Dendrobium huoshanense compound granule as well as preparation method and quality detection method thereof

Abstract

The invention discloses dendrobium huoshanense compound granules as well as a preparation method and a quality detection method thereof, and belongs to the technical field of traditional Chinese medicine preparations. Dendrobium huoshanense, radix rehmanniae recen, radix ophiopogonis and liquorice are used as a formula, granules are selected as a medicine carrying form, the advantages of maintaining property and flavor tropism of each medicine, mainly treating functions, being relatively large in medicine carrying capacity, easy to dissolve, quick in effect taking and simple and convenient in process are exerted, and a decoction process, a concentration drying process and a granulation process in pharmacy are optimized; the preparation method which is simple and convenient in granulation, high in yield and high in stability is obtained, and the obtained compound granules are uniform in granules, small in angle of repose and relatively good in flowability. Quality control experiment results show that the dendrobium huoshanense compound granules prepared according to the preparation method provided by the invention are high in content of effective components such as total flavonoids and rehmannia D, the process is stable, and technical support is provided for treatment of oral mucositis.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine preparations, and particularly to a compound granule of Dendrobium huoshanense and its preparation method and quality detection method. Background Art

[0002] Oral mucositis (OM) is a disease in which the normal color, shape, integrity and function of the mucosa in a certain part of the oral cavity change. It is a common complication during the radiotherapy and chemotherapy of malignant tumor diseases. It is mostly manifested as ulcers, erythema, bleeding, pain reactions, and often accompanied by symptoms such as dry mouth, bad breath, and constipation.

[0003] In conventional radiotherapy and chemotherapy, the incidence and severity of OM vary depending on the treatment plan. The incidence of oral mucositis induced by radiotherapy and chemotherapy regimens for tumors in different parts ranges from 6.6% to 65%. At present, the drugs for treating OM have a limited scope of action, obvious side effects and high prices. Therefore, active prevention and treatment are particularly important.

[0004] Traditional Chinese medicine believes that most tumor patients are people with yin deficiency in the body. Chemotherapy drugs belong to the pathogenic factors of "drug toxins" and hot-toxic substances. Their nature is hot and they directly consume the qi, blood and body fluids of the human body. After patients undergo multiple courses of chemotherapy, the balance of yin and yang in the body is disordered, the functions of the zang-fu organs are disrupted, yin and yang, qi and blood are deficient, and heat toxins are hidden inside. When the heat toxins are even more severe, mucosal damage occurs.

[0005] Therefore, there is an urgent need to provide a traditional Chinese medicine preparation with the main therapeutic principles of clearing heat and detoxifying, nourishing yin and promoting fluid production and relieving sore throat. Summary of the Invention

[0006] The purpose of the present invention is to provide a compound granule of Dendrobium huoshanense and its preparation method and quality detection method to solve the problems existing in the above-mentioned prior art. The present invention selects the granule as the drug-loading form, giving play to its advantages of maintaining the nature, flavor, meridian tropism, and functions and indications of each medicinal flavor, relatively large drug-loading capacity, easy dissolution, quick effect, and simple process. The compound granule prepared has a high content of active ingredients and a stable process, providing technical support for the treatment of oral mucositis.

[0007] The present invention formulates a compound of Dendrobium huoshanense by adding and subtracting medicinal flavors based on the ancient formula Zengye Jiedu Decoction. The prescription composition: Dendrobium huoshanense, Rehmannia glutinosa, Ophiopogon japonicus, Glycyrrhiza uralensis. The efficacy is to increase fluid and moisten dryness. Explanation of the formula: In the formula, Dendrobium huoshanense is used as the monarch drug, which is slightly cold in nature, sweet in taste, enters the stomach and kidney meridians, and has the functions of promoting the production of body fluid to nourish the stomach and nourishing yin and clearing heat. Ophiopogon japonicus and Rehmannia glutinosa are sweet and cold-natured and moistening yin products, assisting the monarch drug to increase fluid and moisten dryness, and together they are the ministerial drugs. Glycyrrhiza uralensis has the functions of clearing heat and detoxifying, relieving spasm and pain, and coordinating the actions of various drugs, and is the adjuvant drug. The combination of various drugs clears heat and detoxifies, nourishes yin and increases fluid, promotes the flow of water by increasing fluid, and moistens dryness and relieves constipation.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a preparation method of a compound granule of Dendrobium huoshanense, comprising the following steps:

[0010] Weigh 10 parts of Dendrobium huoshanense, 6 parts of Rehmannia glutinosa, 6 parts of Ophiopogon japonicus, and 5 parts of Glycyrrhiza uralensis by weight;

[0011] Mix the Rehmannia glutinosa, the Ophiopogon japonicus, and the Glycyrrhiza uralensis to obtain a water decoction raw material. After soaking in water, decoct, combine the water decoction liquid, and concentrate to obtain a concentrated liquid;

[0012] Dry the concentrated liquid, pulverize it, and pass it through a No. 6 sieve to obtain a fine powder of dry extract;

[0013] Pulverize the Dendrobium huoshanense, pass it through a No. 6 sieve, combine it with the fine powder of dry extract, and mix evenly to obtain a medicinal powder;

[0014] Mix the medicinal powder with auxiliary materials to obtain a mixed powder, mix it with a wetting agent to make a soft material, granulate it using a No. 1 sieve, collect it and dry it, and screen it with a No. 1 sieve to obtain the compound granule of Dendrobium huoshanense.

[0015] Preferably, the mass ratio of the water decoction raw material to water is 1:16; the soaking time is 1 h; the decoction is carried out 3 times, and the single decoction time is 1 h.

[0016] Preferably, the concentration is reduced pressure concentration to a relative density of 1.21 g / mL at a temperature of 75 °C; the drying of the concentrated liquid is vacuum drying at a temperature of 80 °C.

[0017] Preferably, the auxiliary materials are a mixture of starch and powdered sugar, and the mass ratio of the starch to the powdered sugar is 3:1; the mass ratio of the auxiliary materials to the medicinal powder is 2:3.

[0018] Preferably, the wetting agent is ethanol with a volume fraction of 70%, and the mass of the wetting agent is 15% of the mass of the mixed powder;

[0019] The drying after collection is vacuum drying for 1 h at a temperature of 70 °C.

[0020] The present invention also provides a compound granule of Dendrobium huoshanense obtained according to the above preparation method.

[0021] The present invention also provides a method for detecting the quality of a compound granule of Dendrobium huoshanense, comprising the steps of measuring the total flavonoid content and the content of rehmannioside D in the compound granule of Dendrobium huoshanense.

