CN102068649B - Quality control method for gastric condition-regulating pill as traditional Chinese preparation - Google Patents
Quality control method for gastric condition-regulating pill as traditional Chinese preparation Download PDFInfo
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- CN102068649B CN102068649B CN200910228360A CN200910228360A CN102068649B CN 102068649 B CN102068649 B CN 102068649B CN 200910228360 A CN200910228360 A CN 200910228360A CN 200910228360 A CN200910228360 A CN 200910228360A CN 102068649 B CN102068649 B CN 102068649B
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Abstract
The invention relates to a quality control method for a gastric condition-regulating pill as traditional Chinese preparation, which is prepared from Chinese herbal medicines as raw materials, belonging to the technical field of Chinese patent medicine; the method comprises the steps that: a microscope is arranged for quality standard to identify whether the formula of the gastric condition-regulating pill contains betel nut, immature bitter orange, magnolia bark and cinnamon; with synephrine, dehydrocostus lactone and liquorice as reference substances, thin-layer chromatography is adopted to identify whether the formula of gastric condition-regulating pill contains the ingredients of synephrine, dehydrocostus lactone and liquorice; and with magnolol and honokiol as reference substances, high efficiency liquid chromatography is adopted to detect the quantitative index and the inspection method of the content of the magnolia bark in the formula of gastric condition-regulating pill, thereby relatively perfecting the quality standard, strictly controlling the quality of medicines, ensuring the improvement of product quality and improving the quality control for medicines owing to the revised quality standard.
Description
Technical field
The invention belongs to the technical field of Chinese patent drug, relating to the Chinese crude drug is that a kind of Chinese medicine preparation that raw material is processed is transferred the red detection method of stomach.
Background technology
Transferring the stomach pellet is a kind of classical Chinese patent drug, the standard WS of the Ministry of Public Health
3-B-0395-90 record prescription and quality standard:
Prescription: banksia rose 75g fructus amomi 75g Radix Glycyrrhizae 75g
The betel nut 75g dried immature fruit of citron orange (stir-fry) the 75g bark of official magnolia (ginger system) 75g
Rhizoma cyperi (vinegar system) 75g cardamom 75g spirit fat (vinegar system) 50g
High langal 25g cloves 25g Chinese cassia tree 25g
Method for making: above 12 flavors, be ground into fine powder, sieve, mixing is used water pill, drying, the dried ball of every 500g is used Cinnabar 16g, peach gum 3gization water dressing, polishing, drying promptly gets.
Inspection: should meet each item regulation (2 pages of appendix) relevant under the pill item.
More than fill a vacancy
The relevant at present red document of stomach of transferring has a lot, but Shang Weijian and the above-mentioned disclosed standard WS of the Ministry of Public Health
3-B-0395-90 transfers the red prescription of stomach similar or identical; Proportioning is identical, the document of the product standard that effect is similar, but also there are shortcomings such as discriminating project, discriminating means, content assaying method imperfection in the standard of prior art; For improving the product quality of Chinese medicine, need improve target level of product quality.
Summary of the invention
The objective of the invention is to overcome the red standard weak point of accent stomach of prior art, a kind of red detection method of accent stomach that can the qualitative and quantitative analysis drug ingedient is provided.
In order to reach the technical scheme that the object of the invention adopts be: a kind of Chinese medicine preparation is transferred the red detection method of stomach, and wherein said pharmaceutical formulation is by Chinese crude drug:
Banksia rose 75g fructus amomi 75g Radix Glycyrrhizae 75g
The betel nut 75g dried immature fruit of citron orange (bran stir-fry) the 75g bark of official magnolia (ginger system) 75g
Rhizoma cyperi (vinegar system) 75g cardamom 75g excrementum pteropi (vinegar system) 50g
Galangal 25g cloves 25g Chinese cassia tree 25g forms,
Method for making; More than 12 the flavor, be ground into fine powder, sieve, mixing, the water pill, polishing, drying promptly gets, and it is characterized in that: the step of its method is:
(1) microscopic features with betel nut, the dried immature fruit of citron orange, the bark of official magnolia, Chinese cassia tree are contrast, and microscope differentiates to transfer in the red prescription of stomach whether contain betel nut, the dried immature fruit of citron orange, the bark of official magnolia, Chinese cassia tree; With synephrine, dehydro-, Radix Glycyrrhizae is reference substance, and thin-layered chromatography differentiates to transfer in the red prescription of stomach whether contain synephrine, dehydro-, licorice ingredient;
(2) be reference substance with magnolol, honokiol, high performance liquid chromatography detects the content of transferring the bark of official magnolia in the red prescription of stomach.
