CN110954645B - Detection method of high-quality Sihuang dysentery stopping granules - Google Patents
Detection method of high-quality Sihuang dysentery stopping granules Download PDFInfo
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Abstract
The invention discloses a detection method of high-quality Sihuang dysentery stopping granules, which is characterized in that a thin-layer chromatography is used for identifying coptis chinensis, golden cypress, rhubarb, scutellaria baicalensis, isatis root and liquorice in a preparation, and the content of baicalin in the preparation is determined by adopting HPLC. The content of the baicalin is obviously improved after the quality process is changed, and the detection method provided by the invention is combined, so that the detection result is stable and reliable, the specificity is strong, the reproducibility is good, the quality of the Sihuang dysentery stopping granules can be comprehensively and effectively controlled, the product quality can be stabilized in the market, the safety and the effectiveness of veterinary drugs can be ensured, and the market requirements can be better met.
Description
Technical Field
The invention belongs to the technical field of veterinary drug detection, and relates to a detection method for the quality of high-quality Sihuang dysentery stopping granules.
Background
The Sihuang Zhizhui granule is prepared by extracting and processing 6 traditional Chinese medicines of coptis chinensis, phellodendron, rhubarb, astragalus, isatis root and liquorice into Sihuang Zhizhui extract, and mixing the extract with cane sugar and dextrin according to a certain proportion, has the effects of clearing heat, purging intense heat and stopping dysentery, and is mainly used for treating damp-heat dysentery and chicken colibacillosis clinically, such as damp-heat dysentery, white dysentery, yellow-white dysentery, watery dysentery and the like caused by escherichia coli, pasteurellosis and salmonella infection of chicken and secondary infection of viral diseases; diarrhea caused by pig escherichia coli and salmonella, yellow and white dysentery, transmissible gastroenteritis, epidemic diarrhea, rotavirus diarrhea and other viral diseases, and severe jet-shaped watery diarrhea and the like. It is recorded in P587 of 2010 version 2 of the veterinary dictionary of the people's republic of china due to its definite therapeutic effect and good public praise.
The quality control of the Sihuang dysentery stopping granules only has a second standard of ' pharmacopoeia of the people's republic of China ' 2010 edition, wherein the records are as follows: the prescription of the Sihuang Zhili granule is as follows: 200g of coptis root, 200g of phellodendron bark, 100g of rhubarb, 200g of scutellaria root, 200g of isatis root and 100g of liquorice
The preparation method comprises the following steps: decocting the 6 medicines in water for 2 times, the first time lasts for 2 hours, the second time lasts for 1 hour, combining the decoctions, filtering, concentrating the filtrate to thick paste with the relative density of 1.32-1.35, adding a proper amount of sucrose and dextrin, granulating, and drying to prepare 1000 g.
The final product was a yellow to tan colored granule.
The identification method comprises the following steps: (1) taking 10g of the product, adding 2.5g of diatomite, uniformly grinding, adding 50mL of methanol, placing the mixture on a water bath for refluxing for 1 hour, cooling the mixture, filtering the mixture, and concentrating the filtrate to 5mL to be used as a sample solution to be detected. Taking 2g of rhizoma Coptidis as reference material, adding 20mL of methanol, and making into reference material solution by the same method. Taking berberine hydrochloride reference substance, adding methanol to make into 1mg solution per 1mL, and using as reference substance solution. Testing by thin layer chromatography (appendix 32 page), sucking 5 μ l of each of the three solutions, dropping on the same silica gel G thin layer plate, placing in a developing tank saturated with ammonia vapor with benzene-ethyl acetate-isopropanol-methanol-concentrated ammonia solution (6:3:1.5:1.5:0.5) as developing agent, presaturating for 30min, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the sample to be detected, fluorescent spots with the same color are displayed at the corresponding positions of the chromatogram of the reference medicinal material; the same yellow fluorescent spot appeared in the corresponding position of the control chromatogram.
(2) Taking 2g of the product, adding 50mL of methanol, carrying out ultrasonic treatment for 20min, filtering, evaporating the filtrate to dryness, adding 10mL of water into the residue to dissolve the residue, adding 1mL of hydrochloric acid, heating on a water bath for 30min, immediately cooling, extracting with diethyl ether twice, extracting 20mL each time, combining the diethyl ether solutions, evaporating the mixed ethyl ether solutions on the water bath to dryness, and adding 1mL of trichloromethane into the residue to dissolve the residue to obtain a sample solution to be detected. 0.1g of rhubarb reference drug is prepared and prepared into reference drug solution in the same way. Performing thin layer chromatography (appendix 32 page), sucking the two solutions to 5 μ l each, dropping on the same silica gel H thin layer plate with sodium carboxymethylcellulose solution as binder, spreading with petroleum ether (30-60 deg.C) -ethyl formate-formic acid (15:5:1) upper solution as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the sample to be detected, five same orange-yellow fluorescent spots are displayed at the positions corresponding to the chromatogram of the reference medicinal material; after fumigating in ammonia vapor, the spots turned red.
There are many patents on "Sihuang Zhili" (for example:
chinese patent 200910066083.3 discloses a preparation method of a Sihuang dysentery stopping effervescent granule, which is prepared from the following raw materials and auxiliary materials in parts by weight: raw materials: 200 parts of coptis chinensis, 200 parts of golden cypress, 100 parts of rheum officinale, 200 parts of scutellaria baicalensis, 200 parts of isatis root and 100 parts of liquorice; auxiliary materials: 108-132 parts of sodium bicarbonate, 90-110 parts of fumaric acid, 549-671 parts of diluent and 18-22 parts of flavoring agent. Meanwhile, the effervescent granules are prepared by adopting a method of separately granulating by acid and alkali. The invention adopts sodium bicarbonate as an alkaline agent, fumaric acid as an acidic agent and other auxiliary materials to prepare the Sihuang dysentery stopping effervescent granule, can reduce the consumption of the auxiliary materials, and has the advantages of simple preparation process, easy forming, difficult moisture absorption, high effervescence speed, good effervescence effect, good dissolvability, good clarity of liquid medicine after effervescence, no coke foreign matter and the like.
