Dendrobium officinale polysaccharide in-situ gel, and preparation method and use thereof

Abstract

The application discloses a dendrobium officinale polysaccharide in-situ gel and a preparation method and application thereof. The in-situ gel is prepared by mixing dendrobium officinale polysaccharide, low-acetyl gellan gum and a dissolving aid in a certain proportion and crushing. The application is first based on 'combination of medicine and adjuvant', utilizes the thickening characteristics of the dendrobium officinale polysaccharide, is compounded with the low-acetyl gellan gum, and adds the dissolving aid, so that the gelation characteristics and the dissolution speed of the dendrobium officinale polysaccharide are improved. The compounded gel can be quickly dissolved into sol in hot water, can be expanded into a gel in gastric juice, can form a gel protective film adhered to an ulcer, can utilize the viscous property of the high molecular material of the dendrobium officinale polysaccharide to play a physical protection role, can play the prebiotic effect of the dendrobium officinale polysaccharide, can play the functions of benefiting the stomach and generating saliva, regulating immunity and the like, the dendrobium officinale polysaccharide in-situ gel prolongs the action time of the dendrobium officinale polysaccharide in the stomach, further improves the efficacy, and has a good treatment effect on gastritis and gastric ulcer.

Description

Technical Field

[0001] This invention relates to the technical field of traditional Chinese medicine preparations, and more specifically, to an in-situ gel powder of Dendrobium officinale polysaccharide, its preparation method, and its uses. Background Technology

[0002] Dendrobium officinale is an orchid species, commonly known as Dendrobium officinale or Dendrobium nobile. It is the top of the nine great Chinese medicinal herbs. In 2019, Dendrobium officinale was included in the list of medicinal and edible plants, which greatly promoted the development of the Dendrobium officinale industry.

[0003] Dendrobium officinale is traditionally used to benefit the stomach and promote the production of body fluids, as well as to strengthen the digestive system. Its main active ingredient is polysaccharide. The 2020 edition of the Chinese Pharmacopoeia stipulates that the polysaccharide content of Dendrobium officinale, calculated as anhydrous glucose in the dried product, shall not be less than 25.0%.

[0004] Due to the large molecular weight of polysaccharides, they cannot be administered via intravenous, intramuscular, or subcutaneous injection. Furthermore, they are difficult to absorb directly from the intestines after oral administration. Therefore, the protective mechanism of Dendrobium officinale polysaccharides on the intestines remains unclear. Some scholars have begun to attempt to elucidate the mechanism of pharmacological activity of Dendrobium officinale polysaccharides based on intestinal flora analysis. Literature reports that after continuous gavage administration of Dendrobium officinale polysaccharides to mice for 7 days, it increased the total number of intestinal bacteria, increased the number of Lactobacillus colonies, and decreased the number of Escherichia coli and Bifidobacterium colonies. It also had a positive regulatory effect on the activities of amylase, xylanase, and cellulase (Deng Chenzhe. Effects of Dendrobium officinale polysaccharides on intestinal bacterial diversity and related enzyme activities in constipated mice. Chinese Journal of Clinical Pharmacology). Dendrobium officinale polysaccharides may inhibit the growth and colonization of pathogenic bacteria by promoting the growth of probiotics and reshaping the intestinal microecological balance, thus exerting an immunomodulatory effect on the stomach.

[0005] Dendrobium officinale polysaccharides are large molecules; direct ingestion can lead to slow dissolution, poor absorption, and low bioavailability. In-situ gels, also known as in-situ gels, are formulations that, after being administered in solution form, undergo an immediate phase transition at the application site, transforming from a liquid state to a non-chemically cross-linked semi-solid gel. Based on their phase transition mechanisms, in-situ gels can be classified into temperature-sensitive, pH-sensitive, and ion-sensitive types. As a novel drug formulation, they exhibit good tissue compatibility and a long residence time at the administration site; they also serve to store drugs and prevent environmental impacts, making them a hot research topic in pharmaceutics and biotechnology. To protect the stability of traditional Chinese medicine extracts in gastric acid and enhance their protective effect on the stomach, some technologies currently employ pH-sensitive in-situ gel encapsulation to create formulations. The common method involves adding the active ingredient of the drug as the content material into a pre-constructed in-situ gel matrix, forming an encapsulated in-situ gel formulation (e.g., Chinese patents CN114288243B and CN113694144A). While this method can successfully construct in-situ gel formulations that exist as sols in vitro but undergo a phase transition in the acidic conditions of the stomach to form a gel with a certain strength and viscosity, better covering the ulcer surface and slowly dissolving and releasing the drug for more effective efficacy, these encapsulated formulations use a large amount of the in-situ gel matrix as an excipient, resulting in a relatively low content of the active ingredient. To improve the efficacy of the formulation and prolong its duration of action in the stomach, further research on its therapeutic effects is necessary, and new formulations with higher active ingredient content and greater safety and effectiveness should be developed. Summary of the Invention

[0006] The purpose of this invention is to overcome the aforementioned defects and deficiencies in the prior art and to provide a drug-excipient integrated gel based on the principle of "drug-excipient integration". This gel uses the effective part of Dendrobium officinale polysaccharide as the main body of the drug and ion-sensitive low-acetylated gellan gum as the excipient. It utilizes the pharmacological effects and physical properties of Dendrobium officinale polysaccharide to form an in-situ gel. This gel serves as both the active pharmaceutical ingredient and the main matrix of the gel, prolonging its action time in the stomach and enhancing its efficacy, thereby achieving an effective protective effect on the stomach.

[0007] The second objective of this invention is to provide a method for preparing the above-mentioned Dendrobium officinale polysaccharide in situ gel powder.

[0008] The third objective of this invention is to provide an in-situ gel formulation of Dendrobium officinale polysaccharides.

[0009] The fourth objective of this invention is to provide the application of the above-mentioned Dendrobium officinale polysaccharide in-situ gel powder and Dendrobium officinale polysaccharide in-situ gel preparation.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] A Dendrobium officinale polysaccharide in situ gel powder comprises the following components in the following mass ratio: Dendrobium officinale polysaccharide: low acetylated gelatin: cosolvent = 5-20: 4-8: 5-15.