Described Chinese medicine preparation is transferred the red detection method of stomach; It is characterized in that: the described microscope of putting differentiates that the method that does not contain betel nut, the dried immature fruit of citron orange, the bark of official magnolia, Chinese cassia tree in the red prescription of accent stomach is: get these article; Putting microscopically observes: thin ancient piece of jade, round, flat and with a hole in its centre cell contains prism of calcium oxalate around the fibrous bundle, forms crystal fiber; The endosperm cell fragment is colourless, and wall is thicker, and more great similar round pit is arranged; Prism of calcium oxalate is present in the Bao Bi tissue in flakes; Lithocyte divides dendritic, and behind the wall, laminated striation is obvious; Square or the similar round of lithocyte class, wall are simultaneously poor;
Described thin-layered chromatography is differentiated and transferred the method that whether contains the synephrine composition in the red prescription of stomach to be: get these article 5g, porphyrize adds methyl alcohol 15ml, and sonicated 20 minutes filters, the filtrating evaporate to dryness, and residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the synephrine reference substance, adds methyl alcohol and processes the solution that every 1ml contains 0.5mg, as reference substance solution; According to the thin-layered chromatography test, draw each 2～5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With normal butyl alcohol-glacial acetic acid-water is developping agent, with normal butyl alcohol: glacial acetic acid: the upper solution of water=4: 1: 5, launch; Take out; Dry, spray is with 0.5% ethanol solution of ninhydrin, and it is clear to be heated to spot colour developing; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
Described thin-layered chromatography is differentiated and transferred the method that whether contains the dehydro-composition in the red prescription of stomach to be: get these article 5g, porphyrize adds methenyl choloride 10ml, and sonicated 30 minutes filters, and filtrating is as need testing solution; Other removes hydrogen constuslactone reference substance, adds methenyl choloride and processes the solution that every 1ml contains 0.5mg, as reference substance solution; According to the thin-layered chromatography test, draw each 4～6 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be developping agent with cyclohexane-acetone; With cyclohexane: acetone=10: 2, launch, take out; Dry, spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to spot colour developing; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
Described thin-layered chromatography is differentiated and is transferred the method that whether contains licorice ingredient in the red prescription of stomach to be: get these article 5g, porphyrize, the 40ml that adds diethyl ether, reflux 20 minutes; Filter, discard filtrating, the dregs of a decoction volatilize ether, add methyl alcohol 30ml; Reflux 1 hour filters, the filtrating evaporate to dryness, and residue adds water 40ml makes dissolving; Extract 3 times with water saturated normal butyl alcohol, each 20ml merges normal butyl alcohol liquid, water 20ml washing; Discard water liquid, normal butyl alcohol liquid evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution; Extracting liquorice control medicinal material 1g in addition, the 10ml that adds diethyl ether, reflux 20 minutes filters, and discards filtrating; The dregs of a decoction volatilize ether, add methyl alcohol 10ml, and reflux 1 hour filters, the filtrating evaporate to dryness; Residue adds water 10ml makes dissolving, extracts 3 times with water saturated normal butyl alcohol, and each 10ml merges normal butyl alcohol liquid; Water 10ml washing discards water liquid, and normal butyl alcohol liquid evaporate to dryness shines medicinal material solution in pairs with legal system.According to the thin-layered chromatography test, draw each 2～4 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With methenyl choloride-methanol-water is developping agent, methenyl choloride: methyl alcohol: water=40: 10: 1, launch; Take out; Dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to spot colour developing; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
Described Chinese medicine preparation is transferred the red detection method of stomach, and it is characterized in that: with magnolol, honokiol is reference substance, and liquid phase chromatography detects transfers the method for the content of the bark of official magnolia in the red prescription of stomach to be:
(1) chromatographic condition and system suitability test: chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filling agent; Methyl alcohol: water=78: 22 is moving phase; The detection wavelength is 294nm; Number of theoretical plate calculates by the magnolol peak should be not less than 3800;
(2) preparation of reference substance solution: get the magnolol reference substance, the honokiol reference substance is an amount of, accurately claim surely, add methyl alcohol and process the solution that every 1ml contains magnolol 130 μ g, honokiol 45 μ g, promptly get;
(3) preparation of need testing solution: it is an amount of to get these article, is ground into fine powder, gets powder 0.5g, and accurate the title decides, the accurate methyl alcohol 25ml that adds; Claim decide weight, sonicated 30 minutes is put coldly, and weight decided in title again, supplies the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate, promptly get;
(4) determination method: determination method is drawn above-mentioned reference substance solution and each 5 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets; The every 1g of these article contains the bark of official magnolia with magnolol (C
18H
18O
2) and honokiol (C
18H
18O
2) the total amount meter, must not be less than 1.6mg.
