CN108614039A - The detection method of quercetin content in a kind of Chinese medicine composition that treating colitis - Google Patents
The detection method of quercetin content in a kind of Chinese medicine composition that treating colitis Download PDFInfo
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- CN108614039A CN108614039A CN201611164584.1A CN201611164584A CN108614039A CN 108614039 A CN108614039 A CN 108614039A CN 201611164584 A CN201611164584 A CN 201611164584A CN 108614039 A CN108614039 A CN 108614039A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of detection methods of quercetin content in Chinese medicine composition for treating colitis, the Chinese medicine composition is made of calcite, halite, Clematis aethusifolia, rhododendron micranthum, nutmeg, ceriander seed, rhizoma zingiberis, fructus piperis longi, Radix Ophiopogonis, Gu Yue, raphani,radix, white sand, HALITUM PURPUREUM, elecampane, ten five tastes anaesthetic of nitre by certain weight ratio, is clinically mainly used for treating chronic diarrhea.Quercetin is the common component of Clematis aethusifolia and rhododendron micranthum, and has sizable content in two taste medicinal materials, therefore intends selecting the assay of Quercetin as the method for Control of drug quality.Under selected liquid phase chromatogram condition, Quercetin detaches well with adjacent component in test solution, and negative fluid is noiseless.Through methodological study, the repeatability of method, stability, precision, recovery test comply with relevant regulations.
Description
Technical field
The present invention relates to a kind of drug detection methods, particularly, are related to Mongolian oak in a kind of Chinese medicine composition that treating colitis
The detection method of skin cellulose content belongs to pharmaceutical technology field.
Background technology
At present colitis be it is a kind of mainly invade and rectum colon mucosa, rotten to the corn, ulcer is commonly formed, reason is unknown with abdomen
It rushes down, abdominal pain, pus and blood stool are cardinal symptom, the disease of diffusivity non-specificity large intestine inflammation.This disease involves rectum and distal colorectal more
Then intestines develop to proximal part, or even involve entire colon.Both at home and abroad at present treatment colitis, doctor trained in Western medicine mostly use intravenous nutrition,
Antibiotic, hormone, immunosupress and operative treatment, though there is certain curative effect, side effect is larger, easily recurrence of being discontinued.It grinds at present
The new oral or bowel lavage 5-aminosalicylic acid preparation of system can reach therapeutic effect, but of high cost, price.Therefore, it develops
It is very necessary to go out a kind of affordable, the drug for the treatment of colitis curative for effect.
A kind of Chinese medicine composition for treating colitis, prescription by following weight than raw material form：Calcite 141-165,
Halite 28-33, Clematis aethusifolia 282-329, rhododendron micranthum 282-329, nutmeg 7-8, ceriander seed 7-8, rhizoma zingiberis 7-8, fructus piperis longi
7-8, Radix Ophiopogonis 7-8, Gu Yue 7-8, raphani,radix 7-8, white sand 7-8, HALITUM PURPUREUM 7-8, elecampane 7-8, nitre 14-16.
The Chinese medicine composition of above-mentioned treatment colitis, the prior art, can not there are no the quality standard detecting method of preparation
Effectively ensure product quality in production and use, and then pharmaceutical effectiveness cannot be guaranteed.Therefore, a kind of distinctive inspection is established
Survey method is necessary to current quality research.
Invention content
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of Chinese traditional medicine composition analyte detection side for treating colitis
Method makes the Chinese medicine composition effectively ensure product quality in production and use, and then ensures pharmaceutical effectiveness.
The detection method of content of Quercetin in a kind of Chinese medicine composition that treating colitis, it is characterised in that utilize efficient liquid
Phase chromatography carries out assay to Quercetin.
The assay of the Quercetin, according to high performance liquid chromatography (《Chinese Pharmacopoeia》The one annex VI of version in 2010
D it) measures.
(1) chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler, with methanol -2%
Phosphate aqueous solution (52: 48) is mobile phase, and Detection wavelength 380nm, 25 DEG C of column temperature, theoretical cam curve is based on Quercetin chromatographic peak
It calculates, 4000 must not be less than.
