Method for detecting quality of pinellia ternate, bighead atractylodes rhizome and gastrodia elata granules

Abstract

The invention relates to the technical field of quality detection of traditional Chinese medicine preparations, in particular to a quality detection method of rhizoma pinelliae, rhizoma atractylodis macrocephalae and rhizoma gastrodiae granules, and the rhizoma pinelliae, rhizoma atractylodis macrocephalae and rhizoma gastrodiae granules established by the invention are proved to be safe and effective through quality inspection. The rhizoma pinelliae, rhizoma atractylodis macrocephalae and rhizoma gastrodiae granules not only keep the effects of eliminating dampness, reducing phlegm, calming the liver and calming endogenous wind in an original prescription, but also overcome the defects that a decoction is temporarily decocted, time-consuming and energy-consuming, is easy to mildew and deteriorate after being placed for a long time and the like, are smaller in dosage and more convenient to take, carry, store and transport, and greatly reduce the labor intensity and shorten the labor time, so that the production cost of the rhizoma pinelliae, rhizoma atractylodis macrocephalae and rhizoma gastrodiae granules is reduced. And the burden of people is greatly relieved, the market vacancy is filled, the economic benefit is considerable, and obvious advantages are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of quality detection of traditional Chinese medicine preparations, and in particular to a quality detection method for Pinellia, Atractylodes and Gastrodia elata granules. Background Art

[0002] With the improvement of people's living standards and the enhancement of health awareness, the demand for traditional Chinese medicine industry in the market has increased year by year due to its long history, unique therapeutic effects and ability to prevent and treat many common diseases and chronic diseases.

[0003] Pinellia, Atractylodes, and Gastrodia are mentioned in "Medical Enlightenment," written by Cheng Guopeng, a renowned Qing Dynasty physician. They are known for their ability to resolve phlegm, extinguish wind, strengthen the spleen, and dispel dampness, primarily treating symptoms of wind-phlegm disturbance. On August 1, 2022, the State Administration of Traditional Chinese Medicine published the "Key Information Table of Ancient Classic Prescriptions (25 Prescriptions)" and included Pinellia, Atractylodes, and Gastrodia as Prescription 23. Pinellia, Atractylodes, and Gastrodia are composed of eight herbs: Pinellia (Qing Banxia), Gastrodia, Poria, Tangerine Peel, Atractylodes, Licorice, Ginger, and Jujube. Atractylodes macrocephala, Pinellia ternata, and Tangerine peel are warm in nature, and have the effects of drying dampness and resolving phlegm, strengthening the spleen and invigorating qi, and drying dampness and promoting diuresis; Gastrodia elata has the effects of calming wind and stopping spasms, calming liver yang, and dispelling wind and unblocking meridians; Poria cocos has the effects of promoting diuresis and removing dampness, strengthening the spleen, and calming the mind; Licorice and jujube have the effects of invigorating qi, clearing away heat and detoxifying, nourishing blood and tranquilizing the mind; Ginger belongs to the lung, spleen, and stomach meridians, and has the effects of dispelling exterior pathogens and cold, warming the middle and stopping vomiting.

[0004] Due to numerous issues with traditional decoctions, such as poor taste and inconvenience in portability, the classic "bowl of soup" formula remains limited in clinical practice. Consequently, researchers are exploring modern solutions for decoctions. Traditional Chinese medicine granules, made from slices of Chinese herbal medicine using modern processing techniques, offer the same therapeutic benefits. Granules offer numerous advantages, including controlled quality, clear efficacy, convenient transportation and storage, portability, clear mechanisms of action, and widespread application.

[0005] For example, Chinese patent application number CN115704806A discloses a quality inspection method for Banxia Baizhu Tianma decoction. This method only studies the Banxia Baizhu Tianma decoction and does not examine the Banxia Baizhu Tianma granules. Quality inspection methods vary widely between different production processes, making it impossible to apply the inspection method from one process to products from another.

[0006] For example, the Chinese patent application number: CN108956843A discloses a method for rapid multi-information thin-layer identification of freeze-dried powder of Pinellia, Atractylodes, and Gastrodia elata decoction. This method only qualitatively identifies the four medicinal ingredients of the compound, namely, fried Atractylodes macrocephala, licorice, Gastrodia elata, and dried orange peel, from the perspective of thin-layer qualitative identification, and cannot fully reflect the overall properties of the compound. Although the sample preparation method of this method is simple, the color clarity and separation of the spots on each thin-layer are poor. There is a phenomenon of spot overlap on the dried orange peel and Gastrodia elata identification thin-layer plates, and the amount of characteristic spots of each medicinal ingredient is relatively small. In addition, the thin-layer development method of Gastrodia elata and dried orange peel disclosed in this patent involves secondary development, which takes a long time and is a bit cumbersome, and needs to be further improved.

[0007] For example, Chinese patent application number CN114748599A discloses a composition of Pinellia, Atractylodes, and Gastrodia elata granules, as well as its preparation and use. This method focuses solely on the preparation and research of Pinellia, Atractylodes, and Gastrodia elata granules from the perspective of industrial production. Quality control studies were not conducted on the pilot product, and the extraction process involved was not optimized or validated.

