Quality control method for pharmaceutical composition containing fresh dendrobium extract

Abstract

The invention provides a quality control method for a medicine composition containing a fresh dendrobium extract, and belongs to the technical field of medicine quality control methods. The quality control method for the pharmaceutical composition containing the fresh dendrobium extract comprises one or more of aleppo avens thin layer identification, capillary artemisia thin layer identification, gentiana scabra bunge thin layer identification and gentiopicroside content determination. According to the quality control method of the pharmaceutical composition containing the fresh dendrobium extract, the quality stability of the pharmaceutical composition can be accurately and precisely controlled.

Description

[0001] The application is a divisional application of the invention patent with the application number 202210777444.0, the application date of June 23, 2022, and the invention name of a pharmaceutical composition containing fresh dendrobium extract and its preparation method and application. TECHNICAL FIELD

[0002] The application belongs to the technical field of pharmaceutical quality control methods, and specifically relates to a quality control method for a pharmaceutical composition containing fresh dendrobium extract. BACKGROUND

[0003] Since the quality of fresh dendrobium is affected by its harvesting period, the content of effective components of fresh dendrobium of different harvesting periods has great difference, so it is necessary to harvest as much fresh dendrobium as possible in the appropriate harvesting period, but a large amount of fresh dendrobium will have quality problems in long-term storage. Fresh dendrobium is not easy to store and is extremely susceptible to physiological activity of fresh dendrobium itself and storage conditions. Experimental studies have proved that the conventional storage methods of cool storage and frozen storage will cause the physiological activity of dendrobium to lead to flowering, germination, drying or mildewing of dendrobium, and also cause the metabolism, oxidation, enzymolysis and microbial action of physiological activity to lead to the transformation and loss of components of fresh dendrobium, thereby affecting the effect of the pharmaceutical composition.

[0004] The pharmaceutical composition containing fresh dendrobium extract of the application is mainly used for yin deficiency and body fluid deficiency, nourishing yin to generate body fluid, promoting qi and eliminating accumulation, and needs mild medicinal properties to avoid aggravating the burden of the spleen and stomach. Since raw bitter orange peel is rich in volatile oil and has strong acidity, harsh medicinal properties and strong irritation, the use of raw bitter orange peel will cause the pharmaceutical composition to have certain irritation to the gastric mucosa and gastrointestinal tract, and long-term use may have bad effects on the stomach and physiology, which is not conducive to the function of the pharmaceutical composition.

[0005] The pharmaceutical composition containing fresh dendrobium extract of the application is composed of multiple medicinal materials. Due to the limitations of growth environment, year, variety and other factors of traditional Chinese medicinal materials, the proportion of effective components in the same variety of medicinal materials may change greatly. Without necessary quantitative control, the quality uniformity and consistency of the pharmaceutical composition will be difficult to control, thereby affecting the stability of the curative effect of the pharmaceutical composition. When multiple components of the composition are mixed, the components will interact with each other, causing mutual interference of detection results, ultimately affecting the qualitative and quantitative evaluation of the quality of the pharmaceutical composition, and the quality controllability is poor. Therefore, it is necessary to establish a special, precise and accurate quality control method for the pharmaceutical composition to realize the stable quality performance of the composition, which is a problem to be solved by the application. SUMMARY

[0006] In view of this, the purpose of the present application is to provide a quality control method for a pharmaceutical composition containing fresh dendrobium extract, which is obtained by systematically analyzing the components of the pharmaceutical composition, excluding negative interference of specificity, screening the target of quality control, and determining the quality discrimination and control index of the pharmaceutical composition with blue cloth positive control drug, wormwood control drug, and gentiopicroside. The accuracy of the pharmaceutical components is judged by comprehensive judgment of various detection methods, and the specific indicators in the medicine are quantitatively analyzed to determine whether they meet the set indicators. Thus, the quality of the pharmaceutical composition containing fresh dendrobium extract is scientifically and effectively controlled. Stable and controllable product quality is an important supporting condition for realizing the expected effect of the present application.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides a quality control method for a pharmaceutical composition containing fresh dendrobium extract, which includes one or several of the following thin layer identification and / or content determination: (1) Thin layer identification: 5-20 ml of the pharmaceutical composition is accurately measured and placed in a separatory funnel, and extracted with ethyl acetate for 1-3 times, 5-20 ml each time. The ethyl acetate layer is collected, dried in a water bath, and the residue is dissolved with 2-10 volumes of methanol to obtain a test solution; 1-3 g of blue cloth positive control drug is added to 5-20 ml of water, heated and refluxed for 1-2 hours, filtered, and 5-20 ml of the filtrate is placed in a separatory funnel to prepare a control drug solution in the same way as the pharmaceutical composition; Another appropriate amount of gallic acid control is added to methanol to prepare a control solution of 1 mg / ml, and shaken to obtain a control solution; According to the thin layer chromatography (general) test, 3-5 μl of each of the above three solutions is spotted on the same silica gel G thin layer plate, and a mixture of dimethylbenzene-ethyl acetate-methanol-formic acid in a volume ratio of (4-6):(2-4):(1-2):(1-2) is used as the developing agent. After development, drying, and inspection under 365 nm, the test chromatogram shows the same color fluorescent spots at the corresponding positions of the control drug chromatogram; Then, 5% ferric trichloride-ethanol solution is sprayed for color development, and the sample is inspected under daylight to detect gallic acid; (2) Thin layer identification: 5-20 ml of the pharmaceutical composition is accurately measured and placed in a separatory funnel, and extracted with diethyl ether for 1-3 times, 5-20 ml each time. The diethyl ether liquid is discarded, and then extracted with ethyl acetate for 1-3 times, 5-25 ml each time. The ethyl acetate layer is collected, dried in a water bath, and the residue is dissolved with 1-5 ml of methanol to obtain a test solution; Take another Yincheng control drug 0.5g-1g, add water 5ml-25ml, heat reflux 1h-3h, cool, centrifugal, take 5ml-20ml supernatant in separating funnel, with the same method as the preparation of drug composition control drug solution; According to the thin layer chromatography (general) test, 1μl-3μl of the above two solutions are taken and spotted on the same polyamide film plate, with toluene-ethyl acetate-formic acid-glacial acetic acid-water (1-3):(10-20):(2-4):(1-2):(1-2) as the developing agent, developed, taken out and air dried, and observed under 365nm; In the test sample chromatogram, the same color fluorescent spots appear at the corresponding positions of the control drug chromatogram; (3) Thin layer identification: accurately take 5ml-20ml of the drug composition, and extract with water-saturated n-butanol for 1-3 times, 5ml-30ml each time, combine the n-butanol liquid, evaporate to dryness, dissolve the residue with 5ml-20ml of methanol, add to the neutral alumina column, elute with 50ml-100ml of methanol until colorless, evaporate to dryness, dissolve the residue with 1ml-5ml of methanol to prepare the test sample solution; Take another gentiopicroside control, add methanol to prepare a solution containing 2mg per 1ml to prepare the control solution; According to the thin layer chromatography (general) test, 1μl-3μl of the above two solutions are taken and spotted on the same polyamide film plate, with toluene-ethyl acetate-formic acid-glacial acetic acid-water (1-3):(10-20):(2-4):(1-2):(1-2) as the developing agent, developed, taken out and air dried, and observed under 365nm; In the test sample chromatogram, the same color fluorescent spots appear at the corresponding positions of the control drug chromatogram; 254 On the thin layer plate, dichloromethane-methanol-water (10-30):(5-15):(1-5) as the lower layer solution as the developing agent, developed, taken out, air dried, and observed under UV light 254nm; In the test sample chromatogram, the same color spots appear at the corresponding positions of the control chromatogram; (4) Content determination: Gentiopicroside: according to high performance liquid chromatography; Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as the filler: acetonitrile-0.2% phosphoric acid solution (10-30:70-90) as the mobile phase; detection wavelength is 270nm; theoretical plate number calculated by gentiopicroside should not be less than 3000; Preparation of control solution: take gentiopicroside control product, accurately weigh, add methanol to dissolve and dilute to prepare a solution containing about 0.07mg per 1ml, and obtain; Preparation of test sample solution: take 5-10ml of the drug composition, accurately take and place in a 50ml volumetric flask, accurately add an appropriate amount of methanol, shake and dilute to the mark, filter, and obtain; Determination method: accurately take 10μl of the control solution and the test sample solution respectively, inject into the liquid chromatograph, and determine, and obtain; The drug composition contains gentiopicroside C 16 H20 O9 not less than 0.40 mg / ml.

[0008] Preferably, the thin layer identification of (1) is as follows: 10 ml of the pharmaceutical composition is taken precisely and placed in a separating funnel, extracted with ethyl acetate for 2 times, 20 ml each time, the ethyl acetate layer is collected, dried in a water bath, the residue is dissolved with 2 volumes of methanol to obtain a test solution; 1 g of the blue cloth is taken, 20 ml of water is added, heated and refluxed for 2 hours, filtered, 10 ml of the filtrate is taken and placed in a separating funnel, and a control medicinal material solution is prepared in the same way as the pharmaceutical composition; Another appropriate amount of gallic acid control is taken, and a control solution of 1 mg / ml is prepared by adding methanol; According to the test of thin layer chromatography (general rules), 3-5 μl of each of the above three solutions is taken and spotted on the same silica gel G thin layer plate, a developing agent of dimethylbenzene-ethyl acetate-methanol-formic acid with a volume ratio of 5:2:1:1 is used for development, dried after being taken out, and observed under 365 nm; in the test product chromatogram, the same color fluorescent spots appear at the positions corresponding to the control medicinal material chromatogram; Then, 5% ferric trichloride-ethanol solution is sprayed for color development, and observed under sunlight, and gallic acid can be detected.

[0009] Preferably, the thin layer identification of (2) is as follows: 10 ml of the pharmaceutical composition is taken precisely and placed in a separating funnel, extracted with diethyl ether for 2 times, 20 ml each time, the diethyl ether liquid is discarded, extracted with ethyl acetate for 2 times, 25 ml each time, the ethyl acetate layer is collected, dried in a water bath, the residue is dissolved with 2 ml of methanol to obtain a test solution; Another 0.5 g of yinchen control medicinal material is taken, 25 ml of water is added, heated and refluxed for 3 hours, cooled, centrifuged, and 10 ml of supernatant is taken and placed in a separating funnel, and a control medicinal material solution is prepared in the same way as the pharmaceutical composition; According to the test of thin layer chromatography (general rules), 2 μl of each of the above two solutions is taken and spotted on the same polyamide film plate, a developing agent of toluene-ethyl acetate-formic acid-glacial acetic acid-water with a volume ratio of 1:20:4:1:1 is used for development, dried after being taken out, and observed under 365 nm; in the test product chromatogram, the same color fluorescent spots appear at the positions corresponding to the control medicinal material chromatogram.

[0010] Preferably, the thin layer identification of (3) is as follows: 10 ml of the pharmaceutical composition is taken precisely, extracted with water-saturated n-butanol for 2 times, 25 ml each time, the n-butanol liquid is combined, evaporated to dryness, the residue is dissolved with 10 ml of methanol, added to a neutral alumina column, eluted with 70 ml of methanol until colorless, evaporated to dryness, the residue is dissolved with 2 ml of methanol to obtain a test solution; Another gentalin glycoside control is taken, a solution of 2 mg per 1 ml is prepared by adding methanol, and a control solution is prepared. Perform the thin-layer chromatography (general procedure) test, taking 2 μl of each of the two solutions mentioned above and spotting them separately on the same silica gel GF plate. 254 On a thin-layer plate, the lower layer solution of dichloromethane-methanol-water with a volume ratio of 25:10:3 was used as the developing solvent. After development, the plate was removed, dried, and examined under a UV lamp at 254 nm. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0011] Preferably, the neutral alumina column has the following specifications: 100~200 mesh, 4g, and inner diameter 1.0cm.

[0012] Preferably, the content of (4) is determined as follows: gentiopicrin: by high performance liquid chromatography; Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel as the packing material; acetonitrile-0.2% phosphoric acid solution (10:90) as the mobile phase; detection wavelength of 270 nm; theoretical plate number calculated based on gentiopicroside, should not be less than 3000; Preparation of reference solution: Take an appropriate amount of gentiopicroside reference standard, accurately weigh it, dissolve it in methanol and dilute it to prepare a solution containing about 0.07 mg per ml. Preparation of the test solution: Take 5 ml of the drug composition, accurately measure it into a 50 ml volumetric flask, accurately add an appropriate amount of methanol, shake well and dilute to the mark, filter, and the solution is obtained. Determination method: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result; The pharmaceutical composition contains gentiopicroside C 16 H 20 O9 must be no less than 0.40 mg / ml.

[0013] Preferably, the active pharmaceutical ingredient composition of the pharmaceutical composition is: 25-150 parts of fresh Dendrobium officinale extract and 650-2000 parts of herbal extract. The raw material composition of the herbal extract is as follows: 50-150 parts of stir-fried Citrus aurantium, 3-15 parts of Cinnamomum cassia root, 50-150 parts of Polygonatum sibiricum, 50-150 parts of Rehmannia glutinosa, 15-50 parts of Gentiana scabra, 50-150 parts of Scutellaria baicalensis, 50-100 parts of Indigofera tinctoria, 50-150 parts of Ophiopogon japonicus, 50-150 parts of Asparagus cochinchinensis, 50-150 parts of Eriobotrya japonica leaf, and 50-200 parts of Artemisia capillaris. The fresh Dendrobium extract was prepared by the following method: Take fresh Dendrobium officinale, add water at 6 - 10 times the weight of the said medicinal materials, heat, decoct 1 - 3 times for 1 - 3 hours, filter, combine the filtrates, let stand for 8 - 24 hours, take the supernatant and concentrate it to a clear extract with a relative density not less than 1.03 measured at 70°C, cool, take the clear extract and centrifuge it with a high - speed sedimentation centrifuge, quickly freeze the centrifugate, and place it in a cold storage for freezing to obtain the fresh Dendrobium officinale extract.

[0014] Preferably, the stir - fried Fructus Aurantii is prepared by the following method: First, clean Fructus Aurantii, cut it into thin slices of 2 - 3 mm, and set aside; heat the frying container over medium heat until wheat bran is evenly scattered, the amount of wheat bran: 10% - 15% of the weight of the medicinal materials, immediately when the smoke rises, then put in the evenly sliced Fructus Aurantii, quickly stir - fry evenly, fry until the surface of Fructus Aurantii is light yellow or yellow and the wheat bran is black, immediately take out, sieve off the wheat bran, and let it cool to obtain the stir - fried Fructus Aurantii.

[0015] Preferably, the combined extract of medicinal materials is prepared by the following method: Take stir - fried Fructus Aurantii, Ardisia crenata, Polygonatum sibiricum, Rehmannia glutinosa, Gentiana scabra, Scutellaria baicalensis, Geum aleppicum, Ophiopogon japonicus, Asparagus cochinchinensis, Eriobotrya japonica, Artemisia capillaris, add water at 6 - 10 times the weight, heat, decoct 1 - 3 times for 1 - 3 hours, filter, combine the filtrates, let stand for 8 - 24 hours, and take the supernatant to obtain the combined extract of medicinal materials.

[0016] Preferably, the pharmaceutical composition is prepared by the following method: Take the fresh Dendrobium officinale extract and the combined extract of medicinal materials, combine and concentrate to a clear extract with a relative density not less than 1.05 measured at 80°C, take the clear extract and centrifuge it with a high - speed sedimentation centrifuge, heat the centrifugate, add conventional excipients, sterilize for 2 - 4 hours, cool, then add conventional excipients, mix evenly, fill into containers to obtain the pharmaceutical composition.

[0017] The beneficial effects of the present invention: The pharmaceutical composition of the present invention has the functions of clearing the stomach and purging fire, nourishing yin and promoting the production of body fluid, promoting qi circulation and removing stagnation. It is effective in treating yin deficiency with internal heat, can reduce the concentration of harmful inflammatory factors in the serum, and is effective in treating abnormal blood glucose and lipid levels caused by abnormal serum inflammatory factor concentrations. The prescription and preparation process of the pharmaceutical composition containing fresh Dendrobium officinale extract in the present invention uses fresh Dendrobium officinale extract, which solves the problems of inconvenient large - scale storage and preservation of fresh Dendrobium officinale, solves the accurate dosing of fresh Dendrobium officinale, and greatly improves the stability of the effective components. Using stir - fried Fructus Aurantii in the prescription reduces the irritation of the pharmaceutical composition of the present invention to the gastrointestinal tract and reduces adverse reactions compared with using raw Fructus Aurantii.

[0018] In traditional preparation processes, the medicinal ingredients in the formula of this invention are directly added to water for extraction and decoction, followed by filtration. This leads to the formation of a large number of solid impurities or precipitates, and the adsorption of active ingredients, which is detrimental to the stability of the drug and does not meet the quality requirements of modern pharmaceutical preparations. Furthermore, it lacks operability and continuity in production. This invention employs a high-speed sedimentation centrifugation process, which improves the production stability of the composition and solves the operability problem of traditional Chinese medicine decoction production. Its process and quality are controllable. This invention establishes a specific, precise, and accurate quality control method for this composition, ensuring the controllability and stability of the composition's quality.

[0019] The pharmaceutical composition of this invention uses fresh Dendrobium extract, herbal extract, and stir-fried Citrus aurantium. A high-speed sedimentation centrifugation process is employed, and a proprietary, precise, and accurate quality control method is established to improve and ensure the stability and controllability of the pharmaceutical composition's process and quality. Pharmacodynamic experiments further demonstrate that the formulation, preparation method, and quality control method of the pharmaceutical composition of this invention can improve and ensure the achievement of the expected therapeutic effect, making the pharmaceutical composition of this invention safer and more effective. Attached Figure Description

[0020] Figure 1 This image shows the sprouting of fresh Dendrobium officinale (batch number YZ12-210104) after being stored at room temperature and in a cool, dark place for 6 months.

