Processing technology of braised polygala tenuifolia decoction pieces and construction method and identification method of HPLC fingerprint spectrum of braised polygala tenuifolia decoction pieces

Abstract

The invention belongs to the technical field of processing and quality control of traditional Chinese medicine decoction pieces, and particularly relates to a processing technology of simmered polygala tenuifolia decoction pieces, an HPLC fingerprint spectrum of the simmered polygala tenuifolia decoction pieces, a construction method of the HPLC fingerprint spectrum and an identification method of the HPLC fingerprint spectrum. The processing technology of braised polygala tenuifolia is standardized, parameterization of the processing technology is achieved, and the adopted electromagnetic braised medicine machine is safe, stable in performance, environmentally friendly, controllable in parameter, good in batch-to-batch quality consistency of decoction pieces and suitable for industrial production. The method solves the problems of fragility of containers, poor safety, high labor intensity, uneven quality and the like in the traditional braising method, is green and environment-friendly, meets the GMP (Good Manufacturing Practice) requirements, and can improve the production efficiency by more than 20%. The invention further provides the HPLC fingerprint spectrum, a construction method of the HPLC fingerprint spectrum and an HPLC fingerprint spectrum identification method. The simmered polygala tenuifolia decoction pieces can be scientifically identified through the fingerprint spectrum.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation and quality control of Chinese herbal medicine slices, and specifically relates to a preparation process of stewed Polygala root slices, a HPLC fingerprint spectrum of stewed Polygala root, a method for constructing the HPLC fingerprint spectrum, and a method for identifying the HPLC fingerprint spectrum. Background Art

[0002] Polygala is the dried root of Polygala tenuifolia Willd. or Polygalasibirica L. of the Polygalaceae family. It has the effects of calming the mind and improving intelligence, connecting the heart and kidney, removing phlegm, and reducing swelling. It is a commonly used drug for calming the mind and improving intelligence in clinical Chinese medicine. The Ming Dynasty's "Pharmaceuticals" recorded that "it is pungent, heavy and violent, and it stings the throat and tongue, similar to Arisaema and Pinellia ternata". Yan Jie of the Qing Dynasty recorded in "Depei Materia Medica" that "it hurts the throat when used raw", indicating that medical scientists of all dynasties have noticed that raw Polygala has the side effects of "stinging the throat" and vomiting. Therefore, the herbal books of all dynasties emphasized that Polygala must be processed before use. The processing methods of Polygala in all dynasties mainly include licorice soup soaking, licorice boiling, licorice boiling, licorice stewing, etc. Among them, the licorice stewing method is a unique processing technology of the Jianchang Gang in Jiangxi. After stewing, Polygala can not only eliminate the side effect of "stinging the throat", but also enhance its function of improving intelligence and calming the mind.

[0003] The 2008 edition of the "Jiangxi Province Traditional Chinese Medicine Preparation Specifications" records that the preparation method of stewed Polygala is "Take clean licorice, cut it into sections, flatten it, mix it with clean Polygala, put it in a stewing pot, add an appropriate amount of warm water, and cover it; move the pot into the stove, pile dry bran around it, light the fire, and stew for 4 to 6 hours. When the juice in the pot is basically absorbed, take it out, pick out the licorice, and dry it. For every 100kg of Polygala, use 6kg of licorice." This method gives the general steps, but does not disclose the best stewing time, amount of water added, drying temperature and other stewing process parameters. It is difficult to ensure the consistency of quality between batches of slices in actual production. In addition, in this standard, the description of the color of the slices is subjective evaluation such as "dark brown, yellow brown", and the sensory evaluation is not conducive to controlling the quality of traditional Chinese medicine slices. The item of Polygala only includes the properties, microscopic and thin-layer identification, and inspection items (including moisture, total ash, and acid-insoluble ash) of stewed Polygala. The thin-layer is only used as a control medicinal material of Polygala for comparison. It cannot scientifically identify the stewed Polygala slices and evaluate their quality, making it difficult to ensure the quality and clinical efficacy of the stewed Polygala slices.

[0004] Polygala has the effects of calming the mind, improving intelligence, removing phlegm, and reducing swelling. It is used for insomnia, forgetfulness, palpitations, confusion, coughing and expectoration, sores, swelling and pain in the breast caused by disharmony between the heart and kidney. Stewed Polygala alleviates dryness, eliminates numbness, and is mainly used to calm the mind and improve intelligence. It is used for palpitations. At present, most of the studies on the chemical components of Polygala are mainly based on thin layer identification and content determination of a few compounds. Polygala differs in composition before and after stewing with licorice. Only the content of some of the components is determined, and its quality cannot be effectively, comprehensively, and objectively evaluated or controlled. Based on this, it is necessary to address the problem of limitations of traditional technologies. The main purpose of the present invention is to provide a method for constructing HPLC fingerprints and identification methods for raw Polygala and its stewed products, which can intuitively reflect the characteristics of complex components in raw Polygala and its stewed products as a whole and provide similarity analysis, and then carry out its quality evaluation and identification of raw and finished products, which has certain guiding significance for the quality standard control of raw Polygala and stewed Polygala.