[0008] In view of the above situation, in order to overcome the above technical problems, the present invention designs a quality detection method for Pinellia, Atractylodes and Gastrodia elata granules to solve the above technical problems. Summary of the Invention

[0009] The technical purpose to be achieved by the present invention is: the purpose of the present invention is to provide a quality detection method for Pinellia, Atractylodes and Gastrodia elata granules, which can be qualitatively analyzed as a whole, has a short analysis cycle, and is suitable for the overall quality control of Pinellia, Atractylodes and Gastrodia elata granules preparations.

[0010] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0011] A method for detecting the quality of Pinellia, Atractylodes and Gastrodia elata granules comprises the following steps:

[0012] Step 1: Soak the raw medicinal material mixture of the Banxia Baizhu Tianma granule composition in 1500 ml of water respectively, filter every 30 minutes, and measure the volume;

[0013] Step 2: Nine orthogonal experiments with three factors and three levels were conducted on the extracted mixture. The three factors of the orthogonal experiments were set as the amount of water added, decoction time, and number of decoctions. The gastrodin content and extract rate were the subjects of investigation, with weights of 0.6 and 0.4, respectively. The comprehensive scores were used as the evaluation indicators to determine the extraction conditions. Combined with variance analysis, the optimal extraction process was screened.

[0014] Step 3: Repeat the extraction process three times, extract, filter, and concentrate according to the preferred experimental conditions, and determine the extract yield and gastrodin content according to the above method;

[0015] Step 4: Develop a time / temperature color change curve for the index component, select the index component with the largest loss rate as the primary evaluation criterion, and the index component with relatively small change as the secondary criterion. The characteristic spectrum inspection and evaluation criteria are evaluated based on the similarity of the samples before and after concentration (warm immersion). Gastrodin is used as the object of investigation, and the content of gastrodin after concentration under different temperature conditions is analyzed;

[0016] Step 5: Take 5-8g of the granules obtained by the above extraction and concentration, spread them flat on a tray, place them in a forced air drying oven, and investigate the effects of different drying temperatures and times on moisture and gastrodin content;

[0017] Step 6: Take 358.5g of the paste made from the raw medicinal material mixture, add an appropriate amount of auxiliary material sucrose, mix for different times, and pass through sieves with different mesh sizes to observe the particle formation;

[0018] Step 7: Perform thin layer chromatography identification of Pinellia, Poria and Atractylodes on the Banxia Baizhu Tianma granules to obtain relevant data;

[0019] Step 8: Determine the content of the Banxia Baizhu Tianma granules, determine the chromatographic conditions, prepare the solution, and then conduct a specificity experiment;

[0020] Step 9: Conduct linear relationship experiments on Pinellia, Atractylodes and Gastrodia Granules, including gastrodin linearity experiment, p-hydroxybenzyl alcohol linearity experiment, precision experiment, stability experiment, recovery experiment, method robustness investigation and sample determination.

[0021] Preferably, the Pinellia, Atractylodes and Gastrodia elata granules are composed of 56 grams of Pinellia, 37.3 grams of Gastrodia elata, 37.3 grams of Poria, 37.3 grams of Citrus aurantium, 111.9 grams of Atractylodes, 18.7 grams of Licorice, 30.0 grams of Ginger and 60.0 grams of Jujube.

[0022] Preferably, the thin layer chromatography identification of Poria cocos in step seven includes taking 6 g of the powder of the product, adding 50 ml of ether, ultrasonically treating for 10 minutes, filtering, evaporating the filtrate to dryness, adding 1 ml of methanol to the residue to dissolve it as the test solution, and taking 1 g of Poria cocos control medicinal material as the control medicinal material solution. According to the thin layer chromatography test, 5 μL of each of the above two solutions are taken and spotted on the same silica gel G thin layer plate, using toluene-ethyl acetate-formic acid (20:5:0.5) as the developing agent, developing, taking out, drying, spraying with a 2% vanillin sulfuric acid solution-ethanol (4:1) mixed solution, and heating at 105°C until the spots are clearly colored.

[0023] Preferably, the thin layer chromatography identification of Atractylodes macrocephala in step seven includes weighing 20 g of the formula granules, adding 100 ml of water, ultrasonicating for 30 min, filtering, extracting with ether 3 times, 20 ml each time, combining the ether solution, evaporating to dryness, and adding 1 ml of methanol to the residue to dissolve it as the test solution; taking another 0.5 g of Atractylodes macrocephala control medicinal material as the control medicinal material solution, testing according to the thin layer chromatography method, taking 5 μl of each of the above two solutions, and spotting them on the same silica gel G thin layer plate, using petroleum ether (60-90°C)-ethyl acetate (5:1) as the developing agent, developing, taking out, drying, spraying with 10% sulfuric acid ethanol solution, heating at 105°C until the spots are clearly colored, and placing them under ultraviolet light (365 nm) for inspection.

[0024] Preferably, the linear experiment of gastrodin in step nine includes weighing 20.47 mg of gastrodin reference substance, placing it in a 20 ml volumetric flask, dissolving it with a diluent and diluting it to the scale, shaking it well, and using it as a gastrodin reference substance stock solution; accurately measuring the gastrodin stock solution and placing it in different volumetric flasks to prepare solutions containing different contents of gastrodin, respectively, and measuring them according to the chromatographic conditions, with the gastrodin concentration as the horizontal coordinate and the peak area as the vertical coordinate, to obtain a linear regression equation.

