Gastric retention type traditional Chinese medicine probiotic double-layer floating tablet and preparation method thereof

Abstract

The invention provides an intragastric retention type traditional Chinese medicine probiotic double-layer floating tablet and a preparation method thereof, and belongs to the technical field of pharmaceutical preparation preparation. The traditional Chinese medicine probiotic double-layer floating tablet comprises a dendrobium huoshanense extract layer, a probiotic layer and an adhesive, wherein the dendrobium huoshanense extract layer is prepared from a dendrobium huoshanense extract, hydroxypropyl methyl cellulose, octadecanol, polyacrylic resin II, magnesium stearate microcrystalline cellulose and xylitol; the probiotic layer is prepared from probiotic powder, soluble starch, xylitol, mannitol, magnesium stearate and dextrin. The gastric retention type traditional Chinese medicine probiotic double-layer floating tablet disclosed by the invention can meet the continuous treatment of the dendrobium huoshanense extract on gastric mucosa, can also meet the quick release of the probiotics, provides a continuous carbon source and promotes the proliferation of the probiotics, so that the gastrointestinal synergistic treatment effect is exerted, the medicine taking frequency of a patient is reduced, and the bioavailability of the patient is improved. The bioavailability of the dendrobium huoshanense extract is improved, the survival rate of probiotics is enhanced, and the intestinal flora is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparation, and particularly relates to a gastric retention type traditional Chinese medicine probiotic double-layer floating tablet and a preparation method thereof. Background Art

[0002] Dendrobium huoshanense C.Z.Tang et S.J.Cheng is a perennial herb of the genus Dendrobium in the Orchidaceae family. It was first recorded in "Shennong's Herbal Classic" and is a traditional precious Chinese medicinal material, with the effects of tonifying the spleen and stomach, protecting the liver and gallbladder, and nourishing yin and clearing heat. Dendrobium huoshanense has a high content of water-soluble polysaccharides and a relatively sticky texture, and the low bioavailability of traditional preparations limits its effect of "strengthening the stomach and intestines".

[0003] Probiotics are the general term for microorganisms that colonize the human intestinal tract and maintain the microecology in the intestine. Appropriate intake of probiotics helps to correct intestinal flora disorders, promote the proliferation of beneficial bacteria, improve human gastrointestinal health, and can also enhance the body's immunity, improve the immune regulation effect, and even have the effect of antibiotics. However, oral administration of probiotics is the only daily way to supplement probiotics, but the colonization rate of oral probiotics in the large intestine is relatively low, and the long-term conditioning has little effect.

[0004] Therefore, it is an urgent technical problem to provide a preparation that can not only meet the continuous treatment of Dendrobium huoshanense on the gastric mucosa, but also meet the rapid release of probiotics, promote proliferation, and improve the intestinal flora. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a gastric retention type traditional Chinese medicine probiotic double-layer floating tablet and a preparation method thereof. The gastric retention type traditional Chinese medicine probiotic double-layer floating tablet has a complete finished product and uniform color, can start floating instantaneously in gastric juice, has a floating time > 10h, and can achieve the purpose of slow-release drugs.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a gastric retention type traditional Chinese medicine probiotic double-layer floating tablet, which includes a Dendrobium huoshanense extract layer, a probiotic layer, and a binder;

[0008] The Dendrobium huoshanense extract layer is made of the following raw materials in mass percentages:

[0009] Dendrobium huoshanense extract 25% - 35%, hydroxypropyl methylcellulose 7% - 9%, stearyl alcohol 16% - 20%, polyacrylic resin II 22% - 30%, magnesium stearate 0.8% - 1.5%, and microcrystalline cellulose and xylitol are added up to 100%. The mass ratio of microcrystalline cellulose to xylitol is 12 - 17:4;

[0010] The probiotic layer is made of raw materials in the following mass percentages:

[0011] Probiotic powder 15% - 22%, soluble starch 35% - 42%, xylitol 12% - 16%, mannitol 8% - 10%, magnesium stearate 0.8% - 1.5%, and filled to 100% with dextrin.

[0012] Preferably, the Dendrobium huoshanense extract is the water extract of Dendrobium huoshanense; the probiotic powder includes a fifteen - strain composite probiotic.

[0013] Preferably, the preparation method of the Dendrobium huoshanense extract includes the following steps:

[0014] After drying and crushing the dry stems of Dendrobium huoshanense, sieving is carried out to obtain the crushed Dendrobium huoshanense; the crushed Dendrobium huoshanense is mixed with water, subjected to extraction, and the filtrate after extraction is collected, concentrated and dried to obtain the Dendrobium huoshanense extract.

[0015] Preferably, the mesh number of the sieving is 80 - 120 meshes.

[0016] Preferably, the mass - to - volume ratio of the crushed Dendrobium huoshanense to water is 1 g:15 - 25 mL.

[0017] Preferably, the temperature of the extraction is 65 - 85 °C, the number of extraction times is 1 - 3 times, and the extraction time for each time is 1 - 3 h.

[0018] Preferably, the mass ratio of the Dendrobium huoshanense extract layer to the probiotic layer is 1:1.

[0019] Preferably, the binder is the binder for the Dendrobium huoshanense extract layer and the probiotic layer binder. The binder for the Dendrobium huoshanense extract layer is water, and the probiotic layer binder is malt syrup.

[0020] The present invention also provides a preparation method of the above - mentioned gastric - retention traditional Chinese medicine probiotic double - layer floating tablets, including the following steps:

[0021] Mix the Dendrobium huoshanense extract, hydroxypropyl methylcellulose, stearyl alcohol, polyacrylic resin II, microcrystalline cellulose, and xylitol, add water as a binder, mix and prepare to obtain a soft material, carry out wet granulation, dry and size - reduce, and add magnesium stearate to obtain the Dendrobium huoshanense extract layer;

[0022] Mix the probiotic powder, soluble starch, xylitol, dextrin, and mannitol, add malt syrup as a binder, carry out wet granulation, freeze - dry, and add magnesium stearate to obtain the probiotic layer;

[0023] First, press the Dendrobium huoshanense extract layer for the first time to obtain Dendrobium huoshanense tablets, and then add a probiotic layer to the Dendrobium huoshanense tablets for the second press to obtain a gastric retention type traditional Chinese medicine probiotic double-layer floating tablet.

[0024] Preferably, the hardness of the first press is 40-60 N; the hardness of the second press is 10-15 N.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides a gastric retention type traditional Chinese medicine probiotic double-layer floating tablet and a preparation method thereof. The finished product of the gastric retention type traditional Chinese medicine probiotic double-layer floating tablet of the present invention is complete, has a uniform color, has a smooth finish, the weight difference conforms to the provisions of the Chinese Pharmacopoeia, has an appropriate hardness, can float in gastric juice, the floating time > 10 h, has the effect of slow-release drug, and the disintegration time limit of the Dendrobium huoshanense extract layer and the probiotic layer in the traditional Chinese medicine probiotic double-layer floating tablet conforms to the provisions of the Chinese Pharmacopoeia (2020 Edition). The gastric retention type traditional Chinese medicine probiotic double-layer floating tablet of the present invention can meet the continuous treatment of the gastric mucosa by the Dendrobium huoshanense extract, and can also meet the rapid release of probiotics and provide a continuous carbon source to promote the proliferation of probiotics, so as to play a synergistic gastrointestinal treatment role, reduce the frequency of taking medicine by patients, improve the bioavailability of the Dendrobium huoshanense extract, enhance the survival rate of probiotics, and improve the intestinal flora. Description of the Drawings

[0027] Figure 1 It is the detection result of the floating performance of the Dendrobium huoshanense tablets prepared by the present invention;

[0028] Figure 2 It is the glucose concentration standard curve;

[0029] Figure 3 It is the in vitro release curve of 3 batches of Dendrobium huoshanense tablets prepared in Example 1;

[0030] Figure 4 It is the appearance of the gastric retention type traditional Chinese medicine probiotic double-layer floating tablet prepared in Example 1;

[0031] Figure 5 It is the observation diagram of the gastric tissue injury of mice after treatment in different groups. A is the observation diagram of the gastric tissue injury of mice after treatment in the blank group; B is the observation diagram of the gastric tissue injury of mice after treatment in the model group; C is the observation diagram of the gastric tissue injury of mice after treatment in the positive control group; D is the observation diagram of the gastric tissue injury of mice after treatment in the water extract group of Dendrobium huoshanense; among them, the position pointed by the black arrow is the position of gastric mucosa congestion;

[0032] Figure 6HE staining results of mouse gastric tissues after treatment with different groups (10.0×). a: HE staining results of mouse gastric tissues after treatment in the blank group; b: HE staining results of mouse gastric tissues after treatment in the model group; c: HE staining results of mouse gastric tissues after treatment in the positive control group; d: HE staining results of mouse gastric tissues after treatment in the aqueous extract group of Dendrobium huoshanense.

