loud, high-pitched sound four taste effective site
Get association day loud, high-pitched sound four taste compound recipe pharmaceutical decocting piece 1kg, 70% ethanol 10L reflux, extract, 2 times, each extraction 2 hours, decompression and solvent recovery, obtains extract, adds the water dissolution of certain volume, and after making medical material amount and dispersion, liquor capacity is than being 1:4.By 10L D101 macroporous adsorbent resin, adsorption flow rate 2Bv/h, resin column blade diameter length ratio is 1:12, carry out remove impurity with the water elution of 1.5 times of resin volumes, remove impurity flow velocity is 4Bv/h, with 70% ethanol elution volume of 8 times of resins, elution flow rate is 3Bv/h, collects 70% ethanol elution, recycling design, drying under reduced pressure, again with 90% ethanol elution of 8 times of resin volumes, elution flow rate is 4 ~ 9BV/h, collects 90% ethanol elution, recycling design, drying under reduced pressure; 70% ethanol elution thing is mixed with 90% ethanol elution thing be and assist day loud, high-pitched sound four taste effective site.
Embodiment 4: the preparation-solvent extraction of association's day loud, high-pitched sound four taste effective site
Get association day loud, high-pitched sound four taste compound recipe pharmaceutical decocting piece 1Kg, 75% alcohol reflux 2 times, solvent load is 10L, each extraction 2 hours, and decompression and solvent recovery, obtains extract, is first suspended in water by extract, and after making medical material and dispersion, liquor capacity is than being 1:8(w/v); Then with the solvent petroleum ether of low polarity, solvent load is the aqueous solution of 1-3 times of extract, extracts 3 times, obtains liposoluble constituent; Use the solvent chloroform of middle polarity again, solvent load is the aqueous solution of 1-3 times of extract, through 4 extractions composition wherein; The liposoluble constituent that forward extraction obtains with it is mixed to get association's day loud, high-pitched sound four taste effective site.
Embodiment 5: the preparation-supercritical C0 of association's day loud, high-pitched sound four taste effective site
2fluids extraction
Get association day loud, high-pitched sound four taste compound recipe pharmaceutical decocting piece 1Kg, add 70% methanol, supercritical fluid extraction 2 times, each extraction 2 hours, solvent load is 8L, is suspended in water by the extract of gained, then by the ethyl acetate of low polarity, extraction composition wherein, must assist day loud, high-pitched sound four taste effective site.
Embodiment 6: the preparation-liquid-liquid counter-current extraction of association's day loud, high-pitched sound four taste effective site
Get association day loud, high-pitched sound four taste compound recipe pharmaceutical decocting piece 1Kg, 70% alcohol reflux 3 times, solvent load is 10L, each extraction 2 hours, decompression and solvent recovery, obtains extract, first extract is suspended in water, then be extracted with ethyl acetate and obtain liposoluble constituent, then with chloroform extraction acquisition terpenoid, phenols, alkaloids composition wherein, liposoluble constituent and terpenoid, phenols, alkaloids composition are mixed to get and assist day loud, high-pitched sound four taste extract.
Embodiment 7: the assay of total phenols, total terpene, total alkaloids in association's day loud, high-pitched sound four taste effective site
The assay of total phenols in association's day loud, high-pitched sound four taste effective site
Accurate absorption curcumin reference substance solution (concentration is 23.68 μ g/ml) 0.5,1.0,1.5,2.0,2.5,3.0 and 3.5ml, put in 25ml measuring bottle, add dehydrated alcohol to 5ml, add 0.3% sodium lauryl sulphate 2.0ml and 0.6% ferric chloride-0.9% potassium ferricyanide (1: 0.9) mixed solution 1.0ml, mixing, in the dark place 5min, add 0.1mol/L hydrochloric acid solution to scale, in the dark place 20min, take developer as blank, measure trap at 720nm wavelength place.With curcumin reference substance sampling amount for abscissa, absorbance is vertical coordinate, drawing standard curve.
