CN1895325A - Quality control for sedum sarmentosum and its preparation - Google Patents
Quality control for sedum sarmentosum and its preparation Download PDFInfo
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- CN1895325A CN1895325A CN 200610085972 CN200610085972A CN1895325A CN 1895325 A CN1895325 A CN 1895325A CN 200610085972 CN200610085972 CN 200610085972 CN 200610085972 A CN200610085972 A CN 200610085972A CN 1895325 A CN1895325 A CN 1895325A
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Abstract
A quality control method for Herba seai sarmentosi and the Chinese medicine made of it and in the form of particle, tablet, capsule, injection, etc includes such steps as acid-hydrlyzing the flavonoside contained in specimen to obtain aglycon guercetin, and measuring the content of aglycon quercetin by HPLC method.
Description
Technical field:
The present invention relates to the method for quality control of Herba Sedi and preparation thereof, this method of quality control is the content assaying method of formulating at the Herba Sedi composition, be applicable to the quality control of Herba Sedi medical material, also be applicable to the quality control of the dosage forms of making by the single Herba Sedi such as granule, tablet, capsule and injection, also can be used for containing the quality control of the compound preparation of Herba Sedi medical material, belong to the field of Chinese medicines.
Background technology:
Hepatitis is that a kind of disease of serious harm human health, especially viral hepatitis are popular comparatively extensive in China.The hepatitis B virus carrier rate is 9.75%, and wherein about 1.2 hundred million people carry hepatitis B virus for a long time.Die from relevant about 280,000 examples of hepatopathy number of hepatitis B every year.According to the survey result demonstration nineties in 20th century, the infection with hepatitis C virus number reaches 3,800 ten thousand examples.Hepatitis is threatening popular health just day by day.
Drug therapys such as modern clinical normal employing bifendate and oleanolic acid, it is more obvious that the former falls the enzyme effect, but poor to the protective effect of liver plasma membrane, and easily knock-on and hepatocyte had detrimental effect of drug withdrawal.Acute and chronic hepatitis patients all has damp and hot usually, and chronic hepatitis is particularly damp and hotly especially accumulate for a long time, and the Herba Sedi clearing away heat-damp and promoting diuresis can suppress inflammatory exudation, repairs the hepatocyte of damage, has the effect of falling the enzyme hepatoprotective.Therefore under Chinese medical theory instructs, develop the Chinese medicine traditional advantage, research preparation stabilization, drug safety, hepatoprotective quality controllable, that curative effect is reliable, cheap have great importance.
The Chinese medicine Herba Sedi is the fresh or dry herb of Crassulaceae plant Herba Sedi Sedum sarmentosum Bunge.Its preparation such as granule, capsule etc. have the effect of eliminating damp-heat, are used for acute hepatitis, the treatment of migration hepatitis and chronic hepatitis of active stage.Modern study shows, the chemical constituent of Herba Sedi is mainly one-tenth such as flavone and glycosides thereof, triterpene, sterol, alkaloid, the cyanogen glycosides (Ren Fengxia that grades, Zhao Yimin. Herba Sedi chemical constituent and Advance on Pharmacological Activities. modern Chinese medicine research and practice, 2003,17 (1): 58～60).For Herba Sedi medical material and preparation thereof, there is not good method of quality control so far, its quality is uncontrollable, the curative effect instability.Therefore need make the quality of Herba Sedi from the medical material to the preparation controlled by setting up content assaying method and qualitative identification method thereof, thereby guarantee the homogeneity and the effectiveness of medication raw material and preparation, guarantee the drug effect of preparation.
Summary of the invention:
The method of quality control that the purpose of this invention is to provide a kind of Herba Sedi and preparation thereof, be used to detect its clinical quality control with dosage forms such as raw medicinal material and the Herba Sedi granule of being made by the Herba Sedi raw material, Herba Sedi sheet, sedum sarmentosum capsules and injections, this method also can be used for the quality control of Herba Sedi composition in its compound preparation.
The characteristics of the inventive method are: containing with the Quercetin at sample is the principle that the flavonoid glycoside composition hydrolyzable of aglycon generates aglycon quercetin, will measure the content of Quercetin after the sample hydrolysis with the HPLC method.
Compared with the prior art, beneficial effect of the present invention is embodied in, and the invention provides the content assaying method of Herba Sedi medical material and preparation thereof, has improved the quality control level of medicine, has guaranteed the homogeneity of the quality of the pharmaceutical preparations and the stability of curative effect.
The concrete Herba Sedi medical material and the method for quality control of preparation thereof can be realized by following steps:
Flavonoid glycoside in the sample is hydrolyzed into aglycon quercetin, and reuse HPLC method is measured quercetin content, and its method is:
A. chromatographic condition: with octadecylsilane chemically bonded silica is filler; Detect wavelength 370 ± 2nm; Column temperature: 20～35 ℃; Mobile phase is any in methanol-sour water, acetonitrile-sour water and methanol-acetonitrile-sour water; Theoretical cam curve is calculated with the Quercetin peak should be not less than 2000;
B. the preparation of reference substance solution: it is an amount of to get the Quercetin that is dried to constant weight, and accurate the title decides, and solubilizer is made the reference substance solution that 1ml contains 6～60 μ g;
C. the preparation of need testing solution: sample thief 1～6 weight portion, the accurate title, decide, and adds to extract solvent 50 weight portions, weigh, reflux or supersound extraction, cooling, supply weightlessness, filter, get subsequent filtrate 25 weight portions, add sour water 2～18 weight portions, reflux hydrolysis, cooling, hydrating solution is adjusted pH value to 3～7 with aqueous slkali, extracts solvent and is settled to 50 weight portions, shakes up, sampling is crossed film, promptly;
D. algoscopy: accurate respectively reference substance solution and need testing solution 5～10 μ L of drawing, injection hplc determination;
64～50), acetonitrile-sour water (20～35: 80～65) and methanol-acetonitrile-sour water (15～35: 15～5: any in any 70～60), particular methanol-sour water (45: 55), acetonitrile-sour water (28: 72) and the methanol-acetonitrile-sour water (27: 10: 63) numerical range of above-mentioned chromatographic condition mobile phase can be chosen methanol-sour water (36～50:; Sour water can be that concentration is any in glacial acetic acid aqueous solution, trifluoroacetic acid aqueous solution and the phosphate aqueous solution of 0.02-0.2%; The Quercetin reference substance can select for use in methanol, acetonitrile and the mobile phase any to make the solution that every 1ml contains 6～60 μ g in the preparation of reference substance solution; The test sample preparation, desirable 1～the 6g of sample, 40～90% methanol or ethanol 50ml extract, extraction time was at 0.2～2 hour, during acid hydrolysis, the acid consumption can be 2～18ml, and sour water can be in 5～35% hydrochloric acid, sulphuric acid and the phosphate aqueous solution any for concentration, hydrolysis time 30～80 minutes; Close absorption reference substance solution and molten 5～10 μ l of test sample inject hplc determination respectively.
Description of drawings
Fig. 1 Quercetin reference substance chromatogram, mobile phase is methanol among the figure: 0.1% trifluoroacetic acid water (55: 45).
Quercetin contains mapping in Fig. 2 Herba Sedi medical material, and mobile phase is methanol: 0.1% trifluoroacetic acid water (45: 55).
Quercetin contains mapping in Fig. 3 sedum sarmentosum capsules, and mobile phase is methanol: 0.1% trifluoroacetic acid water (45: 55).