[0033] Figure 7 Results of the absorbance of probiotic suspensions in different groups changing with time during the cultivation of aerobic probiotics.

[0034] Figure 8 Results of the absorbance of probiotic suspensions in different groups changing with time during the cultivation of anaerobic probiotics. Detailed implementation manners

[0035] The present invention provides a gastric-retention type traditional Chinese medicine probiotic double-layer floating tablet, and the traditional Chinese medicine probiotic double-layer floating tablet includes a Dendrobium huoshanense extract layer, a probiotic layer and a binder.

[0036] The Dendrobium huoshanense extract layer is made of raw materials with the following mass percentages:

[0037] Dendrobium huoshanense extract 25% - 35%, hydroxypropyl methylcellulose 7% - 9%, stearyl alcohol 16% - 20%, polyacrylic resin II 22% - 30%, magnesium stearate 0.8% - 1.5%, and microcrystalline cellulose and xylitol are added up to 100%. The mass ratio of microcrystalline cellulose to xylitol is 12 - 17:4.

[0038] The probiotic layer is made of raw materials with the following mass percentages:

[0039] Probiotic powder 15% - 22%, soluble starch 35% - 42%, xylitol 12% - 16%, mannitol 8% - 10%, magnesium stearate 0.8% - 1.5%, and dextrin is added up to 100%.

[0040] In the present invention, the Dendrobium huoshanense extract is preferably the water extract of Dendrobium huoshanense; the probiotic powder comprises a fifteen-strain compound probiotic. The present invention has no special limitation on the source of the fifteen-strain compound probiotic, and a product well-known in the art can be used. The mass ratio of the Dendrobium huoshanense extract layer to the probiotic layer is 1:1. The binder is the binder for the Dendrobium huoshanense extract layer and the binder for the probiotic layer. The binder for the Dendrobium huoshanense extract layer is water, and the binder for the probiotic layer is malt syrup. The present invention has found through research that alcohol-based binders will inhibit the activity of probiotics and cannot enable the probiotics to play the role of regulating the intestinal flora, thereby reducing the efficacy of the probiotics. The present invention uses malt syrup as the binder for the probiotic layer to ensure the activity of the probiotics and the formability of the probiotic layer. The dosage of the binder is such that the Dendrobium huoshanense extract layer or the probiotic layer reaches the state of "forming a ball when held in the hand and dispersing when touched".

[0041] In the present invention, the gastric retention type traditional Chinese medicine probiotic double-layer floating tablet can float in gastric juice, and the floating function mainly depends on the performance of the Dendrobium huoshanense layer auxiliary carrier. The gastric retention type traditional Chinese medicine probiotic double-layer floating tablet will float on the surface of gastric juice. Among them, the probiotic layer conforms to the tablet drug release rule, and the probiotics enter the small intestine and then the large intestine through the pylorus along with gastric juice; while the floating Dendrobium huoshanense layer will slowly release the Dendrobium huoshanense extract with the help of the drug-loading auxiliary: a part of the Dendrobium huoshanense extract released earlier is dispersed in gastric juice, playing the role of protecting the gastric mucosa, and flowing into the small intestine and large intestine along with gastric juice. The Dendrobium huoshanense extract reaching the large intestine can be used as a carbon source for probiotics to promote the proliferation of probiotics; while another part of the Dendrobium huoshanense extract released later continuously replenishes the content of Dendrobium huoshanense extract in gastric juice and continuously repairs the gastric mucosa. The Dendrobium huoshanense extract not completely absorbed then flows into the large intestine as a continuous carbon source for probiotics to promote the proliferation of probiotics.

[0042] In the present invention, the preparation method of the Dendrobium huoshanense extract preferably comprises the following steps:

[0043] After drying and pulverizing the dry stems of Dendrobium huoshanense, sieving is carried out to obtain the pulverized Dendrobium huoshanense; the pulverized Dendrobium huoshanense is mixed with water, subjected to extraction, the filtrate after extraction is collected, concentrated and dried to obtain the Dendrobium huoshanense extract.

[0044] When preparing the extract of Dendrobium huoshanense, the dried stems of Dendrobium huoshanense are crushed and then sieved to obtain the crushed Dendrobium huoshanense. The mesh number of the sieving is preferably 80-120 meshes, more preferably 90-110 meshes, and even more preferably 100 meshes. After obtaining the crushed Dendrobium huoshanense, the crushed Dendrobium huoshanense is mixed with water and subjected to extraction, and the filtrate after extraction is collected. The mass-volume ratio of the crushed Dendrobium huoshanense to water is preferably 1 g:15-25 mL, more preferably 1 g:17-22 mL, and even more preferably 1 g:20 mL. The temperature of the extraction is preferably 65-85 °C, more preferably 70-85 °C, and even more preferably 70, 75, 80 or 85 °C; the number of extraction times is preferably 1-3 times, more preferably 2 times, and the extraction time for each time is preferably 1-3 h, more preferably 1.5-2.5 h, and even more preferably 2 h. Through the above method, the active ingredients of Dendrobium huoshanense, such as the total polysaccharide of Dendrobium huoshanense, can be extracted quickly and in high content. After obtaining the filtrate after extraction, the filtrate after extraction is concentrated and dried to obtain the extract of Dendrobium huoshanense. The present invention has no special limitation on the concentration method, and the methods well-known in the art can be used, such as concentration under reduced pressure at 60 °C. The present invention has no special limitation on the drying method, and the methods well-known in the art can be used, such as drying by baking or freeze-drying. The drying method by baking includes drying at 50-60 °C for 30 min.

[0045] In the present invention, the water extract of Dendrobium huoshanense is sticky in texture and is likely to agglomerate when mixed with other excipients, making it difficult to mix evenly. Through the adjustment of excipients, the prepared gastric retention-type traditional Chinese medicine probiotic double-layer floating tablets have complete tablets, uniform color, gloss, and have a sustained-release effect.

[0046] The present invention also provides a preparation method of the above-mentioned gastric retention-type traditional Chinese medicine probiotic double-layer floating tablets, which includes the following steps:

[0047] Mix the extract of Dendrobium huoshanense, hydroxypropyl methylcellulose, stearyl alcohol, polyacrylic resin II, microcrystalline cellulose, and xylitol, add water as a binder, mix and prepare to obtain a soft material, granulate by wet method, dry and size the granules, and add magnesium stearate to obtain the Dendrobium huoshanense extract layer;

[0048] Mix probiotic powder, soluble starch, xylitol, dextrin, and mannitol, add malt syrup as a binder, granulate by wet method, freeze-dry, and add magnesium stearate to obtain the probiotic layer;

[0049] First, the Dendrobium huoshanense extract layer is subjected to the first pressing to obtain Dendrobium huoshanense tablets, and then the probiotic layer is added on the Dendrobium huoshanense tablets for the second pressing to obtain the gastric retention-type traditional Chinese medicine probiotic double-layer floating tablets.

[0050] When preparing the Dendrobium huoshanense extract layer, the amount of water added is such that the Dendrobium huoshanense extract layer reaches the state of "forming a ball when held in the hand and dispersing immediately when touched". The wet granulation method is to make wet granules through a 24-mesh nylon sieve. The drying can be selected as drying by baking or freeze-drying. Among them, the drying by baking is drying at 50-60°C for 25-35 minutes. After drying, it also includes sizing with 24-mesh and 80-mesh sieves.