[0005] Compared with ordinary Polygala, the cost of stewed Polygala is higher, so sometimes it is easy to mix stewed Polygala with Polygala, and it is inconvenient to distinguish the two with the naked eye. Therefore, distinguishing between stewed Polygala and raw Polygala has certain guiding significance for clinical standardized drug use.

[0006] Patent CN113304194A discloses a process for preparing braised Polygala, which adopts the stewing method to prepare Polygala. The content of key substances contained in the Polygala processed by this method is within the effective range, which reduces the toxic effects of raw Polygala; the processing temperature curve fitted by repeated simulation experiments of imitating the ancient method of processing is used for the processing of Polygala, which is more energy-saving, stable, safe and environmentally friendly compared to the traditional manual operation based on experience; the electric heating jacket assists in heating, and the electric heating jacket contains three groups of heating components, which can control the heating separately according to the amount of liquid medicine in the tank, ensuring accurate heating, while saving energy and being environmentally friendly. In addition, compared with the traditional licorice-assisted stewing, the present invention adopts pretreated licorice and Polygala for stewing together. After the stewing, the Polygala fully absorbs the Polygala juice and the licorice juice, which helps to improve the efficacy of Polygala and reduce the toxic and side effects of raw Polygala. The stewed Polygala obtained by this process, Polygala The effective content of ketone III ranges from 0.18% to 0.25%, the effective content of 3,6-dicercosyl sucrose ester ranges from 0.5% to 0.6%, and the effective content of Polygala tenuifolia saponin is above 2.0%, but the stewed Polygala tenuifolia was not identified.

[0007] In order to solve the above problems, it is urgently necessary to provide a standardized preparation process of stewed Polygala root slices and HPLC fingerprint of stewed Polygala root, a method for constructing HPLC fingerprint and an HPLC fingerprint identification method to ensure the uniformity and controllability of the quality of stewed Polygala root slices, further improve the clinical efficacy of medication, which will greatly promote the industrialization process of stewed Polygala root and its application scope. Summary of the invention

[0008] In view of the fact that the processing technology in the prior art is not standardized, the identification of medicinal pieces is highly subjective, there is no objective evaluation index, and the traditional stewing method has the problems of high labor cost, low safety, fragile containers, unstable product quality, etc., the present invention aims to provide a standardized processing technology for stewed Polygala pieces and an HPLC fingerprint of stewed Polygala, a method for constructing an HPLC fingerprint and a method for identifying the HPLC fingerprint.

[0009] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0010] The invention provides a processing process for stewed Polygala tenuifolia slices, comprising the following steps:

[0011] Take clean licorice segments, flatten them, mix them with clean Polygala segments, put them in an electromagnetic stew pot, add 2 times the amount of water, heat and stew, keep it slightly boiling for 6 hours, until there is no irritation in the throat when chewing, let it cool, take it out, pick out the licorice, and use the juice for later use; dry Polygala to 50% dry, add the juice and mix well, wait until the juice is absorbed, and dry it at 70℃. For every 100kg Polygala, use 6kg of licorice.

[0012] In the present invention, the purified licorice and purified polygala are purified licorice and polygala medicinal materials. Purification is a processing method in which the prescribed medicinal parts of the Chinese medicinal materials are selected before cutting, roasting, mixing or preparing, and the impurities in the non-medicinal parts and moldy, worm-eaten, ash and the like are removed to make them reach the medicinal purity standard.

[0013] Licorice (scientific name: Glycyrrhiza uralensis Fisch) is also known as Guolao, sweet grass, Ural licorice, and sweet root. It is a perennial herb of the Leguminosae family and the genus Glycyrrhiza. Its medicinal parts are the roots and rhizomes. Its medicinal properties are cylindrical. Licorice segments are licorice segments obtained by cutting the medicinal parts of licorice. Its medicinal properties are cylindrical. Flattening refers to beating the cylindrical shape into a flat cylindrical shape.

[0014] The HPLC fingerprint of stewed Polygala tenuifolia slices prepared by the above-mentioned processing process includes 12 characteristic peaks, and the relative retention time RSD values ​​of the characteristic peaks are within 11%. The relative retention times of the 12 characteristic peaks are: peak 1: 0.396-0.416, peak 2: 0.426-0.435, peak 3: 0.753-0.758, peak 4: 0.877-0.881, peak 5: 1.00, peak 6: 1.133-1.137, peak 7: 1.388-1.442, peak 8: 1.444-1.504; peak 9: 1.659-1.674; peak 10: 1.685-1.699; peak 11: 1.699-1.728; peak 12: 2.237-2.249.

[0015] Preferably, in the HPLC fingerprint of the above-mentioned stewed Polygala tenuifolia slices, the RSD value of the relative retention time of the characteristic peaks is within 5%, and the relative retention times of the 12 characteristic peaks are: peak 1: 0.40, peak 2: 0.43, peak 3: 0.75, peak 4: 0.88, peak 5: 1.00, peak 6: 1.14, peak 7: 1.43, peak 8: 1.49; peak 9: 1.67; peak 10: 1.69; peak 11: 1.72; peak 12: 2.25.

[0016] More preferably, in the HPLC fingerprint of the above-mentioned stewed Polygala tenuifolia slices, the relative retention time RSD value of the characteristic peak is within 2%.