[0022] Preferably, the measurement of the total flavonoid content comprises the following steps:

[0023] Weigh 5.03 mg of rutin standard product, dissolve it in 70% ethanol and make up the volume to 25 mL to obtain a reference substance solution;

[0024] Respectively take 0, 0.5, 1, 2, 3, 4, 5, 6 mL of the reference solution and place them in 25-mL volumetric flasks. Add 70% ethanol to 6 mL, and then add 1 mL of 5% NaNO 2 solution, mix well and let stand for 5 min. Then add 1 mL of 10% Al(NO 3 ) 3 solution, let stand for 5 min, and then add 10 mL of 1 mol / L NaOH. After mixing well, make up the volume to 25 mL with 70% ethanol, mix thoroughly, let stand for 10 min, measure the absorbance at a wavelength of 510 nm. Plot the standard curve with absorbance as the ordinate and concentration as the abscissa to obtain the regression equation;

[0025] Take 1.0 g of the Huoshan Dendrobium compound granule, add 50 mL of 70% ethanol, ultrasonicate for 1 h, centrifuge for 20 min, take 10 mL of the supernatant, place it in a 25-mL volumetric flask, and add 1 mL of 5% NaNO 2 solution, mix well and let stand for 5 min. Then add 1 mL of 10% Al(NO 3 ) 3 solution, let stand for 5 min, and then add 10 mL of 1 mol / L NaOH. After mixing well, make up the volume to 25 mL with 70% ethanol, mix thoroughly, let stand for 10 min, measure the absorbance at a wavelength of 510 nm.

[0026] Preferably, the measurement of the content of rehmannioside D includes the following steps:

[0027] Precisely measure 0.95 mg of rehmannioside D reference substance, dissolve it with 10 mL of 25% methanol to obtain a reference solution with a concentration of 0.095 mg / mL;

[0028] Take the reference solution, dilute it to different concentrations, and inject samples respectively. Establish the regression equation with concentration as the abscissa and peak area as the ordinate;

[0029] Take 1 g of the Huoshan Dendrobium compound granule, add 25 mL of 25% methanol, ultrasonicate for 30 min, centrifuge, and filter through a 0.22-μm filter membrane to obtain the test solution; perform liquid chromatography detection on the test solution;

[0030] The conditions for the liquid chromatography detection are as follows: high-performance liquid chromatograph: Agilent 1260 infinity Ⅱ; chromatographic column: Agilent ZORBOX SB-Aq, with dimensions of 4.6 mm × 250 mm, 5 μm; mobile phase: 0.05% phosphoric acid water as solution A, acetonitrile as solution B, isocratic elution for 0 - 30 min, 97% solution A, detection wavelength: 203 nm; flow rate: 1.0 mL / min; injection volume: 20 μL; column temperature: 35 °C.

[0031] The present invention discloses the following technical effects:

[0032] The present invention uses Dendrobium huoshanense, Rehmannia glutinosa, Ophiopogon japonicus and Glycyrrhiza uralensis as the formula, selects granules as the drug-loading form, gives play to the advantages of maintaining the nature, flavor, meridian tropism, functions and indications of each medicinal flavor, having a relatively large drug-loading capacity, being easily soluble, having a quick effect and a simple process, and optimizes the decocting process, concentration and drying process and granulation process in pharmaceutical production, obtaining a preparation method with simple granulation, high yield and high stability. The obtained compound granules have uniform particles, a small angle of repose and good fluidity. Through quality control experiments, the results show that the contents of pharmacodynamic components such as total flavonoids and rehmannioside D in the Dendrobium huoshanense compound granules prepared according to the preparation method provided by the present invention are high and the process is stable. The present invention provides technical support for the treatment of oral mucositis. Description of the Drawings

[0033] 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.

[0034] Figure 1 It is the liquid chromatogram of the specificity investigation experiment for the determination of the content of rehmannioside D in the quality control method. Detailed Embodiments

[0035] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered 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.

[0036] It should be understood that the terms described in the present invention are only for describing specific embodiments 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 can be independently included or excluded from the range.

[0037] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0038] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the description of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of this invention are obvious to those skilled in the art. The description and examples of this invention are merely exemplary.

[0039] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0040] The concentration of ethanol used in this invention is the volume fraction of ethanol.

[0041] The method for determining the total saponin content in this invention is as follows: Refer to the method for determining the total saponin under Ophiopogonis Radix in the Chinese Pharmacopoeia (2020 Edition). Preparation of the reference solution: Weigh an appropriate amount of ruscogenin standard precisely, add methanol to obtain the stock solution.

[0042] Preparation of the standard curve: Precisely measure 0.5, 1, 2, 3, 4, 5, 6 mL of the reference solution respectively, place them in 15 mL centrifuge tubes, evaporate the solvent in a water bath, precisely add 10 mL of perchloric acid, shake well, place in a water bath at 70 °C for 15 min, take out, cool with ice water, use the corresponding reagent as the blank, and determine the absorbance at a wavelength of 397 nm according to the ultraviolet-visible spectrophotometry (General Rule 0401). Draw a standard curve with the absorbance as the ordinate and the concentration as the abscissa.

[0043] Preparation of the test solution: Extraction solution sample: Take 10 mL of each extraction solution, shake and extract 5 times with water-saturated n-butanol solution, 10 mL each time, combine the n-butanol solution, wash it 2 times with ammonia test solution, 5 mL each time, discard the ammonia test solution, evaporate the n-butanol solution to dryness; dissolve the residue with 80% methanol, transfer it to a 50 mL volumetric flask, make up to the mark, shake well, and obtain the solution. Determine the absorbance from "evaporate the solvent in a water bath", calculate the total saponin concentration C1 according to the standard curve, and then calculate the total saponin content according to the following formula. Calculation formula:

[0044] Total saponin content (mg / g) = concentration C1 × total volume V of the extraction solution / weight M of the original medicinal material.

[0045] Concentrate sample: Take 1 mL of the concentrate, dilute it with water to a volume of 25 mL in a volumetric flask, take 10 mL of the diluted solution, shake and extract it 5 times with water-saturated n-butanol solution, and the rest is determined by the above method.

[0046] Dry extract powder sample: Take 0.3 g of the finely ground dry extract powder, add 10 mL of water to dissolve it, shake and extract it 5 times with water-saturated n-butanol solution, and the rest is determined by the above method.

[0047] The method for determining the dry extract yield in the present invention is as follows: Precisely measure 20 mL of the extract after decoction or 2 mL of the concentrate, place them in an evaporating dish that has been dried to a constant weight respectively, evaporate to dryness in a water bath, dry the residue at 105 °C for 3 h, then place it in a desiccator to cool for 30 min, quickly and precisely weigh it, and calculate the dry extract yield according to the following formula:

[0048] Dry extract yield (%) = (dry extract mass / volume of the test sample taken) × (total extract volume / mass of the medicinal materials taken) × 100.

[0049] The method for determining the content of rehmannioside D in the present invention is as follows: Preparation of the reference solution: Precisely measure 0.95 mg of the rehmannioside D reference standard, dissolve it in 10 mL of 25% methanol to obtain a reference solution with a concentration of 0.095 mg / ml.

[0050] Establishment of the standard curve: Dilute the reference stock solution to different concentrations and inject them respectively. Using the concentration as the abscissa and the peak area as the ordinate, establish a regression equation. The obtained regression equation is y = 10627x + 2.5367 (R2 = 0.9999), and there is a good linear relationship for the injection concentration of rehmannioside D in the range of 0.0095 - 0.095 mg / mL.

[0051] Preparation of the test solution: Extract sample: Take 1 g of the granule, add 25 mL of 25% methanol, ultrasonicate for 30 min, centrifuge, and filter through a 0.22 μm filter membrane to obtain the solution.

