Prepared rehmannia root decoction pieces as well as preparation method and quality control method thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine processing and traditional Chinese medicine analysis, in particular to prepared rehmannia root decoction pieces as well as a preparation method and a quality control method thereof. According to the method, the appearance character of'black like paint and bright like oil 'and the taste of'sweet like maltose' (the index is represented by the content of seven main carbohydrate components) in the traditional judgment standard of the prepared rehmannia root decoction pieces and the content of rehmannia D specified by pharmacopeia are used as evaluation indexes, and the steaming pressure, the drying temperature and the drying time in the processing process are optimized; finally, the optimal process for directly processing the fresh rehmannia into the prepared rehmannia root decoction pieces is obtained, so that the contents of seven carbohydrate components in the prepared rehmannia root decoction pieces are all within the content range of the prepared rehmannia root decoction pieces collected in the market, and other indexes all meet the prepared rehmannia root decoction piece standard in the Chinese pharmacopoeia of 2020 edition; while the quality of the prepared rehmannia root decoction pieces is ensured, the production efficiency is improved (the processing time is shortened to 40 hours from the conventional 100 hours), and the production cost is saved.

Description

Technical Field

[0001] The present invention relates to the technical fields of traditional Chinese medicine processing and traditional Chinese medicine analysis, and particularly relates to a processed rehmannia root slice, a preparation method thereof, and a quality control method thereof. Background Art

[0002] Processed rehmannia root is a processed product of Rehmannia glutinosa Libosch. In traditional processes, processed rehmannia root is a processed product of raw rehmannia root medicinal materials. There are two processing methods specified in the pharmacopoeia, namely the stewing method and the steaming method. For the stewing method, take raw rehmannia root, stew it according to the wine stewing method (General Rule 0213) until the wine is completely absorbed, take it out, dry it in the sun until the outer skin mucus is slightly dry, cut it into thick slices or pieces, and dry it to obtain the product. For every 100 kg of raw rehmannia root, 30 - 50 kg of yellow rice wine is used. For the steaming method, take raw rehmannia root, steam it according to the steaming method (General Rule 0213) until it is dark and moist, take it out, dry it in the sun until it is about 80% dry, cut it into thick slices or pieces, and dry it to obtain the product. Raw rehmannia root medicinal materials are processed from fresh rehmannia root. Currently, most raw rehmannia root medicinal materials are processed by farmers in the producing areas, and their processing methods and drying temperatures are different. During this process, the quality is not effectively controlled, and the quality is uneven. Moreover, the fresh rehmannia root is not thoroughly cleaned of sediment during the processing, resulting in unqualified quality of the prepared rehmannia root slices and processed rehmannia root slices. For example, the Henan Institute for Drug Control sampled 187 batches of rehmannia root and processed rehmannia root slices nationwide, and the results showed that: among the 42 unqualified samples, 21 batches were due to the unthorough cleaning of soil during the processing of raw rehmannia root medicinal materials, resulting in excessive total ash and acid-insoluble ash. In addition, when processing raw rehmannia root medicinal materials into processed rehmannia root slices, the medicinal materials need to be sorted according to size because different sizes of medicinal materials require different moistening times and steaming times, but most pharmaceutical enterprises do not conduct grading. The above factors lead to uneven quality of processed rehmannia root slices prepared from raw rehmannia root medicinal materials. At the same time, preliminary investigations found that it takes about 100 hours for a pharmaceutical factory to process raw rehmannia root into processed rehmannia root, which requires multiple steps such as thorough washing - moistening with yellow rice wine - steaming - drying - cutting, etc., resulting in a long processing time, high energy consumption, and high costs. However, in actual ancient methods, fresh rehmannia root was used as the raw material to process and prepare processed rehmannia root slices. For example, "Leigong's Treatise on Processing of Medicinal Herbs" in the Southern and Northern Dynasties recorded: "Collect raw rehmannia root, remove the white skin, steam it in a willow-wood steamer on a porcelain pot, spread it out to let the air out, mix it with wine and steam it again, and then dry it." "Illustrated Classic of Materia Medica" in the Song Dynasty recorded: "Collect the roots in February and August, steam them for two or three days until they are rotten, and dry them in the sun, which is called processed rehmannia root. Those dried in the shade are raw rehmannia root."

[0003] Processing and preparing processed rehmannia root from fresh rehmannia root can not only select high-quality fresh rehmannia root at the raw material stage, simplify the thorough washing process, retain the active ingredients of rehmannia root to the greatest extent, improve the taste and effect of processed rehmannia root, facilitate the quality monitoring of processed rehmannia root, but also greatly shorten the processing time, save costs, and improve production efficiency.

[0004] Rehmannia glutinosa mainly contains carbohydrates (monosaccharides, oligosaccharides and polysaccharides), iridoid glycosides and phenyl ethanol glycosides. Moreover, the total content of carbohydrate components (monosaccharides and oligosaccharides) reaches more than 50% in various types of Rehmannia glutinosa (fresh Rehmannia glutinosa, processed Rehmannia glutinosa and prepared Rehmannia glutinosa), belonging to the components with large quantities. Among them, fresh Rehmannia glutinosa is mainly composed of stachyose, and the single component content of stachyose is greater than 50%. Processed Rehmannia glutinosa mainly contains stachyose, raffinose and sucrose, while prepared Rehmannia glutinosa mainly contains mannotriose, fructose and glucose. The latest research believes that oligosaccharides can promote the reproduction of beneficial bacteria in the intestine, inhibit the growth of harmful bacteria, and can reshape the homeostasis by correcting the imbalanced flora, thus having beneficial effects on the human body. At present, it is added to a variety of foods as a "prebiotic" for use. The oligosaccharide extract enriched from Rehmannia glutinosa (rich in raffinose, mannotriose, etc.) has been proven to have pharmacological effects such as hypoglycemic, antioxidant, inhibiting cancer cell proliferation, treating intestinal inflammation, reducing liver injury, and alleviating the airway pathological changes of rats with chronic obstructive pulmonary disease (COPD). Sucrose can provide energy for brain tissue function, human muscle activity, etc. and maintain body temperature. It can also increase the synthesis of ATP in the body, which is beneficial to the vitality of amino acids and the synthesis of proteins. In addition, monosaccharides also have various pharmacological effects. For example, fructose is called "healthy sugar" because of its low glycemic index, and has functions such as nourishing the stomach, protecting the liver, moistening the intestine, and laxative, and is the sweetest among natural sugars with excellent flavor and taste; glucose is a physiological carbohydrate, with functions of nutrition, detoxification, strengthening the heart and diuresis, and is the main source of energy required by the body. It also has a protective effect and detoxification function on the liver, and can promote the excretion of toxins. In addition, glucose is very important for brain function, can strengthen memory, stimulate calcium absorption and increase communication between cells. Therefore, the carbohydrate components should be an important index for evaluating the quality of prepared Rehmannia glutinosa slices.

