Gastric retention-type traditional chinese medicine probiotic double-layer floating tablet and preparation method therefor
Patent Information
- Application Number
- PCT/CN2026/080328
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-03-12
- Filing Date
- 2026-02-27
- Publication Date
- 2026-09-17
Smart Images
Figure CN2026080328_17092026_PF_FP_ABST
Abstract
Description
A gastric retention type traditional Chinese medicine probiotic double-layer floating tablet and its preparation method Technical Field
[0001] This application belongs to the field of pharmaceutical preparation technology, and in particular relates to a gastric retention type traditional Chinese medicine probiotic double-layer floating tablet and its preparation method. Background Technology
[0002] Dendrobium huoshanense CZTang et SJCheng is a perennial herb belonging to the genus Dendrobium in the Orchidaceae family. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it is a traditional and precious Chinese medicinal herb 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 viscous texture, resulting in low bioavailability in traditional preparations, thus limiting its effect of "strengthening the intestines and stomach."
[0003] Probiotics are a collective term for microorganisms that colonize the human gut and maintain its microecological balance. Appropriate intake of probiotics helps correct gut microbiota imbalance, promotes the proliferation of beneficial bacteria, improves gastrointestinal health, enhances immunity, improves immune regulation, and even has antibiotic-like effects. However, oral probiotics are the only way to supplement probiotics daily, but oral probiotics have a low colonization rate in the large intestine, resulting in minimal long-term benefits.
[0004] Therefore, providing a formulation that can both meet the needs of Dendrobium huoshanense for continuous treatment of the gastric mucosa and meet the needs of rapid release of probiotics, promote proliferation, and improve intestinal flora is an urgent technical problem to be solved. Summary of the Invention
[0005] Therefore, the purpose of this application is to provide a gastric retention type traditional Chinese medicine probiotic double-layer floating tablet and its preparation method. The finished product of the gastric retention type traditional Chinese medicine probiotic double-layer floating tablet is intact and uniform in color. It can float instantly in gastric juice, with a floating time of >10h, and can achieve the purpose of sustained drug release.
[0006] To achieve the above-mentioned objectives, this application provides the following technical solutions:
[0007] This application provides a gastric retention type traditional Chinese medicine probiotic double-layer floating tablet, which includes a Dendrobium officinale extract layer, a probiotic layer and an adhesive;
[0008] The Dendrobium huoshanense extract layer is made from the following raw materials in the indicated mass percentages:
[0009] The mixture contains 25%–35% Dendrobium huoshanense extract, 7%–9% hydroxypropyl methylcellulose, 16%–20% stearyl alcohol, 22%–30% polyacrylic acid resin II, and 0.8%–1.5% magnesium stearate, which are then supplemented to 100% with microcrystalline cellulose and xylitol, wherein the mass ratio of microcrystalline cellulose to xylitol is 12–17:4.
[0010] The probiotic layer is made from the following raw materials in the indicated weight percentages:
[0011] Probiotic powder 15%–22%, soluble starch 35%–42%, xylitol 12%–16%, mannitol 8%–10%, magnesium stearate 0.8%–1.5%, and dextrin to make up to 100%.
[0012] In this application, the *Dendrobium huoshanense* extract is preferably a water extract of *Dendrobium huoshanense*; the probiotic powder includes a fifteen-strain compound probiotic, and this application does not specifically limit the source of the fifteen-strain compound probiotic, as long as a product known in the art is used. The mass ratio of the *Dendrobium huoshanense* extract layer to the probiotic layer is 1:1. The binder is a binder for both the *Dendrobium huoshanense* extract layer and the probiotic layer; the binder for the *Dendrobium huoshanense* extract layer is water, and the binder for the probiotic layer is malt syrup. This application has found that ethanol-based binders inhibit the activity of probiotics, preventing them from regulating the intestinal flora and thus reducing their efficacy. This application 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 amount of binder used is sufficient to achieve a state where the *Dendrobium huoshanense* extract layer or the probiotic layer "can be clumped in the hand but crumbles upon contact."
[0013] In this application, the gastric retention type Chinese herbal probiotic double-layer floating tablet can float in gastric juice, and the floating function mainly depends on the performance of the Dendrobium officinale layer excipient carrier. The gastric retention type probiotic double-layer floating tablet floats on the surface of gastric juice. The probiotic layer follows the drug release pattern of tablets, with the probiotics entering the small intestine and then the large intestine via the pylorus with the gastric juice. The floating Dendrobium huoshanense layer slowly releases Dendrobium huoshanense extract with the help of excipients: some of the earlier released Dendrobium huoshanense extract disperses in the gastric juice, playing a protective role for the gastric mucosa, and flows into the small and large intestines with the gastric juice. The Dendrobium huoshanense extract that reaches the large intestine can serve as a carbon source for probiotics, promoting their proliferation. The other part of the delayed-release Dendrobium huoshanense extract continuously replenishes the Dendrobium huoshanense extract content in the gastric juice, continuously repairing the gastric mucosa. The Dendrobium huoshanense extract that is not fully absorbed then flows into the large intestine as a continuous carbon source for probiotics, promoting their proliferation.
[0014] In this application, the preferred method for preparing the Dendrobium huoshanense extract includes the following steps:
[0015] The dried stems of Dendrobium huoshanense were pulverized and sieved to obtain pulverized Dendrobium huoshanense. The pulverized Dendrobium huoshanense was mixed with water and extracted. The filtrate after extraction was collected, concentrated, and dried to obtain Dendrobium huoshanense extract.
[0016] In preparing Dendrobium huoshanense extract, the dried stems of Dendrobium huoshanense are pulverized and sieved to obtain pulverized Dendrobium huoshanense. The sieve mesh size is preferably 80-120 mesh, more preferably 90-110 mesh, and even more preferably 100 mesh. After obtaining the pulverized Dendrobium huoshanense, it is mixed with water for extraction, and the filtrate is collected. The mass-to-volume ratio of the pulverized Dendrobium huoshanense to water is preferably 1g:15-25mL, more preferably 1g:17-22mL, and even more preferably 1g:20mL. The extraction temperature is preferably 65-85℃, more preferably 70-85℃, and even more preferably 70, 75, 80, or 85℃. The number of extractions is preferably 1-3 times, more preferably 2 times, and the extraction time for each extraction is preferably 1-3 hours, more preferably 1.5-2.5 hours, and even more preferably 2 hours. This application enables the rapid and high-content extraction of active ingredients from *Dendrobium huoshanense*, such as total polysaccharides, using the aforementioned method. After obtaining the filtrate from the extraction, the filtrate is concentrated and dried to obtain the *Dendrobium huoshanense* extract. This application does not specifically limit the concentration method; any method known in the art, such as vacuum concentration at 60°C, is acceptable. This application also does not specifically limit the drying method; any method known in the art, such as oven drying or freeze drying, is acceptable. The oven drying method includes drying at 50–60°C for 30 minutes.
[0017] The water extract of Dendrobium huoshanense in this application has a relatively viscous texture and is prone to clumping when mixed with other excipients, making it difficult to mix evenly. Through the adjustment of excipients, this application prepares a gastric retention type Chinese medicine probiotic double-layer floating tablet product that is intact, uniform in color, and glossy, and also has a sustained-release effect.
[0018] This application also provides a method for preparing the above-mentioned gastric retention type traditional Chinese medicine probiotic double-layer floating tablet, including the following steps:
[0019] The extract of Dendrobium huoshanense, hydroxypropyl methylcellulose, stearyl alcohol, polyacrylic acid resin II, microcrystalline cellulose and xylitol were mixed and water was added as a binder to prepare a soft material. The material was wet granulated, dried and granulated, and magnesium stearate was added to obtain the Dendrobium huoshanense extract layer.