Precision takes in embodiment 2 assists day each 3 parts of loud, high-pitched sound four taste effective site sample, and every part of 30mg, puts in 50mL measuring bottle, add 70% appropriate amount of ethanol, supersound process 5 minutes, is diluted to scale with 70% dissolve with ethanol, shakes up, filter, discard just filtrate, precision measures subsequent filtrate 5.0mL in 10mL measuring bottle, adds 70% ethanol to scale, shake up, as sample solution.
The above-mentioned sample solution 1.0mL of accurate absorption puts in 25ml measuring bottle respectively, add dehydrated alcohol to 5ml, add 0.3% sodium lauryl sulphate 2.0ml and 0.6% ferric chloride-0.9% potassium ferricyanide (1: 0.9) mixed solution 1.0ml, mixing, in the dark place 5min, add 0.1mol/L hydrochloric acid solution to scale, in the dark place 20min, take developer as blank, measure trap at 720nm wavelength place; External standard two-point method calculates content, obtains the present invention and assists total phenols in day loud, high-pitched sound four taste effective site to count 18.91% with curcumin.
The assay of total terpene in association's day loud, high-pitched sound four taste extract
Accurate absorption jasminoidin reference substance solution (0.0948mg/mL) 0.1,0.2,0.4,0.6,0.8 and 1.0mL, put in 10mL tool plug scale test tube.Vanillin-concentrated sulfuric acid solution that concentration is about 1mg/mL is slowly dripped in test tube, and jolting test tube frequently.Finally be settled to 5mL, shake up, room temperature leaves standstill 10min, measures absorbance in 438nm place.With jasminoidin reference substance sampling amount (mg) for abscissa, absorbance is vertical coordinate, drawing standard curve.
Precision takes embodiment 2 and assists day each 3 parts of loud, high-pitched sound four taste effective site sample, and every part of 25mg, puts in 50mL measuring bottle, add 70% appropriate amount of ethanol, supersound process 5 minutes, is diluted to scale with 70% dissolve with ethanol, shakes up, filter, discard just filtrate, precision measures subsequent filtrate 5.0mL in 10mL measuring bottle, adds 70% ethanol to scale, shake up, as sample solution.
The above-mentioned sample solution 1.0mL of accurate absorption puts in 10mL tool plug scale test tube respectively.Vanillin-concentrated sulfuric acid solution that concentration is about 1mg/mL is slowly dripped in test tube, and jolting test tube frequently.Finally be settled to 5mL, shake up, room temperature leaves standstill 10min, measures absorbance in 438nm place; External standard two-point method calculates content, obtains the present invention and assists total terpene in day loud, high-pitched sound four taste effective site to count 27.04% with jasminoidin.
The assay of total alkaloids in association's day loud, high-pitched sound four taste extract
Accurate absorption berberine hydrochloride reference substance solution (0.0592mg/mL) 0.5ml, 1.5ml, 2.5ml, 3.5ml, 4ml, 5.5ml put in 10ml tool plug scale test tube, volatilize solvent, add 6ml bromothymol blue test solution sample dissolution respectively, then add 6ml chloroform, firmly jolting 2min, in impouring separatory funnel, leaves standstill 30min, get chloroform layer, extract three times with method, get in chloroform stratification 25ml volumetric flask, and complement to scale with chloroform.Take corresponding reagent as blank, measure absorbance in 415nm, with berberine hydrochloride reference substance sampling amount (mg) for abscissa, absorbance is vertical coordinate, drawing standard curve.
Precision takes embodiment 2 and assists day each 3 parts of loud, high-pitched sound four taste effective site sample, every part of 30mg, puts in 25mL measuring bottle, adds 70% appropriate amount of ethanol, supersound process 5 minutes, is diluted to scale, shakes up, as sample solution with 70% dissolve with ethanol.