Quercetin contains mapping in Fig. 4 compound recipe Herba Ardisiae Japonicae Herba Sedi granule, and mobile phase is methanol: 0.1% trifluoroacetic acid water (45: 55)
The specific embodiment
Embodiment 1: the preparation of Herba Sedi granule (effervescent, Sugarless type)
Get bright Herba Sedi and decoct with water 1 hour, filter, be evaporated to the clear paste of relative density 1.24 (60～65 ℃).The amount of doubling 92% ethanol stirs evenly, and leaves standstill 8～12 hours.Draw supernatant, be evaporated to the extractum of relative density 1.36～1.38 (60～65 ℃).It is an amount of to get extractum portion, 1.2 parts of poly lactoses, water, mixing, and spray drying, 0.3 part of spray powder and sodium bicarbonate, 0.6 part of sodium carbonate, 0.9 part of citric acid powder, 1.0 parts of poly lactoses, aspartame are an amount of, dry granulation, granulate, packing, promptly.
Embodiment 2: the preparation of sedum sarmentosum capsules
Get bright Herba Sedi, clean, add 4 times of water gagings and decocted 1 hour, filter, filtrate decompression is concentrated into the clear paste of relative density 1.24 (60～65 ℃).The amount of doubling 92% ethanol stirs evenly, and leaves standstill 8～12 hours.Draw the clear paste that supernatant is evaporated to relative density 1.10～1.12 (55～60 ℃), (160 ℃ of intake air temperatures, 90 ℃ of air outlet temperature get spray powder to spray drying, add 1% magnesium stearate, mixing, dry granulation, granulate, incapsulate, make 1000, promptly.
Embodiment 3: the preparation of Herba Sedi sheet
Get bright Herba Sedi, clean, add 4 times of water gagings and decocted 1 hour, filter, filtrate decompression is concentrated into the clear paste of relative density 1.24 (60～65 ℃).The amount of doubling 92% ethanol stirs evenly, and leaves standstill 8～12 hours.Draw the clear paste that supernatant is evaporated to relative density 1.10～1.12 (55～60 ℃), spray drying (160 ℃ of intake air temperatures, 90 ℃ of air outlet temperature), get spray powder, add 1% magnesium stearate, add 5% crosslinked carboxymethyl fecula sodium, stir evenly, granulate, dry (60 ℃), tabletting, promptly.
The preparation of embodiment 4, Herba Sedi injection
Get bright Herba Sedi, clean, add 4 times of water gagings and decocted 1 hour, filter, filtrate decompression is concentrated into the clear paste of relative density 1.12 (60～65 ℃), uses ethanol precipitate with ethanol 2 times, contain the alcohol amount and be respectively 65%, 85%, all filter decompression filtrate recycling ethanol at every turn, thin up is equivalent to dry extract 0.5 to every 1ml and restrains, and regulates pH value to 7, cold preservation, the leaching supernatant adds active carbon and boils, and filters, embedding, sterilization, promptly.
Embodiment 5: the discriminating of Herba Sedi
Thin layer is differentiated: the material powder 4.0g that gets it filled, extract 4 times each 25ml with the ether jolting, discard ether solution, treat that ether volatilizes, add 80% ethanol 50ml, reflux 60 minutes filters, and filtrate is put in the round-bottomed flask, added 25% hydrochloric acid (g/ml) 4ml reflux 40 minutes, filter, 80 ℃ of water-baths of filtrate are steamed to about 10ml, add water 20ml, sodium hydroxide with 25% is transferred pH9.0, extract once with the jolting of 25ml ether, discard ether solution, transfer pH2.0 with 25% hydrochloric acid, extract 3 times with the ethyl acetate jolting, each 30ml, combined ethyl acetate liquid volatilizes, residue adds 1ml methanol makes dissolving, as test sample liquid.Other gets Herba Sedi control medicinal material powder 4.0g, shines medical material solution in pairs with legal system.According to thin layer chromatography (2005 version " an appendix VI of Chinese pharmacopoeia B) test, draw each 10 μ L of above-mentioned two kinds of solution, put respectively in same be on the silica gel H lamellae of adhesive with the sodium carboxymethyl cellulose, with toluene-ethyl acetate-formic acid (5: 2: 1) is developing solvent, launch, take out, dry, spray is put under the ultra-violet lamp (365nm) and is inspected with 3% aluminum chloride alcoholic solution.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color.
The assay of Quercetin in embodiment 6 Herba Sedis
Assay: the photograph high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2005 D) measure.
Chromatographic condition and system suitability test octadecylsilane chemically bonded silica are filler, methanol: 0.1% trifluoroacetic acid aqueous solution (45: 55) is a mobile phase (v/v); The detection wavelength is 371nm.
The preparation precision of reference substance solution takes by weighing in 60 ℃ of drying under reduced pressure an amount of to the Quercetin of constant weight, adds methanol and makes the solution that every 1ml contains 30 μ g, promptly.
The about 4.0g of this product fine powder is got in the preparation of need testing solution, and accurate the title decides, and puts in the round-bottomed flask, the methanol 50ml of accurate adding 60% weighs reflux 1 hour, put cold, claim again to decide weight, supply the weight that subtracts mistake, shake up with 60% methanol, filter, precision is measured subsequent filtrate 25ml, adds 25% hydrochloric acid (g/ml), 4 ml reflux 40 minutes, fast cooling, add 25% sodium hydroxide 2ml, cooling is transferred in the 100ml measuring bottle, is diluted to scale with 60% methanol, shake up, microporous filter membrane (0.45 μ m) filters, and gets filtrate, promptly.
Accurate respectively reference substance solution and each the 5 μ L of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
This product is pressed dry product and is calculated, and contains Quercetin (C
15H
10O
7) must not be less than 0.06%.
Embodiment 7: medical material sample liquid preparation method preferred
In order to propose as much as possible in the Herba Sedi with the Quercetin is the flavonoid glycoside composition of aglycon, and the extracting method of sample is investigated, and in addition the acid hydrolysis process of extracting solution has been carried out preferably.Bright Herba Sedi is dried before this, and measured moisture.
1. the selection of extracting mode
Mainly contain flavonoid glycoside compound in the Herba Sedi medical material, this constituents has medium polarity, can well be extracted by certain density alcoholic solution, selected 60% methanol solution to carry out reflux, extract, and supersound extraction respectively, with the peak area of the Quercetin after the extracting solution hydrolysis and the ratio of samples weighing (A/W) as investigating index, preferred preferable extracting mode.
Get two parts of Herba Sedi (dry product) medical materials after the pulverizing, every part of 4.0g accurately claims surely, puts in the round-bottomed flask, each adds the methanol of 50ml60%, weighs respectively, as following table, extraction time is 30 minutes, and weightlessness is supplied in cooling fast, filter, precision pipettes subsequent filtrate 25ml, adds 25% hydrochloric acid 6ml water-bath back hydrolysis 40 minutes, cooling adds 25% sodium hydroxide 4ml fast, cooling, methanol constant volume with 60% is to 50ml, the film of taking a sample, promptly.Sample liquid HPLC analyzes, and calculates, and the results are shown in Table 1:
The experimental result of table 1 sample extraction mode
| Extracting mode | Methanol concentration (%) | Medical material weight (g) | Peak area (A) | A/W |
| Ultrasonic backflow | 60 60 | 4.0060 4.0030 | 1829000 3436000 | 456565.2 858356.2 |
As shown in Table 2, the content of Quercetin is higher behind the extracting solution acid hydrolysis of alcohol reflux, so select the extracting mode of backflow as medical material for use.