[0051] When preparing the probiotic layer, the amount of maltose syrup added is such that the probiotic layer reaches the state of "forming a ball when held in the hand and dispersing immediately when touched". The invention has no special limitation on the freeze-drying method, and the methods well-known in the art can be adopted.

[0052] During pressing, the hardness of the first pressing is 40-60 N; the hardness of the second pressing is 10-15 N. The invention adopts the method of pressing the Dendrobium huoshanense extract layer first and then pressing the probiotic layer. The prepared gastric retention type traditional Chinese medicine probiotic double-layer floating tablets meet the requirement of the priority release of the probiotic layer, and the Dendrobium huoshanense layer slowly dissolves, and the average disintegration time limit is more than 12 h.

[0053] In the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art.

[0054] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0055] In the following embodiments, the fifteen-strain compound probiotic (Xi'an Jushengyuan Biotechnology Co., Ltd., product batch number 20230801), dextrin (Changling Jilong Biopharmaceutical Co., Ltd., product batch number 20230407), magnesium stearate (Shandong Liujia Pharmaceutical Excipients Co., Ltd., product batch number 20221201), xylitol (Shandong Futian Pharmaceutical Co., Ltd., product batch number 20230601), soluble starch (Tianjin Damao Chemical Reagent Factory, product batch number 20230801), maltose syrup (trade name 5% maltose syrup, Chunyuan Food (Yucheng) Co., Ltd., product batch number 20230701).

[0056] Microcrystalline cellulose (Shandong Liujia Pharmaceutical Excipients Co., Ltd., product batch number 130701), xylitol (Anhui Yishou Jinfang Guoyao Co., Ltd., product batch number C118062803), stearyl alcohol (Jiangxi Alpha High Pharmaceutical Co., Ltd., product batch number 20200201), polyacrylic resin II, stearic acid (both purchased from Huzhou Zhanwang Pharmaceutical Co., Ltd., product batch numbers are 210602 and 210201 respectively), sodium carboxymethyl cellulose (Shanghai Macklin Biochemical Technology Co., Ltd., product batch number 20201007), hypromellose (Hunan Xinlvfang Pharmaceutical Co., Ltd., product batch number 21021804).

[0057] Example 1

[0058] A preparation method of a gastric retention type traditional Chinese medicine probiotic double - layer floating tablet is as follows:

[0059] (1) Preparation of Dendrobium huoshanense extract: Take the dried stems of Dendrobium huoshanense, crush them, and pass through a 100 - mesh sieve. Mix the crushed Dendrobium huoshanense with water at a mass - to - volume ratio of 1 g:20 mL, extract at 70 °C for 2 h, filter to obtain the first filtrate and the first filter residue. Mix the first filter residue with water at a mass - to - volume ratio of 1 g:20 mL, extract at 70 °C for 2 h, filter and collect the second filtrate. Combine the first filtrate and the second filtrate to obtain the extracted filtrate. Concentrate the extracted filtrate under reduced pressure at 60 °C and freeze - dry it to obtain the Dendrobium huoshanense extract;

[0060] (2) 30% of Dendrobium huoshanense extract, 8% of hydroxypropyl methylcellulose (HPMC), 18% of stearyl alcohol, 28% of polyacrylic resin II, 11% of microcrystalline cellulose, 4% of xylitol. Pass through a sieve, mix evenly, use an appropriate amount of water as a binder to reach the state of forming a ball when held in the hand and dispersing when touched, mix to prepare a soft material, pass through a 24 - mesh nylon sieve to make wet granules, dry at 55 °C for 30 minutes, screen with 24 - mesh and 80 - mesh sieves, retain the granules larger than 80 - mesh and smaller than 24 - mesh, and then add 1% of magnesium stearate to obtain the Dendrobium huoshanense extract layer material;

[0061] Among them, each percentage in this step refers to the percentage of the mass of each material in the total mass of the materials, and the total mass of the materials refers to the total mass of Dendrobium huoshanense extract, hydroxypropyl methylcellulose, stearyl alcohol, polyacrylic resin II, magnesium stearate, microcrystalline cellulose and xylitol;

[0062] (3) Take 20% of the fifteen - strain composite probiotic, 40% of soluble starch, 15% of xylitol, 15% of dextrin, 9% of mannitol. Pass through a sieve, mix evenly, use an appropriate amount of malt syrup as a binder to reach the state of forming a ball when held in the hand and dispersing when touched, use wet granulation, freeze - dry, add 1% of magnesium stearate, and mix evenly to obtain the probiotic layer material; Among them, each percentage in this step refers to the percentage of the mass of each material in the total mass of the materials, and the total mass of the materials refers to the total mass of the fifteen - strain composite probiotic, soluble starch, xylitol, dextrin, mannitol and magnesium stearate;

[0063] (4) Take the Dendrobium huoshanense extract layer material and press it at a pressure of 50 N to obtain the Dendrobium huoshanense tablet layer. Then add an equal mass of the probiotic layer material on the Dendrobium huoshanense tablet layer and perform secondary tableting at a pressure of 13 N to obtain the gastric retention type traditional Chinese medicine probiotic double - layer floating tablet.

[0064] Comparative Example 1

[0065] The difference between this comparative example and Example 1 lies in step (2) of this comparative example: the amount of Dendrobium huoshanense extract is 40%, hydroxypropyl methylcellulose (HPMC) is 6.9%, stearyl alcohol is 15.4%, polyacrylic resin II is 24%, microcrystalline cellulose is 9.4%, xylitol is 3.4%. Sieve, mix well, use an appropriate amount of water as a binder to reach the state of being able to form a ball in the hand and falling apart when touched, mix to prepare soft materials, pass through a 24-mesh nylon sieve to make wet granules, dry at 55°C for 30 minutes, size the granules with a 24-mesh and an 80-mesh sieve, retain the granules larger than 80 mesh and smaller than 24 mesh, and then add 0.9% of magnesium stearate to obtain the Dendrobium huoshanense extract layer material. The remaining steps are the same as those in Example 1.

[0066] Comparative Example 2

[0067] The difference between this comparative example and Example 1 lies in step (2) of this comparative example: the amount of Dendrobium huoshanense extract is 50%, hydroxypropyl methylcellulose (HPMC) is 5.7%, stearyl alcohol is 12.9%, polyacrylic resin II is 20%, microcrystalline cellulose is 7.9%, xylitol is 2.9%. Sieve, mix well, use an appropriate amount of water as a binder to reach the state of being able to form a ball in the hand and falling apart when touched, mix to prepare soft materials, pass through a 24-mesh nylon sieve to make wet granules, dry at 55°C for 30 minutes, size the granules with a 24-mesh and an 80-mesh sieve, retain the granules larger than 80 mesh and smaller than 24 mesh, and then add 0.6% of magnesium stearate to obtain the Dendrobium huoshanense extract layer material. The remaining steps are the same as those in Example 1.

[0068] Comparative Example 3

[0069] The difference between this comparative example and Example 1 lies in that in step (2) of this comparative example, 8% of hydroxypropyl methylcellulose (HPMC), 28% of polyacrylic resin II (Eudragit II), and 11% of microcrystalline cellulose are replaced with 32% of hydroxypropyl methylcellulose (HPMC) and 15% of microcrystalline cellulose. The remaining steps are the same as those in Example 1.

[0070] Comparative Example 4

[0071] The difference between this comparative example and Example 1 lies in that in step (2) of this comparative example, 8% of hydroxypropyl methylcellulose, 28% of polyacrylic resin II, and 11% of microcrystalline cellulose are replaced with 32% of polyacrylic resin II and 15% of microcrystalline cellulose. The remaining steps are the same as those in Example 1.

[0072] Comparative Example 5

[0073] The difference between this comparative example and Example 1 lies in that in step (2) of this comparative example, 8% of hydroxypropyl methylcellulose (HPMC), 28% of polyacrylic resin II, and 11% of microcrystalline cellulose are replaced with 32% of sodium carboxymethylcellulose (CMC-Na) and 15% of microcrystalline cellulose. The remaining steps are the same as those in Example 1.

