CN113030344A

CN113030344A - Quality control method of amber mind-tranquilizing pills

Info

Publication number: CN113030344A
Application number: CN202110401428.7A
Authority: CN
Inventors: 谢晓燕; 马全龙; 王浩骅; 孔令冉; 段广场; 王哲; 陈志勇; 国春燕; 邓春燕
Original assignee: SHANDONG XINBAO PHARMACEUTICAL CO Ltd
Current assignee: SHANDONG XINBAO PHARMACEUTICAL CO Ltd
Priority date: 2021-04-14
Filing date: 2021-04-14
Publication date: 2021-06-25

Abstract

The invention discloses a quality control method of amber mind-tranquilizing pills, which are water-honeyed pills, and the microscopic identification method is used for identifying schisandra chinensis, liquorice and spina date seeds in the amber mind-tranquilizing pills; identifying radix Angelicae sinensis, fructus Schisandrae chinensis, Ginseng radix, cortex et radix Polygalae and Glycyrrhrizae radix in Succinum tranquilization pill by thin layer chromatography; and (3) measuring the content of the schisandrin in the amber mind-tranquilizing pills by adopting an HPLC method. The quality control method has strong specificity, stability, reliability and good reproducibility, can comprehensively and effectively control the quality of the amber tranquilizing pills (water-honeyed pills), is beneficial to stabilizing the quality of products, ensures the safety and effectiveness of clinical medication, and better meets the requirements of medical treatment and markets.

Description

Quality control method of amber mind-tranquilizing pills

Technical Field

The invention relates to a quality control method of a Chinese patent medicine amber mind-tranquilizing pill (water-honeyed pill), belonging to the technical field of analysis and quality control of Chinese medicinal preparations.

Background

The Succinum tranquilizing pill (water honeyed pill) has effects of nourishing yin and blood, tonifying heart, and tranquilizing mind. The traditional Chinese medicine composition is used for treating heart-blood deficiency, palpitation and insomnia, dysphoria and uneasiness, and comprises the following main components in percentage by weight:

the preparation method comprises the following steps: pulverizing the above eighteen raw materials into fine powder, sieving, and mixing. Adding 15-20 g of refined honey into every 100g of powder to prepare water-honeyed pills, and drying to obtain the brown to tan water-honeyed pills, which are sweet, slightly sour and pungent in taste.

The original amber tranquilizing pill is a big honeyed pill of 9 g/pill, and the original standard (WS)₃-B-2621-97) only has the microscopic identification of the schisandra, liquorice and spina date seed 3, and no relatively comprehensive quality control method reflects the quality condition of the amber mind-tranquilizing pills (water-honeyed pills), so the production process and the product quality of the amber mind-tranquilizing pills (water-honeyed pills) cannot be realizedEffectively controlled and cannot better ensure the curative effect of the medicine. In order to improve the controllability of product quality and ensure the product quality, it is necessary to research a simple and comprehensive detection method with good specificity and stability for reflecting the quality condition of the amber mind-tranquilizing pills (water-honeyed pills).

Disclosure of Invention

The invention aims to provide a quality control method of amber mind-tranquilizing pills (water-honeyed pills), which has strong specificity, stability, reliability and good reproducibility, can comprehensively and accurately analyze active components of medicaments, is favorable for effectively controlling the production process and the product quality of the amber mind-tranquilizing pills (water-honeyed pills), stabilizing the product quality, ensuring the safety and the effectiveness of clinical medication, ensuring the clinical curative effect of the amber mind-tranquilizing pills and better meeting the requirements of medical treatment and market.

In the research, the invention refers to a thin-layer identification method under the part of 'Chinese pharmacopoeia' 2020 edition and related preparation items, performs thin-layer identification research on 12 raw material medicines of Chinese angelica, Chinese magnoliavine fruit, ginseng, polygala tenuifolia, liquorice (baked with honey), rehmannia glutinosa, spina date seed (fried), dwarf lilyturf tuber, poria cocos, salvia miltiorrhiza, radix scrophulariae and platycodon grandiflorum in a prescription, and finally establishes a microscopic identification method of 3 medicinal materials (Chinese magnoliavine fruit, liquorice and spina date seed) and a thin-layer (TLC) identification method of 5 medicinal materials (Chinese angelica, Chinese magnoliavine fruit, ginseng, polygala tenuifolia and liquorice), and has the advantages of.

High Performance Liquid Chromatography (HPLC) has the advantages of rapidness, sensitivity, high separation efficiency and the like, and in qualitative and quantitative analysis of traditional Chinese medicines, after characteristic components are separated by an HPLC method, a detector is used for detection and analysis, so that the method is an effective method for comprehensively evaluating the quality of a traditional Chinese medicine composition. On the basis of the microscopic identification and the thin-layer identification, the method simultaneously adopts an HPLC method to determine the content of the schizandrol A in the amber tranquilizing pills (water-honeyed pills), introduces the content of the schizandrol A as a quality control index, and better controls the product quality.

The detailed technical scheme of the invention is as follows:

a quality control method of Succinum tranquilizing pill comprises identifying fructus Schisandrae chinensis, Glycyrrhrizae radix, and semen Ziziphi Spinosae in Succinum tranquilizing pill by microscopic identification method; identifying radix Angelicae sinensis, fructus Schisandrae chinensis, Ginseng radix, cortex et radix Polygalae and Glycyrrhrizae radix in Succinum tranquilization pill by thin layer chromatography; and (3) measuring the content of the schisandrin in the amber mind-tranquilizing pills by adopting an HPLC method. Through the organic combination of the three qualitative and quantitative indexes, the quality of the amber mind-tranquilizing pill can be more comprehensively and accurately reflected, and the effective quality control of the amber mind-tranquilizing pill is facilitated.

Further, the microscopic identification method comprises the following operation steps: mixing the amber mind tranquilizing pills with chloral hydrate, tabletting, and observing the properties of the schisandra chinensis, the liquorice and the spina date seeds in the tablets under a microscope.

Further, when observed by a microscope, the epidermoid stone cells of the seed coat have a light yellowish-brown surface appearance similar to a polygon, thicker walls, fine and dense pores and cavities containing dark brown substances (schisandra chinensis); the parenchyma cells around the fiber bundle contain calcium oxalate square crystals to form crystal fibers (liquorice); the seed-coat-grid cells are brownish red, the surface is polygonal, the diameter is about 15 mu m, the wall thickness is thick, the cells are lignified, and the cells are small. In side view, the cells are in a row and are rectangular, and the length of the cells is 60-80 mu m (spina date seeds). The schisandra chinensis, the liquorice and the spina date seeds have prominent microscopic characteristics, strong specificity and no interference on negative, and can simply, conveniently and quickly detect the quality condition of the amber tranquilizing pills.