(2) preparation of reference substance solution：Take Quercetin reference substance appropriate, it is accurately weighed, add methanol that every 1ml is made and contains 50 μ g
Solution to get.
(3) preparation of test solution：Take this product appropriate, it is finely ground, 1g is taken, accurately weighed, 10% dilute hydrochloric acid is added in precision
Ethanol solution 50ml, weighed weight is heated to reflux 45 minutes, lets cool, then weighed weight, anhydrous with 10% dilute hydrochloric acid
Ethanol solution supplies the weight of less loss, shakes up, filtration, take subsequent filtrate to get.
(4) measuring method：It is accurate respectively to draw test solution and each 10 μ l of reference substance solution, liquid chromatograph is injected, is surveyed
Determine to obtain the final product.
In order to further illustrate the present invention in a kind of Chinese medicine composition for treating colitis Quercetin detection method of content
Quality controllability, it is related study it is as follows：
1. the establishment of testing index and assay method
This preparation is made of ten five tastes anaesthetic such as calcite, be clinically mainly used for treat chronic diarrhea, Clematis aethusifolia,
Rhododendron micranthum is main ingredient in side, and through searching related document, Clematis aethusifolia mainly contains saponins, flavonoids, lignanoids, additionally
Including other compounds such as volatile oil, alkaloid, organic acid, alkane, flower, leaf are containing Quercetin (quercetin), Kaempferol
(kaempfer-O1) flavones ingredients such as；Rhododendron micranthum contains saponin, tannin, reducing substances, polysaccharide, flavones, grease and volatilization
Oil etc..Flavonoids has Quercetin, gossypetin, Kaempferol in leaf.Quercetin is the common component of Clematis aethusifolia and rhododendron micranthum,
Quercetin has the effects that antitumor, anti-inflammatory, platelet aggregation-against, antioxidant radical, expands blood vessel.And have in two taste medicinal materials
Sizable content, therefore the quasi- content for measuring Quercetin in this product.Under selected chromatographic condition, quercitrin in test solution
Plain to be detached well with adjacent component, negative fluid is noiseless.Through methodological study, the repeatability of method, precision, is returned at stability
Yield experiment complies with relevant regulations, therefore it is Quercetin to establish this preparation quantitative target, and quantitative approach is high performance liquid chromatography
Method.
2. instrument and agents useful for same
Instrument：Lab Alliance liquid chromatographs, ANASTAR work stations, Lab Alliance detectors.
Reagent and reagent：Methanol is chromatographically pure, and water is double distilled water, and phosphoric acid is to analyze pure, Quercetin reference substance (lot number：
100081-200406, HPLC method measure based on 97.3%, are provided by Nat'l Pharmaceutical ＆ Biological Products Control Institute), Da Gebu -15 intestines
Scorching ball (self-control, lot number：20110516).
3. measuring the establishment of wavelength
Make solvent with mobile phase, the ultraviolet spectrogram of Quercetin reference substance determined in 200~500nm wave-length coverages,
As a result there is absorption maximum at 380nm wavelength, determine that 380nm is to measure wavelength.
4. the screening and establishment of mobile phase
The measurement of Quercetin is mostly using methanol-phosphoric acid solution system as mobile phase.With reference to《Chinese Pharmacopoeia》Version first in 2015
The assay of total flavonoids in portion's ginkgo leaf, screens the flow phase system of different proportion, this experiment is successively adopted
With -2% phosphoric acid of methanol (50: 50) (I), -2% phosphoric acid of methanol (65: 35) (II), -2% phosphoric acid of methanol (58: 42) (III), first
- 0.4% phosphoric acid of alcohol (50: 50) (method in pharmacopeia) is investigated, and under conditions of system (III), Quercetin peak shape is preferable,
Reach good with other components to detach, separating degree meets the requirements, so it is mobile phase to select -2% phosphoric acid of methanol (58: 42).