Invention beneficial effect: microscope is set in the quality standard differentiates that accent stomach pellet does not contain betel nut, the dried immature fruit of citron orange, the bark of official magnolia, Chinese cassia tree in writing out a prescription; With synephrine, dehydro-, Radix Glycyrrhizae is reference substance, and thin-layered chromatography differentiates to transfer in the red prescription of stomach whether contain synephrine, dehydro-, licorice ingredient;
(2) be reference substance with magnolol, honokiol, high performance liquid chromatography detects the content of transferring the bark of official magnolia in the red prescription of stomach, and the every 1g of these article contains the bark of official magnolia with magnolol (C
18H
18O
2) and honokiol (C
18H
18O
2) the total amount meter, must not be less than the quantitative target and the method for inspection of 1.6mg, make quality standard comparatively perfect, revised quality standard, the quality control that has improved medicine.
Description of drawings
Fig. 1 betel nut microscopical identification figure.
Fig. 2 dried immature fruit of citron orange microscopical identification figure.
Fig. 3 bark of official magnolia microscopical identification figure.
Fig. 4 Chinese cassia tree microscopical identification figure.
Fig. 5, Fig. 6 are the thin-layer chromatograms of transferring the dried immature fruit of citron orange in the stomach pellet.
Fig. 7 is a thin-layer chromatogram of transferring the banksia rose in the stomach pellet.
Fig. 8 is a thin-layer chromatogram of transferring Radix Glycyrrhizae in the stomach pellet.
Fig. 9 is honokiol and magnolol reference substance curve map.
Figure 10 transfers the red curve map of stomach.
Figure 11 is the negative sample curve map.
Figure 12 is honokiol and magnolol reference substance curve map (recovery).
Figure 13 transfers the red appearance of stomach mark-on curve map (recovery).
Figure 14 magnolol linear graph.
Figure 15 honokiol linear graph.
Description of reference numerals:
Fig. 5: [left side → right side: synephrine (0727-200105), dried immature fruit of citron orange medicinal material, red (060301) test sample of accent stomach, feminine gender]
Fig. 6: [left side → right side: synephrine (0727-200105), accent stomach red (060301), accent stomach red (060302), accent stomach pellet (060303), feminine gender]
Fig. 7: { left side → right side: reference substance [dehydro-(111525-200404)], accent stomach red (060301), accent stomach red (060302), accent stomach pellet (060303), feminine gender }
Fig. 8: [left side → right side: Radix Glycyrrhizae control medicinal material (120904-200511), accent stomach red (060301), accent stomach red (0603002), accent stomach pellet (060303), feminine gender]
Embodiment
Below in conjunction with embodiment, the present invention is further specified the red quality standard of accent stomach after embodiment 1 improves: prescription:
Banksia rose 75g fructus amomi 75g Radix Glycyrrhizae 75g
The betel nut 75g dried immature fruit of citron orange (bran stir-fry) the 75g bark of official magnolia (ginger system) 75g
Rhizoma cyperi (vinegar system) 75g cardamom 75g spirit fat (vinegar system) 50g
Galangal 25g cloves 25g Chinese cassia tree 25g
Method for making: above 12 flavors, be ground into fine powder, sieve, mixing, the water pill, polishing, drying promptly gets.
Proterties: these article are tawny to the sepia water-bindered pill, gas fragrance, and flavor is arduous.
Differentiate: these article are got in (1), put microscopically and observe: thin ancient piece of jade, round, flat and with a hole in its centre cell contains prism of calcium oxalate around the fibrous bundle, forms crystal fiber.The endosperm cell fragment is colourless, and wall is thicker, and more great similar round pit is arranged.Prism of calcium oxalate is present in the Bao Bi tissue in flakes.Lithocyte divides dendritic, and behind the wall, laminated striation is obvious.Square or the similar round of lithocyte class, wall are simultaneously poor.
(2) get these article 5g, porphyrize adds methyl alcohol 15ml, and sonicated 20 minutes filters, the filtrating evaporate to dryness, and residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Other gets the synephrine reference substance, adds methyl alcohol and processes the solution that every 1ml contains 0.5mg, as reference substance solution.According to thin-layered chromatography (" first appendix VI of Chinese pharmacopoeia version in 2005 B) test, draw each 2～5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; Upper solution with normal butyl alcohol-glacial acetic acid-water=4: 1: 5 is a developping agent; Launch, take out, dry; Spray is with 0.5% ethanol solution of ninhydrin, and it is clear to be heated to the spot colour developing.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.