[0005] Although there are also many literature reports on the process research of directly processing fresh Rehmannia glutinosa into prepared Rehmannia glutinosa slices, the evaluation indexes focus on several components such as catalpol, rehmannioside D, acteoside and 5-hydroxymethylfurfural (5-HMF), and the total content of these components is less than 1%. For example, the current edition of the pharmacopoeia stipulates that the content of rehmannioside D in prepared Rehmannia glutinosa shall not be less than 0.05%, and the remaining 99.5% of the components are unknown? The existing literature does not investigate the carbohydrate components that occupy an absolute dominant position in prepared Rehmannia glutinosa, and cannot truly evaluate the quality of prepared Rehmannia glutinosa slices. The present invention intends to use the appearance traits and taste of prepared Rehmannia glutinosa "black as lacquer, bright as oil" (characterized by the content of 7 carbohydrate components) and the content of rehmannioside D stipulated in the pharmacopoeia as evaluation indexes, and optimize the processing technology of directly processing fresh Rehmannia glutinosa into prepared Rehmannia glutinosa slices, so as to improve production efficiency and save production costs while ensuring the quality of prepared Rehmannia glutinosa slices. Summary of the Invention

[0006] The purpose of the present invention is to provide a prepared Rehmannia glutinosa slice, its preparation method and quality control method to solve the problems existing in the above-mentioned prior art.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a method for preparing prepared rehmannia root slices, comprising the following steps:

[0009] Successively perform atmospheric steaming and drying on fresh rehmannia root slices to obtain dried fresh rehmannia root slices;

[0010] After mixing the dried fresh rehmannia root slices with wine, successively perform moistening, high-temperature and high-pressure steaming, cooling and drying to obtain the prepared rehmannia root slices;

[0011] The time of the atmospheric steaming is 3 h and the temperature is 75 °C;

[0012] The pressure of the high-temperature and high-pressure steaming is 0.1 MPa, the time is 6 h, and the temperature is 120 °C.

[0013] Further preferably, the atmospheric steaming and high-temperature and high-pressure steaming are carried out in a high-pressure sterilization steamer; the pressure of the atmospheric steaming is 0; the pressure of the atmospheric steaming being 0 means that the pressure gauge shows 0 when the temperature is 75 °C; the pressure of the high-temperature and high-pressure steaming being 0.1 MPa means that the pressure gauge shows 0.1 when the temperature is 120 °C.

[0014] Preferably, the wine includes 16° yellow rice wine or 42° white liquor;

[0015] And / or, the mass-volume ratio of the dried fresh rehmannia root slices to the wine is 5:1.

[0016] Preferably, the drying temperature is 60 °C and the time is 5 h.

[0017] Preferably, the drying temperature is 60 °C and the time is 24 h.

[0018] Preferably, the moistening time is 2 h.

[0019] Preferably, the thickness of the fresh rehmannia root slices is 2-3 cm.

[0020] The present invention provides prepared rehmannia root slices prepared by using the above preparation method.

[0021] The present invention provides a quality control method for the above prepared rehmannia root slices, comprising:

[0022] (1) Observe the appearance and properties of the prepared rehmannia root slices;

[0023] (2) Detect the extract, moisture, total ash and acid-insoluble ash of the prepared rehmannia root slices; the extract of the prepared rehmannia root slices ≥ 65.0%, the moisture ≤ 15.0%, the total ash ≤ 8.0%, and the acid-insoluble ash ≤ 3.0%;

[0024] (3) Perform thin-layer chromatography identification on the prepared Rehmanniae Radix Praeparata slices;

[0025] (4) Perform high-performance liquid chromatography detection on the content of rehmannioside D in the prepared Rehmanniae Radix Praeparata slices; the content of rehmannioside D in the prepared Rehmanniae Radix Praeparata slices ≥ 0.050%;

[0026] (5) Perform high-performance liquid chromatography detection on the content of mannotriose in the prepared Rehmanniae Radix Praeparata slices; the content of mannotriose in the prepared Rehmanniae Radix Praeparata slices ≥ 20.0%.