[0021] Figure 2 This image shows fresh Dendrobium officinale (batch number YZ12-210315) sprouting and flowering after being stored at room temperature and in a cool, dark place for 6 months.

[0022] Figure 3 The image shows the cell rupture of fresh Dendrobium officinale (batch number YZ12-210104-1) after it has been frozen for 6 months.

[0023] Figure 4 The image shows the cell rupture of fresh Dendrobium officinale (batch number YZ12-210315) after 6 months of frozen storage.

[0024] Figure 5 The image shows the cell rupture of fresh Dendrobium officinale (batch number YZ12-210329) after 6 months of frozen storage.

[0025] Figure 6 Thin-layer chromatography (TLC) images of dendrobine in fresh Dendrobium extract after 6 months of frozen storage, showing: 1. Dendrobine reference standard; 2. Fresh Dendrobium extract 210304; 3. Fresh Dendrobium extract 210305.

[0026] Figure 7 Thin-layer chromatography (TLC) images of dendrobine in fresh Dendrobium officinale after 6 months of storage at room temperature and in a cool place. 1. Dendrobine reference standard; 2. Fresh Dendrobium officinale YZ12-210104.

[0027] Figure 8Thin-layer chromatography (TLC) images of dendrobine in fresh Dendrobium officinale medicinal materials after 6 months of frozen storage. 1. Dendrobine reference standard; 2. Fresh Dendrobium officinale medicinal material YZ12-210104-1.

[0028] Figure 9 The image shows the corresponding blue cloth thin-layer identification pattern for thin-layer chromatography. The carrier was a pre-fabricated silica gel G plate from Qingdao Marine Chemical Plant; the developing solvent was xylene-ethyl acetate-methanol-formic acid (5:2:1:1); the development method was air drying followed by examination at 365 nm; the temperature was 24℃; and the relative humidity was 70%. In the image: 1. Control medicinal material extracted with n-butanol; 2. Negative sample extracted with n-butanol; 3. Test sample extracted with n-butanol; 4. Control medicinal material extracted with ethyl acetate; 5. Negative sample extracted with ethyl acetate; 6. Test sample extracted with ethyl acetate; 7. Gallic acid reference standard.

[0029] Figure 10 The image shows the corresponding blue cloth thin-layer identification pattern for thin-layer chromatography. The substrate was a pre-fabricated silica gel G plate from Qingdao Marine Chemical Plant. The developing solvent was xylene-ethyl acetate-methanol-formic acid (5:2:1:1). The development method was as follows: after drying, the silica gel G thin-layer plate was sprayed with 5% ferric chloride-ethanol and then examined under sunlight. The temperature was 24℃, and the relative humidity was 70%. From left to right, the images correspond to: 1. Control medicinal material extracted with n-butanol; 2. Negative sample extracted with n-butanol; 3. Test sample extracted with n-butanol; 4. Control medicinal material extracted with ethyl acetate; 5. Negative sample extracted with ethyl acetate; 6. Test sample extracted with ethyl acetate; and 7. Gallic acid reference standard.

[0030] Figure 11 The chromatograms for the durability study of blue cloth for thin-layer identification are shown. The left image is viewed at room temperature (25.6℃) and normal humidity (68%) at 365nm. The right image is viewed at room temperature (25.6℃) and normal humidity (68%) after being sprayed with 5% ferric chloride-ethanol solution for color development and then examined under sunlight.

[0031] Figure 12 The chromatograms for the durability study of thin-layer identification using blue cloth are shown. The left chromatogram is for normal temperature (8℃) and normal humidity (72%), viewed at 365nm. The right chromatogram is for low temperature (8℃) and normal humidity (72%), developed by spraying with 5% ferric chloride-ethanol solution and viewed under sunlight.

[0032] Figure 13 The chromatograms for the durability study of blue cloth for thin-layer identification are shown. The left chromatogram is for high temperature (35℃) and normal humidity (54%), viewed at 365nm. The right chromatogram is for high temperature (35℃) and normal humidity (54%), sprayed with 5% ferric chloride-ethanol solution for color development, and viewed under sunlight.

[0033] Figure 14The chromatograms for the durability study of blue cloth for thin-layer identification are shown. The left chromatogram is for room temperature (26℃) and high humidity (88%), viewed at 365nm. The right chromatogram is for room temperature (26℃) and high humidity (88%), sprayed with 5% ferric chloride-ethanol solution for color development, and viewed under sunlight.

[0034] Figure 15 The chromatograms for the durability study of blue cloth for thin-layer identification are shown. The left image is viewed at 365nm under normal temperature (26℃) and low humidity (18%). The right image is viewed under sunlight under normal temperature (26℃) and low humidity (18%), after being sprayed with 5% ferric chloride-ethanol solution for color development.

[0035] Figure 16 The chromatograms for the robustness study of thin-layer chromatography using the blue cloth positive control are shown below. The left image is of the Shenghai plate, viewed at 365 nm under normal temperature (26℃) and humidity (68%). The right image is of the Shenghai plate, viewed under normal temperature (26℃) and humidity (68%), sprayed with 5% ferric chloride-ethanol solution for color development, and viewed under sunlight. In the chromatograms: 1. Gallic acid reference standard; 2. Negative; 3. 191109; 4. 200326; 5. 200815; 6. Blue cloth positive control medicinal material. The carrier in the figure is silica gel G pre-prepared plate (of which, the Shenghai plate is produced by Qingdao Shenghai Fine Silica Gel Chemical Co., Ltd., batch number: 20200812; the rest are produced by Qingdao Haiyang Chemical Co., Ltd., batch number: 20190831), and the developing solvent is xylene-ethyl acetate-methanol-formic acid (5:2:1:1).

[0036] Figure 17 Chromatograms for the specificity study of thin-layer identification of Artemisia capillaris were obtained at room temperature (25.6℃) and normal humidity (68%). In the chromatograms, 1. Artemisia capillaris [Mianyinchen] reference material; 2. Negative; 3. 191212; 4. 200314; 5. 200928.

[0037] Figure 18 Chromatograms for the durability (temperature) of thin-layer identification of Artemisia capillaris are shown. The left chromatogram represents low temperature (8℃) and normal humidity (72%); the right chromatogram represents high temperature (35℃) and normal humidity (54%). In the chromatograms, 1. Artemisia capillaris [Mianyin Chen] reference material; 2. Negative; 3. 191212; 4. 200314; 5. 200928.

[0038] Figure 19 Chromatograms for the durability (humidity) of thin-layer identification of Artemisia capillaris are shown. The left chromatogram represents high humidity (88%) at room temperature (26℃); the right chromatogram represents low humidity (18%) at room temperature (26℃). In the chromatograms, 1. Artemisia capillaris [Mianyin Chen] reference material; 2. Negative; 3. 191212; 4. 200314; 5. 200928.

[0039] Figure 20The chromatograms of the thin-layer identification of Artemisia capillaris are shown in the figure. The plate is a Luqiao plate, at room temperature (26℃) and normal humidity (68%). In the chromatogram, 1. Artemisia capillaris [Mianyinchen] reference material; 2. Negative; 3. 191212; 4. 200314; 5. 200928.

[0040] Figure 21 and Figure 22 Chromatograms for the specificity study of gentian thin-layer chromatography identification are shown below. In the chromatograms: 1. 200725; 2. Negative; 3. Gentianoside reference standard; 4. 200928; 5. 201015. Figure 21 Medium carrier: silica gel GF254 preform plate; developing solvent: lower layer solution of dichloromethane-methanol-water (25∶10∶3); inspection: under ultraviolet light (254nm); temperature: 16℃; humidity: 30%.

[0041] Figure 23 and Figure 24 Chromatograms for the durability (temperature) of gentian thin-layer identification for thin-layer identification. Figure 23 Low temperature (5℃); Figure 24 High temperature (35℃) and low humidity (12%); chromatogram 1. 200725; 2. Negative; 3. Gentianoside reference standard; 4. 200928; 5. 201015.

[0042] Figure 25 and Figure 26 Chromatograms for the durability (humidity) of gentian thin-layer identification corresponding to thin-layer identification. Figure 25 High temperature (35℃) and low humidity (12%). Figure 26 At room temperature (17.5℃) and high humidity (88%); in the chromatogram: 1. 200725; 2. Negative; 3. Gentianoside reference standard; 4. 200928; 5. 201015.

[0043] Figure 27 The chromatograms of the gentian thin-layer identification plate corresponding to the thin-layer identification are shown in the figure. The plate was operated manually at room temperature (18℃) and normal humidity (52%). In the chromatogram, 1. 200725; 2. Negative; 3. Gentianoside reference standard; 4. 200928; 5. 201015.

[0044] Figure 28 The chromatogram for the specificity of gentiopicrin in the content determination is shown.

[0045] Figure 29 The linear graph of gentiopicroside corresponding to the content determination is shown. Detailed Implementation

[0046] This invention provides a quality control method for pharmaceutical compositions containing fresh Dendrobium officinale extract, comprising one or more of the following thin-layer chromatography identification and / or content determination: (1) Thin-layer identification: Accurately measure 5ml-20ml of the drug composition, place it in a separatory funnel, extract with ethyl acetate 1-3 times, 5ml-20ml each time, collect the ethyl acetate layer, evaporate to dryness in a water bath, add 2-10 parts by volume of methanol to dissolve the residue, and obtain the test solution. Take 1-3g of the blue cloth reference material, add 5ml-20ml of water, heat under reflux for 1-2 hours, filter, take 5-20ml of the filtrate and place it in a separatory funnel, and prepare the reference material solution in the same way as the drug composition. Take an appropriate amount of gallic acid reference standard, add methanol to prepare a 1 mg / ml reference standard solution, shake well to obtain the reference standard solution; According to the thin-layer chromatography method (general rules), take 3-5 μl of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use xylene-ethyl acetate-methanol-formic acid in a volume ratio of (4-6):(2-4):(1-2):(1-2) as the developing solvent, develop, remove and air dry, and examine at 365 nm; in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material; Spray with 5% ferric chloride-ethanol solution for color development, and examine under sunlight to detect gallic acid; (2) Thin-layer identification: Accurately measure 5ml-20ml of the drug composition, place it in a separatory funnel, extract with ether 1-3 times, 5ml-20ml each time, discard the ether solution, extract with ethyl acetate 1-3 times, 5ml-25ml each time, collect the ethyl acetate layer, evaporate to dryness in a water bath, add 1ml-5ml of methanol to dissolve the residue, and prepare the test solution. Take 0.5g-1g of Artemisia capillaris reference material, add 5ml-25ml of water, heat under reflux for 1h-3h, cool, centrifuge, take 5ml-20ml of supernatant and place it in a separatory funnel, and prepare the reference material solution in the same way as the drug composition. According to the thin-layer chromatography method (general rules), take 1-3 μl of each of the above two solutions and spot them separately on the same polyamide film plate. Use toluene-ethyl acetate-formic acid-glacial acetic acid-water in a volume ratio of (1-3):(10-20):(2-4):(1-2):(1-2) as the developing solvent, develop, remove and air dry, and examine at 365 nm; in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material; (3) Thin-layer identification: Accurately measure 5ml-20ml of the drug composition, shake and extract 1-3 times with water-saturated n-butanol, 5ml-30ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 5ml-20ml of methanol, add it to a neutral alumina column, elute with 50ml-100ml of methanol until colorless, evaporate to dryness, dissolve the residue in 1ml-5ml of methanol to obtain the test solution; Separately, prepare a reference solution by adding methanol to a solution containing 2 mg per ml. Perform the thin-layer chromatography (general procedure) test, taking 1-5 μl of each of the two solutions mentioned above, and spotting them separately on the same silica gel GF plate. 254 On a thin-layer plate, the lower layer of a dichloromethane-methanol-water solution with a volume ratio of (10-30):(5-15):(1-5) was used as the developing solvent. After development, the plate was removed, dried, and examined under a UV lamp at 254 nm. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample. (4) Content determination: Gentianin: Analyzed by high performance liquid chromatography; Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel as the packing material; acetonitrile-0.2% phosphoric acid solution (10-30:70-90) as the mobile phase; detection wavelength of 270 nm; theoretical plate number calculated based on gentiopicroside, should not be less than 3000; Preparation of reference solution: Take an appropriate amount of gentiopicroside reference standard, accurately weigh it, dissolve it in methanol and dilute it to prepare a solution containing about 0.07 mg per ml. Preparation of the test solution: Take 5-10 ml of the drug composition, accurately measure it into a 50 ml volumetric flask, accurately add an appropriate amount of methanol, shake well and dilute to the mark, filter, and the solution is obtained. Determination method: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result; The pharmaceutical composition contains gentiopicroside C 16 H 20 O9 must be no less than 0.40 mg / ml.

[0047] In this invention, the preferred method for thin-layer identification of (1) is as follows: 10 ml of the drug composition is accurately measured and placed in a separatory funnel. It is extracted twice with ethyl acetate, 20 ml each time. The ethyl acetate layer is collected, evaporated in a water bath, and the residue is dissolved in 2 parts of methanol to obtain the test solution. Take 1g of blue cloth as a reference herb, add 20ml of water, heat under reflux for 2 hours, filter, take 10ml of the filtrate and place it in a separatory funnel, and prepare the reference herb solution in the same way as the drug composition. Take an appropriate amount of gallic acid reference standard and add methanol to prepare a 1 mg / ml reference standard solution; Perform the thin-layer chromatography (general procedure) test, taking 3-5 μl of each of the above three solutions and spotting them separately on the same silica gel G thin-layer plate. Use xylene-ethyl acetate-methanol-formic acid in a volume ratio of 5:2:1:1 as the developing solvent, develop, remove and air dry, and examine at 365 nm; in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material; Spray with a 5% ferric chloride-ethanol solution for color development, and examine under sunlight to detect gallic acid.

[0048] In this invention, the preferred method for thin-layer identification of (2) is as follows: 10 ml of the drug composition is accurately measured and placed in a separatory funnel. It is extracted twice with 20 ml of ether each time. The ether solution is discarded, and then extracted twice with 25 ml of ethyl acetate each time. The ethyl acetate layer is collected, evaporated in a water bath, and the residue is dissolved in 2 ml of methanol to obtain the test solution. Take another 0.5g of Artemisia capillaris reference material, add 25ml of water, heat under reflux for 3h, cool, centrifuge, take 10ml of supernatant and place it in a separatory funnel, and prepare the reference material solution in the same way as the drug composition. According to the thin-layer chromatography (general rules) test, take 2 μl of each of the above two solutions and spot them separately on the same polyamide film plate. Use toluene-ethyl acetate-formic acid-glacial acetic acid-water with a volume ratio of 1:20:4:1:1 as the developing solvent, develop, remove and dry, and examine at 365 nm; in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

[0049] In this invention, the preferred method for thin-layer identification of (3) is as follows: 10 ml of the drug composition is accurately measured and extracted twice with water-saturated n-butanol, 25 ml each time. The n-butanol solutions are combined, evaporated to dryness, and the residue is dissolved in 10 ml of methanol. The residue is added to a neutral alumina column and eluted with 70 ml of methanol until colorless. The residue is evaporated to dryness, and the residue is dissolved in 2 ml of methanol to obtain the test solution. Separately, prepare a reference solution by adding methanol to a solution containing 2 mg per ml. Perform the thin-layer chromatography (general procedure) test, taking 2 μl of each of the two solutions mentioned above and spotting them separately on the same silica gel GF plate. 254 On a thin-layer plate, the lower layer solution of dichloromethane-methanol-water with a volume ratio of 25:10:3 was used as the developing solvent. After development, the plate was removed, dried, and examined under a UV lamp at 254 nm. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0050] In this invention, the specifications of the neutral alumina column are preferably: 100~200 mesh, 4g, and inner diameter 1.0cm.

[0051] In this invention, the preferred method for determining the content of (4) is: gentiopicrin: by high performance liquid chromatography; Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel as the packing material; acetonitrile-0.2% phosphoric acid solution (10:90) as the mobile phase; detection wavelength of 270 nm; theoretical plate number calculated based on gentiopicroside, should not be less than 3000; Preparation of reference solution: Take an appropriate amount of gentiopicroside reference standard, accurately weigh it, dissolve it in methanol and dilute it to prepare a solution containing about 0.07 mg per ml. Preparation of the test solution: Take 5 ml of the drug composition, accurately measure it into a 50 ml volumetric flask, accurately add an appropriate amount of methanol, shake well and dilute to the mark, filter, and the solution is obtained. Determination method: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result; The pharmaceutical composition contains gentiopicroside C 16 H 20 O9 must be no less than 0.40 mg / ml.

[0052] In this invention, the preferred composition of the pharmaceutical raw materials of the pharmaceutical composition is: 25-150 parts of fresh Dendrobium officinale extract and 650-2000 parts of a combination of medicinal materials extract; it is also preferred to have 33 parts of fresh Dendrobium officinale extract and 1200 parts of a combination of medicinal materials extract; it is also preferred to have 33 parts of fresh Dendrobium officinale extract and 800 parts of a combination of medicinal materials extract; it is also preferred to have 80 parts of fresh Dendrobium officinale extract and 1800 parts of a combination of medicinal materials extract; it is also preferred to have 120 parts of fresh Dendrobium officinale extract and 1800 parts of a combination of medicinal materials extract.

[0053] The raw material composition of the fresh Dendrobium extract is: 150-900 parts of fresh Dendrobium; preferably: 200 parts, 400 parts, 600 parts or 800 parts of fresh Dendrobium.