(3) get these article 5g, porphyrize adds methenyl choloride 10ml, and ultrasonic place 30 minutes filters, and filtrating is as need testing solution.Other removes hydrogen constuslactone reference substance, adds methenyl choloride and processes the solution that every 1ml contains 0.5mg, as reference substance solution.According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2005 B) test, draw each 4～6 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With cyclohexane-acetone=10: 2 was developping agent; Launch, take out, dry; Spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to the spot colour developing.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.
(4) get these article 5g, porphyrize, the 40ml that adds diethyl ether, reflux 20 minutes filters; Discard filtrating, the dregs of a decoction volatilize ether, add methyl alcohol 30ml, and reflux 1 hour filters; The filtrating evaporate to dryness, residue adds water 40ml makes dissolving, extracts 3 times with water saturated normal butyl alcohol, and each 20ml merges normal butyl alcohol liquid; Water 20ml washing discards water liquid, and normal butyl alcohol liquid evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution.Extracting liquorice control medicinal material 1g in addition, the 10ml that adds diethyl ether, reflux 20 minutes filters, and discards filtrating; The dregs of a decoction volatilize ether, add methyl alcohol 10ml, and reflux 1 hour filters, the filtrating evaporate to dryness; Residue adds water 10ml makes dissolving, extracts 3 times with water saturated normal butyl alcohol, and each 10ml merges normal butyl alcohol liquid; Water 10ml washing discards water liquid, and normal butyl alcohol liquid evaporate to dryness shines medicinal material solution in pairs with legal system.According to thin-layered chromatography (" an appendix VI of Chinese pharmacopoeia version in 2005 B) test, draw each 2～4 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With methenyl choloride-methanol-water=40: 10: 1 was developping agent; Launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
Inspection: should meet each item relevant under pill item regulation (" an appendix I of Chinese pharmacopoeia version in 2005 A).Assay: the photograph high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2005 D) measure.
Chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filling agent; Methanol-water=78: 22 is a moving phase; The detection wavelength is 294nm.Number of theoretical plate calculates by the magnolol peak should be not less than 3800.
The magnolol reference substance is got in the preparation of reference substance solution, the honokiol reference substance is an amount of, and accurate the title decides, and adds methyl alcohol and processes the solution that every 1ml contains magnolol 130 μ g, honokiol 45 μ g, promptly gets.
It is an amount of that these article are got in the preparation of need testing solution, is ground into fine powder, gets powder 0.5g, accurately claims surely, and the accurate methyl alcohol 25ml that adds claims decide weight, and sonicated 30 minutes is put coldly, claims to decide weight again, supplies the weight that subtracts mistake with methyl alcohol, shakes up, and subsequent filtrate is got in filtration, promptly gets.
Determination method is drawn above-mentioned reference substance solution and each 5 μ l of need testing solution respectively, injects liquid chromatograph, measures, and promptly gets.The every 1g of these article contains the bark of official magnolia with magnolol (C
18H
18O
2) and honokiol (C
18H
18O
2) the total amount meter, must not be less than 1.6mg.
Function with cure mainly: be good for the stomach wide in, the liver of relaxing is pleasant.Be used for the hydrochloric acid in gastric juice stomach cold, the chest turgor, it is noisy to fall to satisfy, appetite pain.
Usage and consumption; Oral.One time 20 ball, 2 times on the one.Attention: be not taken by pregnant women.Specification: per 10 balls weigh 1.45 grams.Storage: sealing.
Differentiate:
One, instrument and reagent
High performance liquid chromatograph: Tianjin, island LC-2010A
HT
Ultrasound Instrument: KUDOS SK8200LH (Shanghai High Kudos Science Instrument Co., Ltd.);
Electronic balance: Sartorius CP225D/CP224S (Beijing Sai Duolisi instrument system company limited);
Silica gel g thin-layer plate, silica gel H thin layer plate, high-efficient silica gel G thin layer plate (subsidiary factory of Haiyang Chemical Plant, Qingdao);
Reference substance: dehydro-(lot number: 1525-200102)
Synephrine (lot number: 110727-200105)
Above reference substance is all available from Nat'l Pharmaceutical & Biological Products Control Institute;
Agents useful for same is pure for analyzing, and water is double distilled deionized water;
Transfer the red (lot number: 060301,060302,060303) of stomach
Provide by Lerentang Pharmaceutical Factory, Zhongxin Pharmaceutical Group Co., Ltd., Tianj.
