Preparation process of mulberry twig standard decoction

Abstract

The invention discloses a preparation process of a ramulus mori standard decoction, and relates to the technical field of traditional Chinese medicine decoction treatment.The preparation process comprises the following steps that firstly, ramulus mori decoction pieces are weighed, placed in an earthen pot and decocted twice, and the preparation process comprises the following steps that 1, water 10 times that of the ramulus mori decoction pieces is added into the earthen pot for the first time, soaked for 30 min, boiled with strong fire and then decocted with slow fire for 30 min; step 2, adding water which is 8 times of the mulberry twig decoction pieces into the marmite for the second time, boiling with strong fire, and decocting with slow fire for 20 minutes; 3, filtering the liquid medicine with 200 meshes, and cooling the filtrate to room temperature in a cold water bath; step 4, combining the two filtrates, and carrying out vacuum concentration at the temperature of 50 DEG C; and Step 5, concentrating until the weight ratio of the medicinal material to the fluid extract is 1: 1, collecting a concentrated solution, and freeze-drying to obtain a sample. According to the method, the component transfer condition from the decoction pieces to the decoction is measured through objective quality standards, and the quality degree and effectiveness of the mulberry twig decoction are ensured.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine decoction processing technology, and in particular to the preparation process of mulberry twig standard decoction. Background Technology

[0002] Mulberry twigs, a traditional Chinese medicine, are the dried tender branches of the mulberry tree (Morus alba), a plant in the Moraceae family. Harvested in late spring and early summer, the leaves are removed, and the branches are sun-dried, or sliced ​​while fresh and sun-dried. It is a deciduous shrub or small tree, 3-15m tall. The bark is grayish-white with shallow, linear fissures; the root bark is yellowish-brown or reddish-yellow, highly fibrous; leaves are simple and alternate; petioles are 1-62.5px long; leaf blades are ovate or broadly ovate, 5-500px long and 4-250px wide, with an acute or acuminate apex, a rounded or nearly cordate base, and coarsely serrated or crenate margins, sometimes irregularly lobed. The upper surface is glabrous and glossy, while the lower surface has short hairs on the veins and hairs in the axils. Three basal veins intertwine with fine veins to form a reticulate pattern, more prominent on the abaxial surface; stipules are lanceolate and deciduous. Flowers unisexual, dioecious; both male and female inflorescences arranged in spicate catkins, axillary; female inflorescences 1-50px long, pubescent, peduncle 5-10mm long; male inflorescences 1-62.5px long, drooping, slightly pubescent; male flowers with 4 perianth segments, 4 stamens, with a sterile pistil in the center; female flowers with 4 perianth segments, connate at the base, stigma 2-lobed. Achenes, numerous densely clustered into an ovoid or oblong aggregate fruit, 1-62.5px long, initially green, turning fleshy, blackish-purple or red when mature. Seeds small. Flowering period April-May, fruiting period May-June;

[0003] Since hospitals and the market currently lack quality research and standardization for traditional Chinese medicine decoctions to measure their quality and efficacy after preparation, this study takes mulberry twig decoction as an example. It specifies key quality indicators such as the main content, characteristic spectrum, and extract of mulberry twig as a single herb after decoction preparation, examines the transfer rate, and defines the range of transfer rate of the main content of mulberry twig as a whole. This ensures the effectiveness of mulberry twig decoction after preparation according to standard methods. Summary of the Invention

[0004] The main objective of this invention is to provide a preparation process for a standard mulberry twig decoction to solve the problems mentioned in the background art.

[0005] In order to derive the preparation process of the standard mulberry twig decoction, the present invention conducted relevant pharmaceutical research, such as screening 15 batches of mulberry twig medicinal materials from major producing areas across the country for quality comparison research, and preparing 15 batches of standard decoction according to the "Management Standards for Traditional Chinese Medicine Decoction Rooms in Medical Institutions".

[0006] To achieve the above objectives, the technical solution adopted by this invention is as follows: the preparation process of the standard mulberry twig decoction first involves weighing mulberry twig slices, placing them in a clay pot, and decocting them twice, including the following steps:

[0007] Step 1: Add 10 times the amount of water to the mulberry twig slices into the clay pot for the first time, soak for 30 minutes, bring to a boil over high heat, then simmer over low heat for 30 minutes.

[0008] Step 2: Add 8 times the amount of water to the mulberry twig slices into the clay pot for the second time, bring to a boil over high heat, then simmer over low heat for 20 minutes.