[0074] Comparative Example 6

[0075] The difference between this comparative example and Example 1 lies in that in step (2) of this comparative example, 8% hydroxypropyl methylcellulose (HPMC), 28% polyacrylic resin II, and 11% microcrystalline cellulose are replaced with 8% hydroxypropyl methylcellulose (HPMC), 24% sodium carboxymethylcellulose (CMC-Na), and 15% microcrystalline cellulose, and the remaining steps are the same as those in Example 1.

[0076] Comparative Example 7

[0077] The difference between this comparative example and Example 1 lies in that in step (2) of this comparative example, 8% hydroxypropyl methylcellulose (HPMC), 28% polyacrylic resin II, and 11% microcrystalline cellulose are replaced with 24% hydroxypropyl methylcellulose (HPMC), 8% sodium carboxymethylcellulose (CMC-Na), and 15% microcrystalline cellulose, and the remaining steps are the same as those in Example 1.

[0078] Comparative Example 8

[0079] The difference between this comparative example and Example 1 lies in that in step (2) of this comparative example, 18% stearyl alcohol is replaced with 18% stearic acid, and the remaining steps are the same as those in Example 1.

[0080] Comparative Example 9

[0081] The difference between this comparative example and Example 1 lies in that in step (2) of this comparative example, 11% microcrystalline cellulose and 4% xylitol are replaced with 15% microcrystalline cellulose, and the remaining steps are the same as those in Example 1.

[0082] Comparative Example 10

[0083] The difference between this comparative example and Example 1 lies in that in step (2) of this comparative example, the amount of Dendrobium huoshanense extract is 30.24%, hydroxypropyl methylcellulose (HPMC) is 8.06%, stearyl alcohol is 18.15%, polyacrylic resin II is 28.23%, microcrystalline cellulose is 11.09%, xylitol is 4.03%, sieved, mixed evenly, and an appropriate amount of water is used as a binder to reach the state of being able to form a ball by hand and falling apart when touched, mixed to prepare soft materials, passed through a 24-mesh nylon sieve to make wet granules, dried at 55°C for 30 minutes, sized with 24-mesh and 80-mesh sieves, retaining particles larger than 80 mesh and smaller than 24 mesh, and then 0.2% magnesium stearate is added to obtain the Dendrobium huoshanense extract layer material, and the remaining steps are the same as those in Example 1.

[0084] Comparative Example 11

[0085] The difference between this comparative example and Example 1 lies in that in step (2) of this comparative example, the amount of Dendrobium huoshanense extract is 30.25%, hydroxypropyl methylcellulose (HPMC) is 8.04%, stearyl alcohol is 18.09%, polyacrylic resin II is 28.14%, microcrystalline cellulose is 11.06%, and xylitol is 4.02%. They are sieved, mixed evenly, and an appropriate amount of water is used as a binder to reach the state of being able to form a ball when held in the hand and falling apart when touched. The mixture is prepared into soft materials, passed through a 24-mesh nylon sieve to make wet granules, dried at 55 °C for 30 minutes, sized with 24-mesh and 80-mesh sieves, and the granules larger than 80 mesh and smaller than 24 mesh are retained. Then 0.4% of magnesium stearate is added to obtain the Dendrobium huoshanense extract layer material. The remaining steps are the same as those in Example 1.

[0086] Comparative Example 12

[0087] The difference between this comparative example and Example 1 lies in that in step (4) of this comparative example, the probiotic layer material is first pressed at a pressure of 10 N to obtain probiotic layer tablets, and then an equal mass of Dendrobium huoshanense extract layer material is added to the probiotic layer tablets and pressed twice at a pressure of 50 N to obtain traditional Chinese medicine probiotic double-layer tablets. The remaining steps are the same as those in Example 1.

[0088] Example 2 Research on Prescription Screening and Preparation Process of Dendrobium huoshanense Extract Layer Material

[0089] 2.1 Process Verification and Quality Evaluation Method for Dendrobium huoshanense Tablets

[0090] ①Appearance

[0091] Randomly select 3 batches of Dendrobium huoshanense layer samples of double-layer floating tablets and observe their colors and appearances with the naked eye.

[0092] ②Hardness

[0093] Detect the hardness of the samples under a hardness detector. The hardness of the Dendrobium huoshanense layer of double-layer floating tablets should be 40 - 60 N.

[0094] ③Tablet Weight Variation

[0095] According to the provisions of "Tablet Weight Variation" under "General Rules for Preparations" in the fourth part of the 2020 edition of the Chinese Pharmacopoeia, randomly select 10 tablets of this product from each batch for inspection.

[0096] ④In Vitro Floating Performance

[0097] Using 900 mL of 0.1 mol / L hydrochloric acid solution as the release medium, at a temperature of (37.0 ± 0.5) °C and a rotation speed of (100 ± 1) r / min. Using the floating start time (i.e., the time from when the tablet is put into the medium and floats from the bottom of the cup to the liquid surface) and the continuous floating time (i.e., the time when the tablet continuously floats on the surface of the medium without sinking) as indicators to investigate the in vitro floating performance of the floating tablets.

[0098] ⑤In vitro cumulative release rate

[0099] The ultraviolet spectrophotometry was used in combination with the second method for the determination of dissolution and release rate in the 2020 edition of Chinese Pharmacopoeia (Volume IV). Six Huoshan Dendrobium tablets were placed in 500 mL of artificial gastric juice (0.1 mol / L hydrochloric acid solution), the rotation speed was set at (100±1) r / min, and the temperature was (37.0±0.5) °C. 2 mL of samples were taken at 2, 4, 6, 8, and 10 h (while adding 2 mL of isothermal artificial gastric juice at the same time), filtered through a 0.45 μm microporous filter membrane, and the absorbance was measured at 490 nm to calculate the cumulative release rate of the drug.

[0100] 2.2 Screening of types and ratios of excipients in the material of Huoshan Dendrobium extract layer

[0101] (1) Investigation of the dosage of the main drug

[0102] The soft materials prepared in step (2) of Example 1 and the soft materials prepared in step (2) of Comparative Example 1 or 2 were used to investigate the effect of the dosage of Huoshan Dendrobium extract on the tablet formability.

[0103] The results showed that when the mass percentage of Huoshan Dendrobium extract was 40% or 50%, the prepared soft materials were too sticky and not easy to form; while when the mass percentage of Huoshan Dendrobium extract was 30%, the prepared soft materials could reach the state of "forming a ball when held in the hand and dispersing when touched", and the prepared wet granules were of uniform size and not sticky. Therefore, the addition amount of Huoshan Dendrobium extract was 30%.

[0104] (2) Investigation of hydrophilic gel materials

[0105] Different amounts of hydroxypropyl methylcellulose (HPMC), sodium carboxymethylcellulose (CMC-Na), or polyacrylic resin II were taken to investigate the types and dosages of hydrophilic gel materials, that is, the Huoshan Dendrobium tablet layers prepared in Example 1 and the Huoshan Dendrobium tablet layers prepared in Comparative Examples 3-7 were used to investigate the sustained-release effect, tablet appearance, and hardness of the Huoshan Dendrobium tablet layers. The specific investigation methods are referred to in Section 2.1.

[0106] The results showed that when each skeleton material was used alone, there was no sustained-release effect or the sustained-release effect was not significant. When two skeleton materials were combined, it was found that when HPMC:CMC-Na = 1:3 or 3:1, the tablet hardness did not meet the requirements of Section 2.1; when HPMC:polyacrylic resin II = 1:3, the tablet appearance was smooth and the hardness met the requirements of Section 2.1. Therefore, HPMC and polyacrylic resin II were selected and combined as the skeleton materials to achieve the expected long-acting and sustained-release effects.

[0107] (3) Investigation of the buoyancy aid

[0108] The Dendrobium huoshanense tablets prepared in step (4) of Example 1 and Comparative Example 8 were used to investigate the types and dosages of floating aids according to the in vitro floating performance described in Section 2.1.

