Optimization method of preparation process of donkey-hide gelatin oral liquid capable of prolonging life

Abstract

The invention discloses a preparation process optimization method of donkey-hide gelatin oral liquid capable of prolonging life, and relates to the technical field of traditional Chinese medicine preparations, the method comprises two key steps of an intermediate clarification process and a donkey-hide gelatin solubilization process: before a liquid medicine and donkey-hide gelatin are combined, adding 1.5-2.5% of chitosan into the liquid medicine, stirring at 20-40 DEG C for 10-30 minutes for flocculation clarification, and filtering to obtain the donkey-hide gelatin oral liquid capable of prolonging life. Tannin and impurities are removed; 0.5-2.0% of Tween 80 is added after the colla corii asini is melted by heat and is fully and uniformly mixed, so that the solubility and the stability of the colla According to the method disclosed by the invention, the technical problem that precipitates are generated by reaction of the donkey-hide gelatin and tannin is fundamentally solved through a two-step optimization strategy, the turbidity of the product is reduced by 54.6%, the filtering performance is remarkably improved, the retention rate of effective components reaches 94% or above, the stability of the product is greatly improved, and the medicinal effect is equivalent; good industrial application values are realized.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine preparation technology, specifically to an optimized preparation process method for Ejiao Yishou Oral Liquid, and particularly to a technical problem of precipitation caused by the reaction of Ejiao with tannins in the medicinal liquid through chitosan flocculation to clarify the medicinal liquid and Tween 80 to solubilize Ejiao. Background Technology

[0002] Ejiao Longevity Oral Liquid is a compound traditional Chinese medicine oral liquid preparation composed of ginseng, roasted astragalus, licorice, costus root, tangerine peel, prepared he shou wu, prepared rehmannia root, and donkey-hide gelatin, among other Chinese medicinal herbs. It is believed to replenish qi and blood, and promote longevity. Donkey-hide gelatin, a renowned blood-tonifying herb, is rich in collagen, amino acids, and polypeptides, making it one of the product's core active ingredients. However, traditional production processes present numerous technical challenges in preparing Ejiao Longevity Oral Liquid, severely impacting product quality and production efficiency.

[0003] The existing production process of donkey-hide gelatin longevity oral liquid usually adopts the following steps: ginseng, roasted astragalus, processed he shou wu, prepared rehmannia root, costus root, tangerine peel, licorice and other medicinal materials are decocted together, filtered to obtain medicinal liquid, concentrated and combined with melted donkey-hide gelatin, sucrose syrup and preservatives are added, and after standing and filtering, the finished product is bottled. The main problems with this process include: First, the prescription contains processed Polygonum multiflorum and Rehmannia glutinosa, which will dissolve a large amount of tannins during decoction. The collagen in the donkey-hide gelatin will react with the tannins in the decoction to produce precipitation, resulting in high turbidity and poor clarity after the decoction and donkey-hide gelatin are combined. Second, the precipitate cannot be completely removed by filtration. Even with enhanced filtration, some of the donkey-hide gelatin that forms the precipitate will be lost, thereby reducing the content of the effective components of donkey-hide gelatin in the product and affecting product quality and efficacy. Third, the product is prone to bottom sedimentation during storage. In severe cases, the sediment will harden, affecting product stability and patient medication compliance. Fourth, filtration is difficult. When the decoction and donkey-hide gelatin are combined and filtered through a 400-mesh sieve, the filtration is in droplets, extremely slow, and leaves a large amount of filter residue after filtration, resulting in low production efficiency.

[0004] To address the aforementioned issues, a process optimization method is needed that can significantly improve product clarity and stability while simultaneously enhancing filtration performance, ensuring no loss of the product's active ingredients. Existing technologies have explored various clarification methods, such as alcohol precipitation, chitosan flocculation, and clarification with natural clarifying agents. However, these methods all have shortcomings when applied to donkey-hide gelatin oral liquid. While alcohol precipitation can reduce turbidity, it significantly reduces the retention rate of active ingredients such as stilbene glycosides and requires heating and concentration to remove ethanol, resulting in high energy consumption and a complex process. Although ZTC1+1Ⅲ type natural clarifying agent retains active ingredients well, its clarification effect is unsatisfactory, with only a slight reduction in turbidity. Directly adding surfactants such as Tween 80 can improve the solubility of donkey-hide gelatin, but it does not solve the fundamental problem of the reaction between tannins and donkey-hide gelatin in the liquid.

[0005] Therefore, there is an urgent need for a process optimization method that removes tannins and impurities from the medicinal liquid before combining it with donkey-hide gelatin, and adds a suitable solubilizer after the donkey-hide gelatin is melted to improve its solubility and stability in water. This would fundamentally solve the precipitation problem in the production of donkey-hide gelatin longevity oral liquid and improve product quality, stability, and production efficiency. Summary of the Invention

[0006] In view of the above-mentioned technical problems existing in the production process of Ejiao Yishou Oral Liquid, the purpose of this invention is to provide an optimized method for the preparation process of Ejiao Yishou Oral Liquid. By establishing a two-step optimization strategy of intermediate clarification process and Ejiao solubilization process, the product clarity and stability are significantly improved, filtration performance is improved, and production efficiency is increased while ensuring that the active ingredients are not lost.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The optimized preparation process of Ejiao Yishou Oral Liquid includes two key steps: intermediate clarification and Ejiao solubilization. The intermediate clarification process removes tannins and impurities from the medicinal liquid before combining it with Ejiao. The Ejiao solubilization process adds a solubilizer after Ejiao is melted to improve its solubility and stability. Specifically, it includes the following steps: Step 1, Medicinal herb extraction: Take ginseng, roasted astragalus, prepared rehmannia root, prepared he shou wu, tangerine peel, costus root, and licorice root, add water and decoct three times. For the first decoction, add 6 times the amount of water and simmer for 2 hours. For the second decoction, add 5 times the amount of water and simmer for 2 hours. For the third decoction, add 5 times the amount of water and simmer for 1 hour. Filter, combine the decoctions, and obtain the medicinal liquid. Step 2, intermediate clarification process: Chitosan is added to the drug solution obtained in Step 1 for flocculation and clarification. The amount of chitosan added is 1.5-2.5% of the drug solution volume, preferably 2.0%; the mixture is stirred for 10-30 minutes, preferably 20 minutes, at a temperature of 20-40℃, preferably 30℃; after standing for 1-3 hours, the mixture is centrifuged, and the supernatant is collected to obtain the clarified drug solution. Step 3, drug concentration: The clarified drug solution obtained in Step 2 is concentrated to a relative density of 1.05-1.15, preferably 1.10; Step 4, donkey-hide gelatin solubilization process: Take donkey-hide gelatin powder, add water and melt it at 60-70℃, preferably 65℃; take Tween 80, dissolve it in water, and mix it thoroughly with the melted donkey-hide gelatin at 60-70℃, preferably 65℃. The amount of Tween 80 added is 0.5-2.0% of the total liquid volume, preferably 1.0%; filter to obtain donkey-hide gelatin solubilization solution; Step 5, Merging and Refining: Combine the clarified and concentrated medicinal liquid obtained in Step 3 with the donkey-hide gelatin solution obtained in Step 4, add syrup made from sucrose and preservatives, adjust the total amount, let stand, filter, and obtain donkey-hide gelatin longevity oral liquid.