[0025] Preferably, the linearity test of p-hydroxybenzyl alcohol in step nine includes: accurately weighing 15.48 mg of a p-hydroxybenzyl alcohol reference substance, placing it in a 20 ml volumetric flask, dissolving it with a diluent and diluting it to the scale, shaking it well, and using it as a p-hydroxybenzyl alcohol reference substance stock solution; accurately measuring the p-hydroxybenzyl alcohol stock solution and placing it in different volumetric flasks to prepare solutions containing different contents of gastrodin, respectively, and measuring them according to chromatographic conditions, with the p-hydroxybenzyl alcohol concentration as the horizontal axis and the peak area as the vertical axis.

[0026] Preferably, the recovery experiment in step nine includes: accurately weighing 14.23 mg of the gastrodin reference substance, placing it in a 50 ml volumetric flask, dissolving it with a diluent and diluting it to the scale as a stock solution (1); accurately weighing 10.06 mg of the p-hydroxybenzyl alcohol reference substance, placing it in a 500 ml volumetric flask, dissolving it with a diluent and diluting it to the scale as a stock solution (2); making an acidic blank excipient solution, accurately measuring 3 parts of each of the different weights of the stock solution (1), placing them in the above-mentioned 9 20 ml volumetric flasks, diluting them to the scale with a diluent, shaking them well, filtering them, using them as test solutions, injecting them into HPLC, and substituting them into the regression equation to calculate the recovery of gastrodin; accurately measuring 3 parts of each of the different weights of the stock solution (2), placing them in the other 9 20 ml volumetric flasks mentioned above, diluting them to the scale with a diluent, shaking them well, filtering them, using them as test solutions, injecting them into HPLC, and substituting them into the regression equation to calculate the recovery of p-hydroxybenzyl alcohol.

[0027] The beneficial effects of the present invention are as follows:

[0028] (1) The present invention takes the industrially optimized Banxia Baizhu Tianma granules as the research object, and establishes a quality detection method for the Banxia Baizhu Tianma granules, which provides a research basis for the rational development and application of the granules. On the one hand, the quality detection method for the Banxia Baizhu Tianma granules provided by the present invention can be used for overall qualitative research. On the other hand, in view of the problems such as the complex chemical composition of the traditional Chinese medicine compound, the many interfering components in thin layer identification, and the poor spot recognition and separation, the impurity removal and optimization of the test sample processing method are carried out in a targeted manner according to the polarity characteristics of different components in the medicinal taste. The selected developing agent is simple to prepare and helps to obtain a chromatogram with clear and rich spots. The color developer used has a fast color development speed, obvious color development, and good separation effect.

[0029] (2) The Banxia Baizhu Tianma granules prepared by the present invention have been proven to be safe and effective through quality inspection. Taking full account of practicality and effectiveness and based on previous research, the present invention not only retains the effects of the original prescription on drying dampness and resolving phlegm, calming the liver and relieving wind, but also overcomes the shortcomings of the decoction, such as temporary decoction, time-consuming and energy-consuming, and easy to mold and spoil after long-term storage. The dosage is also reduced, and the administration, carrying, storage, and transportation are all more convenient. The Banxia Baizhu Tianma granules greatly reduce labor intensity, shorten working hours, greatly alleviate people's burden, fill a market gap, have considerable economic benefits, and have obvious advantages.

[0030] (3) In terms of characteristic spectrum research, the present invention establishes a characteristic spectrum for Banxia Baizhu Tianma Granules based on the importance of prescription drug compatibility and the key indicators of each drug. The characteristic spectrum is characterized by high water-soluble and high-content specific components in each drug, such as gastrodin and p-hydroxybenzyl alcohol in Gastrodia elata. Furthermore, the present invention simply and quickly implements the overall evaluation of Banxia Baizhu Tianma Granules, with a short analysis cycle and strong practical operability, which is beneficial for cost savings and efficiency improvements. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] The above and other aspects of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0033] Figure 1 It is a schematic flow chart of the method of the present invention;

[0034] Figure 2 (A) is a daylight inspection view of the present invention;

[0035] Figure 2 (B) is an inspection view under 366nm ultraviolet light of the present invention;

[0036] (Note: In the figure, 1. Poria cocos control medicinal material; 2. Test sample 1; 3. Test sample 2; 4. Test sample; 5. Poria cocos negative control is missing.)

[0037] Figure 3 This is a specific experimental diagram of the reference solution of the present invention;

[0038] Figure 4 This is a diagram showing the specificity of the test solution of the present invention;

[0039] Figure 5 This is a graph showing the specificity of the blank control solution of the present invention;

[0040] Figure 6 This is a standard curve diagram of the gastrodin reference substance of the present invention;

[0041] Figure 7 This is a standard curve diagram of the p-hydroxybenzyl alcohol reference substance of the present invention;

[0042] Figure 8 (A) is a daylight inspection view of the present invention;

[0043] Figure 8 (B) is an inspection view under 366nm ultraviolet light of the present invention.