[0054] The raw material composition of the herbal extract is as follows: 50-150 parts of stir-fried Citrus aurantium, 3-15 parts of cinnabar root, 50-150 parts of Polygonatum sibiricum, 50-150 parts of Rehmannia glutinosa, 15-50 parts of Gentiana scabra, 50-150 parts of Scutellaria baicalensis, 50-100 parts of Indigofera tinctoria, 50-150 parts of Ophiopogon japonicus, 50-150 parts of Asparagus cochinchinensis, 50-150 parts of Eriobotrya japonica, and 50-200 parts of Artemisia capillaris. Preferably, the composition is: 83.3 parts of stir-fried Citrus aurantium, 5.0 parts of cinnabar root, 83.3 parts of Polygonatum sibiricum, 83.3 parts of Rehmannia glutinosa, 33.3 parts of Gentiana scabra, 83.3 parts of Indigofera tinctoria, 83.3 parts of Ophiopogon japonicus, 83.3 parts of Asparagus cochinchinensis, 83.3 parts of Eriobotrya japonica, and 100 parts of Artemisia capillaris. Alternatively, the following ingredients can be preferred: 100 parts of stir-fried Citrus aurantium, 4 parts of cinnabar root, 55 parts of Polygonatum sibiricum, 55 parts of Rehmannia glutinosa, 35 parts of Gentiana scabra, 55 parts of Scutellaria baicalensis, 100 parts of Indigofera tinctoria, 100 parts of Ophiopogon japonicus, 100 parts of Asparagus cochinchinensis, 100 parts of Eriobotrya japonica leaf, and 80 parts of Artemisia capillaris. Another preferred ingredient is: 100 parts of stir-fried Citrus aurantium, 13 parts of cinnabar root, 100 parts of Polygonatum sibiricum, 100 parts of Rehmannia glutinosa, 40 parts of Gentiana scabra, 80 parts of Scutellaria baicalensis, 80 parts of Indigofera tinctoria, 90 parts of Ophiopogon japonicus, 80 parts of Asparagus cochinchinensis, 80 parts of Eriobotrya japonica leaf, and 120 parts of Artemisia capillaris.

[0055] The preparation method of the fresh Dendrobium extract is as follows: Take fresh Dendrobium, add 6-10 times the weight of water, heat, decoct for 1-3 hours, decoct 1-3 times, filter, combine the filtrates, let stand for 8-24 hours, take the supernatant and concentrate to a clear extract with a relative density of not less than 1.03 at 70℃, stir evenly, cool to room temperature, take the clear extract and centrifuge with a high-speed sedimentation centrifuge, rapidly freeze the centrifuged liquid and freeze it in a cold storage to obtain fresh Dendrobium extract.

[0056] The preparation method of the stir-fried immature bitter orange peel in the herbal extract is as follows: First, clean the bitter orange peel and cut it into thin slices of about 2-3 mm. Then, heat the stir-frying container over medium heat until wheat bran is evenly sprinkled in (wheat bran amount: 10%-15% of the weight of the medicinal material). Smoke will rise immediately. Then, add the evenly sliced ​​bitter orange peel and stir-fry quickly and evenly until the surface of the bitter orange peel turns light yellow or yellow and the wheat bran turns black. Immediately remove it, sift out the wheat bran, and let it cool to obtain stir-fried bitter orange peel.

[0057] The preparation method of the medicinal material composition is as follows: Take stir-fried Citrus aurantium, cinnabar root, Polygonatum sibiricum, Scutellaria baicalensis, Rehmannia glutinosa, Gentiana scabra, Sophora flavescens, Ophiopogon japonicus, Asparagus cochinchinensis, Eriobotrya japonica leaf, and Artemisia capillaris. Add 6-10 times the weight of water, heat, and decoct 1-3 times for 1-3 hours. Filter, combine the filtrates, let stand for 8-24 hours, and take the supernatant to obtain the herbal extract.

[0058] The preparation method of the drug combination of the present invention is as follows: take fresh Dendrobium extract and herbal combination extract, combine and concentrate to a clear paste with a relative density of not less than 1.05 at 80°C, take the clear paste and centrifuge it with a high-speed sedimentation centrifuge, heat the centrifuged liquid, add conventional excipients, sterilize for 2-4 hours, cool, add other conventional excipients, mix well, and fill into the package to obtain the drug composition.

[0059] The preferred method for preparing the fresh Dendrobium extract is as follows: Take fresh Dendrobium officinale, add 8 times the amount of water and decoct for 1.5 hours for the first time, add 6 times the amount of water and decoct for 1.5 hours for the second and third times respectively, filter, combine the filtrates, let stand for 12 hours, take the supernatant and concentrate it to a clear extract with a relative density of not less than 1.03 at 70℃, cool to room temperature, take the clear extract and centrifuge it with a high-speed sedimentation centrifuge, place the centrifuged liquid in a -25℃ cold storage for rapid freezing for 12 hours, and then place it in a -10℃ cold storage to freeze, thus obtaining fresh Dendrobium officinale extract.

[0060] The preferred method for preparing the stir-fried immature bitter orange peel in the herbal extract is as follows: First, clean the bitter orange peel and cut it into thin slices of about 3mm. Then, heat the stir-frying container over medium heat until wheat bran (wheat bran amount: 15% of the weight of the medicinal material) is evenly sprinkled in. As soon as smoke rises, add the evenly cleaned bitter orange peel slices and stir-fry quickly and evenly until the surface of the bitter orange peel turns light yellow or yellow and the wheat bran turns black. Immediately remove it, sift out the wheat bran, and let it cool.

[0061] The preferred method for preparing the herbal extract is as follows: Fried Citrus aurantium, Cinnamomum cassia root, Polygonatum sibiricum, Rehmannia glutinosa, Gentiana scabra, Scutellaria baicalensis, Ophiopogon japonicus, Asparagus cochinchinensis, Eriobotrya japonica leaf, and Artemisia capillaris were decocted for 1.5 hours with 8 times the amount of water for the first time. The second and third decoctions were decocted for 1.5 hours with 6 times the amount of water for the second and third times, respectively. The decoctions were filtered, the filtrates were combined, and the mixture was allowed to stand for 12 hours. The supernatant was then collected to obtain the herbal extract.

[0062] The preferred method for preparing the pharmaceutical composition of the present invention is as follows: Fresh Dendrobium extract and herbal extract are combined and concentrated to a clear extract with a relative density of not less than 1.05 at 80°C. The clear extract is centrifuged using a high-speed sedimentation centrifuge, the centrifuged liquid is heated, conventional excipients are added, sterilization is carried out for 2 hours, cooling is performed, and other conventional excipients such as ethylparaben and benzoic acid are added, mixed well, and filled into containers to obtain the pharmaceutical composition.

[0063] The preferred method for preparing the drug combination of the present invention is as follows: Fresh Dendrobium extract and herbal combination extract are combined and concentrated to a clear extract with a relative density of not less than 1.05 at 80°C. The clear extract is centrifuged using a high-speed sedimentation centrifuge, the centrifuged liquid is heated, conventional excipients are added, sterilization is carried out for 2 hours, and then cooled. Conventional excipients such as ethylparaben, benzoic acid, and sucrose are added, mixed well, and filled into containers to obtain the drug composition.

[0064] The preparation method of the pharmaceutical composition of the present invention is as follows: take the fresh dendrobium extract and the medicinal material combination extract, add conventional excipients, and prepare into clinically acceptable dosage forms according to conventional processes: tablets, capsules, oral liquids, syrups, granules; the excipients include preservatives, flavoring agents, disintegrants, colorants, binders, lubricants, etc.

[0065] The formula of the pharmaceutical composition of the present invention is the fresh dendrobium extract and the medicinal material combination extract. The raw materials of the medicinal material combination extract include stir-fried fructus aurantii, ardisia crenata, polygonatum sibiricum, scutellaria baicalensis, rehmannia glutinosa, gentiana scabra, geum japonicum thunb., ophiopogon japonicus, asparagus cochinchinensis, eriobotrya japonica, artemisia capillaris. The formula of the comparative sample 1 mentioned in the present invention is dendrobium, fructus aurantii, ardisia crenata, polygonatum sibiricum, scutellaria baicalensis, rehmannia glutinosa, gentiana scabra, geum japonicum thunb., ophiopogon japonicus, asparagus cochinchinensis, eriobotrya japonica, artemisia capillaris. Compared with the comparative sample 1, in the formula of the present invention, the fresh dendrobium extract and the medicinal material combination extract are used, and stir-fried fructus aurantii is used in the medicinal material combination extract instead of the unprepared fructus aurantii in the comparative sample 1. Using stir-fried fructus aurantii in the formula of the present invention can reduce the irritation of the pharmaceutical composition of the present invention to the gastrointestinal tract.

[0066] The pharmaceutical composition containing the fresh dendrobium extract of the present invention has the functions of clearing the stomach and purging fire, nourishing yin and promoting the production of body fluid, promoting qi circulation and removing accumulation. Compared with the comparative sample 1 mentioned in the present invention, the pharmaceutical composition of the present invention using the fresh dendrobium extract, the medicinal material composition, the high-speed sedimentation centrifugation process and the exclusive precise quality control method can better ensure quality safety, improve production efficiency, and the preparation method of the present invention realizes the operability of production and stabilizes the quality. The continuous production of the composition can be achieved. Compared with the comparative sample 1 mentioned in the present invention, using stir-fried fructus aurantii in the prescription of the pharmaceutical composition of the present invention can reduce irritation and reduce adverse reactions. Compared with the comparative sample 1 mentioned in the present invention, the pharmaceutical composition has a better effect on small intestine propulsion and gastric juice secretion function, so it has a better effect on heartburn pain and promoting qi circulation and removing accumulation. It is expected to have an effect on the abnormal concentration of serum inflammatory factors causing yin deficiency and internal heat diseases, and an effect on the abnormal blood glucose and lipid levels caused by the abnormal concentration of serum inflammatory factors.

[0067] To make the pharmaceutical composition of the present invention more suitable for administration, reduce the irritation of the pharmaceutical composition to the gastrointestinal tract, reduce adverse reactions, and improve the compliance of users. Fructus Aurantii Immaturus stir-fried with bran is used in the prescription of this patent. Because according to traditional Chinese medicine theory, traditional processing theory and processing principles, after Fructus Aurantii Immaturus is stir-fried with bran, its irritation can be reduced, and its dryness and acidity are milder than those of the raw product. At the same time, Fructus Aurantii Immaturus can enhance the therapeutic effects of strengthening the stomach and relieving distension, and strengthening the stomach and regulating the middle-jiao with the help of the efficacy of wheat bran in benefiting the spleen, so as to achieve the purpose of "reducing dryness and enhancing efficacy". In "Ben Cao Meng Quan" in the Ming Dynasty, it was mentioned that the processing of Fructus Aurantii Immaturus "To control its dryness and assist its digestion and induction, it can be stir-fried and used", "To promote digestion and remove stagnation, use stir-fried with bran, otherwise its qi is strong and may damage the vital qi". In "Chinese Medicine Processing Science", it is considered that "Wheat bran inhibits the sourness and does not damage the upper diaphragm: the upper diaphragm, that is, above the diaphragm, where the primordial qi is stored. It means that when traditional Chinese medicine is processed with bran, the dry and intense nature of traditional Chinese medicine can be alleviated without damaging the primordial qi". In the article "Overview of the Chemical Composition and Gastrointestinal Motility Research of Fructus Aurantii Immaturus and Fructus Aurantii" published by Xu Huan, Chen Haifang, Jie Lei, etc., it is mentioned that the main effective components of Fructus Aurantii Immaturus change quantitatively and qualitatively after stir-frying with bran, the content of volatile oil can be reduced, the stimulating components can be removed, and the dryness can be alleviated, thus alleviating its side effect of severely damaging the vital qi with its intense nature.

[0068] Luo Zhouyan in the Ming Dynasty also recorded in "Medical Essence and Sayings": "To promote digestion and remove stagnation with Fructus Aurantii Immaturus, use stir-fried with bran, otherwise its qi is strong and may damage the vital qi." The medicinal properties of raw Fructus Aurantii Immaturus are intense, and the medicinal properties are mild after stir-frying with bran, which greatly reduces the irritation to the stomach. It has the effects of strengthening the spleen and promoting appetite, regulating the five zang-organs, descending qi, and stopping vomiting and nausea. Therefore, stir-fried Fructus Aurantii Immaturus can achieve the purpose of "removing dryness and harmonizing the stomach", enhancing the functions of ascending the clear and descending the turbid, clearing stomach heat, treating rebellious qi and stomach burning, and facilitating qi movement and dissipating accumulation. Therefore, compared with Comparative Sample 1, the use of stir-fried Fructus Aurantii Immaturus in the prescription of the present invention plays an important role in improving the efficacy of this prescription, reducing adverse reactions, and improving drug safety. Through experimental research, it is confirmed that stir-fried Fructus Aurantii Immaturus in this pharmaceutical composition can reduce the irritation of the drug to the gastrointestinal tract, reduce adverse reactions, is beneficial to improving the curative effect, and is significantly superior to Comparative Sample 1, which is consistent with traditional Chinese medicine theory.

[0069] To ensure the true manifestation of the prescription efficacy under traditional Chinese medicine theory, the traditional preparation method of traditional Chinese medicine decoction is followed. The refining process that may affect the effective components of the drug is not adopted to prevent the possible removal of effective components in the extract. Therefore, there are a large number of solid impurities and suspended substances in the extraction process of the pharmaceutical composition of the present invention that are difficult to remove, resulting in the inability to continuously and normally carry out production. Moreover, the solid impurities may adsorb the medicinal components in the decoction and form precipitates, seriously affecting the efficacy of the pharmaceutical composition and making it not meet the quality requirements of modern pharmaceutical preparations.

[0070] This invention employs high-speed sedimentation centrifugation to filter and purify the concentrated pharmaceutical composition, achieving the removal of precipitates and impurities without affecting the efficacy of the medicinal components. This technology solves the problems of time-consuming and inefficient natural sedimentation for removing impurities and precipitates, which leads to rapid microbial growth in the concentrated solution and makes it impractical for production. It also solves the problems of mechanical filters, such as easy clogging of filter channels, slow speed, low efficiency, inconvenient operation, discontinuous production, and difficulty in quality control. The high-speed sedimentation centrifugation method used in this invention achieves the process design requirements of the pharmaceutical composition, effectively ensuring the quality of the composition and the continuous operability of production. It also preserves the characteristics of traditional Chinese medicine decoctions.

[0071] Modern TCM pathological and pharmacological studies have demonstrated that Yin deficiency with internal heat is closely related to the concentration of inflammatory factors in the body. Among these, the inflammatory cytokines C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) are three core proteins that trigger inflammation. Abnormal or imbalanced concentrations of these inflammatory factors are one of the mechanisms leading to Yin deficiency with internal heat. For example, Yin deficiency with internal heat can result in abnormalities in serum levels of CRP, TNF-α, and IL-6. These abnormal serum inflammatory cytokine concentrations can further cause abnormalities in biochemical parameters such as blood glucose and blood lipids, thereby damaging organs and the endocrine system. Adiponectin, an adipokine, can exert its anti-inflammatory effect by stimulating the release of anti-inflammatory cytokines and inhibiting their production. Therefore, whether the composition of this invention has a better therapeutic effect on Yin deficiency with internal heat can be confirmed by studying its intervention on serum inflammatory factors and its ability to increase adiponectin levels. Pharmacological studies have shown that the pharmaceutical composition of this invention can significantly reduce the concentration of harmful serum inflammatory factors, and its effect is significantly better than that of comparative sample 1, thus achieving the expected better therapeutic effect. Multiple studies have shown that metformin has certain anti-inflammatory effects. Metformin can significantly reduce the levels of TNF-α and IL-6 and can be used as a positive control group.

[0072] To address the issues of fresh Dendrobium being difficult to preserve and its efficacy being affected by component loss, the formulation of this invention uses fresh Dendrobium extract. This solves the problem of fresh Dendrobium easily flowering, sprouting, or becoming moldy during storage, leading to the loss of effective components. It also solves the problem of fresh Dendrobium changing in weight due to moisture loss and drying during storage, which prevents accurate quantitative dosing. Therefore, the use of fresh Dendrobium extract in this invention is necessary to ensure the quality, safety, and expected efficacy of the pharmaceutical composition.

[0073] This invention specifically investigates the impact of freezing conditions on the quality of fresh Dendrobium. The freezing techniques commonly used in the food and pharmaceutical industries for preserving fresh Dendrobium do not comply with the Chinese Pharmacopoeia's regulations on preventing freezing during storage. During freezing, the cells of fresh Dendrobium expand and crack due to freezing, leading to further cracking. After thawing, the material softens, and its juices are lost, potentially resulting in the loss of its active ingredients and affecting its quality. Furthermore, the active ingredients in fresh Dendrobium are not stably preserved during freezing. This impacts the quality and efficacy of the pharmaceutical composition of this invention. Therefore, this invention extracts fresh Dendrobium separately, removes solid impurities through high-speed sedimentation centrifugation, and then preserves it through low-temperature freezing. This provides a method that better ensures the stability of the active ingredients in fresh Dendrobium, which is essential for achieving a rational process and the expected efficacy of the pharmaceutical composition. This invention, through research on the storage of fresh Dendrobium and pharmacodynamic experiments, also demonstrates that the use of fresh Dendrobium extract can ensure the stability of the active ingredients and achieve the expected therapeutic effect.

[0074] The relationship between the preparation method and application of this invention patent is a g / ml relationship between weight parts and volume parts.

[0075] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0076] Unless otherwise specified, the following embodiments are all conventional methods.