[0009] Step 3: Then filter the above medicine solution through a 200-mesh filter and cool the filtrate to room temperature in a cold water bath;

[0010] Step 4: Combine the two filtrates and concentrate them under vacuum at 50°C;

[0011] Step 5: Concentrate the extract to a 1:1 weight ratio of medicinal material to fluid extract, collect the concentrate, and freeze-dry to obtain the sample.

[0012] Preferably, in Step 4, vacuum concentration is performed under an absolute vacuum of 30–40 mbar.

[0013] The present invention has the following beneficial effects:

[0014] The preparation process of the mulberry twig standard decoction of the present invention has objective quality standards to measure the component transfer of mulberry twig slices into decoction, and to ensure the quality and effectiveness of the decoction.

[0015] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0016] Figure 1 This is a flowchart illustrating the preparation process of the mulberry twig standard decoction of the present invention.

[0017] Figure 2 This is a comparative characteristic chromatogram of the test solution of the mulberry twig standard decoction of the present invention;

[0018] Figure 3 This is a comparative characteristic spectrum of the mulberry twig standard decoction of the present invention. Detailed Implementation

[0019] The technical solutions in this utility model will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0020] Example 1:

[0021] like Figure 1Shown: The preparation process of the standard decoction of mulberry branches. First, weigh an appropriate amount of Sarcandrae Glabrae Herba slices, place them in a sand pot, and decoct twice, including the following steps:

[0022] Step1: Add 12 times the amount of water of Sarcandrae Glabrae Herba slices to the sand pot for the first time. After soaking for 30 minutes, bring to a boil over high heat and then change to low heat and decoct for 30 minutes; Step2: Add 10 times the amount of water of Perillae Fructus slices to the sand pot for the second time, bring to a boil over high heat and then change to low heat and decoct for 20 minutes; Step3: Then filter the above-mentioned medicinal liquid through a 200-mesh sieve, and cool the filtrate to room temperature in a cold water bath; Step4: Combine the two filtrates and perform vacuum concentration at a temperature of 50 °C; Step5: Concentrate to a fluid extract with a weight ratio of medicinal materials to fluid extract of 1:1, collect the concentrated liquid, and freeze-dry to obtain the sample.

[0023] Yield of extract: After the aqueous extract of mulberry branches is vacuum concentrated, the concentrated liquid is freeze-dried to dryness, the dry extract powder is collected and weighed, and the yield of extract is calculated. The formula for the yield of extract is as follows:

[0024]

[0025] Range of yield of extract: 5.8% - 9.4%.

[0026] Extractives: Determined by the hot extraction method under the alcohol-soluble extractives determination method (General Principles 2201, Volume IV, Chinese Pharmacopoeia 2020 Edition) with ethanol as the solvent. The range of extractives of the standard decoction is 27.5% - 51.9%.

[0027] Content determination <​​​​​​​​​​​​Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0033] Table 1 shows the transfer rate of morin content in the decoction of medicinal materials:

[0034]

[0035] The content of morin A per 1g of this product should be 9.0mg / g to 23.6mg / g;

[0036] The transfer rate of morin A content in the decoction of medicinal materials ranged from 22.5% to 44.5%.

[0037] Characteristic chromatogram: determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512).

[0038] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the detection wavelength was 320 nm. The theoretical plate number, calculated based on the peak of morin A, should not be less than 6000.

[0039] Table 2 shows the gradient elution table:

[0040]

[0041] Preparation of reference solution: Take about 0.5g of mulberry twig reference material, place it in a stoppered conical flask, add 25ml of 50% methanol, seal tightly, sonicate (600W power, 40kHz frequency) for 15 minutes, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of morin A reference standard, add 50% methanol to prepare a solution containing 40μg per ml, and use this as the reference solution.

[0042] Preparation of test solution: Take an appropriate amount of this product, grind it into a fine powder, and take about 0.2g to prepare the test solution using the same method as the preparation of the reference solution of the control medicinal material.

[0043] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0044] The chromatogram of the test sample should show chromatographic peaks corresponding to the retention times of the six main characteristic peaks in the reference medicinal material, and peak 5(S) should be consistent with the retention time of the reference peak; in the chromatogram of the test sample, the retention times of characteristic peaks 2, 3, and 8 should be within ±4% of the corresponding peaks in the reference medicinal material, and the retention times of characteristic peaks 4, 6, and 7 should be within ±1% of the corresponding peaks in the reference medicinal material.