The above-mentioned sample solution 1.0mL of accurate absorption puts in 10ml tool plug scale test tube respectively, volatilize solvent, add 6ml bromothymol blue test solution sample dissolution respectively, then add 6ml chloroform, firmly jolting 2min, in impouring separatory funnel, leave standstill 30min, get chloroform layer, extract three times with method, get in chloroform stratification 25ml volumetric flask, and complement to scale with chloroform.Take corresponding reagent as blank, measure absorbance in 415nm; External standard two-point method calculates content, obtains the present invention and assists total alkaloids in day loud, high-pitched sound four taste effective site to count 14.29% with berberine hydrochloride.
Embodiment 8: the assay of jasminoidin, curcumin, berberine hydrochloride in association's day loud, high-pitched sound four taste effective site
Chromatographic condition: chromatographic column: Elite C18 (4.6mm × 250mm, 5 μm) chromatographic column; Mobile phase: acetonitrile (A) – 0.4% potassium dihydrogen phosphate aqueous solution (B), gradient elution (0 ~ 15min, 8%A → 18%A; 15 ~ 18min, 18%A → 45%A; 18 ~ 22min, 45%A → 27%A; 22 ~ 35min, 27%A → 12%A; 35 ~ 40min, 12%A → 45%A; 40 ~ 55min, 48%A → 52%A), flow velocity 1.0mLmin
-1; Determined wavelength is respectively 238nm, 346nm, 346nm; Column temperature 30 DEG C; Sample size 20 μ L.
Specification Curve of Increasing: precision takes in drying under reduced pressure to the jasminoidin reference substance 0.89mg and curcumin reference substance 0.98mg to 5ml measuring bottle of constant weight, precision takes berberine hydrochloride 0.81mg in 10ml volumetric flask, draw 2ml in the 5ml volumetric flask being mixed with above-mentioned two reference substances, being mixed with concentration is respectively 0.178, the mixing reference substance storing solution of 0.0392,0.0324mgmL-1, is settled to scale with 50 ethanol, shake up, obtain mixing reference substance solution.Get mixing reference substance solution sample introduction 2.0 respectively respectively, 4.0,6.0,8.0,10.0,20.0,25.0 μ l carry out HPLC analysis under above-mentioned chromatographic condition, with peak area Y for vertical coordinate, the quality X (μ g) of solution is abscissa drawing standard curve, obtains the regression equation of jasminoidin, curcumin, berberine.
Assay: precision takes each 3 parts of 3 batches of embodiment 3 gained association day loud, high-pitched sound four taste effective site samples, every part of about 25mg, in 25ml volumetric flask, 70% ethanol standardize solution, shakes up rear use 0.45 μm of microporous filter membrane and filters, get subsequent filtrate, obtain need testing solution.
Accurate draw above-mentioned need testing solution 20 μ L, injection liquid chromatography, measures each chromatograph peak-to-peak area, calculates content, obtains the present invention and assists in day loud, high-pitched sound four taste effective site and be respectively 1.86%, 12.50%, 1.39% containing curcumin, jasminoidin, berberine hydrochloride.
Embodiment 9: the preparation of tablet
Association's day loud, high-pitched sound four taste effective site prepared by Example 1, adds customary adjuvant, conveniently technique, makes tablet.
Embodiment 10: the preparation of capsule
Association's day loud, high-pitched sound four taste effective site prepared by Example 4, adds customary adjuvant, conveniently technique, makes capsule.
Embodiment 11: the preparation of oral liquid
Association's day loud, high-pitched sound four taste effective site prepared by Example 5, adds customary adjuvant, conveniently technique, makes oral liquid.
Embodiment 12: the preparation of granule
Association's day loud, high-pitched sound four taste effective site prepared by Example 5, adds customary adjuvant, conveniently technique, makes granule.