[0077] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0078] Experimental Example 1 Study on the Effects of Different Harvesting Periods of Fresh Dendrobium on Dendrobine Content The fresh Dendrobium extract used in this invention is obtained by direct extraction from fresh Dendrobium medicinal materials, and its quality stability is directly related to the quality of the fresh Dendrobium medicinal materials. Although Dendrobium can be harvested year-round, it has been found in actual harvesting that the content of effective components in fresh Dendrobium is affected by its harvesting period. Fresh Dendrobium harvested at different times has different contents of effective components, resulting in inconsistent quality. This has a significant impact on the quality stability of the pharmaceutical composition of this invention and on the achievement of the expected therapeutic effect.

[0079] To determine the optimal harvesting period for fresh Dendrobium and ensure the stable quality of fresh Dendrobium medicinal materials and extracts, this invention uses fresh Dendrobium nobile as an example to study the impact of different harvesting periods on the content of its active ingredients.

[0080] Experimental samples

[0081] Experimental methods: The experiment was designed with four harvesting periods: December 2020, March 2021, July 2021, and August 2021. Three batches of samples were collected in each harvesting period. Dendrobine was used as the quality assessment index to determine the dendrobine content of each batch of samples in different harvesting periods.

[0082] Detection method: Refer to the method for determining the content of dendrobine under the Dendrobium section of the 2020 edition of the Chinese Pharmacopoeia. The content is specified as not less than 4.0% of the dendrobine content on a dried basis. The results are shown in Table 1.

[0083] Table 1. Results of the investigation on dendrobine content in fresh Dendrobium nobile at different harvesting periods

[0084] Experimental results show that the dendrobine content of fresh Dendrobium nobile varies greatly depending on the harvesting period. Fresh Dendrobium nobile should be harvested in large quantities between December of the current year and March of the following year to ensure that it meets quality requirements and is of stable quality. Therefore, harvesting a large quantity of fresh Dendrobium nobile during the appropriate harvesting period and ensuring the safe storage of fresh Dendrobium nobile is essential to ensuring the quality stability of the fresh Dendrobium nobile extract and plays an important role in achieving the expected therapeutic effect of the composition of this invention.

[0085] Experiment Example 2 Comparative study on the quality of fresh Dendrobium extract stock solution under different storage methods.

[0086] Currently, fresh Dendrobium faces technical challenges in preservation. Fresh Dendrobium itself possesses inherent physiological activity, making it susceptible to oxidation, metabolism, and enzymatic hydrolysis, which can lead to sprouting, flowering, drying, or mold growth, resulting in component loss. During storage, drying and moisture reduction can cause weight loss in the fresh product, ultimately preventing proper dosage in the prescription and contradicting the original intention of this invention to obtain fresh Dendrobium extract from fresh materials. Furthermore, conventional freezing preservation techniques violate the 2020 edition of the Chinese Pharmacopoeia's requirement for freezing protection during Dendrobium storage. To address these issues, this invention utilizes fresh Dendrobium extract and conducts a comparative quality study on the stock solution of fresh Dendrobium extract, fresh Dendrobium stored in a cool place, and fresh Dendrobium stored frozen.

[0087] Experimental sample:

[0088] Experimental Methods: Three batches of fresh Dendrobium officinale extract stock solution were stored frozen, fresh Dendrobium officinale medicinal materials were stored in a cool place, and fresh Dendrobium officinale medicinal materials were stored frozen, respectively, for a storage period of 6 months. After 6 months, samples were taken from each batch, and their appearance, weight loss, rot, mold, sprouting, frost heave, thin-layer chromatography identification, and dendrobine content were compared. (See attached document) Figure 1 Appendix Figure 2 Appendix Figure 3 Appendix Figure 3 AppendixFigure 4 Appendix Figure 5 Appendix Figure 6 Appendix Figure 7 Appendix Figure 8 .

[0089] Detection methods: Thin-layer chromatography identification and dendrobine content determination were performed according to the identification and content determination methods under the Dendrobium section of the 2020 edition of the Chinese Pharmacopoeia. The results are shown in Table 2.

[0090] Table 2. Comparative Study Results of Main Quality Indicators of Fresh Dendrobium Extract Stock Solution, Fresh Dendrobium Herbs in a Cool Place, and Fresh Dendrobium Herbs Frozen for 6 Months.

[0091] Experimental results show that after 6 months of frozen storage of fresh Dendrobium officinale extract stock solution, its appearance remained unchanged, the dendrobine content was essentially unaffected, and there was no water loss. The amount of fresh Dendrobium officinale used can be precisely controlled, ensuring stable quality. However, after 6 months of cool storage of fresh Dendrobium officinale, its appearance changed, with flowering, sprouting, and mold growth observed. Water loss and weight loss occurred, and the dendrobine content decreased, indicating that cool storage of fresh Dendrobium officinale cannot guarantee its quality. The experiment also demonstrated that after 6 months of frozen storage, freezing of fresh Dendrobium officinale caused deformation and cracking, sap leakage, and a significant decrease in dendrobine content, failing to meet the quality requirements of fresh Dendrobium officinale and consistent with the pharmacopoeia's requirement to protect fresh Dendrobium officinale from freezing. Therefore, the use of stock solution of fresh Dendrobium officinale extract in this invention solves the problem of actual fresh Dendrobium officinale storage in production, effectively ensuring the quality stability of the fresh Dendrobium officinale extract and the pharmaceutical composition.

[0092] Experimental Example 3 Research on extraction process of fresh Dendrobium extract To clarify the extraction process of fresh Dendrobium officinale extract and ensure the feasibility of this invention, this invention studies the extraction process of fresh Dendrobium officinale extract, including specific extraction methods, extraction temperature, extraction time, extraction temperature, relative density, etc.

[0093] An orthogonal experimental design was used to investigate four factors: different extraction times, different extraction temperatures, different extraction times, and different amounts of water added. Each factor had three levels. The experimental results showed that the order of influence of each factor level was: extraction time > amount of water added > extraction temperature > number of extractions. The p-values ​​of each level were all greater than 0.05, indicating that there was no significant difference between each factor level. To save production costs while ensuring that the effective components of fresh Dendrobium are fully extracted, the experimental results of this invention selected extraction at a temperature above 70°C for three extractions. The first extraction used 8 times the amount of water, and the second and third extractions used 6 times the amount of water respectively. Each extraction lasted 1.5 hours.

[0094] Three batches of orthogonal experiments were conducted to verify the results, which proved that the process selected by the orthogonal experiments was stable.

[0095] Experiment Example 4 Research on Concentration Process of Fresh Dendrobium Extract To clarify the concentration process of fresh Dendrobium officinale extract and ensure the feasibility of this invention, the concentration temperature and relative density of fresh Dendrobium officinale extract were studied, and the effects of different concentration temperatures on the dendrobine content were investigated. The results showed that the above concentration temperatures had little effect on the dendrobine content. To ensure that the dendrobine in the fresh Dendrobium officinale extract is not destroyed during concentration, the concentration temperature was chosen to be no higher than 70℃.

[0096] Given that the present invention uses fresh Dendrobium extract to solve the problem of fresh Dendrobium storage, in order to demonstrate that decocting fresh Dendrobium extract alone has no effect on the quality of the composition of the present invention, the present invention has studied the quality indicators of fresh Dendrobium extract to make the quality of fresh Dendrobium extract more controllable, so as to ensure the quality stability of the pharmaceutical composition of the present invention.

[0097] Experimental Example 5 Quality control study of fresh Dendrobium extract Experimental methods: Taking Dendrobium nobile as an example, fresh Dendrobium nobile extract was prepared by the method in step one of Experiment Example 6. Seven batches were prepared. After preparation, the quality indicators of fresh Dendrobium nobile extract, such as yield, relative density, pH value, n-butanol extract and dendrobine content, were studied. The results are shown in Table 3.

[0098] Formula for calculating yield: Relative density and pH value were determined according to General Chapters 0601 and 0631 of Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0099] Determination of n-butanol extract: Accurately measure 10 ml of fresh Dendrobium officinale extract, place it in a separatory funnel, and extract 4 times with 25 ml of water-saturated n-butanol each time. Shake thoroughly, let stand, separate the n-butanol solution, place it in an evaporating dish that has been dried to constant weight, evaporate to dryness, dry at 105℃ for 3 hours, transfer to a desiccator, cool for 30 minutes, quickly and accurately weigh, and calculate to obtain the result.

[0100] Table 3. Results of quality index study of 7 batches of fresh Dendrobium officinale extract.

[0101] Experimental results show that the main quality indicators of the fresh Dendrobium extract stock solution are stable and controllable, meeting the quality requirements of the extract, and can ensure the quality stability and controllability of the pharmaceutical composition of the present invention.

[0102] Based on Experiment 5, to further confirm that the fresh Dendrobium extract of the present invention does not affect the quality of the pharmaceutical composition of the present invention, the present invention uses a concentrated solution of fresh Dendrobium extract and herbal extract combination to prepare the pharmaceutical composition of the present invention, and conducts a quality comparison study with Comparative Sample 1, which is prepared by direct decoction and concentration of fresh Dendrobium and other herbs. The main quality indicators, pH value, relative density, n-butanol extract, gentiopicrin content, and microorganisms are compared.

[0103] Experimental Example 6 Study on the effect of decocting fresh Dendrobium alone on the quality of the pharmaceutical composition of the present invention.

[0104] Experimental samples The pharmaceutical composition of this invention consists of: 1 part fresh Dendrobium extract, 36.4 parts herbal extract, and other conventional excipients.

[0105] Preparation method of the pharmaceutical composition of the present invention: Step 1: Preparation of fresh Dendrobium extract: Take 200 parts of fresh Dendrobium, add water and decoct 3 times. Add 8 times the amount of water for the first time, and 6 times the amount of water for the second and third times. Each decoction lasts for 1.5 hours. Filter, combine the filtrates, let stand for 12 hours, take the supernatant and concentrate it to a clear extract with a relative density of not less than 1.03 at 70℃. Stir evenly, cool to room temperature, take the clear extract and centrifuge it with a high-speed sedimentation centrifuge. Store the centrifuged liquid in a -25℃ cold storage for 12 hours, and then store it in a -10℃ cold storage to obtain fresh Dendrobium extract for later use.

[0106] Step 2: Preparation of the medicinal material composition of the present invention: Take 83.3 parts of stir-fried bitter orange peel, 5.0 parts of cinnabar root, 83.3 parts of polygonatum, 83.3 parts of rehmannia root, 33.3 parts of gentian, 83.3 parts of scutellaria, 83.3 parts of indigo naturalis, 83.3 parts of ophiopogon root, 83.3 parts of asparagus root, 83.3 parts of loquat leaf, and 100 parts of artemisia capillaris. Add water and decoct 3 times. For the first decoction, add 8 times the amount of water and decoct for 1.5 hours. For the second and third decoctions, add 6 times the amount of water and decoct for 1.5 hours each time. Filter, combine the filtrates, let stand for 12 hours, and take the supernatant to obtain the medicinal material composition extract.

[0107] Step 3: Take the fresh Dendrobium extract from Step 1 and the herbal combination extract from Step 2, combine and concentrate them to a clear extract with a relative density of not less than 1.05 at 80℃. Centrifuge the clear extract using a high-speed sedimentation centrifuge. Heat the centrifuged liquid, stir and add 166 parts of sucrose, 0.5 parts of ethylparaben, 3.0 parts of benzoic acid and 0.83 parts of citric acid. Sterilize for 2 hours, cool to 40℃~50℃, add 0.11 parts of menthol, mix well, filter, and fill into containers to obtain the pharmaceutical composition.

[0108] Comparative Sample 1: 600 parts fresh Dendrobium, 249.9 parts Citrus aurantium, 15 parts Cinnamomum cassia, 249.9 parts Polygonatum sibiricum, 249.9 parts Rehmannia glutinosa, 99.9 parts Gentiana scabra, 249.9 parts Scutellaria baicalensis, 249.9 parts Indigofera tinctoria, 249.9 parts Ophiopogon japonicus, 249.9 parts Asparagus cochinchinensis, 249.9 parts Eriobotrya japonica, and 300 parts Artemisia capillaris.

[0109] Preparation method of comparison sample 1: Step 1: Boil the above twelve medicinal materials in water three times, each time for 1.5 hours. Combine the decoctions, filter, let stand for 12 hours, and take the supernatant to concentrate into a clear extract with a relative density of 1.05 (80℃).

[0110] Step 2: Place the clear extract obtained in Step 1 into a mixing tank, heat it, add 498 parts of sucrose, 1.5 parts of ethylparaben, 9.0 parts of benzoic acid, and 2.49 parts of citric acid, sterilize for 2 hours, cool to 40℃~50℃, add 0.25 parts of menthol, mix well, fill into the container, and obtain the control sample 1.

[0111] Experimental methods In this experiment, three batches of samples were prepared, with batch numbers of the pharmaceutical composition of this invention being 210401, 210402, and 210403, respectively; and batch numbers of control samples 210404, 210405, and 210406, respectively. The main focus of the comparative study was on quality indicators such as pH value, relative density, n-butanol extract, dendrobine content, gentiopicrin content, and microorganisms. The results are shown in Table 4.

[0112] pH value and relative density were determined according to General Chapters 0601 and 0631 of Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0113] Determination of n-butanol extract: Accurately measure 10 ml of fresh Dendrobium officinale extract, place it in a separatory funnel, and extract 4 times with 25 ml of water-saturated n-butanol each time. Shake thoroughly, let stand, separate the n-butanol solution, place it in an evaporating dish that has been dried to constant weight, evaporate to dryness, dry at 105℃ for 3 hours, transfer to a desiccator, cool for 30 minutes, quickly and accurately weigh, and calculate to obtain the result.

[0114] Dendrobine content determination: Refer to the Dendrobine content determination under the Dendrobium entry in Part I of the 2020 edition of the Chinese Pharmacopoeia.

[0115] Gentianoside content determination: determined according to the method for determining the gentianoside content of the pharmaceutical composition according to the present invention.

[0116] Table 4. Results of quality comparison study of three batches of the pharmaceutical compositions of the present invention and comparative sample 1 Experimental results show that when fresh Dendrobium is decocted separately and then the drug composition is prepared, the quality indicators are basically unchanged compared with the control sample 1. This indicates that the use of fresh Dendrobium extract has little effect on the quality of the drug composition and does not affect the expected efficacy of the drug composition.

[0117] Experimental Example 7 Research on high-speed sedimentation centrifugal filtration process of the pharmaceutical composition of this invention To ensure the efficacy of traditional decoctions, this product uses the traditional decoction preparation process, which is beneficial for the full retention of the extract's components. However, this preparation process easily generates solid impurities and suspended particulate matter, which are difficult to separate, making the production process uncontrollable and difficult to continue. Furthermore, the drug composition is difficult to stabilize within its shelf life and meet quality standards. Therefore, this invention employs high-speed sedimentation centrifugation filtration for the drug composition, and studies the high-speed sedimentation centrifugation process and its effects. This invention also investigates the impact of centrifugation on the quality of the drug composition before and after centrifugation.

[0118] (1) This experimental example studies the effect of high-speed sedimentation centrifugation on the removal of precipitates from the pharmaceutical composition. The results are shown in Table 5.

[0119] Experimental sample: Concentrated pharmaceutical composition of the present invention, prepared in 3 batches by the preparation method of pharmaceutical composition of the present invention in Experiment Example 6, with batch numbers 200813, 200814 and 200815 respectively.

[0120] Experimental methods: Take 50 ml of each of the three batches of the concentrated pharmaceutical composition of the present invention and place them in containers. Allow them to settle naturally without high-speed sedimentation centrifugation. Let them stand for 48 hours and observe the precipitation state of the concentrated solution.

[0121] Take three batches of the concentrated pharmaceutical composition of the present invention, centrifuge at high speed for 5 minutes (2500 rpm, 600 mm rotation radius, 4.0 kW power) to obtain centrifuged liquid, take 50 ml of the centrifuged liquid and place it in a container, let it stand for 48 hours, and observe the precipitation state of the centrifuged liquid.

[0122] Table 5. Effects of natural sedimentation and high-speed sedimentation centrifugation processes on the precipitation of pharmaceutical compositions.

[0123] Experimental results show that the concentrate obtained through natural sedimentation is in a suspension state, with no clear boundary between the supernatant and the precipitate, and a large amount of precipitate is suspended, making separation difficult. Furthermore, natural sedimentation is time-consuming and uncontrollable, hindering normal production processes and resulting in poor feasibility. In contrast, the concentrate obtained using high-speed sedimentation centrifugation can quickly and effectively remove precipitates from the composition, leaving no suspended precipitate in the solution and preventing precipitate aggregation. This ensures the appearance quality of the drug composition and allows for continuous production.

[0124] To further demonstrate the effect of high-speed sedimentation centrifugation on the removal of precipitates from this pharmaceutical composition, this experimental example used three different methods to remove precipitates: natural sedimentation, filter bag filtration, and high-speed sedimentation centrifugation. The precipitates were then centrifuged using a laboratory high-speed centrifuge to obtain precipitates under uniform conditions. This was used to study the effectiveness of the three different methods in removing precipitates. The results are shown in Table 6.

[0125] Experimental sample: Concentrated pharmaceutical composition of the present invention, prepared in 3 batches by the preparation method of pharmaceutical composition of the present invention in Experiment Example 6, with batch numbers 200819, 200820 and 200821 respectively.

[0126] Experimental equipment: centrifuge, model: TDC-40B.

[0127] Experimental methods: Natural sedimentation removes sediment: Take 50 kg of the concentrated solution of this drug composition (total amount ①), shake it well and place it in a container. Let it stand for 3 days. After standing for 3 days, observe that the upper layer of liquid is in a suspension state without a clear dividing line. Pour the upper layer of liquid out gently to separate it from the sediment at the bottom. There is only a small amount of sediment at the bottom of the container. Weigh it and take the weight as total amount ②. The weight of the upper layer of liquid is total amount ③ = total amount ① - total amount ②.