Further, the step of identifying the angelica in the amber tranquilizing pill by thin layer chromatography comprises the following steps:

1) preparation of a test solution: taking 10g of amber mind-tranquilizing pills, grinding, adding 50ml of ethyl acetate, carrying out ultrasonic treatment for 15 minutes, filtering to obtain a filtrate, volatilizing the solvent from the filtrate, and adding ethyl acetate lml to the residue to dissolve the residue to obtain a sample solution;

2) preparation of control solutions: taking 0.5g of angelica sinensis control medicinal material, grinding, adding 10ml of ethyl acetate, carrying out ultrasonic treatment for 15 minutes, filtering to obtain filtrate, volatilizing the solvent from the filtrate, and adding ethyl acetate lml to the residue to dissolve the filtrate to obtain a control solution;

3) spotting and developing: according to a thin layer chromatography (general 0502 of 2020 edition of Chinese pharmacopoeia) test recorded in Chinese pharmacopoeia, 10 mu l of each of a test solution and a reference solution is absorbed, the test solution and the reference solution are respectively spotted on the same silica gel G thin layer plate, n-hexane-ethyl acetate with the volume ratio of 4:1 is taken as a developing agent for development, the silica gel G thin layer plate is taken out after the development, the silica gel G thin layer plate is dried, the test solution is inspected under ultraviolet light (365nm), and fluorescent spots with the same color are displayed on the positions corresponding to the reference solution chromatogram.

Further, the step of identifying the schisandra chinensis in the amber mind-tranquilizing pills by using the thin-layer chromatography comprises the following steps:

1) preparation of a test solution: taking 10g of amber mind-tranquilizing pills, grinding, adding 100ml of trichloromethane, heating and refluxing for 30 minutes, filtering to obtain a filtrate, volatilizing the solvent from the filtrate, and adding trichloromethane lml to the residue to dissolve the chloroform lml to obtain a test solution;

2) preparation of control solutions: grinding fructus Schisandrae control material lg, adding chloroform 100ml, heating under reflux for 30min, filtering to obtain filtrate, volatilizing solvent from the filtrate, and dissolving the residue in chloroform lml to obtain control solution;

3) spotting and developing: referring to thin layer chromatography (0502 of 2020 th edition of Chinese pharmacopoeia) test recorded in Chinese pharmacopoeia, sucking 2 μ l of each of test solution and control solution, and respectively dropping on the same silica gel GF₂₅₄Developing the thin-layer plate by using petroleum ether-ethyl formate-formic acid upper-layer solution with the boiling range of 30-60 ℃ in a volume ratio of 15: 5: 1 as a developing agent, and taking out the silica gel GF after development₂₅₄And (4) inspecting the thin-layer plate under an ultraviolet lamp (254nm), wherein spots with the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution.

Further, the step of identifying the ginseng in the amber tranquilizing pill by thin-layer chromatography comprises the following steps:

1) preparation of a test solution: taking 20g of amber mind-tranquilizing pills, grinding, adding 100ml of trichloromethane, heating and refluxing for 1 hour, filtering to obtain medicine residues, volatilizing a solvent from the medicine residues, adding 2ml of water, stirring for moistening, adding 50ml of saturated n-butyl alcohol, carrying out ultrasonic treatment for 30 minutes, sucking supernatant, adding 3 volumes of ammonia test solution (prepared by adding water into 400ml of concentrated ammonia solution and dissolving into 1000 ml), shaking uniformly, standing for layering, taking supernatant, evaporating to dryness, and adding methanol lml to residues for dissolving to obtain a test solution;

2) preparation of control solutions: taking 0.5g of ginseng control medicinal material, grinding, adding 100ml of trichloromethane, heating and refluxing for 1 hour, filtering to obtain medicine residue, volatilizing the solvent from the medicine residue, adding 2ml of water, stirring and wetting, adding 50ml of water-saturated n-butyl alcohol, carrying out ultrasonic treatment for 30 minutes, absorbing supernatant, adding 3 volumes of ammonia test solution, shaking uniformly, standing for layering, taking supernatant, evaporating to dryness, and adding methanol lml to residues to dissolve the residues to obtain a control solution;

3) spotting and developing: referring to the thin layer chromatography (general 0502 of 2020 edition of Chinese pharmacopoeia) test recorded in Chinese pharmacopoeia, sucking 10 μ l of each of a test solution and a reference solution, respectively dropping the test solution and the reference solution on the same silica gel G thin layer plate, taking out the silica gel G thin layer plate after developing by using a lower layer solution of trichloromethane-ethyl acetate-methanol-water at a volume ratio of 15: 40: 22: 10 and placed below 10 ℃ as a developing agent, drying in the air, spraying 10wt% sulfuric acid ethanol solution, heating at 105 ℃ until spots are clear, and observing under sunlight, wherein spots with the same color are displayed on the positions corresponding to the reference chromatogram in the test chromatogram.

Further, the thin-layer chromatography method for identifying polygala tenuifolia in the amber tranquilization pill comprises the following steps:

1) preparation of a test solution: taking 20g of amber mind-tranquilizing pills, grinding, adding 100ml of 70wt% methanol, carrying out ultrasonic treatment for 30 minutes, filtering to obtain a filtrate, volatilizing the solvent from the filtrate, and adding 2ml of methanol to the residue to dissolve the residue to obtain a sample solution;

2) preparation of control solutions: taking 0.5g of polygala tenuifolia as a reference material, grinding, adding 100ml of 70wt% methanol, carrying out ultrasonic treatment for 30 minutes, filtering to obtain filtrate, volatilizing the solvent from the filtrate, and adding 2ml of methanol to dissolve the residue to obtain a reference solution;

3) spotting and developing: referring to the thin layer chromatography (general 0502 of 2020 version of Chinese pharmacopoeia) test recorded in Chinese pharmacopoeia, respectively taking 10 μ l of each of the test solution and the reference solution, respectively dropping the solution on the same silica gel G thin layer plate, taking the lower layer solution of trichloromethane-methanol-water with the volume ratio of 7: 3: 1 as a developing agent, taking out the silica gel G thin layer plate after development, drying the silica gel G thin layer plate, placing the silica gel G thin layer plate under an ultraviolet lamp (365nm) for inspection, and displaying fluorescent spots with the same color on the positions corresponding to the reference chromatogram in the test chromatogram.

Further, the step of identifying the liquorice in the amber tranquilizing pill by thin-layer chromatography comprises the following steps:

1) preparation of a test solution: taking 20g of amber mind-tranquilizing pills, grinding, adding 100ml of diethyl ether, heating and refluxing for 1 hour, filtering to obtain medicine residues, adding 50ml of methanol into the medicine residues, heating and refluxing for 1 hour, filtering to obtain filtrate, volatilizing the solvent from the filtrate, adding 40ml of water into residues to dissolve the residues, extracting with n-butanol for 3 times, 20ml each time, combining n-butanol extracts, washing with water for 3 times, discarding water solution, evaporating the n-butanol solution to dryness, and adding 1ml of methanol to dissolve the residues to obtain a sample solution;

2) preparation of control solutions: taking 0.5g of liquorice as a reference medicinal material, grinding, adding 100ml of diethyl ether, heating and refluxing for 1 hour, filtering to obtain medicine residues, adding 50ml of methanol into the medicine residues, heating and refluxing for 1 hour, filtering to obtain filtrate, volatilizing the solvent from the filtrate, adding 40ml of water into the residues to dissolve the residues, extracting with n-butanol for 3 times, 20ml each time, combining n-butanol extract, washing with water for 3 times, discarding water solution, evaporating the n-butanol solution to dryness, and adding 1ml of methanol into the residues to dissolve the residues to obtain a reference solution;

3) spotting and developing: according to the thin layer chromatography (general 0502 of 2020 version of Chinese pharmacopoeia) test recorded in Chinese pharmacopoeia, 10 mul of each of a test solution and a reference solution is absorbed and respectively spotted on the same silica gel G thin layer plate, a lower layer solution which is placed at the temperature of below 10 ℃ in the volume ratio of 13: 7: 2 trichloromethane-methanol-water is taken as a developing agent, the silica gel G thin layer plate is taken out after being developed, the silica gel G thin layer plate is dried in the air, 10wt% sulfuric acid ethanol solution is sprayed, the silica gel G thin layer plate is heated at 105 ℃ until the spots are clearly developed, the test solution is inspected under sunlight, and the spots with the same color are displayed on the positions corresponding to the reference solution chromatogram in the test solution chromatogram.