[0008] Furthermore, the chitosan used in the intermediate clarification process is a chitosan solution with a mass fraction of 1.0-2.0%. The chitosan solution is prepared by dissolving chitosan in 1% acetic acid and then refrigerated and allowed to stand for 24 hours before use.

[0009] Furthermore, the stirring speed in the intermediate clarification process is 80-120 rpm, preferably 100 rpm.

[0010] Furthermore, the centrifugation conditions in the intermediate clarification process are 4000-6000 rpm for 15-25 minutes, preferably 5000 rpm for 20 minutes, and the number of centrifugations is 2-4 times, preferably 3 times.

[0011] Furthermore, the weight ratio of ginseng, roasted astragalus, prepared rehmannia root, prepared he shou wu, tangerine peel, costus root, and licorice is 1:10:5:5:2:1:1.

[0012] Furthermore, the weight ratio of the donkey-hide gelatin to the ginseng in step one is 5:1.

[0013] Furthermore, the preservative is sodium benzoate, and its addition amount is 0.2-0.4% of the total volume of the oral liquid, preferably 0.3%.

[0014] The optimized preparation process of the donkey-hide gelatin longevity oral liquid of the present invention has the following beneficial effects: First, it significantly improves product clarity: Through an intermediate clarification process, tannins and impurities are removed before the medicinal liquid and donkey-hide gelatin are combined. This, combined with a donkey-hide gelatin solubilization process, improves the solubility of the donkey-hide gelatin, reducing the turbidity of the final product from 1004 NTU in the original production process to 456 NTU, a reduction of 54.6% and an improvement in clarity by 2.1 times. The product diluted 5 times appears noticeably clearer and more transparent.

[0015] Secondly, the filtration performance is greatly improved: After the process optimization, the medicine liquid and donkey-hide gelatin are combined and filtered in a straight flow, the filtration is smooth, the filtration speed is significantly accelerated, and there is less residual filter residue after filtration. Compared with the original production process of drip filtration and a large amount of filter residue, the production efficiency is significantly improved.

[0016] Third, effective preservation of active pharmaceutical ingredients: After process optimization, the retention rates of stilbene glycosides reached 95%, hesperidin 96%, and glycyrrhizic acid 99%. The total amounts of astragaloside A, glycyrrhizin, and donkey-derived peptides A1 and A2 were slightly higher than in the original production process. After optimization using Box-Behnken response surface methodology, the retention rate of stilbene glycosides remained stable at over 94%, ensuring maximum retention of active pharmaceutical ingredients.

[0017] Fourth, significantly improve product stability: After 14 days of storage at 40℃ and 75% humidity, the product with optimized process still has no sediment at the bottom of the bottle, while the sample prepared by the original production process has sediment at the bottom after 1 day. As the storage time is extended, the sediment gradually thickens and is adsorbed at the bottom of the bottle, thus greatly improving product stability.

[0018] Fifth, the optimized process parameters were clearly defined: Through single-factor investigation and Box-Behnken response surface methodology, a mathematical model was established between turbidity and stilbene glycoside retention rate and chitosan addition amount, clarification temperature, and clarification time. The optimal process parameters were determined to be 2.0% chitosan addition, 30℃ clarification temperature, and 20 minutes clarification time. The regression model R² was 0.9558, which has high reliability and good process reproducibility.

[0019] Sixth, efficacy equivalence verification: Through evaluation using a phenylhydrazine-induced zebrafish anemia model, both the modified and unmodified Ejiao Yishou oral liquid significantly improved phenylhydrazine-induced zebrafish anemia. The staining intensity of cardiac erythrocytes was significantly increased compared with the model control group (P<0.01), and there was no significant difference before and after the process improvement, proving that the process optimization did not affect the efficacy of the product.

[0020] Seventh, the process is highly innovative: This invention is the first to combine chitosan flocculation clarification process with Tween 80 solubilization process in the preparation of donkey-hide gelatin oral liquid. By first clarifying the medicinal liquid to remove tannins and then solubilizing the donkey-hide gelatin to improve stability, the fundamental problem of precipitation caused by the reaction between donkey-hide gelatin and tannins is solved from a mechanistic perspective. The technical route is clear and highly innovative.

[0021] Eighth, it has industrial application value: the optimized process can be implemented using conventional pharmaceutical equipment without the need for special equipment investment. Chitosan and Tween 80 are both pharmaceutical excipients with high safety and limited cost increase, while product quality and production efficiency are significantly improved, showing good prospects for industrial application and economic benefits. Attached Figure Description

[0022] Figure 1 The diagram shows the process flow of the prescription, illustrating the technical problems of precipitation and filtration difficulties that easily occur when the medicinal liquid and donkey-hide gelatin are combined in the traditional production process of donkey-hide gelatin longevity oral liquid.

[0023] Figure 2 This is a schematic diagram of the process improvement strategy, illustrating the overall approach to solving the precipitation problem through a two-step optimization strategy involving intermediate clarification and donkey-hide gelatin solubilization.

[0024] Figure 3 The improved production process flow diagram shows in detail the optimized complete production process flow, including intermediate clarification process and donkey-hide gelatin solubilization process.

[0025] Figure 4 The graph shows the effect of different volume fractions of ethanol precipitation on the turbidity of the drug solution. The horizontal axis represents the volume fraction of ethanol, and the vertical axis represents the turbidity NTU value. It shows the changes in the turbidity of the drug solution after precipitation with 40%, 50%, 60%, 70%, and 80% ethanol.

[0026] Figure 5 The graph shows the effect of different volume fractions of ethanol precipitation on the retention rate of stilbene glycosides. The horizontal axis represents the volume fraction of ethanol, and the vertical axis represents the retention rate of stilbene glycosides, illustrating the effect of different concentrations of ethanol on the retention of the active ingredient.

[0027] Figure 6 The bar chart shows the effect of different chitosan mass fractions on turbidity. The horizontal axis represents the chitosan mass fractions of 0.5%, 1.0%, and 2.0%, and the vertical axis represents the turbidity NTU value.

[0028] Figure 7 The graph shows the effect of different chitosan mass fractions on the stilbene glycoside retention rate. The horizontal axis represents the chitosan mass fraction, and the vertical axis represents the stilbene glycoside retention rate.

[0029] Figure 8 The graph shows the effect of three clarification methods on the turbidity of the drug solution. The clarification effects of 70% ethanol precipitation, 1% chitosan flocculation, and ZTC1+1Ⅲ type natural clarifying agent were compared.