[0012] Furthermore, the components include the following mass ratio: Dendrobium officinale polysaccharide; low-acetylated gellan gum; cosolvent = 10:4:6.

[0013] Furthermore, the co-solvent is any one or more of sucrose, lactose, mannitol, or PVP-K30.

[0014] Furthermore, the co-solvent is a mixture of PVP-K30 and sucrose, wherein the mass ratio of the two is: PVP-K30: sucrose = 1:2.

[0015] This invention is based on the principle of "drug and excipient integration." It utilizes the pharmacological effects of Dendrobium officinale polysaccharides in improving gastrointestinal inflammation and their physical properties as thickening and gelling polymers to prepare an oral in-situ gel for gastric protection. The effective polysaccharide fraction is extracted from fresh Dendrobium officinale, compounded with a gelling polysaccharide (low-acetylated gellan gum), and a solubilizer is added to improve the gelling properties and dissolution rate of the Dendrobium officinale polysaccharide. The compounded gel contains hydrophilic groups, which maintain its network structure and improve the dissolution rate of the polysaccharide. It can be rapidly dissolved into a sol in hot water above 80°C. In vitro, it exists as a sol; upon entering the stomach, it undergoes a phase transition under the acidic conditions of the stomach, forming a gel with a certain strength and viscosity. This gel can better cover the ulcer surface, forming a protective film for physical protection, and can also slowly dissolve and release the drug, thus more effectively exerting its efficacy and further improving the therapeutic effect. Currently, similar stomach medications on the market include: Jieweile (aluminum phosphate gel), sucralfate, and colloidal bismuth pectin. Their mechanism of action involves aluminum and bismuth forming complexes or chelates with proteins on the ulcer surface, thus providing protection. However, long-term use can cause dementia, chronic kidney damage, and intestinal obstruction. This invention selects *Dendrobium officinale*, a plant with both medicinal and edible properties, and extracts its effective polysaccharide component. Utilizing the pharmacological effects and viscous, gelling properties of *Dendrobium officinale* polysaccharides, a "drug-excipient combination" oral in-situ gastric protective gel is prepared, providing patients with a safe and effective option. Furthermore, this invention demonstrates that *Dendrobium officinale* polysaccharides promote the proliferation and growth of beneficial gastric bacteria, *Lactobacillus acidophilus*. Simultaneously, the protective effect of the *Dendrobium officinale* polysaccharide in-situ gel on acute gastric ulcers was investigated using an anhydrous ethanol-induced acute gastric ulcer model.

[0016] The present invention also provides a method for preparing the above-mentioned Dendrobium officinale polysaccharide in situ gel powder: Dendrobium officinale polysaccharide and low acetylated gellan gum are compounded in the above proportion, a co-solvent is added and mixed, and then pulverized and sieved to obtain Dendrobium officinale polysaccharide in situ gel powder.

[0017] Furthermore, the preparation method of Dendrobium officinale polysaccharide is as follows: fresh Dendrobium officinale slices are washed, the cell walls are broken and homogenized, water is added in an amount of 2 to 4 times the total mass of fresh Dendrobium officinale, centrifuged, the supernatant is taken and ethanol is added to make the alcohol concentration 75 to 85%, the precipitate is taken, and freeze-dried to obtain Dendrobium officinale polysaccharide with a percentage content of 65% to 90%.

[0018] Furthermore, the pulverization and sieving in the preparation method refers to pulverizing the preparation using ball milling or air jet milling and passing it through an 80-120 mesh sieve.

[0019] Furthermore, the pulverization and sieving in the preparation method refers to pulverizing the preparation using ball milling and passing it through an 80-mesh sieve.

[0020] The present invention also provides an in-situ gel formulation of Dendrobium officinale polysaccharide, which is an aqueous solution containing 2 wt% of any of the above-mentioned Dendrobium officinale polysaccharide in-situ gel powder.

[0021] According to the research of this invention, Dendrobium officinale polysaccharide is a shear-thinned pseudoplastic fluid. From Figure 1 and Figure 2 It is known that the G” of the polysaccharide solution is always higher than G', and tanδ is greater than 1. The degree of intermolecular association in the system is low, and it cannot spontaneously form a network to cross-link into a gel. The polysaccharide solution exhibits the viscous characteristics of a fluid. When adjusting the acidity of the solution, the state of the polysaccharide solution does not differ much in an environment with pH 1–3. Therefore, it is necessary to add a gelling polysaccharide. Experiments show that low-acetylated gelling polysaccharide is preferred. A mixture of Dendrobium officinale polysaccharide and low-acetylated gelling polysaccharide at concentrations of 0.5–2.0 wt% and 0.4–0.8 wt% respectively in hot water can form a sol in warm water, which is beneficial in acidic gastric juices. Under certain conditions, it can expand to form a gel, with a gel strength of 7.95–92.23 N and a gel viscosity of 7.95–128.20 g·sec. Based on the expected cumulative dissolution of 50% and 80% after 2 hours and 6 hours respectively, the preferred optimal concentration ratio is: polysaccharide concentration: 1.0 wt%; low-acetylated gelling agent concentration: 0.4 wt%. The corresponding gel strength is 10.39 N, gel viscosity is 24.91 g·sec, and the dissolution time is 11 minutes and 29 seconds. Adding a co-solvent at a preferred ratio of 0.6 wt% can accelerate the dissolution rate to 4–5 minutes.

[0022] Furthermore, the contents of each component in the preparation are as follows: Dendrobium officinale polysaccharide: 0.5-2.0 wt%, low-acetylated gellan gum: 0.4-0.8 wt%, and solubilizer: 0.5-1.5 wt%.

[0023] Furthermore, the contents of each component are as follows: Dendrobium officinale polysaccharide: 1.0 wt%, low acetylated gellan gum: 0.4 wt%, and solubilizer: 0.6 wt%.