[0017] The above-mentioned method for constructing the HPLC fingerprint comprises the following steps:

[0018] Chromatographic conditions: The chromatographic column was filled with octadecylsilane bonded silica gel; acetonitrile was used as mobile phase A, 0.05% phosphoric acid solution was used as mobile phase B, the detection wavelength was 320 nm; the column temperature was 30°C; the flow rate was 1 ml per minute;

[0019] The gradient elution procedure was as follows: 0 min → 10 min, 10% mobile phase A, 90% mobile phase B linearly changed to 12% mobile phase A, 88% mobile phase B;

[0020] 10min→25min, 12% mobile phase A, 88% mobile phase B linearly changed to 25% mobile phase A, 75% mobile phase B;

[0021] 25min→40min, 25% mobile phase A, 75% mobile phase B linearly changed to 26% mobile phase A, 74% mobile phase B;

[0022] 40 min → 41 min, 26% mobile phase A, 74% mobile phase B linearly changed to 31% mobile phase A, 69% mobile phase B;

[0023] 41 min → 51 min, 31% mobile phase A, 69% mobile phase B;

[0024] 51min→52min, 31% mobile phase A, 69% mobile phase B linearly changed to 41% mobile phase A, 59% mobile phase B;

[0025] 52min→66min, 41% mobile phase A, 59% mobile phase B linearly changed to 46% mobile phase A, 54% mobile phase B;

[0026] Preparation of reference solution Take an appropriate amount of 3,6'-dicerinoyl sucrose reference substance, weigh accurately, and add methanol to make a solution containing 0.1 mg per 1 ml.

[0027] Preparation of test solution: About 0.3 g of stewed Polygala powder (passed through No. 3 sieve) was accurately weighed and placed in a stoppered conical flask. 20 ml of 70% methanol was accurately added, the flask was sealed, and the weight was determined. Ultrasonic treatment (500 W, 40 kHz) was performed for 30 min. The flask was taken out and cooled, and 70% methanol was added to make up for the lost weight. The filtrate was obtained by shaking well and filtering.

[0028] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, measure, and record the chromatogram.

[0029] Preferably, in the above-mentioned method for constructing HPLC fingerprint, the specification of the chromatographic column is 250mm×4.6mm, 5μm.

[0030] Preferably, in the above-mentioned method for constructing the HPLC fingerprint, the conditions for the ultrasonic treatment are 500W, 40kHz.

[0031] According to the HPLC fingerprint identification method of stewed Polygala tenuifolia slices obtained by the above-mentioned HPLC fingerprint construction method, the test sample fingerprint should present chromatographic peaks with the same retention time as the reference chromatographic peaks; calculated according to the Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity between the test sample fingerprint and the control fingerprint shall not be less than 0.90.

[0032] In the processing process of stewed Polygala tenuifolia slices of the present invention, the electromagnetic stewing medicine jar used is a QWY300-00 type automated electromagnetic stewing medicine jar. The QWY300-00 type automated electromagnetic stewing medicine jar is rolled up with a stainless steel plate, the interior of which is sprayed with Teflon, the bottom is a fan-shaped round bottom, and the top is an outward-turned trumpet mouth, which is convenient for loading and unloading. The outer ring of the jar is wrapped with high-temperature mica wire, and the jar body is wrapped with thermal insulation cotton. The jar body is heated by an electromagnetic controller. The characteristics are that the temperature of the stewing process is controlled in sections, which can be adjusted and set; the quantitative addition of water or auxiliary liquid can be automatically controlled during the stewing process to realize intelligent process control, which meets the requirements of GMP specifications; after the stewing is completed, the material can be automatically dumped out without dead angles, the stewing effect is good, and it is easy to clean.

[0033] The beneficial effects of the present invention are:

[0034] (1) The present invention standardizes the processing technology of stewed Polygala tenuifolia and realizes the parameterization of the processing technology. The electromagnetic stewing machine used is safe, stable, environmentally friendly, and has controllable parameters. The quality consistency between batches of decoction pieces is good and suitable for industrial production. It solves the problems of fragile containers, poor safety, high labor costs, and uneven quality in the traditional stewing method. It is environmentally friendly and meets GMP requirements. The production efficiency can be increased by more than 20%, and the labor force is reduced from 2 to 3 people to 1 person.

[0035] (2) The present invention also provides the HPLC fingerprint of the present invention, the method for constructing the HPLC fingerprint and the method for identifying the HPLC fingerprint. The fingerprint can be used to scientifically identify the stewed Polygala tenuifolia slices and evaluate their quality, thereby improving and ensuring the stable quality of the stewed Polygala tenuifolia slices. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The invention provides an HPLC fingerprint of stewed Polygala tenuifolia, wherein 1 is Siberian Polygala tenuifolia A5 peak; 2 is Siberian Polygala tenuifolia A6 peak; 4 is Polygala tenuifolia sucrose ester B peak; 5 is 3,6'-dicercosyl sucrose peak; 6 is Polygala tenuifolia sucrose ester A peak; 10 is Polygala tenuifolia oligosaccharide H peak; and 11 is Polygala tenuifolia oligosaccharide A peak. DETAILED DESCRIPTION

[0037] The following is a further detailed introduction to the processing technology of the braised Polygala tenuifolia slices and its HPLC fingerprint, the construction method of the HPLC fingerprint and the HPLC fingerprint identification method described in the present invention in conjunction with specific embodiments. The technical solutions of the present invention include but are not limited to the following embodiments.