Two, differentiate
2.1 transfer the red microscopical identification of stomach
These article of getting, put microscopically and observe: thin ancient piece of jade, round, flat and with a hole in its centre cell contains prism of calcium oxalate around the fibrous bundle, forms crystal fiber (dried immature fruit of citron orange).The endosperm cell fragment is colourless, and wall is thicker, and more great similar round pit (betel nut) is arranged.Prism of calcium oxalate is present in the Bao Bi tissue in flakes.Lithocyte divides dendritic, and behind the wall, laminated striation is (bark of official magnolia) obviously.Square or the similar round of lithocyte class, wall be poor (Chinese cassia tree) simultaneously, (result sees Fig. 1, Fig. 2, Fig. 3, Fig. 4)
2.2 transfer the qualitative identification of the dried immature fruit of citron orange in the stomach pellet
The preparation of medicinal material solution: get dried immature fruit of citron orange medicinal powder 0.5g, add methyl alcohol 10mL, sonicated 20 minutes filters, the filtrating evaporate to dryness, and residue adds methyl alcohol 0.5mL, makes dissolving, as the medicinal material need testing solution.
The preparation of need testing solution: get different lot numbers and transfer the red powder of stomach (crossing 80 mesh sieves) 5g, add methyl alcohol 15mL, sonicated 20 minutes filters, as need testing solution.
The preparation of reference substance solution: get the synephrine reference substance, add methyl alcohol and process the solution that every 1mL contains 0.5mg, as reference substance solution.
The preparation of negative solution: except that the dried immature fruit of citron orange,,, get 5g,, be prepared as negative solution according to the operation down of need testing solution preparation method item according to the operation of test sample preparation process by all medicines of prescription preparation.
Differential method: according to thin-layered chromatography (first appendix VIB of Chinese Pharmacopoeia version in 2005) test, draw each 2～5 μ L of above-mentioned solution, put respectively on same silica gel H thin layer plate; Upper solution with normal butyl alcohol-glacial acetic acid-water=4: 1: 5 is a developping agent; Launch, take out, dry; Spray is with 0.5% ethanol solution of ninhydrin, and it is clear to be heated to the spot colour developing at 105 ℃.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.Then there is not this spot in the negative control chromatogram.Explain that this method is exclusive, reliable.(result sees Fig. 5, Fig. 6)
2.3 transfer the qualitative identification of the banksia rose in the stomach pellet: the preparation of medicinal material solution: get banksia rose medicinal powder 0.5g, add methenyl choloride 10mL, sonicated 30 minutes filters, and filtrating is as the medicinal material need testing solution.
The preparation of need testing solution: red each 5g of accent stomach that gets different lot numbers pulverizes, and adds methenyl choloride 10mL, and sonicated 30 minutes filters, and filtrating is as need testing solution.
The preparation of reference substance solution: it is an amount of to remove hydrogen constuslactone reference substance, adds methenyl choloride and processes the solution that every 1mL contains 0.5mg, as reference substance solution.
The preparation of negative solution: except that the banksia rose,,, get 5g,, be prepared as negative solution according to the operation down of need testing solution preparation method item according to the operation of test sample preparation process by all medicines of prescription preparation.
Differential method: the photograph thin-layered chromatography (" appendix VIB of Chinese pharmacopoeia version in 2005) test, draw each 4～6 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With cyclohexane-acetone=10: 2 was developping agent; Launch, take out, dry; Spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to the spot colour developing.In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color.Then there is not this spot in the negative control chromatogram.Explain that this method is exclusive, reliable.(result sees Fig. 7)
2.4 transfer the qualitative identification of Radix Glycyrrhizae in the stomach pellet
The preparation of need testing solution: red each 5g of accent stomach that gets different lot numbers pulverizes, the 40mL that adds diethyl ether, and reflux 20 minutes filters, and filtrating discards; The dregs of a decoction volatilize ether, add methyl alcohol 30mL, and reflux 1 hour filters, the filtrating evaporate to dryness; Residue adds water 40mL makes dissolving, extracts 3 times with water saturated normal butyl alcohol, and each 20mL merges normal butyl alcohol liquid, water 20mL washing; Discard water liquid, normal butyl alcohol liquid evaporate to dryness, residue add methyl alcohol 2mL makes dissolving, as need testing solution.
The preparation of control medicinal material solution: extracting liquorice control medicinal material 1g, according to the operation down of need testing solution preparation method item, process control medicinal material solution.