[0128] Shake well the total precipitate at the bottom of the container (②). Weigh 50g from total ② into a centrifuge tube to make two parallel samples. Centrifuge the above samples in a laboratory centrifuge (4000 rpm). After centrifugation for 10 minutes, a clear boundary line will appear between the wet cake and the supernatant, indicating that the precipitate has separated. Remove the sample and calculate the amount of precipitate in the centrifuge tube after centrifugation as precipitate A. Then calculate the amount of precipitate in total ② as precipitate ①.

[0129] Thoroughly mix the total volume of the supernatant ③. Take 50g of the total volume ③ and place it in a centrifuge tube to make two parallel samples. Place the above sample in a laboratory centrifuge and centrifuge (4000 rpm) for 10 minutes. After centrifugation, remove the sample and separate the precipitate. Calculate the amount of precipitate in the centrifuge tube after centrifugation as precipitate B. Then calculate the amount of precipitate in the total volume ③ as precipitate ②.

[0130] Based on the above results, the total amount of precipitate (the sum of precipitate ① and precipitate ②) and its proportion to the total sample amount were calculated. Then, the precipitate removal rate and the unremoved precipitate rate of the concentrated drug combination solution of the present invention after 3 days of natural sedimentation were calculated.

[0131] Calculation formula: (1) Precipitate ① = Total amount ② / 50 × Precipitate A (2) Precipitate ② = Total amount ③ / 50 × Precipitate B (3) Total precipitate amount = precipitate ① + precipitate ② (4) The proportion of removed precipitate to the total sample amount = precipitate ① / total amount ① (5) The proportion of unremoved precipitate in the upper layer to the total sample volume = precipitate ② / total volume ① (6) Precipitation removal rate = Precipitated ① / Total amount of precipitate (7) Unremoved precipitate rate = 1 - Precipitation removal rate Bag filtration removes sediment: Take 50 kg of the concentrated solution of this drug composition (total amount ①), shake it well and place it in a container. Because the filter press used in the experiment had a long filtration time and serious clogging, filter bag filtration (1 μm) was used. Then, the total amount of filter bag and filter residue (precipitate) was calculated as total amount ②. Total amount ② was placed in a laboratory high-speed centrifuge and centrifuged (4000 rpm) for 10 minutes. It was then taken out and weighed as total amount ③. Take another filter bag, wet it with water, and centrifuge it in a laboratory high-speed centrifuge (4000 rpm) for 10 minutes. Remove the bag and weigh the wet filter bag; this is the total weight ④. Calculate the precipitate on the filter bag as precipitate ① (precipitate ① = total weight ③ - total weight ④). Weigh the total amount of filtrate as total amount ⑤. Stir the total amount of filtrate ⑤ thoroughly. Take 50g of filtrate from total amount ⑤ and place it in a centrifuge tube to make two parallel samples. Centrifuge the samples in a laboratory centrifuge (4000 rpm) for 10 minutes. Remove the samples and separate the precipitate. Calculate the amount of precipitate in the centrifuge tube after centrifuging the 50g sample as precipitate A. Then calculate the amount of precipitate in total amount ② as precipitate ②.

[0132] Based on the above results, the total amount of precipitate (the sum of precipitate ① and precipitate ②) and its proportion to the total sample amount were calculated. Then, the precipitate removal rate and the unremoved precipitate rate of the drug combination concentrate of the present invention after filtration with a filter bag were calculated.

[0133] Calculation formula (1) Precipitation ① = Total ③ - Total ④ (Total weight of filter bag and precipitate - Wet filter bag weight); (2) Precipitation ② = Total ② / 50 × Precipitation A; (3) Total precipitate amount = Precipitation ① + Precipitation ②; (4) Percentage of removed precipitate in total sample amount = Precipitation ① / Total ①; (5) Percentage of unremoved precipitate in filtrate in total sample amount = Precipitation ② / Total ①; (6) Precipitation removal rate = Precipitation ① / Total precipitate amount; (7) Unremoved precipitate rate = 1 - Precipitation removal rate.

[0134] High-speed sedimentation centrifugation removes precipitates: Take 50 kg of the concentrated solution of this drug composition (total amount ①) and centrifuge it at high speed. The precipitate and supernatant are clearly separated. After centrifugation, weigh the amount of precipitate in total amount ① as precipitate ①. Take 50 g of precipitate ① and place it in a centrifuge tube. Centrifuge it in a laboratory centrifuge (4000 rpm) for 10 minutes. Take it out. If there is supernatant, separate it and weigh it as precipitate ①. If there is no supernatant, you can directly weigh it as precipitate ①.

[0135] The total weight is the weight of the centrifuged liquid after high-speed sedimentation centrifugation. Thoroughly mix the total volume of centrifuged liquid ②. Take 50g of the centrifuged liquid from total volume ② and place it in a centrifuge tube to make two parallel samples. Place the tubes in a laboratory centrifuge and centrifuge (4000 rpm). After centrifugation for 10 minutes, take them out and calculate the amount of precipitate in the centrifuge tube after centrifugation as precipitate A. Then calculate the amount of precipitate in total volume ② as precipitate ②.

[0136] Based on the above results, the total amount of precipitate (the sum of precipitate ① and precipitate ②) and its proportion to the total sample amount were calculated. Then, the precipitate removal rate and the unremoved precipitate rate of the drug combination concentrate of the present invention after high-speed sedimentation centrifugation were calculated.

[0137] Calculation formula (1) Precipitation ①: Weigh the precipitate directly after high-speed sedimentation centrifugation; (2) Precipitation ② = Total amount ② / 50 × Precipitate A; (3) Total amount of precipitate = Precipitate ① + Precipitate ②; (4) Percentage of precipitate removed to the total amount of sample = Precipitate ① / Total amount ①; (5) Percentage of precipitate not removed in the centrifuged liquid to the total amount of sample = Precipitate ② / Total amount ①; (6) Precipitate removal rate = Precipitate ① / Total amount of precipitate; (7) Precipitate not removed rate = 1 - Precipitate removal rate.

[0138] Table 6. Results of laboratory high-speed centrifugation studies on three batches of concentrated drug compositions after natural sedimentation, filter bag filtration, high-speed sedimentation centrifugation to remove precipitates, and subsequent treatment.

[0139] The above research results demonstrate that after 3 days of natural sedimentation, the pharmaceutical composition of this invention can only remove a small amount of precipitate, leaving a significant amount unremoved. Furthermore, the process is time-consuming and inefficient, making it impractical for production. (Experiments showed that even after 3-7 days of prolonged storage of the concentrate, precipitate separation remains ineffective, and prolonged storage leads to microbial growth, which violates pharmaceutical production regulations and hinders production feasibility. The quality of the concentrated pharmaceutical composition cannot be effectively controlled.) This indicates that this method is unsuitable for removing solid impurities from the pharmaceutical composition of this invention. Such solid impurities may also adsorb components from the medicinal solution during storage, gradually forming solid precipitates that cannot be dispersed by shaking, thus affecting the properties and flavor of the medicinal materials and the functional effects of the pharmaceutical composition.

[0140] The above research results demonstrate that the filter bag filtration method used in the drug composition of this invention can only remove a small portion of the solid impurities. The research also shows that the filtration effect and filtration time are affected by the temperature of the drug composition, making it impractical for production. Therefore, this method is unsuitable for removing solid impurities from the drug composition of this invention. (The physical method of filter bag filtration was chosen to remove solid impurities because mechanical filtration channels are easily clogged, leading to discontinuous production. Chemical refining was not used to maintain the natural medicinal properties and functional characteristics of the drug composition of this invention, ensuring the medicinal properties of traditional Chinese medicine decoctions. Chemical refining might alter or destroy the effective components or medicinal properties of the drug composition, violating the original intention of this invention to adhere to traditional Chinese medicine decoctions.) The above research results demonstrate that the high-speed sedimentation centrifugation method used in the pharmaceutical composition of this invention utilizes the solid-liquid specific gravity difference and the centripetal acceleration generated by the high-speed rotation of substances around the center. Impurities are rapidly separated and settled to the outer edge under dynamic conditions, while the centrifuged liquid gathers to the inner edge, continuously accelerating the sedimentation and separation speed, thus achieving the purpose of removing solid impurities without affecting the medicinal components, which is consistent with the characteristics of traditional Chinese medicine decoctions. High-speed sedimentation centrifugation can essentially remove solid impurities from this pharmaceutical composition, meeting the quality requirements of the pharmaceutical production process and ensuring that this pharmaceutical composition meets the basic conventional requirements of pharmaceutical preparations. This preparation method is production-ready, achieves stable quality of the concentrate, and can be used for large-scale production, making it suitable for removing solid impurities from the pharmaceutical composition of this invention.

[0141] (3) To demonstrate the effect of high-speed sedimentation centrifugation filtration on the quality of this pharmaceutical composition, the effect of the precipitate after high-speed sedimentation centrifugation on the components of this pharmaceutical composition was studied. The effect of centrifugation on the content of gentiopicroside in the pharmaceutical composition of this invention was selected for study. The results are shown in Table 7.

[0142] Experimental method: In this invention, 10 batches of 100ml concentrated drug composition solutions were taken, and the gentiopicroside content of the concentrated solution was directly determined; the concentrated solution was centrifuged at high speed at 2500 rpm, with a rotation radius of 600 mm and a power of 4.0 kW for 5 minutes, and the gentiopicroside content of the centrifuged solution was determined.

[0143] Table 7. Content determination results of 10 batches of concentrated extract of this pharmaceutical composition (n=10) Experimental results show that high-speed sedimentation centrifugation has virtually no effect on the content of gentiopicroside in this pharmaceutical composition. It can be expected that high-speed sedimentation centrifugation will not significantly affect the composition of the pharmaceutical composition, thus ensuring its intended efficacy.

[0144] Experimental Example 8 Research on the quality control method of the pharmaceutical composition of the present invention To ensure greater quality control and stable efficacy of the pharmaceutical composition of this invention, a quality control method for the pharmaceutical composition was studied. Qualitative and quantitative analyses and judgments were performed on the quality control indicators of the composition. Various components in the composition were systematically screened and tested. Specifically, studies were conducted on the developing solvent system, sample preparation methods, specificity, detection wavelength, mobile phase, chromatographic column, precision, reproducibility, robustness, and accuracy.

[0145] The specific steps are as follows: Thin-layer chromatography identification: Step (1) Accurately measure 10 ml of the drug composition and place it in a separatory funnel. Extract twice with ethyl acetate, 20 ml each time. Collect the ethyl acetate layer, evaporate to dryness in a water bath, and dissolve the residue in 2 volumes of methanol to prepare the test solution. Take 1 g of the blue cloth reference material, add 20 ml of water, heat under reflux for 2 hours, filter, and take 10 ml of the filtrate in a separatory funnel. Prepare the reference material solution using the same method as the drug composition. Separately, take an appropriate amount of gallic acid reference standard and add methanol to prepare a 1 mg / ml reference solution. Perform the test according to the thin-layer chromatography method (general rules). Take 3-5 μl of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use xylene-ethyl acetate-methanol-formic acid (5:2:1:1) as the developing solvent, develop, remove and air dry, and examine at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference material. Spray with a 5% ferric chloride-ethanol solution for color development, and examine under sunlight; gallic acid can be detected. See attached chromatogram. Figure 9 and attached Figure 10 .

[0146] Test method: Take three different batches of test samples, negative samples and control materials, and process all the corresponding test solutions simultaneously according to the requirements in step (1), spot them on the same silica gel G thin layer plate, develop and examine them.

[0147] Specificity: Take the drug composition sample and the negative control (using a blue cloth), and prepare the test solution and negative test solution according to the method described in step (1). Spot the test solution, negative test solution, reference drug solution, and reference solution, develop, and air-dry them respectively. After viewing at 365nm, spray with 5% ferric chloride-ethanol solution for color development and view under sunlight. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference drug. The target spots are well separated, and there is no interference from the negative control. See the attached chromatogram. Figure 11 .

[0148] Durability: Temperature: Following the chromatographic conditions described in step (1), the test solution, negative test solution, reference medicinal material solution, and reference solution were spotted onto the same silica gel G plate. The plates were developed and dried at low temperature (8℃, inside a refrigerator) and high temperature (35℃, inside an oven). The plates were then examined at 365 nm, and finally sprayed with 5% ferric chloride-ethanol solution for color development. The results were examined under sunlight, showing good spot separation and no interference from the negative test. The chromatograms are shown in Figure 12 and Appendix 13. Figure 13 .

[0149] Humidity: Following the chromatographic conditions described in step (1), the test solution, negative test solution, and reference herb solution were spotted onto the same silica gel G plate. The plates were developed at high humidity (88%) and low humidity (18%), dried, and examined at 365 nm. The results showed good spot separation and no interference from the negative test. The chromatogram is shown in the appendix. Figure 14 and attached Figure 15 .

[0150] Thin-layer plate: Following the chromatographic conditions described in step (1), spot the test solution, negative test solution, and reference herb solution onto the same silica gel G plate (change the silica gel G plate manufacturer). Develop and dry the plate under normal temperature and humidity conditions. After drying, examine the plate at 365 nm. The results showed good spot separation and no interference from the negative test. See attached chromatogram. Figure 16 .

[0151] Step (2): Accurately measure 10 ml of the drug composition and place it in a separatory funnel. Extract twice with 20 ml of ether each time, discard the ether solution, and then extract twice with 25 ml of ethyl acetate each time. Collect the ethyl acetate layer, evaporate to dryness in a water bath, and dissolve the residue in 2 ml of methanol to obtain the test solution. Separately, take 0.5 g of Artemisia capillaris reference material, add 25 ml of water, heat under reflux for 3 h, cool, centrifuge, and place the supernatant in a separatory funnel to prepare the reference material solution using the same method as the product. Perform the thin-layer chromatography (general rule) test, take 2 μl of each of the above two solutions, and spot them separately on the same polyamide film plate. Use toluene-ethyl acetate-formic acid-glacial acetic acid-water (1:20:4:1:1) as the developing solvent, develop, remove and dry, and examine at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0152] Test method: Take three different batches of test samples, negative samples and control materials, and process all the corresponding test solutions simultaneously according to the requirements in step (2), spot them on the same polyamide film plate, unfold and inspect.

[0153] Specificity: Take the drug composition sample and the negative control lacking Artemisia capillaris, and prepare the test solution and negative test solution according to the method described in step (2). Spot the test solution, negative test solution, and control herbal solution, develop, and air-dry them respectively, and examine them at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the control herbal material. The target spots are well separated, and there is no interference from the negative control. See the attached chromatogram. Figure 17 .

[0154] Durability Temperature: Under the chromatographic conditions described in step (2), the test solution, negative test solution, and reference herb solution were spotted separately onto the same polyamide film plate. The plates were developed and dried at low temperature (8℃, inside a refrigerator) and high temperature (35℃, inside an oven), and examined at 365nm. The results showed good spot separation and no interference from the negative test. See attached chromatogram. Figure 18 .

[0155] Humidity: Under the chromatographic conditions described in step (2), the test solution, negative test solution, and reference herb solution were spotted separately onto the same polyamide film plate. The plates were developed and dried under high humidity (88%) and low humidity (18%) conditions, and then examined at 365 nm. The results showed good spot separation and no interference from the negative test. The chromatogram is attached. Figure 19 .

[0156] Thin-layer plate: Under the chromatographic conditions described in step (2), spot the test solution, negative test solution, and reference herb solution onto the same polyamide thin-layer plate (change the polyamide thin-layer plate manufacturer). Develop and dry under normal temperature and humidity conditions, and examine at 365 nm. The results showed good spot separation and no interference from the negative test. See attached chromatogram. Figure 20 .

[0157] Step (3) Accurately measure 10 ml of the drug composition, extract twice with 25 ml of water-saturated n-butanol each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in 10 ml of methanol, add to a neutral alumina column (100-200 mesh, 4 g, inner diameter 1.0 cm), elute with 70 ml of methanol until colorless, evaporate to dryness, dissolve the residue in 2 ml of methanol to obtain the test solution. Separately, take gentiopicroside reference standard, add methanol to prepare a solution containing 2 mg per ml, as the reference solution. Perform the thin-layer chromatography (general rule) test, take 2 μl of each of the above two solutions, and spot them separately on the same silica gel GF. 254 On a thin-layer plate, the lower layer of a dichloromethane-methanol-water (25:10:3) solution was used as the developing solvent. The plate was then developed, removed, and air-dried before being examined under a UV lamp at 254 nm. The test sample chromatogram showed spots of the same color at the corresponding positions as the reference sample chromatogram.

[0158] Test method: Take three different batches of test sample, negative sample and control medicinal material respectively, and process all the corresponding test solutions simultaneously according to the requirements in step (3), and spot them on the same silica gel GF. 254 On a thin-layer plate, unfold and examine.

[0159] Specificity: Take the drug composition sample and the gentian-deficient negative control, and prepare the test solution and negative test solution according to the method described in step (3). Spot, develop, and air-dry the test solution, negative test solution, and control solution respectively, and examine them under a UV lamp at 254 nm. In the chromatogram of the test sample, a main spot of the same color appears at the corresponding position as in the chromatogram of the control, indicating good separation and no interference from the negative control. See Figures 21 and 22 for the chromatograms.

[0160] Durability Temperature: Under the chromatographic conditions described in step (3), spot the test solution, negative test solution, and reference solution onto the same silica gel GF plate. 254 On the plate, the samples were developed and dried at low temperature (5°C, inside a refrigerator) and high temperature (35°C, inside an oven), and then examined under ultraviolet light (254nm). The results showed good spot separation and no negative interference. See the chromatogram below. Figure 23 and Figure 24 .

[0161] Humidity: Under the chromatographic conditions described in step (3), spot the test solution, negative test solution, and reference solution onto the same silica gel GF plate. 254 On the plate, the samples were developed and dried at high humidity (88%) and low humidity (12%), and then examined under ultraviolet light (254nm). The results showed good spot separation and no negative interference. The chromatograms are shown in Figures 25 and 26.

