Xiangshenrong composition as well as preparation and quality detection method of Xiangshenrong composition

Abstract

The invention discloses a nutshell stomach-nourishing composition as well as a preparation method and a quality detection method of the nutshell stomach-nourishing composition. The preparation method comprises the following steps: taking the medicinal materials according to the formula ratio, adding 7.5 times of water for soaking for 1 hour for the first time, adding 6 times of water for the second time, decocting for two times (30 minutes each time), filtering, drying, crushing, sieving, and packaging. And adding a proper amount of auxiliary materials to prepare a corresponding dosage form. The Xiangbao Weirong composition can obviously improve clinical symptoms such as gastral cavity fullness and pain, belching and sour regurgitation, nausea and anorexia, abnormal stool and poor sleep of chronic gastritis patients, and no obvious toxic or side effect is found in the treatment period.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a Xiangbei Weirong composition and a preparation method and quality detection method thereof. Background Art

[0002] Chronic gastritis, gastric and duodenal ulcer, functional dyspepsia, chronic atrophic gastritis, and gastric polyps belong to the category of "stomach pain" and "fullness" in traditional Chinese medicine, and the main manifestations are pain in the epigastric region near the heart, fullness in the epigastric region, and acid reflux and heartburn. The name of "epigastric pain" was first seen in the Yellow Emperor's Classic of Internal Medicine, which states that "stomach disease causes abdominal distension and pain in the epigastric region". Zhang Zhongjing pointed out in Treatise on Febrile Diseases that "fullness without pain is fullness". This disease is mostly caused by spleen and stomach weakness, dysfunction of transportation and transformation; or external cold, heat, and dampness, which invade the stomach; or irregular diet, irregular hunger and satiety, and qi stagnation; as well as emotional disorders, worry, anger, and other reasons that cause stomach qi stagnation, failure to harmonize and descend, and pain if it is not unblocked, or spleen and stomach failure to nourish, which causes pain if it is not nourished. Ye Tianshi's book "Clinical Guide Medical Cases: Epigastric Pain" mentioned that "long-term and recurrent stomach pain must be caused by phlegm and blood stasis". Stomach pain lasts for a long time, and the qi in the middle jiao is not smooth, which is easy to produce tangible evils such as phlegm and turbidity, and it is difficult to cure, forming a mixed syndrome of deficiency and excess. Therefore, stomach pain is always based on stagnation of stomach qi and failure to harmonize and descend. The basic pathogenesis is pain when it is not smooth and pain when it is not nourished. The pathological factors are mainly qi stagnation, and there are also food accumulation, cold stagnation, heat stagnation, dampness obstruction, phlegm stagnation, blood stasis, etc. The lesion site of stomach pain is in the stomach, which is closely related to the liver and spleen.

[0003] At present, Western medicine mainly uses anti-Helicobacter pylori infection, acid suppression, and gastric mucosal protection to treat chronic gastritis, gastric and duodenal ulcers, and functional dyspepsia. However, in recent years, the drug resistance of anti-Helicobacter pylori infection often leads to the failure of Helicobacter pylori eradication. Emotional disorders, worry and anger, work pressure, and improper diet often lead to repeated attacks of epigastric fullness and pain. The glandular atrophy and intestinal metaplasia of atrophic gastritis are difficult to reverse, and the recurrence of gastric polyps is also a common clinical problem. Epigastric pain and fullness are the dominant diseases of traditional Chinese medicine. Professor Xiao Zhenghua, a young Qihuang scholar, has a definite clinical effect of Xiangbei Weirong Decoction. Professor Xiao Zhenghua believes that spleen and stomach weakness and liver and stomach disharmony are important pathological bases for stomach pain. The middle-jiao qi movement is unfavorable, and the spleen and stomach are incompetent, which can easily lead to qi stagnation and phlegm obstruction, and it is difficult to heal. Therefore, it is advocated that the treatment should be based on soothing the liver and strengthening the spleen, harmonizing the stomach and relieving pain, and resolving phlegm and dispersing knots. The self-made "Xiangbei Weirong Soup" consists of 16 Chinese medicinal herbs including Codonopsis pilosula, stir-fried Atractylodes macrocephala with bran, Poria cocos, tangerine peel, costus root, Fritillaria thunbergii, Pinellia ternata, Curcuma aromatica, stir-fried chicken gizzard lining, lily, processed Polygala tenuifolia, vinegar-soaked Corydalis yanhusuo, Artemisia capillaris, vinegar-soaked turtle shell, cuttlebone, and licorice tablets, with significant clinical efficacy.

[0004] In the formula, Codonopsis pilosula is the main drug for strengthening the spleen and replenishing qi, tonifying the middle and replenishing the stomach, Atractylodes macrocephala is the main drug for replenishing qi and strengthening the spleen, Poria cocos is the main drug for replenishing qi and strengthening the spleen, and they are complementary to Atractylodes macrocephala. The main drugs for replenishing qi and strengthening the spleen are more prominent, and they are all ministerial drugs. Tangerine peel regulates qi and harmonizes the stomach; Aucklandia lappa soothes the liver and promotes qi, harmonizes the stomach and relieves pain; Pinellia ternata dries dampness and resolves phlegm, harmonizes the stomach and reduces adverse reactions; Fritillaria thunbergii clears heat and resolves phlegm, relieves depression and resolves knots; Curcuma zedoaria promotes blood circulation and promotes qi, relieves depression and resolves knots; Stir-fried chicken gizzard lining strengthens the stomach and promotes digestion; Lilium and Polygala tenuifolia are the main drugs for strengthening the spleen and harmonizing the stomach, calming the mind and tranquilizing the spirit; Vinegar Corydalis promotes blood circulation and promotes qi and relieves pain, and Artemisia capillaris clears dampness and heat; Vinegar turtle shell and cuttlebone can soften and disperse knots, Vinegar turtle shell nourishes yin and clears heat, and cuttlebone reduces acid and relieves pain, and they are all adjuvant drugs. Licorice harmonizes all the drugs and is the guiding drug. The whole formula is both attacking and replenishing, and the liver and spleen are coordinated, and together they play the effects of soothing the liver and strengthening the spleen, resolving phlegm and resolving knots, and reducing acid and relieving pain.

[0005] Modern pharmacological studies have shown that costus root contains a large number of sesquiterpenoid components, among which cynaropicrin and costus lactone can effectively inhibit the production of inflammatory factors. At the same time, costus root extract can effectively inhibit gastric mucosal damage without affecting the secretion of gastric acid. Fritillaria thunbergii contains rich alkaloids and sugar components, which can scavenge tissue oxygen free radicals and inhibit gastrointestinal inflammatory reactions. Codonopsis pilosula in Codonopsis pilosula can protect the gastric mucosa by increasing the level of prostaglandins, reducing gastric acid secretion, and stimulating the gastric mucosa to secrete epidermal growth factor. Hesperidin, nobiletin and tangeretin, as the flavonoid compounds with the highest content in tangerine peel, can promote the elimination of gastric juice, increase the activity of pepsin, and enhance the digestive function of the stomach. Pinellia is rich in a large number of alkaloids and organic acids, which can improve nausea, vomiting, and upper abdominal distension symptoms, and has a protective effect on the gastric mucosa. The effective ingredients of Corydalis yanhusuo have an improvement effect on chronic atrophic gastritis and gastric ulcers, among which the total alkaloids of Corydalis yanhusuo can inhibit gastric acid secretion and pepsin activity and protect the gastric mucosa.

[0006] To this end, the present invention provides a Xiangbei Weirong recipe, which can improve the symptoms of abdominal distension, epigastric pain, noisy acid reflux and the like caused by spleen and stomach weakness, liver and stomach disharmony, and phlegm and dampness accumulation. Summary of the invention

[0007] The present invention aims to provide a fragrant and delicious stomach composition.

[0008] Another object of the present invention is to provide a method for preparing the Xiangbei Weirong composition.

[0009] Another object of the present invention is to provide a quality detection method for the Xiangbei Weirong composition.

[0010] The formula of the Xiangbeiweirong composition of the present invention is: 130-170 parts of Codonopsis pilosula, 100-140 parts of Atractylodes macrocephala fried with bran, 80-120 parts of Poria cocos, 80-120 parts of dried tangerine peel, 50-70 parts of costusroot, 80-120 parts of Fritillaria thunbergii, 105-125 parts of Pinellia ternata, 80-120 parts of Curcuma aromatica, 80-120 parts of stir-fried chicken gizzard lining, 100-140 parts of lily, 80-120 parts of processed Polygala tenuifolia, 80-120 parts of vinegar Corydalis yanhusuo, 130-170 parts of Artemisia capillaris, 180-220 parts of vinegar turtle shell, 80-120 parts of cuttlebone and 50-70 parts of liquorice tablets.

[0011] Preferably, the formula of the Xiangbei Weirong composition of the present invention is: 140-160 parts of Codonopsis pilosula, 110-130 parts of Atractylodes macrocephala fried with bran, 90-110 parts of Poria cocos, 90-110 parts of dried tangerine peel, 55-65 parts of costus root, 90-110 parts of Fritillaria thunbergii, 110-120 parts of Pinellia ternata, 90-110 parts of Curcuma aromatica, 90-110 parts of stir-fried chicken gizzard lining, 110-130 parts of lily, 90-110 parts of processed Polygala tenuifolia, 90-110 parts of vinegar Corydalis yanhusuo, 140-160 parts of Artemisia capillaris, 190-210 parts of vinegar turtle shell, 90-110 parts of cuttlebone, and 55-65 parts of licorice tablets.

[0012] Further preferably, the formula of the Xiangbei Weirong composition of the present invention is: 150 parts of Codonopsis pilosula, 120 parts of Atractylodes macrocephala fried with bran, 100 parts of Poria cocos, 100 parts of dried tangerine peel, 60 parts of costus root, 100 parts of Fritillaria thunbergii, 117 parts of Pinellia ternata, 100 parts of Curcuma aromatica, 100 parts of stir-fried chicken gizzard lining, 120 parts of lily, 100 parts of processed Polygala tenuifolia, 100 parts of vinegar Corydalis yanhusuo, 150 parts of Artemisia capillaris, 200 parts of vinegar turtle shell, 100 parts of cuttlebone, and 60 parts of liquorice tablets.

[0013] The preparation method of the Xiangbei Weirong composition of the present invention is as follows: take a prescribed amount of medicinal materials, add 7-8 times of water for the first time to soak for 1-2 hours, add 6-10 times of water for the second time, decoct for 1-4 times, each time for 20-60 minutes, filter, concentrate the filtrate to a thick paste with a relative density of 1.24-1.35 at 70°C, dry, crush, add appropriate amount of auxiliary materials, and prepare corresponding dosage forms.

[0014] Preferably, the preparation method of the Xiangbei Weirong composition of the present invention is: take the formulated amount of medicinal materials, add 7.5 times of water for the first time to soak for 1 hour, add 6 times of water for the second time, decoct twice, each time for 30 minutes, filter, concentrate the filtrate to a thick paste with a relative density of 1.28-1.32 at 70°C, dry, crush, add appropriate amount of auxiliary materials, and make corresponding dosage forms.

[0015] The quality detection method of the Xiangbeiweirong composition of the invention comprises thin layer chromatography identification of dried tangerine peel, costusroot, thunbergia thunbergii and vinegar corydalis and detection of the content of hesperidin.

[0016] The thin layer chromatography identification method of dried tangerine peel of the present invention is: take 5g of this product, add 25mL of methanol, ultrasonically treat for 20 minutes, filter, and concentrate the filtrate to 2mL as the test solution. Separately take 5g of the negative preparation lacking dried tangerine peel, and prepare a negative control solution in the same way; then take 1g of dried tangerine peel control medicinal material, and make a control medicinal material solution in the same way; according to the thin layer chromatography test recorded in the general rules 0502 of the four parts of the 2020 edition of the Chinese Pharmacopoeia, draw 2μL of each of the above three solutions, and point them on the same silica gel G thin layer plate, with ethyl acetate-methanol-water 100:17:13 as the developing agent, develop, take out, dry, spray with aluminum chloride test solution, and put under ultraviolet light 365nm for inspection.

[0017] The thin layer chromatography identification method of costus root described in the present invention is: take 10g of this product, add 50mL of water to dissolve, filter, extract the filtrate with 40mL of ethyl acetate, take the ethyl acetate layer, evaporate to dryness, and add 1mL of ethyl acetate to the residue to dissolve as the test solution. Take 10g of the negative preparation lacking costus root, and prepare the negative control solution in the same way; take 0.5g of costus root control medicinal material, add 50mL of water, decoct for 30min, filter, and prepare the control medicinal material solution with the filtrate in the same way as the test solution preparation method; according to the thin layer chromatography test recorded in the 2020 edition of the Chinese Pharmacopoeia, 5μL of the control medicinal material solution, 5-10μL of the test solution and the negative control solution, respectively, are applied to the same silica gel G thin layer plate, and the upper layer solution of cyclohexane-ethyl formate-formic acid 15:5:1 is used as the developing agent, developed, taken out, dried, sprayed with 5% vanillin sulfuric acid solution, and heated at 105°C until the spots are clearly colored.

[0018] The thin layer identification method of Fritillaria thunbergii described in the present invention is: take 10g of the product, add 2mL of concentrated ammonia test solution and 50mL of chloroform, heat and reflux for 1h, filter, recover the solvent in the filtrate to dryness, add 2mL of chloroform to the residue to dissolve it as a test solution; take another 10g of a negative preparation lacking Fritillaria thunbergii, and prepare a negative control solution in the same way; take 1g of Fritillaria thunbergii control medicinal material, and prepare a control medicinal material solution in the same way; according to the thin layer chromatography test recorded in the general rules 0502 of the fourth volume of the 2020 edition of the "Chinese Pharmacopoeia", 10 to 20μL of each of the above three solutions are absorbed and spotted on the same silica gel G thin layer plate, and ethyl acetate-methanol-concentrated ammonia test solution 17:2:1 is used as the developing agent, developed, taken out, dried, and sprayed with dilute potassium bismuth iodide test solution.