Chinese patent 201310739482.8 discloses a method for testing the quality of Sihuang Zhili particles, which relates to a method for testing the quality of Sihuang Zhili particles, and the method is obtained by revising the product quality standard by combining the actual situation on the basis of the original standard of the Sihuang Zhili particles, thereby increasing the identification of scutellaria and simultaneously providing a High Performance Liquid Chromatography (HPLC) determination method of berberine hydrochloride. The method is verified by methodology, so that the inherent quality of the product can be more effectively controlled, and the stability of the product quality is ensured. The method is simple to operate, and can control the types of effective components as much as possible according to the content determination item of the traditional Chinese medicine product, improve the quality level of the product, and increase the market competitiveness of the product.
In the technology, the common water boiling concentration is carried out on the four-yellow dysentery stopping particles, the degradation of effective substances is obvious after the effective substances are high temperature, and in addition, no detection method for comprehensively checking or detecting the quality of the four-yellow dysentery stopping particles exists at present, so that the search for a perfect detection method in the whole formula is very important.
Disclosure of Invention
In view of the defects of the prior art, the invention provides a method for comprehensively detecting the Sihuang dysentery stopping granules, thereby controlling the quality level of veterinary drug products which are on the market or have improved product processes, and effectively having the effects of clearing heat, purging pathogenic fire and stopping dysentery.
Specifically, the invention is realized by the following steps:
a quality detection method for SIHUANG ZHILI KE LI (diarrhea relieving granule) comprises identifying Coptidis rhizoma, cortex Phellodendri, radix et rhizoma Rhei, Scutellariae radix, radix Isatidis and Glycyrrhrizae radix in preparation by thin layer chromatography, and measuring baicalin content in preparation by HPLC. Wherein the Sihuang Zhili particles are prepared by extracting and processing 6 traditional Chinese medicines of coptis chinensis, phellodendron, rhubarb, astragalus, isatis root and liquorice.
Further, the thin-layer chromatography method for identifying coptis chinensis in the four-yellow dysentery stopping granules comprises the following steps:
1) preparing a test solution: taking 10g of the product, adding 2.5g of diatomite, grinding uniformly, adding 50mL of methanol, carrying out ultrasonic extraction for 30min, shaking, filtering, evaporating the filtrate to dryness, adding 20mL of water into the residue to dissolve, extracting with diethyl ether for 2 times, 20mL each time, combining the diethyl ether solutions, volatilizing at low temperature, adding 2mL of methanol into the residue to dissolve to obtain a sample solution;
2) preparation of a reference solution: adding ethanol into berberine hydrochloride reference substance to obtain 1 μ L solution per 1mL as reference substance solution; taking 2g of a coptis root reference medicinal material, adding 20mL of methanol, and preparing a reference medicinal material solution by the same method;
3) spotting and developing: sucking 2-5 μ L of sample solution and 2 μ L of reference solution, respectively dropping on the same silica gel G thin layer plate, adding carbon tetrachloride at 60-90 deg.C: ethyl acetate: isopropyl alcohol: formic acid: water 3: 3:1.5:1.5: 1 is developing agent, developing, taking out, airing, and viewing under a 365nm ultraviolet lamp; the test chromatogram shows fluorescent spots of the same color at the positions corresponding to those of the control chromatogram.
Further, the identification of the phellodendron amurense in the Sihuang dysentery stopping granules by the thin layer chromatography comprises the following steps:
1) preparing a test solution: taking 10g of the product, grinding, adding 50mL of methanol, carrying out ultrasonic extraction for 30min, filtering, evaporating the filtrate to dryness, adding 20mL of water into the residue to dissolve, extracting the extracted water solution with ethyl acetate for 3 times, each time with 20mL, adjusting the pH value of the extracted water solution to 10-11 with ammonia test solution, extracting with 20mL of trichloromethane for 1 time, evaporating the trichloromethane solution to dryness, and adding 2mL of methanol into the residue to dissolve to obtain a test solution;
2) preparation of a reference solution: taking phellodendrine hydrochloride reference substance, adding methanol to prepare solution containing 0.5mg per 1mL as reference substance solution;
3) spotting and developing: sucking the two solutions 1-2 μ L each, and respectively dropping on the same silica gel G thin layer plate with carbon tetrachloride: methanol: ammonia water 7:0.5:0.3 as developing agent, developing, taking out, drying in the air, spraying improved bismuth potassium iodide test solution; spots of the same color appear in the chromatogram of the test solution at positions corresponding to those in the chromatogram of the control solution.
Further, the identification of rhubarb in the Sihuang Zhili granules by the thin-layer chromatography comprises the following steps:
1) preparing a test solution: taking 10g of the product, grinding, adding 50mL of methanol, performing ultrasonic extraction for 30min, filtering, evaporating filtrate to dryness, adding 20mL of methanol into residue to dissolve, adjusting pH to 1-2 with dilute hydrochloric acid, extracting with diethyl ether for 2 times, each time 20mL, extracting the extracted water solution with ethyl acetate for 2 times, each time 20mL, adding 2mL of trichloromethane into residue to dissolve to obtain a sample solution;
2) preparation of a reference solution: adding methanol into rhein reference substance to obtain 1mg solution per 1mL as reference substance solution;
3) spotting and developing: sucking 2-5 μ L of the sample solution and 5 μ L of the reference solution, respectively dropping on the same silica gel G thin layer plate, adding n-hexane: tetrachloromethane: ethyl acetate: water 5: 4.5: 2: developing the lower layer solution of 0.5 as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under 365nm ultraviolet lamp; spots of the same color appear in the chromatogram of the test solution at positions corresponding to those in the chromatogram of the control solution.