[0030] Figure 9 The graph shows the effect of three clarification methods on the retention rate of stilbene glycosides in the drug solution.

[0031] Figure 10 This is a surface plot showing the response of chitosan addition amount and clarification temperature to OD value.

[0032] Figure 11 The response surface plot shows the chitosan addition amount and clarification time to the OD value.

[0033] Figure 12 This is a surface plot showing the response of clarification time and clarification temperature to OD values.

[0034] Figure 13 The images show a comparison of the filtration results of the original and improved filtration processes. On the left, the original filtration process resulted in difficult filtration, producing droplets, while on the right, the improved filtration process resulted in smooth filtration, producing a straight flow.

[0035] Figure 14 These are photos comparing the filter residue after filtration using the original process and the improved process. The left side shows more filter residue from the original process, while the right side shows less filter residue from the improved process.

[0036] Figure 15The images show a comparison of the properties of samples from the original formulation and the improved formulation. The left column shows the original sample solution, and the right column shows the sample diluted 5 times. The improved formulation sample is clearly clearer.

[0037] Figure 16 A bar chart comparing the content of effective ingredients in the original formulation and the improved formulation was used to compare the contents of six ingredients: stilbene glycoside, hesperidin, astragaloside A, donkey-derived polypeptide, glycyrrhizin, and glycyrrhizic acid.

[0038] Figure 17 This is a series of photos comparing the precipitation of the original and improved process samples at different time points under conditions of 40℃ and 75% humidity, including photos before, after 1 day, 3 days, 7 days, and 14 days.

[0039] Figure 18 These are stained images of red blood cells from the hearts of zebrafish in different groups of a phenylhydrazine-induced zebrafish anemia model, including the normal control group, the model group, the positive control group, the prescription process group, and the improved process group.

[0040] Figure 19 A bar chart evaluating the efficacy of Ejiao Yishou Oral Liquid in improving anemia before and after process improvement, with the vertical axis representing the pixel value of cardiac erythrocyte staining intensity. Detailed Implementation

[0041] Please refer to the attached document. Figures 1-19 The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0042] Example 1: Extraction of the medicinal liquid from donkey-hide gelatin longevity oral liquid Take 127g of ginseng, 1270g of roasted astragalus, 635g of prepared rehmannia root, 635g of prepared fleeceflower root, 254g of dried tangerine peel, 127g of costus root, and 127g of licorice root, a total of seven herbs. Decoct three times with water. For the first decoction, add 6 times the amount of water (relative to the total weight of the herbs) and simmer for 2 hours. For the second decoction, add 5 times the amount of water and simmer for 2 hours. For the third decoction, add 5 times the amount of water and simmer for 1 hour. Filter after each decoction, combine the three decoctions to obtain 20,000mL of liquid for later use. Due to the presence of prepared fleeceflower root and prepared rehmannia root, a large amount of tannins dissolves during the decoction process, resulting in a dark brown liquid with high turbidity.

[0043] Example 2 Comparison of different clarification methods 2.1 Clarification by alcohol precipitation Five 50 mL portions of the extract from Example 1 were precisely measured, and ethanol was added to each portion to achieve a volume fraction of 40%, 50%, 60%, 70%, and 80%, respectively. The solutions were then allowed to stand at 4°C in the dark for 24 hours and filtered. The filtrate was concentrated at 60°C until no alcohol odor remained, and the turbidity and stilbene glycoside content were measured. The stilbene glycoside retention rate was calculated.

[0044] like Figure 4 As shown, the turbidity of the drug solution first increases and then decreases with the increase of ethanol volume fraction. The turbidity after precipitation with 40% ethanol is 199 NTU, and the turbidity reaches the highest value of 248 NTU after precipitation with 50% ethanol. Subsequently, the turbidity gradually decreases, reaching 129 NTU after precipitation with 60% ethanol, 92 NTU after precipitation with 70% ethanol, and the lowest value of 84 NTU after precipitation with 80% ethanol.

[0045] like Figure 5 As shown, the retention rate of stilbene glycosides exhibits a complex change with increasing ethanol volume fraction, first decreasing, then increasing, and then decreasing again. The retention rate is 54.2% with 40% ethanol, drops to a minimum of 42.8% with 50% ethanol, rebounds to 48.6% with 60% ethanol, reaches a peak of 88.9% with 70% ethanol, and then decreases again to 61.5% with 80% ethanol. While the turbidity is similar to that of 80% ethanol precipitation, the retention rate of stilbene glycosides is significantly higher with 70% ethanol. Considering both turbidity and the retention rate of effective components, 70% ethanol volume fraction is determined to be the optimal ethanol volume fraction for precipitation.

[0046] 2.2 Clarification by Chitosan Flocculation Chitosan mass fraction screening: Take 1g of chitosan and prepare chitosan flocculant solutions of 0.5%, 1.0%, and 2.0% using 1% acetic acid solution. After fully dissolving with magnetic stirring, refrigerate and let stand for 24 hours for later use. Accurately measure 50mL of the extract from Example 1, add 1mL of each of the above 3 concentrations of chitosan solution, stir at 50℃ for 20 minutes (stirring speed 100 rpm), let stand at room temperature for 1 hour, then centrifuge at 5000 rpm for 20 minutes, centrifuge 3 times, take the supernatant, and determine the turbidity and stilbene glycoside content, and calculate the stilbene glycoside content retention rate.

[0047] like Figure 6 As shown, the turbidity of the drug solution after flocculation with 0.5% chitosan was 121 NTU, the turbidity after flocculation with 1.0% chitosan was reduced to a minimum of 112 NTU, and the turbidity after flocculation with 2.0% chitosan was increased to 174 NTU. This is because when the chitosan concentration is too high, the excess chitosan itself will increase the turbidity of the solution.

[0048] like Figure 7 As shown, the effect of chitosan mass fraction on the retention rate of stilbene glycosides exhibits a trend of first increasing and then plateauing. The retention rate is 89.6% with 0.5% chitosan, rises to 94.8% with 1.0% chitosan, and reaches 95.1% with 2.0% chitosan. The stilbene glycoside retention rates of 1.0% and 2.0% chitosan are relatively close, and both are significantly higher than those of 0.5%.

[0049] Considering both turbidity and stilbene glycoside retention rate, 1.0% chitosan flocculation resulted in the lowest turbidity, while 2.0% chitosan flocculation showed a slightly higher stilbene glycoside retention rate than 1.0%, but also higher turbidity. For subsequent response surface methodology optimization, this experiment tentatively selected 1.0% chitosan as the preliminary optimal concentration.

[0050] 2.3 Clarification with ZTC1+1Ⅲ type natural clarifying agent Accurately measure 50 mL of the extract from Example 1, add component A solution according to the instructions for ZTC1+1Ⅲ type natural clarifying agent, stir at 80°C for 1 hour, add component B solution, stir at a constant temperature for another 30 minutes, remove and cool to room temperature, and let stand at room temperature for 2 hours. Measure the turbidity and stilbene glycoside content, and calculate the stilbene glycoside retention rate.