[0020] Probiotic powder, soluble starch, xylitol, dextrin, and mannitol are mixed and malt syrup is added as a binder. The mixture is then wet-granulated, freeze-dried, and magnesium stearate is added to obtain the probiotic layer.
[0021] First, the Dendrobium huoshanense extract layer is pressed for the first time to obtain Dendrobium huoshanense tablets. Then, a probiotic layer is added to the Dendrobium huoshanense tablets and pressed for the second time to obtain gastric retention type Chinese medicine probiotic double-layer floating tablets.
[0022] In preparing the Dendrobium huoshanense extract layer, the amount of water added is sufficient to achieve a state where the extract layer "can be clumped when squeezed in the hand, but crumbles easily when touched." The wet granulation method involves passing the granules through a 24-mesh nylon sieve. The drying process can be either oven drying or freeze drying, wherein the oven drying is performed at 50–60°C for 25–35 minutes. After drying, the granules are further sieved using 24-mesh and 80-mesh sieves.
[0023] When preparing the probiotic layer, the amount of maltose syrup added is sufficient to achieve a state where the probiotic layer "can be clumped together when squeezed in the hand, but crumbles upon contact." The invention does not specifically limit the freeze-drying method; any method known in the art may be used.
[0024] During compression, the hardness of the first compression is 40-60 N; the hardness of the second compression is 10-15 N. This application employs a method of first compressing the Dendrobium huoshanense extract layer and then compressing the probiotic layer. The resulting gastric retention type traditional Chinese medicine probiotic double-layer floating tablet meets the requirement of preferential release of the probiotic layer, while the Dendrobium huoshanense layer dissolves slowly, with an average disintegration time of over 12 hours.
[0025] Compared with the prior art, this application has the following beneficial effects:
[0026] This application provides a gastric retention-type traditional Chinese medicine probiotic double-layer floating tablet and its preparation method. The finished gastric retention-type traditional Chinese medicine probiotic double-layer floating tablet of this application is intact, uniform in color, and has a smooth finish. Its weight variation conforms to the requirements of the Chinese Pharmacopoeia, and it has suitable hardness. It can float in gastric juice with a floating time >10 hours, exhibiting a sustained-release drug effect. The disintegration time of the *Dendrobium huoshanense* extract layer and the probiotic layer in the double-layer floating tablet conforms to the requirements of the 2020 edition of the Chinese Pharmacopoeia. This gastric retention-type traditional Chinese medicine probiotic double-layer floating tablet can meet the requirements of continuous treatment of the gastric mucosa by *Dendrobium huoshanense* extract, rapid release of probiotics, and provides a continuous carbon source to promote probiotic proliferation, thereby exerting a synergistic gastrointestinal therapeutic effect, reducing the frequency of medication for patients, improving the bioavailability of *Dendrobium huoshanense* extract, enhancing the survival rate of probiotics, and improving the intestinal flora. Attached Figure Description
[0027] Figure 1 shows the test results of the floating properties of the Dendrobium huoshanense tablets prepared in this application;
[0028] Figure 2 shows the standard curve for glucose concentration;
[0029] Figure 3 shows the in vitro release curves of three batches of Dendrobium huoshanense tablets prepared in Example 1;
[0030] Figure 4 shows the appearance of the gastric retention type double-layer floating tablet of traditional Chinese medicine probiotics prepared in Example 1;
[0031] Figure 5 shows the observation of gastric tissue damage in mice after different treatment groups. A is the observation of gastric tissue damage in mice after treatment with the blank group; B is the observation of gastric tissue damage in mice after treatment with the model group; C is the observation of gastric tissue damage in mice after treatment with the positive control group; D is the observation of gastric tissue damage in mice after treatment with Dendrobium huoshanense water extract. The black arrows indicate the locations of gastric mucosal congestion.
[0032] Figure 6 shows the HE staining results of mouse gastric tissue after different treatment groups (10.0×). a is the HE staining result of mouse gastric tissue after treatment with blank group; b is the HE staining result of mouse gastric tissue after treatment with model group; c is the HE staining result of mouse gastric tissue after treatment with positive control group; d is the HE staining result of mouse gastric tissue after treatment with Dendrobium huoshanense water extract group.
[0033] Figure 7 shows the changes in absorbance of different groups of probiotic suspensions over time during aerobic probiotic culture.
[0034] Figure 8 shows the changes in absorbance of different groups of probiotic suspensions over time during anaerobic probiotic culture. Detailed Implementation
[0035] Unless otherwise specified, all raw material components in this application are commercially available products well known to those skilled in the art.
[0036] The technical solutions provided in this application will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of this application.
[0037] In the following examples, the following ingredients were used: Fifteen-strain compound probiotics (Xi'an Jushengyuan Biotechnology Co., Ltd., batch number 20230801), dextrin (Changling Jilong Biopharmaceutical Co., Ltd., batch number 20230407), magnesium stearate (Shandong Liujia Pharmaceutical Excipients Co., Ltd., batch number 20221201), xylitol (Shandong Futian Pharmaceutical Co., Ltd., batch number 20230601), soluble starch (Tianjin Damao Chemical Reagent Factory, batch number 20230801), and malt syrup (trade name 5% malt syrup, Chunyuan Food (Yucheng) Co., Ltd., batch number 20230701).
[0038] Microcrystalline cellulose (Shandong Liujia Pharmaceutical Excipients Co., Ltd., batch number 130701), xylitol (Anhui Yishou Jinfang Pharmaceutical Co., Ltd., batch number C118062803), stearyl alcohol (Jiangxi Alpha Pharmaceutical Co., Ltd., batch number 20200201), polyacrylic acid resin II, stearic acid (both purchased from Huzhou Zhanwang Pharmaceutical Co., Ltd., batch numbers 210602 and 210201 respectively), sodium carboxymethyl cellulose (Shanghai Maclean Biochemical Technology Co., Ltd., batch number 20201007), hydroxypropyl methylcellulose (Hunan Xinlvfang Pharmaceutical Co., Ltd., batch number 21021804).
[0039] Example 1
[0040] A method for preparing a gastric retention type traditional Chinese medicine probiotic double-layer floating tablet, the steps of which are as follows:
[0041] (1) Preparation of Dendrobium huoshanense extract: Take the dried stem of Dendrobium huoshanense, crush it, pass it through a 100-mesh sieve, and mix the crushed Dendrobium huoshanense with water at a mass-volume ratio of 1g:20mL. Soak it at 70℃ for 2h, filter it to obtain the first filtrate and the first filter residue. Mix the first filter residue with water at a mass-volume ratio of 1g:20mL, soak it at 70℃ for 2h, filter it to collect the second filtrate, combine the first filtrate and the second filtrate to obtain the filtrate after extraction, concentrate the filtrate after extraction under reduced pressure at 60℃, freeze dry it to obtain Dendrobium huoshanense extract;
[0042] (2) 30% of Dendrobium huoshanense extract, 8% of hydroxypropyl methylcellulose (HPMC), 18% of stearyl alcohol, 28% of polyacrylic acid resin II, 11% of microcrystalline cellulose, and 4% of xylitol were sieved, mixed, and a suitable amount of water was used as a binder to achieve a state where the mixture could be formed into a ball by hand and crumbled upon touch. The mixture was then used to prepare a soft material, which was sieved through a 24-mesh nylon sieve to make wet granules. The granules were dried at 55°C for 30 minutes and then sized using 24-mesh and 80-mesh sieves. Particles larger than 80-mesh and smaller than 24-mesh were retained. Then, 1% of magnesium stearate was added to obtain the Dendrobium huoshanense extract layer material.