5. the selection of extracting method
The chemical property of, methanol soluble easily in water according to Quercetin, ethyl alcohol has investigated ethyl alcohol, Diluted Alcohol, methanol, 50% first
5 kinds of solvents such as alcohol, absolute ethyl alcohol-dilute hydrochloric acid mixed solution as solvent, and investigated reflux, ultrasound, three kinds of Soxhlet extraction carry
Mode is taken, finds to be apparently higher than other 4 kinds of solvents using absolute ethyl alcohol-dilute hydrochloric acid mixed solution extraction efficiency, and in chromatogram
Impurity peaks are less, and peak shape is good；Reflux extraction method content is compared with ultrasonic extraction height, and Soxhlet extraction method reduces instead, it may be possible to
Be conducive to the dissolution of Quercetin after returned acid hydrolysis under the high temperature conditions, and the Soxhlet extraction time too long makes containing for Quercetin instead
Amount reduces.Therefore it is Extraction solvent to have selected absolute ethyl alcohol-dilute hydrochloric acid mixed solution, using reflux extraction method.
6. reflux extracting time is investigated
By extracting method under " assay " item in quality standard draft, four parts of same test sample (lot number 20110516) is taken
Respectively refluxing extraction 30,45,60,90 minutes, measure content, the results show that extraction 45 minutes substantially extraction completely, and with carrying
Take the extension of time, content to be declined, thus select 45 minutes for this product extraction time.
7. negative fluid interference test
It is operated by method under " assay " item in quality standard draft, to the feminine gender without Clematis aethusifolia and rhododendron micranthum
Sample is measured, and precision draws reference substance solution, test solution, each 10 μ l of negative solution, injects in liquid chromatograph, paints
Liquid chromatogram processed, is prompted by chromatogram, and on the corresponding position of reference substance chromatography, test solution has identical retention time
Chromatographic peak, negative fluid without absorption, it is noiseless to show that other compositions measure quercetin content, method specificity is good in this peak position
It is good.
8. the range of linearity is investigated
Precision weighs Quercetin reference substance 7.52mg, sets in 50ml measuring bottles, adds methanol to dissolve and is settled to scale, shakes up,
Accurate 1ml, 3ml, 5ml, 7ml, 9ml of drawing is set in 10ml measuring bottles respectively, is added methanol dilution to scale, is shaken up, 15.04 μ are made
The gradient series reference substance solution of g/ml, 45.12 μ g/ml, 75.20 μ g/ml, 105.28 μ g/ml, 135.36 μ g/ml, it is smart respectively
The close 10 μ L that draw inject liquid chromatograph, and peak area is measured by the chromatographic condition drafted.
With quercetin concentration (μ g/ml) for abscissa, peak area is ordinate, draws standard curve, calculates to obtain recurrence side
Journey：Y=21980.63x+34550.05, r=0.9996.
Test result shows Quercetin within the scope of 15.04 μ of μ g/ml~135.36 g/ml, and peak area is with sample size in good
Good linear relationship.
9. precision test is investigated
Take this product appropriate, it is finely ground, 1g is taken, it is accurately weighed, by legal system available test solution below text assay item.Essence respectively
10 μ l of close absorption, continuous sample introduction 6 times, by aforementioned chromatographic condition, sample introduction is analyzed, measures chromatographic peak area, and average peak area is
662466, RSD (%) are 0.33%, and this law precision is good.
10. repetitive test is investigated
6 parts of test sample is taken, method under assay item is pressed respectively and operates, measure, average content 1.52mg/g, RSD (%)
Be 1.81%, this law it is reproducible.
11. stability test is investigated
Take this product appropriate, it is finely ground, 1g is taken, it is accurately weighed, by legal system available test solution below text assay item, respectively at
0th, 2,4,6,8,10h measure Quercetin peak area, RSD (%) be 1.19%, test liquid prepare after measured in 10 hours, chromatography
Peak area is without significant change, and within this time, component chemical property to be measured is stablized.
12. recovery test is investigated
6 parts of this product (lot number 20110516, content 1.52mg/g) is taken, every part takes in right amount, and finely ground, precision weighs about 1g, essence
The close solution 1ml that Quercetin reference substance 1.012mg/ml is added, is operated by method under text assay item, is measured, this method
The rate of recovery is between 98.24~101.17%, RSD 1.10%, and this method rate of recovery is good.