[0162] Manual plate: Under the chromatographic conditions described in step (3), spot the test solution, negative test solution, and reference solution onto the same manually prepared silica gel GF plate. 254 On the plate, the chromatogram was developed and dried at room temperature (18℃) and normal humidity (52%), and then examined under ultraviolet light (254nm). The results showed good spot separation and no negative interference. The chromatogram is shown in Figure 27.

[0163] Determination of gentiopicrin content: Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel as the packing material; acetonitrile-0.2% phosphoric acid solution (10:90) as the mobile phase; detection wavelength of 270 nm; theoretical plate number calculated based on gentiopicrin, should not be less than 3000.

[0164] Preparation of reference solution: Take an appropriate amount of gentiopicroside reference standard, accurately weigh it, dissolve it in methanol and dilute it to prepare a solution containing about 0.07 mg per ml.

[0165] Preparation of the test solution: Take 5 ml of the drug composition, accurately measure it into a 50 ml volumetric flask, accurately add an appropriate amount of methanol, shake well and dilute to the mark, filter, and the solution is obtained.

[0166] Determination method: Accurately pipette 10 μl each of the reference solution and the test solution into the chromatograph and determine the result.

[0167] More specifically, the content of gentiopicroside in the drug composition is determined by high performance liquid chromatography.

[0168] Instruments: High-performance liquid chromatograph: Thermo-fisher U-3000 / Agilent 1100 liquid chromatograph; KQ-500DB CNC ultrasonic extraction instrument (Kunshan Ultrasonic Instrument Co., Ltd.); Reagents and reagents: Samples were provided by the present invention unit; gentiopicroside reference standard was provided by the National Institutes for Food and Drug Control (batch number 110770-201013, content calculated as 96.9%); reagents: methanol, acetonitrile, and phosphoric acid were all chromatographic grade, and water was prepared using an UPW-50N ultrapure water purifier.

[0169] Examine accuracy, precision, specificity, linear range, etc.

[0170] Specificity: Sample solution and negative control solution were prepared according to the test solution preparation method. Under the chromatographic conditions described above, the reference solution, sample solution, and negative control solution were injected into the liquid chromatograph. The sample solution chromatogram showed corresponding peaks at the same positions as the reference solution chromatogram, and the negative control solution showed no interference. See attached chromatogram for details. Figure 28 .

[0171] Repeatability: Six samples from the same batch (batch number: 191212) were prepared according to the test solution preparation method, and the content of gentiopicrin was determined under the above chromatographic conditions. The results are shown in Table 8, indicating good repeatability.

[0172] Table 8 Results of repeatability tests (n=6)

[0173] Precision: The same gentiopicroside reference solution containing 0.72 μg of gentiopicroside was precisely pipetted and injected 6 times consecutively. The results are shown in Table 9, indicating good precision.

[0174] Table 9 Precision test results (n=6)

[0175] Linearity assessment: Under the chromatographic conditions described above, reference solutions containing 0.14 μg, 0.36 μg, 0.72 μg, 1.05 μg, 1.49 μg, and 1.79 μg of gentiopicrin were precisely injected sequentially into the liquid chromatograph, and the peak areas were measured. Linear regression was performed with the injection volume X as the abscissa and the peak area Y as the ordinate, and a working curve was plotted. The linear regression equation was: Y = 1285X - 6.202, R0 2 =0.9999 (Results are shown in Table 10). Gentianiin showed a good linear relationship between the injection amount and peak area of ​​the reference standard in the range of 0.1430 μg to 1.7939 μg. See the attached graph for the linearity diagram. Figure 29 .

[0176] Table 10 Linearity of Gentianoside Reference Standard

[0177] Accuracy: Accurately measure 5 ml of the sample (batch number: 191212) with the determined content (average content 0.7592 mg / ml), place it in a 50 ml volumetric flask, accurately add 25 ml of gentiopicrin reference solution (0.1494876 mg / ml), shake to mix, prepare the sample solution according to the test solution preparation method, and determine the content of gentiopicrin according to the above chromatographic conditions. Calculate the recovery rate. The results are shown in Table 11, indicating a good recovery rate.

[0178] Table 11 Recovery Rate Test Data (n=6)

[0179] Durability The Influence of Different Brands of Chromatographic Columns and Different Detection Equipment The content of gentiopicroside in the same batch of samples (batch number: 191212, sample solution prepared using the method for processing the test solution of this product) was determined using chromatographic columns of different brands (at least three types) with octadecylsilane-bonded silica gel as the packing material, with the same detection equipment; or with different detection equipment (at least two models). The influence of differences in chromatographic column brands and detection equipment was investigated. The results are shown in Tables 12 and 13. The results indicate that chromatographic columns of different brands with octadecylsilane-bonded silica gel as the packing material had no effect on the content determination of this product; different detection equipment also had no effect on the content determination of this product.

[0180] Table 12 Determination results of chromatographic columns of different brands

[0181] Table 13 Measurement results from different testing devices

[0182] Stability test: Take the sample (batch number: 191212), prepare the test solution according to the preparation method of the test solution, and determine the content of gentiopicroside under the above chromatographic conditions. The results are shown in Table 14.

[0183] Table 14 Sample stability test

[0184] The experimental results show that the quality control method of the present invention has simple sample treatment, less sample consumption, high precision, high accuracy, high stability, good repeatability, and is simple and easy to operate. This method can comprehensively, reliably and effectively control the quality of this pharmaceutical composition.

[0185] Compared with the formulation of Comparative Sample 1, the formulation of the present invention uses stir-fried Fructus Aurantii Immaturus processed with bran, while the formulation of Comparative Sample 1 uses raw Fructus Aurantii Immaturus without processing. Compared with raw Fructus Aurantii Immaturus, the stir-fried Fructus Aurantii Immaturus used in the formulation of the present invention has a reduced content of volatile oil, removes the stimulating components, alleviates the dryness property, reduces the irritation of the pharmaceutical composition of the present invention to the gastrointestinal tract, and helps to reduce the adverse reactions of the pharmaceutical composition of the present invention; the stir-fried Fructus Aurantii Immaturus in the formulation of the present invention can enhance its effect of strengthening the spleen and relieving distension, and is very important for improving the effect of promoting qi circulation and relieving accumulation of the composition of the present invention. Therefore, the present invention studies the changes in the content of chemical components before and after stir-frying Fructus Aurantii Immaturus.

[0186] Experimental Example 9 Study on the changes in the content of main chemical components before and after stir-frying Fructus Aurantii Immaturus This experimental example studies the changes in the content of main chemical components before and after stir-frying Fructus Aurantii Immaturus, and mainly examines the changes in the contents of naringin, neohesperidin, and hesperidin before and after stir-frying Fructus Aurantii Immaturus.

[0187] Materials: Thermo Fisher U3000 high performance liquid chromatograph (Thermo Fisher (China) Technology Co., Ltd.), AB104-N ten-thousandth electronic balance (Mettler Toledo Group), XSE105DU one-hundred-thousandth electronic analytical balance (Mettler Toledo Group). Neohesperidin, hesperidin, naringin, reference substances (National Institutes for Food and Drug Control, Shanghai Standard Technology Service Co., Ltd., batch numbers are 112055, 112054, 82373-94-2 respectively), acetonitrile and methanol are chromatographically pure, water is purified water, and the rest of the reagents are of analytical grade. Fructus Aurantii Immaturus was identified by Associate Professor Liushao Huan of the Department of Pharmacognosy of Guizhou Medical University as the dried immature fruit of the Rutaceae plant Citrus aurantium Citrus aurantium L The Fructus Aurantii Immaturus slices and stir-fried slices were prepared according to the method in the present invention.

[0188] Methods and Results Chromatographic conditions: Diamonsil C18 column (4.6 mm × 150 mm, 5 μm), mobile phase methanol (A) - water (B), gradient elution (0 - 6 min, 49% A; 6 - 15 min, 49% - 75% A; 15 - 26 min, 75% - 100% A; 26 - 30 min, 100% A; 30 - 32 min, 100% - 49% A; 32 - 35 min, 49% A), flow rate 1.0 mL·min -1 , detection wavelength 324 nm, column temperature 25 °C, injection volume 10 μL.

[0189] Preparation of reference substance solution Weigh appropriate amounts of marmin, nobiletin, and tangeretin reference substances accurately, and prepare a mixed reference substance solution with the mass concentrations of marmin, nobiletin, and tangeretin all being 80 mg / L by adding methanol.

[0190] Preparation of test sample solution Take about 0.5 g of the crude powder of the sample, accurately weigh it and place it in a stoppered conical flask, accurately add 50 mL of methanol, weigh it, heat it under reflux in a water bath for 1 h, cool it, weigh it again, make up the lost weight with methanol, shake well, filter, accurately transfer 25 mL of the continued filtrate into a volumetric flask, and make it up to the mark with methanol, shake well to obtain the solution.

[0191] Sample determination Inject 10 μL of 6 batches of Fructus Aurantii and stir-fried Fructus Aurantii with bran samples into the liquid chromatograph respectively, and the results are shown in Table 15.

[0192] Table 15 Determination of the contents of main chemical components before and after stir-frying Fructus Aurantii with bran (n = 2)

[0193] The experimental results show that there are differences in the main chemical components of Fructus Aurantii before and after stir-frying with bran, especially the volatile components. Among them, marmin is a fat-soluble component. After stir-frying Fructus Aurantii with bran, the content of marmin decreases, which is consistent with the traditional Chinese medicine processing theory that stir-frying Fructus Aurantii with bran can reduce the content of volatile oil and the irritation of Fructus Aurantii to the gastrointestinal tract to achieve "removing dryness and harmonizing the stomach". Therefore, it is necessary and of great significance to use stir-fried Fructus Aurantii in the drug combination of the present invention to improve the drug effect and reduce adverse reactions.

[0194] Stir-fried Fructus Aurantii with bran is used in the prescription of the present invention. Stir-fried Fructus Aurantii with bran has the functions of regulating qi and relieving fullness, promoting qi circulation and relieving distension. After stir-frying Fructus Aurantii with bran, it can reduce the content of volatile oil, reduce dryness, relieve the medicinal properties, and reduce the irritation to the gastrointestinal tract. The results of traditional Chinese medicine pharmacological research prove that stir-fried Fructus Aurantii with bran can increase the small intestine propulsion rate. After processing Fructus Aurantii, its effect of relieving fullness and distension in the middle-jiao can be enhanced, and the effect of stir-fried Fructus Aurantii with bran is the best. Raw Fructus Aurantii can reduce the small intestine propulsion rate and reduce the therapeutic effect of the drug. Therefore, the present invention has carried out experimental research on the small intestine propulsion movement of the drug composition of the present invention and comparative sample 1.

[0195] Experimental Example 10 Study on the effect of the pharmaceutical composition of the present invention and comparative sample 1 on the small intestinal propulsion of mice 1. Experimental animals: 84 Kunming mice (20g±2g), half male and half female.

[0196] 2. Experimental Samples: Model group: 0.2ml / 10g physiological saline Neostigmine: 0.02 mg / 10 g Pharmaceutical composition 210401 of the present invention (prepared in Experimental Example 6): 200 mg / 10 g Comparative Sample 1 210404 (prepared in Experiment 6): 200 mg / 10 g 3. Experimental Methods Eighty-four Kunming mice (half male and half female) were randomly divided into four groups of 21 mice each. After fasting (but not water), the mice were administered the drugs listed in Table 14 via gavage. One hour after gavage, each mouse was given 0.2 ml of ink. Twenty minutes after ink administration, the mice were euthanized by cervical dislocation. The abdomen was opened, and the gastrointestinal propulsion rate was measured. The experimental results are expressed as mean ± standard deviation (X±SD). Multiple group comparisons were performed using analysis of variance and pairwise comparisons of mean values ​​for multiple groups using LSD. The experimental results are shown in Table 16.

[0197] Table 16 Effects of the pharmaceutical composition of the present invention and comparative sample 1 on the small intestinal propulsion rate in mice (X±SD)

[0198] Compared with the control group: P < 0.05; P < 0.01.

[0199] Experimental results show that the pharmaceutical composition of the present invention and the comparative sample 1 have the effect of promoting gastrointestinal propulsion. This further confirms that the use of stir-fried bitter orange peel in the pharmaceutical composition of the present invention can increase the small intestinal propulsion rate and improve the intestinal propellant effect. Moreover, the effect of the pharmaceutical composition of the present invention in promoting gastrointestinal propulsion is significantly better than that of the comparative sample 1, and enhances the effect of the pharmaceutical composition of the present invention in promoting qi circulation and eliminating food stagnation.

[0200] This invention uses fresh Dendrobium officinale extract, stir-fried Citrus aurantium, and a combination of herbal extracts. A high-speed centrifugal sedimentation process is employed, and a proprietary, precise, and accurate quality control method is established to ensure more stable and safer processing and quality of the composition, resulting in a more effective therapeutic effect. Therefore, this invention conducted pharmacodynamic studies comparing the drug composition with comparative sample 1. Specifically, the study investigated the effect of the drug composition on gastric secretion function in rats compared to comparative sample 1; and the study investigated the mechanism of action of the drug composition and comparative sample 1 in treating yin deficiency and internal heat, specifically its effects on the levels of serum inflammatory cytokines C-reactive protein, tumor necrosis factor, and adiponectin in rats.

[0201] Experimental Example 11 This invention relates to a study of the effects of the pharmaceutical composition of the present invention and comparative sample 1 on the gastric juice secretion function of rats.

[0202] 1. Experimental animals: 48 SD rats (220g±30g), half male and half female. 2. Experimental Samples: Normal control group: physiological saline: 10 mL / kg.

[0203] Model group: physiological saline: 10 mL / kg.

[0204] Aluminum magnesium carbonate tablets (Bayer Healthcare Co., Ltd., batch number 208169): 500 mg / kg Pharmaceutical composition 210401 of the present invention (prepared in Experimental Example 6): 600 mg / kg Comparative sample 1 210404 (prepared in Experiment 6): 600 mg / kg 3. Experimental Methods SPF-grade SD rats were randomly divided into a normal control group, a model group, the drug composition group of this invention, a control sample group, and an aluminum magnesium carbonate tablet group. The drug composition group, control sample group, aluminum magnesium carbonate tablet group, and blank group were administered the corresponding drugs by gavage daily. The normal control group and model group were administered an equal volume of physiological saline by gavage for 3 consecutive days, with fasting and water restriction for 12 hours after the last administration. Preoperatively, rats were anesthetized with ether, a small incision was made along the midline of the abdomen, the stomach was located, and the pylorus was ligated. The drug was administered once more via the duodenum according to Table 15. Gastric fluid was collected after 5 hours and centrifuged at 3000 rpm for 15 minutes to measure gastric fluid volume, total gastric acidity, and pepsin activity. Experimental results are expressed as X±SD. Multiple group comparisons were performed using analysis of variance and pairwise comparisons of means within multiple groups using LSD. Experimental results are shown in Table 17.

[0205] Table 17 Effects of the pharmaceutical composition of the present invention and comparative sample 1 on gastric secretion in rats (X±SD)

[0206] Compared with the control group: P < 0.05; P < 0.01. Experimental results show that the pharmaceutical composition of the present invention and comparative sample 1 have the effects of inhibiting gastric juice secretion, reducing total gastric acidity and total acid excretion, and increasing pepsin activity. The pharmaceutical composition of the present invention has a significantly better effect on gastric juice secretion than comparative sample 1. This indicates that the pharmaceutical composition of the present invention has a better effect on clearing stomach heat and purging fire.

[0207] Experimental Example 12 The mechanism of action of the pharmaceutical composition of the present invention and comparative sample 1 in treating yin deficiency and internal heat was investigated, and therefore its effects on the levels of inflammatory cytokines C-reactive protein, tumor necrosis factor and adiponectin in rat serum were studied.

[0208] 1. Experimental Materials Experimental animals: 52 male SD clean-grade rats, 208±10g. The animals were housed separately in an animal room at 20±2℃, with free access to food and water, and a 12h light / dark cycle.

[0209] Experimental samples: Drug composition 210401 of this invention, control sample 1 210404, and positive control drug metformin tablets (Shanghai Shibao Pharmaceutical Co., Ltd.), prepared to 10 mg / ml before use; Rat C-reactive protein, adiponectin, TNF-α, and insulin ELISA kits were purchased from Nanjing Dizhao Biotechnology Co., Ltd., batch number: 20200803A; C-reactive protein (CRP), TNF-α, and adiponectin levels were detected according to the ELISA kit method.

[0210] 2. Experimental methods and results Serum inflammatory cytokine detection Three days later, the rats were fasted but allowed free access to water for 12 hours. Blood was collected from the orbital venous plexus using a glass capillary tube. The blood was divided into two parts: one part was allowed to stand for 15 minutes, then centrifuged at 3000 rpm, 4°C for 15 minutes to separate the serum, which was then aliquoted and stored at -70°C. The other part was anticoagulated with heparin sodium, allowed to stand, then centrifuged at 500 rpm for 10 minutes, discarding the supernatant and retaining the red blood cells. The red blood cells were washed three times with physiological saline, centrifuged again, and the red blood cells were used to prepare hemolysate for hemoglobin (GHB) measurement. C-reactive protein (CRP), TNF-α, and adiponectin levels were measured according to the kit instructions.

[0211] Experimental data are expressed as mean ± standard deviation (mean ± SD). Student's two tailed t-test or one-way ANOVA combined with the Newman-Keuls multiple comparison post-hoc test was used to compare differences between groups. p < 0.05 was considered statistically significant. Experimental results are shown in Table 18.