[0038] Example 1

[0039] Study on the optimization of processing technology of stewed Polygala tenuifolia

[0040] The 2008 edition of the "Jiangxi Province Traditional Chinese Medicine Preparation Specifications" records that the preparation method of stewed Polygala is "Take clean licorice, cut it into sections, flatten it, mix it with clean Polygala, put it in a stewing pot, add an appropriate amount of warm water, and cover it; move the pot into the stove, pile dry bran around it, light the fire, and stew it for 4 to 6 hours. When the juice in the pot is basically absorbed, take it out, pick out the licorice, and dry it. For every 100kg of Polygala, use 6kg of licorice", but there are no clear regulations on the stewing time, amount of water added, and drying temperature. It is difficult to ensure the consistency of quality between batches of decoction pieces in actual production. Under the Polygala medicinal material item of the 2020 edition of the "Chinese Pharmacopoeia", the hydrolysis product of Polygala saponin components, Polygala tenuifolia saponin, is used as a quality control indicator. However, there are no reports of Polygala tenuifolia saponin being detected in Polygala medicinal materials, so the quality of Polygala tenuifolia cannot be fully controlled. Polygala root saponin B is one of the main active ingredients of Polygala root that can calm and hypnotize the body, and it is also one of the ingredients that can produce toxicity. Ketone III and 3,6′-dicerinoyl sucrose are the main active ingredients of Polygala tenuifolia. The 2020 edition of the Chinese Pharmacopoeia stipulates that Polygala tenuifolia in Polygala tenuifolia decoction pieces Ketoacetyl III, 3,6'-dicerinoyl sucrose, shall not be less than 0.15% and 0.5% respectively. The contents of ketone III, 3,6′-diesinapoylsucrose and polygala saponin B as well as the degree of irritation reduction were used as evaluation indicators, and the different processing parameters of stewed Polygala were compared to optimize and determine the best processing technology.

[0041] 1.1 Experimental equipment

[0042] Experimental instruments: The instruments used in the experiment are shown in Table 1.

[0043] Table 1 List of instruments used

[0044]

[0045] Trial drugs: Polygala root slices were purchased from Shandong Baiweitang Chinese Medicine Slices Co., Ltd., batch number 180611; Polygala root ketone III (China Food and Drug Inspection Institute, batch number 11850-201203, purity 95.7%), 3,6′-diesinapoylsucrose (Chengdu Kroma Biotechnology Co., Ltd., batch number CHB170314, purity 98.0%), Polygala root saponin B (Chengdu Purifa Technology Development Co., Ltd., batch number PRF10053042, purity 98.3%).

[0046] Reagents: Wahaha purified water, acetonitrile (chromatographic grade), sodium chloride injection (Chenxin Pharmaceutical Co., Ltd., batch number 1910120721); 75% medical alcohol (Shandong Lierkang Disinfection Technology Co., Ltd., batch number 1903104A), and the remaining reagents were of analytical grade.

[0047] Animals: Large-eared white rabbits of either sex, with a body weight of 1.5±0.2 kg and normal eyes (purchased from Shandong Lukang Pharmaceutical Co., Ltd., license number: SCXK-Lu20080002).

[0048] 1.2 Optimization of the processing technology of stewed Polygala tenuifolia

[0049] Orthogonal test: Through single factor investigation, it was determined that the amount of water added (A), stewing time (B), and drying temperature (C) were the influencing factors, and the The contents of ketone III, 3,6′-dicerinoylsucrose, and polygala saponin B, and the degree of irritation reduction were used as the evaluation indicators. 4 )Orthogonal experimentThe orthogonal experiment design table is shown in Table 2.

[0050] Table 2. Table of factors and levels of orthogonal experiment of stewed Polygala tenuifolia

[0051]

[0052] Sample preparation: Take 50g of clean Polygala root pulp, divide it into 9 parallel portions, add 3g of licorice and clean Polygala root pulp to each portion, mix well, put it in a stewing medicine jar, add the corresponding amount of clean water according to the level in Table 2, cover the jar, move the electric heating jacket and stew for the corresponding time, pour out the jar, pick out the licorice, and dry at the corresponding temperature to obtain 9 portions of processed products.

[0053] Polygala Determination of the contents of ketone III, 3,6′-dicerinoylsucrose and polygalaponin B

[0054] Preparation of mixed reference solution: Accurately weigh Polygala tenuifolia Ketone III, 3,6′-bisinapoyl sucrose, and polygala saponin B are added with methanol to prepare a polygala containing A mixed reference solution of ketone III 0.0600 mg / mL, 3,6′-dicerinoylsucrose 0.2000 mg / mL, and polygalaponin B 0.7300 mg / mL.