2. extract choice of Solvent:
Get three parts of Herba Sedi (dry product) medical materials after the pulverizing, every part of 4.0g, accurate claim fixed, put in the round-bottomed flask, according to the form below adds Different concentrations of alcohol 50ml, weighs respectively, reflux, extract, 30 minutes, weightlessness is supplied in cooling fast, filter, precision pipettes subsequent filtrate 25ml, adds 25% hydrochloric acid 6ml water-bath back hydrolysis 40 minutes, cooling adds 25% sodium hydroxide 4ml fast, cooling, be settled to 50ml, the film of taking a sample, promptly.Sample liquid HPLC analyzes, and calculates, and the results are shown in Table 2:
The experimental result of table 2 sample extraction solvent
| Sample number | Methanol concentration (%) | Medical material weight (g) | Peak area (A) | A/W |
| 1 2 3 | 40 60 80 | 4.1253 4.1350 4.0674 | 741400 3492000 3349000 | 179720.3 844498.2 823376.1 |
As shown in Table 2, higher with the content of Quercetin behind the extracting solution acid hydrolysis of 60% ethanol extraction, so select for use 60% alcoholic solution as extracting solvent.
3. extract the preferred of volume
Get three parts of Herba Sedi (dry product) medical materials after the pulverizing, every part of 4.0g, accurate claim fixed, put in the round-bottomed flask, add 600% ethanol 30ml, 50ml, 70ml respectively, weigh respectively, reflux, extract, 30 minutes, weightlessness is supplied in cooling fast, filter, precision pipettes subsequent filtrate 25ml, adds 25% hydrochloric acid 6.0ml water-bath back hydrolysis 40 minutes, cooling adds 25% sodium hydroxide 4.0ml fast, cooling, be settled to 50ml, the film of taking a sample, promptly.Sample liquid HPLC analyzes, and calculates, and the results are shown in Table 3:
The experimental result of table 3 sample extraction volume
| Sample number | Extract volume (ml) | Medical material weight (g) | Peak area (A) | A/W |
| 1 2 3 | 30 50 70 | 4.1037 4.1350 4.0673 | 1803000 3492000 3260000 | 439359.6 844498.2 801514.5 |
As shown in Table 3, when extracting volume and being 30ml after the extracting solution hydrolysis content of Quercetin lower, and extract volume and be 〉=during 50ml, the content of Quercetin is higher and tend towards stability after the extracting solution hydrolysis, the extraction volume of prompting 50ml can extract the glycoside composition that with the Quercetin is aglycon fully, selects 50ml for use so extract volume.
4. the selection of reflux extracting time
Get three parts of Herba Sedi (dry product) medical materials after the pulverizing, every part of 4.0g, accurate claim fixed, put in the round-bottomed flask, add 60% ethanol 50ml respectively, weigh, reflux, extract, is 0.5 hour, 1.0 hours, 1.5 hours respectively, and weightlessness is supplied in cooling fast, filter, precision pipettes subsequent filtrate 25ml, adds 25% hydrochloric acid 6ml water-bath back hydrolysis 40 minutes, cooling adds 25% sodium hydroxide 4ml fast, cooling, be settled to 50ml, the film of taking a sample, promptly.Sample liquid HPLC analytical calculation the results are shown in Table 4:
The experimental result of table 4 back flow of sample extraction time
| Sample number | Extraction time (h) | Medical material weight (g) | Peak area (A) | A/W |
| 1 2 3 | 0.5 1.0 1.5 | 4.1235 4.1021 4.0935 | 3367000 4844000 4565000 | 816539.3 1180858.6 1115182.6 |
As shown in Table 4, extraction time 〉=1 hour, the content of Quercetin is very approaching after the extracting solution hydrolysis, therefore, can think that reflux, extract, 1 hour can comparatively fully extract the glycoside composition that in the medical material with the Quercetin is aglycon, so extraction time selected for use 1 hour.
Brief summary: above result shows that the Herba Sedi of 4.0g (dry product) medical material adds 60% ethanol 50ml, and reflux, extract, 1.0 hours can be extracted the flavonoid glycoside composition that in the medical material with the Quercetin is aglycon fully.
5. sour consumption preferred in the acid hydrolysis
Get five parts of Herba Sedi (dry product) medical materials after the pulverizing, every part of accurate title of 4.0g is fixed, put in the round-bottomed flask, add 60% ethanol 50ml respectively, weigh reflux, extract, 1.0 hours, cooling fast, supply weightlessness, filter, precision pipettes five parts of subsequent filtrates, every part of 25ml, added 25% hydrochloric acid 1.0ml, 2.0ml, 4.0ml, 6.0ml, 8.0ml water-bath back hydrolysis respectively 40 minutes, cooling fast, what wherein add acid 4,6,8ml adds 25% sodium hydroxide solution 2,4,6ml more respectively, cooling, be settled to 50ml, the film of taking a sample, promptly.Sample liquid HPLC analyzes, and calculates, and the results are shown in Table 5:
The selection experimental result of the sour consumption of table 5
| Sample number | 25% hydrochloric acid (ml) | Medical material weight (g) | Peak area (A) | A/W |
| 1 2 3 4 5 | 1.0 2.0 4.0 6.0 8.0 | 4.0214 4.0735 4.0038 4.1024 4.1354 | 1131000 3647000 6267000 5174000 4451000 | 281245.3 895298.9 1565236.0 1261212.9 1076316.7 |
By peak area that table 5 is surveyed as can be known, in the 25ml subsequent filtrate, add acid amount＜4.0ml, and to fail to make with the Quercetin be that the flavonoid glycoside hydrolysis of aglycon is complete, and add acid during amount 〉=4.0.0ml, the peak area of Quercetin is tending towards descending gradually in the hydrolyzed solution of being surveyed, illustrate that adding acid amount 〉=4.0ml can make the Quercetin of hydrolysis partly destroyed, therefore, selected to add the acid amount and carried out acid hydrolysis for 4.0ml.
6. the selection of acid hydrolysis time
Get three parts of Herba Sedi (dry product) medical materials after the pulverizing, every part of 4.0g accurately claims surely, puts in the round-bottomed flask, add 60% ethanol 50ml respectively, weigh reflux, extract, 1.0 hours, weightlessness is supplied in cooling fast, filters, precision pipettes subsequent filtrate 25ml, adds 25% hydrochloric acid 4.0ml, and the water-bath back hydrolysis is 20 minutes, 40 minutes, 60 minutes respectively, cooling adds 25% sodium hydroxide 2.0ml fast, cooling, be settled to 50ml, the film of taking a sample, promptly.Sample liquid HPLC analyzes, and calculates, and the results are shown in Table 6:
The experimental result of table 6 acid hydrolysis selection of time
| Sample number | The acid hydrolysis time (minute) | Medical material weight (g) | Peak area (A) | A/W |
| 1 2 3 | 20 40 60 | 4.1352 4.1459 4.0965 | 3742000 5759000 5361000 | 904913.9 1389083.2 1308678.1 |
As shown in Table 6, water-bath returned acid hydrolysis 40 minutes, can make with the Quercetin is that the flavonoid glycoside hydrolysis of aglycon is complete, is 40 minutes so select hydrolysis time.
Brief summary: above result shows, can make in 40 minutes in the Herba Sedi with the Quercetin with 25ml extracting solution adding 4ml 25% hydrochloric acid water-bath back hydrolysis is that the flavonoid glycoside hydrolysis of aglycon is complete.