The preparation of negative solution: except that Radix Glycyrrhizae,,, get 5g,, process negative solution according to the operation down of need testing solution preparation method item according to the operation of test sample preparation process by all medicines of prescription preparation.
Differential method: the photograph thin-layered chromatography (" appendix VIB of Chinese pharmacopoeia version in 2005) test, draw each 2～4 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With methenyl choloride-methanol-water=40: 10: 1 was developping agent; Launch, take out, dry; Spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the spot colour developing.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.Then there is not this spot in the negative control chromatogram.(result sees Fig. 8) assay:
One, instrument and reagent:
1. instrument condition
Instrument: high performance liquid chromatograph: Tianjin, island LC-2010AHT detecting device: UV/VIS; Detect wavelength: 294nm.
2. chromatographic condition:
Chromatographic column:
C185um 4.6 * 150mm Column; Moving phase: methanol-water (78: 22); Flow velocity: 0.8mL/min; Column temperature: 25 ℃; Sample size: 5 μ L
3. reagent and reagent
Reference substance: magnolol (lot number: 110729-200309), honokiol (lot number: 110730-200206) provide by the biological calibrating institute that makes of Chinese medicine;
Reagent: methyl alcohol (chromatographically pure)
Test sample: transfer the red (lot number: 060301,060302,060303) provide of stomach by Lerentang Traditional Chinese Medicine Factory, Tianjin.
Two, the investigation of experimental technique
1. the investigation of extraction conditions
1.1 the selection of extraction solvent: this experiment is to 30%, 50%, 70%, 100% methyl alcohol;
1.2 the selection of method for distilling: hot reflux twice 45 minutes/inferior, dipping 24 hours, ultrasonic.
1.3 the preparation of reference substance solution: precision takes by weighing the magnolol reference substance, the honokiol reference substance is an amount of, adds methyl alcohol and processes the solution that every 1mL contains magnolol 135.68 μ g, honokiol 44.896 μ g respectively, promptly gets.
1.4 the preparation of need testing solution: get transfer stomach red (lot number: 060301) powder (crossing 80 mesh sieves), about 1.0 grams, accurate claim fixed; Put in the tool plug conical flask, the accurate methyl alcohol 25mL that adds above-mentioned four kinds of concentration shakes up respectively; Close plug; Weigh respectively and to handle, mend heavy back and filter (hot reflux handle after, the filtration constant volume is 50mL) as need testing solution with above-mentioned three kinds of methods.The result sees table 1,2,3:
Table 1 hot reflux method is investigated the result
Table 2 dipping method is investigated the result
2. the selection of condition determination
2.1 the selection of wavelength: select 294 down for detecting wavelength according to first the 176th page of bark of official magnolia assay item of Chinese Pharmacopoeia version in 2005.
The investigation result of table 3 ultrasonic method
Experimental result shows the ultrasonic optimum extracting method that is, 100% methyl alcohol is the optimum extraction solvent.
2.2 the selection of moving phase
Selecting moving phase down according to first the 176th page of bark of official magnolia assay item of Chinese Pharmacopoeia version in 2005 is methanol-water (78: 22)
3. blank assay
Flavour of a drug by sample prescription takes by weighing except that the bark of official magnolia are an amount of, and the preparation method processes blank by test sample, use the chromatographic condition identical with sample to measure, the while working sample, and the result shows that negative sample goes out the place, peak at magnolol and honokiol and do not have the peak.See accompanying drawing
4. linear relationship is investigated
The preparation of magnolol and honokiol reference substance solution: precision takes by weighing magnolol and the honokiol reference substance is an amount of, processes the reference substance solution that every 1mL contains magnolol 135.68 μ g, honokiol 44.896 μ g with methyl alcohol.Draw above-mentioned solution 1.0,2.0,4.0,5.0,6.0,8.0,10ul, peak area is measured in sample introduction analysis successively, and the result sees table 4-5:
Table 4 magnolol linear relationship is investigated the result
With the sample size is horizontal ordinate, and peak area is an ordinate, the drawing standard curve, and Figure 14 magnolol is linear as follows.Obtain regression equation through statistical calculations: Y=1.396 * 10
7X-3.293 * 10
4R=0.9998
Table 5 honokiol linear relationship is investigated the result
With the sample size is horizontal ordinate, and peak area is an ordinate, the drawing standard curve, and Figure 15 honokiol is linear as follows.Obtain regression equation through statistical calculations: Y=2.191 * 10
6X-8.636 * 10
3R=0.9998
5. precision test