[0043] In this step, each percentage refers to the percentage of the mass of each material to the total mass of the materials. The total mass of the materials refers to the total mass of Dendrobium officinale extract, hydroxypropyl methylcellulose, stearyl alcohol, polyacrylic acid resin II, magnesium stearate, microcrystalline cellulose and xylitol.
[0044] (3) Take 20% of the 15-strain compound probiotic, 40% of soluble starch, 15% of xylitol, 15% of dextrin, and 9% of mannitol. Sieve and mix well. Add an appropriate amount of malt syrup as a binder to achieve a state where it can be formed into a ball by hand but crumbles when touched. Perform wet granulation, freeze-dry, add 1% of magnesium stearate, and mix well to obtain the probiotic layer material. In this step, each percentage refers to the percentage of the mass of each material to the total mass of the material. The total mass of the material refers to the total mass of the 15-strain compound probiotic, soluble starch, xylitol, dextrin, mannitol, and magnesium stearate.
[0045] (4) Take the Dendrobium huoshanense extract layer material and press it under a pressure of 50N. ， The Dendrobium huoshanense tablets were obtained, and then an equal mass of probiotic material was added to the Dendrobium huoshanense tablets and compressed again under a pressure of 13N to obtain gastric retention type Chinese medicine probiotic double-layer floating tablets.
[0046] Comparative Example 1
[0047] The difference between this comparative example and Example 1 is that in this comparative example, step (2) is as follows: the amount of Dendrobium huoshanense extract is 40%, hydroxypropyl methylcellulose (HPMC) is 6.9%, stearyl alcohol is 15.4%, polyacrylic acid resin II is 24%, microcrystalline cellulose is 9.4%, xylitol is 3.4%, sieve, mix, and add an appropriate amount of water as a binder to achieve a state where it can be formed into a ball by hand and crumbles when touched. Mix to prepare a soft material, pass it through a 24-mesh nylon sieve to make wet granules, dry it at 55°C for 30 minutes, and granulate it through 24-mesh and 80-mesh sieves, retaining particles larger than 80 mesh and smaller than 24 mesh. Then add 0.9% magnesium stearate to obtain the Dendrobium huoshanense extract layer material. The remaining steps are the same as in Example 1.
[0048] Comparative Example 2
[0049] The difference between this comparative example and Example 1 is that in this comparative example, step (2) is as follows: the amount of Dendrobium huoshanense extract is 50%, hydroxypropyl methylcellulose (HPMC) is 5.7%, stearyl alcohol is 12.9%, polyacrylic acid resin II is 20%, microcrystalline cellulose is 7.9%, xylitol is 2.9%, sieve, mix, and add an appropriate amount of water as a binder to achieve a state where it can be formed into a ball by hand and crumbles when touched. Mix to prepare a soft material, pass it through a 24-mesh nylon sieve to make wet granules, dry it at 55°C for 30 minutes, and granulate it through 24-mesh and 80-mesh sieves, retaining particles larger than 80 mesh and smaller than 24 mesh. Then add 0.6% magnesium stearate to obtain the Dendrobium huoshanense extract layer material. The remaining steps are the same as in Example 1.
[0050] Comparative Example 3
[0051] The difference between this comparative example and Example 1 is that in step (2) of this comparative example, 8% hydroxypropyl methylcellulose (HPMC), 28% polyacrylic acid resin II (Eudragit II), and 11% microcrystalline cellulose are replaced with 32% hydroxypropyl methylcellulose (HPMC) and 15% microcrystalline cellulose. The remaining steps are the same as in Example 1.
[0052] Comparative Example 4
[0053] The difference between this comparative example and Example 1 is that in step (2) of this comparative example, 8% hydroxypropyl methylcellulose, 28% polyacrylic acid resin II, and 11% microcrystalline cellulose are replaced with 32% polyacrylic acid resin II and 15% microcrystalline cellulose. The remaining steps are the same as in Example 1.
[0054] Comparative Example 5
[0055] The difference between this comparative example and Example 1 is that in step (2) of this comparative example, 8% hydroxypropyl methylcellulose (HPMC), 28% polyacrylic acid resin II, and 11% microcrystalline cellulose are replaced with 32% sodium carboxymethyl cellulose (CMC-Na) and 15% microcrystalline cellulose. The remaining steps are the same as in Example 1.
[0056] Comparative Example 6
[0057] The difference between this comparative example and Example 1 is that in step (2) of this comparative example, 8% hydroxypropyl methylcellulose (HPMC), 28% polyacrylic acid resin II, and 11% microcrystalline cellulose are replaced with 8% hydroxypropyl methylcellulose (HPMC), 24% sodium carboxymethyl cellulose (CMC-Na), and 15% microcrystalline cellulose. The remaining steps are the same as in Example 1.
[0058] Comparative Example 7
[0059] The difference between this comparative example and Example 1 is that in step (2) of this comparative example, 8% hydroxypropyl methylcellulose (HPMC), 28% polyacrylic acid resin II, and 11% microcrystalline cellulose are replaced with 24% hydroxypropyl methylcellulose (HPMC), 8% sodium carboxymethyl cellulose (CMC-Na), and 15% microcrystalline cellulose. The remaining steps are the same as in Example 1.
[0060] Comparative Example 8
[0061] The difference between this comparative example and Example 1 is that in step (2) of this comparative example, 18% octadecyl alcohol is replaced with 18% stearic acid, and the remaining steps are the same as in Example 1.
[0062] Comparative Example 9
[0063] The difference between this comparative example and Example 1 is that in step (2) of this comparative example, 11% microcrystalline cellulose and 4% xylitol are replaced with 15% microcrystalline cellulose, while the remaining steps are the same as in Example 1.
[0064] Comparative Example 10
[0065] The difference between this comparative example and Example 1 is that in step (2) of this comparative example, the amount of Dendrobium huoshanense extract is 30.24%, hydroxypropyl methylcellulose (HPMC) 8.06%, stearyl alcohol 18.15%, polyacrylic acid resin II 28.23%, microcrystalline cellulose 11.09%, xylitol 4.03%, sieved, mixed, and an appropriate amount of water is used as a binder to achieve a state where it can be formed into a ball by hand and crumbles when touched. The mixture is then used to prepare a soft material, which is sieved through a 24-mesh nylon sieve to make wet granules. The granules are dried at 55°C for 30 minutes, and then sized using 24-mesh and 80-mesh sieves, retaining particles larger than 80 mesh and smaller than 24 mesh. Then, 0.2% magnesium stearate is added to obtain the Dendrobium huoshanense extract layer material. The remaining steps are the same as in Example 1.
[0066] Comparative Example 11
[0067] The difference between this comparative example and Example 1 is that in step (2) of this comparative example, the amount of Dendrobium huoshanense extract is 30.25%, hydroxypropyl methylcellulose (HPMC) 8.04%, stearyl alcohol 18.09%, polyacrylic acid resin II 28.14%, microcrystalline cellulose 11.06%, xylitol 4.02%, sieved, mixed, and an appropriate amount of water is used as a binder to achieve a state where it can be formed into a ball by hand and crumbles when touched. The mixture is then used to prepare a soft material, which is sieved through a 24-mesh nylon sieve to make wet granules. The granules are dried at 55°C for 30 minutes, and then sized using 24-mesh and 80-mesh sieves, retaining particles larger than 80 mesh and smaller than 24 mesh. Then, 0.4% magnesium stearate is added to obtain the Dendrobium huoshanense extract layer material. The remaining steps are the same as in Example 1.