[0052] Concentrate sample: Take 1 mL of the concentrate, dilute and make up the volume to 25 mL with water, transfer 3 mL of the diluted solution, make up the volume to 5 mL with 25% methanol, ultrasonicate for 30 min, and filter through a 0.22 μm filter membrane to obtain the solution.

[0053] Dry extract powder sample: Take 0.3 g of the finely ground dry extract powder, add 10 mL of water to dissolve it, take 3 mL of the solution, make up the volume to 5 mL with 25% methanol, ultrasonicate for 30 min, and filter through a 0.22 μm filter membrane to obtain the solution.

[0054] Granule sample: Take 1 g of the granule, add 25 mL of 25% methanol, ultrasonicate for 30 min, centrifuge, and filter through a 0.22 μm filter membrane to obtain the solution.

[0055] Chromatographic conditions: High performance liquid chromatograph: Agilent 1260 infinity Ⅱ; Chromatographic column: Agilent ZORBOX SB-Aq (4.6 mm × 250 mm, 5 μm); Mobile phase: 0.05% phosphoric acid water (A) - acetonitrile (B), isocratic elution (0 - 30 min, 97% A), detection wavelength: 203 nm; Flow rate: 1.0 mL / min; Injection volume: 20 μL; Column temperature: 35 °C.

[0056] Inject the test sample solution according to the above chromatographic conditions.

[0057] The method for determining the yield in the present invention is as follows: Collect the dried compound dry extract powder, accurately weigh it (m1), the total mass of the Chinese medicinal materials in the prescription is m2, and calculate the yield (%) of the dry extract powder according to the formula:

[0058] Yield (%) = m1 / m2 × 100.

[0059] The method for determining the water content in the present invention is as follows: Take an appropriate amount of the dry extract powder or granule sample, place it in a flat weighing bottle (m1) dried to a constant weight, accurately weigh it (m2), open the bottle cap and dry it at 105 °C for 3 h, cover the bottle cap, transfer it to a desiccator, cool for 30 min, accurately weigh it, then dry it at 105 °C for 1 h, cool it, and accurately weigh it (m3) until the difference between two consecutive weighings does not exceed 5 mg. According to the weight loss, calculate the water content (%) according to the formula.

[0060] Water content (%) = (m2 - m3) / (m2 - m1) × 100%.

[0061] Example 1

[0062] 1. Dosage form selection

[0063] When selecting the dosage form, it is necessary to comprehensively consider the properties of the Chinese medicinal materials in the prescription, the dosage, safety, effectiveness, and convenience of production and transportation after the preparation is formed. The compound of Huoshan Dendrobium is administered orally clinically, and dosage forms such as tablets, granules, capsules, oral liquids, and syrups are preferably selected. Liquid preparations such as oral liquids are inconvenient to use and carry, and when the amount of crude drug per unit dose is high, the stability of the finished product and the transfer rate of active ingredients are also difficult to meet the requirements. Therefore, solid preparations are considered as the suitable dosage form for this prescription.

[0064] The prescription provided by the present invention consists of four traditional Chinese medicines. Among them, there is a precious medicinal material, Huoshan Dendrobium. To avoid losses during extraction, it is advisable to grind it into powder for use in medicine. For medicinal materials such as Rehmannia glutinosa in the prescription, they contain relatively more sugar, and the extract has a large viscosity. Considering that a large amount of excipients need to be added to assist in the formation of the preparation, therefore, a dosage form with a relatively large drug-loading capacity needs to be selected. Granules are a commonly used drug-loading form for traditional Chinese medicine compounds, which can better maintain the nature, flavor, meridian tropism, and functions and indications of each medicinal flavor, have a relatively large drug-loading capacity, are easily soluble, have a quick effect, and the process is simple and suitable for large-scale production operations. Therefore, granules are selected as the dosage form of this prescription.

[0065] 2. Selection of Extraction Method

[0066] According to the "Special Provisions on the Administration of Traditional Chinese Medicine Registration": For innovative traditional Chinese medicine prescriptions derived from ancient classic famous prescriptions or traditional Chinese medicine clinical experience prescriptions, if the prescription composition, clinical positioning, dosage and administration, etc. are basically the same as those in previous clinical applications, using traditional processes that are basically the same as the drugs used clinically, and the functions and indications, applicable populations, dosing regimens, and clinical benefits, etc. can be initially determined through human experience, non-clinical efficacy studies may not be carried out. Therefore, when selecting the process route of the present invention, the clinical usage of the original prescription is followed, that is, water decoction method is used as its extraction process.

[0067] 3. Prescription of Granule Raw Materials

[0068] 10 g of Huoshan Dendrobium, 6 g of Rehmannia glutinosa, 6 g of Ophiopogon japonicus, and 5 g of Glycyrrhiza uralensis.

[0069] 4. Preparation Process

[0070] The process for preparing the compound granules of Huoshan Dendrobium of the present invention is briefly described as follows:

[0071] Take a portion of the prescription medicinal materials without Huoshan Dendrobium (Rehmannia glutinosa, Ophiopogon japonicus, Glycyrrhiza uralensis), soak them in water, decoct, combine the water decoction liquid, concentrate it to a relative density of about 1.21 g / mL, dry the concentrated liquid, and the obtained dried product is pulverized and passed through a No. 6 sieve (100 mesh) to obtain fine powder of dry extract; pulverize Huoshan Dendrobium and pass it through a No. 6 sieve to obtain fine powder. Combine and mix the fine powder of Huoshan Dendrobium with the fine powder of dry extract, add excipients, mix well, add ethanol to make soft materials, granulate with a No. 1 sieve (10 mesh), collect and dry, and screen with a No. 1 sieve to obtain the product.

[0072] Example 2 Investigation of Extraction Process

[0073] 1. Investigation of Soaking Time

[0074] In the water decoction process, the factor of soaking time has a certain influence on other factors, so it is investigated separately, and the optimal soaking time is determined through the correlation between the water absorption of the medicinal materials and the soaking time.

[0075] Weigh 15 portions of the prescription medicinal materials without Dendrobium huoshanense, add water with a mass 10 times that of the medicinal materials. Place them at room temperature and take them out at 30, 40, 50, 60, and 70 minutes respectively. Filter out the moisture of the medicinal materials, weigh the weight of the medicinal materials after water absorption, calculate the water absorption rate of the medicinal materials at each time, and have 3 parallels for each soaking time. Calculate the water absorption rate according to the following formula:

[0076] Water absorption rate (%) = (weight of the medicinal materials after water absorption - original weight of the medicinal materials) / original weight of the medicinal materials.

[0077] The results are shown in Table 1, indicating that the water absorption rate is the highest when soaking for 60 minutes, and the water absorption rate of the medicinal materials decreases after soaking for 70 minutes. Therefore, the soaking time is determined to be 60 minutes.

[0078] Table 1 Investigation of soaking time ( n = 3)

[0079]

[0080]

[0081] 2. Investigation of decocting time

[0082] Weigh 9 portions of the prescription medicinal materials without Dendrobium huoshanense, add water with a mass 10 times that of the medicinal materials, soak for 30 minutes, and decoct for 30 minutes, 45 minutes, and 60 minutes respectively. After decocting once, calculate the total saponin content and the dry extract yield, and have 3 parallels for each decocting time.