Further, the thin-layer chromatography method for identifying the scutellaria baicalensis in the four-yellow dysentery stopping granules comprises the following steps of:
1) preparing a test solution: taking 10g of the product, grinding, adding 50mL of methanol, extracting with 30% ethanol under reflux for 3 times, adding 12 times of the product for the first time, 10 times of the product for the second time, 8 times of the product for the third time, 1.5 hours each time, filtering, mixing, drying the filtrate by distillation, dissolving the residue with 20mL of water, adjusting the pH value to 1-2 with dilute hydrochloric acid, extracting with diethyl ether for 2 times, 20mL each time, drying the chloroform solution by distillation, and dissolving the residue with 2mL of methanol to obtain a sample solution;
2) preparation of a reference solution: taking baicalin, adding methanol into control to obtain 1mg solution per 1mL as control solution;
3) spotting and developing: sucking the two solutions, each 5 mu L of the two solutions, respectively dropping the solutions on the same silica gel G thin layer plate, and adding carbon tetrachloride: n-butanol: formic acid: water 13: 8: 2: 1, developing the lower layer solution as a developing agent, taking out, airing, and respectively inspecting under a 365nm ultraviolet lamp; in the chromatogram of the test solution, spots or fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the reference solution.
Further, the identification of isatis root in the Sihuang dysentery stopping granules by the thin layer chromatography comprises the following steps:
1) preparing a test solution: taking 10g of the product, grinding, adding 50mL of methanol, reflux-extracting with ethyl acetate-methanol (3:1) for 30min, evaporating filtrate to dryness, dissolving residue with 20mL of water, extracting with 20mL of chloroform for 1 time, evaporating chloroform solution to dryness, dissolving residue with 2mL of methanol to obtain sample solution;
2) preparation of a reference solution: adding diluted ethanol into arginine control to obtain solution containing 0.5mg per 1mL as control solution;
3) spotting and developing: sucking the two solutions 1-2 μ L each, and dropping on the same silica gel G thin layer plate respectively, adding n-hexane: n-butanol: methanol: formic acid 8: 2: 1: 1 is developing agent, taking out, drying with hot air, spraying ninhydrin test solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution and the reference solution.
Further, the identification of the liquorice in the Sihuang dysentery stopping granules by the thin-layer chromatography comprises the following steps:
1) preparing a test solution: taking 10g of the product, grinding, adding 50mL of methanol, performing ultrasonic extraction for 30min, shaking, filtering, evaporating filtrate, dissolving residue with 20mL of water, adjusting pH to 1-2 with dilute hydrochloric acid, extracting with diethyl ether for 2 times (20 mL each time), mixing diethyl ether solutions, volatilizing at low temperature, dissolving residue with 2mL of methanol to obtain sample solution;
2) preparation of a reference solution: the ammonium glycyrrhizinate control solution is prepared by adding methanol to obtain a solution containing 2mg per lm l.
3) Spotting and developing: sucking the two solutions 2-5 μ L each, and dropping on the same silica gel G thin layer plate prepared from 1% sodium hydroxide solution, adding n-butanol: ammonia water: formic acid: water 7: 1: developing with developer 1, taking out, air drying, heating with 10% ethanol sulfate solution at 105 deg.C until color development of spots is clear, and inspecting under 365nm ultraviolet lamp; the main fluorescent spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution.
Further, the high performance liquid chromatography method for determining the content of the baicalin in the Sihuang dysentery stopping granules comprises the following steps:
1) preparing a test solution: taking a proper amount of Sihuang dysentery stopping granules, grinding to obtain 2g of the Sihuang dysentery stopping granules, precisely weighing, placing the ground Sihuang dysentery stopping granules in a conical flask, precisely adding ethyl acetate: weighing 50mL of methanol solution 1:50, carrying out ultrasonic treatment for 30min, cooling, weighing, complementing the weight loss amount with the methanol solution, shaking up, filtering, precisely absorbing 3mL of subsequent filtrate, adding the subsequent filtrate on a 200-mesh neutral alumina column, eluting with 50mL of methanol, collecting the eluent, evaporating by distillation in a water bath, dissolving the residue with methanol, and standing in a 10mL volumetric flask, shaking up and filtering to obtain the product;
2) preparation of a reference solution: drying under reduced pressure at 60 deg.C for 4 hr to obtain baicalin reference substance, adding methanol to obtain solution containing 0.06mg per 1mL, and shaking;
3) HPLC chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler; gradient elution is carried out by taking acetonitrile (A) -0.1 percent phosphoric acid solution (B) as a mobile phase; the detection wavelength is 280nm, and the number of theoretical plates is not less than 2500 calculated according to baicalin peak;
4) the determination method comprises the following steps: respectively and precisely sucking 10uL of the reference solution and the test solution, injecting into a high performance liquid chromatograph, and measuring according to the chromatographic conditions in the step 3).
Wherein, the gradient elution procedure in the step 3) of the high performance liquid phase method is as follows:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0-10 | 15 | 85 |
| 10-20 | 28 | 72 |
| 20-30 | 28 | 72 |
The second objective of the present invention is to provide a process for increasing the content of active ingredients in the four-yellow dysentery stopping granules, wherein the four-yellow dysentery stopping granules only contain six traditional Chinese medicine components, but the active ingredients are relatively complex, and the traditional method, especially the high temperature decoction, causes part of the active ingredients to denature and lose the effect. Therefore, the Chinese medicinal materials such as the coptis root and the liquorice powder are extracted at low temperature, the high-temperature damage is avoided, and the high-quality Sihuang dysentery stopping granules are finally obtained. The preparation process of the Sihuang dysentery stopping granules comprises the following steps:
(1) preparing the coptis chinensis and liquorice remote paste:
(2) preparing an isatis root volatile oil inclusion compound, and distilling isatis root to obtain a decoction residue I aqueous solution for later use;
(3) mixing the decoction of Scutellariae radix, cortex Phellodendri and radix et rhizoma Rhei with the water solution of residue I obtained after distillation, concentrating, drying, mixing with pharmaceutically acceptable adjuvants, drying, granulating, and sieving to obtain Chinese medicinal granule.