Claims (5)
1. association's day loud, high-pitched sound four taste effective site, it is characterized in that described association day loud, high-pitched sound four taste effective site is made up of Flavonoids by Macroporous Adsorption Resin, comprise the steps: to get association day loud, high-pitched sound four taste compound recipe pharmaceutical decocting piece 0.5-1.5 weight portion, 50%-95% alcohol reflux 2-3 time, solvent load is 5 ~ 15 parts by volume, each extraction 1-2 hour, decompression and solvent recovery, obtains extract, is first suspended in water by extract, make the rear liquor capacity of medical material amount and dispersion than being 1:10-1:2.5, in w/v, by the 6-10 volume macroporous adsorbent resin doubly of suitable crude drug amount, adsorption flow rate 2BV/h, resin column blade diameter length ratio is 1:4 ~ 1:13, carries out remove impurity with the water elution of 1-5 times of resin volume, and remove impurity flow velocity is 2 ~ 6BV/h, with the 50%-70% ethanol elution of 5-12 times of resin volume, elution flow rate is 1 ~ 3BV/h, collect 50%-70% ethanol elution, recycling design, drying under reduced pressure, again with the 71%-90% ethanol elution of 5-12 times of resin volume, elution flow rate is 4 ~ 9BV/h, collect 71%-90% ethanol elution, recycling design, drying under reduced pressure, 50%-70% ethanol elution thing is mixed with 71%-90% ethanol elution thing be and assist day loud, high-pitched sound four taste effective site, wherein total terpenoid in effective site, total phenols, the summation of total alkaloids constituents percentage composition is 50 ~ 99%, percentage composition is in w/w,
The crude drug of wherein said association day loud, high-pitched sound four taste compound recipe pharmaceutical decocting piece consists of:
Rhizoma Curcumae Longae 150 weight portion, Cortex Phellodendri 90 weight portion, Fructus Gardeniae 120 weight portion, Fructus Tribuli 150 weight portion, described Fructus Tribuli is that the dry mature fruit of zygophyllaceae plant Fructus Tribuli TribulusterrestrisL. obtains through micro-parch;
Described macroporous adsorbent resin is low pole or nonpolar resin.
2. the preparation method of association as claimed in claim 1 day loud, high-pitched sound four taste effective site, is characterized in that described preparation method comprises the steps:
Get association day loud, high-pitched sound four taste compound recipe pharmaceutical decocting piece 0.5-1.5 weight portion, 50%-95% alcohol reflux 2-3 time, solvent load is 5 ~ 15 parts by volume, each extraction 1-2 hour, decompression and solvent recovery, obtains extract, is first suspended in water by extract, make the rear liquor capacity of medical material amount and dispersion than being 1:10-1:2.5, in w/v; By the 6-10 volume macroporous adsorbent resin doubly of suitable crude drug amount, adsorption flow rate 2BV/h, resin column blade diameter length ratio is 1:4 ~ 1:13, carries out remove impurity with the water elution of 1-5 times of resin volume, and remove impurity flow velocity is 2 ~ 6BV/h; With the 50%-70% ethanol elution of 5-12 times of resin volume, elution flow rate is 1 ~ 3BV/h, collect 50%-70% ethanol elution, recycling design, drying under reduced pressure, again with the 71%-90% ethanol elution of 5-12 times of resin volume, elution flow rate is 4 ~ 9BV/h, collects 71%-90% ethanol elution, recycling design, drying under reduced pressure, mixes 50%-70% ethanol elution thing with 71%-90% ethanol elution thing to be and assists day loud, high-pitched sound four taste effective site;
The crude drug of wherein said association day loud, high-pitched sound four taste compound recipe pharmaceutical decocting piece consists of:
Rhizoma Curcumae Longae 150 weight portion, Cortex Phellodendri 90 weight portion, Fructus Gardeniae 120 weight portion, Fructus Tribuli 150 weight portion, described Fructus Tribuli is that the dry mature fruit of zygophyllaceae plant Fructus Tribuli TribulusterrestrisL. obtains through micro-parch;
Described macroporous adsorbent resin is low pole or nonpolar resin.
3. the preparation method of association as claimed in claim 2 day loud, high-pitched sound four taste effective site, is characterized in that described 50%-70% ethanol elution thing and 71%-90% ethanol elution thing are for directly to mix.