The Herba Sedi medical material 4.0g after the pulverizing is got in the preparation of need testing solution, [get simultaneously Herba Sedi medical material after the pulverizing in addition and measure moisture (" an appendix IX of Chinese pharmacopoeia version in 2005 H second method)] accurately claims surely, puts in the round-bottomed flask, the methanol 50ml of adding 60%, weigh reflux, extract, 1 hour, cooling fast, supply weightlessness, filter, precision is measured subsequent filtrate 25ml, adds 25% hydrochloric acid (g/ml) 4.0ml water-bath back hydrolysis 40 minutes, cooling fast, add 25% sodium hydroxide 2ml, cooling is transferred in the 100ml measuring bottle, with 60% methanol constant volume to scale, sampling is crossed film, promptly.
The chromatographic condition of Quercetin is preferred in the embodiment eight HPLC methods mensuration Herba Sedi
1. instrument and reagent
Waters Dlta 600 high pressure pumps, Waters 2487 detectors, Waters 600 controllers, methanol, acetonitrile are chromatographically pure (Huaiyin Han Bang Science and Technology Ltd.), trifluoroacetic acid is chromatographically pure (Tedia company), and water is sub-boiling distillation water, and all the other reagent are analytical pure.
The Quercetin reference substance provides (for assay usefulness, lot number is 0081-9304) by Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
Chromatographic column: selecting octadecylsilane chemically bonded silica for use is filler, Huaiyin Han Bang Science and Technology Ltd., C
18Post: Lichrospher C
18(4.6 * 250mm, 5 μ m); Dalian Chemical Physics institute of the Chinese Academy of Sciences, anti-phase C
18Post: Kromasil C
18(4.6 * 250mm, 5 μ m).The result shows that the chromatographic column of two different manufacturers all can reach good separation, but better with the Kromasil chromatographic column.Measure wavelength: according to ultraviolet spectra, the maximum absorption wavelength of Quercetin in methanol is 371nm, is defined as 371nm so measure wavelength.
2. the selection of mobile phase condition
The HPLC method is measured the flow phase system of kaempferol in the Herba Sedi and selected, selected with (1) methanol: sour water, (2) acetonitrile: sour water, (3) methanol: acetonitrile: the sour water system, test.
At (1) methanol: in the sour water system, methanol: the ratio of sour water is to 64: 60 between time in sample Quercetin peak energy can be accessed separation at 36: 50, but methanol: appearance time is about 20 minutes during sour water (45: 55), and the peak separates better.
At (2) acetonitrile: in the sour water system, acetonitrile: the sour water ratio is to 80: 65 between time in sample Quercetin peak energy can be accessed separation at 20: 35, but acetonitrile: appearance time is about 20 minutes during sour water (28: 72), and the peak separates better.
At (3) methanol: acetonitrile: in the sour water system, methanol: acetonitrile: when the ratio of sour water was (27: 10: 63), the Quercetin peak energy accessed better separation.
Different acid is compared, selected for use trifluoroacetic acid, glacial acetic acid, phosphoric acid and phosphoric acid, phosphate sodium dihydrogen buffer solution to test, the result shows, it is 2-5 that mobile phase is controlled its acid ph value, adds 0.02% biphosphate sodium water solution as 0.1% trifluoroacetic acid aqueous solution, 0.2% glacial acetic acid aqueous solution, 0.06% phosphate aqueous solution and 0.05% phosphoric acid and all the Quercetin peak can be separated well.So acid can be selected trifluoroacetic acid, glacial acetic acid and other organic acid or weak inorganic acid such as phosphoric acid and buffer solution thereof for use.
The research of embodiment nine medical material sample detection chromatographic processes
1. the investigation of linear relationship
The standard curve of Quercetin is drawn: precision takes by weighing the Quercetin reference substance 5.05mg that is dried to constant weight in 60 ℃, places the 50ml volumetric flask, adds dissolve with methanol and is diluted to scale, makes Quercetin standard stock solution (0.101mg/ml).Precision pipettes 0.5ml, 1ml, 2ml, 3ml, 4ml, 5ml, 6ml in the 10ml volumetric flask respectively again, add methanol and be diluted to scale, be mixed with serial reference substance liquid, draw above-mentioned solution 5 μ l respectively, inject chromatograph of liquid, measure, with the peak area is vertical coordinate (Y), reference substance concentration (μ g/ml) is abscissa (X), and the drawing standard curve gets regression equation: Y=94131 X-23497 (R
2=0.9998)
Quercetin concentration is good linear relationship between 5.05-60.6 μ g/ml.The results are shown in Table 7 and Fig. 1:
Table 7 Quercetin standard curve
| Sequence number | Quercetin (μ g/ml) | Peak area | The peak area meansigma methods |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 | 5.05 10.1 20.2 30.3 40.4 50.5 60.6 | 467300 455300 940800 929900 1855000 1820000 2857000 2859000 3771000 3796000 4707000 4700000 5706000 5688000 | 461300 935400 1838000 2858000 3784000 4704000 5697000 |
2. replica test
With a collection of medical material, prepare 3 parts in sample by the need testing solution preparation method, to measure in accordance with the law, the average content of repeated Quercetin as a result is 0.070%, RSD=1.14% sees Table 8:
The replica test result of Quercetin in table 8 medical material
| Sequence number | (g) weighs | Peak area value | Quercetin (%) | Average content (%) | RSD ％ |
| 1 2 3 4 5 6 | 4.0319 4.1521 4.1897 | 1312000 1321000 1331000 1349000 1349000 1340000 | 0.071 0.071 0.070 0.070 0.070 0.069 | 0.070 | 1.14 |
3. average recovery test
Precision takes by weighing totally 6 parts of each 4.0g of medical material that measure content, and 2 parts one group, each group has added Quercetin contrast liquid (1.01mg/ml) 2.56ml, 3.20ml, 3.84ml respectively.The Quercetin of mensuration, and calculating in accordance with the law average recovery rate is 101.2%, RSD=0.87%.The results are shown in Table 9:
Average recovery result of the test in table 9 medical material
| Sequence number | The heavy g of sample | Peak area | Average area | Add scalar mg | Sample size mg | Detected level mg | Response rate % | Meansigma methods % | RSD ％ |
| 1 2 3 4 5 6 | 4.3215 4.2890 3.8543 3.8628 4.4716 4.4235 | 1463000 1438000 1453000 1402000 1293000 1300000 1266000 1337000 1609000 1601000 1516000 1544000 | 1451000 1428000 1297000 1302000 1605000 1530000 | 3.23 3.23 3.88 3.88 2.58 2.58 | 3.14 3.12 2.83 2.84 3.51 3.34 | 6.43 6.35 6.78 6.79 6.14 5.95 | 101.6 99.9 101.7 101.8 101.9 100.7 | 101.2 | 0.87 |
4. medical material assay
Get three batches of medical materials, measure as method.Measurement result sees Table 10:
The assay result of Quercetin in the table 10 Herba Sedi medical material
| Lot number | Weight (g) | Peak area (A) | Content (%) | Average content (%) | Water content (%) | Dry product content (%) |
| 040901 040902 040903 | 4.1025 4.1032 4.4987 4.4993 4.4231 4.2750 | 1318000 1310000 1471000 1479000 1232000 1233000 | 0.068 0.068 0.069 0.070 0.060 0.061 | 0.068 0.070 0.060 | 0.70 0.64 0.81 | 0.068 0.070 0.061 |
Content limit: this product is pressed dry product and is calculated, and contains Quercetin (C
15H
10O
6) must not count and be less than 0.06%.