[0019] The thin layer chromatography identification of vinegar Corydalis yanhusuo described in the present invention: take 5g of the product, add 50mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, add 10mL of water to the residue to dissolve it, add concentrated ammonia test solution to adjust to alkalinity, shake and extract with ether 3 times, 10mL each time, combine the ether solution, evaporate to dryness, add 1mL of methanol to the residue to dissolve it as the test solution; take another 5g of the negative preparation lacking vinegar Corydalis yanhusuo, and prepare a negative control solution in the same way; take 1g of Corydalis yanhusuo control medicinal material, and prepare a control medicinal material solution in the same way; according to the thin layer chromatography test recorded in the general rules of the fourth part of the 2020 edition of the "Chinese Pharmacopoeia" 0502, absorb 4 to 5μL of each of the above three solutions, respectively spot them on the same silica gel G thin layer plate, use toluene-acetone 9:2 as the developing agent, develop, take out, dry, put it in an iodine cylinder for about 3 minutes and then take it out, after wafting off the iodine adsorbed on the plate, place it under a UV lamp at 365nm for inspection.

[0020] The hesperidin content detection method of the present invention is as follows: chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as a filler, the mobile phase is methanol: 2% acetic acid = 35:65, the flow rate is 1 mL / min, the column temperature is 30°C, the detection wavelength is 283 nm, the injection volume is 10 μL, and the theoretical plate number should be not less than 2000 calculated based on the hesperidin peak; preparation of reference solution: take an appropriate amount of hesperidin reference, accurately weigh it, add methanol to make a solution containing 40 μg per 1 mL, and obtain the solution; preparation of test solution: take 1 g of Xiangbei Weirong composition, accurately weigh it, put it in a stoppered conical bottle, accurately add 25 mL of 75% methanol, seal it, weigh the weight, ultrasonically treat it for 15 minutes, let it cool, weigh the weight again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution; determination method: accurately aspirate 10 μL of reference solution and test solution respectively, inject them into a liquid chromatograph, and determine the solution to obtain the solution.

[0021] The Xiangbei Weirong composition of the present invention or the Xiangbei Weirong composition prepared by the preparation method is used in preparing medicines for treating chronic gastritis.

[0022] The parts described in the formulation of the present invention represent mass weight units such as g and kg.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The prescription of the present invention has significant efficacy. Clinical observation results show that Xiangbei Weirong Prescription can significantly improve the clinical symptoms and signs of patients with chronic gastritis, such as epigastric distension and pain, belching and acid reflux, nausea and loss of appetite, abnormal stool, and poor sleep (P < 0.001), with a total effective rate of 94%.

[0025] 2. The preparation of the present invention is easy to use. The extracted formula is made into granules, which can meet the medication needs of patients and is convenient for patients to take compared with the original method of decoction.

[0026] 3. The extraction method of the preparation of the present invention is scientific and reasonable. Through experimental investigation, it was determined that the best combination of water extraction process is to add 6 times the amount of water, extract twice, each time for 30 minutes. The RSD of the hesperidin content and the paste yield of the water extraction verification sample was less than 3.0%, indicating that the extraction process is stable and feasible.

[0027] 4. The present invention carries out qualitative identification on 8 medicinal materials in the preparation, namely, Codonopsis pilosula, stir-fried Atractylodes macrocephala with bran, Poria cocos, dried orange peel, costus root, thunbergii bulb, vinegar-corydalis and curcuma, wherein the negative control of the qualitative identification of 4 medicinal materials, namely, dried orange peel, costus root, thunbergii bulb and vinegar-corydalis has no interference, and the method has good repeatability after verification by 3 batches of pilot samples, so it is included in the draft text of the quality standard; Codonopsis pilosula, stir-fried Atractylodes macrocephala with bran, Poria cocos and curcuma all adopt two extraction methods and carry out systematic experiments, and are not included in the quality standard text due to negative interference.

[0028] 5. The present invention inspected the particle size, moisture, filling volume difference, and microbial limit of three batches of Xiangbei Weirong granules. The results of various inspections showed that this product complies with the relevant provisions of the General Rules of Part IV of the 2020 Edition of the "Chinese Pharmacopoeia" under 0114 Granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Specific HPLC chromatogram of water extract (where A is the reference solution, B is the extract test solution, and C is the extract negative sample solution);

[0030] Figure 2 Linear regression plot of hesperidin;

[0031] Figure 3 Chromatogram of flow rate investigation results (from top to bottom, flow rates are 0.8mL / min, 1.0mL / min, and 1.2mL / min);

[0032] Figure 4 Chromatogram of column temperature investigation results (from top to bottom, column temperatures are 25°C, 30°C, and 35°C);

[0033] Figure 5 The chromatographic column was Elite Hypersil BDS C18 chromatogram;

[0034] Figure 6 The chromatographic column was Welch Ultimate XB-C18 chromatogram;

[0035] Figure 7 Moisture absorption balance curve of Xiangbei Weirong granules;

[0036] Figure 8 Moisture absorption curve of Xiangbei Weirong granules;

[0037] Fig. 9TLC result of Codonopsis pilosula (the left one is the result obtained by method 1, and the right one is the result obtained by method 2; 1-2 is the control medicinal material of Codonopsis pilosula, 3-4 is the test sample of Xiangbei Weirong Granules, and 5-6 is the negative control solution (lacking Codonopsis pilosula));

[0038] Fig.10 Atractylodes macrocephala TLC result diagram (the left is the result obtained by method 1, and the right is the result obtained by method 2; 1-2 is the control herbal material of Atractylodes macrocephala, 3-4 is the test sample of Xiangbei Weirong Granules, and 5-6 is the negative control solution (fried Atractylodes macrocephala without bran));

[0039] Fig.11 Poria cocos TLC result diagram (the left one is the result obtained by method 1, and the right one is the result obtained by method 2; 1 is the Poria cocos control medicinal material, 2-4 are the Xiangbei Weirong granules test products, and 5 is the negative control solution (lacking Poria cocos));

[0040] Fig.12 Tangerine peel TLC result diagram (left is the result obtained by method 1, the middle is the result obtained by the upper layer solution of toluene-ethyl acetate-formic acid-water as the developing solvent, and the right is the result obtained by ethyl acetate-methanol-water as the developing solvent; 1. Tangerine peel as the control medicinal material; 2-4. Xiangbei Weirong granules as the test sample; 5. Negative control solution (without tangerine peel));

[0041] Fig.13 The results of costus root TLC (the left is the results obtained by method 1, where 1-2 are costus root control medicinal materials, 3-4 are Xiangbei Weirong granules test products, and 5 is the negative control solution (lacking costus root); the right is the results obtained by method 2, where 1 is costus root control medicinal materials, 2-4 are three batches of Xiangbei Weirong granules test products, and 5 is the negative control solution (lacking costus root));

[0042] Fig.14 TLC result of Fritillaria thunbergii (1 is the control medicinal material of Fritillaria thunbergii, 2-4 are the test samples of Xiangbei Weirong Granules, and 5 is the negative control solution (lacking Fritillaria thunbergii));

[0043] Fig.15 Vinegar Corydalis TLC result diagram (1 is the Corydalis control medicinal material, 2-4 are the Xiangbei Weirong granules test samples, and 5 is the negative control solution (lacking vinegar Corydalis));

[0044] Fig.16 Curcuma TLC result diagram (the left one is the result obtained by method 1, and the right one is the result obtained by method 2; 1-2 are the control herbs of Curcuma, 3-4 are the test samples of Xiangbei Weirong Granules, and 5-6 are the negative control solutions (lacking Curcuma));

[0045] Fig.17 Chromatogram of TLC durability study of dried orange peel and costusroot in Xiangbei Weirong granules;

[0046] Fig.18Chromatogram of TLC durability study of Fritillaria thunbergii and vinegar-corydalis in Xiangbei Weirong granules;

[0047] Fig.19 Full wavelength scanning of hesperidin;

[0048] Fig. 20 The mobile phase was acetonitrile-water (22:78) chromatogram;

[0049] Fig.21 The mobile phase was methanol-2% acetic acid (35:65) chromatogram;

[0050] Fig. 22 The mobile phase was methanol-1% acetic acid (35:65) chromatogram;

[0051] Fig.23 The mobile phase was acetonitrile-2% acetic acid (22:78) chromatogram;

[0052] Fig.24 Chromatogram with acetonitrile-1% acetic acid (22:78) as the mobile phase

[0053] Fig.25 The mobile phase was acetonitrile-0.05% aqueous phosphoric acid (22:78) chromatogram;

[0054] Fig.26 The mobile phase was acetonitrile-2% acetic acid in water (22:78) chromatogram;

[0055] Fig. 27 HPLC chromatogram (a is hesperidin, b is the test sample, and c is the negative control sample);

[0056] Fig.28 Linear regression plot of hesperidin;

[0057] Fig.29 Chromatogram of flow rate investigation results (from top to bottom, flow rates are 0.8mL / min, 1.0mL / min, and 1.2mL / min);

[0058] Fig.30 Chromatogram of column temperature investigation results (from top to bottom, column temperatures are 25°C, 30°C, and 35°C);

[0059] Fig.31 The chromatogram of Yuexu with serial number 60230803026 and column number 2101.113;

[0060] Fig.32 The chromatogram of Yuexu with serial number 60210901093 and column number 2101.109;

[0061] Fig.33 The detection instrument is Agilent 1260 chromatogram;

[0062] Fig.34 The detection instrument was an Agilent 1260infinity II chromatograph. DETAILED DESCRIPTION

[0063] The technical solution of the present invention is further described in detail below through specific embodiments.

[0064] Embodiment 1 Xiangbei Weirong composition formula

[0065] 150g of Codonopsis pilosula, 120g of Atractylodes macrocephala fried with bran, 100g of Poria cocos, 100g of dried tangerine peel, 60g of costus root, 100g of Fritillaria thunbergii, 117g of Pinellia ternata, 100g of Curcuma aromatica, 100g of stir-fried chicken gizzard lining, 120g of lily, 100g of processed Polygala tenuifolia, 100g of Corydalis yanhusuo with vinegar, 150g of Artemisia capillaris, 200g of turtle shell with vinegar, 100g of cuttlebone, and 60g of licorice slices.

[0066] Embodiment 2 Xiangbei Weirong composition formula

[0067] 170g of Codonopsis pilosula, 140g of Atractylodes macrocephala fried with bran, 120g of Poria cocos, 120g of dried tangerine peel, 70g of costus root, 120g of Fritillaria thunbergii, 125g of Pinellia ternata, 120g of Curcuma aromatica, 120g of stir-fried chicken gizzard lining, 140g of lily, 120g of processed Polygala tenuifolia, 120g of Corydalis yanhusuo cooked with vinegar, 170g of Artemisia capillaris, 220g of turtle shell cooked with vinegar, 120g of cuttlebone, and 70g of licorice slices.

[0068] Embodiment 3 Xiangbei Weirong composition formula

[0069] 130g of Codonopsis pilosula, 100g of Atractylodes macrocephala fried with bran, 80g of Poria cocos, 80g of dried tangerine peel, 50g of costus root, 80g of Fritillaria thunbergii, 105g of Pinellia ternata, 80g of Curcuma aromatica, 80g of stir-fried chicken gizzard lining, 100g of lily, 80g of processed Polygala tenuifolia, 80g of Corydalis yanhusuo cooked with vinegar, 130g of Artemisia capillaris, 180g of turtle shell cooked with vinegar, 80g of cuttlebone, and 50g of licorice slices.

[0070] Embodiment 4 Xiangbei Weirong composition formula

[0071] 160g of Codonopsis pilosula, 130g of Atractylodes macrocephala fried with bran, 110g of Poria cocos, 110g of dried tangerine peel, 650g of costusroot, 110g of Fritillaria thunbergii, 120g of Pinellia ternata, 110g of Curcuma aromatica, 110g of stir-fried chicken gizzard lining, 130g of lily, 110g of processed Polygala tenuifolia, 110g of Corydalis yanhusuo cooked with vinegar, 160g of Artemisia capillaris, 210g of turtle shell cooked with vinegar, 110g of cuttlebone, and 65g of licorice slices.

[0072] Example 5: Formula of Xiangbei Weirong Composition

[0073] 140g of Codonopsis pilosula, 110g of Atractylodes macrocephala fried with bran, 90g of Poria cocos, 90g of dried tangerine peel, 55g of costus root, 90g of Fritillaria thunbergii, 110g of Pinellia ternata, 90g of Curcuma aromatica, 90g of stir-fried chicken gizzard lining, 110g of lily, 90g of processed Polygala tenuifolia, 90g of Corydalis yanhusuo fried with vinegar, 140g of Artemisia capillaris, 190g of turtle shell fried with vinegar, 90g of cuttlebone, and 55g of licorice slices.

[0074] The formula of the Xiangbei Weirong composition of Examples 1-5 is used in the following preparation methods

[0075] Example 6 Preparation method of Xiangbeiweirong composition

[0076] Take the prescribed amount of medicinal materials, add 7.5 times of water for the first time, soak for 1 hour, add 6 times of water for the second time, decoct twice, each time for 30 minutes, filter, concentrate the filtrate to a thick paste with a relative density of 1.30 at 70°C, add 0.8 times of microcrystalline cellulose to mix, granulate through a 14-mesh sieve, dry at 70°C, and sieve through a 10-mesh sieve to obtain the product.

[0077] Example 7 Preparation method of Xiangbeiweirong composition

[0078] Take the prescribed amount of medicinal materials, add 7.5 times of water for the first time, soak for 1 hour, add 6 times of water for the second time, decoct twice, each time for 30 minutes, filter, concentrate the filtrate to a thick paste with a relative density of 1.28 at 80°C, add 0.9 times of microcrystalline cellulose to mix, granulate through a 14-mesh sieve, dry at 80°C, and sieve through a 10-mesh sieve to obtain the product.

[0079] Example 8 Preparation method of Xiangbeiweirong composition

[0080] Take the prescribed amount of medicinal materials, add 8 times of water for the first time, soak for 2 hours, add 10 times of water for the second time, decoct for 4 times, each time for 60 minutes, filter, concentrate the filtrate to a thick paste with a relative density of 1.35 at 70°C, add 1 times of microcrystalline cellulose as the extract, mix well, granulate through an 18-mesh sieve, dry at 80°C, and sieve through a 10-mesh sieve to obtain the product.

[0081] Example 9 Preparation method of Xiangbeiweirong composition

[0082] Take the prescribed amount of medicinal materials, add 7 times the water for the first time, soak for 1 hour, add 6 times the water for the second time, decoct once, each time for 20 minutes, filter, concentrate the filtrate to a thick paste with a relative density of 1.24 at 70°C, add 0.7 times the amount of microcrystalline cellulose to mix, granulate through a 14-mesh sieve, dry at 60°C, and sieve through a 10-mesh sieve to obtain the product.