[0083] Table 2 Investigation of decocting time ( n = 3)

[0084]

[0085] Note: a Indicates that compared with decocting for 30 minutes, P < 0.05; b Indicates that compared with decocting for 45 minutes, P < 0.05.

[0086] The results are shown in Table 2. When the decocting time of the medicinal materials is 30 minutes, the dry extract yield is 37.81%, and the total saponin content is 0.78 mg / mL; when the decocting time is 45 minutes and 60 minutes, the dry extract yields are 38.85% and 36.94% respectively, and the total saponin contents are 0.88 mg / mL and 0.92 mg / mL respectively; the results of variance analysis show that there are significant differences in the dry extract yields at 45 minutes and 60 minutes (P < 0.05), and there are significant differences in the total saponin contents at 30 minutes, 45 minutes, and 60 minutes (P < 0.05), and they show a gradually increasing trend. Therefore, select the 3 levels of decocting times of 30, 45, and 60 minutes for the subsequent orthogonal experiment.

[0087] 3. Water addition amount

[0088] Take 18 portions of the prescription medicinal materials without Dendrobium huoshanense, add water with 10, 12, 14, 16, 18, and 20 times the mass respectively, soak for 30 min, decoct for 30 min, and decoct once. Then calculate the total saponin content and the dry extract yield. Each water addition amount has 3 parallels.

[0089] Table 3 Investigation of water addition amount( n = 3)

[0090]

[0091] Note: a Indicates P < 0.05 compared with adding water with 10 times the amount; b Indicates P < 0.05 compared with adding water with 12 times the amount; c Indicates P < 0.05 compared with adding water with 14 times the amount; d Indicates P < 0.05 compared with adding water with 16 times the amount.

[0092] The results are shown in Table 3. When the water addition amount is 10, 12, 14, 16, 18, and 20 times that of the extracted medicinal materials, the dry extract yield shows a trend of first increasing and then decreasing. Among them, the dry extract yield with 16 times water is the highest, and the dry extract yields with 18 times and 20 times water start to decline; the total saponin content shows an upward trend. The results of variance analysis show that there is no statistical difference between 18 times water and 20 times water. Therefore, 14, 16, and 18 times are selected as 3 levels for the orthogonal experiment.

[0093] 4. Decoction times

[0094] Take 9 portions of the prescription medicinal materials without Dendrobium huoshanense, add water with 10 times the mass respectively, soak for 30 min, decoct for 30 min, and decoct 1, 2, and 3 times respectively. Then calculate the total saponin content and the dry extract yield. Each decoction time has 3 parallels.

[0095] Table 4 Investigation of decoction times( n = 3)

[0096]

[0097] Note: a Indicates P < 0.05 compared with decocting 1 time; b Indicates P < 0.05 compared with decocting 2 times.

[0098] The results are shown in Table 4. When the number of decoction times was 1, the dry extract yield was 37.81% and the total saponin content was 7.809 mg / L. When the number of decoction times was 2, the dry extract yield was 51.15% and the total saponin content was 13.54 mg / mL, both of which increased significantly compared with that of 1 decoction time. After the third decoction, the dry extract yield and the total saponin content further increased, reaching 51.50% and 16.03 mg / mL respectively. The results of one-way ANOVA showed that there were significant differences in the dry extract yield and the total saponin content between 1 decoction time and 2 or 3 decoction times (P<0.05). Therefore, three levels of 1, 2, and 3 decoction times were selected for the orthogonal experiment.

[0099] 5. Orthogonal experiment

[0100] On the basis of the single-factor experiment, taking the number of decoction times, the amount of water added, and the decoction time as the investigation factors, the experiment was designed according to the L9(3 4 ) orthogonal table. Three levels were set for each factor, and the factor level table is shown in Table 5.

[0101] Table 5 Factor level table of extraction process

[0102]

[0103] Weigh 6 g of Rehmanniae Radix, 6 g of Ophiopogonis Radix, and 5 g of Glycyrrhizae Radix respectively, for a total of 9 portions. Extract according to the three-factor and three-level orthogonal table (Table 5), filter, and combine the extraction solutions of each time, and record the volume. Take an appropriate amount of the above solution, and measure and calculate the dry extract yield (%), the total saponin content (mg / g), and the content of Rehmannioside D (mg / g). The orthogonal experiment design and results are shown in Table 6, and the ANOVA results are shown in Table 7.

[0104] Table 6 Orthogonal experiment design and results

[0105]

[0106]

[0107] Note: Comprehensive score = dry extract yield / maximum dry extract yield * 30 + total saponin content / maximum total saponin content * 35 + content of Rehmannioside D / maximum content of Rehmannioside D * 35

[0108] Table 7 ANOVA results

[0109]

[0110] It can be seen from the intuitive analysis results in Table 6 that the influence of each factor on the comprehensive score is A>B>C, that is, the number of decoction times > the amount of water added > the decoction time, and the optimized process is A3B3C3.

[0111] As can be seen from the results of the variance analysis in Table 7, the number of decocting times has a significant effect on the comprehensive score (P<0.05), while the amount of added water and the decocting time have no significant effect, indicating that the amount of added water and the decocting time can be adjusted according to the actual production conditions. Considering the subsequent concentration time-consuming and resource conservation in the actual production process, the added water quality is adjusted to 16 times the quality of the medicinal materials. Therefore, the optimal extraction process is screened as adding 16 times the amount of water, soaking for 60 minutes, decocting 3 times, and each time for 1 hour.

[0112] 6. Process verification

[0113] Weigh three portions of the medicinal materials and extract them according to the optimal extraction process screened above to conduct the verification of the water extraction process, with three parallel runs.

[0114] Table 8 Results of process verification

[0115]

[0116] The results are shown in Table 8. The comprehensive scores obtained by extracting with the optimized process are 99.18, 99.03, and 97.95 respectively, with an average value of 98.72 and an RSD of 0.68%, indicating that the optimized process is stable and feasible.

[0117] Research results of the concentration process and drying process in Example 3

[0118] 1. Investigation of the concentration process

[0119] The concentration methods include atmospheric pressure concentration and reduced pressure concentration. Considering that the atmospheric pressure concentration takes too long and may cause relatively large losses, reduced pressure concentration is selected.

[0120] Weigh 9 portions of the prescription medicinal materials without Dendrobium huoshanense, extract them according to the optimal process screened in Example 2, and then divide the water decoction equally into 9 portions, with 3 portions in parallel at each temperature. Concentrate them under reduced pressure to a ratio of medicinal materials to liquid of 1:1 (relative density is about 1.21 g / mL) at 65°C, 75°C, and 85°C respectively, and measure and calculate the total saponin content and the content of rehmannioside D.