[0068] Comparative Example 12
[0069] The difference between this comparative example and Example 1 is that in step (4) of this comparative example, the probiotic layer material is first pressed at a pressure of 10N to obtain a probiotic layer tablet, and then an equal mass of Dendrobium officinale extract layer material is added to the probiotic layer tablet and pressed again at a pressure of 50N to obtain a double-layer tablet of traditional Chinese medicine probiotics. The remaining steps are the same as in Example 1.
[0070] Example 2: Screening of Dendrobium huoshanense extract layer material formulation and preparation process study
[0071] 2.1 Verification and Quality Evaluation Methods for Dendrobium huoshanense Layer Processing
[0072] ① Properties
[0073] Three batches of double-layered floating Dendrobium officinale samples were randomly selected, and their color and appearance were observed with the naked eye.
[0074] ② Hardness
[0075] The hardness of the sample was tested using a hardness tester. The hardness of the Dendrobium officinale layer in the double-layer floating sheet should be 40-60 N.
[0076] ③ Difference in tablet weight
[0077] In accordance with the provisions on "Tablet Weight Variation" under the "General Rules for Preparations" in the 2020 edition of the Chinese Pharmacopoeia (Volume IV), 10 tablets of this product were randomly selected from each batch for inspection.
[0078] ④ External buoyancy performance
[0079] The release medium was 900 mL of 0.1 mol / L hydrochloric acid solution, at a temperature of (37.0 ± 0.5) ℃ and a rotation speed of (100 ± 1) r / min. The in vitro flotation performance of the tablets was evaluated using the initial floating time (the time it takes for the tablet to float from the bottom of the container to the surface of the liquid after being placed in the medium) and the continuous floating time (the time it takes for the tablet to float on the surface of the medium without sinking) as indicators.
[0080] ⑤ Cumulative release rate in vitro
[0081] The dissolution and release rate determination method was performed using ultraviolet spectrophotometry combined with the second method of the 2020 edition of the Chinese Pharmacopoeia (Volume IV). Six Dendrobium huoshanense tablets were placed in 500 mL of artificial gastric fluid (0.1 mol / L hydrochloric acid solution). The rotation speed was set to (100±1) r / min and the temperature to (37.0±0.5) ℃. 2 mL samples were taken at 2, 4, 6, 8 and 10 h (2 mL of isothermal artificial gastric fluid was added simultaneously). The samples were filtered through a 0.45 μm microporous membrane and the absorbance was measured at 490 nm. The cumulative drug release rate was calculated.
[0082] 2.2 Screening of excipient types and ratios in Dendrobium huoshanense extract layer materials
[0083] (1) Investigation of the dosage of the main drug
[0084] The effect of the amount of Dendrobium officinale extract prepared in step (2) of Example 1 and in step (2) of Comparative Example 1 or 2 on tablet formability.
[0085] The results showed that when the mass percentage of Dendrobium huoshanense extract was 40% and 50%, the resulting soft material was too sticky and difficult to form; while when the mass percentage of Dendrobium huoshanense extract was 30%, the resulting soft material could be formed into a ball when squeezed in the hand and dispersed when touched, and the resulting wet particles were uniform in size and did not stick together. Therefore, the addition amount of Dendrobium huoshanense extract was 30%.
[0086] (2) Investigation of hydrophilic gel materials
[0087] Different amounts of hydroxypropyl methylcellulose (HPMC), sodium carboxymethyl cellulose (CMC-Na), or polyacrylic acid resin II were used to investigate the type and amount of hydrophilic gel materials, namely, the Dendrobium huoshanense slices prepared in Example 1 and the Dendrobium huoshanense slices prepared in Comparative Examples 3-7. ， The sustained-release effect, tablet appearance, and hardness of Dendrobium huoshanense tablets were investigated. For specific investigation methods, please refer to section 2.1.
[0088] The results showed that when any of the matrix materials were used alone, they had no sustained-release effect or the sustained-release effect was not significant. Combining two matrix materials revealed that when HPMC:CMC-Na = 1:3 or 3:1, the tablet hardness did not meet the requirements of section 2.1; however, when HPMC:polyacrylic acid resin II = 1:3, the tablet appearance was smooth, and the hardness met the requirements of section 2.1. Therefore, HPMC and polyacrylic acid resin II were selected as a combination of matrix materials to achieve the expected long-acting, sustained-release effect.
[0089] (3) Investigation of bleaching aids
[0090] The Dendrobium huoshanense tablets prepared in step (4) of Example 1 and Comparative Example 8 were subjected to an in vitro flotation performance test to examine the type and amount of flotation aid as described in section 2.1.
[0091] The results showed that the Dendrobium huoshanense slices containing octadecanol (Example 1) could quickly start floating and maintain the float for 8-10 hours, while the Dendrobium huoshanense slices containing stearic acid (Comparative Example 8) did not float or maintained the float for less than 30 minutes. Therefore, octadecanol was selected as the floatation aid.
[0092] (4) Investigation of fillers
[0093] The Dendrobium huoshanense slices prepared in step (4) of Example 1 and Comparative Example 9 were examined for their formability and compressibility in accordance with the properties described in section 2.1.
[0094] The results showed that the Dendrobium huoshanense tablets prepared in Comparative Example 9 were prone to sticking and punching, resulting in a rough surface. In contrast, the addition of xylitol to the Dendrobium huoshanense tablets prepared in Example 1 significantly improved the appearance and formability of the tablets. The combined use of microcrystalline cellulose and xylitol resulted in tablets with uniform texture and an attractive appearance.
[0095] (5) Investigation of Lubricant Dosage
[0096] The Dendrobium huoshanense tablets prepared in step (4) of Example 1, Comparative Example 10 and Comparative Example 11 were examined for their formability according to the properties described in section 2.1.
[0097] The results showed that if the amount of magnesium stearate was too low, the tablets were still prone to sticking. Therefore, after adding 1% magnesium stearate and mixing evenly, the granules had good compressibility and the tablet surface was smooth.
[0098] Table 1. Selection of Main and Auxiliary Materials by Type and Ratio
[0099] Therefore, through single-factor investigation (see Table 1), the amount of Dendrobium huoshanense extract added was initially determined to be 30%. HPMC and polyacrylic acid resin II were selected as the skeleton material to achieve the expected long-lasting and sustained-release effect. Octadecyl alcohol was selected as the bleaching aid, and appropriate amounts of microcrystalline cellulose and xylitol were added as fillers. After adding 1% magnesium stearate and mixing evenly, tablets were compressed.
[0100] 2.3 Evaluation results of Dendrobium huoshanense lamellae quality
[0101] 2.3.1 External buoyancy performance
[0102] The Dendrobium huoshanense tablets prepared in Example 1 were tested for in vitro flotation properties according to section 2.1.
[0103] The results in Figure 1 show that the Dendrobium huoshanense tablets prepared in this application floated instantly after being placed in water.
[0104] 2.3.2 Optimization Experiment of Formula Excipients
[0105] Based on single-factor experiments, an L9(3) design was created. 4 An orthogonal experiment was conducted to investigate the effects of the dosage of HPMC, stearyl alcohol, and polyacrylic acid resin II on tablet performance and to screen the optimal formulation.