[0024] The present invention also provides the application of the above-mentioned Dendrobium officinale polysaccharide in-situ gel powder or Dendrobium officinale polysaccharide in-situ gel preparation, specifically in the preparation of drugs or health products for protecting gastric mucosa and treating chronic erosive gastritis and / or gastric ulcers.

[0025] This invention demonstrates that Dendrobium officinale polysaccharide promotes the proliferation and growth of Lactobacillus acidophilus, a beneficial probiotic in the stomach. Therefore, this invention also seeks protection for the use of Dendrobium officinale polysaccharide in the preparation of growth promoters that promote the proliferation of Lactobacillus acidophilus, a beneficial probiotic in the stomach.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention utilizes the gastric protective effect and thickening properties of Dendrobium officinale polysaccharides to provide an in-situ gel based on a "drug-excipient integration" approach. This reduces the amount of excipients needed, and product performance is improved through compounding and micronization, enhancing the solubility of the polysaccharides. This provides a simplified operating process for industrial production and offers new ideas and methods for developing this precious "food and medicine homology" resource. When taken orally at a certain concentration, this Dendrobium officinale polysaccharide in-situ gel expands to form a gel upon entering the gastric juice, covering the ulcer site to form a protective film, providing not only physical protection but also slow dissolution and release of the polysaccharides in the stomach, further enhancing efficacy. It leverages the gastric protective and prebiotic effects of Dendrobium officinale polysaccharides, promoting the proliferation and growth of Lactobacillus acidophilus in the stomach and improving the tolerance of Lactobacillus acidophilus to acidic gastric conditions. Attached Figure Description

[0028] Figure 1 Steady-state shear flow curves of Dendrobium polysaccharide solutions at different concentrations.

[0029] Figure 2 The results of the frequency scanning experiment of fresh polysaccharides from Dendrobium officinale.

[0030] Figure 3 The cumulative dissolution curves are for 9 sets of tests.

[0031] Figure 4 The diagram shows the properties of the in-situ gel (A: original powder; B: after dissolving in water; C: after adding artificial gastric juice).

[0032] Figure 5 This is a comparison chart of textural properties.

[0033] Figure 6 The cumulative dissolution curve is the optimal formulation.

[0034] Figure 7 The growth curves of Lactobacillus acidophilus with different carbon sources are shown.

[0035] Figure 8 The effect of culture time on viable bacterial count under artificial gastric fluid conditions (n=3).

[0036] Figure 9 The extent of gastric damage in different groups.

[0037] Figure 10 Pathological observation of gastric tissues from different groups. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0039] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0040] Example 1: Screening of the preparation process for Dendrobium officinale in-situ gel powder

[0041] 1. Rheological property testing of Dendrobium officinale polysaccharides

[0042] (1) Effect of different mass concentrations on the rheological properties of polysaccharide solutions

[0043] Fresh polysaccharides from Dendrobium officinale were prepared into solutions with mass concentrations of 1%, 5%, and 10% using deionized water. The relationship between apparent viscosity η and shear rate γ (0–100 / s) was investigated at 25°C.

[0044] (2) Dynamic rheological measurement

[0045] At 25℃, frequency scanning experiments were conducted at frequencies ranging from 0.1 to 100 rad / s to determine the dependence of G' and G” on angular frequency.

[0046] The rheological properties of Dendrobium officinale polysaccharides at different mass concentrations were determined by Figure 1 As shown, the viscosity of polysaccharides is dependent on the solution concentration. When the solution concentration increases to a certain level, molecules overlap, leading to tighter inter-molecular connections, restricting their movement and stretching, resulting in a significant increase in viscosity. With increasing shear rate (from 0 / s to 100 / s), the viscosity of the polysaccharide solution decreases to varying degrees. Fresh polysaccharide solutions exhibit shear-thinned pseudoplastic fluid characteristics, but remain relatively stable within the concentration range of 1%–5%. The dynamic rheological test results of *Dendrobium officinale* polysaccharides are presented by… Figure 2As shown, the G” of the polysaccharide solution is always higher than G', and tanδ is greater than 1. The degree of intermolecular association in the system is low, and network cross-linking cannot be formed. The fresh polysaccharide solution of Dendrobium officinale exhibits the viscous characteristics of a fluid. It can be seen that Dendrobium officinale polysaccharide is normally a viscous fluid and cannot form a gel on its own.

[0047] 2. Screening of gel matrices

[0048] The above tests show that Dendrobium officinale polysaccharides cannot form a gel on their own; therefore, a gelling polysaccharide needs to be added as a gel matrix for the formulation. Common gelling polysaccharides in this field include low-acetylated gellan gum and sodium alginate, both of which are ion-sensitive gelling polysaccharides. Experiments showed that low-acetylated gellan gum dissolves in hot water as a clear solution, and its dissolution rate is not affected when combined with Dendrobium officinale polysaccharides. In contrast, sodium alginate dissolves in water to form a viscous colloidal solution, and its dissolution rate is even slower when combined with Dendrobium officinale polysaccharides. Since the main component of this formulation is the effective part of Dendrobium officinale polysaccharides, which inherently possesses viscosity and provides the necessary adhesive properties after gelation, low-acetylated gellan gum was chosen as the gel matrix.

[0049] 3. Screening of Dendrobium officinale polysaccharide / low acetylglenoid concentration ratio

[0050] (1) In vitro erosion rate determination

[0051] Different ratios of Dendrobium officinale / low-acetylated gellan gum mixtures were placed in 50 mL stoppered test tubes containing a pre-weighed mass (W0). Boiling water was added according to the ratio to dissolve the mixtures, and the tubes were preheated in a 37°C water bath shaker for 15 min. Artificial gastric fluid was added dropwise until a complete gel was formed. The initial mass W1 was recorded. The tubes were then transferred to a (37.0±0.5)°C water bath shaker, and 5 mL of artificial gastric fluid at the same temperature was added. The tubes were then shaken at a constant temperature (140 r·min). －1 Immediately after 60 minutes, remove the tube, pour off the supernatant, dry the tube, and quickly weigh it (Wt). Place the stoppered tube back into a constant-temperature water bath to equilibrate the temperature, add 5 mL of artificial gastric fluid at the same temperature, and continue shaking for 60 minutes. The difference in the mass of the remaining gel in the bottle at adjacent time points represents the amount of gel erosion during this period. The gel erosion rate = gel erosion amount / initial mass, calculated using the formula Q = (W1 - Wt) / (W1 - W0) × 100%. Plot the cumulative erosion curve of the in-situ gel of *Dendrobium officinale* with gel erosion rate (Y) as the ordinate and time (X) as the abscissa.