Further, the step of measuring the content of the schizandrol A by an HPLC method comprises the following steps:

1) preparation of a test solution: taking a proper amount of amber mind-tranquilizing pills, grinding, mixing uniformly, precisely weighing 1.5-2g, placing in a mortar, adding 4g of diatomite, grinding uniformly, transferring to a conical flask with a plug, precisely adding 20ml of methanol, sealing the plug, weighing, carrying out ultrasonic treatment for 30 minutes, cooling, weighing again, complementing the lost weight with methanol, shaking uniformly, filtering, and taking filtrate to obtain a sample solution;

2) preparation of control solutions: taking a proper amount of schizandrol A reference substance, precisely weighing, and adding methanol to obtain a solution of each 20 μ g/ml, to obtain a reference substance solution;

3) HPLC chromatographic conditions: octadecylsilane chemically bonded silica is used as a filling agent; methanol-water with the volume ratio of 55: 45 is taken as a mobile phase; the detection wavelength is 250 nm; the flow rate is 1 ml/min; the column temperature is room temperature; the number of theoretical plates is not less than 6000 calculated according to the schizandrol A peak;

4) the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring, and calculating schizandrol A content according to peak area.

Furthermore, in the detection by the HPLC method, the ultrasonic power is 100W, and the ultrasonic frequency is 40 kHz.

Further, the amber sedative pill contains schisandra chinensis and schizandrol A (C)₁₁H₆O₃) Calculated by every 1g, the content of the active ingredients is not less than 0.18 mg.

In order to comprehensively detect the quality of the amber mind-tranquilizing pills (water-honeyed pills) and effectively control the quality of the amber mind-tranquilizing pills (water-honeyed pills), the inventor carries out qualitative and quantitative detection on traditional Chinese medicinal materials and effective components of the medicinal materials through a large number of tests, and establishes a new quality control method of the traditional Chinese medicinal amber mind-tranquilizing pills (water-honeyed pills). The invention adopts a microscopic identification method to identify the schisandra chinensis, the liquorice and the spina date seeds in the preparation; identifying radix Angelicae sinensis, fructus Schisandrae chinensis, Ginseng radix, cortex et radix Polygalae, and Glycyrrhrizae radix by thin layer chromatography; meanwhile, HPLC is adopted to determine the schizandrol A content of the schisandra chinensis in the preparation, so that comprehensive evaluation and control on the quality of the amber mind-tranquilizing pills (water-honeyed pills) are realized, comprehensive and reliable bases are provided for authenticity identification and internal quality detection of the amber mind-tranquilizing pills (water-honeyed pills), the stability of product quality and the safety and effectiveness of clinical medication are ensured, and the requirements of medical treatment and market are better met.

Drawings

FIG. 1 is a UV scanning chart of schizandrol A in Succinum tranquilizing pill (water honeyed pill);

FIG. 2 is an HPLC chromatogram of Schisandra chinensis;

FIG. 3 an HPLC chromatogram without a Schisandra chinensis negative sample;

FIG. 4 is an HPLC chromatogram of a sample of Succinum tranquilizing pill (water-honeyed pill);

FIG. 5 is an HPLC chromatogram of a schizandrol A control;

FIG. 6 is a linear relationship graph of schizandrol A peak area and concentration of Succinum tranquilizing pill (water-honeyed pill);

FIG. 7 is a microscopic identification chart of fructus Schisandrae chinensis for Succinum tranquilizing pill (water-honeyed pill);

FIG. 8 is a microscopic identification chart of radix Glycyrrhizae for Succinum tranquilizing pill (water-honeyed pill);

FIG. 9 is a microscopic identification picture of semen Ziziphi Spinosae of Succinum tranquilization pill (water honeyed pill);

FIG. 10 shows TLC chromatogram of radix Angelicae sinensis of Succinum tranquilization pill (water-honeyed pill);

FIG. 11 is a TLC chromatogram of fructus Schisandrae chinensis of Succinum tranquilization pill (water-honeyed pill);

FIG. 12 is a TLC chromatogram of Ginseng radix of Succinum tranquillizing pill (water-honeyed pill);

FIG. 13 is TLC chromatogram of cortex et radix Polygalae of Succinum tranquilization pill (water honeyed pill);

FIG. 14 is a TLC chromatogram of radix Glycyrrhizae of Succinum tranquillizing pill (water-honeyed pill);

FIG. 15 is a TLC chromatogram of amber sedative pill (honeyed pill) containing rehmanniae radix;

FIG. 16 is a chromatogram of TLC of semen Ziziphi Spinosae of Succinum tranquilization pill (water honeyed pill);

FIG. 17 is a TLC chromatogram of radix Ophiopogonis in Succinum tranquillizing pill (water-honeyed pill);

FIG. 18 is a chromatogram of Poria TLC of Succinum tranquilizing pill (water-honeyed pill);

FIG. 19 is a TLC chromatogram of Saviae Miltiorrhizae radix of Succinum tranquilization pill (water-honeyed pill);

FIG. 20 is a TLC chromatogram of Platycodon grandiflorum of Succinum tranquilization pill (water-honeyed pill);

FIG. 21 is a TLC chromatogram of radix scrophulariae of Succinum tranquillizing pill (water-honeyed pill).

Detailed Description

The present invention is further described in detail by the following specific examples, which are carried out on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of the present invention is not limited to the following examples.

Example 1

HPLC methodological study of Schizandrol A

1. Instruments and reagents

The instrument comprises the following steps: AG135 type electronic analytical balance (METTLER, Switzerland), Shimadzu high performance liquid chromatograph LC-15C type pump, SPD-15C detector, Agilent HC-C₁₈(250 mm. times.4.6 mm, 5 μm) chromatography column; the number of theoretical plates is not less than 6000 calculated according to the schizandrol A peak.

Comparison products: schisandrin (for content determination, China institute for food and drug testing, 20mg, 110857-.

And (3) testing the sample: succinum tranquilizing pill (water honeyed pill).

2. Selection of chromatographic conditions

2.1 detection wavelength selection: accurately weighing appropriate amount of schizandrol A reference substance, adding methanol to obtain solution containing 20 μ g per 1ml, and scanning with ultraviolet spectrophotometry (in "Chinese pharmacopoeia" 0401 in 2020 edition of the general rules of four departments) at wavelength of 200-400 nm to obtain schizandrol A with maximum absorption at wavelength of 222nm and 250nm (figure 1), so that the detection wavelength is 250 nm.

2.2 determination of mobile phase: referring to the content determination method of schisandrin in Tianwang Buxin pill in the first department of the 2020 edition of Chinese pharmacopoeia, methanol-water (55: 45) is determined as a mobile phase.