[0027] Preferably, the thin-layer chromatography identification includes Method 1 and Method 2;

[0028] Method 1 includes the steps of spotting a test solution and a acteoside reference solution on a silica gel G thin-layer plate, using a mixed solution of ethyl acetate, methanol, and formic acid as the developing agent, developing, dipping the plate in a 0.1% ethanolic solution of 2,2-diphenyl-1-picrylhydrazyl, air-drying, and observing under daylight; the preparation method of the test solution includes mixing the powder of the sample to be tested and an ethanol solution, performing ultrasonic treatment and n-butanol extraction in sequence to obtain the test solution; the preparation method of the acteoside reference solution includes mixing acteoside reference substance with methanol to obtain the acteoside reference solution; the concentration of acteoside in the acteoside reference solution is 1 mg / mL;

[0029] Method 2 includes the steps of spotting a test solution and a reference solution containing mannotriose and fructose on a silica gel G thin-layer plate, using a mixed solution of glacial acetic acid, anhydrous formic acid, water, and ethyl acetate as the developing agent, developing, spraying with a 10% sulfuric acid ethanol solution, heating, and observing under a 365 nm ultraviolet lamp; the preparation method of the test solution includes mixing the powder of the sample to be tested and an ethanol solution, performing ultrasonic treatment and filtration in sequence to obtain the test solution; the preparation method of the reference solution containing stachyose, raffinose, and sucrose includes mixing mannotriose reference substance and fructose reference substance with methanol to obtain the reference solution containing mannotriose and fructose; the concentration of mannotriose in the reference solution containing mannotriose and fructose is 1 mg / mL, and the concentration of fructose is 1 mg / mL.

[0030] Preferably, in step (4), the high-performance liquid chromatography detection uses octadecylsilane chemically bonded silica as the filler; the chromatographic column for the high-performance liquid chromatography detection is XB-C 18 , the column temperature is 30 °C, the flow rate is 1 mL / min, and the injection volume is 10 μL; the high-performance liquid chromatography detection uses methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and the detection wavelength is 203 nm;

[0031] In step (5), the high performance liquid chromatography (HPLC) detection uses tertiary amine alkyl-bonded silica gel as the filler; the chromatographic column for the HPLC detection is a COSMOSIL Sugar-D chromatographic column, the column temperature is 30°C, the flow rate is 1 mL / min, and the injection volume is 10 μL; acetonitrile is used as mobile phase A and water is used as mobile phase B in the HPLC detection.

[0032] The present invention discloses the following technical effects:

[0033] The present invention takes the appearance traits and taste of Rehmanniae Radix Praeparata "black as lacquer, bright as oil" (characterized by the content of 7 main sugar components) and the content of rehmannioside D specified in the pharmacopoeia as evaluation indicators, optimizes the steaming pressure, drying temperature and drying time during the processing, and finally obtains the best process for directly processing fresh Rehmanniae Radix into Rehmanniae Radix Praeparata slices. As a result, the contents of 7 sugar components in the prepared Rehmanniae Radix Praeparata slices all fall within the content range of the Rehmanniae Radix Praeparata slices collected from the market. While ensuring the quality of Rehmanniae Radix Praeparata slices (i.e., effectively solving the problem of unstable quality of Rehmanniae Radix Praeparata slices caused by uneven quality of raw Rehmanniae Radix materials), the production efficiency is greatly improved (the processing time is shortened from the conventional 100 h to 40 h), and the production cost is saved. Moreover, the quality control of sugar components is introduced into the quality standard of Rehmanniae Radix Praeparata slices, the evaluation indicators are more reasonable, and the quality standard can better reflect the quality of Rehmanniae Radix Praeparata slices.

[0034] The preparation method and quality control method provided by the present invention can provide Rehmanniae Radix Praeparata slices with stable and controllable quality for clinical use. The present invention provides a theoretical basis for the quality standard of Rehmanniae Radix Praeparata slices. Description of the Drawings

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0036] Figure 1 It is a picture of Rehmanniae Radix Praeparata slices;

[0037] Figure 2 It is a thin layer chromatography identification diagram of Rehmanniae Radix Praeparata slices; among them, A is acteoside, and 1-3 are 3 batches of Rehmanniae Radix Praeparata slices;

[0038] Figure 3 It is a thin layer chromatography identification diagram of Rehmanniae Radix Praeparata slices; among them, A is the reference substance, 1-3 are 3 batches of Rehmanniae Radix Praeparata slices; a is mannotriose, and b is fructose;

[0039] Figure 4HPLC-ELSD chromatograms of 7 saccharide components in the prepared Rehmanniae Radix Praeparata pieces and the commercially available Rehmanniae Radix Praeparata pieces of the present invention; among them, A is the 7-saccharide mixed standard; B is the commercially available Rehmanniae Radix Praeparata pieces; C is the Rehmanniae Radix Praeparata pieces prepared by the present invention; 1-7 are D-fructose, glucose, sucrose, melibiose, raffinose, mannotriose and stachyose in sequence. Detailed implementation manners

[0040] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing particular implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0043] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and embodiments of the present invention are only exemplary.

[0044] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0045] This research group determined 22 batches of Rehmanniae Radix Praeparata pieces collected from the market, and clarified the distribution ranges of 7 main saccharide components (D-fructose, glucose, sucrose, melibiose, raffinose, mannotriose and stachyose) therein. The results showed that the total content of the above 7 saccharide components in the commercially available Rehmanniae Radix Praeparata pieces was between 54.7% and 70.9%, belonging to absolutely large components and being the main material basis of Rehmanniae Radix Praeparata.

[0046] The fresh Rehmanniae Radix used in this invention is purchased from Wuzhi County, Henan Province and is identified as the fresh tuberous root of Rehmannia glutinosa Libosch. of the family Scrophulariaceae.