The particulate qualitative identification of embodiment ten Herba Sedis
Differentiate
The preparation of need testing solution: get this product powder 6g, add water 50ml, supersound process 20min filters, filtrate is put in the separatory funnel, the jolting that adds diethyl ether is extracted 4 times, and each 25ml discards ether solution, water liquid added 25% hydrochloric acid (g/ml) 10ml reflux 40 minutes, filter, add 25% sodium hydroxide accent pH9.0, put in the separatory funnel, the ether jolting that adds 25ml is extracted once, discard ether solution, the hydrochloric acid of water liquid reuse 25% is transferred pH2.0, extracts three times with the ethyl acetate jolting, each 30ml, volatilize, residue adds methanol 1ml makes dissolving, as need testing solution.
The preparation of reference substance solution: get control medicinal material powder 4.0g, extract 4 times, each 25ml with the ether jolting, discard ether solution, treat that ether volatilizes, add 80% ethanol 50ml, reflux 60 minutes filters, and filtrate is put in the round-bottomed flask, added 25% hydrochloric acid (g/ml) 4ml reflux 40 minutes, filter, 80 ℃ of water-baths of filtrate are steamed to about 10ml, add water 20ml, sodium hydroxide with 25% is transferred pH9.0, extract once with the jolting of 25ml ether, discard ether solution, transfer pH2.0 with 25% hydrochloric acid, extract 3 times with the ethyl acetate jolting, each 30ml, combined ethyl acetate liquid volatilizes, residue adds 1ml methanol makes dissolving, in contrast medical material solution.
Algoscopy: according to thin layer chromatography (2005 version " an appendix VI of Chinese pharmacopoeia B) test, draw each 5 μ L of above-mentioned two kinds of solution, put respectively in same be on the silica gel H lamellae of adhesive with the sodium carboxymethyl cellulose, with toluene-ethyl acetate-formic acid (5: 2: 1) is developing solvent, launch, take out, dry, spray is put under the ultra-violet lamp (365nm) and is inspected with 3% aluminum chloride alcoholic solution.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color.
The assay of Quercetin in the embodiment 11 Herba Sedi granules
Assay is measured according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).
Chromatographic condition and system suitability test octadecylsilane chemically bonded silica are filler, methanol: 0.1% trifluoroacetic acid aqueous solution (45: 55) is a mobile phase (v/v); The detection wavelength is 371nm.
The Quercetin reference substance that the preparation precision of reference substance solution takes by weighing in 60 ℃ of drying under reduced pressure to constant weight is an amount of, adds methanol and makes the solution that every 1ml contains 30 μ g, promptly.
This product granule is got in the preparation of need testing solution, and porphyrize is got powder 5.0g, the accurate title, decide, put in the 100ml tool plug conical flask, add 60% methanol 50ml, weigh, (ultrasonic power is 250w to supersound extraction 30min, frequency is 50KHz), put coldly, supply weightlessness, shake up after-filtration, precision is measured subsequent filtrate 25ml, adds 25% hydrochloric acid 6ml water-bath back hydrolysis 60min, fast cooling, be transferred in the 50ml measuring bottle, add 25% sodium hydroxide 4ml, 60% methanol constant volume shakes up to scale, the film of taking a sample, promptly.
Accurate respectively reference substance solution and each the 5 μ L of need testing solution of drawing of algoscopy inject hplc determination, calculate, promptly.
This product contains Herba Sedi with Quercetin (C for every bag
15H
10O
7) meter, must not be less than 1.56mg.
The qualitative identification of embodiment 12 sedum sarmentosum capsules
Differentiate
The preparation of need testing solution: get this product content 2g, add water 50ml, supersound process 20min filters, filtrate is put in the separatory funnel, the jolting that adds diethyl ether is extracted 4 times, and each 25ml discards ether solution, water liquid added 25% hydrochloric acid (g/ml) 10ml reflux 40 minutes, filter, add 25% sodium hydroxide accent pH9.0, put in the separatory funnel, the ether jolting that adds 25ml is extracted once, discard ether solution, the hydrochloric acid of water liquid reuse 25% is transferred pH2.0, extracts three times with the ethyl acetate jolting, each 30ml, volatilize, residue adds methanol 1ml makes dissolving, as need testing solution.
The preparation of reference substance solution: get control medicinal material powder 4.0g, extract 4 times, each 25ml with the ether jolting, discard ether solution, treat that ether volatilizes, add 80% ethanol 50ml, reflux 60 minutes filters, and filtrate is put in the round-bottomed flask, added 25% hydrochloric acid (g/ml) 4ml reflux 40 minutes, filter, 80 ℃ of water-baths of filtrate are steamed to about 10ml, add water 20ml, sodium hydroxide with 25% is transferred pH9.0, extract once with the jolting of 25ml ether, discard ether solution, transfer pH2.0 with 25% hydrochloric acid, extract 3 times with the ethyl acetate jolting, each 30ml, combined ethyl acetate liquid volatilizes, residue adds 1ml methanol makes dissolving, in contrast medical material solution.
Algoscopy: according to thin layer chromatography (2005 version " an appendix VI of Chinese pharmacopoeia B) test, draw each 5 μ L of above-mentioned two kinds of solution, put respectively in same be on the silica gel H lamellae of adhesive with the sodium carboxymethyl cellulose, with toluene-ethyl acetate-formic acid (5: 2: 1) is developing solvent, launch, take out, dry, spray is put under the ultra-violet lamp (365nm) and is inspected with 3% aluminum chloride alcoholic solution.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color.
The assay of Quercetin in embodiment 13 sedum sarmentosum capsules
Assay is measured according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).
Chromatographic condition and system suitability test octadecylsilane chemically bonded silica are filler, methanol: 0.1% trifluoroacetic acid aqueous solution (45: 55) is a mobile phase (v/v); The detection wavelength is 371nm.
The Quercetin reference substance that the preparation precision of reference substance solution takes by weighing in 60 ℃ of drying under reduced pressure to constant weight is an amount of, adds methanol and makes the solution that every 1ml contains 30 μ g, promptly.
This product content under the content uniformity item is got in the preparation of need testing solution, and porphyrize is got about 1.5g, the accurate title, decide, and puts in the 100ml tool plug conical flask, adds 60% methanol 50ml, claim to decide weight, supersound process (ultrasonic power is 250w, and frequency is 50KHz) 30 minutes is put cold, claim to decide weight again, supply the weight that subtracts mistake with 60% methanol, shake up, filter, precision is measured subsequent filtrate 25ml and is put in the round-bottomed flask, add 25% hydrochloric acid (g/ml) 6ml and shake up, put in the water-bath reflux 60 minutes, be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, add 25% sodium hydroxide 4ml, be cooled to room temperature, be diluted to scale with 60% methanol, shake up, microporous filter membrane (0.45 μ m) filters, and gets filtrate, promptly.
Accurate respectively reference substance solution and each the 5 μ L of need testing solution of drawing of algoscopy inject hplc determination, calculate, promptly.
Every of this product contains Herba Sedi with Quercetin (C
15H
10O
7) meter, must not be less than 0.39mg.
Embodiment 14 sedum sarmentosum capsules sample preparation methods preferred
In order to propose to be the glycoside composition of aglycon with the Quercetin in the preparation as much as possible, the extracting method of sample is investigated, in addition the acid hydrolysis process of extracting solution has been carried out preferably.