[0051] The results showed that the turbidity of the drug solution after clarification with ZTC1+1Ⅲ type natural clarifying agent was 268 NTU, and the stilbene glycoside retention rate was 84.9%.

[0052] 2.4 Comparison of Three Clarification Methods like Figure 8 As shown, comparing the effects of three clarification methods on the turbidity of the drug solution, the turbidity of the unclarified original drug solution was 530 NTU, the turbidity after clarification with ZTC1+1Ⅲ type natural clarifying agent was 268 NTU, showing the worst clarification effect; the turbidity after precipitation with 70% ethanol was 92 NTU, showing the best clarification effect; the turbidity after flocculation with 1% chitosan was 112 NTU, showing a clarification effect slightly inferior to 70% ethanol precipitation, but significantly better than ZTC1+1Ⅲ type natural clarifying agent.

[0053] like Figure 9 As shown, comparing the effects of three clarification methods on the retention rate of stilbene glycosides in the drug solution, with the stilbene glycoside content of the unclarified original drug solution as the baseline value of 100%, the retention rate after clarification with ZTC1+1Ⅲ type natural clarifying agent was 84.9%, the retention rate after precipitation with 70% ethanol was 88.9%, and the retention rate after flocculation with 1% chitosan was 94.8%. The retention rate of stilbene glycosides by chitosan flocculation method was significantly higher than that of the other two methods.

[0054] Considering both turbidity reduction and active ingredient retention, 70% ethanol precipitation yielded the lowest turbidity, but its stilbene glycoside retention rate was significantly lower than that of chitosan flocculation. Furthermore, ethanol precipitation requires heating and concentration to remove ethanol, making the process complex and energy-intensive. ZTC1+1Ⅲ type natural clarifying agent showed the worst clarification effect, with unsatisfactory stilbene glycoside retention. 1% chitosan flocculation, while providing good clarification, maximizes the retention of active ingredients in the solution, and is simple to operate. Therefore, chitosan flocculation was selected as the preferred method for intermediate clarification, and key process parameters such as chitosan dosage, clarification temperature, and clarification time were further optimized.

[0055] Example 3 Single-factor investigation of chitosan flocculation process 3.1 Effect of chitosan dosage on the drug solution Accurately measure 50 mL of the extract from Example 1, dividing it into six portions. Add 0.5 mL, 1.0 mL, 2.0 mL, 3.0 mL, and 4.0 mL of 1% chitosan solution to each portion, respectively, to achieve final chitosan concentrations of 1.0%, 2.0%, 4.0%, 6.0%, and 8.0%. Place each portion of the extract in a 50°C water bath and stir at 100 rpm for 20 minutes. After standing for 1 hour, centrifuge at 5000 rpm for 20 minutes, repeating the centrifugation three times. Collect the supernatant. Measure the turbidity and stilbene glycoside content, and calculate the stilbene glycoside retention rate.

[0056] Turbidity values ​​and stilbene glycoside retention rates were normalized to a standard of 0 to 1, and the overall OD value was calculated with each value weighted at 50%. The calculation formula is as follows: dmax = (Yi - Ymin) / (Ymax - Ymin); dmin=(Ymax-Yi) / (Ymax-Ymin); OD value = 50% × dmax + 50% × dmin; Where dmax is a factor whose value is better the larger it is (such as stilbene glycoside retention rate), dmin is a factor whose value is better the smaller it is (such as turbidity), Ymax is the maximum value of the evaluation index, and Ymin is the minimum value of the evaluation index.

[0057] With increasing chitosan dosage, the overall OD value showed a trend of first increasing and then decreasing. The OD value was 0.52 when the chitosan dosage was 1.0%, reaching a peak of 0.96 at 2.0%, and then gradually decreased: 0.73 at 4.0%, 0.48 at 6.0%, and dropping to 0.15 at 8.0%. This indicates that a chitosan dosage of 2.0% yielded the best overall effect.

[0058] 3.2 Effect of chitosan clarification temperature on the drug solution Accurately measure 50 mL of the extract from Example 1, dividing it into 5 portions. Add 1 mL of 1% chitosan solution to each portion and place them at 20°C, 30°C, 40°C, 50°C, and 60°C respectively. Stir at 100 rpm for 20 minutes, let stand for 1 hour, and then centrifuge at 5000 rpm for 20 minutes three times. Collect the supernatant. Measure the turbidity and stilbene glycoside content, and calculate the overall OD value.

[0059] The effect of clarification temperature on the overall OD value showed a complex trend of first increasing, then decreasing, and then slightly increasing again. The OD value was 0.53 at 20℃, reached a peak of 0.88 at 30℃, decreased to 0.45 at 40℃, slightly increased to 0.58 at 50℃, and reached 0.52 at 60℃. The overall effect was best at 30℃, possibly because the solubility, flocculation activity, and viscosity of the solution of chitosan reached an optimal balance at 30℃.

[0060] 3.3 Effect of chitosan clarification time on the drug solution Accurately measure 50 mL of the extract from Example 1, dividing it into 5 portions. Add 1 mL of 1% chitosan solution to each portion and place them at 50°C. Stir at 100 rpm for 5 minutes, 10 minutes, 20 minutes, 30 minutes, and 40 minutes, respectively. After standing for 2 hours, centrifuge at 5000 rpm for 20 minutes, repeating the centrifugation three times. Collect the supernatant. Measure the turbidity and stilbene glycoside content, and calculate the overall OD value.

[0061] The effect of clarification time on the overall OD value showed a trend of first increasing and then decreasing. The OD value was 0.81 at 5 minutes, slightly increased to 0.83 at 10 minutes, reached a peak of 0.88 at 20 minutes, significantly decreased to 0.66 at 30 minutes, further decreased to 0.12 at 40 minutes, and slightly rebounded to 0.18 at 50 minutes. A clarification time of 20 minutes yielded the best overall effect; excessively long clarification times may lead to redispersing of flocculated substances or degradation of effective components.

[0062] Based on the results of the single-factor investigation, the optimal values ​​for chitosan addition, clarification temperature, and clarification time are 2.0%, 30℃, and 20 minutes, respectively. Three levels are selected above and below these optimal values ​​for subsequent Box-Behnken response surface methodology optimization.

[0063] Example 4: Optimization of Box-Behnken Response Surface Experiment Response surface methodology (RSM) was designed using Design-Expert 10.0 software. The chitosan addition amount (A), clarification temperature (B), and clarification time (C) in the clarification process were considered as factors. The overall OD value, weighted by turbidity and stilbene glycoside retention rate, was used as the evaluation index. A three-factor, three-level RSM experiment was designed. The code values ​​for each factor level were -1, 0, and +1, respectively, to determine the optimal parameters for the clarification process. The factor level design is shown in Table 1.