[0044] Note: Figure 8 (A) 1: Pinellia ternata control (from top to bottom, leucine control, valine control, alanine control); 2, 4, 6: test sample granules; 3: Pinellia ternata control; 5: negative control without Pinellia ternata; DETAILED DESCRIPTION

[0045] In order to better understand the above technical solution, the above technical solution will be described in detail below with reference to the accompanying drawings and specific implementation methods.

[0046] like Figure 1-8 As shown, a method for detecting the quality of Pinellia, Atractylodes and Gastrodia elata granules comprises the following steps:

[0047] Step 1: Soak the raw medicinal material mixture of the Banxia Baizhu Tianma granule composition in 1500 ml of water respectively, filter every 30 minutes, and measure the volume;

[0048] Step 2: Nine orthogonal experiments with three factors and three levels were conducted on the extracted mixture. The three factors of the orthogonal experiments were set as the amount of water added, decoction time, and number of decoctions. The gastrodin content and extract rate were the subjects of investigation, with weights of 0.6 and 0.4, respectively. The comprehensive scores were used as the evaluation indicators to determine the extraction conditions. Combined with variance analysis, the optimal extraction process was screened.

[0049] Step 3: Repeat the extraction process three times, extract, filter, and concentrate according to the preferred experimental conditions, and determine the extract yield and gastrodin content according to the above method;

[0050] Step 4: Develop a time / temperature color change curve for the index component, select the index component with the largest loss rate as the primary evaluation criterion, and the index component with relatively small change as the secondary criterion. The characteristic spectrum inspection and evaluation criteria are evaluated based on the similarity of the samples before and after concentration (warm immersion). Gastrodin is used as the object of investigation, and the content of gastrodin after concentration under different temperature conditions is analyzed;

[0051] Step 5: Take 5-8g of the granules obtained by the above extraction and concentration, spread them flat on a tray, place them in a forced air drying oven, and investigate the effects of different drying temperatures and times on moisture and gastrodin content;

[0052] Step 6: Take 358.5g of the paste made from the raw medicinal material mixture, add an appropriate amount of auxiliary material sucrose, mix for different times, and pass through sieves with different mesh sizes to observe the particle formation;

[0053] Step 7: Perform thin layer chromatography identification of Pinellia, Poria and Atractylodes on the Banxia Baizhu Tianma granules to obtain relevant data;

[0054] Step 8: Determine the content of gastrodin and p-hydroxybenzyl alcohol in the Banxia Baizhu Tianma granules, and after determining the chromatographic conditions, prepare the solution and then conduct a specificity experiment;

[0055] Step 9: Conduct linear relationship experiments on Pinellia, Atractylodes and Gastrodia Granules, including gastrodin linearity experiment, p-hydroxybenzyl alcohol linearity experiment, precision experiment, stability experiment, recovery experiment, method robustness investigation and sample determination.

[0056] The Pinellia, Atractylodes and Gastrodia elata granules are composed of 56 grams of Pinellia, 37.3 grams of Gastrodia elata, 37.3 grams of Poria, 37.3 grams of Citrus aurantium, 111.9 grams of Atractylodes, 18.7 grams of Licorice, 30.0 grams of Ginger and 60.0 grams of Jujube.

[0057] Weigh the prescription amount of 358.5g of Pinellia (Qing Banxia) 56g, Gastrodia elata 37.3g, Poria 37.3g, Tangerine peel 37.3g, Atractylodes macrocephala 111.9g, Licorice 18.7g, and Jujube 60g as sample 1, and then weigh 717g of 112g Pinellia (Qing Banxia), 74.6g Gastrodia elata, 74.6g Poria 37.3g, Tangerine peel 74.6g, Atractylodes macrocephala 223.8g, Licorice 37.4g, and Jujube 120g as sample 2.

[0058] The following are the processing parameters and qualification standards of relevant medicinal materials:

[0059]

[0060]

[0061] Add 1500 ml of water to the medicinal material mixture prepared above, soak, filter every 30 minutes, and measure the volume.

[0062]

[0063] It can be seen from the data in the table that the water absorption decreases after immersion for 1 hour and almost no water is absorbed, so the immersion time is set to 1 hour.

[0064] L9(33) was used to design 9 groups of orthogonal experiments with 3 factors and 3 levels. The water addition amount was 6 times, 8 times, and 10 times, the decoction time was 1 hour, 1.5 hours, and 2 hours, and the decoction times were 1 time, 2 times, and 3 times as the three factors of the extraction orthogonal experiment. The gastrodin content and extract rate were taken as the investigation objects, with weights of 0.6 and 0.4 respectively. The comprehensive score was used as the investigation index to determine the extraction conditions. Combined with variance analysis, the best extraction process was screened out.

[0065] Orthogonal optimization of the extraction process revealed that the number of decoctions (C) had the greatest impact on the overall score, followed by the amount of water added (A), with decoction time (B) having the least impact. Considering factors such as actual production and economic costs, the final selection criteria were three decoctions, 10-fold water addition, and one hour of decoction time.

[0066] Extraction process orthogonal factor level table

[0067]

[0068] Orthogonal experimental design of extraction process and result table

[0069]

[0070]

[0071] Orthogonal experiment variance analysis results table

[0072]

[0073] Note: F 0.05 (2, 2) = 19.00

[0074] The extraction process was verified and the analysis results showed that the gastrodin content in three parallel experiments was stable, and the difference was small compared with the optimization results of the orthogonal experiment, that is, the initially determined extraction process was stable and feasible.