The sample of the same lot number of accurate absorption (lot number: 060301) 5 μ L, repeat sample introduction 6 times, measure the peak area integrated value by above-mentioned chromatographic condition, calculating relative standard deviation is 0.95%, 0.92%.The result sees table 6:
Table 6 precision experimental result
6. reappearance test
Get same lot number sample (lot number: 060301) powder (cross 80 mesh sieves), take by weighing 6 parts, every part of about 0.5g, the content assaying method by drafting extracts, and analyzes, and records the peak area integrated value, the calculating relative standard deviation is 0.71%, 1.13%.The result sees table 7:
Table 7 reappearance experimental result
7. recovery test
Take by weighing sample (transferring stomach red 060301) the about 0.25g of powder, 6 parts, the accurate title, decide; Put in the 50mL tool plug triangular flask, (2mL is 0.848mg/mL) with honokiol (1mL to add the magnolol reference substance respectively; 0.5612mg/mL); Press the test sample preparation and measure an operation down, calculate recovery rate, the result sees table 8-9:
8. stability experiment
With the need testing solution sample introduction analysis for preparing, every at a distance from 1 hour sample introduction, measure the peak area integrated value by above-mentioned chromatographic condition, calculating relative standard deviation is 1.43%, 1.40%.Show that sample is basicly stable in 8h.The result sees table 10:
9. different lot numbers are transferred magnolol and honokiol assay in the stomach pellet:
9.1 the preparation precision of reference substance solution takes by weighing the magnolol reference substance, the honokiol reference substance is an amount of, adds methyl alcohol and processes the solution that every 1mL contains magnolol 135.68ug, honokiol 44.896ug respectively, promptly gets.
Table 8 honokiol recovery experimental result
Table 9 magnolol recovery experimental result
Table 10 stability experiment result
9.2 the preparation of need testing solution is got little of the about 0.5g of these article of lot number powder, the accurate title, decide, and to 50mL tool plug triangular flask, adds methyl alcohol (100%) 25mL; Weigh, sonicated 30 minutes is taken out, and is placed to room temperature; Weigh again, mend heavy, promptly get after miillpore filter filters.
According to drafting method, draw each 5 μ L sample introduction of reference substance solution and need testing solution, measure.Measured 3 batches altogether and transferred magnolol and honokiol content in the stomach pellet, the result sees table 11:
Magnolol and honokiol assay result in table 11 sample
Magnolol and honokiol assay result in table 11 sample
Limit: the every gram of these article contains the bark of official magnolia with magnolol (C
18H
18O
2) and honokiol (C
18H
18O
2) the total amount meter, must not be less than 1.6mg.
Claims (1)
1. a Chinese medicine preparation is transferred the red detection method of stomach, and wherein said pharmaceutical formulation is by Chinese crude drug:
Method for making; More than 12 the flavor, be ground into fine powder, sieve, mixing, the water pill, polishing, drying promptly gets, and it is characterized in that: the step of its method is:
(1) microscopic features with betel nut, the dried immature fruit of citron orange, the bark of official magnolia, Chinese cassia tree are contrast; The method of betel nut in the red prescription of microscope discriminating accent stomach, the dried immature fruit of citron orange, the bark of official magnolia, Chinese cassia tree is: get these article; Putting microscopically observes: thin ancient piece of jade, round, flat and with a hole in its centre cell contains prism of calcium oxalate around the fibrous bundle, forms crystal fiber; The endosperm cell fragment is colourless, and wall is thicker, and more great similar round pit is arranged; Prism of calcium oxalate is present in the Bao Bi tissue in flakes; Lithocyte divides dendritic, and behind the wall, laminated striation is obvious; Square or the similar round of lithocyte class, wall are simultaneously poor;
(2) be reference substance with synephrine, dehydro-, Radix Glycyrrhizae, thin-layered chromatography is differentiated synephrine, dehydro-, the licorice ingredient of transferring in the red prescription of stomach;
Differentiate and transfer the method for synephrine composition in the red prescription of stomach to be: get these article 5g, porphyrize adds methyl alcohol 15ml, and sonicated 20 minutes filters, the filtrating evaporate to dryness, and residue adds methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the synephrine reference substance, adds methyl alcohol and processes the solution that every 1ml contains 0.5mg, as reference substance solution; According to the thin-layered chromatography test, draw each 2～5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With normal butyl alcohol-glacial acetic acid-water is developping agent, with normal butyl alcohol: glacial acetic acid: the upper solution of water=4: 1: 5, launch; Take out; Dry, spray is with 0.5% ethanol solution of ninhydrin, and it is clear to be heated to spot colour developing; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color;