[0212] Table 18 Effects of the pharmaceutical composition of the present invention and comparative sample 1 on serum inflammatory markers and adiponectin levels in rats (n=8)

[0213] # p<0.05 vs Blank control p<0.05 vs Model comparison. Experimental results demonstrate that the pharmaceutical composition of this invention can significantly reduce the concentrations of two inflammatory factors, C-reactive protein (CRP) and TNF-α, and increase the serum adiponectin level in rats (p < 0.05 compared with the model group), revealing that the pharmaceutical composition of this invention exhibits a significant reversal effect on serum inflammatory cytokines. Furthermore, the pharmaceutical composition of this invention is superior to that of comparative sample 1. It has a predictable effect in treating yin deficiency and internal heat.

[0214] Based on Experiment 12, abnormal concentrations of inflammatory factors in the blood can lead to abnormal blood glucose and lipid biochemical parameters. To further confirm that the pharmaceutical composition of the present invention improves blood glucose and lipid biochemical parameters while improving serum inflammatory factor concentrations, the present invention conducted a study on the effects of the pharmaceutical composition and control sample 1 on glucose tolerance, fasting blood glucose, insulin levels, insulin resistance index, and glycated hemoglobin levels in rats; and conducted a study on the effects of the pharmaceutical composition of the present invention and control sample 1 on the levels of total cholesterol (TC), triglycerides (TG), free fatty acids (FFA), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) in rat serum.

[0215] Experimental Example 13 Based on Experiment 12, the mechanism of action of the pharmaceutical composition of the present invention and comparative sample 1 in treating yin deficiency and internal heat was investigated. Therefore, the effect of the composition on the expression of rat adipokines IL-6 (interleukin-6) and MCP-1 (macrophage chemotactic protein-1) genes was studied.

[0216] 1. Experimental Materials Experimental animals: 62 male SD clean-grade rats, weighing 205±10g, were housed separately in an animal room at 20±2℃ with free access to food and water and a 12h light / dark cycle.

[0217] Experimental samples: the pharmaceutical composition 210401 of this invention and the control sample 1 210404, the positive drug metformin tablets (Shanghai Shibao Pharmaceutical Co., Ltd.), prepared to 10mg / ml before use; the insulin ELISA kit, all purchased from Nanjing Dizhao Biotechnology Co., Ltd., batch number: 20210801A.

[0218] 2. Experimental methods and results 100 mg of periepididymal adipose tissue from rats was weighed, and RNA was extracted according to the RANiso Plus (Total RNA Extraction Reagent) procedure. The 260 / 280 ratio was measured using a UV spectrophotometer. RNA was reverse transcribed into cDNA using the EasyScript First-Strand cDNA Synthesis SuperMix kit and stored at -70°C. Real-time quantitative PCR (qPCR) detection was then performed using the Thermo Scientific Maxima SYBR Green / ROX qPCR Master Mix (2X) kit. -ΔΔCt The expression levels of IL-6 (interleukin-6) and MCP-1 (macrophage chemokine-1) genes in adipose tissue were calculated.

[0219] Experimental data are expressed as mean ± standard deviation (mean ± SD). Student's two tailed t-test or one-way ANOVA combined with Newman-Keuls multiple comparison post-hoc test was used to compare differences between groups. p < 0.05 was considered statistically significant. Experimental results are shown in Table 19.

[0220] Table 19 Effects of the pharmaceutical composition of the present invention and comparative sample 1 on rat adipokinin gene expression. (n=4)

[0221] # p <0.05 vs Blank control p <0.05 vs Model comparison Experimental results showed that the expression levels of rat adipokines genes were further detected using qPCR. The levels of MCP-1 and IL-6 in the model control group were significantly higher than those in the blank group. p <0.05%, the pharmaceutical composition of the present invention can significantly reduce the concentration of these two inflammatory factors of fat ( p <0.05), and its effect was stronger than that of control sample 1.

[0222] Experimental Example 14 Study on the effects of the pharmaceutical composition of the present invention and comparative sample 1 on glucose tolerance, fasting blood glucose, insulin level, insulin resistance index, and glycated hemoglobin (HbA1c) level in rats 1. Experimental Materials Experimental animals: 54 male SD clean-grade rats, 208±10 g.

[0223] Experimental samples: Pharmaceutical composition 210401 and control sample 1 210404 (both prepared from Experimental Example 6), positive control drug metformin tablets (Shanghai Shibao Pharmaceutical Co., Ltd.), prepared to 10 mg / ml immediately before use; glucose, analytical grade, Nanjing Chemical Reagent Co., Ltd., batch number: 20041820340, prepared to 0.2 g / ml with distilled water immediately before use; glucose assay kit, Nanjing Jiancheng Bioengineering Institute, batch number: 20200101145; fructose, Amresco product, batch number: 3768B037; rat glycated hemoglobin test kit, Nanjing Jiancheng Bioengineering Institute, batch number: 20200917.

[0224] Experimental instruments: Enzyme-linked immunosorbent assay (ELISA) reader, model: 1500, Thermo Electron Corporation; Ultra-low temperature freezer, model: 702, Thermo Electron Corporation; Cell culture incubator, model: 3111, Thermo Electron Corporation; Clean bench, model: SW-CJ-IF, Suzhou Antai Air Technology Co., Ltd.; Inverted microscope, model: XAZ-DZ, Chongqing Optical Instrument Factory; Low-temperature centrifuge, model: 1-15K, Sigma Corporation; Ultrapure water system, model: ROLAB, Hitech Instruments Co., Ltd.; Consumables: Glass capillaries, 6-well and 96-well flat-bottom cell culture plates, pipette tips, EP tubing, etc., purchased from Nanjing Weiwo Biotechnology Co., Ltd. and other companies.

[0225] 2. Experimental methods and results Preparation of rat insulin resistance model and glucose tolerance test Rats were randomly divided into four groups: a blank group (treated with normal drinking water) and a control group (treated with 10% fructose solution instead of normal drinking water). All rats were fed a normal diet. Four weeks after modeling, glucose tolerance was tested: after 12 hours of fasting with free access to water, zero-point blood was collected from the orbital venous plexus of the rats using a glass capillary tube. Each group of rats was administered 2 g / kg of glucose solution by gavage, and blood was collected from the orbital venous plexus at 30 min, 60 min, and 120 min after gavage. One end of the capillary tube was sealed, and after the blood had stood for 15 min, it was centrifuged at 3000 rpm, 4℃ for 15 min. Reagents were prepared according to the glucose assay kit instructions, and 300 μL of the solution was added to each well of a 96-well plate. 3 μL of the centrifuged serum was then added to each well, and the plate was gently shaken to ensure thorough mixing. Incubate at 37°C for 15 minutes to allow the reaction to complete, and then scan the absorbance value at 505 nm using an ELISA reader.

[0226] Glucose tolerance test Rats were administered glucose tolerance by gavage for 2 and 4 weeks. Rats were fasted but allowed free access to water for 12 hours. Zero-point blood samples were collected from the orbital venous plexus using a glass capillary tube. Each group of rats was administered glucose solution (2 g / kg) by gavage, and blood samples were collected from the orbital venous plexus at 30, 60, and 120 minutes post-gavage. After the blood was allowed to stand for 15 minutes, it was centrifuged at 3000 rpm, 4°C for 15 minutes to separate serum. Serum glucose concentration was measured according to the glucose assay kit, and the area under the glucose curve was calculated.

[0227] Fasting blood glucose, insulin level testing and insulin resistance index calculation Three days after oral administration of the drug for 4 weeks, following a glucose tolerance test, rats were fasted but allowed free access to water for 12 hours. Blood was collected from the orbital venous plexus using a glass capillary tube. After the blood had stood for 15 minutes, it was centrifuged at 3000 rpm, 4°C for 15 minutes to separate the serum. Blood glucose levels were measured using a glucose kit, and insulin levels were detected using a rat insulin ELISA kit. The insulin resistance index (HOMA-IR) was calculated. HOMA-IR = (fasting blood glucose concentration × fasting insulin concentration) / 22.5. Glycated hemoglobin (HbA1c) levels were measured using the kit method.

[0228] Experimental data are expressed as mean ± standard deviation (mean ± SD). Student's two tailed t-test or one-way ANOVA combined with the Newman-Keuls multiple comparison post-hoc test was used to compare differences between groups. p < 0.05 was considered statistically significant. Experimental results are shown in Tables 20, 21, 22, and 23.

[0229] Table 20. Glucose tolerance in rats 4 weeks after modeling (n=10) p<0.05 vs Blank control group. Table 21 Results of the effect of the pharmaceutical composition of the present invention and comparative sample 1 on glucose tolerance in rats after 2 weeks of administration. (n=8) # p<0.05 vs Blank control p<0.05 vs Model group comparison. Table 22 Results of the effect of the pharmaceutical composition of the present invention on glucose tolerance in rats after 4 weeks of administration compared with comparative sample 1 (n=8) # p<0.05 vs Blank control p<0.05 vs Model comparison. p<0.05 vs Comparison sample 1.

[0230] Table 23 Results of the effects of the pharmaceutical composition of the present invention and comparative sample 1 on fasting blood glucose, insulin level, insulin resistance index and glycated hemoglobin level in rats. (n=8)

[0231] # p<0.05 vs Blank control p<0.05 vs Model comparison.

[0232] Experimental results demonstrate that after 2 weeks of administration, the pharmaceutical composition of this invention significantly reduced fasting blood glucose and significantly reduced blood glucose levels 30-120 minutes after glucose infusion. The area under the glucose curve (AUC) also confirms this effect. After 4 weeks of administration, both the pharmaceutical composition and control sample 1 significantly reduced fasting blood glucose and insulin levels, significantly reduced the insulin resistance index, and significantly reduced the level of glycated hemoglobin (HbA1c) in the blood (p < 0.05 compared to the model group). The pharmaceutical composition of this invention is superior to control sample 1.

[0233] Experimental Example 15 Study on the effects of the pharmaceutical composition of the present invention and comparative sample 1 on the levels of total cholesterol (TC), triglycerides (TG), free fatty acids (FFA), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) in rat serum. 1. Experimental Materials Experimental animals: 50 male SD clean-grade rats, 209±10g.

[0234] Experimental samples: Pharmaceutical composition 210401 and control sample 1 210404 (both prepared from Experimental Example 6), positive control drug metformin tablets (Shanghai Shibao Pharmaceutical Co., Ltd.), prepared to 10 mg / ml before use; rat insulin and rat free fatty acids, Nanjing Jiancheng Bioengineering Institute, batch number: 20200814; total cholesterol, Beijing Beihua Kangtai Clinical Reagent Co., Ltd., batch number: 20200115; low-density lipoprotein cholesterol, Beijing Beihua Kangtai Clinical Reagent Co., Ltd., batch number: 20200403; high-density lipoprotein cholesterol, Beijing Beihua Kangtai Clinical Reagent Co., Ltd., batch number: 20200507; triglyceride reagent kit, Beijing Beihua Kangtai Clinical Reagent Co., Ltd., batch number: 20200408.

[0235] 2. Experimental methods and results Three days later, the rats were fasted but allowed free access to water for 12 hours. Blood was collected from the orbital venous plexus using a glass capillary tube. The blood was divided into two parts: one part was allowed to stand for 15 minutes, then centrifuged at 3000 rpm and 4°C for 15 minutes to separate the serum, which was then aliquoted and stored at -70°C. The other part was anticoagulated with heparin sodium, allowed to stand, then centrifuged at 500 rpm for 10 minutes, discarding the supernatant and retaining the red blood cells.

[0236] Red blood cells were washed three times with physiological saline, centrifuged, and then used to prepare hemolysate for hemoglobin (GHB) assay. Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C, LDL-C), free fatty acids (FFA), and triglycerides (TG) were measured and calculated according to the corresponding kit methods.

[0237] Experimental data are expressed as mean ± standard deviation (mean ± SD). Student's two-tailed t-test or one-way ANOVA combined with Newman-Keuls Multiple Comparison post-hoc test was used to compare differences between groups. p < 0.05 was considered statistically significant. Experimental results are shown in Table 24.

[0238] Table 24. Effects of the pharmaceutical composition of the present invention on serum lipid metabolism indicators in rats compared with comparative sample 1. (n=8) Note: #p < 0.05 vs. blank control; p < 0.05 vs. model comparison.

[0239] Experimental results showed that after 5 weeks of oral administration, the drug composition of the present invention significantly reduced the levels of total cholesterol, low-density lipoprotein cholesterol, free fatty acids and triglycerides in rat serum, while significantly increasing the level of high-density lipoprotein (p < 0.05 compared with the control group), revealing that the drug composition of the present invention has a good anti-lipid metabolism effect, and the drug composition of the present invention is superior to the control sample 1, and has a predictable effect on the symptoms of yin deficiency and internal heat.

[0240] Experimental Example 16 The mechanism of action of the pharmaceutical composition of the present invention and comparative sample 1 in treating yin deficiency and internal heat was investigated, and therefore its effects on rat adipose tissue IKKβ and skeletal muscle tissue AMPK and insulin signaling were studied.

[0241] 1. Experimental Materials Experimental animals: 62 male SD clean-grade rats, 200±10 g each. The animals were housed separately in an animal room at 20±2℃, with free access to food and water, and a 12-hour light / dark cycle.

[0242] Experimental drugs: the present invention drug composition 210401, the control sample 1210404, and the positive drug metformin tablets (Shanghai Shibao Pharmaceutical Co., Ltd.), prepared at 10 mg / m² before use.

[0243] 2. Experimental methods and results Immunoblotting was used to detect the phosphorylation level of IKK beta in rat adipose tissue and the activation levels of AMPK and insulin signaling in skeletal muscle.

[0244] Experimental data are expressed as mean ± standard deviation (mean ± SD). Student's two tailed t-test or one-way ANOVA combined with the Newman-Keuls multiple comparison post-hoc test was used to compare differences between groups. p < 0.05 was considered statistically significant. Experimental results are shown in Tables 25 and 26.

[0245] Table 25 Results of the effect of the pharmaceutical composition of the present invention and comparative sample 1 on the phosphorylation level of periepididymal lipoprotein IKKβ in rats (western blotting)

[0246] p<0.05 vs Model,# p <0.05 vs blank Experimental results showed that the phosphorylation level of IKKβ in rat fat, AMPK activation and insulin signal activation in skeletal muscle were further detected by immunoblotting. The phosphorylation level of IKKβ in fat in the model group was significantly enhanced compared with the blank group. The drug composition of the present invention can significantly reduce the phosphorylation level of lipoprotein IKKβ, and its effect is better than that of the control sample 1.

[0247] Table 26 Results of the effects of the pharmaceutical composition of the present invention and comparative sample 1 on the phosphorylation levels of insulin signaling pathway proteins and AMPK in rat skeletal muscle tissue (x±SD, n=3)

[0248] p <0.05 vs Model group, # p <0.05 vs Blank 1 Experimental results demonstrate that in skeletal muscle tissue, the phosphorylation level of AMPK in the model rats was significantly reduced compared to the control group, indicating that the pharmaceutical composition of this invention exhibits an antagonistic effect. Conversely, the model group showed enhanced phosphorylation at serine sites of IRS-1 and weakened phosphorylation at tyrosine residues, correspondingly reducing the phosphorylation level of Akt downstream of insulin IRS-1. These pathological changes can be enhanced or reversed by the pharmaceutical composition of this invention.

[0249] Conclusion: The above research results indicate that the pharmaceutical composition of the present invention can significantly reduce serum inflammatory cytokines C-reactive protein (CRP) and TNF-α concentrations, MCP-1 and IL-6 adipokine gene expression levels, and increase adiponectin levels; it can reduce fasting blood glucose, insulin levels, insulin resistance index, and glycated hemoglobin (HbA1c) levels; it can significantly reduce serum total cholesterol (TC), triglycerides (TG), and free fatty acids (FFA), increase high-density lipoprotein cholesterol (HDL-C) levels, and decrease high-density lipoprotein cholesterol (LDL-C) levels; it can significantly reduce lipoprotein IKKβ phosphorylation levels and improve or reverse insulin signaling pathway protein and AMPK phosphorylation levels. This demonstrates that the pharmaceutical composition of the present invention exhibits a more comprehensive improvement effect on the above-mentioned abnormal levels caused by yin deficiency and internal heat, and the effects of the pharmaceutical composition of the present invention are superior to those of comparative sample 1.

[0250] In summary, compared with comparative sample 1, the pharmaceutical composition containing fresh Dendrobium officinale extract of this invention can reduce the irritation of the composition, decrease adverse reactions, and improve compliance. Both the pharmaceutical composition containing fresh Dendrobium officinale extract of this invention and comparative sample 1 have certain effects on small intestinal propulsion, gastric juice secretion, abnormal serum inflammatory factor concentrations, blood glucose, insulin, lipid metabolism abnormalities, and pancreatic signal transduction. However, the effect of the pharmaceutical composition of this invention is significantly better than that of comparative sample 1. It can be expected that the pharmaceutical composition of this invention will have better efficacy in clearing stomach heat, promoting qi circulation and eliminating stagnation, and relieving yin deficiency and internal heat.

[0251] Example 1 Fresh Dendrobium extract of this invention Take 200g of fresh Dendrobium, add water and decoct 3 times. For the first decoction, add 8 times the weight of the medicinal material in water, and for the second and third decoctions, add 6 times the weight of the medicinal material in water. Each decoction is for 1.5 hours. Combine the filtrates and let stand for 12 hours. Take the supernatant and concentrate it to a clear extract with a relative density of not less than 1.03 at 70℃. Cool it to room temperature and centrifuge the clear extract using a high-speed sedimentation centrifuge. Place the centrifuged liquid in a -25℃ cold storage for rapid freezing for 12 hours, and then freeze it in a -10℃ cold storage to obtain the final product.