[0083] The Xiangbeiweirong composition prepared in Examples 6-9 was used in the following quality testing methods

[0084] Example 10 Quality testing method of Xiangbeiweirong composition

[0085] (1) TLC identification of dried orange peel, costusroot, Fritillaria thunbergii, and vinegar-corydalis yanhusuo

[0086] Thin layer chromatography identification of tangerine peel: Take 5g of this product, add 25mL of methanol, ultrasonically treat for 20 minutes, filter, and concentrate the filtrate to 2mL as the test solution. Take another 5g of the negative preparation lacking tangerine peel, and prepare the negative control solution in the same way. Take 1g of tangerine peel control medicinal material and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the Fourth Part of the 2020 Edition of the Chinese Pharmacopoeia), 2μL of each of the above three solutions was taken and spotted on the same silica gel G thin layer plate, and ethyl acetate-methanol-water (100:17:13) was used as the developing agent. Develop, take out, dry, spray with aluminum chloride test solution, and examine under ultraviolet light (365nm). Results In the chromatogram of the test product, fluorescent spots of the same color appeared at the corresponding position of the chromatogram of the control medicinal material, and there was no interference with the negative control.

[0087] Thin layer chromatography identification of costus root: take 10g of this product, add 50mL of water to dissolve, filter, extract the filtrate with 40mL of ethyl acetate, take the ethyl acetate layer, evaporate to dryness, add 1mL of ethyl acetate to the residue to dissolve, as the test solution. Take another 10g of the negative preparation lacking costus root, and prepare the negative control solution in the same way. Take 0.5g of costus root control medicinal material, add 50mL of water, decoct for 30min, filter, and prepare the control medicinal material solution with the same method as the test solution preparation method. According to the thin layer chromatography method (General Rules 0502 of the Fourth Part of the 2020 Edition of the Chinese Pharmacopoeia), take 5μL of the control medicinal material solution, 5-10μL of the test solution and the negative control solution, respectively, and spot them on the same silica gel G thin layer plate, use the upper layer solution of cyclohexane-ethyl formate-formic acid (15:5:1) as the developing agent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, and heat at 105°C until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material, and there is no interference with the negative control.

[0088] Thin layer identification of Fritillaria thunbergii: Take 10g of this product, add 2mL of concentrated ammonia test solution and 50mL of chloroform, heat and reflux for 1h, filter, recover the solvent in the filtrate until dry, and add 2mL of chloroform to dissolve the residue as the test solution. Take another 10g of negative preparation without Fritillaria thunbergii, and prepare a negative control solution in the same way. Take 1g of Fritillaria thunbergii control medicinal material, and prepare a control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the Fourth Part of the 2020 Edition of the Chinese Pharmacopoeia), take 10-20μL of each of the above three solutions and spot them on the same silica gel G thin layer plate, use ethyl acetate-methanol-concentrated ammonia test solution (17:2:1) as the developing agent, develop, take out, dry, and spray with dilute potassium bismuth iodide test solution. In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material.

[0089] Thin layer chromatography identification of vinegar Corydalis: Take 5g of this product, add 50mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, add 10mL of water to the residue to dissolve, add concentrated ammonia test solution to adjust to alkalinity, shake and extract with ether 3 times, 10mL each time, combine the ether solution, evaporate to dryness, and add 1mL of methanol to the residue to dissolve as the test solution. Take another 5g of the negative preparation lacking vinegar Corydalis, and prepare the negative control solution in the same way. Take 1g of Corydalis control medicinal material, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the Fourth Part of the 2020 Edition of the Chinese Pharmacopoeia), take 4-5μL of each of the above three solutions, spot them on the same silica gel G thin layer plate, use toluene-acetone (9:2) as the developing agent, develop, take out, dry, put in the iodine cylinder for about 3 minutes, take out, and after wafting out the iodine adsorbed on the plate, place it under ultraviolet light (365nm) for inspection. After verification by three batches of pilot samples, this method has good repeatability, so it is included in the text of the draft quality standard.

[0090] (2) Detection of hesperidin content

[0091] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler; methanol-2% acetic acid (35:65); flow rate of 1 mL / min; column temperature of 30°C; detection wavelength of 283 nm; injection volume of 10 μL. The theoretical plate number calculated based on the hesperidin peak should not be less than 2000.

[0092] Preparation of reference solution: Take an appropriate amount of hesperidin reference substance, weigh it accurately, and add methanol to make a solution containing 40 μg per 1 mL.

[0093] Preparation of test solution: Take 1 g of Xiangbei Weirong composition, weigh accurately, put it in a stoppered conical flask, add 25 mL of 75% methanol accurately, stopper it, weigh it, treat it with ultrasound for 15 minutes, cool it, weigh it again, make up the lost weight with 75% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0094] Sample determination: accurately weigh 1 g of sample, prepare it according to the preparation method of the test solution, inject 10 μL of each sample, record the chromatogram, determine the peak area, and calculate the hesperidin content (μg / g) in each sample.

[0095] Calculated based on the actual 10g per bag, each bag of this product contains tangerine peel as hesperidin (C 23 H 28 O 11 ) shall not be less than 4.0 mg. In order to verify the effectiveness of the present invention, the invention team conducted a series of experiments, as follows:

[0096] 1. Preparation process research

[0097] (I) Water extraction process research

[0098] 1. Instruments and reagents

[0099] 1.1 Instrument

[0100] Electronic balance (YHM-20002, Huizhou Yingheng Electronic Technology Co., Ltd.), decoction pot, high performance liquid chromatograph (Agilent1260, Agilent Technologies, Inc., USA), electronic balance (AUW220D, Shimadzu Philippines Factory), centrifuge (H2-16K, Hunan Kecheng Instrument Equipment Co., Ltd.).

[0101] 1.2 Drug testing

[0102] Costusroot, Fritillaria thunbergii, turtle shell with vinegar, processed Polygala tenuifolia, dried orange peel, licorice slices, stir-fried chicken gizzard lining, cuttlebone, Artemisia capillaris, vinegar-corydalis, Codonopsis pilosula, Poria, lily, Pinellia ternata, stir-fried Atractylodes macrocephala with bran, and Curcuma aromatica were purchased from Guizhou Tongjitang Chinese Medicine Co., Ltd. (batch numbers are 23033002, 230630, 230805, 2207468171, 230727, 230317, 231010, 230428, 230603, 230708, 230919, 230909, 230417, 230 822, 230917, 230508) all comply with the standards of Part I of the Chinese Pharmacopoeia; hesperidin reference substance (batch number: 110721-202220, content 97.2%) was purchased from China Food and Drug Inspection Institute; methanol (chromatographic grade, TEDIA, USA); water (purified water, manufactured by Guiyang Wahaha Food Co., Ltd.); acetic acid (HPLC grade, Tianjin Komio Chemical Reagent Co., Ltd.); Xiangbei Weirong water extract and Xiangbei Weirong water extract without tangerine peel were homemade by the laboratory of the Second Affiliated Hospital of Guizhou University of Chinese Medicine.

[0103] 2 Methods and Results

[0104] 2.1 Screening of inspection indicators

[0105] The main drug in the Xiangbei Weirong prescription is Codonopsis pilosula, and the auxiliary drugs are Atractylodes macrocephala and Poria cocos. There is no content determination item recorded in the "Chinese Pharmacopoeia", so hesperidin in the adjuvant drug Tangerine peel is selected as the detection indicator for content determination.

[0106] 2.2 Determination of hesperidin content in water extract of Xiangbeiweirong

[0107] 2.2.1 Chromatographic conditions and system suitability test

[0108] The chromatographic column was Welch Ultimate XB-C18 (250 mm × 4.6 mm, 5 μm); the mobile phase was methanol-2% acetic acid (35:65); the column temperature was 30°C; the detection wavelength was 283 nm; the injection volume was 10 μL. The theoretical plate number calculated based on the hesperidin peak should not be less than 2000.

[0109] 2.2.2 Solution preparation

[0110] 2.2.2.1 Preparation of reference solution

[0111] Accurately weigh an appropriate amount of hesperidin reference substance and add methanol to prepare a reference substance solution of about 40 μg / mL.

[0112] 2.2.2.2 Preparation of test solution

[0113] Accurately measure 5 mL of the aqueous extract, place it in a 25 mL volumetric flask, add methanol to the mark, shake well, filter, and filter the filtrate through a 0.22 μm microporous filter membrane to obtain the product.

[0114] 2.2.2.3 Preparation of negative sample solution

[0115] Accurately measure 1 mL of the water extract of the fragrant beetle without tangerine peel, and prepare it according to the method under "2.2.2.2".

[0116] 2.2.3 Exclusivity

[0117] Accurately measure the above-mentioned reference solution, test solution, and negative control solution respectively, and measure them according to the chromatographic conditions. The results are shown in Figure 1 .

[0118] 2.2.4 Linearity and range

[0119] 1 mL, 3 mL, 5 mL, 10 mL, and 20 mL of the hesperidin reference substance stock solution (concentration of 194.40 μg / mL) were accurately pipetted into a 25 mL volumetric flask, and methanol was added to the volume to obtain a series of reference substance solutions with concentrations of 7.78 μg / mL, 23.33 μg / mL, 38.88 μg / mL, 77.76 μg / mL, and 155.52 μg / mL, respectively. The above-mentioned reference substance solutions and 194.40 μg / mL of the reference substance stock solution were accurately pipetted respectively, injected into a high performance liquid chromatograph, and measured to obtain the peak area. The hesperidin injection amount (X) was used as the abscissa and the peak area (Y) was used as the ordinate. The linear regression equation was y=1602.3x+1.7522(R 2 =1), hesperidin has a good linear range between 0.078μg and 1.944μg. The linear regression graph is shown in Figure 2 , the results are shown in Table 1.

[0120] Table 1 Hesperidin linear data

[0121]

[0122] 2.2.5 Precision test

[0123] Accurately pipette 10 μL of hesperidin reference solution (38.88 μg / mL), repeat the injection 6 times, measure the peak area, and calculate the average peak area value and relative standard deviation (RSD), see Table 2.

[0124] Table 2 Hesperidin precision

[0125]

[0126] Results: The average peak area of ​​hesperidin was 627.26, and the RSD value was 0.09%, indicating that the instrument had good precision.

[0127] 2.2.6 Repeatability test

[0128] Take about 5 mL of Xiangbeiweirong water extract, accurately pipette, prepare 6 test solutions in parallel according to the preparation method of the test solution, inject 10 μL of each, measure the peak area, and calculate the content, average content and RSD. The results are shown in Table 3.

[0129] Table 3 Repeatability test

[0130]

[0131]

[0132] Results: The average content of hesperidin in the sample was 0.194 mg / mL, and the RSD was 1.38% (n=6), indicating good repeatability.

[0133] 2.2.7 Stability test

[0134] Take the test solution under the repeatability test item, inject at 0, 2.5, 5, 7.5, 10.5, measure the hesperidin peak area, calculate the average hesperidin peak area and RSD, see Table 4. The results show that RSD = 0.42%, and the sample solution is stable within 10.5h.

[0135] Table 4 Stability test results

[0136]

[0137] 2.2.8 Sample recovery rate

[0138] Preparation of reference substance stock solution: Accurately weigh 9.54 mg of hesperidin reference substance, place in a 100 mL volumetric flask, add methanol to the mark, shake well, and obtain 0.0927 mg / mL reference substance stock solution.

[0139] Using the sample recovery method, 3 mL of each of 6 portions of the determined Xiangbei Weirong water extract (content 0.194 mg / mL) were taken, accurately weighed, and placed in a stoppered conical flask, and 5 mL of hesperidin reference solution (concentration 0.0927 mg / mL) was accurately added to 6 portions; according to the test solution preparation method, 10 μL of the reference solution and the test solution were respectively aspirated, injected into the high performance liquid chromatograph, the peak area was determined, and the sample recovery rate was calculated. The results are shown in Table 5.

[0140] The recovery rate was calculated as follows: recovery rate = (measured amount - sample amount) / added amount * 100%.

[0141] Table 5 Hesperidin recovery test results (n = 6)

[0142]

[0143] The results showed that the average recovery rate measured by this method was 98.30%, RSD% = 0.73%, indicating that the recovery rate of hesperidin was good.

[0144] 2.2.9 Durability test

[0145] The effects of different flow rates, column temperatures and chromatographic column models on the determination of hesperidin content in the water extract of Xiangbei Weirong were investigated.

[0146] 2.2.9.1 Investigation of different flow rates

[0147] Welch Ultimate XB-C18 (250 mm × 4.6 mm, 5 μm) was used as the chromatographic column, the column temperature was 30 ° C, and the detection wavelength was 283 nm. The chromatographic separation effects of different flow rates (0.8 mL / min, 1.0 mL / min, and 1.2 mL / min) on hesperidin were investigated. The results are shown in Table 6. Figure 3 .

[0148] Table 6 Investigation results of different flow rates

[0149]

[0150]

[0151] The results showed that the flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min had little effect on the separation of the hesperidin chromatographic peak. According to the actual laboratory use, the flow rate was selected to be 1.0 mL / min.

[0152] 2.2.9.2 Investigation of different column temperatures

[0153] Welch Ultimate XB-C18 (250 mm × 4.6 mm, 5 μm) was used as the chromatographic column to investigate the chromatographic separation effect of hesperidin at different column temperatures (25 °C, 30 °C, 35 °C). The results are shown in Table 7. Figure 4 .

[0154] Table 7 Results of investigation at different column temperatures

[0155]

[0156] The results show that the column temperature of 25℃, 30℃ and 35℃ has no difference in the separation effect of the chromatographic peaks. Considering the actual use, the column temperature of 30℃ was selected.

[0157] 2.2.9.3 Investigation of different chromatographic columns

[0158] The effects of different types of chromatographic columns on the determination of hesperidin content were investigated. Column 1: Elite Hypersil BDS C18 (4.6×250mm, 5μm), column number E2821784; Column 2: Welch Ultimate XB-C18 (4.6×250mm, 5μm), column number: 00201-31043; Instrument: Agilent 1260LC high performance liquid chromatograph, DAD ultraviolet detector. The results are shown in Table 8. Figure 5-6 The results showed that the hesperidin peak was baseline-separated from the adjacent chromatographic peaks with good resolution and good experimental reproducibility.