[0075] Extraction process validation results table

[0076]

[0077] According to the prescription ratio, the extraction process was repeated 3 times, and the extraction, filtration and concentration were carried out according to the preferred experimental conditions. The paste yield and gastrodin content were determined according to the above method.

[0078] A time / temperature color change curve for the index component was developed, selecting the index component with the greatest loss rate as the primary evaluation criterion, and those with relatively small changes as the secondary criterion. A content loss rate of less than 10% was preferred. The characteristic spectrum was evaluated based on the similarity between the samples before and after concentration (warm immersion), requiring a similarity of 0.9 or higher. Gastrodin was used as the investigational subject, and its content after concentration at different temperature conditions was analyzed.

[0079] Take an appropriate amount of the granules obtained by the above extraction and concentration, spread them flat on a tray, and place them in a forced air drying oven to investigate the effects of different drying temperatures and times on the moisture and gastrodin content, and determine the optimal granule drying process parameters.

[0080] The drying process was investigated and analyzed, and it was found that the drying temperature below 90°C had no significant effect on the gastrodin content. In summary, the drying temperature was selected to be 70-90°C to ensure complete drying without burning.

[0081] Granule Drying Process Investigation Results Table

[0082]

[0083] Take a prescribed amount of clear paste, add an appropriate amount of auxiliary material sucrose, mix for different times, pass through sieves with different mesh sizes, and observe the particle formation.

[0084] The results of the molding process showed that the ratio of clear paste to auxiliary materials was 1:6, the mixing time of soft materials was 3 min, and the granulation was performed through a 16-mesh sieve, and the granules had good moldability.

[0085] Molding process inspection results table

[0086]

[0087] like Figure 8 (A) and Figure 8 As shown in (B), thin-layer chromatography identification of Pinellia ternata is as follows: Weigh one bag of formula granules (3.4722 g), add 50 ml of water, heat and reflux for 1 hour, filter, evaporate to dryness, dissolve the residue in 20 ml of methanol, filter, evaporate to dryness, and concentrate to 1 ml to prepare the test solution. Separately, take 0.5 g of Pinellia ternata control medicinal material, add 20 ml of water, heat and reflux for 1 hour, filter, evaporate to dryness, dissolve the residue in 10 ml of methanol, filter, evaporate to dryness, and concentrate to 1 ml to prepare the control medicinal material solution. Separately, take alanine reference substance, valine reference substance, and leucine reference substance, add methanol to prepare a mixed solution containing 1 mg of each per 1 ml, and prepare the control solution. According to the thin layer chromatography method (General Method 0502), 5 μl each of the test sample solution and the control medicinal material solution, and 1 μl of the reference solution, are spotted separately on the same silica gel G thin layer plate. Develop with n-butanol-glacial acetic acid-water (8:3:1) as the developing solvent. Remove, air dry, spray with ninhydrin test solution, and heat at 105°C until the spots are clearly colored. In the chromatogram of the test sample, a spot of the same color will appear at the corresponding position in the chromatogram of the reference sample.

[0088] Poria cocos TLC Identification: Take 6g of this product powder, add 50ml of ether, sonicate for 10min, filter, evaporate the filtrate to dryness, and dissolve the residue in 1ml of methanol as the test solution. Separately, take 1g of Poria cocos control medicinal material, add 20ml of water, decoct for 1h, filter, evaporate to dryness, and dissolve the residue in 1ml of methanol as the control medicinal material solution. Test according to the thin layer chromatography method (General Method 0502). Take 5μl of each of the above solutions and spot them on the same silica gel G thin layer plate. Use toluene-ethyl acetate-formic acid (20:5:0.5) as the developing solvent, develop, remove, dry, and spray with a mixture of 2% vanillin sulfuric acid solution and ethanol (4:1). Heat at 105°C until the spots are clearly colored.

[0089] Identification of Atractylodes macrocephala by thin-layer chromatography: Weigh 20 g (3.4722 g) of the formula granules, add 100 ml of water, sonicate for 30 min, filter, extract with ether three times, 20 ml each time, combine the ether solutions, evaporate to dryness, dissolve the residue in 1 ml of methanol, and use this as the test solution. Separately, take 0.5 g of Atractylodes macrocephala control medicinal material, add 10 ml of water and boil for 30 min, cool, filter, extract with ether three times, 20 ml each time, combine the ether solutions, evaporate to dryness, dissolve the residue in 1 ml of methanol, and use this as the control medicinal material solution. Test according to the thin-layer chromatography method (General Method 0502). Take 5 μl of each of the above two solutions and spot them on the same silica gel G thin-layer plate. Use petroleum ether (60-90°C)-ethyl acetate (5:1) as the developing solvent, develop, remove, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine under ultraviolet light (365 nm).

[0090] Content determination: Chromatographic conditions, welch1timateC184.6×250mm, 5μm; mobile phase: acetonitrile-0.05% phosphoric acid solution (3:97); isocratic elution; flow rate: 1.0ml / min; column temperature: 30℃; injection volume: 20μl; detection wavelength: 220nm; the theoretical plate number, calculated based on the gastrodin peak, should be greater than or equal to 5000.