[0109] The results showed that the Dendrobium huoshanense tablet layer containing stearyl alcohol (Example 1) could float quickly and had a floating time of 8 - 10 hours, while the Dendrobium huoshanense tablet layer containing stearic acid (Comparative Example 8) did not float or had a floating time of less than 30 minutes. Therefore, stearyl alcohol was selected as the floating aid.

[0110] (4) Investigation of fillers

[0111] The Dendrobium huoshanense tablet layers prepared in step (4) of Example 1 and Comparative Example 9 were used to investigate the formability and compressibility according to the properties described in Section 2.1.

[0112] The results showed that the Dendrobium huoshanense tablet layer prepared in Comparative Example 9 was extremely prone to sticking to the punch and showed a pitted surface, while the addition of xylitol to the Dendrobium huoshanense tablet layer prepared in Example 1 could significantly improve the appearance and formability of the tablets. When microcrystalline cellulose and xylitol were used in combination, the prepared tablets had a uniform texture and a beautiful appearance.

[0113] (5) Investigation of lubricant dosage

[0114] The Dendrobium huoshanense tablets prepared in step (4) of Example 1, Comparative Example 10 and Comparative Example 11 were used to investigate the formability according to the properties described in Section 2.1.

[0115] The results showed that when the dosage of magnesium stearate was too low, it was still prone to sticking to the punch. Therefore, after adding 1% of magnesium stearate and mixing evenly, the compressibility of the granules was good and the tablet surface was smooth.

[0116] Table 1 Screening of the types and ratios of main and auxiliary materials

[0117]

[0118] Therefore, through single - factor investigation (see Table 1), it was preliminarily determined that the addition amount of Dendrobium huoshanense extract was 30%, HPMC and polyacrylic resin II were jointly used as the matrix materials to achieve the expected long - acting and sustained - release effects, stearyl alcohol was selected as the floating aid, an appropriate amount of microcrystalline cellulose and xylitol were added as fillers, and after adding 1% of magnesium stearate and mixing evenly, tableting was carried out.

[0119] 2.3 Quality evaluation results of Dendrobium huoshanense tablet layer

[0120] 2.3.1 In vitro floating performance

[0121] The Dendrobium huoshanense tablets prepared in Example 1 were tested according to the in vitro floating performance described in Section 2.1.

[0122] Figure 1The results show that the prepared Dendrobium huoshanense tablets start to float immediately after being put into water.

[0123] 2.3.2 Optimization experiment of prescription excipients

[0124] Based on the single-factor experiments, an L 9 (3 4 ) orthogonal experiment was designed to investigate the effects of the dosages of HPMC, stearyl alcohol, and polyacrylic resin II on the tablet properties and to screen the optimal prescription.

[0125] Table 2 Factor-level table of excipient dosages

[0126] Horizontal factor AHPMC % B Stearyl alcohol % C Polyacrylic resin II % 1 4 14 20 2 8 18 24 3 12 22 28

[0127] Refer to the method of Example 1 to prepare the gastric-retention traditional Chinese medicine probiotic bilayer floating tablets. The differences from Example 1 are as follows: the dosages of HPMC, stearyl alcohol, polyacrylic resin II, microcrystalline cellulose, and xylitol. Among them, the dosages of HPMC, stearyl alcohol, and polyacrylic resin II refer to the factor-level table of excipient dosages (see Table 2) and the orthogonal test result table (see Table 4). The remaining dosages are made up to 100% with microcrystalline cellulose and xylitol, and the mass percentage of microcrystalline cellulose to xylitol is 15:4.

[0128] According to the in vitro cumulative release rate in Section 2.1, the cumulative release rate of the Dendrobium huoshanense tablet layer was used as an index to comprehensively score the drug release performance of the tablets (see Table 3). The drug release performance was investigated with the comprehensive score (Q) as an index. The orthogonal test results and the in vitro floating performance results are shown in Table 4, and the variance analysis is shown in Table 5.

[0129] Table 3 Tablet scoring criteria

[0130]

[0131] Table 4 Orthogonal test result table

[0132]

[0133]

[0134] Table 5 Variance analysis table

[0135] Factor Sum of squares of deviations Degree of freedom Mean square F Significance A 1386.889 2 693.444 19.203 P<0.05 B 441.556 2 220.778 6.114 P>0.05 C 66.889 2 33.444 0.923 P>0.05 Error 72.222 2 36.111

[0136] Note: F 0.05 (2,2) = 19.00.

[0137] The results in Table 4 show that the comprehensive scores of Experiment Nos. 5 and 9 are both greater than 90 points. Therefore, the in vitro drug release models of Experiment Nos. 5 and 9 were investigated to further determine the optimal prescription.

[0138] 2.3.3 Fitting of in vitro drug release model

[0139] The cumulative release degrees of in vitro drug release of No. 5 and No. 9 were respectively fitted to time according to the first-order equation, and the results are shown in Table 6.

[0140] Table 6 Fitting Table of In Vitro Drug Release Model

[0141]

[0142] The results in Table 6 show that the first-order equation fitting degree of No. 5 is good, the floating time is long, the cumulative dissolution rate at 10 h reaches 87.5%, the drug release performance is good, and it is the best prescription, namely 8% HPMC, 18% stearyl alcohol, and 28% polyacrylic resin II.

[0143] 2.3.4 Process Verification of Dendrobium huoshanense Tablets

[0144] Determination Method for Total Polysaccharide Content

[0145] (1) Preparation of Standard Curve

[0146] Precisely weigh 1 mg of anhydrous glucose standard product dried to constant weight at 105 °C into a 10 mL volumetric flask, and prepare a 0.1 mg / mL glucose standard solution. Precisely measure 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the reference solution, and place them in 10 mL stoppered test tubes respectively. Add water to each test tube to make up to 1.0 mL, use distilled water as the blank control, and determine the glucose content by the phenol-sulfuric acid method. Add 1 mL of the prepared 5% phenol solution and 5 mL of concentrated sulfuric acid solution to each test tube respectively, shake well quickly, heat in a boiling water bath for 15 min to allow the sample to react fully, then place the test tube in an ice-water bath to stand and cool, and finally measure the absorbance at 490 nm. Draw a standard curve with the absorbance as the ordinate and the concentration as the abscissa.

[0147] The standard curve of glucose concentration is shown in Figure 2 , and the regression equation is obtained according to the standard curve: y = 12.749x - 0.0099, R 2 = 0.9959 (unit: mg / mL).

[0148] (2) Determination of Total Polysaccharide Content in Extract

[0149] Precisely weigh 0.05 g of the Dendrobium huoshanense extract prepared in Example 1, dissolve it in water and dilute to a 10 mL volumetric flask, make up the volume, mix well, take 1 mL, and dilute to a 100 mL volumetric flask, shake well to obtain. Precisely measure 1.0 mL of the sample solution, use distilled water as the blank control, and measure the absorbance according to the "phenol-sulfuric acid method" described in (1), and substitute it into the regression equation in (1) to calculate the total polysaccharide content in the Dendrobium huoshanense extract.

[0150] According to Figure 2The standard curve showed that the total polysaccharide content in the extract of Dendrobium huoshanense was 33.71%.

[0151] (3) Determination of the total polysaccharide content in tablets

[0152] The 3 batches of Dendrobium huoshanense tablets prepared in Example 1 were pulverized, an appropriate amount of fine powder was taken, added to 500 mL of 0.1 mol / L hydrochloric acid, fully dissolved and filtered to obtain the solution. 1.0 mL of the sample solution was precisely taken, and the total polysaccharide content in the extract was determined by the "phenol-sulfuric acid method".

[0153] The 3 batches of Dendrobium huoshanense tablets prepared in Example 1 were inspected according to the process verification and quality evaluation method of Dendrobium huoshanense tablets in Section 2.1, and at the same time, the cumulative release rate of Dendrobium huoshanense tablets in Section 2.3.2 was investigated. The results are shown in Tables 7 - 8 and Figure 3 .