1. the selection of extracting mode
This preparation mainly contains flavonoid glycoside compound, this constituents has medium polarity, can well be extracted by certain density alcoholic solution, selected 60% methanol solution to carry out reflux, extract, and supersound extraction respectively, with the peak area of the Quercetin after the extracting solution hydrolysis and the ratio of samples weighing (A/W) as investigating index, preferred preferable extracting mode.
Get two parts of contents under this product content uniformity item, every part of 1.5g accurately claims surely, puts in the round-bottomed flask, each adds the methanol of 50ml60%, weighs respectively, as following table, extraction time is 30 minutes, and weightlessness is supplied in cooling fast, filter, precision pipettes subsequent filtrate 25ml, adds 25% hydrochloric acid 6ml water-bath back hydrolysis 40min, cooling adds 25% sodium hydroxide 4ml fast, cooling, methanol constant volume with 60% is to 50ml, the film of taking a sample, promptly.Sample liquid is analyzed through HPLC, calculates, and the results are shown in Table 11:
The experimental result of table 11 sample extraction mode
| Extracting mode | Concentration of alcohol (%) | Weight of formulation (g) | Peak area (A) | A/W |
| Ultrasonic backflow | 60 60 | 1.5703 1.5849 | 1861000 598600 | 1185123.8 377689.4 |
As shown in Table 11, the content of Quercetin is higher behind the extracting solution acid hydrolysis of 60% ethanol ultrasonic extraction, so select ultrasonic extracting mode as preparation for use.
2. extract choice of Solvent:
The glycosides and the aglycon that mainly contain flavone compound in this preparation, this constituents has medium polarity, can well be extracted by certain density alcoholic solution, selected 40%, 60%, 80% methanol to carry out supersound extraction respectively, with the peak area of the Quercetin after the extracting solution hydrolysis and the ratio of samples weighing (A/W) as investigating index, preferred preferable extraction solvent.
Get three parts of contents under this product content uniformity item, every part of 1.5g, accurate claim fixed, put in the tool plug conical flask, the methanol 50ml that according to the form below adds variable concentrations weighs respectively, ultrasonic 30 minutes (ultrasonic power is 250w, and frequency is 50KHz) puts cold, supply weightlessness, filter, precision pipettes subsequent filtrate 25ml, add 25% hydrochloric acid 6ml water-bath back hydrolysis 40min, cooling adds 25% sodium hydroxide 4ml fast, cooling, the methanol constant volume with 60% is to the 50ml film of taking a sample, promptly.Sample liquid is analyzed through HPLC, calculates, and the results are shown in Table 12:
The experimental result of table 12 sample extraction solvent
| Sample number | Methanol concentration (%) | Weight of formulation (g) | Peak area (A) | A/W |
| 1 2 3 | 40 60 80 | 1.5706 1.5010 1.5061 | 1643000 2653000 2580000 | 1046097.0 1767488.3 1713033.7 |
As shown in Table 12, higher with the content of Quercetin behind the extracting solution acid hydrolysis of 60% methanol extraction, so select for use 60% methanol solution as extracting solvent.
3. extract the preferred of volume
Get three parts of contents under this product content uniformity item, every part of 1.5g accurately claims surely, puts in the tool plug conical flask, add 60% methanol 30ml, 50ml, 70ml respectively, weigh respectively, (ultrasonic power was 250w in ultrasonic 30 minutes, frequency is 50KHz), put coldly, supply weightlessness, filter, precision pipettes subsequent filtrate 15ml, 25ml, 35ml respectively, adds 25% hydrochloric acid 6ml water-bath back hydrolysis 40min, cooling adds 25% sodium hydroxide 4ml fast, cooling, be settled to 50ml, the film of taking a sample, promptly.Sample liquid is analyzed through HPLC, calculates, and the results are shown in Table 13:
The experimental result of table 13 sample extraction volume
| Sample number | Extract volume (ml) | Weight of formulation (g) | Peak area (A) | A/W |
| 1 2 3 | 30 50 70 | 1.4967 1.5061 1.5010 | 1259000 2327000 1847000 | 841183.9 1545050.1 1230513.0 |
As shown in Table 13, when extracting volume and being 30ml after the extracting solution hydrolysis content of Quercetin lower, and when extracting volume and being 50ml and 70ml, the content of Quercetin is higher, and content is very approaching.The extraction volume that 50ml is described can extract composition fully, selects 50ml for use so extract volume.
4. the selection of supersound extraction time
Get three parts of contents under this product content uniformity item, every part of 1.5g, accurate claim fixed, put in the tool plug conical flask, add 60% methanol 50ml respectively, (ultrasonic power was 250w in ultrasonic 20 minutes, 30 minutes, 40 minutes, frequency is 50KHz), put coldly, supply weightlessness, filter, precision pipettes subsequent filtrate 25ml, adds 25% hydrochloric acid 6ml water-bath back hydrolysis 40min, cooling adds 25% sodium hydroxide 4ml fast, cooling, be settled to 50ml, the film of taking a sample, promptly.Sample liquid is analyzed through HPLC, calculates, and the results are shown in Table 14:
The experimental result of table 14 sample extraction time
| Sample number | Extraction time (min) | Weight of formulation (g) | Peak area (A) | A/W |
| 1 2 3 | 20 30 40 | 1.5090 1.5703 1.5272 | 893400 1643000 1249000 | 592047.7 1044501.0 817836.6 |
As shown in Table 14, during extraction time 〉=30 minute, the content of Quercetin is very approaching after the extracting solution hydrolysis, therefore, can think that the glycoside composition that will in the preparation with the Quercetin be aglycon comparatively fully extracts, so extraction time selected for use 30 minutes.
Brief summary: above result shows that the preparation of 1.5g adds 60% methanol 50ml, and supersound extraction 30 minutes can be extracted the flavonoid glycoside composition that in the preparation with the Quercetin is aglycon fully.
5. sour consumption preferred in the acid hydrolysis
Because the composition that contains in the preparation is to be the glycoside composition of aglycon with the Quercetin, therefore, after extracting solution needs hydrolysis, carries out HPLC and measure.Content assaying method with reference to Folium Ginkgo
[3], selected acid-hydrolyzed method for use, sour consumption and acid hydrolysis time are investigated.
Get five parts of contents under this product content uniformity item, every part of 1.5g, the accurate title, decide, and puts in the tool plug conical flask, adds 60% methanol 50ml respectively, (ultrasonic power was 250w in ultrasonic 30 minutes, frequency is 50KHz), put coldly, supply weightlessness, filter, precision pipettes five parts of subsequent filtrates, and every part of 25ml adds 25% hydrochloric acid 2.0ml respectively, 4.0ml, 6.0ml, 8.0ml, 10.0ml water-bath back hydrolysis 40min, cooling fast, all the other add 25% sodium hydroxide 2.0ml successively except that the sample that adds sour 2.0ml, 4.0ml, 6.0ml, 8.0ml, cooling, 60% methanol constant volume is to 50ml, the film of taking a sample, promptly.Sample liquid is analyzed through HPLC, calculates, and the results are shown in Table 15:
The selection experimental result of the sour consumption of table 15
| Sample number | Add acid amount (ml) | Weight of formulation (g) | Peak area (S) | S/W |
| 1 2 3 4 5 | 2.0 4.0 6.0 8.0 10.0 | 1.5039 1.5026 1.5061 1.5173 1.5077 | 135200 2249000 2580000 2420000 2168000 | 89899.6 1496739.0 1713033.7 1594938.4 1437951.8 |
By peak area that table 15 is surveyed as can be known, in the 25ml subsequent filtrate, add acid amount＜6.0ml, and to fail to make with the Quercetin be that the flavonoid glycoside hydrolysis of aglycon is complete, and add acid during amount 〉=6.0ml, the peak area of Quercetin reduces gradually in the hydrolyzed solution of being surveyed, the existing destroyed explanation of part of prompting adds acid amount 6.0ml, and can to make Quercetin be that the flavonoid glycoside hydrolysis of aglycon is complete, therefore, selected to add the acid amount and carried out acid hydrolysis for 6.0ml.