[0091] Investigation of the concentration process showed that under 100°C, the fluctuation of gastrodin was relatively large, and there was no obvious difference under 80°C and 90°C. Therefore, the extraction and concentration temperature was set at 90°C, and the concentration time was not more than 10 hours.

[0092] Table 2: Gastrodin content determination results at different concentration temperatures and time points

[0093]

[0094] Solution preparation: (1) Preparation of mixed reference solution: Accurately weigh appropriate amounts of gastrodin reference substance and p-hydroxybenzyl alcohol reference substance to prepare stock solutions, accurately measure 5 ml of gastrodin reference substance stock solution and p-hydroxybenzyl alcohol reference substance stock solution, place them in the same 20 ml volumetric flask, dilute to the scale, shake well, and use as mixed reference solution. The mass concentration of gastrodin reference substance is 49.23 μg / ml, and the mass concentration of p-hydroxybenzyl alcohol is 25.03 μg / ml; (2) Preparation of test solution: Take the gastrodin reference substance and p-hydroxybenzyl alcohol as the reference solution. The test sample particles were ground into powder, 4.1667 g were weighed accurately, and placed in a 25 ml volumetric flask. An appropriate amount of diluent was added and ultrasonicated for 30 min to dissolve the particles. The mixture was cooled, diluted to the mark, shaken, filtered, and the filtrate was used as the test sample solution. (3) Preparation of negative test sample solution: Gastrodia elata test sample particles were ground into powder, 4.1667 g were weighed accurately, and placed in a 25 ml volumetric flask. An appropriate amount of diluent was added and ultrasonicated for 30 min to dissolve the particles. The mixture was cooled, diluted to the mark, shaken, filtered, and the filtrate was used as the negative test sample solution.

[0095] Specificity experiment: Take mixed reference solution, test solution and negative test solution and measure according to the above chromatographic conditions.

[0096] Linear relationship experiment: For the linear experiment of gastrodin, accurately weigh 20.47 mg of gastrodin reference substance, place it in a 20 ml volumetric flask, dissolve it with diluent and dilute it to the scale, shake it well, and use it as the gastrodin reference substance stock solution; accurately measure the gastrodin stock solution and place it in different volumetric flasks to make solutions containing 488.72 μg / ml, 195.49 μg / ml, 97.74 μg / ml, 48.87 μg / ml, 24.44 μg / ml, and 9.77 μg / ml of gastrodin, respectively. Determine them according to the chromatographic conditions under "Item 2.3.1", with the gastrodin concentration as the horizontal axis and the peak area as the vertical axis, to obtain the linear regression equation.

[0097] Linearity test of p-hydroxybenzyl alcohol: Accurately weigh 15.48 mg of p-hydroxybenzyl alcohol reference substance, place it in a 20 ml volumetric flask, dissolve it with diluent and dilute to the scale, shake well, and use it as the p-hydroxybenzyl alcohol reference substance stock solution; Accurately measure the p-hydroxybenzyl alcohol stock solution and place it in different volumetric flasks to prepare solutions containing 7.54 μg / ml, 3.77 μg / ml, 1.88 μg / ml, 0.754 μg / ml, 0.377 μg / ml, and 0.188 μg / ml of gastrodin, respectively. Determine them according to the chromatographic conditions under "Item 2.3.1", with the p-hydroxybenzyl alcohol concentration as the horizontal axis and the peak area as the vertical axis.

[0098] Precision experiment: Prepare the mixed reference solution and test solution according to the above steps, inject the sample 6 times continuously according to the above chromatographic conditions, and calculate the retention time and peak area of 6 injections.

[0099] Precision test results table

[0100]

[0101] Stability test: Mix the reference solution and the test solution as described above, and place them at room temperature for 0, 2, 4, 8, 12, and 24 hours before injection according to the above chromatographic conditions.

[0102] Stability test results table

[0103]

[0104] Recovery rate experiment: Accurately weigh 14.23 mg of the gastrodin reference substance, place it in a 50 ml volumetric flask, dissolve it with a diluent and dilute it to the scale as a stock solution (1); accurately weigh 10.06 mg of the p-hydroxybenzyl alcohol reference substance, place it in a 500 ml volumetric flask, dissolve it with a diluent and dilute it to the scale as a stock solution (2); accurately weigh about 3.3336 g, a total of 18 portions, place it in a 20 ml volumetric flask, dissolve it with an appropriate amount of diluent, and dilute it to the scale, shake well, filter, and prepare an acidic blank excipient solution. Accurately measure 3 portions of the stock solution (1) (0.8 ml, 1.0 ml, and 1.2 ml), respectively, and place them in the above 9 20 ml volumetric flasks, dilute to the scale with diluent, shake well, filter, and use them as test solutions. Inject them into HPLC, and substitute the results into the regression equation to calculate the recovery rate of gastrodin. Accurately measure 3 portions of the stock solution (2) (0.8 ml, 1.0 ml, and 1.2 ml), respectively, and place them in the other 9 20 ml volumetric flasks, dilute to the scale with diluent, shake well, filter, and use them as test solutions. Inject them into HPLC, and substitute the results into the regression equation to calculate the recovery rate of p-hydroxybenzyl alcohol.