[0055] Preparation of test solution: Weigh about 0.5 g of sample powder (passed through No. 3 sieve), accurately weigh, place in a stoppered conical flask, accurately add 25 mL of 70% methanol, weigh the weight, ultrasonically treat (power 400 W, frequency 40 kHz) for 60 min, take out, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, take the filtrate, filter with 0.45 μm microporous filter membrane as the test solution.

[0056] Chromatographic conditions: Kromasil C 18 Chromatographic column (250mm×4.6mm, 5μm); mobile phase is acetonitrile (A)-0.05% phosphoric acid solution, gradient elution (0-20min: 10% A-26% A; 20-30min: 26% A; 30-31min: 26% A-31% A; 31-40min: 31% A; 40-41min: 31% A-41% A; 41-55min: 41% A-46% A); column temperature 30°C; detection wavelength 320nm, volume flow rate 1.0mL / min, injection volume 20μL.

[0057] Determination method: Accurately pipette 20 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0058] Rabbit conjunctival irritation test

[0059] Sample preparation: Take the raw and stewed powders of Polygala tenuifolia (pass through a No. 3 sieve), weigh accurately, and add physiological saline to prepare a suspension with a mass concentration of 30%. Adjust the osmotic pressure of each suspension with NaCl to the osmotic pressure equivalent to that of 0.9% physiological saline, and adjust the pH to 7 with acid and alkali solution for use. Use blank physiological saline as a control.

[0060] Grouping and administration: Take 3 healthy rabbits in each group, check both eyes 24 hours before the experiment, and remove animals with eye irritation symptoms, corneal defects and conjunctival damage. Fix the rabbit in a rabbit cage, gently pull open the rabbit's eyelids to make it away from the eyeball, and drip each suspension into the eyelid. During the experiment, 50μL of each suspension was dripped into each rabbit eye. After administration, gently close the upper and lower eyelids for about 10 seconds to prevent the test solution from flowing out, so that the sample suspension and the eye are fully in contact. After 2 minutes, rinse with about 30mL of normal saline until there is no foreign matter in the rabbit's naked eyes. Observe the rabbit's eyes after 1 hour, and use a blank of normal saline as the control group. Take pictures to record the irritation reaction of the rabbit's conjunctiva.

[0061] Scoring and evaluation criteria for eye irritation reaction: The total score of a rabbit's eye irritation reaction is the sum of the scores of congestion, edema, and secretions. The scoring criteria for the rabbit conjunctival irritation test and the irritation degree scoring criteria are shown in Table 3 and Table 4, respectively.

[0062] Table 3 Scoring criteria for rabbit conjunctival irritation test

[0063]

[0064] Table 4 Scoring standard for conjunctival irritation degree of rabbit eyes

[0065]

[0066] Scoring criteria: The weighted method is used to comprehensively evaluate the cooking process of Polygala tenuifolia. The weight coefficients of the contents of ketone III, 3,6′-dicerinoylsucrose, and polygala saponin B and the irritation to the conjunctiva of rabbits were 20%, 20%, 20%, and 40%, respectively. The comprehensive quality score = 20×(the content of polygala in each sample Actual content of ketone III / polygala in the sample The total score was calculated by adding the scores of each index, and then performing visual analysis and variance analysis. The results are shown in Tables 5 and 6.

[0067] Table 5 Orthogonal test results

[0068]

[0069]

[0070] Table 6 Analysis of variance

[0071]

[0072] Note: F 0.05 (2, 6) = 5.14.

[0073] Conclusion: It can be seen from the table that the amount of water added (A) and the stewing time (B) have an effect on the The content of ketone III, 3,6′-bisinacoylsucrose, and polygala saponin B, as well as the degree of conjunctival irritation of rabbits, had a significant effect (P<0.05), while the drying temperature (C) had no significant effect (P>0.05). From the range R, it can be seen that the primary and secondary factors affecting the quality of stewed Polygala are A>B>C, and the optimal processing technology is A2B2C3, that is, the clean licorice slices and clean Polygala are mixed evenly, placed in the stewing pot, and 2 times the amount of water is added. The stewing time is set to 6h (the temperature of the medicine liquid is 97℃), and it is simmered for 3h to cool to room temperature. When the juice in the pot is basically absorbed, take it out, pick out the licorice, and dry it at 70℃. For every 100kg of Polygala, the amount of licorice is 6kg.

[0074] Verification test of the processing technology of stewed Polygala: 500g of clean Polygala were taken respectively, and three batches of stewed Polygala were prepared according to the optimal orthogonal test process. The average contents of ketone III, 3,6′-diesinapoylsucrose, and polygala saponin B were 0.27%, 0.77%, and 1.74%, respectively, and the RSDs were 1.00%, 1.29%, and 1.01%, respectively. The results showed that the processing technology of stewed polygala was stable and feasible.

[0075] Example 2

[0076] Pilot study on the processing technology of stewed Polygala tenuifolia

[0077] 2.1 Experimental equipment

[0078] Instruments and equipment: QWY300-00 electromagnetic stewing pot (Hangzhou Jinzhu Machinery Co., Ltd.); FYJ-C tunnel drying room (Jiangsu Yanneng Electric Heating Equipment Co., Ltd.); L-2000Elite high performance liquid chromatograph (Hitachi, Japan); DS-T100 pulverizer (1 / 100000) (Shanghai Dingshuai Industry and Trade Co., Ltd.); S-054 drying oven (Jinan Bohang Biotechnology Co., Ltd.).