Further, the preparation process of the coptis chinensis and liquorice sweet paste comprises the following steps:
(1) crushing the coptis and the liquorice into particles or powder of 20-80 meshes;
(2) adding 10-60 times of water into the granules or powder of the coptis chinensis and the liquorice in the step (1), adjusting the pH value to 2.0-3.5, soaking for 3-6 hours at 20-60 ℃, and filtering to obtain filtrate;
(3) concentrating and drying the filtrate obtained in the step (2) to obtain the coptis chinensis and liquorice cleaning paste.
The preparation process of the isatis root volatile oil inclusion compound comprises the following steps: soaking radix Isatidis in water for 1-4 hr, distilling for 6-8 hr, collecting volatile oil, and collecting residue and water solution after distillation; obtaining isatis root volatile oil, adding beta-cyclodextrin to prepare an inclusion compound; and distilling to obtain a decoction residue I aqueous solution for later use.
The process of the step (3) is as follows: decocting the residue I obtained after distillation with Scutellariae radix, cortex Phellodendri and radix et rhizoma Rhei in water for 1-3 times, each for 1-2 hr, mixing the decoction with the residue I water solution, filtering, concentrating the filtrate to obtain fluid extract with relative density of 1.08-1.20(80 deg.C), adding ethanol to make ethanol content reach 45%, standing for 24 hr, filtering, recovering ethanol under reduced pressure, concentrating to obtain soft extract with relative density of 1.28-1.33(80 deg.C), mixing with the above extracts, adding sucrose, drying, and sieving with 20 mesh sieve to obtain the final product.
On the basis of the quality detection method of the Sihuang dysentery stopping granules, the new quality detection method of the Sihuang dysentery stopping granules is established through optimization, upgrading and reconstruction. The method adopts thin-layer chromatography to qualitatively identify Coptidis rhizoma, cortex Phellodendri, radix et rhizoma Rhei, Scutellariae radix, radix Isatidis and Glycyrrhrizae radix in the granule respectively, so as to achieve multi-component combined control, and simultaneously uses HPLC to quantitatively determine baicalin content of Scutellariae radix in the preparation, so as to further effectively control quality of the granule for treating dysentery, and realize comprehensive evaluation of the quality of the preparation, thereby ensuring stability of product quality and safety and effectiveness of veterinary drugs to the utmost extent.
The detection method adopted by the invention is scientific and reasonable, has controllable conditions, strong specificity and high stability, and has strong practicability; the application of the quality detection method can ensure the clinical curative effect of the Sihuang dysentery stopping granules and better meet the requirements of livestock raising and breeding markets.
The method optimizes and upgrades the identification of the original detection method of the four-yellow dysentery stopping granules, reduces the use of harmful reagents, increases the identification of the whole-class TLC detection and improves the accuracy. Compared with the original quality detection method, the isatis root volatile oil is included by cyclodextrin, so that the quality is more stable, the isatis root volatile oil is more suitable for storage, but the extraction efficiency in the detection process is not high.
Drawings
FIG. 1 is a high performance liquid chromatogram of baicalin in Scutellariae radix, which is a baicalin control chromatogram, a negative control chromatogram, and a chromatogram of a test sample from top to bottom.
FIG. 2 is a thin-layer chromatogram of Coptidis rhizoma in the granule for treating dysentery, wherein 1, 4 are berberine reference substances, 2 are test substances, and 3 are berberine-deficient negative preparation samples, in order from left to right.
FIG. 3 is a thin layer chromatogram of radix Isatidis in Sihuang ZHILI granule, in which 1, 4 are radix Isatidis control, 2 is radix Isatidis-deficient negative preparation sample, and 3 is sample, in order from left to right.
FIG. 4 is a thin-layer chromatogram of radix et rhizoma Rhei in the granule for treating dysentery, wherein 1 and 4 are radix et rhizoma Rhei control materials, 3 is a sample of negative preparation lacking radix et rhizoma Rhei, and 2 is a sample, in order from left to right.
FIG. 5 is a thin layer chromatogram of Scutellariae radix in the granule for treating dysentery, wherein 1 is a baicalin-deficient negative preparation sample, 3 is a sample, and 2 and 4 are baicalin control, in order from left to right.
FIG. 6 is a chromatogram of Phellodendri cortex in the granule for treating dysentery, wherein 1, 4 are phellodendrine control, 2 is sample, and 3 is sample of negative preparation without Phellodendri cortex.
FIG. 7 is a thin layer chromatogram of radix Glycyrrhizae in the granule for treating dysentery, wherein 1 and 4 are glycyrrhizic acid control materials, 2 is sample, and 3 is negative preparation sample without radix Glycyrrhizae.
Detailed Description
Example 1 HPLC methodological study of baicalin in Scutellaria baicalensis Georgi
1) Instruments and reagents: agilent 1100 hplc, VWD detector. Acetonitrile is chromatographically pure, the other reagents are analytically pure, and water is double distilled water.
2) Chromatographic conditions are as follows: kromasil C18Chromatographic column (4.6mm × 250mm, 5 μm), mobile phase acetonitrile (A) -0.1% phosphoric acid solution (B) detection wavelength is 346nm, flow rate is 1 mL/min.