3. The extraction method comprises the following steps:

3.1 extraction method selection test:

preparation of a reference solution: taking 10.11mg of a schisandrin reference substance (110857-201815), putting the reference substance into a 50ml measuring flask, adding methanol to dissolve and diluting to a scale mark to be used as a reference substance storage solution; precisely measuring 1ml of the reference stock solution, placing into a 10ml measuring flask, diluting with methanol to scale, and shaking to obtain reference solution.

Preparing a test solution: taking about 2g of amber mind-tranquilizing pills (batch number: 20201101), grinding, precisely weighing 4 parts, placing in a mortar, adding 4g of diatomite, uniformly grinding, transferring to a conical flask with a plug, precisely adding 20ml of methanol, sealing the plug, weighing the weight, performing two parts of ultrasonic treatment (power of 100W and frequency of 40kHz) for 30min, performing two parts of reflux extraction for 30min, cooling, weighing again, complementing the lost weight with methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the medicine.

Respectively and precisely sucking 10 mul of reference solution and test solution, injecting into a liquid chromatograph for determination, wherein the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; methanol-water with the volume ratio of 55: 45 is taken as a mobile phase; the detection wavelength is 250 nm; the flow rate is 1 ml/min; the column temperature is room temperature; the number of theoretical plates is not less than 6000 calculated according to the schizandrol A peak. The schizandrol A content was calculated from the peak area, and the results are shown in Table 1.

TABLE 1 comparison of different extraction methods

Extraction method	Ultrasonic extraction	Reflux extraction
			Content (mg/g)	0.22	0.22

As a result: the ultrasonic treatment and the reflux extraction content are basically consistent, and the ultrasonic treatment method is simple and convenient, so the extraction method is determined to be ultrasonic treatment.

3.2 extraction time selection test:

preparation of a reference solution: precisely measuring 1ml of the reference stock solution under 3.1 items, placing in a 10ml measuring flask, diluting with methanol to scale, and shaking.

Preparing a test solution: taking about 2g of amber mind-tranquilizing pills (batch number: 20201101), grinding, precisely weighing 6 parts, placing in a mortar, adding 4g of diatomite, uniformly grinding, transferring into a conical flask with a plug, precisely adding 20ml of methanol, sealing the plug, weighing, ultrasonically treating two parts (power 100W and frequency 40kHz) for 20min, ultrasonically treating two parts (power 100W and frequency 40kHz) for 30min, ultrasonically treating two parts (power 100W and frequency 40kHz) for 40min, cooling, weighing again, complementing weight loss by methanol, uniformly shaking, filtering, and taking a subsequent filtrate to obtain the medicine.

Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring, and obtaining the result under the same chromatographic conditions as above, wherein the result is shown in Table 2.

TABLE 2 Schizandrol A content determination extraction time selection results table

Extraction time (min)	20	30	40
				Content (mg/g)	0.20	0.22	0.21

As a result: the ultrasonic treatment has basically consistent contents in 20, 30 and 40 minutes, and the ultrasonic treatment time is determined to be 30 minutes in order to ensure complete extraction of the schizandrol A.

3.3 extraction solvent selection test:

Preparing a test solution: taking about 2g of amber mind-tranquilizing pills (batch number: 20201101), grinding, precisely weighing 6 parts, placing in a mortar, adding 4g of diatomite, uniformly grinding, transferring to a conical flask with a plug, precisely adding 20ml of ethanol into two parts, precisely adding 20ml of 70% methanol into two parts, precisely adding 20ml of methanol into two parts, sealing the plug, weighing, carrying out ultrasonic treatment (power 100W and frequency 40kHz) for 30min, cooling, weighing again, respectively supplementing the weight loss by using corresponding solvents, uniformly shaking, filtering, and taking the subsequent filtrate.

Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring, and obtaining the result under the same chromatographic conditions as above, wherein the results are shown in Table 3.

TABLE 3 Schizandrol A content determination extraction solvent selection results table

Extraction solvent	Ethanol		70% methanol	Methanol
					Average content (mg/g)	0.13	0.16	0.22

As a result: the extraction can be completed by using methanol, and the extraction can not be completed by using ethanol and 70% methanol, so that the methanol is selected as an extraction solvent.

3.4 solvent amount selection test:

Preparing a test solution: taking about 2g of amber mind-tranquilizing pills (batch number: 20201101), grinding, precisely weighing 6 parts, placing in a mortar, adding 4g of diatomite, uniformly grinding, transferring to a conical flask with a plug, precisely adding 10ml of methanol into two parts, precisely adding 20ml of methanol into two parts, precisely adding 30ml of methanol into two parts, sealing the plug, weighing, carrying out ultrasonic treatment (power 100W and frequency 40kHz) for 30min, cooling, weighing again, complementing the weight loss by methanol, uniformly shaking, filtering, and taking the subsequent filtrate to obtain the weight loss pill.

Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring, and obtaining the result under the same chromatographic conditions as above, wherein the results are shown in Table 4.

TABLE 4 Schizandrol A content solvent dosage selection results table

Amount of solvent (ml)	10	20	30
				Average content (mg/g)	0.18	0.21	0.22

As a result: the extraction solvent is 20ml, the content of 30ml is not changed greatly, the content of 10ml is low, the extraction is not sufficient, and 20ml is selected for the convenience of test.

3.5 the preparation method of the best test solution determined by the screening comprises the following steps:

taking 1.5-2g of the product, grinding, precisely weighing, placing in a mortar, adding 4g of diatomite, uniformly grinding, transferring to a conical flask with a plug, precisely adding 20ml of methanol, sealing the plug, weighing, ultrasonically treating (with the power of 100W and the frequency of 40kHz) for 30 minutes, cooling, weighing again, complementing the lost weight with methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the product.

4. And (3) stability test:

preparation of a reference solution: taking 9.85mg of a schisandrin A reference substance (110857-.

Test solution: weighing about 2g of Succinum tranquilizing pill (batch number: 20201101), precisely weighing, placing in a mortar, adding 4g of diatomaceous earth, grinding, transferring to a conical flask with a plug, precisely adding 20ml of methanol, sealing the plug, weighing, ultrasonically treating (power 100W, frequency 40kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with methanol, shaking, filtering, and collecting the subsequent filtrate.

Precisely sucking 10 μ l of each of the control solution and the sample solution, injecting into a liquid chromatograph at 0 hr, 2 hr, 4 hr, 6 hr, 8 hr, and 12 hr, and subjecting to chromatographic conditions: octadecylsilane chemically bonded silica is used as a filling agent; methanol-water with the volume ratio of 55: 45 is taken as a mobile phase; the detection wavelength is 250 nm; the flow rate is 1 ml/min; the column temperature is room temperature; the number of theoretical plates is not less than 6000 calculated according to the schizandrol A peak. The results are shown in Table 5.

TABLE 5 Schizandrol A content determination stability test result table

As a result: the peak areas RSD of the reference solution and the test solution are respectively 1.32 percent and 1.68 percent within 12 hours, which proves that the reference solution and the test solution have good stability within 12 hours.

5. And (3) precision test:

preparation of a reference solution: precisely sucking 1ml of the reference stock solution under the stability term, placing in a 10ml measuring flask, diluting with methanol to scale, and shaking up to obtain the final product.

Precisely sucking 10 μ l of schizandrol A reference solution, injecting into a liquid chromatograph, performing stability test under the same chromatographic conditions, and recording chromatogram. The sample introduction was carried out 6 times in succession, and the RSD of the peak area integral value was calculated, and the results are shown in Table 6.