Specific implementation mode
Embodiment 1
Prescription：
Calcite 156.74g, halite 31.35g, Clematis aethusifolia 313.48g, rhododendron micranthum 313.48g, nutmeg
7.84g, ceriander seed 7.84g, rhizoma zingiberis 7.84g, fructus piperis longi 7.84g, Radix Ophiopogonis 7.84g, Gu Yue 7.84g, raphani,radix 7.84g, white sand
7.84g, HALITUM PURPUREUM 7.84g, elecampane 7.84g, nitre 15.67g
Preparation process：
Above ten five tastes, calcite, halite, white sand, HALITUM PURPUREUM, nitre are individually ground into impalpable powder；Parsley
Clematis, rhododendron micranthum are ground into fine powder；Eight taste such as remaining nutmeg is ground into fine powder；12 taste such as nutmeg and calcite, celery
Leaf clematis, rhododendron micranthum powder facing-up, sieving, mixing.About 10% microcrystalline cellulose is added, 1% hypromellose is added in mixing
Plain solution, mixing are made pill, it is dry to get.
Quercetin content detection method：
Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler, with -2% phosphorus of methanol
Aqueous acid (52: 48) is mobile phase, and Detection wavelength 380nm, 25 DEG C of column temperature, theoretical cam curve is based on Quercetin chromatographic peak
It calculates, 4000 must not be less than.
The preparation of reference substance solution：Take Quercetin reference substance appropriate, it is accurately weighed, add methanol that every 1ml is made and contains 50 μ g's
Solution to get.
The preparation of test solution：Take this product appropriate, it is finely ground, 1g is taken, accurately weighed, the nothing of 10% dilute hydrochloric acid is added in precision
Hydrous ethanol solution 50ml, weighed weight are heated to reflux 45 minutes, let cool, then weighed weight, with the absolute ethyl alcohol of 10% dilute hydrochloric acid
Solution supplies the weight of less loss, shakes up, filtration, take subsequent filtrate to get.
Measuring method：It is accurate respectively to draw test solution and each 10 μ l of reference substance solution, liquid chromatograph is injected, is measured,
To obtain the final product.
As a result：By detection, the content of Quercetin is about 1.02mg/g in pill.
Embodiment 2
Prescription：
Calcite 156.74g, halite 31.35g, Clematis aethusifolia 313.48g, rhododendron micranthum 313.48g, nutmeg
7.84g, ceriander seed 7.84g, rhizoma zingiberis 7.84g, fructus piperis longi 7.84g, Radix Ophiopogonis 7.84g, Gu Yue 7.84g, raphani,radix 7.84g, white sand
7.84g, HALITUM PURPUREUM 7.84g, elecampane 7.84g, nitre 15.67g
Preparation process：
Above ten five tastes, calcite, halite, white sand, HALITUM PURPUREUM, nitre are individually ground into impalpable powder；Parsley
Clematis, rhododendron micranthum are ground into fine powder；Eight taste such as remaining nutmeg is ground into fine powder；12 taste such as nutmeg and calcite, celery
Leaf clematis, rhododendron micranthum powder facing-up, sieving, mixing.About 10% microcrystalline cellulose is added, 2% hypromellose is added in mixing
0.5% magnesium stearate, mixing is added in plain solution, mixing, and tablet is made in tabletting.
Quercetin content detection method：
Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler, with -2% phosphorus of methanol
Aqueous acid (52: 48) is mobile phase, and Detection wavelength 380nm, 25 DEG C of column temperature, theoretical cam curve is based on Quercetin chromatographic peak
It calculates, 4000 must not be less than.
The preparation of reference substance solution：Take Quercetin reference substance appropriate, it is accurately weighed, add methanol that every 1ml is made and contains 50 μ g's
Solution to get.