[0047] Study on the Preparation and Quality Standard of Prepared Rehmanniae Radix in Example 1

[0048] 1. Determination of 7 Kinds of Carbohydrate Components in Multiple Batches of Market-Sold Prepared Rehmanniae Radix Slices

[0049] Collect 12 batches of prepared Rehmanniae Radix slice samples from major Chinese medicine slice factories. Using the method in the literature "Qualitative and Quantitative Analysis of Rehmannia glutinosa Libosch. and Its Slices Based on Glycomics" (Qualitative and Quantitative Analysis of Rehmannia glutinosa Libosch. and Its Slices Based on Glycomics [J]. Chinese Journal of Experimental Traditional Medical Formulae, 2024, 30(18): 136 - 142.), determine the contents of 7 main carbohydrate components (D-fructose, glucose, sucrose, melibiose, raffinose, mannotriose, and stachyose) in the 12 batches of samples, clarify their distribution ranges and means, and provide a control basis for the subsequent screening of the processing technology of prepared Rehmanniae Radix slices. The results are shown in Table 1. The subsequent determinations all use this method and will not be specifically described again. The results show that the distribution range of the sum of the 7 carbohydrate components is between 53.0% and 70.9%, and the mean value is 61.9%. Among them, the content of mannotriose is the highest, with a distribution range of 20.8% - 31.6% and a mean value of 28.2%; followed by glucose, with a content range of 6.88% - 13.8% and a mean value of 11.0%; the third is D-fructose, with a content range of 7.02% - 15.9% and a mean value of 10.6%; the fourth is stachyose, with a content range of 1.29% - 12.5% and a mean value of 6.39%; the fifth is melibiose, with a content range of 2.44% - 5.02% and a mean value of 3.36%; the sixth is sucrose, with a content range of 0.208% - 3.22% and a mean value of 1.25%; the seventh is raffinose, with a content range of 0.161% - 2.50% and a mean value of 1.12%.

[0050] Table 1 Determination Results of 7 Carbohydrate Components in 12 Batches of Market-Sold Prepared Rehmanniae Radix Slices / %

[0051]

[0052] 2. Study on the Processing Technology of Prepared Rehmanniae Radix

[0053] 2.1 Comparison of Prepared Rehmanniae Radix Dried at Different Drying Temperatures

[0054] Take fresh Rehmanniae Radix of the same batch, remove impurities, wash away the sediment, slice it into pieces 2 - 3 cm thick while it is still fresh, steam it in boiling water bath at normal pressure (0) and 100 °C for 8 h, take it out after cooling, divide it into 3 equal parts, and dry them in an oven at 50 °C, 60 °C and 80 °C for 24 h respectively. Then determine the contents of 7 kinds of saccharide components in each sample, and the results are shown in Table 2.

[0055] Table 2 Determination results of 7 kinds of saccharide components in processed Rehmanniae Radix slices with different drying temperatures / %

[0056]

[0057] Note: "-" indicates not detected.

[0058] The results show that the total sugar contents of the samples dried at 60 °C and 80 °C are comparable, and the drying time is 24 h for both. Although the total sugar content of the sample dried at 50 °C is higher, the drying time reaches 30 h, which is too long and consumes a large amount of energy. Therefore, drying at 60 °C is finally selected (Table 2).

[0059] 2.2. Comparison of processed Rehmanniae Radix steamed at normal pressure for different times under the condition of adding yellow rice wine

[0060] Take fresh Rehmanniae Radix of the same batch, remove impurities, wash away the sediment, slice it into pieces 2 - 3 cm thick while it is still fresh, divide it into 3 equal parts, add 9° yellow rice wine according to the mass ratio of medicinal material to yellow rice wine of about 10:1, moisten it for 12 h, and steam it at normal pressure (0) and 100 °C for 7 h, 8 h and 10 h respectively. Take it out after cooling, dry them in an oven at 60 °C for 24 h respectively. Then determine the contents of 7 kinds of saccharide components in each sample, and the results are shown in Table 3.

[0061] Table 3 Determination results of 7 kinds of saccharide components in processed Rehmanniae Radix slices steamed at normal pressure for different times under the condition of adding yellow rice wine / %

[0062]

[0063] From the determination results of multiple batches of processed Rehmanniae Radix slices collected from the market in the early stage, it can be known that the composition characteristic of saccharide components in processed Rehmanniae Radix is that a large amount of stachyose is converted into mannotriose, D - fructose and glucose. Compared with fresh / dried Rehmanniae Radix, the content of stachyose in processed Rehmanniae Radix decreases significantly, while the contents of the 3 kinds of sugars generated by conversion increase significantly. Based on the above characteristics, the results in Table 3 show that yellow rice wine can accelerate the conversion of saccharide components in Rehmanniae Radix samples. Therefore, yellow rice wine should be added during the processing of Rehmanniae Radix.

[0064] 2.3. Comparison of processed Rehmanniae Radix steamed at high temperature and high pressure for different times under the condition of not adding yellow rice wine

[0065] Take fresh Rehmanniae Radix of the same batch, remove impurities, wash away the sediment, cut into slices 2 - 3 cm thick while still fresh, divide them into 3 equal parts, and steam them at high temperature (120 °C) and high pressure (0.1 MPa) for 3 h, 5 h, and 7 h respectively. After cooling, take them out and dry them in an oven at 60 °C for 24 h. Then measure the contents of 7 kinds of saccharide components in each sample, and the results are shown in Table 4.

[0066] Table 4 Determination results of 7 kinds of saccharide components in the prepared Rehmanniae Radix Praeparata slices steamed at high temperature and high pressure for different times without adding yellow rice wine / %

[0067]

[0068] The results show that steaming at high temperature and high pressure can accelerate the conversion of saccharide components in the Rehmanniae Radix sample. The method of steaming at high temperature and high pressure should be adopted in the processing of Rehmanniae Radix Praeparata (Table 4).

[0069] 2.4. Comparison of the prepared Rehmanniae Radix Praeparata slices with yellow rice wine of different alcohol contents

[0070] Take fresh Rehmanniae Radix of the same batch, remove impurities, wash away the sediment, cut into slices 2 - 3 cm thick while still fresh, divide them into 3 equal parts, and add yellow rice wine or white liquor with different alcohol contents (9 °, 16 °, and 42 °) according to the mass ratio of the medicinal material to yellow rice wine of about 10:1. Moisten them for 12 h, then place them at high temperature (120 °C) and high pressure (0.1 MPa) and steam them for 6 h (for 42 ° white liquor, steam for 5 h). After cooling, take them out and dry them in an oven at 60 °C for 24 h. Then measure the contents of 7 kinds of saccharide components in each sample, and the results are shown in Table 5.