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0-10 | 15 | 85 |
| 10-20 | 28 | 72 |
| 20-30 | 28 | 72 |
3) Preparing a test solution: preparing a test solution: taking a proper amount of Sihuang dysentery stopping granules, grinding to obtain 2g of the Sihuang dysentery stopping granules, precisely weighing, placing the ground Sihuang dysentery stopping granules in a conical flask, precisely adding ethyl acetate: weighing methanol at a ratio of 1:50 in 50mL, carrying out ultrasonic treatment for 30min, cooling, weighing, complementing the weight loss by using a methanol solution, shaking uniformly, filtering, precisely absorbing 3mL of subsequent filtrate, adding the subsequent filtrate on a 200-mesh neutral alumina column, eluting by using 50mL of methanol, collecting eluent, evaporating by using a water bath, dissolving the residue by using methanol, and carrying out volume determination in a 10mL volumetric flask, shaking uniformly, and filtering to obtain the product.
4) Preparation of a reference solution: drying under reduced pressure at 60 deg.C for 4 hr to obtain baicalin control, adding methanol to obtain solution containing 0.06mg per 1mL, and shaking.
5) Negative control solution preparation: taking the negative control containing no Scutellariae radix, and performing the same method under the test solution preparation to obtain negative control solution, wherein the chromatogram shows that no interference exists at baicalin position. See fig. 1.
6) And (3) linear relation investigation: an appropriate amount of baicalin control was precisely weighed, and added with methanol to prepare 0.0529mg/mL solution, and the solution was subjected to measurement under the above chromatographic conditions, and 0.4. mu.L, 0.6. mu.L, 0.8. mu.L, 1. mu.L, 1.2. mu.L, and 1.5. mu.L were injected, respectively, and the peak areas were measured, as shown in Table 1. And drawing a standard curve by taking the measured peak area as an ordinate and the sample amount (mu g) of a reference substance as an abscissa, wherein the regression equation y is 5024.8x-0.69, R2 is 0.9998, and the baicalin sample amount is good in linearity within the range of 0.02115-0.0794 mu g.
TABLE 1 results of linear relationship examination
7) And (3) precision test: the control solution was precisely aspirated, and the sample injection was repeated 6 times, 5 μ L each time, with a peak area RSD of 0.45%, indicating good instrument precision. The results are shown in Table 2.
TABLE 2 results of precision test
8) And (3) stability test: and taking the same sample solution, and measuring for 0h, 2h, 4h, 6h and 8h after preparation respectively, wherein the result shows that the sample is stable within 8 h. The results are shown in Table 3.
TABLE 3 stability test results
9) And (3) repeatability test: 6 parts of samples of the same batch are taken respectively, the operation is carried out according to the sample preparation method under the item of sample determination, the content is determined respectively, and the RSD is 0.96 percent. The results are shown in Table 4.
TABLE 4 results of the repeatability tests
10) Recovery rate test: precisely weighing 6 parts of sample with known content, respectively adding appropriate amount of reference substance, and measuring with the preparation method under the item of "preparation of test solution" to obtain baicalin with average recovery rate of 0.594% and RSD. The results are shown in Table 5
TABLE 5 recovery test results
Example 2: the thin-layer chromatography method for identifying coptis chinensis in the four-yellow dysentery stopping granules comprises the following steps:
1) preparing a test solution: taking 10g of the product, adding 2.5g of diatomite, grinding uniformly, adding 50mL of methanol, carrying out ultrasonic extraction for 30min, shaking, filtering, evaporating the filtrate to dryness, adding 20mL of water into the residue to dissolve, extracting with diethyl ether for 2 times, 20mL each time, combining the diethyl ether solutions, volatilizing at low temperature, adding 2mL of methanol into the residue to dissolve to obtain a sample solution;
2) preparation of a reference solution: adding ethanol into berberine hydrochloride reference substance to obtain 1 μ L solution per 1mL as reference substance solution; taking 2g of a coptis root reference medicinal material, adding 20mL of methanol, and preparing a reference medicinal material solution by the same method;
3) spotting and developing: sucking 2-5 μ L of sample solution and 2 μ L of reference solution, respectively dropping on the same silica gel G thin layer plate, adding carbon tetrachloride at 60-90 deg.C: ethyl acetate: isopropyl alcohol: formic acid: water 3: 3:1.5:1.5: 1 is developing agent, developing, taking out, airing, and viewing under a 365nm ultraviolet lamp; the test chromatogram shows fluorescent spots of the same color at the positions corresponding to those of the control chromatogram.
Example 3: the identification of phellodendron amurense in the four-yellow dysentery stopping granules by the thin layer chromatography comprises the following steps:
1) preparing a test solution: taking 10g of the product, grinding, adding 50mL of methanol, carrying out ultrasonic extraction for 30min, filtering, evaporating the filtrate to dryness, adding 20mL of water into the residue to dissolve, extracting the extracted water solution with ethyl acetate for 3 times, each time with 20mL, adjusting the pH value of the extracted water solution to 10-11 with ammonia test solution, extracting with 20mL of trichloromethane for 1 time, evaporating the trichloromethane solution to dryness, and adding 2mL of methanol into the residue to dissolve to obtain a test solution;
2) preparation of a reference solution: taking phellodendrine hydrochloride reference substance, adding methanol to prepare solution containing 0.5mg per 1mL as reference substance solution;
3) spotting and developing: sucking the two solutions 1-2 μ L each, and respectively dropping on the same silica gel G thin layer plate with carbon tetrachloride: methanol: ammonia water 7:0.5:0.3 as developing agent, developing, taking out, drying in the air, spraying improved bismuth potassium iodide test solution; spots of the same color appear in the chromatogram of the test solution at positions corresponding to those in the chromatogram of the control solution.