[0121] The results are shown in Table 9. As the concentration temperature increases, the total saponin and rehmannioside D contents gradually decrease. During the experiment, it is found that the time required for concentration at different temperatures varies greatly. The concentration time at 65°C is about 4.0 hours, the concentration time at 75°C is about 2.5 hours, and the concentration time at 85°C is significantly reduced compared to that at 65°C, about 2.0 hours, with a relatively high concentration efficiency. Considering comprehensively, the extraction solution of the medicinal materials is concentrated at 75°C.

[0122] Table 9 Investigation results of the concentration process ( n = 3)

[0123]

[0124] 2. Investigation of the drying method

[0125] Weigh 9 portions of the prescription medicinal materials without Dendrobium huoshanense, extract and concentrate them according to the method in "1. Investigation of the concentration process", then divide the concentrated liquid into 3 equal portions, each portion being 14 mL. Dry them in a forced-air oven at 70 °C, in a vacuum oven at 70 °C, and by vacuum freeze-drying respectively. Collect the dry extract powder and measure the yield (%), water content (%), total saponin content, and rehmannioside D content.

[0126] The results are shown in Table 10. The dry extracts obtained by vacuum drying and freeze-drying are relatively loose in texture and have a higher yield of dry extract powder; the dry extract obtained by forced-air drying is relatively hard and has more losses. However, in comparison, freeze-drying consumes a large amount of energy and has a lower efficiency in large-scale production. According to the analysis of the measurement results, among the three drying methods, the dry extract powder obtained by vacuum drying has a higher yield, higher total saponin content and rehmannioside D content, lower water content, and a shorter drying time. Therefore, it is considered more appropriate to adopt vacuum drying at 70 °C.

[0127] Table 10 Results of the investigation of drying methods ( n = 3)

[0128]

[0129] 3. Investigation of drying temperature

[0130] Weigh 9 portions of the prescription medicinal materials without Dendrobium huoshanense, extract and concentrate them according to the method in "1. Investigation of the concentration process", then divide the concentrated liquid into 3 equal portions, each portion being 14 mL. Dry them in a vacuum oven at 60 °C, 70 °C, and 80 °C respectively. Collect the dry extract powder and measure the yield (%), water content (%), total saponin content, and rehmannioside D content.

[0131] The results are shown in Table 11. The yields of dry extract powder at 60 °C, 70 °C, and 80 °C are 49.84%, 46.89%, and 53.17% respectively, and the yield of dry extract powder at 80 °C is the highest. The water contents are 3.49%, 3.45%, and 4.14% respectively. The water content of the dry extract powder by vacuum drying at 80 °C is relatively high, but there is no significant difference. As the temperature increases, there is a significant difference in the drying time, and the drying time at 80 °C is the shortest, which is 12 h. Therefore, vacuum drying at 80 °C for 12 h is selected as the optimal drying process.

[0132] Table 11 Results of the investigation of vacuum drying temperature ( n = 3)

[0133]

[0134] Example 4 Forming process of the preparation

[0135] 1. Selection of granulation method

[0136] The dry extract powder obtained in Example 3 was ground and passed through a No. 6 sieve (100-mesh sieve). The powder of Dendrobium huoshanense was ground and passed through a No. 6 sieve, and then reserved for use.

[0137] Wet granulation: Take 4.5 g of half the amount of the fine dry extract powder, weigh 5 g of the fine powder of Dendrobium huoshanense, mix them evenly, then add 9.5 g of dextrin in a ratio of 1:1 to the amount of medicinal powder, and mix well. Add 70% ethanol (volume fraction) accounting for 25% of the total mass in small amounts and multiple times to make soft materials. Granulate with a No. 1 sieve, dry in a forced-air dryer at 70 °C for 1 h, and then screen with a No. 1 sieve to obtain the granules.

[0138] Thick extract granulation: Take one portion of the thick extract concentrated to 1.275 g / mL, add 10 g of the fine powder of Dendrobium huoshanense, add 9.5 g of dextrin, mix well to make soft materials, granulate with a No. 1 sieve, dry in a forced-air dryer at 70 °C for 1 h, and then screen with a No. 1 sieve to obtain the granules.

[0139] The investigation results are shown in Table 12. The granules prepared by the wet method are uniform, with a high granulation rate, a repose angle < 30°, and good fluidity. While the soft materials prepared by the thick extract method are more sticky, the color of the granules is relatively light, the granules are uneven, and the granulation rate is low. Through comprehensive comparison, the wet granulation method should be selected.

[0140] Table 12 Investigation Results of Granulation Methods

[0141]

[0142] 2. Investigation of Types of Excipients

[0143] Take 4.5 g of half the amount of the fine dry extract powder and 5 g of the fine powder of Dendrobium huoshanense respectively, and add different excipients (dextrin, starch, powdered sugar, lactose, powdered sugar - dextrin, powdered sugar - starch, powdered sugar - lactose, dextrin - starch, dextrin - lactose) in an amount of 9.5 g according to the mass ratio of medicine to excipient of 1:1. After mixing evenly, add the wetting agent (70% ethanol) accounting for 20% of the total mass in small amounts and multiple times to make soft materials. Granulate with a No. 1 sieve, dry in a forced-air drying oven at 70 °C for 1 h, and then screen with a No. 1 sieve to obtain the granules.

[0144] The investigation results are shown in Table 13. Among the pure excipients (dextrin, starch, powdered sugar, lactose), when using dextrin, the soft materials are loose, the obtained granules contain more fine powder, and the granulation rate is 93.82%; when using starch, the soft materials are moderate, the obtained granules are uniform, and the granulation rate is 92.71%; when using powdered sugar, the soft materials are moderate, the obtained granules are slightly hard, the color is dark brownish yellow, and the granulation rate is the highest, which is 95.62%. The repose angles of the granules prepared with the above three excipients are all < 30°, and the fluidity is good. When using lactose, the soft materials are slightly sticky, the obtained granules are slightly hard, the color is dark brown, and the granulation rate is 94.83%.

[0145] Since the sugar powder has the highest forming rate and the particles obtained from dextrin have moderate hardness, the granulation of the mixed excipients (sugar powder-dextrin, sugar powder-starch, sugar powder-lactose, dextrin-starch, dextrin-lactose) with a mass ratio of 1:1 was investigated. Among them, the soft material obtained from sugar powder-dextrin was moderate, the resulting particles were light brownish-yellow, the particles were uniform, and the forming rate was 91.66%; the soft material obtained from sugar powder-starch was moderate, the resulting particles were brownish-yellow, the particles were uniform, and the forming rate was the highest, at 97.11%; the soft material obtained from sugar powder-lactose was moderate, the resulting particles were dark brownish-yellow, the particles were relatively hard, and the forming rate was 93.54%; the soft material obtained from dextrin-starch was moderate, the resulting particles were light brownish-yellow, there were more fine powders in the particles, and the forming rate was 89.63%; the soft material obtained from dextrin-lactose was moderate, the resulting particles were light brownish-yellow, the particles were uniform, and the forming rate was 89.80%. Therefore, sugar powder-starch was selected as the best excipient for the next investigation.