[0064] Table 1. Three-Factor, Three-Level Design Table

[0065] Seventeen experiments were conducted according to the experimental protocol generated by Design-Expert 10.0 software, including 12 factorial experiments and 5 center point replication experiments. The experimental protocol and results are shown in Table 2.

[0066] Table 2 Box-Behnken test protocol and results

[0067] The binomial equation was fitted using Design-Expert 10.0 software to establish a multiple linear regression equation between each factor and the overall OD value: Y = 0.8080 - 0.1525A + 0.0975B - 0.03C - 0.1425AB + 0.0475AC +0.0025BC - 0.1653A² - 0.2433B² - 0.1403C²; Where Y is the overall OD value, A is the amount of chitosan added, B is the clarification temperature, and C is the clarification time.

[0068] The regression model was subjected to significance tests and analysis of variance, and the results are shown in Table 3.

[0069] Table 3. Significance tests and analysis of variance for regression models

[0070] Note: ** indicates P < 0.01, meaning the difference is highly significant; This indicates that P > 0.05, meaning the difference is not significant.

[0071] The regression model has an R² of 0.9558 and an adjusted R² of 0.8991, indicating that the model can explain 89.91% of the response value variation, demonstrating a good fit. The model's P-value < 0.01 indicates that the model is highly significant. The lack-of-fit term, P = 0.7514 > 0.05, is not significant, further demonstrating the relatively high reliability of this regression model.

[0072] Analysis of variance showed that the chitosan addition amount (A) and clarification temperature (B) had extremely significant effects on the overall OD value (P<0.01), while the effect of clarification time (C) was not significant (P>0.05). The interaction term AB had an extremely significant effect (P<0.01), while the interaction terms AC and BC had no significant effect. The quadratic terms A², B², and C² were all extremely significant (P<0.01). According to the F-value, the order of influence of each factor on the clarification effect of the drug solution was: chitosan addition amount > clarification temperature > clarification time.

[0073] like Figure 10As shown, the response surface of chitosan addition amount and clarification temperature to OD value exhibits a distinct peak shape, with the OD value reaching its highest point when the chitosan addition amount is approximately 2.0% and the clarification temperature is approximately 30℃. With increasing or decreasing chitosan addition amount and increasing or decreasing clarification temperature, the OD value shows a decreasing trend, indicating that there is an optimal combination point between the two factors.

[0074] like Figure 11 As shown, the response surface of chitosan addition amount and clarification time to OD value also exhibits a peak shape, but the peak shape is relatively flat, indicating that the effect of clarification time on OD value is relatively small. The OD value reaches its highest value when the chitosan addition amount is about 2.0% and the clarification time is about 20 minutes.

[0075] like Figure 12 As shown, the response surface peaks of clarification time and clarification temperature to OD values ​​are not obvious, and the surface is relatively flat, further confirming that the effect of clarification time is small, while the effect of clarification temperature is relatively large.

[0076] Based on optimization using Design-Expert 10.0 software, the optimal process parameters for this model are: chitosan addition of 2.0%, clarification temperature of 30℃, and clarification time of 20 minutes. Under these process conditions, the theoretically predicted total OD value is 0.8080.

[0077] Example 5: Optimal Process Validation Three batches of verification experiments were conducted according to the optimal process parameters obtained in Example 4 (chitosan addition amount 2.0%, clarification temperature 30℃, clarification time 20 minutes). For each batch, 50 mL of the extract from Example 1 was taken, and 1 mL of 1% chitosan solution was added to make the final chitosan addition amount 2.0%. The mixture was stirred at 100 rpm for 20 minutes at 30℃, allowed to stand for 1 hour, and then centrifuged at 5000 rpm for 20 minutes. This process was repeated three times, and the supernatant was collected. The turbidity and stilbene glycoside content were measured, and the overall OD value was calculated.

[0078] Validation results: The turbidity of the three batches of samples were 72.1 NTU, 73.8 NTU, and 71.5 NTU, respectively, with an average turbidity of 72.5 NTU and an RSD of 1.6%. The stilbene glycoside retention rates were 94.2%, 95.1%, and 94.8%, respectively, with an average retention rate of 94.7% and an RSD of 0.5%. The overall OD values ​​were 0.82, 0.81, and 0.83, respectively, with an average OD value of 0.82, which is close to the theoretical prediction value of 0.8080, with a deviation of only 1.5%. The validation results indicate that the optimized process parameters are accurate, reliable, and have good reproducibility.

[0079] Example 6: Effects of different solubilizers on the presence of donkey-hide gelatin in medicinal liquid Take a block of donkey-hide gelatin, crush and grind it into powder using a traditional Chinese medicine pulverizer, take 5 portions, each 1.5g, add 10mL of water to each portion, and place them in a 65℃ water bath to melt. Take the first portion and combine it directly with 30mL of concentrated medicinal solution (prepared according to Example 1 and concentrated to a relative density of 1.10), add water to a total volume of 50mL, mix well, refrigerate and let stand for 24 hours, and filter through a 400-mesh sieve.

[0080] Weigh out 0.5g each of Tween 80, polyethylene glycol 400, polyoxyethylene 40 stearate, and vitamin E succinate polyethylene glycol ester. Add 10mL of water to each and dissolve completely in a 65℃ water bath. Slowly add each of these solutions to the melted donkey-hide gelatin (parts 2 to 5) and mix thoroughly. After mixing, add 30mL of concentrated medicinal solution to each solution and mix well. Adjust the total volume to 50mL, refrigerate and let stand for 24 hours, then filter through a 400-mesh sieve.

[0081] The solubility and stability of donkey-hide gelatin in the medicinal solution were investigated using turbidity, L-hydroxyproline, glycine, alanine, L-proline, and the total amount of donkey-derived polypeptides A1 and A2 as indicators. The results are shown in Table 4.

[0082] Table 4. Effects of adding different solubilizers on the content of donkey-hide gelatin in the medicinal solution.

[0083] As shown in Table 4, compared with the gelatin solution without added solubilizer, the turbidity of the gelatin solution with added Tween 80 decreased from 1126 NTU to 498 NTU, a reduction of 55.8%. Furthermore, the content of L-hydroxyproline increased from 1.4 mg / mL to 1.5 mg / mL, glycine from 3.4 mg / mL to 4.1 mg / mL, alanine from 1.4 mg / mL to 1.7 mg / mL, L-proline from 2.8 mg / mL to 3.0 mg / mL, and the total amount of donkey-derived polypeptides A1 and A2 increased from 44.9 μg / mL to 49.3 μg / mL. All characteristic components of donkey-hide gelatin were significantly improved.

[0084] Although the turbidity of the adhesive solution with added polyethylene glycol 400, polyoxyethylene 40 stearate, and vitamin E succinate was lower than that of the adhesive solution without these additives, it was still higher than that of the adhesive solution with added Tween 80. Furthermore, the total amounts of hydroxyproline, glycine, alanine, proline, and donkey-derived peptides A1 and A2 were all lower than those of the adhesive solution with added Tween 80.