Example 4: the identification of rhubarb in the Sihuang dysentery stopping granules by the thin layer chromatography comprises the following steps:
1) preparing a test solution: taking 10g of the product, grinding, adding 50mL of methanol, performing ultrasonic extraction for 30min, filtering, evaporating filtrate to dryness, adding 20mL of methanol into residue to dissolve, adjusting pH to 1-2 with dilute hydrochloric acid, extracting with diethyl ether for 2 times, each time 20mL, extracting the extracted water solution with ethyl acetate for 2 times, each time 20mL, adding 2mL of trichloromethane into residue to dissolve to obtain a sample solution;
2) preparation of a reference solution: adding methanol into rhein reference substance to obtain 1mg solution per 1mL as reference substance solution;
3) spotting and developing: sucking 2-5 μ L of the sample solution and 5 μ L of the reference solution, respectively dropping on the same silica gel G thin layer plate, adding n-hexane: tetrachloromethane: ethyl acetate: water 5: 4.5: 2: developing the lower layer solution of 0.5 as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under 365nm ultraviolet lamp; spots of the same color appear in the chromatogram of the test solution at positions corresponding to those in the chromatogram of the control solution.
Example 5: the thin-layer chromatography for identifying the scutellaria baicalensis in the four-yellow dysentery stopping granules comprises the following steps:
1) preparing a test solution: taking 10g of the product, grinding, adding 50mL of methanol, extracting with 30% ethanol under reflux for 3 times, adding 12 times of the product for the first time, 10 times of the product for the second time, 8 times of the product for the third time, 1.5 hours each time, filtering, mixing, drying the filtrate by distillation, dissolving the residue with 20mL of water, adjusting the pH value to 1-2 with dilute hydrochloric acid, extracting with diethyl ether for 2 times, 20mL each time, drying the chloroform solution by distillation, and dissolving the residue with 2mL of methanol to obtain a sample solution;
2) preparation of a reference solution: taking baicalin, adding methanol into control to obtain 1mg solution per 1mL as control solution;
3) spotting and developing: sucking the two solutions, each 5 mu L of the two solutions, respectively dropping the solutions on the same silica gel G thin layer plate, and adding carbon tetrachloride: n-butanol: formic acid: water 13: 8: 2: 1, developing the lower layer solution as a developing agent, taking out, airing, and respectively inspecting under a 365nm ultraviolet lamp; in the chromatogram of the test solution, spots or fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the reference solution.
Example 6: the thin-layer chromatography method for identifying the isatis root in the Sihuang dysentery stopping granules comprises the following steps:
1) preparing a test solution: taking 10g of the product, grinding, adding 50mL of methanol, reflux-extracting with ethyl acetate-methanol (3:1) for 30min, evaporating filtrate to dryness, dissolving residue with 20mL of water, extracting with 20mL of chloroform for 1 time, evaporating chloroform solution to dryness, dissolving residue with 2mL of methanol to obtain sample solution;
2) preparation of a reference solution: adding diluted ethanol into arginine control to obtain solution containing 0.5mg per 1mL as control solution;
3) spotting and developing: sucking the two solutions 1-2 μ L each, and dropping on the same silica gel G thin layer plate respectively, adding n-hexane: n-butanol: methanol: formic acid 8: 2: 1: 1 is developing agent, taking out, drying with hot air, spraying ninhydrin test solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution and the reference solution.
Example 7: the identification of the liquorice in the Sihuang dysentery stopping granules by the thin-layer chromatography comprises the following steps:
1) preparing a test solution: taking 10g of the product, grinding, adding 50mL of methanol, performing ultrasonic extraction for 30min, shaking, filtering, evaporating filtrate, dissolving residue with 20mL of water, adjusting pH to 1-2 with dilute hydrochloric acid, extracting with diethyl ether for 2 times (20 mL each time), mixing diethyl ether solutions, volatilizing at low temperature, dissolving residue with 2mL of methanol to obtain sample solution;
2) preparation of a reference solution: the ammonium glycyrrhizinate control solution is prepared by adding methanol to obtain a solution containing 2mg per lm l.
3) Spotting and developing: sucking the two solutions 2-5 μ L each, and dropping on the same silica gel G thin layer plate prepared from 1% sodium hydroxide solution, adding n-butanol: ammonia water: formic acid: water 7: 1: developing with developer 1, taking out, air drying, heating with 10% ethanol sulfate solution at 105 deg.C until color development of spots is clear, and inspecting under 365nm ultraviolet lamp; the main fluorescent spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution.
Example 8: a Sihuang Zhili granule for treating dysentery comprises the following components by weight percent:
200g of coptis root, 200g of phellodendron bark, 100g of rhubarb, 200g of scutellaria root, 200g of isatis root and 100g of liquorice
The preparation process comprises the following steps:
(1) crushing the coptis and the liquorice into particles or powder of 20-80 meshes;
(2) adding 10-60 times of water into the granules or powder of the coptis chinensis and the liquorice in the step (1), adjusting the pH value to 2.0-3.5, soaking for 3-6 hours at 20-60 ℃, and filtering to obtain filtrate;
(3) concentrating and drying the filtrate obtained in the step (2) to obtain the coptis chinensis and liquorice cleaning paste.
(4) Soaking radix Isatidis in water for 1-4 hr, distilling for 6-8 hr, collecting volatile oil, and collecting residue and water solution after distillation; obtaining isatis root volatile oil, adding beta-cyclodextrin to prepare an inclusion compound; and distilling to obtain a decoction residue I aqueous solution for later use.
(5) Decocting the residue I obtained after distillation and the prescribed amount of radix Scutellariae, cortex Phellodendri and radix et rhizoma Rhei in water for 1-3 times, each time for 1-2 hours, mixing the decoction with the residue I water solution, filtering, concentrating the filtrate to obtain fluid extract with relative density of 1.08-1.20(80 deg.C), adding ethanol to make ethanol content reach 45%, standing for 24 hours, filtering, recovering ethanol under reduced pressure, concentrating to obtain soft extract with relative density of 1.28-1.33(80 deg.C), adding sucrose, drying, and sieving with 20 mesh sieve to obtain 1000 g.
By detection and calculation, the product contains baicalin (C) per 1g Scutellariae radix21H18O11) The content was 6.69mg, and the dissolution was 98.5% in 5min and 100.1% in 10min, as determined by the method of example 1 of the present invention.