[0079] Test drugs: 3,6'-biserucyl sucrose, Polygala tenuifolia Ketone III, Polygala tenuifolia saponins (batch numbers H25M6L1, 20170512, R23N8F49013, Shanghai Yuanye Biotechnology Co., Ltd., purity greater than 98.0%), Wahaha purified water, acetonitrile (chromatographic grade), and other reagents were analytically pure. The original Polygala root was identified by Professor Li Feng of the Chinese Medicine Identification Teaching and Research Office of Shandong University of Traditional Chinese Medicine as the dried root of Polygala tenuifolia Willd. or Polygala sibirica L. of the Polygalaceae family.

[0080] 2.2 Processing technology verification

[0081] In order to verify the stability and feasibility of the above-mentioned preferred processing technology and determine its industrial processing technology, the newly developed QWY300-00 electromagnetic stewing medicine pot complete set of equipment was used for pilot testing in Jiangxi Jiangzhong Chinese Medicine Pieces Co., Ltd. Due to the increase in the amount of pilot medicine, according to the processing conditions provided by the laboratory combined with the optimized process, after repeated verification, the best processing technology of stewed Polygala was finally determined as follows: take the clean licorice segment, flatten it, mix it with the clean Polygala segment, put it in the electromagnetic stewing medicine pot, add 2 times the amount of water, heat and stew it, keep it slightly boiling for 6h, until there is no throat tingling when chewing, cool it, take it out, pick out the licorice, and use the juice for later use; Polygala is dried to 50% dry, added to the juice and mixed well, and dried at 70°C after the juice is absorbed. For every 100kg of Polygala, 6kg of licorice is used. According to the above-mentioned preferred processing technology, 10 batches of pilot products of stewed Polygala slices were prepared, and the weight of raw Polygala slices and stewed Polygala slices were recorded, and the yield of the finished product was calculated. Yield = weight of stewed Polygala root slices / weight of raw Polygala root slices × 100%. The pilot production data is shown in Table 7.

[0082] Table 7 Pilot production data of stewed Polygala root slices

[0083]

[0084]

[0085] 2.3 Quality evaluation of pilot preparation process

[0086] Three batches of stewed Polygala were produced from the same batch of Polygala for pilot product quality evaluation to evaluate the stability and feasibility of the processing technology and the quality consistency between batches of decoction pieces. The test indicators include the properties of the decoction pieces, inspection (water content, total ash content, acid-insoluble ash content), alcohol-soluble extracts, and the content of active ingredients (polygala tenuifolia saponins, polygala tenuifolia saponins, Ketone III, 3,6′-dicerinoyl sucrose). The test results are shown in Tables 8 and 9.

[0087] 2.3.1 Identification of properties

[0088] According to the observation of the properties of the three batches of samples, it is consistent with the description of the properties of stewed Polygala in the 2008 edition of the "Jiangxi Province Traditional Chinese Medicine Preparation Specifications": This product is a cylindrical or grooved cylindrical small segment with a dark brown surface, a slightly sweet taste, and no throat irritation when chewed. The results show that the properties of the three batches of pilot samples are consistent, and there is no significant difference between the batches.

[0089] 2.3.2 Determination of moisture, total ash and acid-insoluble ash

[0090] The moisture content was determined according to the drying method 0832 of the 2020 edition of the Chinese Pharmacopoeia, and the total ash and acid-insoluble ash were determined according to item 2302 of the 2020 edition of the Chinese Pharmacopoeia. The results are shown in Table 8.

[0091] Table 8 Determination results of moisture content, total ash content and acid-insoluble ash content of three batches of stewed Polygala tenuifolia (n=4)

[0092]

[0093] The results showed that the three batches of pilot samples all met the requirements of the 2008 edition of "Jiangxi Province Traditional Chinese Medicine Preparation Specifications", which stipulated that the moisture content of stewed Polygala should not exceed 12%, the total ash content should not exceed 6%, and the acid-insoluble ash content should not exceed 1.5%.

[0094] 2.3.3 Determination of alcohol-soluble extract and active ingredient content

[0095] According to the hot soaking method under Item 2201 of the 2020 edition of the Chinese Pharmacopoeia, 70% ethanol was used as the solvent to determine the extract content of 10 batches of samples. The content of active ingredients in 10 batches of samples was determined by the method for determining the content of ketone III and 3,6'-dicerinoylsucrose. The results are shown in Table 9.

[0096] Table 9 Results of determination of alcohol-soluble extracts and active ingredient contents of three batches of stewed Polygala tenuifolia (n=4)

[0097]

[0098] The results showed that the contents of the three batches of samples were similar, all of which met the requirements of the 2020 edition of the Chinese Pharmacopoeia: the extract of Polygala tenuifolia should not be less than 30.0%, the saponin content of Polygala tenuifolia should not be less than 2.0%, and the The limit requirements are that the ketone III content shall not be less than 0.10% and the 3,6'-dicerinoylsucrose content shall not be less than 0.30%.