[0045] Example 2:

[0046] 1. Raw material selection

[0047] According to the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules," the samples used in the research should be representative, covering the main producing areas or authentic production areas of the medicinal materials to be harvested, with no fewer than three batches from each producing area. Mulberry branches are cultivated and distributed throughout my country. They are mainly produced in Sichuan, Guizhou, Guangxi, and Hubei provinces, where cultivation has reached a large scale and they are a major source of commercial mulberry branch products. Based on a comprehensive evaluation of raw material quality and sustainable resource supply, the preliminary production areas for raw materials used in large-scale production were determined to be Jingyan County (Sichuan), Zhongjiang County (Sichuan), Yashan County (Guizhou), Anshun County (Guizhou), Yunlin County (Guangxi), and Enshi Prefecture (Hubei). Ultimately, 18 qualified batches were selected for research on standard decoctions.

[0048] 2. Research and standard setting on extract yield and transfer rate

[0049] Table 3 shows the transfer rate of 18 batches of mulberry twig standard decoction.

[0050]

[0051]

[0052] Based on the content of mulberry twig slices, the content of standard decoction, and the yield of extract in 18 batches, the average content transfer rate of standard decoction was calculated to be 33.5%.

[0053] The range calculated using the mean plus or minus 3 times the SD is 22.5% to 45.5%.

[0054] The range calculated using 70% to 130% of the mean is 23.4% to 43.5%.

[0055] Based on the actual range, it is 28.6% to 39.4%;

[0056] It is recommended to take 22.5% to 45.5% as the upper and lower limits of the standard decoction transfer rate.

[0057] 3. Content research and standard setting

[0058] Modern research indicates that *Gnaphalium affine* contains various components, such as sesquiterpenes, flavonoids (including dihydrochalcones, dihydroflavonoids, and anthocyanins), coumarins (isozidine), and organic acids (fatty acids and phenolic acids). *Gnaphalium affine* possesses a variety of pharmacological activities, including antitumor, anticancer, antibacterial, anti-inflammatory, effects on leukocytes and platelets, anti-gastric ulcer, antiasthmatic, and expectorant effects. The 2020 edition of the Chinese Pharmacopoeia lists isozidine and rosmarinic acid as quality control indicators for *Gnaphalium affine*. Combined with the content detection requirements in the *Gnaphalium affine* formula granules YBZ-PFKL-2022157, its quality control components are isozidine and rosmarinic acid. Therefore, this study selected isozidine and rosmarinic acid as the quality control indicators for the standard *Gnaphalium affine* decoction.

[0059] Based on the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules", and combined with data from 15 batches of standard decoctions for Swelling Joint Wind, the limits and upper and lower limits of isozymine content in the standard decoctions were calculated.

[0060] The range calculated using the mean plus or minus 3 times the SD is: 1.3mg to 3.6mg;

[0061] The range calculated using 70%–130% of the mean is 1.7 mg–3.2 mg.

[0062] Based on the actual range of 2.0mg to 3.1mg, it is recommended to use the average value of 70% to 130%, i.e., 1.7mg to 3.2mg, as the upper and lower limits of the standard decoction content.

[0063] Based on the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules", and combined with data from 15 batches of standard decoctions for Swelling Joint Wind, the limits and upper and lower limits of rosmarinic acid content in the standard decoctions were calculated.

[0064] The range calculated using the mean plus or minus 3 times the SD is: 1.5mg to 3.6mg;

[0065] The range calculated using 70%–130% of the mean is 1.8 mg–3.3 mg.

[0066] Based on the actual range of 2.0mg to 3.1mg, it is recommended to use the mean plus or minus 3 times the SD and the mean of 70% to 130%, i.e., 1.8mg to 3.6mg, as the upper and lower limits of the standard decoction content.

[0067] Based on the content of 15 batches of *Euphorbia helioscopia* decoction pieces, the content of standard decoction (isopyridinium chloride), and the yield of extract, the average transfer rate of the standard decoction content was calculated to be 73.0%.

[0068] The range calculated using the mean plus or minus 3 times the SD is 38.4% to 107.6%.

[0069] The range calculated using 70% to 130% of the mean is 51.1% to 94.9%.

[0070] Based on the actual range, it is 50.4% to 99.2%;

[0071] It is recommended to combine the mean plus or minus 3 times the SD and the mean of 70% to 130%, i.e., 38.4% to 99.2%, as the upper and lower limits of the standard decoction transfer rate.

[0072] Based on the content of 15 batches of *Euphorbia humifusa* decoction pieces, the content of standard decoction (rosmarinic acid), and the yield of extract, the average transfer rate of standard decoction content was calculated to be 45.9%.

[0073] The range calculated using the mean plus or minus 3 times the SD is 24.7% to 69.5%.