The preparation of test solution：Take this product appropriate, it is finely ground, 1g is taken, accurately weighed, the nothing of 10% dilute hydrochloric acid is added in precision
Hydrous ethanol solution 50ml, weighed weight are heated to reflux 45 minutes, let cool, then weighed weight, with the absolute ethyl alcohol of 10% dilute hydrochloric acid
Solution supplies the weight of less loss, shakes up, filtration, take subsequent filtrate to get.
Measuring method：It is accurate respectively to draw test solution and each 10 μ l of reference substance solution, liquid chromatograph is injected, is measured,
To obtain the final product.
As a result：By detection, the content of Quercetin is about 1.07mg/g in tablet.
Embodiment 3
Prescription：
Calcite 156.74g, halite 31.35g, Clematis aethusifolia 313.48g, rhododendron micranthum 313.48g, nutmeg
7.84g, ceriander seed 7.84g, rhizoma zingiberis 7.84g, fructus piperis longi 7.84g, Radix Ophiopogonis 7.84g, Gu Yue 7.84g, raphani,radix 7.84g, white sand
7.84g, HALITUM PURPUREUM 7.84g, elecampane 7.84g, nitre 15.67g
Preparation process：
Above ten five tastes, calcite, halite, white sand, HALITUM PURPUREUM, nitre are individually ground into impalpable powder；Parsley
Clematis, rhododendron micranthum are ground into fine powder；Eight taste such as remaining nutmeg is ground into fine powder；12 taste such as nutmeg and calcite, celery
Leaf clematis, rhododendron micranthum powder facing-up, sieving, mixing.About 10% microcrystalline cellulose and 0.5% magnesium stearate, mixing, dress is added
Capsule is made in capsule.
Quercetin content detection method：
Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler, with -2% phosphorus of methanol
Aqueous acid (52: 48) is mobile phase, and Detection wavelength 380nm, 25 DEG C of column temperature, theoretical cam curve is based on Quercetin chromatographic peak
It calculates, 4000 must not be less than.
The preparation of reference substance solution：Take Quercetin reference substance appropriate, it is accurately weighed, add methanol that every 1ml is made and contains 50 μ g's
Solution to get.
The preparation of test solution：Take this product appropriate, it is finely ground, 1g is taken, accurately weighed, the nothing of 10% dilute hydrochloric acid is added in precision
Hydrous ethanol solution 50ml, weighed weight are heated to reflux 45 minutes, let cool, then weighed weight, with the absolute ethyl alcohol of 10% dilute hydrochloric acid
Solution supplies the weight of less loss, shakes up, filtration, take subsequent filtrate to get.
Measuring method：It is accurate respectively to draw test solution and each 10 μ l of reference substance solution, liquid chromatograph is injected, is measured,
To obtain the final product.
As a result：By detection, the content of Quercetin is about 1.15mg/g in capsule.
Embodiment 4
Prescription：
Calcite 156.74g, halite 31.35g, Clematis aethusifolia 313.48g, rhododendron micranthum 313.48g, nutmeg
7.84g, ceriander seed 7.84g, rhizoma zingiberis 7.84g, fructus piperis longi 7.84g, Radix Ophiopogonis 7.84g, Gu Yue 7.84g, raphani,radix 7.84g, white sand
7.84g, HALITUM PURPUREUM 7.84g, elecampane 7.84g, nitre 15.67g
Preparation process：
Above ten five tastes, calcite, halite, white sand, HALITUM PURPUREUM, nitre are individually ground into impalpable powder；Parsley
Clematis, rhododendron micranthum are ground into fine powder；Eight taste such as remaining nutmeg is ground into fine powder；12 taste such as nutmeg and calcite, celery
Leaf clematis, rhododendron micranthum powder facing-up, sieving, mixing.About 10% sucrose is added, it is molten that 1% hydroxypropyl methylcellulose is added in mixing
Liquid, granulation are dry to get granule.
Quercetin content detection method：
Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler, with -2% phosphorus of methanol
Aqueous acid (52: 48) is mobile phase, and Detection wavelength 380nm, 25 DEG C of column temperature, theoretical cam curve is based on Quercetin chromatographic peak
It calculates, 4000 must not be less than.