TABLE 6 measurement of Schizandrol A content and precision test results table

The results show that: the RSD of the reference substance is 1.32 percent, which proves that the precision of the instrument is good.

6. And (3) linear relation investigation:

preparation of linear solution: taking 9.85mg (with the purity of 99.7%) of a schisandrin reference substance (110857-.

Each 10. mu.l of the solution was precisely pipetted, injected into a liquid chromatograph, subjected to the same stability test under the same chromatographic conditions, and the chromatographic peak area was recorded as shown in Table 7.

TABLE 7 measurement of the Linear Range of the Schizandrol A control

Taking the peak area value (A) as a vertical coordinate and the concentration (c) of the reference solution as a horizontal coordinate to perform linear regression to obtain a linear regression equation: a 19680c +6974.3 and r 0.99905 (fig. 6). The results show that: the linear relation between the schizandrol A and the peak area is good within the range of 3.9282-58.9227 mu g/ml.

7. And (3) repeatability test:

preparation of a reference solution: precisely weighing 1ml of the reference stock solution under the term of stability, placing in a 10ml measuring flask, diluting with methanol to scale, and shaking.

Preparing a test solution: about 2g of amber sedative pill (batch number: 20201101) is prepared according to the preparation method of the test solution under the item of stability, and 6 parts are prepared by the same method.

Precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring under the same chromatographic conditions. The results are shown in Table 8.

TABLE 8 repeatability test results table for schizandrol A content determination

The results show that: the test method has good repeatability.

8. Sample recovery rate test:

preparing a test solution: precisely weighing about 1g of the same batch of amber mind-tranquilizing pills (batch number: 20201101, content: 0.22mg/g) with known content, precisely weighing 6 parts in total, placing in a mortar, adding 2g of diatomite, uniformly grinding, precisely adding 1ml of schizandrol A reference substance solution (196.409 mu g/ml), precisely adding 19ml of methanol, sealing, weighing, ultrasonically treating for 30 minutes (power 100W, frequency 40kHz), cooling, weighing again, supplementing the weight lost by methanol, shaking uniformly, and filtering to obtain the medicine. The results are shown in Table 9.

Calculating the formula: recovery (%). percent (measured-sample content)/control addition × 100%

TABLE 9 test results of sample recovery for schizandrol A content determination

The results show that: the recovery rate of the test method was good.

9. The specificity is as follows:

preparing a test solution: weighing about 2g of Succinum tranquilizing pill (lot number: 20201101), precisely weighing, and processing according to preparation method of test solution under the term of "stability".

Preparing a schisandra chinensis medicinal material solution: taking 0.1g of schisandra chinensis, precisely weighing, and preparing according to a preparation method of a test solution under the item of stability to obtain the schisandra chinensis.

Preparation of a negative test solution: taking the fructus Schisandrae chinensis removed water-honeyed pill as negative sample, taking about 2g, precisely weighing, and preparing according to the preparation method of test solution under the item of "stability".

Precisely sucking 10 μ l each of fructus Schisandrae solution, negative sample solution, sample solution and reference solution, injecting into liquid chromatograph under the chromatography conditions as above, and recording chromatogram.

As a result: schizandrol A can be effectively detected, and negative test sample without interference (figure 2-figure 5) after fructus Schisandrae chinensis is removed.

10. And (3) sample content determination: taking 3 batches of Succinum tranquilizing pill (water honeyed pill) samples, determining the content of schisandrin according to the method, and the determination results are shown in Table 10.

TABLE 10 measurement results of schizandrol A content in Succinum tranquilizing pill sample

The average content of schizandrol A in 3 batches of samples of the product by a high performance liquid chromatography method is 0.22 mg/g.

According to the above test results, considering the source of the herbs, the production and storage of the preparation, etc., one gram of the product contains Schisandra chinensis and schisandrin (C)₁₁H₆O₃) Calculated, the content of the active ingredient should not be less than 0.18 mg.

Example 2 microscopic identification of Schisandra chinensis, Glycyrrhiza uralensis and Zizyphi Spinosae semen in Succinum tranquilizing pill (water-honeyed pill)

Taking an appropriate amount of Succinum tranquilizing pill (water-honeyed pill), making into tablet with chloral hydrate, and observing the properties of each medicine in the preparation under microscope, wherein the shape of fructus Schisandrae chinensis, Glycyrrhrizae radix and semen Ziziphi Spinosae under microscope is more prominent.

1) Shape of fructus Schisandrae under microscope: the stone cells of the epidermis of the seed coat have a yellowish-brown surface appearance similar to a polygon, thicker walls, fine and dense pores, and dark brown substances in the cell cavities, as shown in figure 7.

2) Microscopic shape of licorice: parenchymal cells around the fiber bundle contained calcium oxalate cristobalite, forming crystalline fibers, as shown in fig. 8.

3) The shape of the wild jujube seed under a microscope is as follows: the seed-coat-grid cells are brownish red, the surface is polygonal, the diameter is about 15 mu m, the wall thickness is thick, the cells are lignified, and the cells are small. In side view, the cells are in a row, rectangular and 60-80 μm long, as shown in FIG. 9.

As a result: fig. 7-9 are microscopic identification images of schisandra chinensis, liquorice and spina date seeds in the amber mind-tranquilizing pills (water-honeyed pills), and the results show that the microscopic identification method is simple and convenient, has strong specificity, no interference in negative and prominent microscopic characteristics, and can be used as a quality control index in the amber mind-tranquilizing pills (water-honeyed pills).

Example 3 identification of Dang Gui in amber Anshen Wan (Water-honeyed pill)

1) Preparation of a test solution: taking 10g of amber mind-tranquilizing pills (water-honeyed pills), grinding, adding 50ml of ethyl acetate, carrying out ultrasonic treatment for 15 minutes, filtering, volatilizing filtrate, and adding ethyl acetate lml into residues for dissolving to obtain a test solution.

2) Preparation of negative test solution: the angelica in the amber sedative pill is removed and is used as a negative test sample. A negative sample solution was prepared by the above method from 10g of the negative sample.

3) Preparation of control solutions: collecting radix Angelicae sinensis control material 0.5g, grinding, adding ethyl acetate 10ml, ultrasonic treating for 15 min, filtering, volatilizing filtrate, and dissolving residue with ethyl acetate lml to obtain control solution.

4) Spotting and developing: testing by thin layer chromatography (general 0502 of 2020 edition of Chinese pharmacopoeia), sucking 10 μ l of each of the 3 solutions, respectively dropping on the same silica gel G thin layer plate, developing with n-hexane-ethyl acetate (volume ratio 4: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots with the same color are displayed at the positions corresponding to those of the chromatogram of the reference drug, and fluorescent spots with the same color are not displayed at the positions corresponding to those of the chromatogram of the negative test sample (FIG. 10), so that the specificity is strong.

Example 4 identification of Schisandra chinensis in Succinum tranquilizing pill (Water-honeyed pill)

1) Preparation of a test solution: taking 10g of amber mind tranquilizing pills (water honeyed pills), grinding, adding 100ml of trichloromethane, heating and refluxing for 30 minutes, filtering, evaporating filtrate to dryness, and adding trichloromethane lml to residues for dissolving to obtain a test solution;

2) preparation of negative test solution: removing fructus Schisandrae chinensis from Succinum tranquillizing pill, and making into negative test sample. A negative sample solution was prepared by the above method from 10g of the negative sample.