[0074] The range calculated using 70% to 130% of the mean is 33.0% to 61.2%.

[0075] Based on the actual range, it is 35.4% to 54.7%;

[0076] It is recommended to take the average plus or minus 3 times the SD, i.e., 24.7% to 69.5%, as the upper and lower limits of the standard decoction transfer rate.

[0077] 4. Characteristic spectrum research and standard setting

[0078] The chromatogram of the mulberry twig standard decoction with experimental batch number BT(SZ)220301 was selected as the reference chromatogram to establish a control characteristic chromatogram. Based on the data analysis results, five common peaks were finally selected as characteristic peaks. For the generated standard chromatogram, morin A was selected as the reference peak, and the relative retention times of the characteristic peaks in the chromatogram were calculated. By comparing with the chromatogram of the reference standard, according to the "Characteristic Chromatogram" section of the National Drug Standard YBZ-PFKL-2021109 for "Mulberry Twig Formula Granules," peak 1 (S) of the mulberry twig standard decoction characteristic chromatogram is identified as morin A.

[0079] Table 4 shows the retention time of 18 batches of mulberry twig standard decoction.

[0080]

[0081]

[0082] Table 5 shows the relative retention times of 18 batches of mulberry twig standard decoction.

[0083]

[0084]

[0085] The results showed that the characteristic chromatograms of the 18 batches of mulberry twig standard decoction samples all showed chromatographic peaks corresponding to the retention times of the six main characteristic peaks in the reference medicinal materials. The relative retention time RSD of peaks 2 to 6 was less than 2%, indicating that the elution times of each peak were consistent among the batches.

[0086] The national standard YBZ-PFKL-2021109, "Mulberry Twig Formula Granules," only specifies that the retention times of peaks 1-6 should correspond to those of the reference medicinal material, and the relative retention times of peaks 2-6 with peak 1 (S). Combining the test results of 18 batches of mulberry twig standard decoctions and the retention time data from the repeatability and precision tests in the methodology (see Tables 4-18 and 4-19 below), it can be seen that the retention times of peaks 1-6 are stable and correspond to the retention times of the corresponding reference standards. The relative peak drift of peaks 2-6 with peak 1 (S1) is very small, basically within ±10%, and their RSDs are all less than 5%. Therefore, it is stipulated that the test sample chromatogram should show 6 characteristic peaks, and their retention times should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the peak of morin A reference standard is the S peak. The relative retention times of each characteristic peak and the S peak should be calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 1.103 (peak 2), 1.367 (peak 3), 1.837 (peak 4), 2.672 (peak 5), and 3.282 (peak 6).

[0087] 5. Leachate research and standard setting

[0088] Grind the product into a fine powder, take approximately 2g, accurately weigh it, and accurately add 100ml of ethanol. Determine the soluble extract using the hot extraction method as described in the General Chapter 2201 of the Chinese Pharmacopoeia 2020. The test results for 18 batches of mulberry twig standard decoction are shown in Table 6.

[0089] Table 6 shows the results of extract determination of 18 batches of mulberry branches.

[0090]

[0091]

[0092] The alcohol-soluble extract of 18 batches of standard decoctions was determined, with an average value of 39.3%. The range calculated using the mean plus or minus 3 times the SD was 36.7%–41.9%; the range calculated using the mean of 70%–130% was 27.5%–51.9%; and the actual range was 37.5%–40.7%.

[0093] It is recommended to take 27.5% to 51.9% as the upper and lower limits of the standard decoction extract.

[0094] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0095] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to any specific implementation. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. The preparation process of the standard mulberry twig decoction, firstly, weigh out mulberry twig slices, place them in a clay pot, and decoct twice, characterized in that... Includes the following steps: Step 1: Add 10 times the amount of water to the mulberry twig slices into the clay pot for the first time, soak for 30 minutes, bring to a boil over high heat, then simmer over low heat for 30 minutes. Step 2: Add 8 times the amount of water to the mulberry twig slices into the clay pot for the second time, bring to a boil over high heat, then simmer over low heat for 20 minutes. Step 3: Then filter the above medicine solution through a 200-mesh filter and cool the filtrate to room temperature in a cold water bath; Step 4: Combine the two filtrates and concentrate them under vacuum at 50°C; Step 5: Concentrate the extract to a 1:1 weight ratio of medicinal material to fluid extract, collect the concentrate, and freeze-dry to obtain the sample.

2. The preparation process of the mulberry twig standard decoction according to claim 1, characterized in that, In Step 4, vacuum concentration is carried out under an absolute vacuum of 30–40 mbar.

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