[0159] Table 8 Durability test

[0160]

[0161] In summary, the chromatographic conditions for hesperidin were determined as follows: octadecylsilane bonded silica gel as filler; methanol-2% acetic acid (35:65); flow rate 1 mL / min; column temperature 30°C; detection wavelength 283 nm; injection volume 10 μL.

[0162] 2.3 Determination of the extract yield of Xiangbeiweirong in water extract

[0163] Accurately pipette 5 mL of the water extract, repeat twice, transfer to a constant weight evaporating dish, evaporate to dryness in a water bath, dry at 105°C for 3 hours, transfer to a desiccator for 30 minutes, accurately weigh, and calculate the paste yield. The formula for calculating the paste yield is as follows:

[0164]

[0165] W is the weight of the solid matter in 5 mL of filtrate, V is the constant volume, and Wt is the sample feed weight.

[0166] 2.4 Comprehensive score calculation

[0167] The results of the cream yield and hesperidin content test were comprehensively scored. The weight of the cream yield was 0.3, and the weight of the hesperidin content was 0.7. The comprehensive score calculation formula was: cream yield / maximum cream yield × 0.3 + hesperidin content / maximum hesperidin content × 0.7.

[0168] 2.5 Investigation of water absorption rate of medicinal materials

[0169] Weigh about 87.5g of the slices according to the 1 / 2 prescription ratio, place in a 2000mL beaker, add 870mL of water to soak, soak for different time periods (1h, 3h, 5h, 7h, 24h, 30h), filter, weigh the wet medicine weight, measure the water absorption of the slices at each time point, and use the final water absorption rate when the water absorption rate does not change significantly. Calculate the water absorption rate according to water absorption rate (%) = [wet weight of medicinal materials - dry weight of medicinal materials (mL)] / dry weight of medicinal materials (g) × 100%. Parallel 3 times. The results are shown in Table 9.

[0170] Table 9 Water absorption test results of decoction pieces

[0171]

[0172] As shown in Table 6, the average water absorption rate of the slices is 141.2%. For the convenience of operation, the water absorption rate is calculated as 1.5 times, that is, the water absorption of the slices is 1.5 times the weight of the slices. Therefore, 1.5 times the weight of the slices should be added in water for the first extraction.

[0173] 2.6 Investigation of immersion time

[0174] Weigh the slices according to the 1 / 2 prescription ratio, a total of 10 portions, add 6 times the amount of water, soak for 0h, 0.5h, 1h, 1.5h and 2h, decoct twice, 30 minutes each time, filter, concentrate the liquid, and adjust the volume to 250mL to obtain the extract. Prepare the test solution according to the method under "2.2.2.2", inject it into the liquid chromatograph for detection, investigate the effects of different soaking times on the hesperidin content and the paste yield, and calculate the comprehensive score. The results are shown in Table 10.

[0175] Table 10 The results of the investigation on the water extract yield and hesperidin content at different soaking times (n=2)

[0176]

[0177] The results showed that there was no difference in the comprehensive score after immersion for 1 hour, so the immersion time was selected as 1 hour.

[0178] 2.7 Investigation of decoction frequency

[0179] Weigh the medicinal pieces according to the 1 / 2 prescription ratio, add 6 times the amount of water while meeting the water absorption rate of the medicinal materials, soak for 60 minutes, decoct for 1, 2, 3, and 4 times, 30 minutes each time, filter, concentrate the filtrate, and adjust the volume to 250mL. Prepare the test solution according to the method under "2.2.2.2", inject it into the liquid chromatograph for detection, examine the effects of different decoction times on the hesperidin content and the paste yield, and calculate the comprehensive score. See Table 11.

[0180] Table 11 The results of the investigation on the water extract yield and hesperidin content at different decoction times (n=2)

[0181]

[0182] The results showed that the comprehensive score was the highest after decocting for 3 times. Therefore, the extraction times were selected as 1, 2, and 3 times after comprehensive consideration.

[0183] 2.8 Investigation of water addition amount

[0184] Weigh the medicinal pieces according to the 1 / 2 prescription ratio, add 6, 8, and 10 times of water respectively while meeting the water absorption rate of the medicinal materials, soak for 60 minutes, decoct twice, 30 minutes each time, filter, concentrate the filtrate, and adjust the volume to 250mL. Prepare the test solution according to the preparation method under "2.2.2.2", inject and measure, examine the effect of water addition on the hesperidin content and paste yield of Xiangbei Weirong water extract, and calculate the comprehensive score. See Table 12.

[0185] Table 12 The results of the investigation on the effect of different water addition amounts on the water extract yield and hesperidin content (n=2)

[0186]

[0187] The results showed that when the amount of water added was 8 times, the comprehensive score was the highest. When the water was added more times, the concentration time was longer, resulting in a decrease in the hesperidin content. After comprehensive consideration, the final water addition amount was 6 times, 8 times, and 10 times.

[0188] 2.9 Decoction time study

[0189] Weigh the medicinal pieces according to the 1 / 2 prescription ratio, add 9.5 times water for the first time to soak for 60 minutes, add 8 times water for the second time, decoct twice, decoct for 20, 30, 40, 50, 60 minutes respectively, filter, concentrate the filtrate, and make the volume to 250mL. Prepare the test solution according to the method under "2.2.2.2", inject it into the liquid chromatograph for determination, investigate the effect of different decoction time on the hesperidin content and the paste yield, and calculate the comprehensive score. See Table 13.

[0190] Table 13 The results of the investigation on the water extract yield and hesperidin content at different decoction times (n=2)

[0191]

[0192] The results showed that after 30 min of decoction, there was no difference in the comprehensive score. Considering pharmacoeconomics, the extraction time was selected as 30 min, 45 min, and 60 min.

[0193] 2.10 Orthogonal experiment and result analysis

[0194] Combined with the results of the single factor test, about 87.5g was weighed according to the 1 / 2 prescription ratio, a total of 18 portions, and the extraction time, water addition amount, and extraction times were used as the investigation factors. L9(3) was selected. 4 The orthogonal experimental design table was used to conduct the experiment, and the comprehensive score of hesperidin content and paste yield was used as the evaluation index to select the optimal extraction process conditions. The factor levels are shown in Table 14. The results are shown in Tables 15 and 16.

[0195] Table 14 Orthogonal test factor level table

[0196]

[0197] Table 15 Orthogonal test results (n = 2)

[0198]

[0199]

[0200] Note: K is the mean; R is the range.

[0201] Table 16 Variance analysis results of comprehensive scores of extracts

[0202]

[0203] Note: F0.01(1,2)=99, F0.05(1,2)=19.00.

[0204] Through the orthogonal test results (Table 15), it can be intuitively analyzed that the influence of the three factors on the content of hesperidin is C>A>B, and the best combination is A3B2C3. From the variance analysis (Table 16), it can be concluded that there is a very significant difference in the number of extractions (P<0.01), and there is no significant difference in the water addition multiple and decoction time (P>0.05). Combined with the actual clinical use, the best combination of water extraction process is determined to be A1B1C2, that is, add 6 times the amount of water, extract twice, each time for 30 minutes.

[0205] 2.11 Extraction process verification experiment

[0206] 1 / 2 of the clinical prescription pieces were taken and extracted according to the optimal extraction process conditions (i.e., adding 7.5 times the water for the first time and soaking for 60 minutes, adding 6 times the water for the second time, decocting twice, each time for 30 minutes), and the paste yield and hesperidin content were determined. The results showed (see Table 17): the RSD of the hesperidin content and paste yield of the water extraction verification samples were both less than 3.0%, indicating that the extraction process was stable and feasible.

[0207] Table 17 Extraction process validation test

[0208]

[0209] (II) Research on formulation molding process

[0210] 1. Instruments and Materials

[0211] Electronic balance (YHM-20002, Huizhou Yingheng Electronic Technology Co., Ltd.), electronic balance (Shimadzu Philippines Factory, model AUW220D), electronic balance (Shanghai Jinghai Instrument Co., Ltd., model FA2204N), electric heating blast drying oven (Tianjin Test Instrument Co., Ltd., model 101-2AB), moisture meter (Ohaus Instrument (Changzhou) Co., Ltd., model MB27ZH), pharmacopoeia sieve (Zhejiang Shangyu Jinding Standard Sieve Factory). Soluble starch (Anhui Shanhe Pharmaceutical Excipients Co., Ltd., 210401), dextrin (Guangrao Lifeng Biotechnology Co., Ltd., 2022062301), corn starch (Qufu Tianli Pharmaceutical Excipients Co., Ltd., 20230227), microcrystalline cellulose (Anhui Shanhe Pharmaceutical Excipients Co., Ltd., B220925).

[0212] 2 Methods and Results

[0213] This experiment uses mechanical method for wet granulation. First, prepare Xiangbei Weirong extract, then add a certain proportion of auxiliary materials and mix well to make a soft material to achieve the degree of "holding into a ball, and kneading to disperse". Then pass the soft material through a 14-mesh sieve to make wet granules, dry at 80°C, and control the moisture content of the granules within 5%. The following four aspects are investigated from the selection of auxiliary materials, the ratio of extract to auxiliary materials, the relative density of the extract, and the drying temperature. The quality of Xiangbei Weirong granules is evaluated in terms of soft materials and granulation conditions, formability, moisture absorption rate and fluidity, and the best forming process of Xiangbei Weirong granules is selected.

[0214] 2.1 Evaluation indicators

[0215] 2.1.1 Determination of Formation Rate: According to the particle size regulations of granules (General Rule 0104), granules refer to those that can pass through the No. 1 sieve and cannot pass through the No. 5 sieve. The formula for calculating the formation rate is: (total mass of granules - mass of granules that can pass through the No. 5 sieve - mass of granules that cannot pass through the No. 1 sieve) / total mass of granules × 100%.

[0216] 2.1.2 Determination of moisture absorption rate: Take about 1g of particles and put them into a weighing bottle that has been dried to a constant weight, shake gently to spread the particles evenly, and then weigh them accurately. Then open the lid of the weighing bottle and place it in a desiccator with a saturated sodium chloride solution at the bottom. After 72 hours, take it out and weigh it immediately to calculate the moisture absorption rate. The moisture absorption rate calculation formula is: (mass of particles after 72 hours - initial particle mass) / initial particle mass × 100%.

[0217] 2.1.3 Determination of angle of repose

[0218] The fixed funnel method is used to determine the angle of repose. Qualified particles are naturally dropped from a funnel at a certain height onto a watch glass. After all the particles have been drained, the height of the cone formed is h, and the diameter of the cone is d. According to the trigonometric function α = degrees (atan (2h / d)), the angle of repose α is calculated. The smaller α is, the better the fluidity.

[0219] 2.2 Screening of different types of excipients

[0220] The investigation was mainly based on single-flavor excipients to screen the most suitable excipients. About 15g of Xiangbei Weirong extract with a relative density of 1.28 (70℃) was weighed from the same batch, and a total of 4 portions were mixed evenly with soluble starch, dextrin, corn starch, and microcrystalline cellulose. After wet granulation, the soft material and granulation conditions, particle formation rate, moisture absorption rate, repose angle, and excipient dosage were evaluated to determine the best excipient. The results are shown in Table 18.

[0221] Table 18 Granulation screening results of different excipient types

[0222]

[0223] The results show that the granulation rate of the four excipients is good, all greater than 95%, and the angle of repose is less than 40°. From the perspective of soft materials and granulation, the soft material obtained by mixing soluble starch, dextrin, and corn starch is slightly sticky, with sticky screen or long particles, while the soft material is moderate and easy to granulate when mixed with microcrystalline cellulose. And from the perspective of the amount of excipients, the amount of corn starch, dextrin, and soluble starch is too large compared to microcrystalline cellulose, which is about 2 times that of microcrystalline cellulose, which will cause patients to take too much and have poor compliance. Therefore, in the selection of excipients, microcrystalline cellulose is selected as an excipient.

[0224] 2.3 Screening of extract and excipient ratio

[0225] According to the screening results of single-flavor excipients, the extract and microcrystalline cellulose were mixed for granulation. About 15 g of Xiangbei Weirong extract with a relative density of 1.29 (70°C) was weighed, and a total of 7 portions were made. The extract and excipients were mixed in the ratios of 1:0.7, 1:0.75, 1:0.8, 1:0.85, 1:0.9, 1:0.95, and 1:1, respectively. The effects of different ratios of extract and excipients on the soft material, granulation, molding rate, and particle properties were observed, and the optimal ratio of extract and excipients was selected. The results are shown in Table 19.

[0226] Table 19 Granulation screening results of different extract and microcrystalline cellulose ratios

[0227]

[0228] As shown in Table 19, when the drug-excipient ratio is 1:0.70, the prepared soft material is sticky and cannot be granulated; when the drug-excipient ratio is 1:0.75, the prepared soft material is relatively wet and sticky, and the prepared granules are uneven and have long strips; when the drug-excipient ratio is 1:0.8-0.9, the obtained soft material is moderate and the granules are uniform; when the drug-excipient ratio is 1:0.95-1, the prepared granules are more fine powder. Therefore, the ratio of extract to excipient is determined to be 1:0.8-0.9.

[0229] 2.4 Determination of relative density of extract

[0230] Take 20 times the amount of clinical prescription medicinal materials, extract according to the best water extraction process and concentrate to 2000ml for use. Measure the same weight of extract and further concentrate to relative densities of 1.24, 1.26, 1.28, 1.30, 1.32, and 1.35 respectively. Take about 20g of extracts of different relative densities respectively, add microcrystalline cellulose according to the ratio of extract to excipients (1:0.8), mix well and perform wet granulation. The appropriate relative density range of the extract is screened out by indicators such as soft material condition, granulation condition, molding rate and particle properties. The results are shown in Table 20.

[0231] Table 20 Effect of different relative density extracts on granule preparation

[0232]

[0233]

[0234] As shown in Table 20, when the relative density of Xiangbei Weirong extract is 1.18-1.24 (70℃), the obtained granular powder is more and the amount of auxiliary materials used is too large; when the relative density is 1.28-1.32 (70℃), the soft material state is moderate and the particles are uniform;

[0235] At 1.35-1.40 (70℃), the soft material is slightly sticky and there is a phenomenon of sticking to the sieve, and there are coarse and hard particles in the particles. Therefore, the relative density range of the extract is determined to be 1.28-1.32 (70℃).