3) Preparation of control solutions: grinding fructus Schisandrae control material lg, adding chloroform 100ml, heating under reflux for 30min, filtering, evaporating filtrate, and dissolving residue with chloroform lml to obtain control solution;

4) spotting and developing: performing thin layer chromatography (China pharmacopoeia 2020 edition general rule 0502) test, sucking 2 μ l of the above 3 solutions, and respectively dropping on the same silica gel GF₂₅₄And (3) taking an upper layer solution of petroleum ether (with a boiling range of 30-60 ℃) -ethyl formate-formic acid (with a volume ratio of 15: 5: 1) as a developing agent on the thin-layer plate, developing, taking out, airing, and inspecting under an ultraviolet lamp (254 nm). In the chromatogram of the test sample, spots of the same color appear at the positions corresponding to those of the chromatogram of the control drug, and fluorescent spots of the same color do not appear at the positions corresponding to those of the chromatogram of the negative test sample (FIG. 11), and the specificity is high.

Example 5 identification of Ginseng radix in Succinum tranquilizing pill (Water-honeyed pill)

1) Preparation of a test solution: taking 20g of amber mind-tranquilizing pills (water-honeyed pills), grinding, adding 100ml of trichloromethane, heating and refluxing for 1 hour, removing trichloromethane liquid, volatilizing solvent from medicine residues, adding 2ml of water, stirring and wetting, adding 50ml of saturated n-butyl alcohol, carrying out ultrasonic treatment for 30 minutes, sucking supernatant, adding 3 times of ammonia test solution, shaking uniformly, standing for layering, taking supernatant, evaporating to dryness, and adding methanol lml into residues to dissolve the residues to obtain a test solution;

2) preparation of negative test solution: removing Ginseng radix from Succinum tranquilizing pill, and making into negative test sample. 20g of the negative test sample was taken and prepared into a negative test sample solution according to the above-mentioned method.

3) Preparation of control solutions: taking 0.5g of ginseng control medicinal material, grinding, adding 100ml of trichloromethane, heating and refluxing for 1 hour, removing trichloromethane liquid, volatilizing solvent from medicine residue, adding 2ml of water, stirring and moistening, adding 50ml of water-saturated n-butyl alcohol, carrying out ultrasonic treatment for 30 minutes, sucking supernatant, adding 3 times of ammonia test solution, shaking uniformly, standing for layering, taking supernatant, evaporating to dryness, and adding methanol lml into residue to dissolve the residue to obtain a control solution;

4) spotting and developing: performing thin layer chromatography (0502 of China pharmacopoeia 2020 edition), sucking 10 μ l of the above 3 solutions, respectively dropping on the same silica gel G thin layer plate, spreading with chloroform-ethyl acetate-methanol-water (volume ratio 15: 40: 22: 10) lower layer solution at below 10 deg.C as developing agent, taking out, air drying, spraying 10wt% sulphuric acid ethanol solution, heating at 105 deg.C until the color of spots is clear, and inspecting in sunlight. In the chromatogram of the test sample, spots of the same color appear at the positions corresponding to those of the chromatogram of the control drug, and spots of the same color do not appear at the positions corresponding to those of the chromatogram of the negative test sample (FIG. 12), and the specificity is high.

Example 6 identification of Polygala tenuifolia in amber Anshen Wan (Water-honeyed pill)

1) Preparation of a test solution: taking 20g Succinum tranquilization pill (water honeyed pill), grinding, adding 100ml 70wt% methanol, ultrasonic treating for 30min, filtering, evaporating filtrate, dissolving residue with 2ml methanol to obtain sample solution;

2) preparation of negative test solution: the polygala root in the amber mind tranquilizing pill is removed and is used as a negative test sample. 20g of the negative test sample was taken and prepared into a negative test sample solution according to the above-mentioned method.

3) Preparation of control solutions: grinding cortex et radix Polygalae 0.5g, adding 70wt% methanol 100ml, ultrasonic treating for 30min, filtering, evaporating filtrate, dissolving residue with methanol 2ml to obtain reference solution;

4) spotting and developing: performing thin layer chromatography (general 0502 of 2020 version of Chinese pharmacopoeia) test, sucking 10 μ l of each of the 3 solutions, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (volume ratio 7: 3: 1) lower layer solution as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color are displayed at the positions corresponding to those of the chromatogram of the control drug, and fluorescent spots of the same color are not displayed at the positions corresponding to those of the chromatogram of the negative test sample (FIG. 13), so that the specificity is high.

Example 7 identification of Glycyrrhiza in amber Anshen Wan (Water-honeyed pill)

1) Preparation of a test solution: taking 20g Succinum tranquilization pill (water honeyed pill), grinding, adding ether 100ml, heating and refluxing for 1 hr, filtering, discarding ether solution, adding methanol 50ml into residue, heating and refluxing for 1 hr, filtering, evaporating filtrate to dryness, dissolving residue with 40ml water, extracting with n-butanol for 3 times, 20ml each time, mixing n-butanol solutions, washing with water for 3 times, discarding water solution, evaporating n-butanol solution to dryness, dissolving residue with 1ml methanol to obtain sample solution.

2) Preparation of negative test solution: the liquorice in the amber mind tranquilizing pill is removed and is used as a negative test sample. 20g of the negative test sample was taken and prepared into a negative test sample solution according to the above-mentioned method.

3) Preparation of control solutions: collecting Glycyrrhrizae radix control material 0.5g, grinding, adding ether 100ml, heating and refluxing for 1 hr, filtering, discarding ether solution, adding methanol 50ml into residue, heating and refluxing for 1 hr, filtering, evaporating filtrate to dryness, dissolving residue with water 40ml, extracting with n-butanol for 3 times, 20ml each time, mixing n-butanol solutions, washing with water for 3 times, discarding water solution, evaporating n-butanol solution to dryness, dissolving residue with methanol 1ml, and making into control solution.

4) Spotting and developing: testing by thin layer chromatography (general 0502 of 2020 version of Chinese pharmacopoeia), sucking 10 μ l of each of the 3 solutions, respectively dropping on the same silica gel G thin layer plate, spreading with lower layer solution of chloroform-methanol-water (volume ratio 13: 7: 2) at 10 deg.C or below as developing agent, taking out, air drying, spraying with 10wt% sulphuric acid ethanol solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under sunlight. In the chromatogram of the test sample, spots of the same color appear at the positions corresponding to those of the chromatogram of the control drug, and spots of the same color do not appear at the positions corresponding to those of the chromatogram of the negative test sample (FIG. 14), and the specificity is high.

Example 8 identification of rehmannia in amber Anshen Wan (Water-honeyed pill)

Referring to the identification item (3) in the rehmannia glutinosa medicinal material of the first part of the year 2020 edition of Chinese pharmacopoeia, a rehmannia glutinosa reference medicinal material (121225-. Collecting 7g Succinum tranquilization pill (water honeyed pill), cutting, adding 80 wt% methanol 60ml, ultrasonic treating for 30min, filtering, evaporating filtrate to dryness, dissolving residue with 5ml water, extracting with water saturated n-butanol under shaking for 4 times, 10ml each time, mixing n-butanol solutions, evaporating to dryness, and dissolving residue with 2ml methanol to obtain sample solution. 7g of negative test sample lacking rehmanniae radix is prepared into a negative test sample solution by the same method. Taking rehmanniae radix reference material, and making into reference material solution by the same method.