[0085] Tween 80 (polysorbate 80) is a nonionic surfactant with an HLB value of 15.0. It possesses excellent hydrophilicity and emulsifying ability, inhibiting protein denaturation, autopolymerization, and surface adsorption. It is a commonly used emulsifier, solubilizer, wetting agent, and stabilizer in the biopharmaceutical field and has been widely applied in pharmaceuticals and food, exhibiting high safety. Therefore, Tween 80 was chosen as the solubilizer for donkey-hide gelatin in Ejiao Yishou Oral Liquid.

[0086] Example 7: Effect of Tween 80 dosage on the concentration of donkey-hide gelatin in medicinal liquid. Take a block of donkey-hide gelatin, crush and grind it into powder using a traditional Chinese medicine pulverizer, take 4 portions, each 1.5g, add 10mL of water to each portion, and place them in a 65℃ water bath to melt. Take the first portion and combine it directly with 30mL of concentrated medicinal solution (the clarified concentrated medicinal solution prepared according to the methods of Example 1 and Example 5, with a relative density of 1.10), add water to a total volume of 50mL, mix well, refrigerate and let stand for 24 hours, and filter through a 400-mesh sieve.

[0087] Weigh out 0.25g, 0.5g, and 1.0g of Tween 80 respectively, add 10mL of water to each, and dissolve completely in a 65℃ water bath. Slowly add these solutions to the melted donkey-hide gelatin (parts 2 to 4), mixing thoroughly. After mixing, add 30mL of concentrated medicinal solution to each solution, mix well, adjust the total volume to 50mL, refrigerate and let stand for 24 hours, then filter through a 400-mesh sieve. Medicinal solutions containing 0.5%, 1.0%, and 2.0% Tween 80 were prepared, and the effect of different amounts of Tween 80 on the content of donkey-hide gelatin in the medicinal solution was investigated. The results are shown in Table 5.

[0088] Table 5. Effects of different dosages of Tween 80 on the amount of donkey-hide gelatin in the medicinal solution.

[0089] As shown in Table 5, the solution with 1.0% Tween 80 had the lowest turbidity (498 NTU), and the total amounts of L-hydroxyproline, glycine, alanine, L-proline, and donkey-derived polypeptides A1 and A2 were all higher than those with 0.5% and 2.0% Tween 80, as well as the solution without Tween 80. With 0.5% Tween 80, the turbidity was 544 NTU, indicating an unsatisfactory solubilization effect. With 2.0% Tween 80, the turbidity actually increased to 584 NTU, possibly due to the formation of micelles from the excessive Tween 80, leading to increased turbidity, and the content of various donkey-hide gelatin components decreased slightly.

[0090] Taking into account both turbidity and the retention of the effective components of donkey-hide gelatin, the optimal addition amount of Tween 80 was determined to be 1.0%.

[0091] Example 8: Preparation of Ejiao Longevity Oral Liquid using Prescription Process Take 400 mL of the extract from Example 1 and concentrate it to a relative density of 1.10 to obtain a concentrated medicinal solution. Take 6.35 g of pulverized donkey-hide gelatin, add water and dissolve it in a 65°C water bath, filter, and obtain a melted donkey-hide gelatin solution. Mix the melted donkey-hide gelatin solution with the concentrated medicinal solution. Take 10 g of sucrose and add water to make a syrup, add 0.6 g of sodium benzoate, combine it with the above medicinal solution, adjust the total volume to 200 mL, refrigerate and let stand for 24 hours, filter through a 400-mesh sieve, and obtain the donkey-hide gelatin longevity oral liquid prepared by the prescription process.

[0092] like Figure 13 As shown, when the medicinal liquid prepared by the prescription process is combined with donkey-hide gelatin and filtered through a 400-mesh sieve, it forms droplets, and the filtration is very slow and difficult. Figure 14 As shown, after filtration, a large amount of dark brown filter residue remains on the filter paper. The filter residue is thick and viscous.

[0093] Example 9: Improved process for preparing donkey-hide gelatin longevity oral liquid Take 400 mL of the extract from Example 1, and add 8 mL of 1% chitosan solution (to make the final chitosan addition amount 2.0%) according to the optimized process parameters of Example 5. Place the mixture in a 30°C water bath and stir at 100 rpm for 20 minutes. After standing for 1 hour, centrifuge at 5000 rpm for 20 minutes, repeating the centrifugation three times. Collect the supernatant to obtain a clear drug solution. Concentrate the clear drug solution to a relative density of 1.10 to obtain a clear concentrated drug solution.

[0094] Take 6.35g of crushed donkey-hide gelatin, add water and dissolve in a 65℃ water bath. Weigh 2.0g of Tween 80, add 20mL of water, dissolve completely in a 65℃ water bath, then slowly add to the dissolved donkey-hide gelatin, mix thoroughly, filter, and obtain a donkey-hide gelatin solution.

[0095] The clarified and concentrated medicinal solution was combined with the gelatin-enhancing solution and mixed thoroughly. 10g of sucrose was added to water to make a syrup, and 0.6g of sodium benzoate was added. The syrup was then combined with the above medicinal solution, and the total volume was adjusted to 200mL. The mixture was refrigerated and allowed to stand for 24 hours. It was then filtered through a 400-mesh sieve to obtain the improved gelatin-enhancing oral liquid.

[0096] like Figure 13 As shown, when the medicinal liquid prepared by the improved process is combined with donkey-hide gelatin and filtered, it flows in a straight line, resulting in smooth and fast filtration. Figure 14 As shown, very little filter residue remains on the filter paper after filtration, appearing as a light-colored thin layer, which contrasts sharply with the formulation process.

[0097] Example 10: Comparison of Product Quality Between Prescription Process and Improved Process 10.1 Comparison of Product Characteristics The samples prepared in Examples 8 and 9 were observed and photographed. Figure 15As shown, the sample solution prepared using the original formulation process is dark brown, opaque, and highly turbid. The sample solution prepared using the improved process is lighter in color, has better light transmittance, and significantly improved clarity. The difference becomes even more pronounced after diluting both samples five times with water; the sample prepared using the original formulation process remains relatively turbid, while the sample prepared using the improved process is clearer and more transparent.

[0098] The turbidity of the two samples was measured. The turbidity of the sample with the original process was 1004 NTU, while the turbidity of the sample with the improved process was 456 NTU. The turbidity was reduced by 54.6%, and the clarity was improved by 2.1 times.

[0099] 10.2 Comparison of active ingredient content The contents of stilbene glycoside, hesperidin, astragaloside A, glycyrrhizin, and glycyrrhizic acid were determined by high performance liquid chromatography (HPLC), and the total amounts of donkey-derived polypeptide A1 and donkey-derived polypeptide A2 were determined by UPLC-MS / MS. The results are shown in Table 6.