Differentiate and transfer the method for dehydro-composition in the red prescription of stomach to be: get these article 5g, porphyrize adds methenyl choloride 10ml, and sonicated 30 minutes filters, and filtrating is as need testing solution; Other removes hydrogen constuslactone reference substance, adds methenyl choloride and processes the solution that every 1ml contains 0.5mg, as reference substance solution; According to the thin-layered chromatography test, draw each 4～6 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be developping agent with cyclohexane-acetone; With cyclohexane: acetone=10: 2, launch, take out; Dry, spray is with 5% vanillic aldehyde sulfuric acid solution, and it is clear to be heated to spot colour developing; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on, show the spot of same color; Thin-layered chromatography is differentiated and is transferred the method for licorice ingredient in the red prescription of stomach to be: get these article 5g, and porphyrize, the 40ml that adds diethyl ether, reflux 20 minutes filters; Discard filtrating, the dregs of a decoction volatilize ether, add methyl alcohol 30ml, and reflux 1 hour filters; The filtrating evaporate to dryness, residue adds water 40ml makes dissolving, extracts 3 times with water saturated normal butyl alcohol, and each 20ml merges normal butyl alcohol liquid; Water 20ml washing discards water liquid, and normal butyl alcohol liquid evaporate to dryness, residue add methyl alcohol 2ml makes dissolving, as need testing solution; Extracting liquorice control medicinal material 1g in addition, the 10ml that adds diethyl ether, reflux 20 minutes filters, and discards filtrating; The dregs of a decoction volatilize ether, add methyl alcohol 10ml, and reflux 1 hour filters, the filtrating evaporate to dryness; Residue adds water 10ml makes dissolving, extracts 3 times with water saturated normal butyl alcohol, and each 10ml merges normal butyl alcohol liquid; Water 10ml washing discards water liquid, and normal butyl alcohol liquid evaporate to dryness shines medicinal material solution in pairs with legal system; According to the thin-layered chromatography test, draw each 2～4 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate; With methenyl choloride-methanol-water is developping agent, methenyl choloride: methyl alcohol: water=40: 10: 1, launch; Take out; Dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to spot colour developing; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color;
(3) be reference substance with magnolol, honokiol, high performance liquid chromatography detects transfers the method for the content of the bark of official magnolia in the red prescription of stomach to be:
Chromatographic condition and system suitability test: chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filling agent; Methyl alcohol: water=78: 22 is moving phase; The detection wavelength is 294nm; Number of theoretical plate calculates by the magnolol peak should be not less than 3800;
The preparation of reference substance solution: get the magnolol reference substance, the honokiol reference substance is an amount of, accurately claim surely, add methyl alcohol and process the solution that every 1ml contains magnolol 130 μ g, honokiol 45 μ g, promptly get;
The preparation of need testing solution: it is an amount of to get these article, is ground into fine powder, gets powder 0.5g, and accurate the title decides, the accurate methyl alcohol 25ml that adds; Claim decide weight, sonicated 30 minutes is put coldly, and weight decided in title again, supplies the weight that subtracts mistake with methyl alcohol; Shake up, filter, get subsequent filtrate, promptly get;
Determination method: draw each 5 μ l of above-mentioned reference substance solution and need testing solution respectively, inject liquid chromatograph, measure, promptly get.
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| CN106404939A (en) * | 2016-08-30 | 2017-02-15 | 天津中新药业集团股份有限公司乐仁堂制药厂 | Content determination method of stomach conditioning pill |
| CN107202856B (en) * | 2017-05-23 | 2018-07-31 | 四川逢春制药有限公司 | A kind of detection method of Chinese medicine preparation that treating flu |
| CN115144251A (en) * | 2021-03-30 | 2022-10-04 | 山东新时代药业有限公司 | Method for processing biological sample |
| CN114487251B (en) * | 2021-12-02 | 2023-11-03 | 津药达仁堂集团股份有限公司隆顺榕制药厂 | Thin-layer chromatography detection method for agilawood Shu Yupian |
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| 封士兰等.中药质量标准研究新进展.《兰州医学院学报》.2002,第28卷(第04期), * |
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Address after: 300380 Yumen Road, Xiqing District, Tianjin Patentee after: Lerentang Pharmaceutical Factory of Jinyao Darentang Group Co.,Ltd. Address before: 300380 Yumen Road, Xiqing District, Tianjin Patentee before: Letrentang pharmaceutical factory of Tianjin Zhongxin Pharmaceutical Group Co.,Ltd. |