[0052] (2) Determination of gel strength and gel viscosity

[0053] The in-situ gel of Dendrobium officinale polysaccharide that has undergone phase transition was taken, and the maximum force required for gel rupture was determined using a texture analyzer; this force is the gel strength. Measurement conditions: P / 0.5 cylindrical probe, pre- and mid-measurement velocity 1.00 mm / s, probe rise rate 10.00 mm / s, displacement 10.00 mm, trigger force 5.0 g. Each experiment was repeated in triplicate.

[0054] (3) Preliminary screening of Dendrobium officinale polysaccharide and low acetylglenoid concentration

[0055] Selection of Dendrobium officinale polysaccharide concentration: Based on the dosage and the solubility of the polysaccharide, the concentration of Dendrobium officinale polysaccharide is selected to be 0.5% to 2%.

[0056] Selection of low-acetylated gellan gel concentration: 1% of *Dendrobium officinale* polysaccharide was mixed with low-acetylated gellan gel at concentrations ranging from 0.1% to 0.8%. Artificial gastric fluid was added after mixing, and the vials were inverted for 30 seconds. The gels from different ratios of *Dendrobium officinale* polysaccharide / low-acetylated gellan gel were observed to slide or separate into layers. The results showed that when the low-acetylated gellan gel concentration was within the range of 0.1% to 0.3%, the mixed solutions of *Dendrobium officinale* polysaccharide / low-acetylated gellan gel all showed sliding after inversion, while a concentration of 0.4% did not show sliding along the vial wall. Therefore, low-acetylated gellan gel with a concentration range of 0.4% to 0.8% was selected for further screening.

[0057] (4) Optimize the concentration ratio of Dendrobium officinale polysaccharide and low-acetylglenoid using factorial design method.

[0058] A two-factor, three-level factorial experimental design was used to screen the optimal concentration ratio of Dendrobium officinale polysaccharide and low-acetylated gellan gum using the in vitro erosion rate at 2 h and 6 h as the response value. The expected cumulative erosion values ​​at 2 h and 6 h were 50% and 80%, respectively. The factorial design was used to investigate the correlation between gel erosion rate, gel strength, viscosity, dissolution rate and polysaccharide and low-acetylated gellan gum.

[0059] In this model, two factors will vary at three levels, coded as -1, 0, and 1, respectively. The concentrations (%) of Dendrobium officinale polysaccharide and low-acetylated gellan gum are used as independent variables. The Dendrobium officinale polysaccharide concentration ranges from 0.5% to 2%, and the low-acetylated gellan gum concentration is taken as the commonly used amount of 0.4% to 0.8%. The resulting mixtures are used to determine the 2-hour in vitro erosion rate (Y1), 6-hour in vitro erosion rate (Y2), gel strength (Y3), viscosity (Y4), and dissolution time (Y5) as response values. The factorial design experimental factor levels are shown in Table 1. The results for gel strength and gel viscosity are shown in Table 2, and the in vitro erosion rate results are shown in Table 3. Figure 3 As shown.

[0060] Table 1. Factor Levels in Factorial Design Experiments

[0061]

[0062] Table 2 Results of Factor Levels in Factorial Design Experiments

[0063]

[0064]

[0065] The regression equations for the corresponding indicators were obtained by using a multiple regression model for fitting and optimization:

[0066] 2h dissolution rate (Y1) = 40.71 - 13.31 (A) - 14.24 (B);

[0067] 6h dissolution rate (Y2) = 77.15 - 13.52 (A) - 14.16 (B) - 0.9 (AB) - 8.64 (A2) - 4.86 (B2);

[0068] Gel strength (Y3) = 11.96 + 5.91 (A) + 3.94 (B) + 0.45 (AB) + 15.32 (A2) + 1.64 (B2);

[0069] Viscosity (Y4) = 35.61 + 38.23 (A) + 4.30 (B);

[0070] Dissolution time (Y5) = 11.36 + 5.49(A) + 0.095(B) - 0.041(AB) - 0.59(A2) + 0.058(B2)

[0071] As can be seen from the various binomial equations, the concentrations of Dendrobium officinale polysaccharide and low-acetylated gellan gum are negatively correlated with the dissolution rates at 2h and 6h, while the concentrations of Dendrobium officinale polysaccharide and low-acetylated gellan gum are positively correlated with gel strength and viscosity. Among these, the concentration of Dendrobium officinale polysaccharide is the main influencing factor on gel strength and viscosity, while the concentration of low-acetylated gellan gum has a slightly greater impact on the dissolution rates at 2h and 6h. The dissolution time is determined by the concentration of Dendrobium officinale polysaccharide.

[0072] Higher concentrations of Dendrobium officinale polysaccharide and low-acetylated gellan gum resulted in slower dissolution, hindering drug release and efficacy. Therefore, using the expected cumulative dissolution rate as an indicator, and combining factorial design and software analysis, the optimal concentration ratio was determined to be: Dendrobium officinale polysaccharide concentration: 0.91%; low-acetylated gellan gum concentration: 0.40%; predicted dissolution rate at 2 hours: 57.31%; predicted dissolution rate at 6 hours: 80.40%. For ease of operation, a Dendrobium officinale polysaccharide concentration of 1% (approximately optimal) was selected.

[0073] 4. Screening of cosolvents

[0074] The concentration of polysaccharides in Dendrobium officinale is positively correlated with dissolution time. At a polysaccharide concentration of 2%, the dissolution time exceeds 20 minutes, and at a concentration of 1%, it exceeds 10 minutes. This poses a challenge for practical applications. Therefore, a co-solvent is needed to improve the dissolution rate. PVP-K30 is a nonionic polymeric surfactant that can be used as a co-solvent and stabilizer for colloidal solutions. Sucrose, lactose, and mannitol are all polyhydroxy compounds with solubilizing effects. Therefore, PVP-K30, sucrose, lactose, and mannitol were selected as co-solvents for screening.