[0154] Table 7 Process verification of 3 batches of Dendrobium huoshanense tablets

[0155]

[0156] Table 8 In vitro release of 3 batches of Dendrobium huoshanense tablets (cumulative release amount: %)

[0157]

[0158]

[0159] The Dendrobium huoshanense tablets prepared in Example 1 of the present invention have a beautiful appearance, uniform color, moderate hardness, and the weight difference of the 3 batches of process verification tablets is less than 5%; they can instantaneously float in the dissolution medium, with a floating time > 10 h, and the release rate uniformly increases within 10 h, and the average release rate is 87.3% (see Figure 3 ), and there is no significant difference in the in vitro release rate between batches, and the process reproducibility is good.

[0160] Example 3

[0161] The appearance, hardness and disintegration time limit of the intragastric retention type traditional Chinese medicine probiotic double-layer floating tablets prepared in Example 1 and the traditional Chinese medicine probiotic tablets prepared in Comparative Example 12 were inspected, and their quality was evaluated.

[0162] Figure 4 The results showed that the intragastric retention type traditional Chinese medicine probiotic double-layer floating tablets prepared in Example 1 had a complete finished product, uniform color, certain smoothness, and the hardness of the prepared probiotic layer was 10.0 N, with appropriate hardness and an average disintegration time limit of 10 min, indicating that they could dissolve in the human digestive tract and be absorbed and utilized by the human body.

[0163] The average disintegration time limit of the traditional Chinese medicine probiotic tablets prepared in Comparative Example 12 was 12 h, which met the slow-release requirements of the Dendrobium huoshanense layer but did not achieve the preferential release of the probiotic layer. For the gastric-retention type traditional Chinese medicine probiotic double-layer floating tablets of Example 1, the Dendrobium huoshanense layer had a greater hardness and the probiotic layer had a lower hardness. The average disintegration time limit of this layer was 15 min, meeting the requirement for the preferential release of the probiotic layer, and the Dendrobium huoshanense layer slowly dissolved with an average disintegration time limit of 12 h. Therefore, the tabletting order of the gastric-retention type traditional Chinese medicine probiotic double-layer floating tablets should be to pre-tablet the Dendrobium huoshanense layer first and then add the probiotic layer for secondary tabletting.

[0164] Example 4

[0165] Quality evaluation of the gastric-retention type traditional Chinese medicine probiotic double-layer floating tablets prepared in Example 1 was carried out according to the 2.1 part of Example 2.

[0166] (1) Appearance: Figure 4 The results showed that the finished gastric-retention type traditional Chinese medicine probiotic double-layer floating tablets prepared in Example 1 were intact, had a uniform color, and had a certain smoothness.

[0167] (2) Weight variation: The results showed that the average tablet weight of 20 gastric-retention type traditional Chinese medicine probiotic double-layer floating tablets was 0.5085 g. According to the weight variation inspection method in Part IV of the Chinese Pharmacopoeia (2020 Edition), when the tablet weight ≥ 0.3 g, the variation limit is ±5.0%. Then, the tablet weight of the traditional Chinese medicine probiotic double-layer floating tablets between 0.4831 g and 0.5339 g meets the requirements. The final results showed that none of the tablets exceeded the weight variation limit, meeting the provisions of the Chinese Pharmacopoeia.

[0168] (3) Hardness: The results showed that the traditional Chinese medicine probiotic double-layer floating tablets prepared in Example 1 all had appropriate hardness, with an average hardness of 65.18 N.

[0169] (4) Disintegration time limit: The results showed that the disintegration time limits of 6 traditional Chinese medicine probiotic double-layer floating tablets prepared in Example 1 were all good. The average disintegration time limit of the probiotics was 19.5 min, and the average disintegration time limit of the Dendrobium huoshanense layer was 12 h 12.5 min, meeting the provisions of the Chinese Pharmacopoeia (2020 Edition).

[0170] Example 5

[0171] Study on the viability stability of live bacteria and their tolerance to simulated gastrointestinal fluids in gastric-retention type traditional Chinese medicine probiotic double-layer floating tablets

[0172] 5.1 Experimental method

[0173] 5.1.1 In vitro simulated digestion

[0174] 5.1.1.1 Preparation of digestive fluids

[0175] (1) In vitro simulated gastric fluid: Adjust the pH value of 0.1 mol / L potassium phosphate buffer to 3.5, sterilize, add pepsin (10 g / L), mix to fully dissolve, and obtain simulated gastric fluid. Preheat to 37°C before use.

[0176] (2) In vitro simulated intestinal fluid: adjust the pH of potassium phosphate buffer to 6.8 and sterilize, add trypsin (10 g / L) and porcine bile salt (3 g / L), mix and fully dissolve to obtain simulated intestinal fluid. Preheat to 37°C before use.

[0177] 5.1.1.2 Simulated gastrointestinal digestion

[0178] Take one tablet of the gastric retention type Chinese medicine probiotic double-layer floating tablet (referred to as double-layer tablet, specification: 0.5g / tablet) prepared in Example 1, and take another 0.1g of the 15-bacteria composite probiotic (equivalent to the amount of probiotic powder added to the double-layer tablet, with a total viable count of ≥1×10 8 CFU / g), accurately weighed, placed in 20.0mL simulated gastric fluid (pH3.5), put in 37℃90r / min water bath for 2h of shaking digestion, then added 80.0mL simulated intestinal fluid, continued to shake digestion in 37℃120r / min water bath for 2h, simulating gastrointestinal digestion process under anaerobic conditions. The plate count method was used to determine the number of viable bacteria, and parallel experiments were performed at the same time, and the survival rate was calculated according to the following formula.

[0179] Survival rate (%) = lgN i ÷lgN 0 ×100, where Ni represents the number of viable bacteria after ih of digestion; N 0 Indicates the initial viable count.

[0180] 5.1.1.3 Live bacteria count

[0181] The live bacteria count is based on the Lactobacillus and Thermophilus Streptococcus counting method in GB 4789.35-2016 “National Food Safety Standard Food Microbiology Examination Lactic Acid Bacteria Examination”, with slight modifications.

[0182] Pipette 1.0mL of the above digestion solution into a test tube containing 9.0mL of sterile physiological saline, shake thoroughly, then make a 10-fold serial dilution, select 2 to 3 appropriate dilution gradients, pipette 100μL into a sterile plate, make 3 plates for each dilution, pour about 15mL of MRS culture medium cooled to 48℃ into the plate, rotate the plate to mix evenly. After the plate solidifies, invert it into an anaerobic bag, and culture it anaerobically at (36±1)℃ for (72±2)h before conducting a live bacteria count experiment.

[0183] 5.2 Experimental Results

[0184] 5.2.1 Tolerance of probiotics to simulated gastrointestinal fluid

[0185] The survival conditions of the bilayer tablets in the simulated gastrointestinal tract are shown in Table 9 as follows.

[0186] Table 9 Tolerance of probiotics in bilayer tablets to simulated gastrointestinal fluids (survival rate / %, n = 6)

[0187] Group 0h Gastric juice for 2 h Gastric juice for 2 h + Intestinal juice for 2 h Double-layer tablet 100±3.74 78.37±1.18 94.54±2.75 Fifteen-strain compound probiotics 100±1.76 76.76±5.26 90.31±0.65

[0188] The results in Table 9 show that in the simulated gastric juice environment, the survival rates of both decreased. Among them, the survival rate of the bilayer tablets after digestion in simulated gastric juice was 78.37%, while the survival rate of the raw material of the fifteen-strain compound probiotics without making tablets after digestion in simulated gastric juice was 76.76%. There was no significant difference between the two, indicating that the probiotic layer in the bilayer tablets could be fully dissolved, enabling the complete release of probiotics.

[0189] The survival rates of probiotics in the bilayer tablets and the fifteen-strain compound probiotics after passing through the simulated gastrointestinal fluids were 94.54% and 90.31% respectively. After passing through the more stringent simulated gastrointestinal fluids, the survival rates of both decreased first and then increased, indicating that after being damaged by gastrointestinal fluids, probiotics could still recover their activity within a certain period of time. In addition, the survival rate of probiotics in the bilayer tablet group was slightly higher than that in the fifteen-strain compound probiotic group, suggesting that as the extract layer of Dendrobium huoshanense in the bilayer tablets was continuously released, it could provide a carbon source for probiotics and promote the proliferation of probiotics.