4. the detection method of association as claimed in claim 1 day loud, high-pitched sound four taste effective site, it is characterized in that the method comprises in following content assaying method any one or a few:
The assay of total phenols in association's day loud, high-pitched sound four taste effective site: accurate absorption concentration is the curcumin reference substance solution 0.5 of 23.68 μ g/ml, 1.0,1.5,2.0,2.5,3.0 and 3.5ml, put in 25ml measuring bottle, add dehydrated alcohol to 5ml, add 0.6% ferric chloride-0.9% potassium ferricyanide mixed solution 1.0ml of 0.3% sodium lauryl sulphate 2.0ml and 1: 0.9, mixing, in the dark place 5min, add 0.1mol/L hydrochloric acid solution to scale, in the dark place 20min, take developer as blank, measure trap at 720nm wavelength place; With curcumin reference substance sampling amount for abscissa, absorbance is vertical coordinate, drawing standard curve; Precision takes each 3 parts of association's day loud, high-pitched sound four taste effective site sample, and every part of 30mg, puts in 50mL measuring bottle, add 70% appropriate amount of ethanol, supersound process 5 minutes, is diluted to scale with 70% dissolve with ethanol, shakes up, filter, discard just filtrate, precision measures subsequent filtrate 5.0mL in 10mL measuring bottle, adds 70% ethanol to scale, shake up, as sample solution; The above-mentioned sample solution 1.0mL of accurate absorption puts in 25ml measuring bottle respectively, add dehydrated alcohol to 5ml, add 0.6% ferric chloride-0.9% potassium ferricyanide mixed solution 1.0ml of 0.3% sodium lauryl sulphate 2.0ml and 1: 0.9, mixing, in the dark place 5min, add 0.1mol/L hydrochloric acid solution to scale, in the dark place 20min, take developer as blank, measure trap at 720nm wavelength place; External standard two-point method calculates content, and total phenols in day loud, high-pitched sound four taste effective site must be assisted to count 15% ~ 26% with curcumin;
The assay of total terpene in association's day loud, high-pitched sound four taste effective site: accurate jasminoidin reference substance solution 0.1,0.2,0.4,0.6,0.8 and the 1.0mL drawing 0.0948mg/mL, puts in 10mL tool plug scale test tube; Vanillin-concentrated sulfuric acid solution that concentration is 1mg/mL is slowly dripped in test tube, and jolting test tube frequently; Finally be settled to 5mL, shake up, room temperature leaves standstill 10min, measures absorbance in 438nm place; With jasminoidin reference substance sampling amount for abscissa, absorbance is vertical coordinate, drawing standard curve; Precision takes each 3 parts of association's day loud, high-pitched sound four taste effective site sample, and every part of 25mg, puts in 50mL measuring bottle, add 70% appropriate amount of ethanol, supersound process 5 minutes, is diluted to scale with 70% dissolve with ethanol, shakes up, filter, discard just filtrate, precision measures subsequent filtrate 5.0mL in 10mL measuring bottle, adds 70% ethanol to scale, shake up, as sample solution; The above-mentioned sample solution 1.0mL of accurate absorption puts in 10mL tool plug scale test tube respectively; Vanillin-concentrated sulfuric acid solution that concentration is 1mg/mL is slowly dripped in test tube, and jolting test tube frequently; Finally be settled to 5mL, shake up, room temperature leaves standstill 10min, measures absorbance in 438nm place; External standard two-point method calculates content, and total terpene in day loud, high-pitched sound four taste effective site must be assisted to count 25% ~ 36% with jasminoidin;