[0075] Take 1g of Dendrobium officinale polysaccharide and 0.4g of low-acetylglucosamine, divide into 4 portions, and add the same amount (1.0g) of sucrose, lactose, mannitol, and PVP-K30 to each portion. Mix well, then add 100mL of boiling water, stir constantly, and record the dissolution time. The results are shown in Table 3 below.

[0076] Table 3. Dissolution phenomena and dissolution time of different co-solvents

[0077]

[0078] It is evident that PVP-K30 has the most significant solubilizing effect, followed by sucrose. Sucrose has a sweet taste, relatively low hygroscopicity among sugars, is economical and safe, and can be used as both a flavoring agent and a filler in products. Therefore, further screening of the effect and dosage of the mixture of sucrose and PVP-K30 is necessary.

[0079] Take 1g of polysaccharide and 0.4g of low-acetylated gellan gum, and add 0.5g, 1g, and 1.5g of a mixture of PVP-K30 and sucrose in equal proportions, respectively. Screen the addition amount, and the results are shown in Table 4.

[0080] Table 4 Results of the screening test for the amount of excipients added

[0081]

[0082] The taste score is 1-10, with higher scores indicating better taste.

[0083] It is evident that the higher the amount added, the faster the dissolution rate, but the difference is not significant. The powder was packaged in 2g packets, with a fixed amount of excipients at 0.6g. The optimal PVP-K30 / sucrose ratio was determined based on dissolution rate and taste scores, and the results are shown in Table 5.

[0084] Table 5 Results of the test on the proportion of excipients added

[0085]

[0086]

[0087] The taste rating is 1-10, with higher scores indicating better taste.

[0088] Considering both dissolution speed and taste, the recommended addition amount is 0.2g of PVP-K30 and 0.4g of sucrose, with a mass ratio of PVP-K30:sucrose = 1:2.

[0089] 5. Micronization treatment

[0090] After adding the solubilizer according to the prescription, the dissolution rate was greatly improved, but it still took at least 4 minutes to dissolve completely. Therefore, the mixture was further micronized.

[0091] Ball milling method: The ratio of large balls (3mm) to small balls (2mm) is 4:6. After experimentation, the optimal ball-to-material ratio is 50:1. The grinding time is 2 hours. After passing through an 80-mesh sieve, the yield is 79.3%.

[0092] Airflow pulverization: Compressed air is used as the pulverizing medium, preferably at a constant pressure of 8 MPa. The material is collected, passed through a 100-mesh sieve, and the yield is 65.2%.

[0093] After comprehensive comparison, ball milling was chosen for micronization.

[0094] 6. Screening Results

[0095] In summary, the optimal formulation for Dendrobium officinale in-situ gel powder is as follows: 100g polysaccharide, 40g low-acetylated gellan gum, 20g PVP-K30, and 40g sucrose, making 100 packets. Each packet contains 2.0g of Dendrobium officinale polysaccharide, 0.4g low-acetylated gellan gum, 0.2g PVP-K30, and 0.4g sucrose.

[0096] Dissolve one sachet (2g) of this product in 100mL of hot water, then add artificial gastric juice. See below for the characteristics at each stage. Figure 4 .

[0097] 7. Validation experiment of optimal formulation process

[0098] According to the optimal formula, take 10g of Dendrobium officinale polysaccharide, 4g of low-acetylated gellan gum, 2g of PVP-K30, and 4g of sucrose, totaling 20g. Mix thoroughly, pulverize, and pass through an 80-mesh sieve to obtain Dendrobium officinale polysaccharide in-situ gel. Accurately weigh Dendrobium officinale polysaccharide and low-acetylated gellan gum according to the optimal concentration ratio, and prepare three batches of parallel samples consecutively. Measure each index and compare with the optimal process formula. The comparison results are shown in Table 6. Figure 5 and Figure 6 As shown in Table 6 and Figure 5 It is evident that the addition of a co-solvent and micronization had no effect on its textural properties. Figure 6The cumulative erosion curves of the optimal formulation show that the erosion rates of the three batches of optimal formulations at 2 hours were 54.2%, 53.1%, and 55%, respectively, and the erosion rates at 6 hours reached 80%, 78.4%, and 81.2%, respectively, which are close to the predicted values.

[0099] Table 6 Comparison of Texture Parameters

[0100]

[0101] Example 2: Preparation of Dendrobium officinale polysaccharide in situ gel powder

[0102] (1) Extraction of polysaccharides from Dendrobium officinale:

[0103] Fresh Dendrobium officinale was washed, dried, and cut into small segments about 1 cm in length. Water was added at a material-to-liquid ratio of 1:2, and the mixture was homogenized for 20 minutes using an ultra-micro pulverizer (Jinan Tianyu TYM-8L). The mixture was then shaken for 6 minutes, centrifuged, and the filtrate was collected. An appropriate amount of ethanol was added to make the ethanol concentration of the solution 80%. The mixture was allowed to stand for 1 hour, centrifuged, and the precipitate was collected. The precipitate was washed twice with 80% ethanol and then freeze-dried under vacuum (-40℃, 0.003MPa) to dry the polysaccharide. The polysaccharide was then pulverized to obtain Dendrobium officinale polysaccharide.

[0104] (2) Mixing: Take Dendrobium officinale polysaccharide, low acetylglucan gum, PVP-K30 and sucrose and mix them in a ratio of 10:4:2:4;

[0105] (3) Micronization: The mixed powder was pulverized by ball milling with a ratio of large balls (3mm) to small balls (2mm) of 4:6 and a ball-to-material ratio of 50:1. After grinding for 2 hours, the powder was passed through an 80-mesh sieve to obtain Dendrobium officinale polysaccharide in situ gel.