[0106] Table 2. Factors affecting the amount of auxiliary materials used
[0107] The gastric retention type probiotic double-layer floating tablets were prepared according to the method of Example 1. The difference from Example 1 is that the amounts of HPMC, stearyl alcohol, polyacrylic acid resin II, microcrystalline cellulose and xylitol are as follows: the amounts of HPMC, stearyl alcohol and polyacrylic acid resin II are shown in the excipient dosage factor level table (see Table 2) and the orthogonal test results table (see Table 4). The remaining amounts are made up to 100% with microcrystalline cellulose and xylitol, wherein the mass percentage of microcrystalline cellulose to xylitol is 15:4.
[0108] Based on the in vitro cumulative release rate in section 2.1, the drug release performance of the tablets was comprehensively scored using the cumulative release rate of Dendrobium huoshanense tablets as an indicator (see Table 3). The drug release performance was examined using the comprehensive score (Q) as an indicator. The results of the orthogonal experiment and the in vitro flotation performance are shown in Table 4, and the analysis of variance is shown in Table 5.
[0109] Table 3 Tablet Scoring Criteria
[0110] Table 4. Results of the Orthogonal Experiment
[0111] Table 5. Analysis of Variance Table Note: F 0.05 (2,2)=19.00.
[0112] The results in Table 4 show that the comprehensive scores of experiments 5 and 9 are both greater than 90 points. Therefore, the in vitro drug release models of experiments 5 and 9 will be examined to further determine the optimal formulation.
[0113] 2.3.3 Fitting of in vitro drug release model
[0114] The cumulative release rates of drugs in vitro for samples 5 and 9 were fitted to time using a first-order equation, and the results are shown in Table 6.
[0115] Table 6 Fitting Table of In Vitro Drug Release Model
[0116] The results in Table 6 show that Equation No. 5 has a good fit, a long holding time, and a cumulative dissolution rate of 87.5% after 10 hours, indicating good drug release performance. It is the optimal formulation, namely HPMC 8%, octadecanol 18%, and polyacrylic acid resin II 28%.
[0117] 2.3.4 Validation of the Dendrobium huoshanense tablet manufacturing process
[0118] Method for determination of total polysaccharide content
[0119] (1) Preparation of standard curve
[0120] Accurately weigh 1 mg of anhydrous glucose standard (dried to constant weight at 105℃) into a 10 mL volumetric flask to prepare a 0.1 mg / mL glucose standard solution. Accurately measure 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the standard solution into separate 10 mL stoppered test tubes. Add water to each tube to bring the volume to 1.0 mL. Use distilled water as a blank control. Determine the glucose content using the phenol-sulfuric acid method. Add 1 mL of prepared 5% phenol solution and 5 mL of concentrated sulfuric acid solution to each test tube, shake quickly, and heat in a boiling water bath for 15 min to ensure complete reaction. Then, place the test tubes in an ice-water bath to cool. Finally, measure the absorbance at 490 nm. Plot a standard curve with absorbance on the ordinate and concentration on the abscissa.
[0121] The standard curve for glucose concentration is shown in Figure 2. Based on the standard curve, the regression equation is: y = 12.749x - 0.0099, R0. 2 =0.9959 (unit: mg / mL).
[0122] (2) Determination of total polysaccharide content in extract
[0123] Accurately weigh 0.05 g of the Dendrobium huoshanense extract prepared in Example 1, dissolve it in water to a 10 mL volumetric flask, make up to volume, mix well, take 1 mL, put it into a 100 mL volumetric flask, make up to volume, shake well, and the product is obtained. Accurately measure 1.0 mL of the sample solution, use distilled water as a blank control, and determine the absorbance according to the "phenol-sulfuric acid method" described in (1). Substitute the absorbance into the regression equation in (1) to calculate the total polysaccharide content in the Dendrobium huoshanense extract.
[0124] According to the standard curve in Figure 2, the total polysaccharide content in the Dendrobium huoshanense extract was determined to be 33.71%.
[0125] (3) Determination of total polysaccharide content in tablets
[0126] Three batches of Dendrobium huoshanense slices prepared in Example 1 were pulverized. An appropriate amount of fine powder was added to 500 mL of 0.1 mol / L hydrochloric acid and dissolved thoroughly before filtration. 1.0 mL of the sample solution was accurately pipetted and the total polysaccharide content of the extract was determined by the phenol-sulfuric acid method.
[0127] Three batches of Dendrobium huoshanense tablets prepared in Example 1 were tested according to the process verification and quality evaluation method of Dendrobium huoshanense tablets in Part 2.1, and the cumulative release rate of Dendrobium huoshanense tablets in Part 2.3.2 was also investigated. The results are shown in Tables 7-8 and Figure 3.
[0128] Table 7 Process Validation of Three Batches of Dendrobium officinale Tablets
[0129] Table 8. In vitro release rate of three batches of Dendrobium officinale tablets (cumulative release: %)
[0130] The Dendrobium huoshanense tablets prepared in Example 1 of this application have an attractive appearance, uniform color, and moderate hardness. The weight difference among the three batches of process verification tablets is less than 5%. They can float instantly in the dissolution medium, with a floating time of >10h. The release rate increases uniformly within 10h, with an average release rate of 87.3% (see Figure 3). There is no significant difference in in vitro release rate between batches, and the process reproducibility is good.
[0131] Example 3
[0132] The appearance, hardness, and disintegration time of the gastric retention type Chinese herbal probiotic double-layer floating tablets prepared in Example 1 and the Chinese herbal probiotic tablets prepared in Comparative Example 12 were examined to evaluate their quality.
[0133] The results in Figure 4 show that the gastric retention type probiotic double-layer floating tablets prepared in Example 1 are complete, uniform in color, and have a certain degree of smoothness. The hardness of the prepared probiotic layer is 10.0N, which is suitable. The average disintegration time is 10 minutes, indicating that it can dissolve in the human digestive tract and can be absorbed and utilized by the human body.
[0134] The average disintegration time of the probiotic tablets prepared in Comparative Example 12 was 12 hours, which meets the sustained-release requirement of the Dendrobium huoshanense layer, but does not achieve preferential release of the probiotic layer. In contrast, the gastric retention type probiotic double-layer floating tablets of Example 1, with a harder Dendrobium huoshanense layer and a softer probiotic layer, had an average disintegration time of 15 minutes, meeting the requirement for preferential release of the probiotic layer. The Dendrobium huoshanense layer dissolved slowly, with an average disintegration time of 12 hours. Therefore, the compression sequence for gastric retention type probiotic double-layer floating tablets should be: first pre-compress the Dendrobium huoshanense layer, then add the probiotic layer for secondary compression.
[0135] Example 4
[0136] The quality evaluation of the gastric retention type probiotic double-layer floating tablets prepared in Example 1 was conducted according to section 2.1 of Example 2.
[0137] (1) Properties: The results in Figure 4 show that the gastric retention type probiotic double-layer floating tablets prepared in Example 1 are complete, have uniform color, and have a certain degree of smoothness.
[0138] (2) Weight Variation: The results showed that the average weight of the 20 gastric retention type probiotic double-layer floating tablets was 0.5085g. According to the weight variation test method in Part IV of the Chinese Pharmacopoeia (2020 edition), the tablet weight ≥ 0.3g has a variation limit of ±5.0%. Therefore, the tablet weight of the probiotic double-layer floating tablets should be between 0.4831g and 0.5339g to meet the requirements. The final results showed that no tablet exceeded the weight variation limit, which complies with the provisions of the Chinese Pharmacopoeia.
[0139] (3) Hardness: The results showed that the double-layer floating tablets of traditional Chinese medicine probiotics prepared in Example 1 all had suitable hardness, with an average hardness of 65.18 N.