[0071] Table 5 Determination results of 7 kinds of saccharide components in the prepared Rehmanniae Radix Praeparata slices steamed at high temperature and high pressure with yellow rice wine / white liquor of different degrees for moistening / %

[0072]

[0073] The results show that under the combined action of high temperature, high pressure, and yellow rice wine or white liquor, the conversion of saccharide components in Rehmanniae Radix is greatly accelerated, and the higher the alcohol content, the faster the conversion of saccharide components. That is, the conversion of saccharide components is the fastest under the combined conditions of 42 ° white liquor, high temperature, and high pressure. However, from the detection results, the effect is the best under the combined conditions of 16 ° yellow rice wine, high temperature, and high pressure, and the total amount of 7 kinds of sugars can reach 67.92%.

[0074] 2.5. Comparison of the prepared Rehmanniae Radix Praeparata slices steamed at high temperature and high pressure for different times after moistening with 16 ° yellow rice wine

[0075] Take fresh Rehmanniae Radix of the same batch, remove impurities, wash away the sediment, slice it into pieces 2 - 3 cm thick while it is still fresh, divide it into 10 equal parts, add yellow rice wine with an alcohol content of 16° according to the mass ratio of medicinal material to yellow rice wine of about 10:1, moisten it for 2 h (previous research found that moistening for 2 - 3 h can reach saturation), then place it under high temperature (120 °C) and high pressure (0.1 MPa) and steam it for different times (3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 12 h, and 13 h), take it out after cooling, place it in an oven at 60 °C and dry it for 24 h, and then determine the contents of 7 kinds of saccharide components in each sample. The results are shown in Table 6.

[0076] Table 6 Determination results of 7 kinds of saccharide components in the prepared Rehmanniae Radix pieces steamed under high temperature and high pressure for different times after moistening with 16° yellow rice wine / %

[0077]

[0078]

[0079] The results show that with the extension of the steaming time, the total content of 7 kinds of sugars in the prepared Rehmanniae Radix pieces gradually decreases, indicating that the steaming time should not be too long. Comparatively, the proportional relationship of 7 kinds of saccharide components to be measured in the prepared Rehmanniae Radix pieces steamed for 6 h is closest to that of the prepared Rehmanniae Radix pieces sold on the market (Table 6). The only shortcoming is that the contents of glucose and D-fructose in the prepared Rehmanniae Radix pieces by this method are on the low side, lower than the lowest value of the commercially available samples.

[0080] 2.6 Comparison of the prepared Rehmanniae Radix pieces steamed under high temperature and high pressure for different times after moistening with 42° white liquor

[0081] Take fresh Rehmanniae Radix of the same batch, remove impurities, wash away the sediment, slice it into pieces 2 - 3 cm thick while it is still fresh, divide it into 3 equal parts, add 42° yellow rice wine according to the mass ratio of medicinal material to yellow rice wine of about 10:1 and moisten it for 2 h respectively, then place it under high temperature (120 °C) and high pressure (0.1 MPa) and steam it for different times (3 h, 4 h, and 5 h), take it out after cooling, place it in an oven at 60 °C and dry it for 24 h, and then determine the contents of 7 kinds of saccharide components in each sample. The results are shown in Table 7.

[0082] Table 7 Determination results of 7 kinds of saccharide components in the prepared Rehmanniae Radix pieces steamed under high temperature and high pressure for different times after moistening with 42° yellow rice wine / %

[0083]

[0084] The results showed that with the extension of steaming time, the conversion of saccharide components in Rehmanniae Radix Preparata was intensified. The total sugar content was the highest when steamed at 42° for 4 h, and the proportions of the 7 sugars were relatively close to those of the commercially available processed Rehmanniae Radix Preparata slices (Table 7). However, the problems still remained that the contents of glucose and D-fructose were lower than the lowest values of the commercially available samples, and currently there is only 42° white liquor but no 42° yellow rice wine on the market, while the Pharmacopoeia stipulates the use of yellow rice wine, so the method of moistening with 42° white liquor was abandoned.

[0085] 2.7. Comparison of processed Rehmanniae Radix Preparata slices steamed at different times with different high-temperature combinations under high pressure after moistening with 16° yellow rice wine

[0086] Take the fresh Rehmanniae Radix Preparata of the same batch, remove impurities, wash the sediment, cut into slices 2 - 3 cm thick while still fresh, divide them into 4 equal parts, add 16° yellow rice wine to moisten for 2 h according to the mass ratio of medicinal material to yellow rice wine of about 10:1, then place them under the conditions of 120 °C and 0.1 MPa and steam for 3 h, and then change to the conditions of 105 °C and 0.1 MPa and steam for 2 h, 3 h, 5 h and 8 h respectively. After cooling, take them out and dry them in an oven at 60 °C for 24 h, and then determine the contents of 7 saccharide components in each sample. The results are shown in Table 8.

[0087] Table 8 Determination results of 7 saccharide components in processed Rehmanniae Radix Preparata slices steamed at different times with different temperature + high-pressure combinations after moistening with 16° yellow rice wine / %

[0088]

[0089]

[0090] The results showed that steaming at 120 °C for 3 h first and then at 105 °C for different times did not increase the contents of glucose and fructose, and the contents of these two saccharide components were still lower than those of glucose and D-fructose in the commercially available processed Rehmanniae Radix Preparata slices (Table 8), so this scheme was still not feasible. Therefore, this method was discarded.