[0190] Example 6

[0191] Effect of Dendrobium huoshanense extract on ethanol-induced gastric injury model in mice

[0192] 6.1 Experimental method

[0193] Twenty-four clean-grade ICR mice were taken. After 3 days of adaptive feeding, they were randomly divided into a blank group, a model group, an omeprazole positive control group, and a water extract group of Dendrobium huoshanense, with 6 mice in each group. Each group was administered by gavage at a dose of 10 mL / kg. The blank group and the model group were given a 0.05% sodium carboxymethylcellulose solution by gavage daily. The positive group was given the content of an omeprazole enteric-coated capsule at a dose of 480 mg / kg by gavage daily. The water extract group of Dendrobium huoshanense was given the crude drug amount of the water extract of Dendrobium huoshanense prepared in step (1) of Example 1 at a dose of 1.56 g / kg (equivalent human dose) by gavage daily. Gavage was continued for 10 days (the gavage samples were all dissolved in a 0.05% sodium carboxymethylcellulose solution). After administration on the 9th day, the mice were fasted but allowed to drink water. One hour after the last administration on the 10th day, except for the blank group, the mice in the other groups were given a 50% ethanol solution by gavage (0.1 mL / 20 g per mouse) to form an acute gastric mucosal injury model 1 hour later. The stomachs of the mice were taken to observe and measure the injury scores, and the gastric tissues were fixed in 4% paraformaldehyde to observe the pathological injuries.

[0194] (1) Determination of the gastric mucosal injury score in mice

[0195] After the mice were killed, the stomach tissue was removed by laparotomy and cut open along the greater curvature of the stomach. The stomach contents were washed with pre-cooled PBS and spread on filter paper to observe the damage of the mouse gastric mucosa. The damaged part was measured with a ruler.

[0196] The gastric mucosal injury score was calculated according to the Guth method:

[0197] No damage was scored as 0 points; mucosal erythema and punctate bleeding (length and width <0.5mm) was scored as 1 point; small erosion was scored as 2 points (<1mm); moderate erosion (1mm-2mm) was scored as 3 points; large erosion (2mm-3mm) was scored as 4 points; large area erosion (≥3mm) was scored as 5 points.

[0198] If the width is >1 mm, the score is doubled, and patchy hemorrhages are scored according to the area.

[0199] (2) Pathological observation of mouse stomach tissue

[0200] The gastric tissue was fixed, dehydrated and transparent, wax-embedded, sliced ​​and mounted, dewaxed, stained with hematoxylin-eosin (HE), dehydrated and transparent, sealed, and observed.

[0201] 6.2 Experimental Results

[0202] Table 10 Effects of different groups of treatment on acute liver and stomach damage in mice (n=6, )

[0203] Group Mouse gastric mucosa injury score (points) Blank group 0.00±0.00 Model group <![CDATA[2.83±1.72 ## > Omeprazole positive group <![CDATA[1.50±0.57 * > Water extract group of Dendrobium huoshanense <![CDATA[1.50±0.83 * >

[0204] Note: ## P<0.01 indicates a significant difference compared with the blank group. * P<0.05 indicated a significant difference compared with the model group.

[0205] The results in Table 10 show that compared with the model group, the degree of gastric mucosal injury in the mice treated with the water extract of Dendrobium huoshanense was significantly reduced, and the degree of gastric mucosal injury reduced in the water extract of Dendrobium huoshanense group was equivalent to that in the omeprazole-positive group. Therefore, the water extract of Dendrobium huoshanense of the present invention can effectively alleviate gastric mucosal injury.

[0206] Figure 5The results showed that there was no exfoliation, congestion, edema or damage in the gastric mucosa of the mice in the blank group. An acute liver injury model was established in the mice of the model group by intragastric administration of 50% ethanol solution. It could be observed that a large number of erosions appeared on the gastric surface of the mice in the model group, and there were multiple hemorrhages in the mucosa. The difference between the model group and the blank group was significant, indicating that the acute liver injury model in mice induced by 50% ethanol solution was successfully constructed. There were a small amount of erythema and hemorrhage on the gastric surface of the mice in the omeprazole positive group, and no significant bleeding lesions were observed, showing a good protective effect. After the intervention of the aqueous extract of Dendrobium huoshanense, the erosion on the gastric surface of the mice was alleviated, and the bleeding width was significantly reduced, indicating that the aqueous extract of Dendrobium huoshanense has the effect of alleviating acute gastric mucosal injury.

[0207] Figure 6 The results showed that the gastric tissue structure of the mice in the model group was abnormal, with large-area ulcers in the mucosal layer, erosion and necrosis of epithelial cells, as shown in the tissue pointed by the black arrow; capillary dilation and congestion, as shown in the tissue pointed by the yellow arrow; and a large number of inflammatory cell infiltrations could be seen, as shown in the tissue pointed by the red arrow. The gastric tissue structures of the mice in the positive control group and the aqueous extract group of Dendrobium huoshanense were basically normal, and the arrangement of epithelial cells in some areas was loose, as shown by the black arrow; no obvious ulcers or inflammatory cell infiltrations were observed in the tissue. It shows that the aqueous extract of Dendrobium huoshanense can repair the tissue structure, relieve inflammatory infiltration and improve acute gastric mucosal injury.

[0208] Example 7

[0209] Effect of Dendrobium huoshanense Extract on Probiotics (Compound Probiotic Powder)

[0210] I. Experimental Methods

[0211] 7.1 Probiotic Proliferation

[0212] Weigh 1 g of fifteen-strain compound probiotics and add it to 10 mL of MRS medium, and suspend it to obtain a mixed probiotic suspension. Prepare 60 mL of MRS medium as the aerobic probiotic culture group and MRS medium containing anhydrous cysteine hydrochloride as the anaerobic probiotic culture group respectively, sterilize them, add 9 mL of the above two media to test tubes respectively, and add 200 μL of the mixed probiotic suspension to each test tube. After shaking the test tubes, place the test tubes of the aerobic probiotic culture group in a constant temperature incubator at 37 °C for 24 h, and place the test tubes of the anaerobic probiotic culture group in an anaerobic jar and in a constant temperature incubator at 37 °C for 24 h.

[0213] After the culture is completed, centrifuge (rotation speed 2000 rpm, time 10 min), discard the supernatant, add 300 μL of 20% glycerol to the precipitate, and store it in a -80 °C ultra-low temperature refrigerator for standby.

[0214] 7.2 Effect of Double-Layer Tablets with Different Concentrations on the Proliferation of Probiotics in Compound Probiotic Powder

[0215] 7.2.1 Strain Cultivation

[0216] Take an appropriate amount of the water extract of Dendrobium huoshanense prepared in step (1) of Example 1 (the preparation process of the water extract of Dendrobium huoshanense is the same as that in the bilayer tablets), and prepare MRS media containing the water extract of Dendrobium huoshanense at concentrations of 0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 g / L respectively, and MRS media containing anhydrous cysteine hydrochloride. Sterilize them. Take 10 mL of the media containing bilayer tablets and add them into test tubes respectively, then add 200 μL of the mixed probiotic suspension to each test tube, shake well. Place the test tubes of the aerobic probiotic culture group in a constant temperature incubator at 37 °C for 24 h, and place the test tubes of the anaerobic probiotic culture group in an anaerobic jar and a constant temperature incubator at 37 °C for 24 h.

[0217] 7.2.2 Dilution Culture

[0218] After the cultivation is completed, take out the test tubes of each group. After mixing the cultivated strains evenly, take an appropriate amount of normal saline for continuous dilution until diluted to 10 7 Then take 900 μL and add it to an empty petri dish. Then pour the prepared and sterilized solid medium into the petri dish containing the bacterial liquid, shake gently and let it stand. After it cools and solidifies, place it in a constant temperature incubator at 37 °C and an anaerobic jar respectively for 24 h.

[0219] 7.2.3 Counting

[0220] Take out the solid medium, conduct colony counting, and perform statistical analysis and graphing.