Performing thin layer chromatography (general rule 0502) test, sucking 5 μ l of the above three solutions, respectively dropping on the same silica gel G thin layer plate, developing with ethyl acetate-methanol-formic acid (volume ratio 16: 0.5: 2) as developing agent, taking out, air drying, soaking the plate with 0.1 wt% 2, 2-diphenyl-1-picrazinyl anhydrous ethanol solution, and air drying. As a result, spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the control solution, but the negative control except rehmanniae radix has interference (FIG. 15), and the specificity is not strong, so that the spot should not be included in the quality control standard.

Example 9 identification of Zizyphi Spinosae semen in Succinum tranquilizing pill (Water-honeyed pill)

Referring to the identification (3) of the spina date seed medicinal material in the first part of the 'Chinese pharmacopoeia' 2020 edition, a spina date seed reference medicinal material (121225-. Collecting 30g Succinum tranquilizing pill (water honeyed pill), cutting, adding 100ml petroleum ether (boiling range 60-90 deg.C), heating and refluxing for 2 hr, filtering, discarding petroleum ether solution, volatilizing residue, adding 60ml methanol, and heating and refluxing for 1 hr. Filtration, evaporation of the filtrate to dryness, and dissolution of the residue in 1ml of methanol to give a test solution. 30g of negative test sample of the jujube kernel lacking the acid is taken to prepare a negative test sample solution in the same way. And preparing 0.5g of spina date seed reference medicinal material into a reference medicinal material solution by the same method.

Sucking 10 μ l of each of the three solutions, respectively dropping on the same silica gel G thin layer plate, developing with water saturated n-butanol as developing agent, taking out, air drying, spraying 1 wt% vanillin sulfuric acid solution, and inspecting under ultraviolet lamp (365 nm). As a result, spots of the same color were not shown at the positions corresponding to the chromatogram of the control drug (FIG. 16), which indicates that the specificity of thin-layer chromatography identification of wild jujube seed is not strong and should not be included in the quality control standards.

Example 10 identification of Ophiopogon japonicus in Succinum tranquilizing pill (Water-honeyed pill)

Referring to the identification of radix ophiopogonis in the first part of the 'Chinese pharmacopoeia' 2020 edition, a radix ophiopogonis reference drug (121225-. Collecting Succinum mind tranquilizing pill (water honeyed pill) 30g, cutting, adding chloroform-methanol (volume ratio of 7: 3) mixed solution 100ml, soaking for 3 hr, ultrasonic treating for 30min, cooling, filtering, evaporating filtrate, and dissolving residue with chloroform 0.5ml to obtain sample solution. 30g of negative test sample lacking radix Ophiopogonis is prepared into negative test sample solution by the same method. 2g of radix Ophiopogonis as reference material is prepared into reference material solution by the same method.

Sucking the three solutions 10 μ l each, dropping on the same silica gel GF₂₅₄Spreading with toluene-methanol-glacial acetic acid (volume ratio 80: 5: 0.1) as developing agent, taking out, air drying, and placing into ultraviolet lamp (2)54 nm). As a result, spots of the same color were observed at the positions corresponding to the chromatogram of the control drug, but the negative control without Ophiopogon japonicus was interfered (FIG. 17), and the specificity was not strong, so it was not suitable to be included in the quality control standards.

Example 11 identification of Poria in Succinum Anshen Wan (Water-honeyed pill)

Referring to the tuckahoe medicinal material identification (3) in the first part of China pharmacopoeia 2020 edition, a tuckahoe reference medicinal material (121225-. Collecting Succinum tranquillizing pill (water honeyed pill) 30g, cutting, adding diethyl ether 100ml, ultrasonic treating for 10 min, filtering, evaporating filtrate, and dissolving residue with methanol 1ml to obtain test solution. Preparing negative sample solution from Poria 30g, and preparing control solution from Poria 0.5 g.

Sucking 10 μ l of each of the three solutions, respectively dropping on the same silica gel G thin layer plate, developing with toluene-ethyl acetate-formic acid (volume ratio 20: 5: 0.5) as developing agent, taking out, air drying, spraying with 2 wt% vanillin sulfuric acid solution-ethanol (volume ratio 4: 1) mixed solution, and heating at 105 deg.C until the spots are clearly developed. As a result, no spot of the same color was observed at the position corresponding to the chromatogram of the control (FIG. 18), so that it was not considered appropriate to be included in the quality control standards.

Example 12 identification of Salvia miltiorrhiza in amber Anshen pills (Water-honeyed pills)

Referring to the identification of salvia miltiorrhiza medicinal material in the first part of China pharmacopoeia 2020 edition, a salvia miltiorrhiza reference medicinal material (121225-. Taking 30g of amber mind-tranquilizing pills (water-honeyed pills), cutting into pieces, adding 60ml of diethyl ether, carrying out ultrasonic treatment for 15 minutes, filtering, volatilizing filtrate, and adding ethyl acetate lml into residues for dissolving to obtain a test solution. 30g of negative test sample lacking Saviae Miltiorrhizae radix is prepared into negative test sample solution by the same method. Taking 0.5g of radix Salviae Miltiorrhizae as reference material, and preparing into reference material solution by the same method.

Sucking 10 μ l of each of the three solutions, respectively dropping on the same silica gel G thin layer plate, developing with n-hexane-ethyl acetate (volume ratio 4: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). As a result, in the chromatogram of the test sample, fluorescent spots of the same color were observed at the positions corresponding to those in the chromatogram of the control drug, but the interference of the negative control (FIG. 19) for removing Salvia miltiorrhiza Bunge was not strong, so this should not be included in the quality control standards.

Example 13 identification of Platycodon grandiflorum in Succinum tranquilizing pill (Water-honeyed pill)

Referring to the identification (3) of the platycodon grandiflorum medicinal material in the first part of the' 2020 edition of the Chinese pharmacopoeia, a platycodon grandiflorum reference medicinal material (121225-. Taking 30g of amber mind tranquilizing pill (water honeyed pill), adding 100ml of 7 wt% sulfuric acid ethanol-water (volume ratio is 1: 3) mixed solution, heating and refluxing for 3 hours, cooling, shaking and extracting with chloroform for 2 times, 20ml each time, combining chloroform solutions, adding water and washing for 2 times, 30ml each time, discarding washing liquid, dehydrating the chloroform solution with anhydrous sodium sulfate, filtering, evaporating filtrate to dryness, and dissolving residue with 1ml of methanol to obtain sample solution. 30g of negative test sample lacking platycodon grandiflorum is prepared into a negative test sample solution by the same method. And preparing 1g of platycodon grandiflorum reference medicinal material into reference medicinal material solution by the same method.

Sucking 10 μ l of each of the three solutions, dropping on the same silica gel G thin layer plate, developing with chloroform-ethyl ether (volume ratio 2: 1) as developing agent, taking out, air drying, spraying 10wt% sulfuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. As a result, no spot of the same color was observed in the chromatogram of the test solution at the position corresponding to the chromatogram of the control solution (FIG. 20), and the method was not feasible, so it was not suitable to be included in the quality control standards.