[0146] Table 13 Investigation results of excipient types

[0147]

[0148] 3. Investigation of excipient dosage

[0149] Take 4.5 g of the fine powder of the semi-dose dry extract and 5 g of the fine powder of Dendrobium huoshanense respectively, add the excipients (dextrin and starch mixed in a mass ratio of 1:1), and add the excipients according to the drug-excipient mass ratios of 1:1, 11:9, 3:2, and 7:3 respectively (that is, the proportions of the excipient dosage in the total mass of the drug powder and excipient are 50%, 45%, 40%, and 30% respectively). After mixing evenly, add 20% of the wetting agent (70% ethanol) in small amounts and multiple times to make the soft material, granulate through a No. 1 sieve, dry in a forced-air drying oven at 70 °C for 1 h, and screen through a No. 1 sieve to obtain the product.

[0150] The investigation results are shown in Table 14. When the excipient dosage was 50%, the soft material prepared was slightly sticky, the resulting particles were dark brownish-yellow, the particles were slightly hard, and the forming rate was 95.78%; when the excipient dosage was 45%, the soft material prepared was slightly sticky, the resulting particles were dark brownish-yellow, the particles were uniform, and the forming rate was 97.34%; when the excipient dosage was 40%, the texture of the soft material prepared was moderate, the resulting particles were dark brownish-yellow, the particles were uniform, and the forming rate was the highest, at 98.02%; when the excipient dosage was 30%, the texture of the soft material prepared was moderate, the resulting particles were dark brownish-yellow, the particles were uniform, and the forming rate was 96.77%. Therefore, the excipient with an addition amount of 40% was selected as the preferred plan.

[0151] Table 14 Investigation results of excipient dosage

[0152]

[0153] 4. Investigation of the ratio of mixed excipients

[0154] Weigh 4.5 g of the fine powder of the dry extract and 5 g of the fine powder of Dendrobium huoshanense separately. Add sugar powder - starch according to the drug - excipient mass ratio of 3:2. The mixing ratios of the two excipients are 1:1, 1:2, 1:3, and 1:4 respectively. After mixing, add the wetting agent (70% ethanol) with a total mass of 20% in small portions and multiple times to prepare soft materials. Granulate through a No. 1 sieve, dry in a forced - air drying oven at 70 °C for 1 h, and screen through a No. 1 sieve to obtain the product.

[0155] The investigation results are shown in Table 15. When the mixing ratio of sugar powder - starch is 1:1, the prepared soft material is moderate. The obtained granules are dark brown - yellow, the granules are uniform, the forming rate is 97.62%, and the angle of repose is 27.70°; when the mixing ratio of sugar powder - starch is 1:2, the prepared soft material is moderate. The obtained granules are dark brown - yellow, the granules are uniform, the forming rate is 97.22%, and the angle of repose is 27.14°; when the mixing ratio of sugar powder - starch is 1:3, the prepared soft material is moderate. The obtained granules are dark brown - yellow, the granules are uniform, the forming rate is 97.57%, and the angle of repose is 26.56°, and the angle of repose is the smallest, with better fluidity; when the mixing ratio of sugar powder - starch is 1:4, the prepared soft material is moderate. The obtained granules are dark brown - yellow, the granules are uniform, the forming rate is 95.57%, and the angle of repose is 28.81°. Considering comprehensively, the mixing ratio of sugar powder - starch of the excipient is selected as 1:3.

[0156] Table 15 Investigation results of the mixing ratio of the mixed excipients

[0157]

[0158] 5. Investigation of the dosage of the wetting agent

[0159] Weigh 4.5 g of the fine powder of the dry extract and 5 g of the fine powder of Dendrobium huoshanense separately. Add sugar powder - starch (the mass ratio of sugar powder and starch is 1:3) according to the drug - excipient mass ratio of 3:2. After mixing, use 70% ethanol as the wetting agent and add 10%, 15%, 20%, and 25% of the total mass of the solids respectively to prepare soft materials. Granulate through a No. 1 sieve, dry in a forced - air drying oven at 70 °C for 1 h, and screen through a No. 1 sieve to obtain the product.

[0160] The investigation results show that when the dosage of the wetting agent is 10%, the prepared soft material is slightly dry. The obtained granules are brown - yellow, there are more fine powder in the granules, and the forming rate is 88.80%; when the dosage of the wetting agent is 15%, the prepared soft material is moderate. The obtained granules are dark brown - yellow, the granules are uniform, and the forming rate is 95.11%; when the dosage of the wetting agent is 20%, the prepared soft material is moderate. The obtained granules are dark brown - yellow, the granules are uniform, and the forming rate is 94.90%; when the dosage of the wetting agent is 25%, the prepared soft material is slightly sticky. The obtained granules are dark brown - yellow, the granules are relatively hard, and the forming rate is 94.96%. Considering comprehensively, the amount of the wetting agent added is selected as 15%.

[0161] Table 16 Investigation results of the dosage of the wetting agent

[0162]

[0163] 6. Investigation on the concentration of wetting agent

[0164] Take 4.5 g of fine powder of half-dose dry extract and 5 g of fine powder of Dendrobium huoshanense respectively. Add sugar powder-starch (the mass ratio of sugar powder to starch is 1:3) according to the mass ratio of drug to excipient of 3:2. After mixing, use 50%, 60%, 70%, and 80% ethanol as wetting agents respectively, add 15% of the wetting agent based on the total mass of solids, prepare soft materials, granulate through a No. 1 sieve, dry in a forced-air drying oven at 70 °C for 1 h, and screen through a No. 1 sieve to obtain the products.

[0165] The investigation results are shown in Table 17. When the concentration of the wetting agent is 50% and 60%, the prepared soft materials are relatively sticky and difficult to granulate. The obtained granules are dark brown, relatively hard and have uneven particle sizes. The forming rates are 91.46% and 92.48% respectively. When the concentration of the wetting agent is 70% and 80%, the prepared soft materials are moderate, easy to granulate. The obtained granules are dark brownish-yellow and have uniform particle sizes. The forming rates are 96.43% and 93.78% respectively. Therefore, 70% ethanol is selected as the wetting agent.

[0166] Table 17 Investigation results of the concentration of wetting agent

[0167]

[0168]

[0169] 7. Investigation on drying methods and time

[0170] Take 4.5 g of fine powder of half-dose dry extract and 5 g of fine powder of Dendrobium huoshanense respectively. Add sugar powder-starch (the mass ratio of sugar powder to starch is 1:3) according to the mass ratio of drug to excipient of 3:2. After mixing, use 70% ethanol as the wetting agent, add 15% of the wetting agent based on the total mass of solids, prepare soft materials, granulate through a No. 1 sieve, and dry in a forced-air drying oven at 70 °C for 1 h, 2 h and in a vacuum drying oven at 70 °C for 1 h, 2 h respectively, and screen through a No. 1 sieve to obtain the products.

[0171] The investigation results show that after drying in a forced-air drying oven for 1 h, the obtained granules are dark brownish-yellow, with uniform particle sizes, the forming rate is 95.71%, and the angle of repose is 25.99°. After drying in a forced-air drying oven for 2 h, the obtained granules are dark brownish-yellow, with uniform particle sizes, slightly more fine powder, the forming rate is 93.28%, and the angle of repose is 27.14°. After vacuum drying for 1 h, the obtained granules are dark brownish-yellow, with uniform particle sizes, the forming rate is 95.39%, and the angle of repose is 26.56°. After vacuum drying for 2 h, the obtained granules are dark brownish-yellow, with uniform particle sizes, the forming rate is 95.36%, and the angle of repose is 27.70°. Considering comprehensively, vacuum drying at 70 °C for 1 h is selected as the drying method for preparing the granule agent.