[0140] (4) Disintegration time: The results showed that the disintegration time of the six Chinese herbal probiotic double-layer floating tablets prepared in Example 1 was good. The average disintegration time of the probiotics was 19.5 min, and the average disintegration time of the Dendrobium huoshanense layer was 12 h 12.5 min, which met the requirements of the 2020 edition of the Chinese Pharmacopoeia.
[0141] Example 5
[0142] Study on the stability and tolerance of live bacteria in gastrointestinal fluid-simulated gastrointestinal fluid-induced double-layer floating tablets of probiotics with gastric retention
[0143] 5.1 Experimental Methods
[0144] 5.1.1 In vitro simulated digestion
[0145] 5.1.1.1 Preparation of digestive fluid
[0146] (1) In vitro simulated gastric juice: Adjust the pH of 0.1 mol / L potassium phosphate buffer to 3.5, sterilize it, and then add pepsin (10 g / L), mix to dissolve it completely, and obtain simulated gastric juice. Preheat to 37°C before use.
[0147] (2) In vitro simulated intestinal fluid: After adjusting the pH of potassium phosphate buffer to 6.8 and sterilizing it, add trypsin (10 g / L) and porcine bile salts (3 g / L), mix and dissolve thoroughly to obtain simulated intestinal fluid. Preheat to 37°C before use.
[0148] 5.1.1.2 Simulated Gastrointestinal Digestion
[0149] Take one gastric retention type probiotic double-layer floating tablet (referred to as double-layer tablet, specification: 0.5g / tablet) prepared in Example 1, and another 0.1g of Fifteen-strain compound probiotics (equivalent to the amount of probiotic powder added to the double-layer tablet, with a total live bacteria count ≥1×10⁻⁶). 8 Accurately weighed CFU / g was added to 20.0 mL of simulated gastric juice (pH 3.5) and placed in a water bath at 37°C and 90 rpm for 2 hours of agitated digestion. Then, 80.0 mL of simulated intestinal juice was added, and the digestion was continued at 37°C and 120 rpm for 2 hours under anaerobic conditions to simulate the gastrointestinal digestion process. The viable bacterial count was determined using the plate count method, and parallel experiments were performed. The survival rate was calculated using the formula below.
[0150] Survival rate (%) = lgN i ÷lgN0×100, where Ni represents the number of viable bacteria after digestion ih; N0 represents the initial number of viable bacteria.
[0151] 5.1.1.3 Viable cell count
[0152] The viable bacteria count was based on the method for counting lactobacilli and thermophilic streptococci in GB 4789.35—2016 "National Food Safety Standard for Microbiological Examination of Food - Examination of Lactic Acid Bacteria", with slight modifications.
[0153] Take 1.0 mL of the above digestion solution and place it in a test tube containing 9.0 mL of sterile physiological saline. Shake thoroughly, then perform a 10-fold serial dilution, selecting 2-3 suitable dilution gradients. Take 100 μL of each dilution into sterile petri dishes, making 3 petri dishes for each dilution. Pour approximately 15 mL of MRS medium cooled to 48°C into each petri dish and rotate the dish to mix thoroughly. After the petri dishes solidify, invert them into an anaerobic bag and anaerobic incubate at (36±1)°C for (72±2) h. Then, perform a viable cell count experiment.
[0154] 5.2 Experimental Results
[0155] 5.2.1 Tolerance of probiotics to simulated gastrointestinal fluid
[0156] The survival of the double-layered sheets after simulating the gastrointestinal tract is shown in Table 9.
[0157] Table 9. Tolerance of probiotics in bilayer tablets to simulated gastrointestinal fluid (survival rate / %, n=6)
[0158] The results in Table 9 show that the survival rates of both decreased under simulated gastric juice conditions. The survival rate of the bilayer tablets after digestion in simulated gastric juice was 78.37%, while the survival rate of the unprepared Fifteen-strain compound probiotic raw material 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 can be fully dissolved, allowing the probiotics to be completely released.
[0159] The survival rates of probiotics in the bilayer tablet and the fifteen-strain compound probiotic group, after simulating gastrointestinal fluid, were 94.54% and 90.31%, respectively. Under harsh simulated gastrointestinal fluid conditions, the survival rates of both groups initially decreased and then increased, indicating that probiotics can recover their activity within a certain period after being damaged by gastrointestinal fluid. Furthermore, the survival rate of probiotics in the bilayer tablet group was slightly higher than that in the fifteen-strain compound probiotic group, suggesting that the continuous release of Dendrobium officinale extract from the bilayer tablet provides a carbon source for the probiotics, promoting their proliferation.
[0160] Example 6
[0161] Effects of Dendrobium huoshanense extract on ethanol-induced gastric injury model in mice
[0162] 6.1 Experimental Methods
[0163] Twenty-four clean-grade ICR mice were acclimatized for 3 days and then randomly divided into a blank control group, a model group, an omeprazole positive control group, and a Dendrobium officinale water extract group, with 6 mice in each group. All groups were administered the drug by gavage at a dose of 10 mL / kg. The blank control group and the model group were administered 0.05% sodium carboxymethyl cellulose solution by gavage daily, the positive control group was administered 480 mg / kg of omeprazole enteric-coated capsule contents by gavage daily, and the Dendrobium officinale water extract group was administered 1.56 g / kg of Dendrobium officinale water extract prepared in step (1) of Example 1 by gavage daily for 10 consecutive days (all gavage samples were dissolved in 0.05% sodium carboxymethyl cellulose solution). After administration on the 9th day, the mice were kept on a fasting but allowed free access to water. One hour after the last administration on the 10th day, all mice except the blank control group were administered 50% ethanol solution (0.1 mL / 20 g per mouse) by gavage. One hour later, an acute gastric mucosal injury model was formed. The stomachs of mice were harvested for observation and damage fraction was measured. The stomach tissue was fixed in 4% paraformaldehyde and the pathological damage was observed.
[0164] (1) Determination of gastric mucosal injury fraction in mice
[0165] After the mice were sacrificed, their stomach tissue was removed via laparotomy. The stomach was cut along the greater curvature, and the contents were washed with pre-cooled PBS. The tissue was then spread onto filter paper to observe the gastric mucosal damage. Measurements were taken at the damaged sites using a ruler. The gastric mucosal damage score was calculated using the Guth method.
[0166] No damage is scored as 0 points; mucosal erythema and punctate bleeding (length and width <0.5mm) are scored as 1 point; small erosions are scored as 2 points (<1mm); moderate erosions (1mm~2mm) are scored as 3 points; large erosions (2mm~3mm) are scored as 4 points; and large-area erosions (≥3mm) are scored as 5 points.
[0167] Scoring is doubled for widths greater than 1mm, and scores are based on area for patchy bleeding.
[0168] (2) Pathological observation of mouse gastric tissue
[0169] Gastric tissue was fixed, dehydrated and cleared, embedded in paraffin, sectioned and mounted, dewaxed, stained with hematoxylin and eosin (HE), dehydrated and cleared, mounted, and observed.
[0170] 6.2 Experimental Results
[0171] Table 10 Effects of different treatment groups on acute liver and stomach injury in mice (n=6, ) Note: ## P<0.01 indicates a highly significant difference compared to the control group. * P<0.05 indicates a highly significant difference compared to the model group.
[0172] The results in Table 10 show that, compared with the model group, the degree of gastric mucosal damage in mice treated with Dendrobium huoshanense water extract was significantly reduced, and the degree of gastric mucosal damage reduced by Dendrobium huoshanense water extract group was comparable to that in the omeprazole positive group. Therefore, the Dendrobium huoshanense water extract of this application can effectively alleviate gastric mucosal damage.