[0106] Take 2.0g of the above polysaccharide in situ gel, add hot water above 80℃, and it can be dissolved into a sol within 2 minutes. Add artificial gastric juice and it expands to form a gel. The gel strength was measured to be 10.78N and the gel viscosity was 24.93g.sec using a texture analyzer.

[0107] Example 3: Preparation of Dendrobium officinale polysaccharide in situ gel powder (sucrose-free type)

[0108] (1) Extraction of polysaccharides from Dendrobium officinale:

[0109] Fresh Dendrobium officinale was washed, dried, and cut into small segments about 1 cm in length. Water was added at a material-to-liquid ratio of 1:4, and the mixture was homogenized for 10 minutes using a high-speed blender (Joyoung JYL-Y92) at 35000 rpm. The filtrate was collected by centrifugation, and an appropriate amount of ethanol was added to make the ethanol concentration of the solution 80%. The mixture was allowed to stand for 1 hour, centrifuged, and the precipitate was collected. The precipitate was washed twice with 80% ethanol, and the polysaccharide was dried by vacuum freeze-drying (-40℃, 0.003MPa). The polysaccharide was then pulverized to obtain Dendrobium officinale polysaccharide.

[0110] (2) Mixing: Take Dendrobium officinale polysaccharide, low acetylglucosamine and PVP-K30 and mix them in a ratio of 10:4:3;

[0111] (3) Micronization: The mixed powder is pulverized by airflow, with compressed air as the pulverizing medium and a constant pressure of 8MPa. The material is collected and passed through a 100-mesh sieve to obtain Dendrobium officinale polysaccharide in situ gel (sucrose-free type).

[0112] Take 1.7g of the above polysaccharide in situ gel, add hot water above 80℃, and it can be dissolved into a sol within 3 minutes. Add artificial gastric juice and it expands to form a gel. The gel strength was measured to be 10.63N and the gel viscosity was 25.12g·sec using a texture analyzer (TA-XT plus, PekinElmer, USA).

[0113] Example 4: Effects of Dendrobium officinale polysaccharides on the growth and acid resistance of beneficial gastrointestinal bacteria, Lactobacillus acidophilus.

[0114] The Lactobacillus acidophilus used in this embodiment was purchased from Guangdong Huankai Biotechnology Co., Ltd.

[0115] 1. Preparation of the test solution

[0116] Accurately weigh 1g of Dendrobium officinale polysaccharide (batch number: 20220612) into a 50mL volumetric flask, dissolve it completely in hot water, and make up to volume to obtain a solution with a mass concentration of 20mg / mL. Filter the solution through a 0.22μm microporous membrane for sterilization and set aside for later use.

[0117] 2. Bacterial activation

[0118] Add 0.4 mL of sterile water to a cryovial and gently shake until dissolved. Inoculate the bacterial suspension onto MRS solid medium, 0.1 mL at a time, and incubate at 37°C for 48 hours. Use an inoculation loop to transfer the first-generation culture to liquid medium and continue the culture, incubating at 37°C for 48 hours to obtain the second-generation culture. Repeat this process three times under the same conditions, then store the bacterial culture for later use.

[0119] 3. Preparation of MRS culture medium

[0120] The composition of MRS medium is shown in Table 7. The monosaccharide composition of Dendrobium officinale polysaccharide is mainly glucose and mannose. The carbon source of MRS medium is glucose. In the experimental medium, Dendrobium officinale polysaccharide and mannose were used instead of glucose as the carbon source, and the other components were the same as those of the basal medium.

[0121] Table 7. Composition of MRS Culture Medium

[0122]

[0123]

[0124] Add 15.7g MRS broth and 75g agar to 300mL of distilled water and heat to dissolve, obtaining a solid culture medium. Separately, add 10.5g MRS broth to 200mL of distilled water, shake well, and agitate to obtain a liquid culture medium. Place the liquid culture medium in an autoclave at 105kPa and 121℃ for 6 minutes, then remove. Pour into sterile plates, cool, and store at 4℃ for later use.

[0125] 4. Growth curve of Lactobacillus acidophilus

[0126] 0.6 mL of activated bacterial solution was added to MRS liquid medium (using Dendrobium officinale polysaccharide, glucose, and mannose as the sole carbon sources, respectively), making the total liquid volume 5 mL. The medium was anaerobically cultured at 37°C for 36 h, and the OD value was measured at 600 nm at different time points to obtain the 36-hour growth curve of Lactobacillus acidophilus. Results are shown below. Figure 7 It can be seen that the exponential growth period of Lactobacillus acidophilus is between 4h and 16h. The experimental group using Dendrobium officinale polysaccharide as carbon source did not have an advantage over the glucose and mannose groups before 20h, but after 20h, with the accumulation of bacterial metabolic products, it still showed a certain proliferation effect, which may be related to the fact that Dendrobium officinale polysaccharide can improve the tolerance of Lactobacillus acidophilus.

[0127] 5. Acid resistance test

[0128] Using *Dendrobium officinale* polysaccharide as the carbon source for MRS liquid medium (concentration 20 mg / mL), *Lactobacillus acidophilus* culture was inoculated into MRS liquid medium (with glucose MRS medium of the same concentration as a control) and incubated at 37℃ for 24 h. After incubation, high-speed centrifugation was performed at 8000 rpm for 10 min at 4℃. After centrifugation, the supernatant was discarded, and the lower bacterial pellet was reconstituted with sterile physiological saline and the cell concentration was adjusted to 10³ CFU / mL. This pellet was then inoculated into prepared artificial gastric fluid and incubated at 37℃ for 6 h. Colony counts of *Lactobacillus acidophilus* were performed every 1 h. After incubation at 37℃ under anaerobic conditions for 48 h, the viable cell count was calculated. Figure 8 .

[0129] It is evident that after inoculating the Lactobacillus acidophilus in the artificial gastric fluid, the number of viable bacteria in both experimental groups gradually decreased. However, the number of viable bacteria in the Dendrobium officinale polysaccharide experimental group was higher than that in the glucose experimental group, and the rate of decrease in the number of viable bacteria was slower in the Dendrobium officinale polysaccharide experimental group. This indicates that the Lactobacillus acidophilus growing in the Dendrobium officinale polysaccharide group had strong tolerance within 6 hours, which can improve the tolerance of probiotics under the acidic conditions of the stomach.