6. the selection of acid hydrolysis time
Get three parts of contents under this product content uniformity item, every part of 1.5g accurately claims surely, puts in the tool plug conical flask, add 60% methanol 50ml respectively, ultrasonic 30min (ultrasonic power is 250w, and frequency is 50KHz) is put cold, supply weightlessness, filter, precision pipettes three parts of subsequent filtrates, every part of 25ml, each adds 25% hydrochloric acid 6.0ml, water-bath back hydrolysis 40 minutes, 60 minutes, 80 minutes, cooling adds 25% sodium hydroxide 4ml fast, cooling, be settled to 50ml, the film of taking a sample, promptly.Sample liquid HPLC analyzes, and calculates, and the results are shown in Table 16:
The experimental result of table 16 acid hydrolysis selection of time
| Sample number | Hydrolysis time (min) | Weight of formulation (g) | Peak area (S) | S/W |
| 1 2 3 | 40 60 80 | 1.5031 1.5008 1.5019 | 893400 1643000 1249000 | 592047.7 1044501.0 817836.6 |
By peak area that table 16 is surveyed as can be known, water-bath returned acid hydrolysis 60 minutes, can make with the Quercetin is that the flavonoid glycoside hydrolysis of aglycon is complete.So hydrolysis time is selected 60 minutes.
Brief summary: above result shows, can make in 60 minutes in the preparation with the Quercetin with the hydrochloric acid water-bath back hydrolysis of 25ml extracting solution adding 6.0ml 25% is that the flavonoid glycoside composition hydrolysis of aglycon is complete.
Conclusion: the preparation method of sample liquid is defined as after preferred: get content 1.5g under this product content uniformity item, the accurate title, decide, and puts in the 100ml tool plug conical flask, adds 60% methanol 50ml, weigh, supersound process 30 minutes (ultrasonic power is 250w, and frequency is 50KHz) is put cold, supply weightlessness, shake up after-filtration, precision is measured subsequent filtrate 25ml, adds 25% hydrochloric acid 6.0ml water-bath back hydrolysis 60 minutes, cooling fast, add 25% sodium hydroxide 4ml, cooling is transferred in the 50ml measuring bottle, with 60% methanol constant volume to scale, shake up, the film of taking a sample, promptly.
The qualitative identification of embodiment 15 Herba Sedi sheets
Thin layer is differentiated the preparation of need testing solution: get 10 of this product, porphyrize is got about 3g, add water 50ml, supersound process 20min filters, filtrate is put in the separatory funnel, the jolting that adds diethyl ether is extracted 4 times, and each 25ml discards ether solution, water liquid added 25% hydrochloric acid (g/ml) 10ml reflux 40 minutes, filter, add 25% sodium hydroxide accent pH9.0, put in the separatory funnel, the ether jolting that adds 25ml is extracted once, discard ether solution, the hydrochloric acid of water liquid reuse 25% is transferred pH2.0, extracts three times with the ethyl acetate jolting, each 30ml, volatilize, residue adds methanol 1ml makes dissolving, as need testing solution.
The preparation of reference substance solution: get control medicinal material powder 4.0g, extract 4 times, each 25ml with the ether jolting, discard ether solution, treat that ether volatilizes, add 80% ethanol 50ml, reflux 60 minutes filters, and filtrate is put in the round-bottomed flask, added 25% hydrochloric acid (g/ml) 4ml reflux 40 minutes, filter, 80 ℃ of water-baths of filtrate are steamed to about 10ml, add water 20ml, sodium hydroxide with 25% is transferred pH9.0, extract once with the jolting of 25ml ether, discard ether solution, transfer pH2.0 with 25% hydrochloric acid, extract 3 times with the ethyl acetate jolting, each 30ml, combined ethyl acetate liquid volatilizes, residue adds 1ml methanol makes dissolving, in contrast medical material solution.
Algoscopy: according to thin layer chromatography (2005 version " an appendix VI of Chinese pharmacopoeia B) test, draw each 5 μ L of above-mentioned two kinds of solution, put respectively in same be on the silica gel H lamellae of adhesive with the sodium carboxymethyl cellulose, with toluene-ethyl acetate-formic acid (5: 2: 1) is developing solvent, launch, take out, dry, spray is put under the ultra-violet lamp (365nm) and is inspected with 3% aluminum chloride alcoholic solution.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color.
The assay of Quercetin in the embodiment 16 Herba Sedi sheets
Assay is measured according to high performance liquid chromatography (2005 editions one appendix VI D of Chinese Pharmacopoeia).
Chromatographic condition and system suitability test are filler with the octadecylsilane chemically bonded silica, are mobile phase (v/v) with methanol-0.1% trifluoroacetic acid aqueous solution (45: 55); The detection wavelength is 371nm.Theoretical cam curve is calculated with the Quercetin peak should be not less than 2000.
The Quercetin reference substance that the preparation precision of reference substance solution takes by weighing in 60 ℃ of drying under reduced pressure to constant weight is an amount of, adds methanol and makes the solution that every 1ml contains 30 μ g, promptly.
The preparation of need testing solution: get 10 of this product, porphyrize is got about 2.0g, the accurate title, decide, and puts in the 100ml tool plug conical flask, adds 60% methanol 50ml, claim to decide weight, supersound process (ultrasonic power is 250w, and frequency is 50KHz) 30 minutes is put cold, claim to decide weight again, supply the weight that subtracts mistake with 60% methanol, shake up, filter, precision is measured subsequent filtrate 25ml and is put in the round-bottomed flask, add 25% hydrochloric acid (g/ml) 6ml and shake up, put in the water-bath reflux 60 minutes, be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, add 25% sodium hydroxide 4ml, be cooled to room temperature, be diluted to scale with 60% methanol, shake up, microporous filter membrane (0.45 μ m) filters, and gets filtrate, promptly.
Accurate respectively reference substance solution and each the 5 μ L of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
Every of this product contains Herba Sedi with Quercetin (C
15H
10O
7) meter, must not be less than 0.39mg.
The assay of Quercetin in the injection of embodiment 17 Herba Sedis
Assay is measured according to high performance liquid chromatography (2005 editions one appendix VI D of Chinese Pharmacopoeia).
Chromatographic condition and system suitability test octadecylsilane chemically bonded silica are filler, methanol: 0.1% trifluoroacetic acid aqueous solution (47: 53) is a mobile phase (v/v); The detection wavelength is 371nm.
The Quercetin reference substance that the preparation precision of reference substance solution takes by weighing in 60 ℃ of drying under reduced pressure to constant weight is an amount of, adds methanol and makes the solution that every 1ml contains 30 μ g, promptly.
The preparation precision of need testing solution is measured Herba Sedi injection 10ml, puts in the round-bottomed flask, adds 25% hydrochloric acid (g/ml) 4ml and shakes up, put in the water-bath reflux 40 minutes, be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, add 25% sodium hydroxide 2ml, be cooled to room temperature, be diluted to scale with 60% methanol, shake up, microporous filter membrane (0.45 μ m) filters, get filtrate, promptly.