[0091] 2.8. Comparison of processed Rehmanniae Radix Preparata slices steamed at different times under high temperature and high pressure after moistening with 16° yellow rice wine after drying by steaming at different temperatures under normal pressure

[0092] In the previous designs, fresh Rehmanniae Radix was directly added with yellow rice wine for moistening and then steamed. As a result, the contents of D-fructose and glucose were always low and could not reach the level of commercially available samples. Therefore, it was considered that the enzymes in Rehmanniae Radix might be instantly destroyed during direct steaming, making it difficult to promote the transformation of its components. Thus, the following scheme was designed for investigation: Take fresh Rehmanniae Radix of the same batch, remove impurities, wash the sediment, cut into slices 2 - 3 cm thick while still fresh, divide them into 4 equal parts. Two parts were steamed at 60 °C under normal pressure for 3 h, and the other two parts were steamed at 75 °C under normal pressure for 3 h. Then all were dried at 60 °C for 5 h. Next, yellow rice wine of 16° was added according to the mass ratio of medicinal material to yellow rice wine of about 5:1 for moistening for 2 h. Then, one part in each group was steamed at 120 °C and 0.1 MPa for 3 h, and the other part was steamed at 120 °C and 0.1 MPa for 6 h. After cooling, they were taken out and dried in an oven at 60 °C for 24 h. Then, the contents of 7 kinds of saccharide components in each sample were determined, and the results are shown in Table 9.

[0093] Table 9 Determination results of 7 saccharide components in the prepared Radix Rehmanniae Praeparata slices by steaming at different temperatures under normal pressure, drying, and then steaming at high temperature and high pressure for different times / %

[0094]

[0095]

[0096] The results showed that the contents of 7 saccharide components in the prepared Radix Rehmanniae Praeparata slices by steaming at 75 °C under normal pressure for 3 h, drying at 60 °C for 5 h, moistening with 16° yellow rice wine for 2 h, and then steaming at 120 °C and 0.1 MPa for 6 h were closest to the average contents of these 7 components in the commercially available Radix Rehmanniae Praeparata slices (Table 9). Therefore, these conditions were selected as the processing conditions for Radix Rehmanniae Praeparata slices.

[0097] 2.9, Three-batch verification experiments under optimized conditions

[0098] Take fresh Rehmanniae Radix of the same batch, remove impurities, wash the sediment, cut into slices 2 - 3 cm thick while still fresh, divide them into 3 equal parts, each part being 1 kg. First, steam at 75 °C under normal pressure for 3 h, then dry at 60 °C for 5 h. Next, yellow rice wine of 16° was added according to the mass ratio of medicinal material to yellow rice wine of about 5:1 for moistening for 2 h. Then, steam at 120 °C and 0.1 MPa for 6 h. After cooling, they were taken out and dried in an oven at 60 °C for 24 h. Then, the contents of 7 saccharide components in each sample were determined, and the results are shown in Table 10.

[0099] Table 10 Determination results of 7 saccharide components in the three batches of prepared Radix Rehmanniae Praeparata slices under optimized conditions / %

[0100]

[0101] The results showed that the contents of the 7 saccharide components measured in the 3 batches of verification experiments were close to the average value of the market samples, and the repeatability among the three groups was good (Table 10), indicating that the established processing method was stable and reliable.

[0102] The pictures of the prepared Rehmanniae Radix Praeparata slices by this method are as Figure 1 shown. It can be Figure 1 seen that the Rehmanniae Radix Praeparata slices are irregular pieces and fragments, with different sizes and thicknesses. The surface is blackish-brown, shiny, and highly viscous. The texture is soft and tough, not easy to break, and the cross-section is blackish-brown and shiny. It has a slight odor and a sweet taste.

[0103] Finally, the processing technology of Rehmanniae Radix Praeparata was formulated as follows: Fresh Rehmanniae Radix, remove impurities, wash with sediment, slice while fresh into pieces 2 - 3 cm thick, first steam at 75 °C under normal pressure for 3 h, then dry at 60 °C for 5 h, then add 16° yellow rice wine at a mass ratio of about 5:1 of the medicinal material to the yellow rice wine, moisten for 2 h, then steam at 120 °C and 0.1 MPa for 6 h, take out after cooling, and dry in an oven at 60 °C for 24 h to obtain.

[0104] 3. Quality Standards of Rehmanniae Radix Praeparata

[0105] 3.1. According to the cold extraction method in General Chapter 2201 of the Fourth Part of the Chinese Pharmacopoeia (2020 Edition), determine the content of water-soluble extractives in 3 batches of self-made Rehmanniae Radix Praeparata. The results of the extractive content are shown in Table 11.

[0106] 3.2. According to the second method in General Chapter 0832 of the Fourth Part of the Chinese Pharmacopoeia (2020 Edition), determine the moisture content in 3 batches of fresh Rehmanniae Radix slices. The results of the moisture content are shown in Table 11.

[0107] 3.3. According to the General Chapter of the Fourth Part of the Chinese Pharmacopoeia (2020 Edition), determine the total ash content in 3 batches of fresh Rehmanniae Radix slices. The results of the total ash content are shown in Table 11.

[0108] 3.4. According to the General Chapter of the Fourth Part of the Chinese Pharmacopoeia (2020 Edition), determine the acid-insoluble ash content in 3 batches of fresh Rehmanniae Radix slices. The results of the acid-insoluble ash content are shown in Table 11.