[0236] 2.5 Screen mesh number inspection

[0237] Take the extract with a relative density of 1.30 (70℃), divide it into three parts, mix it with auxiliary materials, and granulate it through 18 mesh, 16 mesh, and 14 mesh screens respectively. The suitable mesh number of the screen is screened based on the soft material and granulation conditions, forming rate, particle properties and repose angle. The results (see Table 21) show that 14 mesh, 16 mesh and 18 mesh screens can all be used for granulation. Considering the actual use of large-scale production, it is decided to use 14 mesh screen for granulation.

[0238] Table 21 Test results of different sieve mesh numbers

[0239]

[0240] 2.6 Investigation of Granule Drying Process

[0241] 2.6.1 Investigation of particle drying temperature

[0242] The drying time varies with the drying temperature. When the temperature is low, the drying time is long; when the temperature is high, the drying time is short but pseudo-drying is prone to occur. The usual drying temperature range for wet granules is 60-80°C. In this experiment, the drying temperature was set at 60°C, 70°C, and 80°C, and the hesperidin content and water content in the granules were used as evaluation indicators to determine the optimal drying temperature. The results are shown in Table 22. The results show that the drying temperature has no significant effect on the content of hesperidin. Considering that the drying time is long when the temperature is too low, in order to ensure the stability of the drug, this experiment set the drying temperature in the range of 70°C to 80°C. Dry the granules until the moisture content is less than 3.0%.

[0243] Table 22 Effect of different drying temperatures on hesperidin content and water content

[0244]

[0245] 2.7 Forming process verification test

[0246] Take about 100g of the same batch of Xiangbei Weirong extract (relative density of 1.30, 70°C) and repeat the granulation operation three times according to the preferred molding process conditions. The results are shown in 23, indicating that the process has good reproducibility and operability.

[0247] Table 23 Forming process verification results

[0248]

[0249] 2.8 Small test confirmation

[0250] According to the optimized water extraction process and molding process conditions, three batches of 5-fold small-scale production were carried out to investigate the feasibility and rationality of the preparation process of this product. The hesperidin content and water content of three batches of Xiangbei Weirong granules were determined. The results are shown in Table 24. The results show that the process preparation conditions are highly operable and can be used for large-scale production.

[0251] Table 24 Small test confirmation batch test results

[0252]

[0253] (III) Determination of particle flowability and critical relative humidity

[0254] 1 Determination of the angle of repose

[0255] The fluidity of the particles was measured according to the method under "2.1.3" of Part (II), and the measurement was repeated 3 times for each batch of samples. The results are shown in Table 25. The average repose angles of the three batches of particles were all less than 40°, indicating that the particle fluidity was good and the particle loading could meet the requirements.

[0256] Table 25 Repose angle measurement results

[0257]

[0258] 2 Determination of critical relative humidity of particles

[0259] The humidity of the packaging environment has a great influence on the packaging of particles, so it is necessary to determine the critical relative humidity of the particles.

[0260] 2.1 Determination of moisture absorption equilibrium time

[0261] Take a glass desiccator with a supersaturated NaCl solution (relative humidity 74.8%) at the bottom, and a weighing bottle (5×3cm) built into the desiccator to reach constant weight in a constant temperature drying oven at 25℃. Take about 1g of sample, accurately weigh it, put it in a weighing bottle with constant weight, shake it gently to make it evenly distributed, accurately weigh it, open the weighing bottle cap, put it in the upper part of the desiccator, and store it in a constant temperature drying oven at 25℃. Weigh it once at 1, 3, 5, 7, 24, 48, 72, 96, and 120h, and calculate the moisture absorption rate at different times. Calculation formula: Moisture absorption rate = (total weight after moisture absorption - total weight before moisture absorption) / total amount before moisture absorption × 100%. The test results show that the samples of three batches (20240109, 20240110, and 20240111) were tested separately, and the experimental results were similar. The experimental results are now listed for analysis. The moisture absorption equilibrium time of the particles is shown in Table 26, and the moisture absorption equilibrium curve is shown in Figure 7 .

[0262] Table 26 Moisture absorption equilibrium time (n=3)

[0263]

[0264] From Table 26, Figure 7 It can be seen that the moisture absorption of this product basically reaches equilibrium after 72 hours, and the moisture absorption rate no longer increases significantly. The moisture absorption equilibrium time of the particles is 72 hours.

[0265] 2.2 Determination of critical relative humidity of particles

[0266] Weigh 14 portions of granules accurately, 1g each, spread them flat in a weighing bottle (5×3cm) that has been dried and kept constant, weigh them accurately, and place them in different humidity environments (see Table 27). Open the weighing bottle caps, keep them at 25°C for 72 hours, and then weigh them. Measure the weight change of the samples, calculate the moisture absorption rate, and draw a curve with the moisture absorption rate as the ordinate and the relative humidity as the abscissa. Draw tangents from both ends of the curve, and the abscissa corresponding to the intersection of the two tangents is the critical relative humidity. The results show that the critical relative humidity of the granules is about 73% (see Figure 8 ). Therefore, during granulation, packaging and storage, the ambient humidity must be controlled below 73% to reduce the impact of moisture on the properties and stability of the drug.

[0267] Table 27 Determination of critical relative humidity of particles

[0268]

[0269]

[0270] In summary, the preparation process of this product is determined as follows: control the humidity of the mixing and packaging environment to <73%, add water to the prescription of 16 ingredients and boil them twice, add 7.5 times the water for the first time and soak for 60 minutes, add 6 times the water for the second time, boil them twice, boil them for 30 minutes each time, filter, concentrate the filtrate to a thick paste with a relative density of 1.28-1.32 (70°C), add an appropriate amount of microcrystalline cellulose and mix well, make them into granules, dry them, and make them into 1000g.

[0271] 2. Research on Preparation Quality Standards

[0272] 1. Properties: This product is brown to tan granules and powder; slight odor, spicy taste.

[0273] 2. Identification

[0274] 2.1 Instruments and reagents

[0275] Electronic balance (YHM-20002, Huizhou Yingheng Electronic Technology Co., Ltd.); ZF-20D dark box UV analyzer (Gongyi Yuhua Instrument Co., Ltd.); DZKW-4 electronic constant temperature water bath (Shanghai Jinqiao Kexi Instrument Factory); 98-1-B electronic temperature control electric heating mantle (Tianjin Test Instrument Co., Ltd.); SK5010HP ultrasonic cleaning machine (power 200W, frequency 53kHz; Shanghai Kedao Ultrasonic Instrument Co., Ltd.); silica gel G thin layer plate (Qingdao Ocean Chemical Plant); polyamide film sheet (Luqiao Sijia Biochemical Plastic Factory, Taizhou City, Zhejiang Province); methanol, n-butanol, glacial acetic acid, anhydrous ethanol, ethanol, chloroform, ethyl acetate, formic acid, cyclohexane, ethyl acetate, petroleum ether (60-90°C), acetic acid, toluene, ethyl formate, concentrated ammonia test solution, toluene, acetone, ether, n-hexane, 10% sulfuric acid ethanol solution, 2% vanillin sulfuric acid solution-ethanol (4:1) mixed solution, 5% aluminum chloride ethanol solution, dilute potassium bismuth iodide test solution, etc.

[0276] 2.2 Methods and Results

[0277] The preparation is composed of 16 Chinese medicinal materials, including Codonopsis pilosula, Atractylodes macrocephala, and turtle shell with vinegar. With reference to the methods in the Chinese Pharmacopoeia and related literature, qualitative identification was performed on 8 medicinal materials in the preparation, including Codonopsis pilosula, Atractylodes macrocephala, Poria cocos, dried orange peel, costus root, Fritillaria thunbergii, vinegar corydalis, and Curcuma aromatica. Among them, the negative control for qualitative identification of 4 medicinal materials, including dried orange peel, costus root, Fritillaria thunbergii, and vinegar corydalis, had no interference. After verification by 3 batches of pilot samples, this method had good repeatability, so it was included in the text of the draft quality standard; Codonopsis pilosula, Atractylodes macrocephala, Poria cocos, and Curcuma aromatica, all used two extraction methods and carried out systematic experiments, but were not included in the text of the quality standard due to negative interference. The test results are as follows.

[0278] 2.2.1 Thin layer identification of Codonopsis pilosula

[0279] Method 1: Take 5g of this product, add 25mL of 80% methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, and dissolve the residue in 1mL of methanol as the test solution. Take another 5g of the negative preparation lacking Codonopsis pilosula, and prepare the negative control solution in the same way. Take 1g of Codonopsis pilosula control medicinal material, add 50mL of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, and prepare the control medicinal material solution in the same way as the test solution preparation method. According to the thin layer chromatography method (General Rule 0502), 8-10μL of the test solution and negative control solution and 10μL of the control medicinal material solution are taken, and spotted on the same silica gel G thin layer plate, and n-butanol-glacial acetic acid-water (7:1:0.5) is used as the developing agent. Develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under a fluorescent lamp. The results show (see Fig. 9 Left), in the test sample chromatogram, there is no spot of the same color at the corresponding position in the control medicinal material chromatogram.

[0280] Method 2: Take 5g of this product, add 25mL of 50% ethanol, heat and reflux for 30 minutes, filter, evaporate the filtrate, and dissolve the residue in 1mL of methanol as the test solution. Take another 5g of the negative preparation lacking Codonopsis pilosula, and prepare a negative control solution in the same way. Take 1g of Codonopsis pilosula control medicinal material, add 50mL of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, and prepare the control medicinal material solution in the same way as the test solution preparation method. According to the thin layer chromatography method (General Rule 0502), 8μL of the test solution and negative control solution and 6μL of the control medicinal material solution were aspirated and spotted on the same silica gel G thin layer plate, using chloroform-ethyl acetate-formic acid (10:2:0.5) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm). The results show (such as Fig. 9 Right), the test sample chromatogram interferes with the negative control solution.

[0281] 2.2.2 Thin layer identification of Atractylodes macrocephala

[0282] Method 1: Refer to the method of the formula granules of Atractylodes macrocephala with bran stir-fry [National Drug Standard for Formula Granules of Traditional Chinese Medicine (Second Batch)]: Take 5g of this product, add 25mL of water to dissolve, centrifuge, filter, extract twice with ethyl acetate by shaking, 20mL each time, combine the ethyl acetate solution, evaporate to dryness, add 1mL of methanol to the residue to dissolve it, and use it as the test solution. Take another 5g of the negative preparation of Atractylodes macrocephala without bran stir-fry, and prepare the negative control solution in the same way. Take 1g of the control medicinal material of Atractylodes macrocephala, add 50mL of water, boil for 30 minutes, filter, concentrate the filtrate to about 20mL, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 10μL of each of the above three solutions, spot them on the same silica gel G thin layer plate, use cyclohexane-ethyl acetate (1:1) as the developing solvent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and inspect under ultraviolet light (365nm). The results show (see Fig.10 Left), in the test sample chromatogram, the test solution and the negative control solution interfere with each other.

[0283] Method 2: Conduct the experiment with reference to the test solution processing method and development conditions of Atractylodes macrocephala in the literature "Study on the Quality Standard of Luling Anshen Granules": Take 10g of this product, add 40mL of methanol, ultrasonicate for 45min, filter, and concentrate the filtrate to 1mL as the test solution. Take another 5g of the negative preparation of Atractylodes macrocephala stir-fried in the absence of bran, and prepare a negative control solution in the same way. Take another 0.5g of Atractylodes macrocephala control medicinal material, and prepare a control medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 6μL of each of the above three solutions, spot them on the same silica gel G plate, develop them with petroleum ether (60-90℃)-ethyl acetate (5:1), take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and inspect them under ultraviolet light (365nm). The results show (see Fig.10 Right), in the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position in the chromatogram of the control medicinal material, and there is no interference in the negative control, but its repeatability is poor, so it was not included in the main text of the draft quality standard.

[0284] 2.2.3 TLC Identification of Poria

[0285] Method 1 (formulated granule method): Take 5g of this product, add 20mL of water to dissolve, centrifuge, filter, and extract twice with ethyl acetate by shaking, 20mL each time, combine the ethyl acetate solution, evaporate to dryness, and add 1mL of methanol to the residue to dissolve it as the test solution. Take another 5g of the negative preparation without Poria cocos, and prepare the negative control solution in the same way. Take 3g of Poria cocos control medicinal material, add 50mL of water, decoct for 30 minutes, filter, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), 10μL of the test solution and negative control solution and 8μL of the control medicinal material solution are taken and spotted on the same polyamide film, and acetic acid is used as the developing agent. Develop, take out, dry, spray with 5% aluminum chloride ethanol solution, blow dry with hot air, and examine under ultraviolet light (365nm). The results show (see Fig.11 Left), the control medicinal material solution has no chromatographic spots.

[0286] Method 2: Refer to the literature; the thin layer identification method of Poria in "Study on Quality Standard of Uremic Nutrition Mixture" is used for experiment: take 5g of this product, add 20mL of water to dissolve, centrifuge, filter, add n-hexane to the filtrate and shake to extract twice, 20mL each time, combine the n-hexane solution, evaporate to dryness, add 1mL of n-hexane to the residue to dissolve, as the test solution. Take another 5g of the negative preparation without Poria, and prepare the negative control solution in the same way. Take 1g of Poria control medicinal material, add appropriate amount of water, decoct for 30min, cool, filter, and prepare the control medicinal material solution with the filtrate in the same way. According to the thin layer chromatography method (General Rule 0502), 2μL of each of the above three solutions are taken and spotted on the same silica gel G thin layer plate, and toluene-ethyl acetate-formic acid (20:5:0.5) is used as the developing solvent. Develop, take out, dry, spray with 2% vanillin sulfuric acid solution-ethanol (4:1) mixed solution, and heat at 105°C until the spots are clearly colored. The results show (see Fig.11 Right), the control drug solution has no chromatographic spots.

[0287] 2.2.4 Thin layer identification of tangerine peel

[0288] Method 1: Refer to the thin layer identification method of tangerine peel on pages 199-200 of the 2020 edition of the "Chinese Pharmacopoeia" for experimental research: take 5g of this product, add 25mL of methanol, ultrasonically treat for 20 minutes, filter, and concentrate the filtrate to 2mL as the test solution. Take another 5g of the negative preparation lacking tangerine peel, and prepare a negative control solution in the same way. Take 1g of tangerine peel control medicinal material and prepare a control medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), 2μL of each of the above three solutions was taken and spotted on the same silica gel G thin layer plate, using ethyl acetate-methanol-water (100:17:13) as the developing solvent, extending to about 3cm, taking out, drying, and then using the upper layer solution of toluene-ethyl acetate-formic acid-water (20:10:1:1) as the developing solvent, extending to about 8cm, taking out, drying, spraying with aluminum chloride test solution, and inspecting under ultraviolet light (365nm). The results show (see Fig.12 Left), negative control has no interference, but the chromatographic spots have poor resolution.