Example 9: a Sihuang Zhili granule for treating dysentery comprises the following components by weight percent:
200g of coptis chinensis, 200g of golden cypress, 100g of rheum officinale, 200g of scutellaria baicalensis, 200g of isatis root and 100g of liquorice, the 6 medicines are decocted twice with water, the first time is 2 hours, the second time is 1 hour, decoction liquid is combined and filtered, filtrate is concentrated to thick paste with the relative density of 1.32-1.35, a proper amount of cane sugar and dextrin are added, granules are prepared, and the granules are dried to prepare 1000g of the traditional Chinese medicine preparation.
By detection and calculation, the product contains baicalin (C) per 1g Scutellariae radix21H18O11) The content was 5.28mg, and the dissolution was 68.3% in 5min and 90.6% in 10min as determined by the method of example 1.
Example 10: a Sihuang Zhili granule for treating dysentery comprises the following components by weight percent:
200g of coptis root, 200g of phellodendron bark, 100g of rhubarb, 200g of scutellaria root, 200g of isatis root and 100g of liquorice
The preparation process comprises the following steps:
(1) soaking radix Isatidis in water for 1-4 hr, distilling for 6-8 hr, collecting volatile oil, and collecting residue and water solution after distillation; obtaining isatis root volatile oil, adding beta-cyclodextrin to prepare an inclusion compound; and distilling to obtain a decoction residue I aqueous solution for later use.
(2) Decocting the residue I obtained after distillation and the prescribed amount of rhizoma Coptidis, radix Glycyrrhizae, radix Scutellariae, cortex Phellodendri, and radix et rhizoma Rhei in water for 1-3 times, each for 1-2 hours, mixing the decoction with the residue I water solution, filtering, concentrating the filtrate to obtain fluid extract with relative density of 1.08-1.20(80 deg.C), adding ethanol to make ethanol content reach 45%, standing for 24 hours, filtering, recovering ethanol under reduced pressure, concentrating to obtain soft extract with relative density of 1.28-1.33(80 deg.C), adding sucrose, drying, and sieving with 20 mesh sieve to obtain 1000 g.
By detection and calculation, the product contains baicalin (C) per 1g Scutellariae radix21H18O11) The content was 5.33mg, and the dissolution was 59.9% in 5min and 72.7% in 10min, as determined by the method of example 1 of the present invention.
Example 11: a Sihuang Zhili granule for treating dysentery comprises the following components by weight percent:
200g of coptis root, 200g of phellodendron bark, 100g of rhubarb, 200g of scutellaria root, 200g of isatis root and 100g of liquorice
The preparation process comprises the following steps:
(1) crushing the coptis and the liquorice into particles or powder of 20-80 meshes;
(2) adding 10-60 times of water into the granules or powder of the coptis chinensis and the liquorice in the step (1), adjusting the pH value to 2.0-3.5, soaking for 3-6 hours at 20-60 ℃, and filtering to obtain filtrate;
(3) concentrating and drying the filtrate obtained in the step (2) to obtain the coptis chinensis and liquorice cleaning paste.
(4) Decocting Scutellariae radix, radix Isatidis, cortex Phellodendri, and radix et rhizoma Rhei with water for 1-3 times, each for 1-2 hr, to obtain decoction, concentrating the filtrate to obtain fluid extract with relative density of 1.08-1.20(80 deg.C), adding ethanol to make ethanol content reach 45%, standing for 24 hr, filtering, recovering ethanol under reduced pressure, concentrating to obtain soft extract with relative density of 1.28-1.33(80 deg.C), adding sucrose, drying, and sieving with 20 mesh sieve to obtain 1000 g.
By detection and calculation, the product contains baicalin (C) per 1g Scutellariae radix21H18O11) The content was 6.15mg, and the dissolution rate was 65.5% in 5min and 88.3% in 10min, as determined by the method of example 1 of the present invention.
Claims (1)
1. A quality detection method of Sihuang Zhili Keli (four yellow diarrhea stopping granule) is characterized in that the method identifies coptis chinensis, phellodendron, rhubarb, scutellaria baicalensis, isatis root and liquorice in the Sihuang Zhili Keli by thin-layer chromatography, and simultaneously adopts HPLC to determine the content of baicalin in the Sihuang Zhili Keli;
the thin-layer chromatography for identifying the coptis chinensis in the Sihuang dysentery stopping granules comprises the following steps:
1) preparing a test solution: taking 10g of the product, adding 2.5g of diatomite, grinding uniformly, adding 50mL of methanol, carrying out ultrasonic extraction for 30min, shaking, filtering, evaporating the filtrate to dryness, adding 20mL of water into the residue to dissolve, extracting with diethyl ether for 2 times, 20mL each time, combining the diethyl ether solutions, volatilizing at low temperature, adding 2mL of methanol into the residue to dissolve to obtain a sample solution;
2) preparation of a reference solution: adding ethanol into berberine hydrochloride reference substance to obtain 1 μ L solution per 1mL as reference substance solution; taking 2g of a coptis root reference medicinal material, adding 20mL of methanol, and preparing a reference medicinal material solution by the same method;
3) spotting and developing: sucking 2-5 μ L of sample solution and 2 μ L of reference solution, respectively dropping on the same silica gel G thin layer plate, adding carbon tetrachloride at 60-90 deg.C: ethyl acetate: isopropyl alcohol: formic acid: water 3: 3:1.5:1.5: 1 is developing agent, developing, taking out, airing, and viewing under a 365nm ultraviolet lamp; in the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution;
the identification of the phellodendron amurense in the Sihuang dysentery stopping granules by the thin-layer chromatography comprises the following steps:
1) preparing a test solution: taking 10g of the product, grinding, adding 50mL of methanol, ultrasonically extracting for 30min, filtering, evaporating filtrate to dryness, adding 20mL of water into residue to dissolve, extracting the extracted water solution with ethyl acetate for 3 times, each time 20mL, adjusting pH of the extracted water solution to 10-11 with ammonia test solution, extracting with 20mL of trichloromethane for 1 time, evaporating trichloromethane solution to dryness, and adding 2mL of methanol to the residue to dissolve to obtain a sample solution;