[0221] 7.3 Exploration of the Effect of the Water Extract of Dendrobium huoshanense on the Growth Rate of Probiotics

[0222] 7.3.1 Effect of the Water Extract of Dendrobium huoshanense on the Growth Rate of Aerobic Bacteria

[0223] Take an appropriate amount of the water extract of Dendrobium huoshanense prepared in step (1) of Example 1, prepare 50 mL of MRS medium containing 5 g / L of the water extract of Dendrobium huoshanense. After sterilization, add the mixed probiotic suspension. At the same time, use the group without the addition of the water extract of Dendrobium huoshanense but only containing the mixed probiotic suspension as the control group, and place it in a constant temperature incubator at 37 °C for cultivation. Sampling is carried out at 0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 h respectively. Each time, take 200 μL and add 200 μL of the medium. Place the taken 200 μL of the bacterial liquid in a sterile 96-well plate, and use a microplate reader to measure the absorbance of the bacterial liquid at 600 nm at different time points.

[0224] 7.3.2 Effect of the Water Extract of Dendrobium huoshanense on the Growth Rate of Anaerobic Bacteria

[0225] Take an appropriate amount of the water extract of Dendrobium huoshanense prepared in step (1) of Example 1, and prepare 50 mL of MRS medium containing 5 g / L of the water extract of Dendrobium huoshanense and cysteine hydrochloride anhydrous. After sterilization, add the mixed probiotic suspension. At the same time, use the medium containing only the mixed probiotic suspension without adding the water extract of Dendrobium huoshanense as the control group, and place it in a constant temperature incubator at 37 °C for cultivation. Samples are taken at 0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 h respectively. Each time, 200 μL is taken and added to 200 μL of the medium. Place the taken 200 μL of the bacterial solution in a sterile 96-well plate, and use an enzyme-labeled instrument to measure the absorbance of the bacterial solution at 600 nm at different time points.

[0226] II. Experimental Results

[0227] (1) Experimental results of the effect of water extracts of Dendrobium huoshanense at different concentrations on the proliferation of probiotics

[0228] Inoculate the compound probiotic suspension into the medium containing 0.625 - 40 g / L of the extract of Dendrobium huoshanense. After 24 h, record the colony count of the plate count, and respectively count the proliferation of aerobic and anaerobic probiotics (see Table 11).

[0229] Table 11 Effect of the water extract of Dendrobium huoshanense on the proliferation of probiotics (n = 3, n±x)

[0230] Serial number Concentration (g / L) Average colony count of aerobic group (pieces) Average colony count of anaerobic group (pieces) 1 0 104±23 93±16 2 0.625 205±41 172±29 3 1.25 233±36 210±26 4 2.5 260±33 244±21 5 5 304±19 296±32 6 10 383±37 353±27 7 20 397±21 394±12 8 40 412±15 446±18

[0231] The results in Table 11 show that with the increase in the concentration of the water extract of Dendrobium huoshanense, the proliferation of probiotics shows a gradually increasing trend, indicating that the water extract of Dendrobium huoshanense at a concentration of 0.625 - 40 g / L has a proliferative effect on both aerobic and anaerobic bacteria.

[0232] (2) Experimental results of the effect of the water extract of Dendrobium huoshanense on the growth rate of probiotics

[0233] Inoculate the mixed probiotic suspension into the MRS medium containing 5 g / L of the fresh juice of Dendrobium huoshanense, and use the absorbance measurement method to observe the growth conditions at different times within 48 h (see Table 12), and analyze the effect on the growth rate of probiotics ( Figure 7 and Figure 8 ).

[0234] Table 12 Changes in the absorbance of the probiotic suspension over time (n = 3)

[0235]

[0236] The results in Table 12 show that with the extension of time, the proliferation of probiotics shows a gradually increasing trend, indicating that the water extract of Dendrobium huoshanense has a proliferative effect on both aerobic and anaerobic bacteria.

[0237] Figure 7 and Figure 8 The results show that the aqueous extract of Dendrobium huoshanense at a concentration of 5 g / L significantly increases the growth rate of probiotics from 4 to 12 h and then tends to be stable.

[0238] In summary, it can be seen that the aqueous extract of Dendrobium huoshanense can promote the proliferation of probiotics in the compound probiotic powder.

[0239] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A gastric retention type Chinese medicine probiotic double-layer floating tablet, characterized in that: The traditional Chinese medicine probiotic double-layer floating tablet comprises a Huoshan Dendrobium extract layer, a probiotic layer and an adhesive; The Huoshan Dendrobium extract layer is made of the following raw materials in percentage by mass: 25% to 35% of Huoshan Dendrobium extract, 7% to 9% of hydroxypropyl methylcellulose, 16% to 20% of octadecyl alcohol, 22% to 30% of polyacrylic acid resin II, and 0.8% to 1.5% of magnesium stearate, supplemented to 100% with microcrystalline cellulose and xylitol, wherein the mass ratio of the microcrystalline cellulose to the xylitol is 12 to 17:4; The probiotic layer is made of the following raw materials in percentage by mass: Probiotic powder 15%-22%, soluble starch 35%-42%, xylitol 12%-16%, mannitol 8%-10%, magnesium stearate 0.8%-1.5%, supplemented with dextrin to 100%.

2. The gastric retention type Chinese medicine probiotic double-layer floating tablet according to claim 1, characterized in that: The Huoshan Dendrobium extract is a Huoshan Dendrobium water extract; the probiotic powder includes fifteen-bacteria composite probiotics.

3. The gastric retention type Chinese medicine probiotic double-layer floating tablet according to claim 2, characterized in that: The preparation method of the Huoshan Dendrobium officinale extract comprises the following steps: The dried stems of Dendrobium huoshanense are crushed and sieved to obtain the crushed Dendrobium huoshanense; the crushed Dendrobium huoshanense is mixed with water, extracted, and the filtrate after extraction is collected, concentrated and dried to obtain the Dendrobium huoshanense extract.

4. The gastric retention type Chinese medicine probiotic double-layer floating tablet according to claim 3, characterized in that: The mesh number of the sieving is 80 to 120 meshes.

5. The gastric retention type Chinese medicine probiotic double-layer floating tablet according to claim 3, characterized in that: The mass volume ratio of the crushed Huoshan Dendrobium and water is 1g:15-25mL.

6. The gastric retention type Chinese medicine probiotic double-layer floating tablet according to claim 3, characterized in that: The leaching temperature is 65-85° C., the leaching times are 1-3 times, and the leaching time for each time is 1-3 hours.

7. The gastric retention type Chinese medicine probiotic double-layer floating tablet according to claim 1, characterized in that: The mass ratio of the Huoshan Dendrobium extract layer to the probiotic layer is 1:

1.

8. The gastric retention type Chinese medicine probiotic double-layer floating tablet according to claim 1, characterized in that: The adhesive is an adhesive for the Huoshan Dendrobium extract layer and an adhesive for the probiotic layer. The adhesive for the Huoshan Dendrobium extract layer is water, and the adhesive for the probiotic layer is maltose syrup.

9. A method for preparing the gastric retention type Chinese medicine probiotic double-layer floating tablets according to any one of claims 1 to 8, characterized in that: The following steps are involved: The Huoshan Dendrobium extract, hydroxypropyl methylcellulose, octadecyl alcohol, polyacrylic acid resin II, microcrystalline cellulose and xylitol are mixed, water is added as a binder, mixed to prepare a soft material, wet granulated, dried, granulated, and magnesium stearate is added to obtain a Huoshan Dendrobium extract layer; Probiotic powder, soluble starch, xylitol, dextrin, and mannitol are mixed, maltose syrup is added as a binder, wet granulation is performed, freeze drying is performed, and magnesium stearate is added to obtain a probiotic layer; First, the Huoshan Dendrobium extract layer is compressed for the first time to obtain Huoshan Dendrobium tablets, and then a probiotic layer is added to the Huoshan Dendrobium tablets for a second compression to obtain gastric retention type Chinese medicine probiotic double-layer floating tablets.

10. The preparation method according to claim 9, characterized in that: The hardness of the first pressing is 40-60N; the hardness of the second pressing is 10-15N.

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