[0100] Table 6 Comparison of effective ingredient content between the original formulation and the improved formulation

[0101] like Figure 16 As shown, comparing the contents of the main active ingredients in samples from the original and improved processes, the contents of stilbene glycosides, hesperidin, and glycyrrhizic acid in the improved process were slightly lower than those in the original process, but the differences were very small, with retention rates of 95%, 96%, and 99%, respectively. The total contents of astragaloside A, glycyrrhizin, and donkey-derived polypeptides A1 and A2 in the improved process were slightly higher than those in the original process, with retention rates of 101%, 102%, and 111%, respectively.

[0102] The above results indicate that the improved process can significantly improve product clarity while retaining the effective components in the liquid to the greatest extent, thus ensuring product quality.

[0103] 10.3 Product Stability Comparison The samples prepared in Examples 8 and 9 were placed in transparent glass bottles and placed in a constant temperature and humidity chamber at 40°C and 75% humidity to simulate accelerated stability test conditions. The sedimentation at the bottom of the bottles was observed and photographed before placement (day 0), and on days 1, 3, 7, and 14.

[0104] Before storage (day 0), the sample prepared by the formula showed only tiny particles at the bottom of the bottle with no obvious sediment. After one day in a constant temperature and humidity chamber, a distinct dark brown precipitate appeared at the bottom of the sample. The precipitate gradually thickened with prolonged storage, reaching approximately 2 mm after 3 days, 4 mm after 7 days, and 6 mm after 14 days. Even when the bottle was inverted, the precipitate remained adsorbed at the bottom, failing to disperse easily and severely affecting the product's appearance and usability.

[0105] The samples prepared using the improved process showed clean bottoms and no sediment before being placed under the same conditions (day 0). After being placed in a constant temperature and humidity chamber for 1, 3, 7, and 14 days, the bottoms of the bottles remained clean with no visible sediment, indicating a significant improvement in product stability.

[0106] The above results fully demonstrate that the improved process, through two optimization steps of intermediate clarification and donkey-hide gelatin solubilization, fundamentally solves the problem of precipitation caused by the reaction of donkey-hide gelatin with tannins, greatly improves the stability of the product, and extends the product's shelf life.

[0107] Example 11 Evaluation of the efficacy of phenylhydrazine-induced zebrafish anemia model before and after process improvement 11.1 Experimental Materials Experimental drugs: Ejiao Yishou Oral Liquid prepared using the prescribed process (Example 8) and Ejiao Yishou Oral Liquid prepared using the improved process (Example 9). Positive control drug: Ruike® Shengxuening Tablets (batch number 20231015), dissolved in standard dilution water.

[0108] Experimental animals: Wild-type AB strain zebrafish, purchased from Hangzhou Huante Biotechnology Co., Ltd. The zebrafish were cultured in a zebrafish recirculating aquaculture system under the following conditions: temperature 26-29℃, pH 7.2-7.5, dissolved oxygen 6-7.8 mg / L, conductivity 500-550 μS, and a photoperiod of 14 hours of light followed by 10 hours of darkness. Females and males were kept separately and fed zebrafish food and brine shrimp daily.

[0109] 11.2 Collection of Zebrafish Embryos Select healthy zebrafish of suitable age and place them in a spawning tank at a female-to-male ratio of 2:1, separating the males and females with a partition. Remove the partition before the next day's light exposure to allow the zebrafish to fertilize freely. Two hours after removing the partition, collect the fertilized eggs, select those that are developing normally and have good morphology, and place them in a clean culture medium for further cultivation in a 28°C constant temperature incubator.

[0110] 11.3 Establishment of anemia model and drug administration 120 wild-type AB strain zebrafish, fertilized 60 hours after hatching (60 hpf), were randomly selected and placed in 6-well plates, 20 fish per well. They were divided into 5 groups: normal control group, model control group, positive control group (Shengxuening 12.5 μg / mL), prescription process group (Ejiao Yishou oral liquid 1 μg / mL), and improved process group (Ejiao Yishou oral liquid 1 μg / mL). Each well contained 3 mL. After incubating the sample plates at 28℃ for 12 hours, except for the normal control group, all other groups were added with 0.5 μg / mL phenylhydrazine solution and incubated at 28℃ for another 24 hours to establish a zebrafish anemia model.

[0111] 11.4 Erythrocyte Staining and Data Analysis Red blood cells from the heart of zebrafish were stained with o-anisidine staining solution. The cells were observed and photographed under a dissecting microscope. Data were acquired using Image-Pro Plus software, and the staining intensity (S) of the heart red blood cells was analyzed. Statistical analysis was performed using SPSS 26.0 software. Data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely statistically significant.

[0112] 11.5 Experimental Results like Figure 18 As shown, the red blood cells in the hearts of zebrafish in the normal control group showed dark staining, indicating a high number of red blood cells and normal hemoglobin levels. In the model control group, the red blood cells in the hearts of zebrafish showed significantly lighter staining, a reduced number of red blood cells, and a paler color, indicating that phenylhydrazine successfully induced anemia. The red blood cells in the hearts of zebrafish in the positive control group (Shengxuening), the prescription process group, and the improved process group showed significantly darker staining and a higher number of red blood cells compared to the model control group, indicating that all treatment groups had an ameliorative effect on anemia.

[0113] like Figure 19 As shown in Table 7, the staining intensity of erythrocytes in the heart of zebrafish in the normal control group was 25125±3197 pixels, while that in the model control group was only 184±24 pixels, showing a highly significant decrease in erythrocyte staining intensity compared to the normal control group (P<0.01), indicating the successful establishment of the zebrafish anemia model. The staining intensity of erythrocytes in the heart of the positive control group (Shengxuening) was 18812±1722 pixels, significantly higher than that of the model control group (P<0.01). The staining intensity of erythrocytes in the heart of the prescription process group was 11347±1816 pixels, and that in the improved process group was 11028±1734 pixels, both significantly higher than that of the model control group (P<0.01), indicating that both processes of the prepared Ejiao Yishou oral liquid significantly improved phenylhydrazine-induced anemia in zebrafish. There was no significant difference between the prescription process group and the improved process group (P>0.05), indicating that the process improvement did not affect the product's efficacy in improving anemia.

[0114] Table 7 Evaluation of the anemia-improving effects of Ejiao Yishou Oral Liquid before and after process improvement

[0115] Note: Compared with the normal control group ## P<0.01; compared with the model control group, ** P<0.01.

[0116] The above experimental results show that the improved process of the donkey-hide gelatin longevity oral liquid has comparable efficacy in improving anemia to the product prepared by the prescription process. The process optimization significantly improved the product's clarity and stability without affecting its pharmacological activity, thus ensuring clinical efficacy.

[0117] Example 12 Method for Determination of Stilbene Glycoside Content The content of stilbene glycosides in Ejiao Yishou Oral Liquid was determined by high performance liquid chromatography.