[0105] Recovery rate of gastrodin

[0106]

[0107] Method durability investigation: Investigation on the durability of chromatographic columns. This experiment investigated the effects of four different chromatographic column lengths on system suitability: Welch1timate C18 4.6×250mm, 5μm; Eclipse1DB-C18 5μm 4.6mm×250mm; 1BridgeTM C18 5μm 4.6×250mm Colμmn; CAPCELLPAK C18 5μm 4.6mm×250mm.

[0108] Durability investigation of high performance liquid chromatography: This experiment investigated the use of two different brands of liquid chromatography instruments, Agilent 1260 and Waterse 2695, to verify the method.

[0109] Sample determination: Take the Banxia Baizhu Tianma granules of batch numbers 241201, 241202 and 241203, treat and determine them according to the preparation method of the test solution above, and calculate the contents of gastrodin and p-hydroxybenzyl alcohol in the Banxia Baizhu Tianma granules.

[0110] Content determination results table

[0111]

[0112]

[0113] The preparation process route is as follows: weigh eight medicinal materials according to the prescription ratio, add 8 times the amount of water, soak for 1 hour, distill and extract for 2 hours, collect the filtrate and filter it through 200 mesh, set aside, add 6 times the amount of water to the residue and boil it for 1 hour, filter it through 200 mesh, and combine the filtrate with the above distilled aqueous solution; concentrate the above combined aqueous extract (temperature is about 90±5°C) to an extract with a relative density of 1.30-1.35 (60°C), filter it through 100 mesh, and collect the paste; granulate and dry; granulate; mix it; and package it to obtain granules.

[0114] Composition of Banxia Baizhu Tianma Granules

[0115]

[0116] Various modifications to the present disclosure will be apparent to those skilled in the art, and the general principles defined herein may be applied to other variations without departing from the scope of the present disclosure. Therefore, the present disclosure is not limited to the examples and designs described herein, but should be given the widest scope consistent with the principles and novel features disclosed herein. Although one or more exemplary embodiments of the present disclosure have been described with reference to the accompanying drawings, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as defined in the appended claims.

Claims

1. A method for detecting the quality of Pinellia, Atractylodes and Gastrodia elata granules, characterized in that: The following steps are involved: Step 1: Soak the raw medicinal material mixture of the Banxia Baizhu Tianma granule composition in 1500 ml of water respectively, filter every 30 minutes, and measure the volume; Step 2: Nine orthogonal experiments with three factors and three levels were conducted on the extracted mixture. The three factors of the orthogonal experiments were set as the amount of water added, decoction time, and number of decoctions. The gastrodin content and extract rate were the subjects of investigation, with weights of 0.6 and 0.4, respectively. The comprehensive scores were used as the evaluation indicators to determine the extraction conditions. Combined with variance analysis, the optimal extraction process was screened. Step 3: Repeat the extraction process three times, extract, filter, and concentrate according to the preferred experimental conditions, and determine the extract yield and gastrodin content according to the above method; Step 4: Develop a time / temperature color change curve for the index component, select the index component with the largest loss rate as the primary evaluation criterion, and the index component with relatively small change as the secondary criterion. The characteristic spectrum inspection and evaluation criteria are evaluated based on the similarity of the samples before and after concentration (warm immersion). Gastrodin is used as the object of investigation, and the content of gastrodin after concentration under different temperature conditions is analyzed; Step 5: Take 5-8g of the granules obtained by the above extraction and concentration, spread them flat on a tray, place them in a forced air drying oven, and investigate the effects of different drying temperatures and times on moisture and gastrodin content; Step 6: Take 358.5g of the paste made from the raw medicinal material mixture, add an appropriate amount of auxiliary material sucrose, mix for different times, and pass through sieves with different mesh sizes to observe the particle formation; Step 7: Perform thin layer chromatography identification of Pinellia, Poria and Atractylodes on the Banxia Baizhu Tianma granules to obtain relevant data; Step 8: Determine the content of gastrodin and p-hydroxybenzyl alcohol in the Banxia Baizhu Tianma granules, determine the chromatographic conditions, prepare the solution, and then conduct a dedicated specificity experiment; Step 9: Conduct linear relationship experiments on Pinellia, Atractylodes and Gastrodia Granules, including gastrodin linearity experiment, p-hydroxybenzyl alcohol linearity experiment, precision experiment, stability experiment, recovery experiment, method robustness investigation and sample determination.

2. A method for detecting the quality of Pinellia, Atractylodes and Gastrodia elata granules according to claim 1, characterized in that: The Pinellia, Atractylodes and Gastrodia elata granules are composed of 56 grams of Pinellia, 37.3 grams of Gastrodia elata, 37.3 grams of Poria, 37.3 grams of Citrus aurantium, 111.9 grams of Atractylodes, 18.7 grams of Licorice, 30.0 grams of Ginger and 60.0 grams of Jujube.