The assay of total alkaloids in association's day loud, high-pitched sound four taste effective site: the accurate berberine hydrochloride reference substance solution 0.5ml drawing 0.0592mg/mL, 1.5ml, 2.5ml, 3.5ml, 4ml, 5.5ml put in 10ml tool plug scale test tube, volatilize solvent, add 6ml bromothymol blue test solution sample dissolution respectively, then add 6ml chloroform, firmly jolting 2min, in impouring separatory funnel, leaves standstill 30min, get chloroform layer, extract three times with method, get in chloroform stratification 25ml volumetric flask, and complement to scale with chloroform; Take corresponding reagent as blank, measure absorbance in 415nm, with berberine hydrochloride reference substance sampling amount for abscissa, absorbance is vertical coordinate, drawing standard curve; Precision takes association's day each 3 parts of loud, high-pitched sound four taste effective site sample, every part of 30mg, puts in 25mL measuring bottle, adds 70% appropriate amount of ethanol, supersound process 5 minutes, is diluted to scale, shakes up, as sample solution with 70% dissolve with ethanol; The above-mentioned sample solution 1.0mL of accurate absorption puts in 10ml tool plug scale test tube respectively, volatilize solvent, add 6ml bromothymol blue test solution sample dissolution respectively, then add 6ml chloroform, firmly jolting 2min, in impouring separatory funnel, leave standstill 30min, get chloroform layer, extract three times with method, get in chloroform stratification 25ml volumetric flask, and complement to scale with chloroform; Take corresponding reagent as blank, measure absorbance in 415nm; External standard two-point method calculates content, and total alkaloids in day loud, high-pitched sound four taste effective site must be assisted to count 12% ~ 28% with berberine hydrochloride;
The assay of jasminoidin, curcumin, berberine hydrochloride in association's day loud, high-pitched sound four taste effective site: chromatographic condition: chromatographic column: Elite C18 chromatographic column, 4.6mm × 250mm, 5 μm; Mobile phase: Yi Jing – 0.4% potassium dihydrogen phosphate aqueous solution, acetonitrile represents with A, and 0.4% potassium dihydrogen phosphate aqueous solution represents with B, condition of gradient elution: 0 ~ 15min, 8%A → 18%A; 15 ~ 18min, 18%A → 45%A; 18 ~ 22min, 45%A → 27%A; 22 ~ 35min, 27%A → 12%A; 35 ~ 40min, 12%A → 45%A; 40 ~ 55min, 48%A → 52%A; Flow velocity 1.0mLmin
-1; Determined wavelength is respectively 238nm, 346nm, 346nm; Column temperature 30 DEG C; Sample size 20 μ L; Specification Curve of Increasing: precision takes in drying under reduced pressure to the jasminoidin reference substance 0.89mg and curcumin reference substance 0.98mg to 5ml measuring bottle of constant weight, precision takes berberine hydrochloride 0.81mg in 10ml volumetric flask, draw 2ml in the 5ml volumetric flask being mixed with above-mentioned two reference substances, being mixed with concentration is respectively 0.178,0.0392,0.0324mgmL
-1mixing reference substance storing solution, be settled to scale with 50 ethanol, shake up, obtain mixing reference substance solution; Get mixing reference substance solution sample introduction 2.0,4.0,6.0,8.0 respectively respectively, 10.0,20.0,25.0 μ l carry out HPLC analysis under above-mentioned chromatographic condition, with peak area Y for vertical coordinate, the quality X of solution is abscissa drawing standard curve, obtains the regression equation of jasminoidin, curcumin, berberine; Assay: precision takes each 3 parts of 3 batches of association's day loud, high-pitched sound four taste effective site samples, every part of 25mg, in 25ml volumetric flask, 70% ethanol standardize solution, shakes up rear use 0.45 μm of microporous filter membrane and filters, get subsequent filtrate, obtain need testing solution; The above-mentioned need testing solution 20 μ L of accurate absorption, injection liquid chromatography, measures each chromatograph peak-to-peak area, calculates content, must assist in day loud, high-pitched sound four taste effective site and be respectively 1.30-2.50%, 9.25-19.50%, 0.80-2.10% containing curcumin, jasminoidin, berberine hydrochloride.
5. association as claimed in claim 1 day loud, high-pitched sound four taste effective site has the application in antiinflammatory, analgesia, the medicine of bacteriostasis or functional food in preparation.
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