2) preparation of a reference solution: taking phellodendrine hydrochloride reference substance, adding methanol to prepare solution containing 0.5mg per 1mL as reference substance solution;
3) spotting and developing: sucking 1-2 mul of each of the test solution and the reference solution, respectively dropping on the same silica gel G thin layer plate, and adding carbon tetrachloride: methanol: developing with ammonia water at ratio of 7:0.5:0.3 as developing agent, taking out, air drying, spraying improved bismuth potassium iodide solution, and displaying spots of the same color at the position corresponding to the chromatogram of the reference substance;
the thin-layer chromatography for identifying the rhubarb in the Sihuang dysentery stopping granules comprises the following steps:
1) preparing a test solution: taking 10g of the product, grinding, adding 50mL of methanol, performing ultrasonic extraction for 30min, filtering, evaporating filtrate to dryness, adding 20mL of methanol into residue to dissolve, adjusting pH to 1-2 with dilute hydrochloric acid, extracting with diethyl ether for 2 times, each time 20mL, extracting the extracted water solution with ethyl acetate for 2 times, each time 20mL, adding 2mL of trichloromethane into residue to dissolve to obtain a sample solution;
2) preparation of a reference solution: adding methanol into rhein reference substance to obtain 1mg solution per 1mL as reference substance solution;
3) spotting and developing: sucking 2-5 μ L of the sample solution and 5 μ L of the reference solution, respectively dropping on the same silica gel G thin layer plate, adding n-hexane: tetrachloromethane: ethyl acetate: water 5: 4.5: 2: developing the lower layer solution of 0.5 as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under 365nm ultraviolet lamp; spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution;
the thin-layer chromatography method for identifying the scutellaria baicalensis in the Sihuang dysentery stopping granules comprises the following steps of:
1) preparing a test solution: taking 10g of the product, grinding, adding 50mL of methanol, extracting with 30% ethanol under reflux for 3 times, adding 12 times of the product for the first time, 10 times of the product for the second time, 8 times of the product for the third time, 1.5 hours each time, filtering, mixing, drying the filtrate by distillation, dissolving the residue with 20mL of water, adjusting the pH value to 1-2 with dilute hydrochloric acid, extracting with diethyl ether for 2 times, 20mL each time, drying the chloroform solution by distillation, and dissolving the residue with 2mL of methanol to obtain a sample solution;
2) preparation of a reference solution: taking baicalin pure product, adding methanol into control to obtain 1mg solution per 1mL as control solution;
3) spotting and developing: sucking the two solutions, each 5 mu L of the two solutions, respectively dropping the solutions on the same silica gel G thin layer plate, and adding carbon tetrachloride: n-butanol: formic acid: water 13: 8: 2: 1, developing the lower layer solution as a developing agent, taking out, airing, and placing under a 365nm ultraviolet lamp for inspection; in the chromatogram of the test solution, spots or fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material;
the thin-layer chromatography method for identifying the isatis root in the Sihuang dysentery stopping granules comprises the following steps:
1) preparing a test solution: 10g of the product is taken, ground, added with 50mL of methanol, and mixed with ethyl acetate: extracting with 3:1 methanol under reflux for 30min, evaporating the filtrate to dryness, dissolving the residue in 20mL of water, extracting with 20mL of chloroform for 1 time, evaporating the chloroform solution to dryness, and dissolving the residue in 2mL of methanol to obtain a sample solution;
2) preparation of a reference solution: adding diluted ethanol into arginine control to obtain solution containing 0.5mg per 1mL as control solution;
3) spotting and developing: sucking the two solutions 1-2 μ L each, and dropping on the same silica gel G thin layer plate respectively, adding n-hexane: n-butanol: methanol: formic acid 8: 2: 1: 1 is developing agent, developing, taking out, drying with hot air, spraying ninhydrin test solution, heating at 105 deg.C until the color of the spot is clear, and the spot with the same color appears in the chromatogram of the test solution at the position corresponding to the chromatogram of the reference material and the control solution;
the HPLC (high performance liquid chromatography) is used for determining the content of the baicalin in the Sihuang dysentery stopping granules, and comprises the following steps:
1) preparing a test solution: taking a proper amount of Sihuang dysentery stopping granules, grinding to obtain 2g of the Sihuang dysentery stopping granules, precisely weighing, placing the ground Sihuang dysentery stopping granules in a conical flask, precisely adding ethyl acetate: weighing 50mL of methanol solution 1:50, carrying out ultrasonic treatment for 30min, cooling, weighing, complementing the weight loss amount with the methanol solution, shaking up, filtering, precisely absorbing 3mL of subsequent filtrate, adding the subsequent filtrate on a 200-mesh neutral alumina column, eluting with 50mL of methanol, collecting the eluent, evaporating by distillation in a water bath, dissolving the residue with methanol, and standing in a 10mL volumetric flask, shaking up and filtering to obtain the product;
2) preparation of a reference solution: drying under reduced pressure at 60 deg.C for 4 hr to obtain baicalin reference substance, adding methanol to obtain solution containing 0.06mg per 1mL, and shaking;
3) HPLC chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler; taking acetonitrile as a mobile phase A-0.1% phosphoric acid solution as a mobile phase B for gradient elution; the detection wavelength is 280nm, and the number of theoretical plates is not less than 2500 calculated according to baicalin peak;
4) the determination method comprises the following steps: respectively and precisely sucking 10uL of the reference solution and the test solution, injecting into a high performance liquid chromatograph, and measuring according to the chromatographic conditions in the step 3).
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