3. A method for detecting the quality of Pinellia, Atractylodes and Gastrodia elata granules according to claim 1, characterized in that: The thin layer chromatography identification of Pinellia ternata in the step seven includes weighing 1 bag (3.4722g) of formula granules, adding 50ml of water, heating and reflux for 1 hour, filtering, evaporating to dryness, dissolving the residue with 20ml of methanol, filtering, evaporating to dryness, and concentrating to 1ml as the test solution, and taking 0.5g of Pinellia ternata control medicinal material, adding 20ml of water, heating and reflux for 1 hour, filtering, evaporating to dryness, dissolving the residue with 10ml of methanol, filtering, evaporating to dryness, and concentrating to 1ml as the control medicinal material solution, and taking alanine reference substance, valine reference substance, and leucine reference substance, adding methanol to make a mixed solution containing 1mg of each per 1ml, as the reference substance solution. According to the thin layer chromatography method (General Rule 0502), 5 μl of the test sample solution, 5 μl of the control medicinal material solution, and 1 μl of the reference sample solution were respectively spotted on the same silica gel G thin layer plate, and developed with n-butanol-glacial acetic acid-water (8:3:1) as the developing agent. The plate was taken out, dried, sprayed with ninhydrin test solution, and heated at 105°C until the spots were clearly colored. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions in the chromatogram of the reference sample.

4. A method for detecting the quality of Pinellia, Atractylodes and Gastrodia elata granules according to claim 1, characterized in that: The thin layer chromatography identification of Poria cocos in step seven includes taking 6 g of the powder of the product, adding 50 ml of ether, ultrasonically treating for 10 minutes, filtering, evaporating the filtrate, adding 1 ml of methanol to dissolve the residue as the test solution, and taking 1 g of Poria cocos control medicinal material as the control medicinal material solution. According to the thin layer chromatography test, 5 μL of each of the above two solutions are respectively taken and spotted on the same silica gel G thin layer plate, using toluene-ethyl acetate-formic acid (20:5:0.5) as the developing solvent, developing, taking out, drying, spraying with a 2% vanillin sulfuric acid solution-ethanol (4:1) mixed solution, and heating at 105°C until the spots are clearly colored.

5. A method for detecting the quality of Pinellia, Atractylodes and Gastrodia elata granules according to claim 1, characterized in that: The thin layer chromatography identification of Atractylodes macrocephala in step seven includes weighing 20 g of the formula granules, adding 100 ml of water, ultrasonicating for 30 minutes, filtering, extracting with ether three times, 20 ml each time, combining the ether solution, evaporating to dryness, and adding 1 ml of methanol to dissolve the residue as the test solution; taking another 0.5 g of Atractylodes macrocephala control medicinal material as the control medicinal material solution, and testing according to the thin layer chromatography method, taking 5 μl of each of the above two solutions, respectively, and spotting them on the same silica gel G thin layer plate, using petroleum ether (60-90°C)-ethyl acetate (5:1) as the developing agent, developing, taking out, drying, spraying with 10% sulfuric acid ethanol solution, heating at 105°C until the spots are clearly colored, and placing under ultraviolet light (365nm) for inspection.

6. A method for detecting the quality of Pinellia, Atractylodes and Gastrodia elata granules according to claim 1, characterized in that: The linear experiment of gastrodin in step nine includes weighing 20.47 mg of gastrodin reference substance, placing it in a 20 ml volumetric flask, dissolving it with a diluent and diluting it to the scale, shaking it evenly, and using it as a gastrodin reference substance stock solution; accurately measuring the gastrodin stock solution and placing it in different volumetric flasks to prepare solutions containing different contents of gastrodin, respectively, measuring them according to chromatographic conditions, with the gastrodin concentration as the horizontal coordinate and the peak area as the vertical coordinate, to obtain a linear regression equation.

7. A method for detecting the quality of Pinellia, Atractylodes and Gastrodia elata granules according to claim 1, characterized in that: The p-hydroxybenzyl alcohol linearity test in step nine includes: accurately weighing 15.48 mg of a p-hydroxybenzyl alcohol reference substance, placing it in a 20 ml volumetric flask, dissolving it with a diluent and diluting it to the mark, shaking it evenly, and using it as a p-hydroxybenzyl alcohol reference substance stock solution; accurately measuring the p-hydroxybenzyl alcohol stock solution and placing it in different volumetric flasks to prepare solutions containing different contents of gastrodin, respectively, and measuring them according to chromatographic conditions, with the p-hydroxybenzyl alcohol concentration as the horizontal axis and the peak area as the vertical axis.

8. A method for detecting the quality of Pinellia, Atractylodes and Gastrodia elata granules according to claim 1, characterized in that: The recovery experiment in step nine includes: accurately weighing 14.23 mg of the gastrodin reference substance, placing it in a 50 ml volumetric flask, dissolving it with a diluent and diluting it to the scale, as a stock solution (1); accurately weighing 10.06 mg of the p-hydroxybenzyl alcohol reference substance, placing it in a 500 ml volumetric flask, dissolving it with a diluent and diluting it to the scale, as a stock solution (2); preparing an acidic blank excipient solution, accurately measuring 3 portions of the stock solution (1) in different amounts, placing them in the above-mentioned 9 20 ml volumetric flasks, diluting them to the scale with a diluent, shaking them well, filtering them, using them as test solutions, injecting them into HPLC, and substituting them into the regression equation to calculate the recovery of gastrodin; accurately measuring 3 portions of the stock solution (2) in different amounts, placing them in the other 9 20 ml volumetric flasks mentioned above, diluting them to the scale with a diluent, shaking them well, filtering them, using them as test solutions, injecting them into HPLC, and substituting them into the regression equation to calculate the recovery of p-hydroxybenzyl alcohol.

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