The preparation of reference substance solution：Take Quercetin reference substance appropriate, it is accurately weighed, add methanol that every 1ml is made and contains 50 μ g's
Solution to get.
The preparation of test solution：Take this product appropriate, it is finely ground, 1g is taken, accurately weighed, the nothing of 10% dilute hydrochloric acid is added in precision
Hydrous ethanol solution 50ml, weighed weight are heated to reflux 45 minutes, let cool, then weighed weight, with the absolute ethyl alcohol of 10% dilute hydrochloric acid
Solution supplies the weight of less loss, shakes up, filtration, take subsequent filtrate to get.
Measuring method：It is accurate respectively to draw test solution and each 10 μ l of reference substance solution, liquid chromatograph is injected, is measured,
To obtain the final product.
As a result：By detection, the content of Quercetin is about 1.02mg/g in granule.
Claims (4)
1. the detection method of quercetin content in a kind of Chinese medicine composition for treating colitis, which is characterized in that the Chinese medicine group
Close object be by calcite, halite, Clematis aethusifolia, rhododendron micranthum, nutmeg, ceriander seed, rhizoma zingiberis, fructus piperis longi, Radix Ophiopogonis, Gu Yue,
Human skeleton, white sand, HALITUM PURPUREUM, elecampane, ten five tastes anaesthetic of nitre are formed by certain weight ratio.
2. the detection method of quercetin content in a kind of Chinese medicine composition for treating colitis, which is characterized in that use efficient liquid
Phase chromatography is measured the content of Quercetin.
3. detection method according to claim 1, which is characterized in that the assay of the Quercetin, according to efficient liquid
Phase chromatography (《Chinese Pharmacopoeia》The one annex VI D of version in 2010) it measures.
(1) chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler, with -2% phosphoric acid of methanol
Aqueous solution (52: 48) be mobile phase, Detection wavelength 380nm, 25 DEG C of column temperature, theoretical cam curve by Quercetin chromatographic peak calculating,
4000 must not be less than.
(2) preparation of reference substance solution：Take Quercetin reference substance appropriate, it is accurately weighed, add methanol that every 1ml is made molten containing 50 μ g
Liquid to get.
(3) preparation of test solution：Take this product appropriate, it is finely ground, 1g is taken, accurately weighed, the nothing of 10% dilute hydrochloric acid is added in precision
Hydrous ethanol solution 50ml, weighed weight are heated to reflux 45 minutes, let cool, then weighed weight, with the absolute ethyl alcohol of 10% dilute hydrochloric acid
Solution supplies the weight of less loss, shakes up, filtration, take subsequent filtrate to get.
4. the detection method of quercetin content in a kind of Chinese medicine composition for treating colitis according to claim 1,
It is characterized in that, the dosage form of the Chinese medicine composition is capsule, tablet, granule, powder or pill.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102641360A (en) * | 2012-05-03 | 2012-08-22 | 贵州百花医药股份有限公司 | Preparation method and quality detection method of detoxification antidiarrheal tablet |
| CN104383019A (en) * | 2014-10-29 | 2015-03-04 | 内蒙古天奇中蒙制药股份有限公司 | Processing method of Mongolian medicine clematis aethusifolia |
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2016
- 2016-12-09 CN CN201611164584.1A patent/CN108614039A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102641360A (en) * | 2012-05-03 | 2012-08-22 | 贵州百花医药股份有限公司 | Preparation method and quality detection method of detoxification antidiarrheal tablet |
| CN104383019A (en) * | 2014-10-29 | 2015-03-04 | 内蒙古天奇中蒙制药股份有限公司 | Processing method of Mongolian medicine clematis aethusifolia |
Non-Patent Citations (2)
| Title |
|---|
| CHEN FANG 等: "Determination of quercetin in Eriocaulon buergerianum Koern. using HHPLC coupled with four-channel UV-visible detector", 《MEDICINAL PLANT》 * |
| 姚会敏 等: "HPLC法测定复方银杏叶口服液中槲皮素的含量", 《黑龙江科技信息》 * |
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