[0130] In summary, the most common probiotic in the stomach, Lactobacillus acidophilus, can utilize Dendrobium officinale polysaccharide for growth and metabolism. Literature reports that the content of Lactobacillus in the stomach of Helicobacter pylori-infected individuals will decrease, and the colonization amount of Helicobacter pylori can be reduced by reshaping the gastric microecological balance (Song Hanyi. Screening and Intervention Mechanism Research of Probiotics with Antagonistic Effects on Helicobacter pylori [D]. China Medical University, 2019). Dendrobium officinale polysaccharide may play a "stomach-benefiting" role by increasing the survival rate of probiotics and thus reducing the colonization amount of pathogenic bacteria.

[0131] In Vivo Pharmacodynamic Experiment of Polysaccharide In-Situ Gel in Example 5

[0132] 1. Experimental Materials

[0133] (1) Drugs and Reagents

[0134] Dendrobium officinale polysaccharide in-situ gel in Example 1, Dendrobium officinale polysaccharide, sucralfate suspension, SOD kit, NO kit, MDA kit, IL-6 kit, CMC-Na, 4% paraformaldehyde, absolute ethanol, vernier caliper

[0135] (2) Experimental Instruments

[0136] Microplate reader, homogenizer, electronic analytical balance, electrothermal constant temperature water bath

[0137] (3) Animal Source

[0138] SPF-grade Kunming mice, weighing 20 ± 2 g, both male and female, provided by the Guangdong Provincial Center for Medical Experimental Animals, experimental animal license number: SYXK (Guangdong) 2022-0125; animal experiment ethics number gdpulacspf2022096.

[0139] 2. Experimental Methods and Results

[0140] (1) Experimental Grouping

[0141] Take 140 SPF-grade mice, with an equal number of males and females. Divide them into 7 groups according to body weight, namely blank group, model group, positive group (sucralfate suspension), high, medium, and low dose groups of Dendrobium officinale polysaccharide gel, and Dendrobium officinale polysaccharide solution group, with 20 mice in each group.

[0142] (2) Administration Dosage

[0143] Mice in the blank group and the model group were given 0.2 mL / 10 g of pure water, the dosage of the positive group was 60 mg / kg and administered at 0.1 mL / 10 g, the high, medium, and low dosages of the Dendrobium officinale polysaccharide gel administration group were 2%, 1%, and 0.5% respectively, and the administration dosage of the Dendrobium officinale polysaccharide solution group was 1%, all administered at 0.2 mL / 10 g for 7 consecutive days.

[0144] (3) Ethanol-induced acute gastric ulcer model

[0145] Mice were fasted for 24 hours before the last administration of the drug, but were allowed to drink water. One hour after the administration of the drug by gavage, all groups except the blank control group were given 0.3 mL of anhydrous ethanol by gavage. The mice were sacrificed 60 minutes later, and the whole stomach was removed by opening the abdomen. The stomach was cut open along the greater curvature and the stomach contents were gently rinsed with physiological saline. The gastric ulcer index was observed and measured. The stomach tissue was collected immediately and various indicators were measured.

[0146] (4) Appearance and ulcer index score of mouse gastric tissue

[0147] The entire stomach was harvested via laparotomy, cut along the greater curvature, fixed with 40% formaldehyde solution, soaked in physiological saline for 40 minutes, and then dried with filter paper. Gastric damage was observed and measured, and the ulcer index was calculated using the Guth method. Guth method: punctate lesions were scored 0.5 points; linear lesions were scored 1 point per mm; plaque lesions (length and width > 2 mm) were scored by area, and the sum of these scores was the ulcer index.

[0148] Table 8. Statistical analysis of ulcer index results in different groups ( n=10)

[0149]

[0150] Compared with the control group, #P < 0.05, ##P < 0.01; compared with the model group,

[0151] Table 9. Results of analysis on the difference in mean ulcer index among different groups.

[0152]

[0153] Compared with the model group,

[0154] like Figure 9 As shown, all dosage groups of Dendrobium officinale oral in-situ gel had good effects in treating ethanol-induced acute gastric ulcers. All three concentrations of Dendrobium officinale oral in-situ gel exhibited good protective effects on the gastric mucosa.

[0155] As shown in Tables 8 and 9, the positive control group showed the best effect, followed by the medium-dose group. The protective effect of the high-dose group was not as good as that of the medium- and low-dose groups, indicating that an appropriate concentration ratio, forming a gel with a certain dissolution rate in the stomach, can exert a greater therapeutic effect. The polysaccharide solution group also showed a protective effect on the gastric mucosa, but it was slightly weaker than that of the gel group.

[0156] (5) Determination of MDA, SOD activity and interleukin IL-6 content in mouse gastric tissue

[0157] Take 0.1g of gastric tissue and prepare a tissue homogenate at a ratio of gastric tissue mass to extract liquid of 1:5. Centrifuge at 3000 r / min for 20 min, collect the supernatant, prepare working solutions according to the instructions on each kit, mix with the homogenate supernatant, and measure the absorbance using a UV-Vis spectrophotometer. Calculate the MDA content, SOD activity, and interleukin IL-6 in the mouse gastric tissue homogenate according to the calculation methods on the kits. The results are shown in Tables 10-12.

[0158] Table 10 Statistical analysis of MDA content in different groups ( n=10)

[0159]

[0160] Compared with the control group, #P < 0.05, ##P < 0.01; compared with the model group,

[0161] Table 11 Statistical analysis of SOD activity results in different groups ( n=10)

[0162]

[0163] Compared with the control group, #P < 0.05, ##P < 0.01; compared with the model group,

[0164] Combining the MDA content in Table 10 and the SOD content in Table 11, it can be seen that compared with the blank group, the MDA content in the gastric tissue of the model group was significantly increased, and the SOD content in the gastric tissue was significantly decreased (P < 0.01). Specifically, the MDA content in the high, medium, and low dose groups of the treatment group was lower than that in the model group, while the SOD content was higher than that in the model group. This suggests that the in-situ gel of Dendrobium officinale polysaccharides can reduce the MDA content in the gastric tissue of mice, increase SOD activity, improve the antioxidant level of the model mice, and reduce lipid peroxidation.