[0252] Example 2 This invention relates to stir-fried bitter orange peel. Citrus aurantium: 400g Wheat bran: 60g First, clean the bitter orange peel and cut it into 3mm thin slices. Then, heat the frying container over medium heat until 60g of wheat bran is evenly sprinkled in and smoke immediately appears. Then, add 400g of evenly cleaned bitter orange peel slices and stir-fry quickly and evenly until the surface of the bitter orange peel turns light yellow or yellow and the wheat bran turns black. Immediately remove it, sift out the wheat bran, and let it cool.

[0253] Example 3 The herbal extract of this invention Take 83.3g of stir-fried Citrus aurantium, 5.0g of Cinnamomum cassia root, 83.3g of Polygonatum sibiricum, 83.3g of Rehmannia glutinosa, 33.3g of Gentiana scabra, 83.3g of Scutellaria baicalensis, 83.3g of Sophora flavescens, 83.3g of Ophiopogon japonicus, 83.3g of Asparagus cochinchinensis, 83.3g of Eriobotrya japonica leaf, and 100g of Artemisia capillaris. Add water and decoct three times. For the first decoction, add 8 times the amount of water and decoct for 1.5 hours. For the second and third decoctions, add 6 times the amount of water and decoct for 1.5 hours each. Filter, combine the filtrates, let stand for 12 hours, and take the supernatant.

[0254] Example 4 The pharmaceutical composition oral solution of the present invention 33g of fresh Dendrobium officinale extract, 1200g of herbal combination extract.

[0255] The fresh Dendrobium extract was prepared according to Example 1.

[0256] The herbal extract was prepared according to Example 3.

[0257] Fresh Dendrobium extract and herbal combination extract were combined and concentrated to a clear extract with a relative density of not less than 1.05 at 80℃. The clear extract was centrifuged using a high-speed sedimentation centrifuge. The centrifuged liquid was heated, and 166g of sucrose, 0.5g of ethylparaben, 3.0g of benzoic acid, and 0.083g of citric acid were added. The mixture was sterilized for 2 hours, cooled to 40℃~50℃, and 0.11g of menthol was added. The mixture was mixed well, filled into containers, and the product was obtained.

[0258] Example 5 Quality testing method for the pharmaceutical composition described in Example 4 Thin-layer chromatography identification of *Gynostemma pentaphyllum*: Accurately measure 10 ml of the sample and place it in a separatory funnel. Extract twice with ethyl acetate, 20 ml each time. Collect the ethyl acetate layer, evaporate to dryness in a water bath, and dissolve the residue in 2 volumes of methanol to obtain the test sample. Take 1 g of *Gynostemma pentaphyllum* reference material, add 20 ml of water, heat under reflux for 2 hours, filter, and take 10 ml of the filtrate in a separatory funnel. Prepare the reference material solution using the same method as the drug composition. Separately, take an appropriate amount of gallic acid reference standard and add methanol to prepare a 1 mg / ml reference solution. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 3-5 μl of each of the above three solutions to the same silica gel G thin-layer plate. Develop with xylene-ethyl acetate-methanol-formic acid (5:2:1:1) as the developing solvent. Remove and air dry, then examine at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference material. Spray with a 5% ferric chloride-ethanol solution for color development, and examine under sunlight to detect gallic acid.

[0259] Thin-layer chromatography identification of Artemisia capillaris: Accurately measure 10 ml of the sample and place it in a separatory funnel. Extract twice with 20 ml of ether each time, discarding the ether solution. Then extract twice with 25 ml of ethyl acetate each time, collecting the ethyl acetate layer. Evaporate to dryness in a water bath. Dissolve the residue in 2 ml of methanol to prepare the test solution. Separately, take 0.5 g of Artemisia capillaris reference material, add 25 ml of water, heat under reflux for 3 h, cool, centrifuge, and place the supernatant in a separatory funnel. Prepare the reference material solution using the same method as the drug composition. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 2 μl of each of the above two solutions to the same polyamide film plate. Develop using toluene-ethyl acetate-formic acid-glacial acetic acid-water (1:20:4:1:1) as the developing solvent. Remove and air dry, then examine at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0260] Gentian thin-layer chromatography identification: Accurately measure 10 ml of the drug composition, extract twice with 25 ml of water-saturated n-butanol each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in 10 ml of methanol, add to a neutral alumina column (100-200 mesh, 4 g, inner diameter 1.0 cm), elute with 70 ml of methanol until colorless, evaporate to dryness, dissolve the residue in 2 ml of methanol to obtain the test solution. Separately, prepare a reference solution by dissolving gentiopicrin reference standard in methanol to a concentration of 2 mg / ml. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502), applying 2 μl of each of the above two solutions separately to the same silica gel GF column. 254 On a thin-layer plate, the lower layer of a dichloromethane-methanol-water (25:10:3) solution was used as the developing solvent. The plate was then developed, removed, air-dried, and examined under ultraviolet light (254 nm). The test sample chromatogram showed spots of the same color at the corresponding positions as the reference sample chromatogram.

[0261] Content determination Gentianoside (by high performance liquid chromatography: Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel as the packing material; acetonitrile-0.2% phosphoric acid solution (10:90) as the mobile phase; detection wavelength of 270 nm; theoretical plate number calculated based on gentiopicrin, should not be less than 3000.

[0262] Preparation of reference solution: Weigh approximately 3.69 mg of gentiopicroside reference standard accurately, add 50 ml of methanol solution to prepare a solution with a concentration of 0.0715 mg / ml.

[0263] Preparation of the test solution: Take 5-10 ml of the drug composition, accurately measure it into a 50 ml volumetric flask, accurately add an appropriate amount of methanol, shake well and dilute to the mark, filter, and the solution is obtained.

[0264] Determination method: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0265] The pharmaceutical composition contains gentiopicrin (C 16 H 20 O9) shall not be less than 0.40 mg / ml.

[0266] Five batches of the pharmaceutical composition of Example 4 were randomly selected according to the above-described content determination conditions, and the results are shown in Table 27.

[0267] Table 27 Results of testing on 5 random batches of pharmaceutical compositions of the present invention

[0268] Based on the experimental results of the above 5 batches of drug composition samples, the drug compositions of the present invention contain gentiopicrin at a concentration of not less than 0.40 mg / ml, which meets the quality requirements. Furthermore, the quality testing method of the present invention is simple, accurate, and reliable.

[0269] The complexity of the pharmaceutical components in the pharmaceutical composition of this invention means that the effective components and their contents cannot be completely determined. However, within the scope of the disclosure in this embodiment, it is sufficient to determine the components and the contents of the main substances in the pharmaceutical composition of this invention.

[0270] This invention is not limited to the above embodiments. Based on the technical solutions disclosed in this invention, those skilled in the art can make some substitutions and modifications to some of the technical features without creative effort, and all such substitutions and modifications are within the protection scope of this invention.

Claims

1. A method for quality control of pharmaceutical compositions containing fresh Dendrobium officinale extract, characterized in that, Includes one or more of the following thin-layer chromatography identification and / or content determination methods: (1) Thin-layer identification: Accurately measure 5ml-20ml of the drug composition, place it in a separatory funnel, extract with ethyl acetate 1-3 times, 5ml-20ml each time, collect the ethyl acetate layer, evaporate to dryness in a water bath, add 2-10 parts by volume of methanol to dissolve the residue, and obtain the test solution. Take 1-3g of the blue cloth reference material, add 5ml-20ml of water, heat under reflux for 1-2 hours, filter, take 5-20ml of the filtrate and place it in a separatory funnel, and prepare the reference material solution in the same way as the drug composition. Take an appropriate amount of gallic acid reference standard, add methanol to prepare a 1 mg / ml reference standard solution, shake well to obtain the reference standard solution; According to the thin-layer chromatography method (general rules), take 3-5 μl of each of the above three solutions and spot them separately on the same silica gel G thin-layer plate. Use xylene-ethyl acetate-methanol-formic acid in a volume ratio of (4-6):(2-4):(1-2):(1-2) as the developing solvent, develop, remove and air dry, and examine at 365 nm; in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material; Spray with 5% ferric chloride-ethanol solution for color development, and examine under sunlight to detect gallic acid; (2) Thin-layer identification: Accurately measure 5ml-20ml of the drug composition, place it in a separatory funnel, extract with ether 1-3 times, 5ml-20ml each time, discard the ether solution, extract with ethyl acetate 1-3 times, 5ml-25ml each time, collect the ethyl acetate layer, evaporate to dryness in a water bath, add 1ml-5ml of methanol to dissolve the residue, and prepare the test solution. Take 0.5g-1g of Artemisia capillaris reference material, add 5ml-25ml of water, heat under reflux for 1h-3h, cool, centrifuge, take 5ml-20ml of supernatant and place it in a separatory funnel, and prepare the reference material solution in the same way as the drug composition. According to the thin-layer chromatography method (general rules), take 1-3 μl of each of the above two solutions and spot them separately on the same polyamide film plate. Use toluene-ethyl acetate-formic acid-glacial acetic acid-water in a volume ratio of (1-3):(10-20):(2-4):(1-2):(1-2) as the developing solvent, develop, remove and air dry, and examine at 365 nm; in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material; (3) Thin-layer identification: Accurately measure 5ml-20ml of the drug composition, shake and extract 1-3 times with water-saturated n-butanol, 5ml-30ml each time, combine the n-butanol solutions, evaporate to dryness, dissolve the residue in 5ml-20ml of methanol, add it to a neutral alumina column, elute with 50ml-100ml of methanol until colorless, evaporate to dryness, dissolve the residue in 1ml-5ml of methanol to obtain the test solution; Separately, prepare a reference solution by adding methanol to a solution containing 2 mg per ml. Perform the thin-layer chromatography (general procedure) test, taking 1-5 μl of each of the two solutions mentioned above, and spotting them separately on the same silica gel GF plate. 254 On a thin-layer plate, the lower layer of a dichloromethane-methanol-water solution with a volume ratio of (10-30):(5-15):(1-5) was used as the developing solvent. After development, the plate was removed, dried, and examined under a UV lamp at 254 nm. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample. (4) Content determination: Gentianin: Analyzed by high performance liquid chromatography; Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel as the packing material; acetonitrile-0.2% phosphoric acid solution (10-30:70-90) as the mobile phase; detection wavelength of 270 nm; theoretical plate number calculated based on gentiopicroside, should not be less than 3000; Preparation of reference solution: Take an appropriate amount of gentiopicroside reference standard, accurately weigh it, dissolve it in methanol and dilute it to prepare a solution containing about 0.07 mg per ml. Preparation of the test solution: Take 5-10 ml of the drug composition, accurately measure it into a 50 ml volumetric flask, accurately add an appropriate amount of methanol, shake well and dilute to the mark, filter, and the solution is obtained. Determination method: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result; The pharmaceutical composition contains gentiopicroside C 16 H 20 O9 must be no less than 0.40 mg / ml.

2. The quality control method according to claim 1, characterized in that, The thin-layer identification of (1) is as follows: 10 ml of the drug composition is accurately measured and placed in a separatory funnel. It is extracted twice with ethyl acetate, 20 ml each time. The ethyl acetate layer is collected, evaporated in a water bath, and the residue is dissolved in 2 volumes of methanol to obtain the test solution. Take 1g of blue cloth as a reference herb, add 20ml of water, heat under reflux for 2 hours, filter, take 10ml of the filtrate and place it in a separatory funnel, and prepare the reference herb solution in the same way as the drug composition. Take an appropriate amount of gallic acid reference standard and add methanol to prepare a 1 mg / ml reference standard solution; Perform the thin-layer chromatography (general procedure) test, taking 3-5 μl of each of the above three solutions and spotting them separately on the same silica gel G thin-layer plate. Use xylene-ethyl acetate-methanol-formic acid in a volume ratio of 5:2:1:1 as the developing solvent, develop, remove and air dry, and examine at 365 nm; in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material; Spray with a 5% ferric chloride-ethanol solution for color development, and examine under sunlight to detect gallic acid.

3. The quality control method according to claim 1, characterized in that, The thin-layer identification of (2) is as follows: 10 ml of the drug composition is accurately measured and placed in a separatory funnel. It is extracted twice with 20 ml of ether each time. The ether solution is discarded. It is then extracted twice with 25 ml of ethyl acetate each time. The ethyl acetate layer is collected, evaporated in a water bath, and the residue is dissolved in 2 ml of methanol to obtain the test solution. Take another 0.5g of Artemisia capillaris reference material, add 25ml of water, heat under reflux for 3h, cool, centrifuge, take 10ml of supernatant and place it in a separatory funnel, and prepare the reference material solution in the same way as the drug composition. According to the thin-layer chromatography (general rules) test, take 2 μl of each of the above two solutions and spot them separately on the same polyamide film plate. Use toluene-ethyl acetate-formic acid-glacial acetic acid-water with a volume ratio of 1:20:4:1:1 as the developing solvent, develop, remove and dry, and examine at 365 nm; in the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

4. The quality control method according to claim 1, characterized in that, The thin-layer identification of (3) is as follows: 10 ml of the drug composition is accurately measured and extracted twice with water-saturated n-butanol, 25 ml each time. The n-butanol solutions are combined, evaporated to dryness, and the residue is dissolved in 10 ml of methanol. The residue is added to a neutral alumina column and eluted with 70 ml of methanol until colorless. The residue is evaporated to dryness, and the residue is dissolved in 2 ml of methanol to obtain the test solution. Separately, prepare a reference solution by adding methanol to a solution containing 2 mg per ml. Perform the thin-layer chromatography (general procedure) test, taking 2 μl of each of the two solutions mentioned above and spotting them separately on the same silica gel GF plate. 254 On a thin-layer plate, the lower layer solution of dichloromethane-methanol-water with a volume ratio of 25:10:3 was used as the developing solvent. After development, the plate was removed, dried, and examined under a UV lamp at 254 nm. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

5. The quality control method according to claim 4, characterized in that, The specifications of the neutral alumina column are: 100~200 mesh, 4g, inner diameter 1.0cm.

6. The quality control method according to claim 1, characterized in that, The content determination of (4) is as follows: gentiopicrin: according to high performance liquid chromatography; Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel as the packing material; acetonitrile-0.2% phosphoric acid solution (10:90) as the mobile phase; detection wavelength of 270 nm; theoretical plate number calculated based on gentiopicroside, should not be less than 3000; Preparation of reference solution: Take an appropriate amount of gentiopicroside reference standard, accurately weigh it, dissolve it in methanol and dilute it to prepare a solution containing about 0.07 mg per ml. Preparation of the test solution: Take 5 ml of the drug composition, accurately measure it into a 50 ml volumetric flask, accurately add an appropriate amount of methanol, shake well and dilute to the mark, filter, and the solution is obtained. Determination method: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result; The pharmaceutical composition contains gentiopicroside C 16 H 20 O9 must be no less than 0.40 mg / ml.

7. The quality control method according to any one of claims 1-6, characterized in that, The active pharmaceutical ingredient composition of the pharmaceutical composition is: 25-150 parts of fresh Dendrobium officinale extract and 650-2000 parts of herbal extract. The raw material composition of the herbal extract is as follows: 50-150 parts of stir-fried Citrus aurantium, 3-15 parts of Cinnamomum cassia root, 50-150 parts of Polygonatum sibiricum, 50-150 parts of Rehmannia glutinosa, 15-50 parts of Gentiana scabra, 50-150 parts of Scutellaria baicalensis, 50-100 parts of Indigofera tinctoria, 50-150 parts of Ophiopogon japonicus, 50-150 parts of Asparagus cochinchinensis, 50-150 parts of Eriobotrya japonica leaf, and 50-200 parts of Artemisia capillaris. The fresh Dendrobium extract was prepared by the following method: Take fresh Dendrobium, add 6-10 times the weight of the medicinal material in water, heat, decoct 1-3 times, decoct for 1-3 hours, filter, combine the filtrates, let stand for 8-24 hours, take the supernatant and concentrate to a clear extract with a relative density of not less than 1.03 at 70℃, cool, take the clear extract and centrifuge with a high-speed sedimentation centrifuge, rapidly freeze the centrifuged liquid and place it in a cold storage to obtain fresh Dendrobium extract.

8. The quality control method according to claim 7, characterized in that, The stir-fried bitter orange peel is prepared by the following method: First, clean the bitter orange peel and cut it into thin slices of 2-3 mm. Set aside. Heat the stir-frying container over medium heat until wheat bran is evenly sprinkled in (the amount of bran should be 10%-15% of the weight of the medicinal material). As soon as smoke appears, add the evenly sliced ​​bitter orange peel and stir-fry quickly and evenly until the surface of the bitter orange peel turns light yellow or yellow and the wheat bran turns black. Remove it immediately, sift out the wheat bran, and let it cool. This gives you stir-fried bitter orange peel with wheat bran.

9. The quality control method according to claim 7, characterized in that, The herbal extract was prepared by the following method: Take stir-fried Citrus aurantium, cinnabar root, Polygonatum sibiricum, Rehmannia glutinosa, Gentiana scabra, Scutellaria baicalensis, Ophiopogon japonicus, Asparagus cochinchinensis, Eriobotrya japonica leaf, and Artemisia capillaris. Add 6-10 times the weight of water, heat, and decoct 1-3 times for 1-3 hours each time. Filter, combine the filtrates, let stand for 8-24 hours, and take the supernatant to obtain the herbal extract.

10. The quality control method according to claim 7, characterized in that, The pharmaceutical composition is prepared by the following method: Fresh Dendrobium extract and herbal extract were combined and concentrated to a clear paste with a relative density of not less than 1.05 at 80℃. The clear paste was centrifuged using a high-speed sedimentation centrifuge. The centrifuged liquid was heated, conventional excipients were added, and the mixture was sterilized for 2-4 hours. After cooling, conventional excipients were added again, the mixture was mixed, and the mixture was filled into containers to obtain the pharmaceutical composition.

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