[0172] Table 18 Investigation results of drying methods and drying time

[0173]

[0174] 8. Process Validation

[0175] The optimal process screened by the present invention:

[0176] Take a prescription of medicinal materials without Dendrobium huoshanense (Rehmannia glutinosa, Ophiopogon japonicus, Glycyrrhiza uralensis), add 16 times the mass of water, soak for 1 h, decoct for 1 h, decoct 3 times in total, combine the decoction liquid, concentrate under reduced pressure at 75 °C to a relative density of about 1.21 g / mL, place the concentrated liquid in a vacuum drying oven and dry at 80 °C, and the obtained dried product is pulverized and sieved through a No. 6 sieve (100 mesh) to obtain fine powder of dry extract; pulverize Dendrobium huoshanense and sieve through a No. 6 sieve to obtain fine powder, combine and mix the Dendrobium huoshanense powder and the fine powder of dry extract to obtain medicinal powder, add sugar powder - starch (mass ratio of starch to sugar powder is 1:3) according to the mass ratio of medicine to excipient of 3:2, mix evenly, add 70% ethanol accounting for 15% of the total mass of solids to make soft materials, granulate with a No. 1 sieve (10 mesh), collect and dry in a vacuum drying oven at 70 °C for 1 h, and screen with a No. 1 sieve to obtain the product.

[0177] Take 3 portions of medicinal materials respectively, prepare 3 portions of granules in parallel according to the optimal process screened by the present invention, and measure the corresponding parameters. The results are shown in Table 19. The prepared granules are uniform. The calculated granulation rate is 94.78%, and the angle of repose is 26.94°. The RSD value is less than 3%, indicating that the preparation process is stable and feasible.

[0178] Table 19 Results of Process Validation

[0179]

[0180] Quality Control Method of Example 5

[0181] Take the 3 batches of Dendrobium huoshanense compound granule samples prepared above for subsequent content determination.

[0182] 1. Determination of Total Flavonoid Content

[0183] 1.1 Preparation of reference solution: Weigh 5.03 mg of rutin reference standard, dissolve and dilute to 25 mL with 70% ethanol to obtain the reference solution.

[0184] 1.2 Establishment of standard curve: Take 0, 0.5, 1, 2, 3, 4, 5, 6 mL of the reference standard respectively and place them in 25 mL volumetric flasks, add 70% ethanol to 6 mL, add 1 mL of 5% NaNO 2 solution, mix well and let stand for 5 min, then add 1 mL of 10% Al(NO 3 ) 3The solution was allowed to stand for 5 min, then 10 mL of 1 mol / L NaOH was added. After mixing evenly, 70% ethanol was added to make up the volume to 25 mL. After thorough mixing, it was allowed to stand for 10 min, and the absorbance was measured at a wavelength of 510 nm. With the absorbance as the ordinate and the concentration as the abscissa, a standard curve was plotted. The regression equation obtained was y = 4.3319x + 0.0034 (R 2 = 0.9990).

[0185] 1.3 Preparation of the test solution: Take 1.0 g of the granule, add 50 mL of 70% ethanol, ultrasonicate for 1 h, centrifuge for 20 min, take 10 mL of the supernatant, place it in a 25-mL volumetric flask, and add 1 mL of 5% NaNO 2 solution. After mixing evenly and allowing to stand for 5 min, add 1 mL of 10% Al(NO 3 ) 3 solution. Allow to stand for 5 min, then add 10 mL of 1 mol / L NaOH. After mixing evenly, add 70% ethanol to make up the volume to 25 mL. After thorough mixing, allow to stand for 10 min, and measure the absorbance at a wavelength of 510 nm.

[0186] 1.4 Methodology investigation

[0187] 1.4.1 Precision investigation

[0188] Take 1.0 g of the sample of Huoshan Dendrobium compound granule, prepare the test solution according to the above method, and continuously measure it 6 times according to the above method. The calculated RSD is 1.56%, indicating good precision.

[0189] 1.4.2 Stability investigation

[0190] Take 1.0 g of the sample of Huoshan Dendrobium compound granule, prepare the test solution according to the above method, measure the absorbance every 10 min within 1 h, and the calculated RSD is 3.32%, indicating good stability.

[0191] 1.4.3 Repeatability investigation

[0192] Take 6 portions of the same sample of Huoshan Dendrobium compound granule, prepare 6 portions of the test solution according to the above method, and measure them separately. The calculated RSD is 3.18%, indicating good repeatability of the method.

[0193] 1.4.4 Accuracy investigation (spiked recovery test)

[0194] Take 1 g of the granular sample with known total flavonoid content, weigh accurately in 6 parallels, place in a 100 mL stoppered conical flask, respectively pipette 10 mL of rutin reference substance solution with a concentration of 0.2012 mg / mL, add to the above conical flask to prepare the test solution. Calculate the average recovery rate to be 99.62%, and the RSD is 3.88%, indicating that the method is stable and reliable.

[0195] 1.5 Determination of the total flavonoid content in the sample

[0196] Table 20 Results of the determination of the total flavonoid content

[0197]

[0198] The results are shown in Table 20. After determination, the total flavonoid content of 3 batches of granules ranges from 4.015 mg / g to 4.397 mg / g, and the RSD value is 3.83%, indicating that the sample quality is stable and controllable.

[0199] 2. Determination of rehmannioside D content

[0200] 2.1 Preparation of the reference substance solution: Accurately pipette 0.95 mg of rehmannioside D reference substance, dissolve with 10 mL of 25% methanol (volume fraction) to obtain a reference substance solution with a concentration of 0.095 mg / mL.

[0201] 2.2 Establishment of the standard curve: Dilute the reference substance stock solution to different concentrations and inject samples respectively. With the concentration as the abscissa and the peak area as the ordinate, establish the regression equation. The obtained regression equation is y = 10627x + 2.5367 (R 2 = 0.9999), and the linear relationship of the injection concentration of rehmannioside D is good in the range of 0.0095 - 0.095 mg / mL.

[0202] 2.3 Preparation of the test solution: Take 1 g of the granule, add 25 mL of 25% methanol (volume fraction), ultrasonicate for 30 min, centrifuge, and filter through a 0.22 μm filter membrane to obtain the solution.

[0203] 2.4 Chromatographic conditions: High-performance liquid chromatograph: Agilent 1260 infinity Ⅱ; chromatographic column: Agilent ZORBOX SB-Aq (4.6 mm × 250 mm, 5 μm); mobile phase: 0.05% phosphoric acid water (A) - acetonitrile (B), isocratic elution (0 - 30 min, 97% A), detection wavelength: 203 nm; flow rate: 1.0 mL / min; injection volume: 20 μL; column temperature: 35 °C.