[0165] Table 12 Results of IL-6 level determination (inflammatory factor IL-6) n=10)

[0166]

[0167] Compared with the control group, #P < 0.05, ##P < 0.01; compared with the model group,

[0168] As shown in Table 12, compared with the blank group, the IL-6 content in the tissues of mice in the model control group was significantly increased; compared with the model group, the IL-6 content in the tissues of each treatment group and the positive control group was significantly decreased, indicating that both Dendrobium officinale polysaccharide solution and gel showed anti-inflammatory effects, with the gel group showing a slightly stronger effect.

[0169] (6) Pathological morphological observation by He staining method

[0170] Mouse stomach tissue was collected, fixed in 4% paraformaldehyde for 24 hours, and then routinely dehydrated, cleared, embedded in paraffin, sectioned, and dewaxed for later use. After staining with He, the tissue was cleared, mounted, and the pathological changes of the stomach, small intestine, and colon were observed under a light microscope. The results are shown in the table below. Figure 10 .

[0171] Observation and scoring: The epithelial and mucosal tissue structures of the normal group, model group, and positive control group were compared to observe whether the boundaries between tissues were clear and whether the cell morphology was intact. Scoring was performed according to the degree of damage. The scoring criteria were: (1) loss of epithelial cells (0-3), (2) submucosal edema (0-4), (3) hemorrhagic injury and (4) inflammatory cell infiltration (0-3). The results are shown in Table 13.

[0172] Table 13 Grading of Pathological Changes in Mouse Gastric Tissue ( n=10)

[0173]

[0174]

[0175] The results showed that, compared with the control group, the model group mice had very little mucosal coverage on the surface of the ulcer lesions, irregular gland arrangement and defects, extending deep into the muscle layer and even reaching the serosal layer. Microscopic examination under a light microscope revealed significantly increased inflammatory cell infiltration, severe gastric mucosal congestion, and mucosal necrosis in the model group. Compared with the model group, there were significant differences in the mucosal coverage among the drug-treated groups, with the overall protective effect in the following order: medium-dose group > high-dose group > low-dose group ≈ solution group.

[0176] The medium-dose group (i.e. the optimal concentration ratio group) showed the best effect, and its protective effect against gastric mucosal hemorrhagic injury was similar to that of the positive group. This indicates that the gel has a certain dissolution rate after gelation, which can better disperse and cover the gastric mucosa and is conducive to exerting the drug effect. The high-dose group had a slightly worse effect, which may be due to its stronger gel strength, poor fluidity, slow dissolution rate, and relatively smaller area covered by the gastric mucosa.

[0177] 3. Summary

[0178] In summary, the above indicators show that the Dendrobium officinale polysaccharide in situ gel prepared by this invention has a good protective effect against anhydrous ethanol-induced acute gastric ulcers.

[0179] The medium-dose group, with the optimal concentration ratio of Dendrobium officinale polysaccharide and low-acetylated gellan gum, showed better protective effects against gastric mucosal damage and improved SOD activity than the high-dose group. This indicates that when Dendrobium officinale polysaccharide and low-acetylated gellan gum are combined at an appropriate concentration ratio, their gelation rate in vivo is more conducive to the efficacy of the drug.

[0180] The polysaccharide solution group showed certain effects on all indicators, but not as good as the high-dose and medium-dose gel groups. The protective effect was comparable to that of the low-dose group, indicating that Dendrobium officinale polysaccharide itself has medicinal effects. The in-situ gel prepared by this invention can improve the efficacy and greatly increase the dissolution rate of polysaccharide, making it more suitable for clinical application.

Claims

1. A Dendrobium officinale polysaccharide in-situ gel powder, characterized in that, The components include the following mass ratio: Dendrobium officinale polysaccharide: low acetylated gellan gum: co-solvent = 5-20: 4-8: 5-15; the co-solvent is a mixture of PVP-K30 and sucrose, with a mass ratio of PVP-K30: sucrose = 1:

2.

2. The Dendrobium officinale polysaccharide in-situ gel powder according to claim 1, characterized in that, The components include the following mass ratio: Dendrobium officinale polysaccharide; low-acetylated gellan gum; cosolvent = 10:4:

6.

3. The method for preparing the Dendrobium officinale polysaccharide in-situ gel powder according to claim 1 or 2, characterized in that, Dendrobium officinale polysaccharide and low-acetylated gellan gum were compounded in a certain proportion, a solubilizer was added and mixed, and then pulverized and sieved to obtain Dendrobium officinale polysaccharide in situ gel powder.

4. The method for preparing the Dendrobium officinale polysaccharide in-situ gel powder according to claim 3, characterized in that, The method for preparing Dendrobium officinale polysaccharide is as follows: fresh Dendrobium officinale slices are washed, the cell walls are broken and homogenized, centrifuged, the supernatant is collected, precipitated with alcohol, the precipitate is dried and pulverized to obtain Dendrobium officinale polysaccharide.

5. The method for preparing the Dendrobium officinale polysaccharide in-situ gel powder according to claim 3, characterized in that, The aforementioned pulverization and sieving involves pulverizing the preparation using ball milling or air jet milling and passing it through an 80-120 mesh sieve.

6. A Dendrobium officinale polysaccharide in-situ gel preparation, characterized in that, It is an aqueous solution containing 2 wt% of the Dendrobium officinale polysaccharide in situ gel powder according to claim 1 or 2.

7. The use of the Dendrobium officinale polysaccharide in-situ gel powder of claim 1 or 2 or the Dendrobium officinale polysaccharide in-situ gel preparation of claim 6 in the preparation of drugs for protecting gastric mucosa, treating chronic erosive gastritis and / or gastric ulcers, or health products for adjuvant protection of gastric mucosa.

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