[0289] Method 2: Take 2 μL of the control medicinal material solution, test solution and negative control solution of method 1, respectively, and develop them with the upper layer solution of ethyl acetate-methanol-water (100:17:13) and toluene-ethyl acetate-formic acid-water (20:10:1:1), respectively. Take out, dry, spray with aluminum chloride test solution, and examine under ultraviolet light (365nm). The results show that the chromatogram run with the upper layer solution of toluene-ethyl acetate-formic acid-water (20:10:1:1) has no interference between the test solution and the negative control solution (see Fig.12 In the chromatogram of the test sample, the fluorescent spot of the same color was found at the corresponding position of the control medicinal material chromatogram using the developing solvent ethyl acetate-methanol-water (100:17:13), and there was no interference with the negative control. Therefore, the developing solvent of tangerine peel was determined to be ethyl acetate-methanol-water (100:17:13), and it was verified by 3 batches of pilot samples (see Fig.12 Right), this method has good reproducibility, so it is included in the text of the draft quality standard.

[0290] 2.2.5 Thin layer identification of costusroot

[0291] Method 1: Refer to the thin-layer identification method of costus root on pages 63-64 of the 2020 edition of the "Chinese Pharmacopoeia" for experimental research: take 5 g of this product, add 25 mL of methanol, ultrasonically treat for 30 minutes, filter, and concentrate the filtrate to about 1 mL as the test solution. Take another 5 g of the negative preparation lacking costus root, and prepare a negative control solution in the same way. Take another 0.5 g of costus root control medicinal material, and prepare a control medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), 5 μL of each of the above three solutions were taken and spotted on the same silica gel G thin layer plate, and the upper layer solution of cyclohexane-ethyl formate-formic acid (15:5:1) was used as the developing agent. Develop, take out, dry, spray with 5% vanillin sulfuric acid solution, and heat at 105°C until the spots are clearly colored. In the chromatogram of the test sample, there are no spots of the same color at the corresponding position of the chromatogram of the control medicinal material (see Fig.13 Left).

[0292] Method 2: Take 10g of this product, add 50mL of water to dissolve, filter, extract the filtrate with 40mL of ethyl acetate, take the ethyl acetate layer, evaporate to dryness, add 1mL of ethyl acetate to dissolve the residue, and use it as the test solution. Take another 10g of the negative preparation lacking costus root, and prepare the negative control solution in the same way. Take 0.5g of costus root control medicinal material, add 50mL of water, decoct for 30min, filter, and use the filtrate to prepare the control medicinal material solution in the same way as the test solution preparation method. According to the thin layer chromatography method (General Rule 0502), take 5μL of the control medicinal material solution, 5-10μL of the test solution and the negative control solution, and spot them on the same silica gel G thin layer plate, use the upper layer solution of cyclohexane-ethyl formate-formic acid (15:5:1) as the developing solvent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, and heat at 105℃ until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material, and there is no interference with the negative control. Fig.13 Right), this method has good reproducibility, so it is included in the text of the draft quality standard.

[0293] 2.2.6 Thin-layer identification of Fritillaria thunbergii

[0294] Take 10g of this product, add 4mL of concentrated ammonia test solution and 50mL of chloroform, heat and reflux for 1h, filter, evaporate the filtrate, add 2mL of chloroform to the residue to dissolve it, and use it as the test solution. Take another 10g of the negative preparation without Fritillaria thunbergii, and prepare the negative control solution in the same way. Take 1g of Fritillaria thunbergii control medicinal material, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 10-15μL of each of the above three solutions and spot them on the same silica gel G thin layer plate, use ethyl acetate-methanol-concentrated ammonia test solution (17:2:1) as the developing agent, develop, take out, dry, and spray with dilute potassium bismuth iodide test solution. In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material, and there is no interference with the negative control. Verified by 3 batches of pilot samples (see Fig.14 ), this method has good reproducibility, so it is included in the text of the draft quality standard.

[0295] 2.2.7 Thin layer chromatography identification of vinegar Corydalis

[0296] Take 5g of this product, add 50mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, add 10mL of water to the residue to dissolve, add concentrated ammonia test solution to adjust to alkalinity, shake and extract with ether 3 times, 10mL each time, combine the ether solution, evaporate to dryness, add 1mL of methanol to the residue to dissolve, as the test solution. Take another 5g of the negative preparation of Corydalis yanhusuo lacking vinegar, and prepare the negative control solution in the same way. Take 1g of Corydalis yanhusuo control medicinal material, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 4-5μL of each of the above three solutions, spot them on the same silica gel G thin layer plate, use toluene-acetone (9:2) as the developing agent, develop, take out, dry, put in the iodine cylinder for about 3 minutes, take out, after wafting off the iodine adsorbed on the plate, put it under ultraviolet light (365nm) for inspection. In the chromatogram of the test product, at the corresponding position of the chromatogram of the control medicinal material, a fluorescent spot of the same color appears. After 3 batches of pilot samples verification (see Fig.15 ), this method has good reproducibility, so it is included in the text of the draft quality standard.

[0297] 2.2.8 TLC Identification of Curcuma

[0298] Method 1: Take 5g of this product, add 25mL of anhydrous ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, add 1mL of ethanol to the residue to dissolve it, and use it as the test solution. Take another 5g of the negative preparation lacking Curcuma, and prepare the negative control solution in the same way. Take another 0.5g of Curcuma control medicinal material, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 8-10μL of each of the above three solutions, spot them on the same silica gel G thin layer plate, use n-hexane-ethyl acetate-methanol (17:3:1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine under ultraviolet light (365nm). In the chromatogram of the test product, fluorescent spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material, but the negative control has interference (see Fig.16 Left).

[0299] Method 2: Take 5g of this product, add 25mL of methanol, heat and reflux for 30 minutes, filter, evaporate the filtrate, add 1mL of methanol to the residue to dissolve it, and use it as the test solution. Take another 5g of the negative preparation lacking Curcuma, and prepare the negative control solution in the same way. Take another 0.5g of Curcuma control medicinal material, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rule 0502), take 8-10μL of each of the above three solutions, and spot them on the same silica gel G thin layer plate, use n-hexane-ethyl acetate (17:3) as the developing solvent, pre-saturate for 30 minutes, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105°C until the spots are clearly colored. Place under ultraviolet light (365nm) for inspection. In the chromatogram of the test product, fluorescent spots of the same color appear at the corresponding position of the chromatogram of the control medicinal material, but the negative control has interference (see Fig.16 Right), so it was not included in the text of the draft quality standard.

[0300] 2.3 Study on the durability of TLC of dried tangerine peel, costusroot, Fritillaria thunbergii, and vinegar-corydalis

[0301] According to the TLC conditions used in the experiment, two kinds of TLC plates made from different batches of silica gel G (batch numbers: 20221207, 20240409) were investigated. The effects of different temperatures (4°C, 25°C, 35°C) and different humidity (18%, 58%, 88%) on the TLC results of dried tangerine peel, costusroot, thunbergii and vinegar corydalis are shown in the table. Fig.17 , 18 The chromatographic behaviors of the test samples on the two thin layer plates under different temperature and humidity conditions were basically the same, and the qualitative identification methods used in the description were all of good durability.

[0302] 3. Inspection

[0303] Inspection shall be carried out in accordance with the relevant provisions under Granules (General Rules 0114 of Part IV of the 2020 Edition of the Chinese Pharmacopoeia), checking the particle size, moisture, filling volume difference, and microbial limit respectively.

[0304] Particle size: Determined according to the particle size and particle size distribution determination method (General Rule 0982 Method 2 Double Screening Method), the sum of the particles that cannot pass through the No. 1 sieve and the particles that can pass through the No. 5 sieve shall not exceed 15%.

[0305] Moisture content: Determined according to the moisture determination method (General Rules 0832 of Part IV of the 2020 edition of the Chinese Pharmacopoeia), it shall not exceed 8.0%. See Table 28 for the moisture determination results.

[0306] Table 28 Moisture determination results

[0307]

[0308] Filling volume difference: Take 10 bags of this product and check the filling volume difference under the granules (General Rule 0104). The filling volume difference limit should be within ±5% of the labeled filling volume. No more than 2 bags should exceed the filling volume difference limit, and no bag should exceed the limit by 1 times. The results are shown in Table 29.

[0309] Table 29 Results of filling volume difference determination

[0310]

[0311] Microbial limits: The microbial limits of non-sterile products were checked according to the microbial count method (General Rule 1105), the control bacteria test method (General Rule 1106) and the microbial limit standards for non-sterile drugs (General Rule 1107). The results are shown in Table 30. The microbial limit tests all met the requirements.

[0312] Table 30 Microbial limit test results

[0313]

[0314]

[0315] The results of various inspections show that this product complies with the relevant provisions under General Chapter 0114 Granules of Part IV of the 2020 Edition of the Chinese Pharmacopoeia.

[0316] 4. Extractables determination

[0317] 4.1 Selection of extract solvent

[0318] Referring to the solvent selection of the extract of Xiangbei Weirong Granules in the Part 1 of the 2020 edition of the "Chinese Pharmacopoeia", and considering that the chemical components of Xiangbei Weirong Granules are mainly flavonoids, terpenes, alkaloids, etc., water, 50% ethanol, 70% ethanol, and 95% ethanol were used as extraction solvents, respectively, and the hot leaching method was used to determine the contents of water-soluble and alcohol-soluble extracts in Xiangbei Weirong Granules, see Table 31.

[0319] Table 31 Extract solvent selection results

[0320]

[0321] It can be seen from Table 31 that when water is used as the solvent for Xiangbei Weirong Granules, the extract content is the highest, followed by 50% ethanol solvent. However, when filtering the extract solvent extracted with water, there are problems of difficulty and slow filtration, and the operability is poor. Therefore, 50% ethanol was selected as the extraction solvent for extract determination.

[0322] 4.2 Determination of alcohol-soluble extracts of Xiangbei Weirong granules

[0323] Take 2g of the test sample, weigh it accurately, put it in a 250mL conical flask, accurately add 50mL of 50% ethanol, seal it, weigh it, let it stand for 1h, connect the reflux condenser, heat it to boiling, and keep it boiling for 1h. After cooling, remove the conical flask, seal it, weigh it again, make up the lost weight with 50% ethanol, shake it well, filter it with a drying filter, accurately measure 25mL of the filtrate, put it in an evaporating dish that has been dried to constant weight, evaporate it on a water bath, dry it at 105℃ for 3h, move it to a desiccator, cool it for 30min, and quickly weigh it accurately. The calculation formula is as follows:

[0324]

[0325] The results show that the average content of alcohol-soluble extract is 35.2%. According to the difficulty of mass production, the extract content of this product is calculated by reducing the average content of 4 batches of samples by 20%, that is, 35.2%-35.2%×20%=28.2%. The extract content of Xiangbei Weirong Granules is tentatively determined to be no less than 28.2%. The results are shown in Table 32.

[0326] Table 32 Test results of alcohol-soluble extracts from different batches of Xiangbei Weirong granules

[0327]

[0328] 5. Experimental study on the determination method of hesperidin content

[0329] 5.1 Instruments and reagents

[0330] Instruments: Agilent 1260infinity II high performance liquid chromatograph; G4212B 1260DAD diode array detector; AUW220D electronic balance (Shimadzu Philippines Factory); electronic temperature control heating mantle (98-1-B, Tianjin Test Instrument Co., Ltd.); ultrasonic cleaner (SK8210HP, Shanghai Kedao Ultrasonic Instrument Co., Ltd., power 250W, frequency 40kHz).

[0331] Test drugs: acetic acid (HPLC grade, Tianjin Komiou Chemical Reagent Co., Ltd.); methanol (chromatographic grade, TEDIA, USA), water (pure water, manufactured by Chengdu Wahaha Food Co., Ltd.); hesperidin reference substance (provided by China Food and Drug Inspection Institute, content is 97.2%, batch number: 110721-202220); Xiangbei Weirong Granules; Tangerine peel-deficient negative preparation.

[0332] 5.2 Selection of indicator components

[0333] In the prescription of Xiangbei Weirong, Codonopsis pilosula is the main drug, Atractylodes macrocephala and Poria cocos are the auxiliary drugs. There is no content determination item recorded in the current edition of the "Chinese Pharmacopoeia", so hesperidin in the adjuvant drug Tangerine peel is selected as the detection indicator for content determination.

[0334] 5.3 Solution preparation

[0335] The content of hesperidin in tangerine peel was determined by high performance liquid chromatography with reference to the methods for determining the content of hesperidin in tangerine peel in the 2020 edition and the 2015 edition of the Chinese Pharmacopoeia.

[0336] 5.3.1 Preparation of reference solution

[0337] Accurately weigh 10.00 mg of hesperidin reference substance, place it in a 50 mL volumetric flask, add methanol to dissolve and dilute to the mark to prepare a 0.1944 mg / mL stock solution. Accurately measure 5 mL of the stock solution and place it in a 25 mL volumetric flask, add methanol to prepare a 38.88 μg / mL reference substance solution.

[0338] 5.3.2 Preparation of test solution

[0339] Take 1 g of Xiangbei Weirong granules, weigh accurately, put it in a stoppered conical flask, add 25 mL of methanol accurately, stopper it, weigh it, ultrasonicate it for 30 min, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, take the filtrate, filter it with a 0.22 μm microporous filter membrane, and use it as the test solution.

[0340] 5.3.3 Preparation of negative control solution

[0341] Take the negative sample lacking tangerine peel and prepare it according to the method of “5.3.2 Preparation of test solution” to obtain the negative control solution lacking tangerine peel.

[0342] 5.4 Investigation of chromatographic conditions

[0343] 5.4.1 Detection wavelength

[0344] Hesperidin was scanned at a full wavelength of 190-400 nm and found to have a maximum absorption at about 283 nm. Fig.19 The detection wavelength is consistent with the current edition of the Pharmacopoeia, so the detection wavelength is selected as 283nm.