[0204] 2.5 Methodology investigation

[0205] 2.5.1 Specificity investigation

[0206] Samples of compound Dendrobium huoshanense granules and Rehmannioside D negative samples were prepared according to the preparation method of test samples. Rehmannioside D reference substance was taken and injected respectively. As Figure 1 shown, the reference substance and the granule samples both showed peaks around 14 min (Peak 1), and there was no interference from the negative samples, indicating good specificity.

[0207] 2.5.2 Precision investigation

[0208] Take 1.0 g of compound Dendrobium huoshanense granule samples, prepare the test solution according to the above method, and inject continuously for 6 times according to the chromatographic conditions. The RSD of the Rehmannioside D content is 1.75%, indicating good precision of the instrument.

[0209] 2.5.3 Stability investigation

[0210] Take 1.0 g of compound Dendrobium huoshanense granule samples, prepare the test solution according to the above method, and inject according to the chromatographic conditions at 0, 2, 4, 6, 8, 10, and 12 h respectively. Calculate the RSD of the Rehmannioside D content to be 2.01%, indicating good stability.

[0211] 2.5.4 Repeatability investigation

[0212] Take 6 samples of the same compound Dendrobium huoshanense granule samples, prepare 6 test solutions according to the above method, inject respectively, and calculate the RSD to be 5.36%, indicating good repeatability of the method.

[0213] 2.5.5 Accuracy investigation (spiked recovery test)

[0214] Take 1 g of granule powder with known Rehmannioside D content, weigh accurately in parallel for 6 portions, place in 50 mL centrifuge tubes, respectively pipette 3 mL, 5 mL, and 7.5 mL of Rehmannioside D reference substance solution with a concentration of 0.0926 mg / mL and add them to the above centrifuge tubes to prepare test solutions, and calculate the recovery rate (%). The results show that the average spiked recovery rate of Rehmannioside D at high, medium, and low concentrations is 98.13%, and the RSD is 2.05%, meeting the requirements, indicating that the analytical method is accurate and reliable. The results are shown in Table 21.

[0215] Table 21 Results of accuracy investigation for determination of Rehmannioside D content

[0216]

[0217] 2.6 Results of determination of Rehmannioside D content in samples

[0218] Table 22 Results of determination of Rehmannioside D content

[0219]

[0220] The results are shown in Table 22. It was determined that the content range of rehmannioside D in 3 batches of granules was 0.307 mg / g - 0.343 mg / g, and the RSD value was 4.79%, indicating that the sample quality was stable and controllable.

[0221] 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 a compound granule of Dendrobium huoshanense, characterized in that: The following steps are involved: According to weight, weigh 10 parts of Huoshan Dendrobium, 6 parts of Radix Rehmanniae, 6 parts of Radix Ophiopogonis and 5 parts of Radix Glycyrrhizae; The raw rehmannia root, the ophiopogon japonicus and the liquorice are mixed to obtain a decoction material, and then the decoctions are combined and concentrated to obtain a concentrated solution. The concentrated solution is dried, crushed and passed through a No. 6 sieve to obtain a dry extract fine powder; The Huoshan Dendrobium is crushed, passed through a No. 6 sieve, combined with the dry extract powder, and mixed to obtain a medicinal powder; The medicinal powder is mixed with the auxiliary materials to obtain a mixed powder, which is mixed with a wetting agent to prepare a soft material, granulated with a No. 1 sieve, collected and dried, and sieved with a No. 1 sieve to obtain the Huoshan Dendrobium compound granules.

2. The preparation method according to claim 1, characterized in that The mass ratio of the decoction raw material to water is 1:16; the soaking time is 1 hour; the decoction is performed 3 times, and the single decoction time is 1 hour.

3. The preparation method according to claim 1, characterized in that: The concentration is carried out under reduced pressure until the relative density is 1.21 g / mL and the temperature is 75° C.; the drying of the concentrated liquid is carried out under vacuum and the temperature is 80° C.

4. The preparation method according to claim 1, characterized in that: The auxiliary material is a mixture of starch and powdered sugar, and the mass ratio of the starch to the powdered sugar is 3:1; the mass ratio of the auxiliary material to the medicinal powder is 2:

3.

5. The preparation method according to claim 1, characterized in that: The wetting agent is 70% ethanol by volume, and the mass of the wetting agent is 15% of the mass of the mixed powder; The drying after collection is vacuum drying for 1 hour at a temperature of 70°C.

6. A compound granule of Dendrobium Huoshanense obtained according to the preparation method according to any one of claims 1 to 5.

7. A method for detecting the quality of Huoshan Dendrobium Compound Granules, characterized in that: The method comprises the steps of measuring the total flavonoids content and the rehmannia glycoside D content of the Huoshan Dendrobium compound granules.

8. The method according to claim 7, characterized in that The measurement of the total flavonoid content comprises the following steps: Weigh 5.03 mg of rutin standard and add 70% ethanol to make up to 25 mL to obtain the reference solution; Take 0, 0.5, 1, 2, 3, 4, 5, 6 mL of the reference solution respectively and place it in a 25 mL volumetric flask, add 70% ethanol to 6 mL, add 1 mL of 5% NaNO2 solution respectively, mix and place for 5 min, add 1 mL of 10% Al(NO3)3 solution, place for 5 min, then add 10 mL of 1 mol / L NaOH, mix, add 70% ethanol to 25 mL, mix thoroughly, place for 10 min, measure the absorbance at a wavelength of 510 nm, draw a standard curve with absorbance as the ordinate and concentration as the abscissa, and obtain the regression equation; Take 1.0g of the Huoshan Dendrobium compound granules, add 50mL of 70% ethanol, ultrasonicate for 1h, centrifuge for 20min, take 10mL of the supernatant, put it in a 25mL volumetric flask, add 1mL of 5% NaNO2 solution, mix and let stand for 5min, add 1mL10% Al(NO3)3 solution, let stand for 5min, then add 10mL of 1mol / L NaOH, mix, add 70% ethanol to make up to 25mL, mix thoroughly, let stand for 10min, and measure the absorbance at a wavelength of 510nm.

9. The method according to claim 7, characterized in that The measurement of the rehmannia glycoside D content comprises the following steps: Accurately measure 0.95 mg of rehmannoside D standard and dissolve it in 10 mL of 25% methanol to obtain a 0.095 mg / mL reference solution; Take the reference solution, dilute it into different concentrations, and inject it respectively. Use the concentration as the horizontal axis and the peak area as the vertical axis to establish a regression equation; Take 1 g of the Huoshan Dendrobium Compound Granules, add 25 mL of 25% methanol, perform ultrasonication for 30 min, centrifuge, and filter through a 0.22 μm filter membrane to obtain a test solution; perform liquid chromatography on the test solution; The conditions for the liquid chromatography detection are as follows: high performance liquid chromatograph: Agilent 1260infinityⅡ; chromatographic column: Agilent ZORBOX SB-Aq, size 4.6mm×250mm, 5μm; mobile phase: 0.05% phosphoric acid water as liquid A, acetonitrile as liquid B, isocratic elution 0-30min, 97% liquid A, detection wavelength: 203nm; flow rate: 1.0mL / min; injection volume: 20μL; column temperature: 35°C.

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