Accurate respectively reference substance solution and each the 5 μ L of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.
The every 10ml of this product contains Herba Sedi with Quercetin (C
15H
10O
7) meter, must not be less than 0.8mg.
The assay of Quercetin in the embodiment 18 compound Herba Ardisiae Japonicae Herba Sedi granule
Assay is measured according to high performance liquid chromatography (2005 editions one appendix VI D of Chinese Pharmacopoeia).
Chromatographic condition and system suitability test octadecylsilane chemically bonded silica are filler, methanol: 0.1% trifluoroacetic acid aqueous solution (45: 55) is a mobile phase (v/v); The detection wavelength is 371nm.
The Quercetin reference substance that the preparation precision of reference substance solution takes by weighing in 60 ℃ of drying under reduced pressure to constant weight is an amount of, adds methanol and makes the solution that every 1ml contains 30 μ g, promptly.
This product granule is got in the preparation of need testing solution, and porphyrize is got powder 5.0g, the accurate title, decide, put in the 100ml tool plug conical flask, add 60% methanol 50ml, weigh, (ultrasonic power is 250w to supersound extraction 40min, frequency is 50KHz), put coldly, supply weightlessness, shake up after-filtration, precision is measured subsequent filtrate 25ml, adds 25% hydrochloric acid 8ml water-bath back hydrolysis 60min, fast cooling, be transferred in the 50ml measuring bottle, add 25% sodium hydroxide 6ml, 60% methanol constant volume shakes up to scale, the film of taking a sample, promptly.
Accurate respectively reference substance solution and each the 5 μ L of need testing solution of drawing of algoscopy inject hplc determination, calculate, promptly.This product contains Herba Sedi with Quercetin (C for every bag
15H
10O
7) meter, must not be less than 1.56mg
Figure 2 shows that Quercetin contains mapping in the Herba Sedi medical material, mobile phase is methanol: 0.1% trifluoroacetic acid water (45: 55).
Figure 3 shows that Quercetin contains mapping in the sedum sarmentosum capsules, mobile phase is methanol: 0.1% trifluoroacetic acid water (45: 55).
Figure 4 shows that Quercetin contains mapping in the compound recipe Herba Ardisiae Japonicae Herba Sedi granule, mobile phase is methanol: 0.1% trifluoroacetic acid water (45: 55).
Claims (6)
1, the method for quality control of a kind of Herba Sedi and preparation thereof is characterized in that flavonoid glycoside in the sample is hydrolyzed into aglycon quercetin, and reuse HPLC method is measured quercetin content.
2, method of quality control according to claim 1 is characterized in that this method comprises the following steps:
A. chromatographic condition: with octadecylsilane chemically bonded silica is filler; Column temperature: 20～35 ℃; Mobile phase: volume ratio is that 36～50: 64～50 methanol-sour water, volume ratio are that 20～35: 80～65 acetonitrile-sour water and volume ratio are 15～35: 15～5: any in methanol-acetonitrile of 70～60-sour water; Detect wavelength 370 ± 2nm;
B. the preparation of reference substance solution: it is an amount of to get the Quercetin that is dried to constant weight, and accurate the title decides, and solubilizer is made the solution that every 1ml contains 6～60 μ g, promptly;
C. the preparation of need testing solution: sample thief 1～6 weight portion, the accurate title, decide, and adds the methanol or ethanol 50 weight portions of extraction solvent 40～90%, weigh backflow or supersound extraction 0.2～2 hour, cooling, supply weightlessness, filter, get subsequent filtrate 25 weight portions, add sour water 2～18 weight portions, reflux hydrolysis 30～80 minutes, cooling, hydrating solution is adjusted pH value to 3～7 with aqueous slkali, extracts solvent and is settled to 50 weight portions, shakes up, sampling is crossed film, promptly;
D. algoscopy: accurate respectively each 5～10 μ L of reference substance solution and need testing solution that draw, inject hplc determination, calculating, promptly.
3, method of quality control according to claim 2 is characterized in that this method comprises the following steps:
A. chromatographic condition: with octadecylsilane chemically bonded silica is filler; Column temperature: 30 ℃; Mobile phase: volume ratio be methanol-sour water of 45: 55, acetonitrile-sour water that volume ratio is 28: 72 and volume ratio be 27: 10: 63 methanol-acetonitrile-sour water in any; Detect wavelength 371nm;
B. the preparation of reference substance solution: it is an amount of to get the Quercetin that is dried to constant weight, and accurate the title decides, and solubilizer is made the solution that every 1ml contains 30 μ g, promptly;
C. the preparation of need testing solution: sample thief 1.5 weight portions, the accurate title, decide, and adds 60% methanol or ethanol 50 weight portions, weigh backflow or supersound extraction 0.5 hour, cooling, supply weightlessness, filter, get subsequent filtrate 25 weight portions, add sour water 6 weight portions, reflux hydrolysis 60 minutes, cooling, hydrating solution is adjusted pH value to 4 with aqueous slkali, extracts solvent and is settled to 50 weight portions, shakes up, sampling is crossed film, promptly;
D. algoscopy: accurate respectively each the 5 μ L of reference substance solution and need testing solution that draw, inject hplc determination, calculating is promptly.
4, according to claim 2 or 3 described method of quality control, it is characterized in that used sour water in the described chromatographic condition mobile phase be concentration be in glacial acetic acid aqueous solution, trifluoroacetic acid aqueous solution and the phosphate aqueous solution of 0.02-0.2% any.
5, according to claim 2 or 3 described method of quality control, it is characterized in that described acid hydrolysis, its sour water is that concentration is any in 5～35% hydrochloric acid, sulphuric acid and the phosphate aqueous solution.
6, the described stringy stonecrop of claim 1 is Chinese medicine preparation and the compound preparation of being made by the Herba Sedi raw material thereof, comprises Herba Sedi granule, Herba Sedi sheet, sedum sarmentosum capsules, Herba Sedi injection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101574387B (en) * | 2008-05-09 | 2012-07-11 | 北京亚东生物制药有限公司 | Quality control method for stringy stonecrop herb preparation |
| CN102988426A (en) * | 2012-11-12 | 2013-03-27 | 青岛金康生物科技有限公司 | Concentration method of traditional Chinese medicine extracting solution |
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| CN1364517A (en) * | 2001-01-17 | 2002-08-21 | 杨孟君 | Nano stringy stonecrop medicine and its preparing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101574387B (en) * | 2008-05-09 | 2012-07-11 | 北京亚东生物制药有限公司 | Quality control method for stringy stonecrop herb preparation |
| CN102988426A (en) * | 2012-11-12 | 2013-03-27 | 青岛金康生物科技有限公司 | Concentration method of traditional Chinese medicine extracting solution |
| CN102988426B (en) * | 2012-11-12 | 2015-04-08 | 青岛金康生物科技有限公司 | Concentration method of traditional Chinese medicine extracting solution |
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Assignee: Suzhou Industrial Park Tianlong Medical Co., Ltd. Assignor: Chaohu C-Dragon Pharmacy Co., Ltd.Of Shanghai Haihong Industrial Co., Ltd. Contract record no.: 2011320010051 Denomination of invention: Quality control for sedum sarmentosum and its preparation Granted publication date: 20101201 License type: Exclusive License Open date: 20070117 Record date: 20110513 |