[0345] 5.4.2 Investigation of mobile phase

[0346] When selecting the chromatographic conditions for hesperidin, Welch Ultimate XB-C18 (250 mm × 4.6 mm, 5 μm) was used as the chromatographic column, the detection wavelength was 283 nm, the flow rate was 1 mL / min, and the column temperature was 30°C to screen the mobile phase. See Table 33.

[0347] Table 33 Hesperidin chromatographic conditions selection table

[0348]

[0349] By comparing the test results of the above-mentioned different mobile phases, it can be seen that when the mobile phase system is acetonitrile-water, the peak shape of the hesperidin chromatographic peak is poor; when the mobile phase system is methanol-acetic acid aqueous solution, the peak shape of the hesperidin chromatographic peak is improved, but the chromatographic peak is wide; when the mobile phase system is acetonitrile-2% acetic acid aqueous solution, the peak shape of the hesperidin chromatographic peak is good, the peak elution time is short, the separation effect is good, and there is no interference from the negative control; when the mobile phase system is acetonitrile-1% acetic acid aqueous solution or acetonitrile-0.05% phosphoric acid aqueous solution, the separation effect of the hesperidin chromatographic peak is slightly worse. Therefore, the mobile phase was preliminarily determined to be: acetonitrile-2% acetic acid aqueous solution (22:78). However, after many tests, the peak shape of the hesperidin chromatographic peak deteriorated and the durability was poor (see Fig.26 ). Then methanol-2% acetic acid (35:65) was used as the mobile phase for analysis. The results showed that the peak shape of the hesperidin chromatographic peak did not show any difference, so methanol-2% acetic acid (35:65) was finally determined as the mobile phase.

[0350] 5.5 Test method for treating the test solution

[0351] 5.5.1 Investigation of extraction solvent

[0352] Take about 1.0g of this product, weigh it accurately, put it in a conical flask with a stopper numbered (1-6), and accurately add 25mL of solvent, No. 1 and No. 2 solvents are methanol; No. 3 and No. 4 solvents are 75% methanol; No. 5 and No. 6 solvents are 50% methanol; treat according to the preparation method of the test solution, determine according to the above chromatographic conditions, record the peak area and calculate the hesperidin content. The results are shown in Table 34.

[0353] Table 34 Effect of extraction solvent on hesperidin content

[0354]

[0355] The results showed that the hesperidin content extracted by methanol was the lowest, and the hesperidin content extracted by 75% methanol and 50% methanol was equivalent. Considering that the viscosity of 50% methanol was greater than that of 75% methanol, it would cause slightly greater damage to the chromatographic column. Therefore, this method used 75% methanol as the solvent for extracting samples.

[0356] 5.5.2 Investigation of extraction methods

[0357] Take about 1.0g of this product, weigh accurately, a total of 10 portions, put in a stoppered conical bottle, accurately add 25mL of 75% methanol to each, weigh the weight, 2 of which are cold soaked for 30min, 2 are heated to reflux for 30min, and 2 are ultrasonically treated for 30min; let cool, weigh again, make up the reduced weight with 75% methanol, shake well, filter, and filter with a 0.22μm microporous filter membrane to obtain. Accurately take 10μL of the above 6 solutions and hesperidin reference substance, inject into the high performance liquid chromatograph, record the chromatogram, and determine the content of hesperidin in the sample. The results are shown in Table 35.

[0358] Table 35 Investigation of extraction methods

[0359]

[0360] The results showed that the content of hesperidin in Xiangbei Weirong granules extracted by heating reflux for 30 minutes and ultrasonic treatment for 30 minutes was the highest, and there was no difference. Considering the difficulty of operation, this experiment selected ultrasonic treatment for 30 minutes as the extraction method for preparing the test solution.

[0361] 5.5.3 Investigation of extraction time

[0362] Take 1g of this product, weigh accurately, a total of 6 portions, put in a stoppered conical bottle, accurately add 25mL of 75% methanol to each, weigh the weight, ultrasonicate 2 portions for 15min; ultrasonicate 2 portions for 30min; ultrasonicate 2 portions for 45min; let cool, weigh again, make up the reduced weight with 75% methanol, shake well, filter, and filter with a 0.22μm microporous filter membrane to obtain. Determine the content of hesperidin in the sample. The results are shown in Table 36.

[0363] Table 36 Investigation of extraction time

[0364]

[0365] The results showed that different ultrasound times had little effect on the content of hesperidin in Xiangbei Weirong granules. From the perspective of pharmacoeconomics, ultrasound for 15 minutes was selected.

[0366] 5.6 Specificity test

[0367] 5.6.1 Solution preparation

[0368] 5.6.1.1 Preparation of reference solution

[0369] Preparation of reference solution 1: Accurately weigh 10.00 mg of hesperidin reference substance, place it in a 50 mL volumetric flask, add methanol to dissolve and dilute to the mark to prepare a 0.1944 mg / mL stock solution. Accurately measure 5 mL of the stock solution and place it in a 25 mL volumetric flask, add methanol to prepare a 38.88 μg / mL reference solution.

[0370] Preparation of reference solution 2: Accurately weigh 10.02 mg of hesperidin reference substance, place it in a 50 mL volumetric flask, add methanol to dissolve and dilute to the mark to prepare a 0.1948 mg / mL stock solution. Accurately measure 5 mL of the stock solution and place it in a 25 mL volumetric flask, add methanol to prepare a 38.96 μg / mL reference solution.

[0371] 5.6.1.2 Preparation of test solution

[0372] Take 1 g of Xiangbei Weirong granules, weigh accurately, put it in a stoppered conical flask, accurately add 25 mL of 75% methanol, stopper it, weigh it, ultrasonically treat it for 15 min, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, take the filtrate, filter it with a 0.22 μm microporous filter membrane, and use it as the test solution.

[0373] 5.6.1.3 Preparation of negative control solution

[0374] Take the negative sample lacking tangerine peel and prepare it according to the method in “5.6.1.2 Preparation of test solution” to obtain the negative control solution lacking tangerine peel.

[0375] 5.6.1.4 Specificity Experiment

[0376] Accurately pipette 10 μL of reference solution, test solution, and negative sample solution without tangerine peel into the chromatograph and record the chromatogram. Fig. 27 .

[0377] From the chromatogram, it can be seen that for the negative control solution, no corresponding chromatographic peak appears at the same retention time position as the reference substance, indicating that other medicinal materials except hesperidin have no interference with the determination.

[0378] 5.7 Linearity and Range

[0379] 1 mL, 3 mL, 5 mL, 10 mL, and 20 mL of the hesperidin reference substance stock solution (concentration of 194.40 μg / mL) were accurately pipetted into a 25 mL volumetric flask, and methanol was added to the volume to obtain a series of reference substance solutions with concentrations of 7.78 μg / mL, 23.33 μg / mL, 38.88 μg / mL, 77.76 μg / mL, 155.52 μg / mL, and 194.40 μg / mL, respectively. 10 μL of the above reference substance solutions were accurately pipetted, injected into a high performance liquid chromatograph, and measured to obtain the peak area. The hesperidin injection amount (X) was used as the abscissa and the peak area (Y) was used as the ordinate. The linear regression equation was y=1602.3x+1.7522(R 2 =1), hesperidin has a good linear range between 0.078μg and 1.944μg. The linear regression graph is shown in Fig.28 , the results are shown in Table 37.

[0380] Table 37 Hesperidin linear data

[0381]

[0382] 5.8 Precision test

[0383] Accurately pipette 10 μL of hesperidin reference solution (38.88 μg / mL), repeat the injection 6 times, measure the peak area, calculate the average peak area value and relative standard deviation (RSD). The results show (see Table 38) that the average peak area of ​​hesperidin is 618.96, and the RSD value is 0.72%, indicating that the instrument has good precision.

[0384] Table 38 Hesperidin Precision

[0385]

[0386] 5.9 Repeatability test

[0387] About 1 g of the sample (batch number 20240110) was taken and accurately weighed. According to the preparation method of the test solution, 6 test solutions were prepared in parallel, and 10 μL was injected respectively. The peak area was determined, and the content, average content and RSD% were calculated. The results (see Table 39) showed that the average content of hesperidin in the sample was 0.54 mg / g, and the RSD was 0.14% (n=6), indicating good repeatability.

[0388] Table 39 Repeatability test

[0389]

[0390] 5.10 Stability test

[0391] The test solution of the repeatability test was injected at 0, 2.5, 5, 10, 15, 20, and 26 h, and the peak area of ​​hesperidin was determined. The average peak area and RSD (%) of hesperidin were calculated, as shown in Table 40. The results showed that the sample solution was stable within 26 h.

[0392] Table 40 Stability test results

[0393]

[0394] 5.11 Sample recovery rate

[0395] Preparation of reference substance stock solution: Accurately weigh 10.02 mg of hesperidin reference substance, place in a 50 mL volumetric flask, add methanol to the mark, shake well, and obtain 0.1948 mg / mL reference substance stock solution.

[0396] Using the sample recovery method, 6 portions of Xiangbei Weirong granules (batch number: 20240110, hesperidin content 0.54 mg / g) with determined content were taken, 0.5 g each, accurately weighed, and placed in a stoppered conical flask. 6 portions were accurately added with 1.5 mL of hesperidin reference solution (concentration 0.1948 mg / mL); prepared according to the test solution preparation method, 10 μL of the reference solution and the test solution were respectively drawn, injected into the high performance liquid chromatograph, the peak area was determined, and the sample recovery rate was calculated. The results are shown in Table 41. The recovery rate calculation method is as follows:

[0397]

[0398] Table 41 Recovery test results of hesperidin (n=6)

[0399]

[0400]

[0401] The results showed that the average recovery rate measured by this method was 104.51% and the RSD% was 1.58%, indicating that the recovery rate of hesperidin was good.

[0402] 5.12 Durability Examination

[0403] The effects of different flow rates, different column temperatures, different batches of the same type of chromatographic columns and different detection instruments on the determination of hesperidin content in Xiangbei Weirong granules were investigated respectively.

[0404] 5.12.1 Investigation of flow rate

[0405] Welch Ultimate XB-C18 (250mm×4.6mm, 5μm) was used as the chromatographic column, the column temperature was 30℃, and the detection wavelength was 283nm. The chromatographic separation effects of different flow rates (0.8mL / min, 1.0mL / min, 1.2mL / min) on hesperidin were investigated. The results are shown in Table 42. Fig.29 .

[0406] Table 42 Results of investigation at different flow rates

[0407]

[0408] The results showed that the flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min had little effect on the separation of the hesperidin chromatographic peak. According to the actual laboratory use, the flow rate was selected to be 1.0 mL / min.

[0409] 5.12.2 Investigation of column temperature

[0410] Welch Ultimate XB-C18 (250mm×4.6mm, 5μm) was used as the chromatographic column, the column temperature was 30℃, and the detection wavelength was 283nm. The chromatographic separation effect of hesperidin at different column temperatures (25℃, 30℃, 35℃) was investigated. The results are shown in Table 43. Fig.30 .

[0411] Table 43 Results of investigation at different column temperatures

[0412]

[0413] The results show that the column temperature of 25℃, 30℃ and 35℃ has no difference in the separation effect of the chromatographic peaks. Considering the actual use, the column temperature of 30℃ was selected.

[0414] 5.12.3 Examination of chromatographic columns

[0415] The effects of different batches of the same type of chromatographic columns on the determination of hesperidin content were investigated. The effects of different serial numbers and column numbers of Welch UltimateXB-C18 (4.6×250mm, 5μm) chromatographic columns on the determination of hesperidin content were investigated. The results are shown in Table 44 and Figure 31-32 The results showed that the hesperidin peak was baseline-separated from the adjacent chromatographic peaks with good resolution and good experimental reproducibility.

[0416] Table 44 Durability test

[0417]

[0418] In summary, the chromatographic conditions for hesperidin were determined as follows: octadecylsilane bonded silica gel as filler; methanol-2% acetic acid (35:65); flow rate 1 mL / min; column temperature 30°C; detection wavelength 283 nm; injection volume 10 μL.

[0419] 5.12.4 Investigation of different inspection instruments

[0420] The effects of different detection instruments on the determination of hesperidin content were investigated. Instrument 1: Agilent 1260infinity II HPLC; Instrument 2: Agilent 1260 HPLC, DAD UV detector. The results are shown in Table 45 and Figure 33-34 The results showed that the hesperidin peak was baseline-separated from the adjacent chromatographic peaks with good resolution and good experimental reproducibility.

[0421] Table 45 Durability test

[0422]

[0423] 5.13 Sample determination

[0424] Weigh about 1 g of 6 batches of samples accurately, prepare according to the preparation method of the test solution, inject 10 μL of each sample, record the chromatogram, determine the peak area, and calculate. The results are shown in Table 46.

[0425] Table 46 Determination results of hesperidin in six batches of Xiangbei Weirong granules

[0426]

[0427] The results show that the content of hesperidin in the test solution is about 0.50-1.10 mg / g. Considering the actual situation of large-scale production, the limit of hesperidin content is temporarily set at not less than 0.40 mg / g. Calculated based on the actual 10g per bag, each bag of this product contains tangerine peel as hesperidin (C 23 H 28 O 11 ) shall not be less than 4.0 mg.

[0428] 3. Clinical efficacy verification

[0429] In order to verify the efficacy of the preparation of the present invention, we further carried out clinical efficacy verification experiments.

[0430] 1. Experimental Design

[0431] 1.1 Source of cases

[0432] All cases were from outpatients and inpatients diagnosed with chronic gastritis in the Department of Gastroenterology of the Second Affiliated Hospital of Guizhou University of Chinese Medicine, and they were also voluntary clinical researchers to participate in this project.

[0433] 1.2 Collection time

[0434] From April 2022 to February 2024.

[0435] 1.3 Diagnostic criteria

[0436] Refer to the guidelines for clinical research of new Chinese medicines.

[0437] 1.3.1 Western medicine diagnostic criteria

[0438] The Western medicine diagnostic criteria refer to the "Chinese Consensus on Chronic Gastritis (2017)" formulated by the Gastroenterology Branch of the Chinese Medical Association, and meet the following two criteria:

[0439] (1) Clinical manifestations: no symptoms; or symptoms such as upper abdominal pain, abdominal distension, fullness after meals and early satiety.