[0173] Figure 5 shows that the gastric mucosa of the control group mice showed no sloughing, congestion, edema, or damage. An acute liver injury model was established in the model group mice by gavage with 50% ethanol solution. Extensive erosion and multiple hemorrhages were observed on the gastric surface of the model group mice, with significant differences between the model group and the control group, indicating the successful establishment of the 50% ethanol solution-induced acute liver injury model in mice. The omeprazole-positive group mice showed a small amount of erythematous hemorrhage on the gastric surface, but no significant bleeding lesions, indicating a good protective effect. Intervention with Dendrobium huoshanense water extract alleviated the erosion on the gastric surface and significantly reduced the width of bleeding, suggesting that Dendrobium huoshanense water extract can alleviate acute gastric mucosal injury.
[0174] Figure 6 shows that the model group mice exhibited abnormal gastric tissue structure, with extensive mucosal ulceration, epithelial cell erosion and necrosis, as indicated by the black arrows; capillary dilation and congestion, as indicated by the yellow arrows; and abundant inflammatory cell infiltration, as indicated by the red arrows. The positive control group and the Dendrobium officinale aqueous extract group showed basically normal gastric tissue structure, with some areas showing loosely arranged epithelial cells, as indicated by the black arrows; no obvious ulceration or inflammatory cell infiltration was observed. This indicates that Dendrobium officinale aqueous extract can repair tissue structure, alleviate inflammatory infiltration, and improve acute gastric mucosal injury.
[0175] Example 7
[0176] Effects of Dendrobium huoshanense extract on probiotics (compound probiotic powder)
[0177] I. Experimental Methods
[0178] 7.1 Probiotic proliferation
[0179] Weigh 1g of the 15-strain compound probiotic and add it to 10mL of MRS medium, then suspend to obtain a mixed probiotic suspension. Prepare 60mL of MRS medium as the aerobic probiotic culture group and MRS medium containing anhydrous cysteine hydrochloride as the anaerobic probiotic culture group, respectively. Sterilize the medium and add 9mL of each medium to a test tube, followed by 200μL of the mixed probiotic suspension. Shake the test tubes. Place the aerobic probiotic culture group test tubes in a 37℃ incubator for 24h, and place the anaerobic probiotic culture group test tubes in an anaerobic container in a 37℃ incubator for 24h.
[0180] After the culture is completed, centrifuge (2000 rpm, 10 min), discard the supernatant, add 300 μL of 20% glycerol to the precipitate, and freeze it in an ultra-low temperature freezer at -80℃ for later use.
[0181] 7.2 Effects of different concentrations of double-layer tablets on the proliferation of probiotics in compound probiotic powder
[0182] 7.2.1 Microbial Culture
[0183] Take an appropriate amount of Dendrobium huoshanense water extract prepared in step (1) of Example 1 (the preparation process of Dendrobium huoshanense water extract is the same as that in the double-layer tablets), and prepare MRS culture media containing Dendrobium huoshanense water extract at concentrations of 0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 g / L and MRS culture media containing anhydrous cysteine hydrochloride, respectively. Sterilize, take 10 mL of culture media containing double-layer tablets and add them to test tubes, add 200 μL of mixed probiotic suspension to each, shake evenly, place the test tubes of the aerobic probiotic culture group in a constant temperature incubator at 37℃ for 24 h, and place the test tubes of the anaerobic probiotic culture group in an anaerobic tank and incubate at 37℃ for 24 h.
[0184] 7.2.2 Dilution Culture
[0185] After the culture is complete, remove the test tubes from each group, mix the cultured bacteria thoroughly, and then use an appropriate amount of physiological saline to continuously dilute to a concentration of 10. 7 Then, take 900 μL into an empty petri dish, and pour the prepared and sterilized solid culture medium into the petri dish containing the bacterial solution. After gently shaking and standing, let it cool and solidify, and then place it in a 37℃ constant temperature incubator and an anaerobic tank for 24 h.
[0186] 7.2.3 Counting
[0187] Remove the solid culture medium, count the colonies, and analyze the data graphically.
[0188] 7.3 Investigating the effect of Dendrobium huoshanense water extract on the growth rate of probiotics
[0189] 7.3.1 Effects of Dendrobium huoshanense water extract on the growth rate of aerobic bacteria
[0190] An appropriate amount of Dendrobium huoshanense aqueous extract prepared in step (1) of Example 1 was used to prepare 50 mL of MRS medium containing 5 g / L of Dendrobium huoshanense aqueous extract. After sterilization, a mixed probiotic suspension was added. A control group containing only the mixed probiotic suspension and no Dendrobium huoshanense aqueous extract was used as a reference. The medium was incubated at 37℃. Samples were taken at 0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 h, with 200 μL of sample taken each time and 200 μL of medium added. The 200 μL of bacterial solution was placed in a sterile 96-well plate, and the absorbance at 600 nm was measured using a microplate reader at different time points.
[0191] 7.3.2 Effects of Dendrobium huoshanense water extract on the growth rate of anaerobic bacteria
[0192] Take an appropriate amount of the Dendrobium huoshanense water extract prepared in step (1) of Example 1, and prepare 50 mL of anhydrous MRS medium containing 5 g / L of Dendrobium huoshanense water extract and cysteine hydrochloride. After sterilization, add a mixed probiotic suspension. At the same time, use the mixed probiotic suspension without Dendrobium huoshanense water extract as a control group. Incubate in a 37℃ constant temperature incubator. Take samples at 0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 h, taking 200 μL each time and adding 200 μL of medium. Place the 200 μL of bacterial solution in a sterile 96-well plate, and use an ELISA reader to measure the absorbance of the bacterial solution at 600 nm at different time points.
[0193] II. Experimental Results
[0194] (1) Experimental results on the effects of different concentrations of Dendrobium huoshanense aqueous extract on probiotic proliferation
[0195] The compound probiotic suspension was inoculated into a culture medium containing Dendrobium huoshanense extract at a concentration of 0.625–40 g / L. After 24 hours, the number of colonies was recorded by plate count, and the proliferation of aerobic and anaerobic probiotics was statistically analyzed (see Table 11).
[0196] Table 11 Effects of Dendrobium huoshanense water extract on probiotic proliferation (n=3, n±x)
[0197] The results in Table 11 show that the proliferation of probiotics gradually increased with the increase of the concentration of Dendrobium huoshanense water extract, indicating that Dendrobium huoshanense water extract at concentrations of 0.625–40 g / L has a proliferative effect on both aerobic and anaerobic bacteria.
[0198] (2) Experimental results on the effect of Dendrobium huoshanense water extract on the growth rate of probiotics
[0199] The mixed probiotic suspension was inoculated into MRS medium containing 5 g / L fresh Dendrobium huoshanense juice. The growth was observed at different time points within 48 h using absorbance measurement (see Table 12), and the effect on the growth rate of probiotics was analyzed (Figures 7 and 8).
[0200] Table 12. Changes in absorbance of probiotic suspension over time (n=3)
[0201] The results in Table 12 show that the proliferation of probiotics gradually increased over time, suggesting that the water extract of Dendrobium huoshanense has a proliferative effect on both aerobic and anaerobic bacteria.
[0202] The results in Figures 7 and 8 show that the 5 g / L concentration of Dendrobium huoshanense water extract significantly increased the growth rate of probiotics from 4 to 12 hours, and then tended to stabilize.
[0203] In summary, the water extract of Dendrobium huoshanense can promote the proliferation of probiotics in compound probiotic powder.