[0118] Instrument: Agilent 1260 high-performance liquid chromatograph, equipped with a quaternary pump, autosampler, column oven, and diode array detector (DAD). Column: ACE Excel 5 C18-AR column (5μm, 4.6mm × 250mm).

[0119] Chromatographic conditions: mobile phase was acetonitrile-water (17:83, V / V), flow rate was 1.0 mL / min, column temperature was 30℃, detection wavelength was 320 nm, and injection volume was 5 μL.

[0120] Preparation of reference solution: Accurately weigh an appropriate amount of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference standard (China National Institutes for Food and Drug Control, batch number 110844-201610), dissolve and dilute with methanol to prepare a reference solution containing 100 μg per mL.

[0121] Preparation of the test solution: Accurately measure 1 mL of the donkey-hide gelatin longevity oral liquid sample and place it in a 5 mL volumetric flask. Dilute with dilute ethanol to the mark, shake well, filter through a 0.45 μm microporous membrane, and take the filtrate as the test solution.

[0122] Linearity assessment: The reference solution was accurately pipetted and diluted with methanol to prepare a series of reference solutions with concentrations of 0.1, 1, 10, 50, and 100 μg / mL. The peak areas were measured under the chromatographic conditions described above. A linear regression was performed on the peak area (Y) against the concentration (X), and the regression equation was Y = 32.45X + 1.28, with R² = 0.9998, indicating that stilbene glycosides have a good linear relationship in the range of 0.1-100 μg / mL.

[0123] Precision test: The same sample solution was precisely pipetted and injected 6 times consecutively. The peak area was measured. The RSD was 1.3%, indicating that the instrument has good precision.

[0124] Repeatability test: Take 6 samples from the same batch, prepare test solutions according to the test solution preparation method, determine the content of stilbene glycosides under the above chromatographic conditions, and calculate the RSD of 1.8%, indicating that the method has good repeatability.

[0125] Stability test: Take the same test solution and inject it at 0, 2, 4, 6 and 8 hours to measure the peak area. The RSD is 1.5%, which indicates that the test solution is stable within 8 hours.

[0126] Recovery test: Accurately measure 0.5 mL of the sample with known content (0.425 mg / mL stilbene glycoside content), add an appropriate amount of reference standard, prepare the solution according to the test solution preparation method, determine the content, and calculate the recovery rate. The average recovery rate was 98.6%, and the RSD was 2.1%, indicating that the method has good accuracy.

[0127] The formula for calculating the stilbene glycoside retention rate is: Stilbene glycoside retention rate = (Stilbene glycoside content in the clarified solution / Stilbene glycoside content in the solution before clarification) × 100%.

[0128] The above embodiments detail the technical solution, implementation process, and beneficial effects of the optimized preparation process of the donkey-hide gelatin longevity oral liquid of the present invention. However, the present invention is not limited to the above embodiments. Ordinary changes and substitutions made by those skilled in the art within the scope of the technical solution of the present invention should be included within the protection scope of the present invention.

Claims

1. An optimized preparation process method for donkey-hide gelatin longevity oral liquid, characterized in that, Includes the following steps: Step 1, Medicinal herb extraction: Take ginseng, roasted astragalus, prepared rehmannia root, prepared he shou wu, tangerine peel, costus root, and licorice root, add water and decoct three times. For the first decoction, add 6 times the amount of water and simmer for 2 hours. For the second decoction, add 5 times the amount of water and simmer for 2 hours. For the third decoction, add 5 times the amount of water and simmer for 1 hour. Filter, combine the decoctions, and obtain the medicinal liquid. Step 2, intermediate clarification process: Chitosan is added to the drug solution obtained in Step 1 for flocculation and clarification. The amount of chitosan added is 1.5-2.5% of the drug solution volume. Stirring is carried out at a temperature of 20-40℃ for 10-30 minutes, and the mixture is allowed to stand for 1-3 hours. After centrifugation, the supernatant is collected to obtain the clarified drug solution. Step 3, drug concentration: Concentrate the clarified drug solution obtained in Step 2 to a relative density of 1.05-1.15; Step 4, donkey-hide gelatin solubilization process: Take donkey-hide gelatin powder, add water and melt it at 60-70℃. Take Tween 80, dissolve it in water, and mix it thoroughly with the melted donkey-hide gelatin at 60-70℃. The amount of Tween 80 added is 0.5-2.0% of the total liquid volume. Filter to obtain donkey-hide gelatin solubilization solution; Step 5, Merging and Refining: Combine the clarified and concentrated medicinal liquid obtained in Step 3 with the donkey-hide gelatin solution obtained in Step 4, add syrup made from sucrose and preservatives, adjust the total amount, let stand, filter, and obtain donkey-hide gelatin longevity oral liquid.

2. The optimized preparation process of the donkey-hide gelatin longevity oral liquid according to claim 1, characterized in that, In step two, the amount of chitosan added is 2.0% of the volume of the drug solution, the clarification temperature is 30°C, and the clarification time is 20 minutes.

3. The optimized preparation process of the donkey-hide gelatin longevity oral liquid according to claim 1 or 2, characterized in that, The chitosan used in step two is a chitosan solution with a mass fraction of 1.0-2.0%. The chitosan solution is prepared by dissolving chitosan in 1% acetic acid and then refrigerated and left to stand for 24 hours before use.

4. The optimized preparation process of the donkey-hide gelatin longevity oral liquid according to any one of claims 1 to 3, characterized in that, The stirring speed in step two is 80-120 revolutions per minute.

5. The optimized preparation process of the donkey-hide gelatin longevity oral liquid according to any one of claims 1 to 3, characterized in that, The centrifugation conditions in step two are 4000-6000 rpm for 15-25 minutes, and the number of centrifugations is 2-4.

6. The optimized preparation process of the donkey-hide gelatin longevity oral liquid according to any one of claims 1 to 3, characterized in that, In step three, the clarified medicinal solution is concentrated to a relative density of 1.

10.

7. The optimized preparation process of the donkey-hide gelatin longevity oral liquid according to any one of claims 1 to 3, characterized in that, In step four, the melting temperature of the donkey-hide gelatin is 65°C, and the amount of Tween 80 added is 1% of the total volume of the medicinal liquid.

8. The optimized preparation process of the donkey-hide gelatin longevity oral liquid according to any one of claims 1 to 3, characterized in that, In step one, the weight ratio of ginseng, roasted astragalus, prepared rehmannia root, prepared he shou wu, tangerine peel, costus root, and licorice is 1:10:5:5:2:1:

1.

9. The optimized preparation process of the donkey-hide gelatin longevity oral liquid according to any one of claims 1 to 3, characterized in that, In step four, the weight ratio of donkey-hide gelatin to ginseng in step one is 5:

1.

10. The optimized preparation process of the donkey-hide gelatin longevity oral liquid according to any one of claims 1 to 3, characterized in that, In step five, the preservative is sodium benzoate, and its addition amount is 0.2-0.4% of the total volume of the oral liquid.

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