[0440] (2) Endoscopic diagnosis: Endoscopic findings are consistent with the basic manifestations of chronic gastritis. In chronic non-atrophic gastritis, endoscopic findings include mucosal erythema, mucosal hemorrhages or plaques, mucosal roughness with or without edema, congestion and exudation. In chronic atrophic gastritis, endoscopic findings include alternating red and white mucosa, with the white phase being the predominant phase. The folds become flat or even disappear, and some mucosal blood vessels are exposed. Mucosal granules or nodules may be present.

[0441] 1.3.2 TCM diagnostic criteria

[0442] The diagnosis and classification standards for chronic gastritis were formulated with reference to the "Expert Consensus on the Diagnosis and Treatment of Chronic Gastritis in Traditional Chinese Medicine (2017 Edition)" formulated by the Spleen and Stomach Disease Branch of the Chinese Association of Traditional Chinese Medicine, see Table 47.

[0443] Table 47 Chronic gastritis symptom classification and quantification table

[0444]

[0445] 1.4 Inclusion criteria

[0446] (1) Meet the Western medical diagnostic criteria for chronic gastritis and the TCM syndrome diagnostic criteria;

[0447] (2) Patients with other diseases who do not require special treatment during treatment and do not affect the primary diagnosis and treatment;

[0448] (3) Patients’ gender and age are not limited;

[0449] (4) The medical records are complete and the records of prescription medication are detailed.

[0450] 1.5 Exclusion criteria

[0451] (1) did not meet the inclusion criteria;

[0452] (2) Patients with pathological diagnosis of severe gastric mucosal dysplasia;

[0453] (3) Patients with malignant tumors;

[0454] (4) Patients with severe primary diseases such as liver and kidney dysfunction, cardiovascular and cerebrovascular diseases, and hematopoietic system diseases;

[0455] (5) Pregnant or lactating women;

[0456] (6) Patients with various serious mental illnesses;

[0457] (7) The information is incomplete due to various reasons.

[0458] 2. Observation indicators

[0459] 2.1 Safety Observation

[0460] (1) General data analysis

[0461] Statistical analysis of the collected general data of the cases confirmed that there was no statistical difference in age, gender, gastritis type, Hp infection degree, etc. between the treatment group and the control group before treatment, and they were comparable. If adverse reactions occur, the clinical manifestations, time of occurrence, severity, symptomatic treatment methods and results should be recorded to objectively evaluate their safety.

[0462] 2.2 Efficacy observation

[0463] (1) According to the Guiding Principles for Clinical Research of New Chinese Medicines, the improvement of TCM syndrome scores was compared and analyzed. Epigastric or abdominal distension and epigastric pain were taken as the main symptoms, and each symptom was divided into four levels: I, II, III, and IV, with scores of 0, 2, 4, and 6, respectively; belching, acid reflux, nausea and vomiting, poor appetite, constipation, and poor sleep were taken as secondary symptoms, and each symptom was marked with scores of 0, 1, 2, and 3, respectively, from none, mild, moderate, and severe. The symptom level was determined according to the following criteria: Level I was no obvious symptoms; Level II was occasional symptoms, with slight discomfort, which basically did not affect normal work and life; Level III was frequent symptoms, the condition was more serious, had a certain impact on work and life, but was still tolerable; Level IV was more serious, and the patient's work and life were seriously affected. The syndrome scores before and after treatment were calculated to analyze the efficacy.

[0464] (2) Hp eradication rate: (total number of people in each group - number of Hp-positive people in each group) / total number of people in each group.

[0465] 3. Efficacy evaluation

[0466] 3.1 Criteria for evaluating efficacy

[0467] (1) Evaluation basis (refer to the Guiding Principles for Clinical Research of New Chinese Medicines).

[0468] (2) Calculation method (Nimodipine method): (score before treatment - score after treatment) / score before treatment × 100%

[0469] (3) Evaluation criteria:

[0470] ①Clinical control: clinical symptoms and signs disappear, and the score is reduced by ≥95%.

[0471] ② Significantly effective: Clinical symptoms and signs were significantly improved, with the score reduced by ≥70% and <95%.

[0472] ③Effective: The clinical symptoms and signs were reduced by ≥30% and <70%.

[0473] ④ Ineffective: There is no significant improvement in clinical symptoms and signs, or the score decreases by less than 30%.

[0474] 4. Statistical analysis

[0475] SPSS 26.0 software was used for statistical analysis of the data. Kolmogorov-Smirnov test was used to test the normal distribution of continuous variables. Normally distributed quantitative data were expressed in the form of paired sample t test to compare the differences between the two groups before and after treatment. P < 0.05 was considered statistically significant.

[0476] 5. Test results

[0477] 5.1 Comprehensive efficacy

[0478] The total effective rate after treatment was 94%. See Table 48.

[0479] Table 48 Comparison of the efficacy of TCM symptoms before and after treatment (x 2 , n=30)

[0480]

[0481] 5.2 Comparison of clinical symptom and sign scores between groups

[0482] The results are shown in Table 49. Compared with before treatment, the clinical symptom and sign scores after treatment were significantly higher than those before treatment, and the difference was extremely statistically significant (P < 0.001).

[0483] Table 49 Comparison of clinical symptom and sign scores between groups before and after treatment

[0484]

[0485] Note: Intra-group comparison: compared with before treatment, # P<0.001.

[0486] 5.3 Comparison of symptom scores between groups

[0487] The results are shown in Table 50. Compared with before treatment, the scores of clinical symptoms and signs between the groups after treatment were significantly higher than those before treatment, and the difference was extremely statistically significant (P < 0.001).

[0488] Table 50 Comparison of symptoms and signs between groups before and after treatment

[0489]

[0490] Note: Comparison of symptoms and signs within the group: Compared with before treatment, # P<0.001.

[0491] 6. Adverse reactions: No adverse reactions occurred during the observation of this subject.

[0492] 7. Comprehensive efficacy analysis

[0493] The results of this study showed that Xiangbei Weirong Recipe can improve the symptoms and signs of patients with chronic gastritis, with a total effective rate of 94%. This shows that Xiangbei Weirong Recipe can significantly improve the clinical symptoms and signs of patients with chronic gastritis, and is an effective and safe Chinese medicine preparation for the treatment of chronic gastritis, which is worthy of promotion and application in clinical practice.

[0494] Although the present invention has been described in detail above by means of general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A fragrant and delicious composition, characterized in that: The formula is: 130-170 parts of Codonopsis pilosula, 100-140 parts of Atractylodes macrocephala fried with bran, 80-120 parts of Poria cocos, 80-120 parts of dried tangerine peel, 50-70 parts of costus root, 80-120 parts of Fritillaria thunbergii, 105-125 parts of Pinellia ternata, 80-120 parts of Curcuma aromatica, 80-120 parts of stir-fried chicken gizzard lining, 100-140 parts of lily, 80-120 parts of processed Polygala tenuifolia, 80-120 parts of vinegar Corydalis yanhusuo, 130-170 parts of Artemisia capillaris, 180-220 parts of vinegar turtle shell, 80-120 parts of cuttlebone, and 50-70 parts of liquorice tablets.

2. A fragrant and delicious composition according to claim 1, characterized in that: The formula is: 140-160 parts of Codonopsis pilosula, 110-130 parts of Atractylodes macrocephala fried with bran, 90-110 parts of Poria cocos, 90-110 parts of dried tangerine peel, 55-65 parts of costus root, 90-110 parts of Fritillaria thunbergii, 110-120 parts of Pinellia ternata, 90-110 parts of Curcuma aromatica, 90-110 parts of stir-fried chicken gizzard lining, 110-130 parts of lily, 90-110 parts of processed Polygala tenuifolia, 90-110 parts of vinegar-cooked Corydalis yanhusuo, 140-160 parts of Artemisia capillaris, 190-210 parts of vinegar-cooked turtle shell, 90-110 parts of cuttlebone, and 55-65 parts of liquorice tablets.

3. A fragrant and delicious composition according to claim 1, characterized in that: The formula is: 150 parts of Codonopsis pilosula, 120 parts of Atractylodes macrocephala fried with bran, 100 parts of Poria cocos, 100 parts of dried tangerine peel, 60 parts of costus root, 100 parts of Fritillaria thunbergii, 117 parts of Pinellia ternata, 100 parts of Curcuma aromatica, 100 parts of stir-fried chicken gizzard lining, 120 parts of lily, 100 parts of processed Polygala tenuifolia, 100 parts of vinegar-cooked Corydalis yanhusuo, 150 parts of Artemisia capillaris, 200 parts of vinegar-cooked turtle shell, 100 parts of cuttlebone, and 60 parts of liquorice tablets.

4. A method for preparing the fragrant and delicious stomach composition according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: taking a prescribed amount of medicinal materials, adding 7-8 times of water for the first time to soak for 1-2 hours, adding 6-10 times of water for the second time, decocting for 1-4 times, each time for 20-60 minutes, filtering, concentrating the filtrate to a thick paste with a relative density of 1.24-1.35 at 70° C., drying, crushing, adding appropriate amount of auxiliary materials, and preparing a corresponding dosage form.

5. The method for preparing a fragrant and delicious stomach composition according to claim 4, characterized in that: The preparation method is as follows: taking a prescribed amount of medicinal materials, adding 7.5 times of water for the first time to soak for 1 hour, adding 6 times of water for the second time, decocting twice, each time for 30 minutes, filtering, concentrating the filtrate to a thick paste with a relative density of 1.28-1.32 at 70°C, drying, crushing, adding appropriate amount of auxiliary materials, and preparing a corresponding dosage form.

6. A quality testing method for the Xiangbei Weirong composition according to any one of claims 1 to 3 or the Xiangbei Weirong composition prepared by the preparation method according to any one of claims 4 to 5, characterized in that: It includes thin layer chromatography identification of dried tangerine peel, costusroot, Fritillaria thunbergii, and vinegar-cured Corydalis and the detection of hesperidin content.

7. The quality inspection method according to claim 6, characterized in that: The thin layer chromatography identification method is as follows: The thin layer chromatography identification method of dried tangerine peel is as follows: take 5g of this product, add 25mL of methanol, ultrasonically treat for 20 minutes, filter, and concentrate the filtrate to 2mL as the test solution. Take another 5g of the negative preparation without dried tangerine peel, and prepare the negative control solution in the same way; take 1g of dried tangerine peel control medicinal material, and prepare the control medicinal material solution in the same way; according to the general rule 0502 thin layer chromatography test, take 2μL of each of the above three solutions, and spot them on the same silica gel G thin layer plate, use ethyl acetate-methanol-water 100:17:13 as the developing solvent, develop, take out, dry, spray with aluminum chloride test solution, and inspect under ultraviolet light at 365nm; The thin layer chromatography identification method of costus root is as follows: take 10g of the product, add 50mL of water to dissolve it, filter it, extract the filtrate with 40mL of ethyl acetate, take the ethyl acetate layer, evaporate it to dryness, and add 1mL of ethyl acetate to dissolve the residue as the test solution. Take another 10g of the negative preparation lacking costus root, and prepare the negative control solution in the same way; take 0.5g of costus root control medicinal material, add 50mL of water, decoct for 30min, filter it, and prepare the control medicinal material solution with the filtrate in the same way as the test solution preparation method; according to the general rule 0502 thin layer chromatography test, take 5μL of the control medicinal material solution, 5-10μL of the test solution and the negative control solution, respectively, and spot them on the same silica gel G thin layer plate, use the upper layer solution of cyclohexane-ethyl formate-formic acid 15:5:1 as the developing agent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, and heat at 105°C until the spots are clearly colored; The thin layer identification method of Fritillaria thunbergii is as follows: take 10g of the product, add 2mL of concentrated ammonia test solution and 50mL of chloroform, heat and reflux for 1h, filter, recover the solvent from the filtrate until dry, add 2mL of chloroform to the residue to dissolve it, and use it as the test solution; take another 10g of the negative preparation without Fritillaria thunbergii, and prepare the negative control solution in the same way; take 1g of Fritillaria thunbergii control medicinal material, and prepare the control medicinal material solution in the same way; according to the thin layer chromatography test in general rule 0502, take 10 to 20μL of each of the above three solutions, and spot them on the same silica gel G thin layer plate, use ethyl acetate-methanol-concentrated ammonia test solution 17:2:1 as the developing solvent, develop, take out, dry, and spray with dilute potassium bismuth iodide test solution; Thin layer chromatography identification of the vinegar Corydalis: take 5g of the product, add 50mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, add 10mL of water to the residue to dissolve it, add concentrated ammonia test solution to adjust to alkalinity, shake and extract with ether for 3 times, 10mL each time, combine the ether solution, evaporate to dryness, add 1mL of methanol to the residue to dissolve it, as the test solution; take another 5g of the negative preparation lacking vinegar Corydalis, and prepare a negative control solution in the same way; take 1g of Corydalis control medicinal material, and prepare a control medicinal material solution in the same way; according to the general rule 0502 thin layer chromatography test, absorb 4-5μL of each of the above three solutions, respectively spot them on the same silica gel G thin layer plate, use toluene-acetone 9:2 as the developing agent, develop, take out, dry, put it in an iodine cylinder for about 3 minutes, take it out, after evaporating the iodine adsorbed on the plate, put it under a UV lamp at 365nm for inspection.

8. The quality inspection method according to claim 6, characterized in that: The hesperidin content detection method is as follows: chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as filler, the mobile phase is methanol: 2% acetic acid = 35:65, the flow rate is 1 mL / min, the column temperature is 30°C, the detection wavelength is 283 nm, the injection volume is 10 μL, and the theoretical plate number should be not less than 2000 calculated based on the hesperidin peak; preparation of reference solution: take an appropriate amount of hesperidin reference, accurately weigh it, add methanol to make a solution containing 40 μg per 1 mL, and obtain the solution; preparation of test solution: take 1 g of Xiangbei Weirong composition, accurately weigh it, put it in a stoppered conical bottle, accurately add 25 mL of 75% methanol, seal it, weigh the weight, ultrasonically treat it for 15 minutes, let it cool, weigh the weight again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution; determination method: accurately aspirate 10 μL of reference solution and test solution respectively, inject them into a liquid chromatograph, and determine them to obtain the solution.

9. Use of the Xiangbei Weirong composition as described in any one of claims 1 to 3 or the Xiangbei Weirong composition prepared by the preparation method as described in any one of claims 4 to 5 in preparing a medicine for treating chronic gastritis.

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