[0109] 3.5. TLC Identification of Rehmanniae Radix Praeparata Slices

[0110] (1) Refer to the thin-layer chromatography identification method under the item of Rehmanniae Radix Praeparata in the Chinese Pharmacopoeia (2020 Edition). The specific steps are as follows: Take 1 g of the powder of this product, add 50 mL of 80% methanol, ultrasonically treat for 30 min, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of water, extract with water-saturated n-butanol by shaking 4 times, 10 mL each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 2 mL of methanol to obtain the test solution. Separately take acteoside reference substance, and make a solution containing 1 mg per 1 mL with methanol as the reference solution. According to the thin-layer chromatography method (General Principles 0502), take 5 μL of the test solution and 2 μL of the reference solution, respectively spot them on the same silica gel G thin-layer plate, use ethyl acetate - methanol - formic acid (16:0.5:2) as the developing solvent, develop, take out, air dry, impregnate with 0.1% 2,2-diphenyl-1-picrylhydrazyl anhydrous ethanol solution, and air dry. The results are as Figure 2 shown. The results show that: at the position corresponding to the acteoside reference substance chromatogram, spots of the same color appear, that is, in the test chromatogram, at the position corresponding to the reference chromatogram, spots of the same color appear.

[0111] (2) Refer to "Qualitative and Quantitative Analysis of Rehmanniae Radix and Slices Based on Glycomics" (Qualitative and Quantitative Analysis of Rehmanniae Radix and Slices Based on Glycomics [J]. Chinese Journal of Experimental Traditional Medical Formulae, 2024, 30(18): 136 - 142.). The specific steps are as follows: Take 0.2 g of the powder of 30 batches of Rehmanniae Radix Praeparata slices respectively, add 5 mL of methanol, ultrasonically treat for 5 min, filter, and take the filtrate as the test solution. Separately take mannotriose reference substance and fructose reference substance, and make a solution containing 1 mg per 1 mL with pure methanol as the reference solution. According to the thin-layer chromatography method (General Principles 0502), take 2 μL of the above test solution and 10 μL of the reference solution, respectively spot them on the same silica gel G thin-layer plate, use glacial acetic acid - formic anhydride - water - ethyl acetate (volume ratio is 4:5:6:12) as the developing solvent, develop, take out, air dry. Spray with 10% sulfuric acid ethanol solution, heat at 120 °C until the spots are clearly colored, and examine under an ultraviolet lamp (365 nm). The results are as Figure 3 shown. The results show that: in the thin-layer chromatography identification diagram, at the position corresponding to the reference chromatogram in the test chromatogram, spots of the same color appear.

[0112] 3.6 Determination of the sugar content in Rehmanniae Radix Praeparata slices

[0113] In the literature "Qualitative and Quantitative Analysis of Rehmanniae Radix and Slices Based on Glycomics" (Qualitative and Quantitative Analysis of Rehmanniae Radix and Slices Based on Glycomics [J]. Chinese Journal of Experimental Traditional Medical Formulae, 2024, 30(18): 136 - 142.), the contents of 7 kinds of carbohydrate components in fresh Rehmanniae Radix Praeparata slices obtained by the optimized method and fresh Rehmanniae Radix Praeparata slices obtained by the freeze-drying method were determined. The results are as Figure 4 shown.

[0114] Chromatographic conditions and system suitability test:

[0115] Using tertiary amine alkyl-bonded silica gel as the filler, COSMOSIL Sugar-D chromatographic column (4.6 mm × 250 mm, 5 μm); column temperature is 30 °C; flow rate is 1 mL / min; injection volume is 10 μL; acetonitrile-water (75:25) is used as the mobile phase, and detected by evaporative light scattering detector (ELSD). The ELSD conditions are drift tube temperature 100 °C; carrier gas flow rate 2 L / min. The number of theoretical plates calculated based on stachyose should be not less than 3000.

[0116] Preparation of the mixed standard solution of 7 oligosaccharides: Accurately weigh 3.07 mg of reference substance D-fructose, 2.52 mg of reference substance glucose, 3.00 mg of reference substance sucrose, 2.52 mg of reference substance melibiose, 2.48 mg of reference substance raffinose, 6.60 mg of reference substance mannotriose and 10.15 mg of reference substance stachyose respectively, place them in the same 10 mL volumetric flask, dissolve with 70% (V / V) methanol and dilute to the mark, shake well, and it is ready.

[0117] Preparation of the test solution: Take the prepared Rhizoma Rehmanniae pieces, dry them at 45 °C for 48 h, then grind them into coarse powder. Take about 0.2 g, accurately weigh, place it in a stoppered conical flask, accurately add 50 mL of 70% methanol, weigh, ultrasonically treat (power 400 W, frequency 50 kHz) for 30 min, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the subsequent filtrate, and it is ready.

[0118] Assay method: Accurately pipette 5 μL and 20 μL of the mixed standard solution of 7 oligosaccharides and 10 μL of the test solution respectively, inject them into the high performance liquid chromatograph, and calculate by the logarithmic equation of external standard two-point method for determination.

[0119] The HPLC-ELSD chromatograms of the self-prepared Rhizoma Rehmanniae pieces, commercially available Rhizoma Rehmanniae pieces and the mixed reference substances are shown in Figure 4 , and the results are shown in Table 10.

[0120] 3.7. Determine according to the content determination method of rehmannioside D under Rhizoma Rehmanniae in the Chinese Pharmacopoeia (2020 Edition).

[0121] Chromatographic conditions and system suitability test: Using octadecylsilane-bonded silica gel as the filler; the chromatographic column is XB-C18; column temperature is 30 °C; flow rate is 1 mL / min; injection volume is 10 μL; using methanol-0.1% phosphoric acid solution (5:95) as the mobile phase, and the detection wavelength is 203 nm. The number of theoretical plates calculated based on the rehmannioside D peak should be not less than 5000.

[0122] Preparation of reference substance solution: Weigh an appropriate amount of rehmannioside D reference substance accurately, add 25% methanol to make a solution containing 70 μg per 1 mL, and you will get it.

[0123] Preparation of test solution: Take the processed rehmannia root slices, dry them at 45 °C for 48 h, then grind them into coarse powder. Take about 1 g, weigh it accurately, put it into a stoppered conical flask, accurately add 25 mL of 25% methanol, weigh it, ultrasonically treat it (power 400 W, frequency 50 kHz) for 1 h, let it cool, weigh it again, make up the lost weight with 25% methanol, shake well, centrifuge at high speed for 10 min, take the supernatant and filter it, take the continuous filtrate, and you will get it.