[0204] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.
Claims
1. A gastric retention type traditional Chinese medicine probiotic double-layer floating tablet, characterized in that, The herbal probiotic double-layer floating tablet comprises a Dendrobium officinale extract layer, a probiotic layer, and an adhesive; The Dendrobium huoshanense extract layer is made from the following raw materials in the following mass percentages: The mixture contains 25%–35% Dendrobium huoshanense extract, 7%–9% hydroxypropyl methylcellulose, 16%–20% stearyl alcohol, 22%–30% polyacrylic acid resin II, and 0.8%–1.5% magnesium stearate, which are then supplemented to 100% with microcrystalline cellulose and xylitol, wherein the mass ratio of microcrystalline cellulose to xylitol is 12–17:
4. The probiotic layer is made from the following raw materials in the indicated weight percentages: Probiotic powder 15%–22%, soluble starch 35%–42%, xylitol 12%–16%, mannitol 8%–10%, magnesium stearate 0.8%–1.5%, and dextrin to make up to 100%.
2. The gastric retention type probiotic double-layer floating tablet according to claim 1, characterized in that, The Dendrobium huoshanense extract is a water extract of Dendrobium huoshanense; the probiotic powder includes a 15-strain compound probiotic.
3. The gastric retention type probiotic double-layer floating tablet according to claim 2, characterized in that, The preparation method of the Dendrobium huoshanense extract includes the following steps: The dried stems of Dendrobium huoshanense were pulverized and sieved to obtain pulverized Dendrobium huoshanense. The pulverized Dendrobium huoshanense was mixed with water and extracted. The filtrate after extraction was collected, concentrated, and dried to obtain Dendrobium huoshanense extract.
4. The gastric retention type probiotic double-layer floating tablet according to claim 3, characterized in that, The sieve mesh size is 80-120 mesh.
5. The gastric retention type probiotic double-layer floating tablet according to claim 3, characterized in that, The sieve mesh size is 90-110 mesh.
6. The gastric retention type probiotic double-layer floating tablet according to claim 3, characterized in that, The sieve mesh size is 100 mesh.
7. The gastric retention type probiotic double-layer floating tablet according to claim 3, characterized in that, The mass-to-volume ratio of the pulverized Dendrobium huoshanense to water is 1g:15-25mL.
8. The gastric retention type probiotic double-layer floating tablet according to claim 3, characterized in that, The mass-to-volume ratio of the pulverized Dendrobium huoshanense mixed with water is 1g:17-22mL.
9. The gastric retention type probiotic double-layer floating tablet according to claim 3, characterized in that, The mass-to-volume ratio of the pulverized Dendrobium huoshanense to water was 1g:20mL.
10. The gastric retention type probiotic double-layer floating tablet according to claim 3, characterized in that, The extraction temperature is 65–85°C.
11. The gastric retention type probiotic double-layer floating tablet according to claim 3, characterized in that, The extraction temperature is 70–85°C.
12. The gastric retention type probiotic double-layer floating tablet according to claim 3, characterized in that, The extraction temperature is 70, 75, 80 or 85°C.
13. The gastric retention type probiotic double-layer floating tablet according to claim 3, characterized in that, The extraction is performed 1 to 3 times.
14. The gastric retention type probiotic double-layer floating tablet according to claim 3, characterized in that, The extraction was performed twice.
15. The gastric retention type probiotic double-layer floating tablet according to claim 3, characterized in that, Each extraction takes 1 to 3 hours.
16. The gastric retention type probiotic double-layer floating tablet according to claim 3, characterized in that, Each extraction session lasts 1.5 to 2.5 hours.
17. The gastric retention type probiotic double-layer floating tablet according to claim 3, characterized in that, Each extraction session lasted 2 hours.
18. The gastric retention type probiotic double-layer floating tablet according to claim 3, characterized in that, The concentration includes vacuum concentration at 60°C.
19. The gastric retention type traditional Chinese medicine probiotic double-layer floating tablet according to claim 3, characterized in that, The drying process includes oven drying or freeze drying.
20. The gastric retention type probiotic double-layer floating tablet according to claim 19, characterized in that, The drying process includes drying at 50–60°C for 30 minutes.
21. The gastric retention type traditional Chinese medicine probiotic double-layer floating tablet according to claim 1, characterized in that, The mass ratio of the Dendrobium officinale extract layer to the probiotic layer is 1:
1.
22. The gastric retention type probiotic double-layer floating tablet according to claim 1, characterized in that, The adhesive is a binder for the Dendrobium huoshanense extract layer and a 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.
23. A method for preparing a gastric retention type probiotic double-layer floating tablet according to any one of claims 1 to 22, characterized in that, Includes the following steps: The extract of Dendrobium huoshanense, hydroxypropyl methylcellulose, stearyl alcohol, polyacrylic acid resin II, microcrystalline cellulose and xylitol were mixed and water was added as a binder to prepare a soft material. The material was wet granulated, dried and granulated, and magnesium stearate was added to obtain the Dendrobium huoshanense extract layer. Probiotic powder, soluble starch, xylitol, dextrin, and mannitol are mixed and malt syrup is added as a binder. The mixture is then wet-granulated, freeze-dried, and magnesium stearate is added to obtain the probiotic layer. First, the Dendrobium huoshanense extract layer is pressed for the first time to obtain Dendrobium huoshanense tablets. Then, a probiotic layer is added to the Dendrobium huoshanense tablets and pressed for the second time to obtain gastric retention type Chinese medicine probiotic double-layer floating tablets.
24. The preparation method according to claim 23, characterized in that, The hardness of the first pressing is 40-60 N; the hardness of the second pressing is 10-15 N.
25. The preparation method according to claim 23, characterized in that, The wet granulation process involves granulating wet particles through a 24-mesh nylon sieve.
26. The preparation method according to claim 23, characterized in that, During drying and granulation, the drying includes oven drying or freeze drying.
27. The preparation method according to claim 26, characterized in that, The drying process involves drying at 50–60°C for 25–35 minutes.
28. The preparation method according to claim 23, characterized in that, The granulation process uses 24-mesh and 80-mesh sieves.
29. The application of the gastric retention type traditional Chinese medicine probiotic double-layer floating tablet according to any one of claims 1 to 22, or the gastric retention type traditional Chinese medicine probiotic double-layer floating tablet obtained by the preparation method according to any one of claims 23 to 28, or the Dendrobium huoshanense extract, in improving acute gastric mucosal injury, characterized in that, The Dendrobium huoshanense extract is the Dendrobium huoshanense extract as described in any one of claims 1 to 22.
30. The use of the gastric retention type traditional Chinese medicine probiotic double-layer floating tablet according to any one of claims 1 to 22, or the gastric retention type traditional Chinese medicine probiotic double-layer floating tablet obtained by the preparation method according to any one of claims 23 to 28, or the Dendrobium huoshanense extract, in the preparation of a drug for improving acute gastric mucosal injury, characterized in that, The Dendrobium huoshanense extract is the Dendrobium huoshanense extract as described in any one of claims 1 to 22.
31. The use of the gastric retention type traditional Chinese medicine probiotic double-layer floating tablet according to any one of claims 1 to 22, or the gastric retention type traditional Chinese medicine probiotic double-layer floating tablet obtained by the preparation method according to any one of claims 23 to 28, or the Dendrobium huoshanense extract in probiotic proliferation or preparation of probiotic proliferation drugs, characterized in that, The Dendrobium huoshanense extract is the Dendrobium huoshanense extract as described in any one of claims 1 to 22.