[0099] Conclusion: The production verification test of stewed Polygala showed that the processing technology of stewed Polygala and the newly developed stewing equipment had stable performance, controllable parameters, good quality consistency between batches of decoction pieces, and the processing technology of stewed Polygala was reasonable and feasible, and the equipment was safe, green, environmentally friendly, and controllable, which was suitable for industrial production.

[0100] Example 3 Establishment and Identification of Fingerprints of Stewed Polygala Radix Pieces

[0101] 3.1 Fingerprint

[0102] The fingerprint of stewed Polygala was created by HPLC. The chromatograms of 10 batches of stewed Polygala powder in this example were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012A Edition) recommended by the Chinese Pharmacopoeia Committee, and the median vector method was used for multi-point correction to generate the HPLC fingerprint of stewed Polygala and the reference fingerprint. Peak No. 5 (3,6'-dianisopylsucrose) was used as a reference, and finally 12 common peaks of the fingerprint were determined to constitute the characteristic peaks of the fingerprint of stewed Polygala. The reference fingerprint of stewed Polygala is shown in Figure 1 The similarity of fingerprints of 10 batches of stewed Polygala root slices was 0.957-0.995. According to the similarity evaluation system of Chinese medicine chromatographic fingerprints, the similarity between the fingerprints of the test sample and the control should not be less than 0.90.

[0103] The 2008 edition of "Jiangxi Province Traditional Chinese Medicine Preparation Specifications" did not have an item for the fingerprint spectrum of stewed Polygala tenuifolia. The present invention investigated and optimized the detection conditions, and finally established a fingerprint spectrum determination method for stewed Polygala tenuifolia, and the methodological investigation test results were good.

[0104] 3.1.1 Chromatographic conditions and system suitability test

[0105] Kromasil C 18 Chromatographic column (250 mm × 4.6 mm, 5 μm); mobile phase acetonitrile (A)-0.05% phosphoric acid solution (B), elution gradient is as follows:

[0106]

[0107] Flow rate: 1.0 mL / min; column temperature: 30°C; detection wavelength: 320 nm; injection volume: 10 μL, automatic injection.

[0108] 3.1.2 Sample preparation

[0109] Preparation of reference solution: Accurately weigh an appropriate amount of reference solution and place it in a 5 mL volumetric flask, dilute to the mark with methanol, and shake well to prepare a mixed reference solution containing 0.1178 mg / mL Siberian Polygala sugar A5, 0.0880 mg / mL Siberian Polygala sugar A6, 0.032 mg / mL Polygala sucrose ester B, 0.3710 mg / mL 3,6′-dicercosyl sucrose, 0.2170 mg / mL Polygala sucrose ester A, 0.1830 mg / mL Polygala oligosaccharide H, and 0.1450 mg / mL Polygala oligosaccharide A.

[0110] Preparation of test solution: Weigh about 0.3 g of the product powder (passed through No. 3 sieve), place in a stoppered conical flask, add 20 mL of 70% methanol, weigh the weight, treat with ultrasound (500 W, 40 kHz) for 30 min, cool, add 70% methanol to make up for the lost weight, shake well and filter, and take the filtrate to obtain the solution.

[0111] 3.1.3 Determination method

[0112] Accurately pipette 10 μL of the reference solution and the test solution, and inject them into the liquid chromatograph for determination.

[0113] 3.1.4 Common peak determination

[0114] In this experiment, 10 batches of Polygala tenuifolia were measured by HPLC, and the common peaks were calibrated by relative retention time. That is, 3,6'-dicerinoylsucrose was used as a reference, and the peaks with the same retention time ratio with the reference were selected as common peaks. Twelve common peaks were determined, and the retention times of the common peaks are shown in Table 10.

[0115] Table 10 Relative retention time of common peaks in stewed Polygala tenuifolia

[0116]

[0117] 3.1.5 Methodological investigation

[0118] Precision test: Take the same test solution and inject 6 times in succession, 10 μL each time, and calculate the relative retention time and relative peak area of ​​each chromatographic peak. The results show that the instrument has good precision.

[0119] Stability test: Take a sample solution and inject 10 μL at 0, 4, 8, 12, 16, 20, and 24 hours, and calculate the relative retention time and relative peak area of ​​each chromatographic peak. The results show that the sample solution has good stability.

[0120] Repeatability experiment: Take the same batch of Polygala tenuifolia samples, prepare 6 test solutions in parallel according to "3.1.2", inject and measure, and calculate the relative retention time and relative peak area of ​​each chromatographic peak. The results show that the method has good repeatability.

[0121] 3.1.6 Durability inspection and chromatographic peak positioning of the components to be measured

[0122] At the same time, the Shimadzu LC-16 HPLC, Hitachi L-2000 HPLC and three different brands and models of chromatographic columns were tested to investigate the durability of the method. The results showed that the peak order of each component in different brands of HPLC and three different brands and models of chromatographic columns was consistent, and the number of theoretical plates and separation degree met the system applicability, indicating that the chromatographic conditions were durable. The results are shown in Table 11.