[0124] Determination method: Accurately pipette 10 μL of the reference substance solution and the test solution respectively, inject them into the liquid chromatograph for determination, and you will get it. The content results of rehmannioside D are shown in Table 11.

[0125] This product contains rehmannioside D (C 27 H 42 O 20 ) not less than 0.050%.

[0126] Table 11 Determination results of extracts, moisture, and rehmannioside D in three batches of processed rehmannia root slices prepared under optimized conditions / %

[0127]

[0128]

[0129] As can be seen from Table 11, the contents of all index components in the self-made processed rehmannia root slices meet the regulations of the existing pharmacopoeia on processed rehmannia root slices. In addition, based on the determination results of three batches of samples, it is proposed to add the regulation that the content of mannotriose in processed rehmannia root slices should not be less than 20.0%.

[0130] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing Rehmannia glutinosa slices, characterized in that: The following steps are involved: Sequentially steaming and drying the fresh Rehmannia root slices at normal pressure to obtain dried fresh Rehmannia root slices; After mixing the dried fresh Rehmannia root slices and wine, the mixture is sequentially steamed, steamed at high temperature and high pressure, cooled and dried to obtain the Rehmannia root slices; The normal pressure steaming time is 3h and the temperature is 75°C; The pressure of the high pressure and high temperature steaming is 0.1 MPa, the time is 6 hours, and the temperature is 120°C.

2. The preparation method according to claim 1, characterized in that: The wine includes 16° yellow wine and / or 42° white wine; The mass ratio of the dried fresh Rehmannia root slices to the wine is 5:

1.

3. The preparation method according to claim 1, characterized in that: The drying temperature is 60° C. and the drying time is 5 hours.

4. The preparation method according to claim 1, characterized in that: The drying temperature is 60° C. and the drying time is 24 hours.

5. The preparation method according to claim 1, characterized in that: The time of the soaking is 2 hours.

6. The preparation method according to claim 1, characterized in that: The thickness of the fresh Rehmannia root slices is 2 to 3 cm.

7. A prepared Rehmannia root slice prepared by the preparation method according to any one of claims 1 to 6.

8. The quality control method of Rehmannia glutinosa slices according to claim 7, characterized in that: include: (1) observing the appearance and properties of the prepared Rehmannia root slices; (2) The extract, water content, total ash content and acid-insoluble ash content of the prepared Rehmannia root slices are tested; the extract of the prepared Rehmannia root slices is ≥ 65.0%, the water content is ≤ 15.0%, the total ash content is ≤ 8.0%, and the acid-insoluble ash content is ≤ 3.0%; (3) performing thin layer chromatography identification on the prepared Rehmannia root slices; (4) Detecting the content of rehmannia glutinosa glycoside D in the prepared rehmannia glutinosa slices by high performance liquid chromatography; the content of rehmannia glutinosa glycoside D in the prepared rehmannia glutinosa slices is ≥ 0.050%; (5) The content of mannotriose in the prepared Rehmannia root slices is detected by high performance liquid chromatography; the content of mannotriose in the prepared Rehmannia root slices is ≥ 20.0%.

9. The quality control method according to claim 8, characterized in that: The thin layer chromatography identification includes method 1 and method 2; The method 1 comprises the steps of taking a test solution and a verbascoside reference solution and spotting them on a silica gel G thin layer plate, using a mixed solution of ethyl acetate, methanol and formic acid as a developing agent, developing, immersing the plate with a 0.1% 2,2-diphenyl-1-picrylhydrazyl anhydrous ethanol solution, drying, and observing under sunlight; the preparation method of the test solution comprises the steps of mixing a sample powder to be tested with an ethanol solution, performing ultrasonic treatment and n-butanol extraction in sequence, to obtain the test solution; the preparation method of the verbascoside reference solution comprises the steps of mixing a verbascoside reference with methanol, to obtain the verbascoside reference solution; the concentration of verbascoside in the verbascoside reference solution is 1 mg / mL; The method 2 comprises the steps of spotting a test solution and a reference solution containing mannotriose and fructose on a silica gel G thin layer plate, developing the plate using a mixed solution of glacial acetic acid, anhydrous formic acid, water and ethyl acetate as a developing agent, spraying the plate with a 10% sulfuric acid ethanol solution, heating the plate and observing the plate under a 365nm ultraviolet lamp; the preparation method of the test solution comprises the steps of mixing a sample powder to be tested with an ethanol solution, performing ultrasonic treatment and filtering in sequence to obtain the test solution; the preparation method of the reference solution containing stachyose, raffinose and sucrose comprises the step of mixing a mannotriose reference substance and a fructose reference substance with methanol to obtain the reference solution containing mannotriose and fructose; the concentration of mannotriose in the reference solution containing mannotriose and fructose is 1 mg / mL, and the concentration of fructose is 1 mg / mL.

10. The quality control method according to claim 8, characterized in that: In step (4), the HPLC detection uses octadecylsilane bonded silica gel as a filler; the HPLC detection chromatographic column is XB-C 18 , the column temperature is 30°C, the flow rate is 1mL / min, and the injection volume is 10μL; the high performance liquid chromatography detection uses methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and the detection wavelength is 203nm; In the step (5), the high performance liquid chromatography detection uses tertiary amine alkyl bonded silica gel as a filler; the chromatographic column of the high performance liquid chromatography detection is a COSMOSIL Sugar-D chromatographic column, the column temperature is 30°C, the flow rate is 1mL / min, and the injection volume is 10μL; the high performance liquid chromatography detection uses acetonitrile as mobile phase A and water as mobile phase B.

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