Example 14 identification of radix scrophulariae in Succinum tranquilizing pill (water-honeyed pill)

Referring to the identification (2) of radix scrophulariae medicinal material in the first part of the 'Chinese pharmacopoeia' 2020 edition, radix scrophulariae reference medicinal materials (121225-. Taking 30g of Succinum tranquilizing pill (water honeyed pill), adding 100ml of methanol, soaking for 1 hr, performing ultrasonic treatment for 30min, filtering, evaporating filtrate to dryness, adding 25ml of water into residue to dissolve, shaking and extracting with water saturated n-butanol for 2 times, 30ml each time, mixing n-butanol solutions, evaporating to dryness, adding 2ml of methanol into residue to dissolve to obtain test solution, taking 30g of negative test sample lacking radix scrophulariae, and preparing the negative test sample solution by the same method. Taking 1g of radix scrophulariae as reference material, and making into reference material solution by the same method.

Sucking 10 μ l of each of the three solutions, respectively dropping on the same silica gel G thin layer plate, placing the lower layer solution of chloroform-methanol-water (volume ratio 12: 4: 1) as developing agent in a developing tank pre-saturated with developing agent for 15 min, developing, taking out, air drying, spraying 5 wt% vanillin-sulfuric acid solution, and blowing hot air until the spots are clear in color. As a result, spots of the same color were not observed at the positions corresponding to the chromatograms of the control drugs (FIG. 21), and the method was not feasible, so it was not suitable for inclusion in the quality control standards.

While the foregoing is directed to the preferred embodiment of the present invention, which is described in some detail and embodiments, the appended claims are not to be interpreted as limiting, and it is to be understood that various changes, modifications, and improvements may be made without departing from the spirit and scope of the invention.

Claims

1. A quality control method of amber mind-tranquilizing pills is characterized in that the amber mind-tranquilizing pills are water-honeyed pills and are characterized in that: identifying fructus Schisandrae chinensis, Glycyrrhrizae radix, and semen Ziziphi Spinosae in Succinum tranquilization pill by microscopic identification; identifying radix Angelicae sinensis, fructus Schisandrae chinensis, Ginseng radix, cortex et radix Polygalae and Glycyrrhrizae radix in Succinum tranquilization pill by thin layer chromatography; and (4) measuring the content of the schisandrin in the amber mind-tranquilizing pills by an HPLC method.

2. The quality control method according to claim 1, wherein: the microscopic identification method comprises the following operation steps: mixing the amber mind tranquilizing pills with chloral hydrate, tabletting, and observing the properties of the schisandra chinensis, the liquorice and the spina date seeds in the tablets under a microscope.

3. The quality control method according to claim 1, wherein: the method for identifying the angelica in the amber tranquilization pill by using the thin-layer chromatography comprises the following steps:

3) spotting and developing: according to the thin layer chromatography test recorded in Chinese pharmacopoeia, 10 mul of each of the test solution and the reference solution is absorbed and respectively spotted on the same silica gel G thin layer plate, n-hexane-ethyl acetate with the volume ratio of 4:1 is used as a developing agent for development, the silica gel G thin layer plate is taken out after the development, the silica gel G thin layer plate is dried in the air and is inspected under ultraviolet light, and fluorescent spots with the same color are displayed on the positions corresponding to the reference solution chromatogram in the test solution chromatogram.

4. The quality control method according to claim 1, wherein: the step of identifying the schisandra chinensis in the amber tranquilization pill by thin-layer chromatography comprises the following steps:

3) spotting and developing: taking out 2 μ of each of the test solution and the reference solution according to the thin layer chromatography test recorded in Chinese pharmacopoeial, respectively dropping on the same silica gel GF₂₅₄Developing the thin-layer plate by using an upper-layer solution of petroleum ether-ethyl formate-formic acid with a boiling range of 30-60 ℃ in a volume ratio of 15: 5: 1 as a developing agent, and taking out the silica gel GF after development₂₅₄And (4) inspecting the thin-layer plate under an ultraviolet lamp, wherein spots with the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution.

5. The quality control method according to claim 1, wherein: the method for identifying the ginseng in the amber sedative pill by using the thin-layer chromatography comprises the following steps:

1) preparation of a test solution: taking 20g of amber mind-tranquilizing pills, grinding, adding 100ml of trichloromethane, heating and refluxing for 1 hour, filtering to obtain medicine residues, volatilizing a solvent from the medicine residues, adding 2ml of water, stirring and wetting, adding 50ml of saturated n-butyl alcohol, carrying out ultrasonic treatment for 30 minutes, sucking supernatant, adding 3 volumes of ammonia test solution, shaking uniformly, standing for layering, taking supernatant, evaporating to dryness, and adding methanol lml into residues to dissolve the residues to obtain a test solution;

3) spotting and developing: referring to the thin layer chromatography test recorded in Chinese pharmacopoeia, sucking 10 μ l of each of the test solution and the reference solution, respectively dropping on the same silica gel G thin layer plate, using the lower layer solution of chloroform-ethyl acetate-methanol-water at a volume ratio of 15: 40: 22: 10 and placed below 10 ℃ as a developing agent, taking out the silica gel G thin layer plate after development, drying in the air, spraying 10wt% sulfuric acid ethanol solution, heating at 105 ℃ until the color development of the spots is clear, and observing under sunlight, wherein in the chromatogram of the test solution, the spots with the same color appear at the positions corresponding to the chromatogram of the reference solution.

6. The quality control method according to claim 1, wherein: the thin-layer chromatography method for identifying polygala tenuifolia in the amber tranquilization pill comprises the following steps:

3) spotting and developing: according to the thin layer chromatography test recorded in Chinese pharmacopoeia, 10 mul of each of the test solution and the reference solution is absorbed and respectively spotted on the same silica gel G thin layer plate, the lower layer solution of trichloromethane-methanol-water with the volume ratio of 7: 3: 1 is taken as a developing agent, the silica gel G thin layer plate is taken out after being developed, the silica gel G thin layer plate is dried in the air and is placed under an ultraviolet lamp for inspection, and fluorescent spots with the same color appear in the chromatogram of the test solution at the position corresponding to the chromatogram of the reference solution.

7. The quality control method according to claim 1, wherein: the step of identifying the liquorice in the amber sedative pill by thin-layer chromatography comprises the following steps:

3) spotting and developing: according to the thin layer chromatography test recorded in Chinese pharmacopoeia, 10 mul of each of the test solution and the reference solution is absorbed and respectively spotted on the same silica gel G thin layer plate, the lower layer solution which is placed at the temperature of below 10 ℃ by the volume ratio of 13: 7: 2 of chloroform-methanol-water is taken as a developing agent, the silica gel G thin layer plate is taken out after being developed, the silica gel G thin layer plate is dried in the air, 10wt% of sulfuric acid ethanol solution is sprayed, the silica gel G thin layer plate is heated at the temperature of 105 ℃ until the spots are clearly developed, the test solution is inspected under sunlight, and the spots with the same color appear on the positions corresponding to the reference solution chromatogram in the test solution.

8. The quality control method according to claim 1, wherein: the HPLC method for determining the content of the schizandrol A comprises the following steps:

2) preparation of control solutions: taking a proper amount of schizandrol A reference substance, precisely weighing, and adding methanol to obtain a solution of 19.5-20.5 μ g/ml each time, to obtain a reference substance solution;

9. The quality control method according to claim 8, wherein: in the HPLC method, the ultrasonic power is 100W and the ultrasonic frequency is 40 kHz.

10. The quality control method according to claim 1 or 8, wherein: per 1g of Succinum tranquilizing pill, the content of schisandrin is not less than 0.18 mg.