[0123] Table 11 Calculation of relative retention time of common peaks of Polygala tenuifolia and investigation results of chromatographic column durability

[0124]

[0125]

[0126] 3.1.7 Similarity Evaluation

[0127] The chromatographic peak fingerprint data of 10 batches of stewed Polygala tenuifolia slices were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" to calculate the similarity of the fingerprints of stewed Polygala tenuifolia slices. The similarity calculation results are shown in Table 12.

[0128] Table 12 Similarity determination results of fingerprint spectra of 10 batches of stewed Polygala root slices

[0129]

[0130] Results The similarities of fingerprints of 10 batches of stewed Polygala tenuifolia slices were 0.957-0.995. According to the similarity evaluation system of Chinese medicine chromatographic fingerprints, the similarity between the test sample and the control fingerprint should not be less than 0.90.

Claims

1. A processing process for stewed Polygala root slices, comprising the following steps: Take clean licorice segments, flatten them, mix them with clean Polygala segments, put them in an electromagnetic stewing pot, add 2 times the amount of water, heat and stew, keep it slightly boiling for 6 hours, until there is no irritation in the throat when chewing, let it cool, take it out, pick out the licorice, and use the juice for later use; dry the Polygala to 50% dry, add the juice and mix well, after the juice is absorbed, dry it at 70℃; for every 100kg of Polygala, use 6kg of licorice.

2. The HPLC fingerprint of the stewed Polygala root slices prepared by the processing process of claim 1 is characterized by: The invention comprises 12 characteristic peaks, the relative retention time RSD values ​​of the characteristic peaks are within 11%, and the relative retention times of the 12 characteristic peaks are as follows: peak 1: 0.396-0.416, peak 2: 0.426-0.435, peak 3: 0.753-0.758, peak 4: 0.877-0.881, peak 5: 1.00, peak 6: 1.133-1.137, peak 7: 1.388-1.442, peak 8: 1.444-1.504; peak 9: 1.659-1.674; peak 10: 1.685-1.699; peak 11: 1.699-1.728; peak 12: 2.237-2.

249.

3. The HPLC fingerprint of the stewed Polygala root slices according to claim 2, characterized in that: The relative retention time RSD values ​​of the characteristic peaks are within 5%, and the relative retention times of the 12 characteristic peaks are: peak 1: 0.40, peak 2: 0.43, peak 3: 0.75, peak 4: 0.88, peak 5: 1.00, peak 6: 1.14, peak 7: 1.43, peak 8: 1.49; peak 9: 1.67; peak 10: 1.69; peak 11: 1.72; peak 12: 2.

25.

4. The HPLC fingerprint of the stewed Polygala root slices according to claim 3, characterized in that: The relative retention time RSD value of the characteristic peak is within 2%.

5. The method for constructing the HPLC fingerprint of claim 2, 3 or 4, characterized in that: The steps include: Chromatographic conditions: The chromatographic column was filled with octadecylsilane bonded silica gel; acetonitrile was used as mobile phase A, 0.05% phosphoric acid solution was used as mobile phase B, the detection wavelength was 320 nm; the column temperature was 30°C; the flow rate was 1 ml per minute; The gradient elution procedure was as follows: 0min→10min, 10% mobile phase A, 90% mobile phase B linearly changed to 12% mobile phase A, 88% mobile phase B; 10min→25min, 12% mobile phase A, 88% mobile phase B linearly changed to 25% mobile phase A, 75% mobile phase B; 25min→40min, 25% mobile phase A, 75% mobile phase B linearly changed to 26% mobile phase A, 74% mobile phase B; 40 min → 41 min, 26% mobile phase A, 74% mobile phase B linearly changed to 31% mobile phase A, 69% mobile phase B; 41 min → 51 min, 31% mobile phase A, 69% mobile phase B; 51min→52min, 31% mobile phase A, 69% mobile phase B linearly changed to 41% mobile phase A, 59% mobile phase B; 52min→66min, 41% mobile phase A, 59% mobile phase B linearly changed to 46% mobile phase A, 54% mobile phase B; Preparation of reference solution Take an appropriate amount of 3,6'-dicerinoyl sucrose reference substance, weigh accurately, and add methanol to make a solution containing 0.1 mg per 1 ml. Preparation of test solution: About 0.3 g of stewed Polygala powder is accurately weighed and placed in a stoppered conical flask. 20 ml of 70% methanol is accurately added, the flask is sealed, and the weight is determined. Ultrasonic treatment is performed for 30 min. The solution is taken out and cooled. 70% methanol is added to make up for the lost weight. The solution is shaken well, filtered, and the filtrate is obtained. Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, measure, and record the chromatogram.

6. The method for constructing the HPLC fingerprint according to claim 5, characterized in that: The specifications of the chromatographic column are 250 mm×4.6 mm, 5 μm.

7. The method for constructing a HPLC fingerprint according to claim 5, characterized in that: The conditions of the ultrasonic treatment were 500 W and 40 kHz.

8. The HPLC fingerprint identification method of stewed Polygala tenuifolia slices obtained by the HPLC fingerprint construction method of claim 5, characterized in that: The fingerprint of the test sample should present chromatographic peaks with the same retention time as the chromatographic peaks of the reference; calculated according to the similarity evaluation system of Chinese medicine chromatographic fingerprints, the similarity between the fingerprint of the test sample and the fingerprint of the reference shall not be less than 0.90.

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