Quality control method and preparation process optimization method of Yulingling paste

Abstract

The invention discloses a quality control method and a preparation process optimization method of Yulingling ointment, which are characterized in that three key components for benefiting qi and nourishing blood, namely 6-Paradol (zingiberol), Eupafolin (eupatorin) and Chrysophanic Acid (chrysophanol), are screened by utilizing a network pharmacology technology and molecular docking analysis; and then quality control and preparation process optimization are performed on the Yulingling ointment through the three key components, so that favorable support is provided for industrial production of the Yulingling ointment.

Description

Technical Field

[0001] The present invention relates to a quality control method for Yuling Paste, and also relates to a method for optimizing the preparation process of Yuling Paste and the preparation process, belonging to the technical field of traditional Chinese medicine research and development. Background Art

[0002] Qi and blood deficiency is a common syndrome in traditional Chinese medicine, also known as deficiency of both qi and blood. It refers to the pathological state in which the qi and blood in the human body do not circulate smoothly or are insufficiently generated, resulting in the decline of visceral functions and the decrease of the body's disease resistance. Qi and blood deficiency generally presents symptoms such as fatigue and dizziness. Yuling Paste is an ancient recipe for dietary therapy in traditional Chinese medicine, originating from "Dietetic Recipes for a Leisurely Life" by Wang Mengying, a famous traditional Chinese medicine expert in the Qing Dynasty. It is mainly made of longan meat and American ginseng, and is made through a steaming process. Its core efficacy is to replenish qi and blood and nourish the heart and spleen, and it is suitable for symptoms such as fatigue, insomnia, and pale complexion caused by qi and blood deficiency. Yuling Paste is one of the classic prescriptions for qi and blood deficiency syndrome, and is currently widely used in the treatment of blood deficiency and qi deficiency, poor complexion, easy fatigue, palpitations, and insomnia and dreaminess.

[0003] Qi and blood deficiency is a common syndrome in traditional Chinese medicine, also known as deficiency of both qi and blood. It refers to the pathological state in which the qi and blood in the human body do not circulate smoothly or are insufficiently generated, resulting in the decline of visceral functions and the decrease of the body's disease resistance. Qi and blood deficiency generally presents symptoms such as fatigue and dizziness. Currently, the treatment of this disease mainly includes dietary therapy, acupuncture, and drug treatment. Among them, dietary therapy combines the synergistic effects of nutritional components and bioactive components to achieve the treatment effect. Specifically, medicinal foods are processed and taken with qi-tonifying medicinal materials such as astragalus membranaceus, ginseng, and codonopsis pilosula, and blood-tonifying medicinal materials such as angelica sinensis, longan meat, and donkey-hide gelatin. The medicinal materials used are all raw materials of both medicine and food that tonify blood and promote blood circulation, strengthen the spleen and nourish the kidney, and regulate the spleen and stomach.

[0004] In the ancient recipe, the preparation method of Yuling Paste is as follows: Peel the longan and put it in a bamboo tube-shaped porcelain bowl. For every one tael of longan meat, add one qian of white granulated sugar. For those with a naturally fire-prone constitution, add American ginseng slices in the same amount as the sugar. Cover the bowl mouth with a layer of silk cotton and steam it on the rice cooker every day for 100 times. However, the preparation method of the ancient recipe of Yuling Paste cannot meet the needs of modern, standardized, and large-scale production. In historical records, it only states "steam for 100 times", and no specific description of the steaming duration is found. At present, there is also no unified standard for this important influencing factor of steaming duration in process research. In addition, the pharmacodynamic substance basis, action targets, and mechanisms of Yuling Paste in exerting the effect of replenishing qi and nourishing blood have not been elucidated, and there is temporarily a lack of a quality control method based on active ingredients. Therefore, it is necessary to systematically and deeply explore the appropriate steaming duration, active ingredients, and pharmacological effects of Yuling Paste, improve and establish the quality control standard of Yuling Paste, and provide a reference for further exploration of the disease treatment mechanism. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a quality control method for Yuling Ointment. This invention explores the potential qi-tonifying and blood-nourishing substances in Yuling Ointment through network pharmacology and molecular docking, predicts its molecular targets and related pathways, and speculates on its potential mechanism for treating qi and blood deficiency, providing support for future research on the molecular mechanisms of traditional Chinese medicine and the development of new therapeutic targets. Simultaneously, experiments demonstrate the significant impact of these potential qi-tonifying and blood-nourishing substances on the efficacy of Yuling Ointment, and a quality control method based on the effective components is established, providing favorable support for the industrial production of Yuling Ointment.

[0006] The specific technical solution of this invention is as follows: A quality control method for Yuling Ointment, wherein the method uses potential substances for replenishing qi and nourishing blood as quality control components, and the higher the content of the quality control components, the better the quality of Yuling Ointment; the potential substances for replenishing qi and nourishing blood are 6-Paradol (gingerol), Eupafolin (eupatorin) and Chrysophanic Acid (chrysophanol).

[0007] Furthermore, the potential substances for replenishing Qi and nourishing blood were obtained through network pharmacology and molecular docking analysis. Specifically, the network pharmacology analysis involved: ① Using a score ≥10 as a screening criterion in the GeneCards database to obtain target genes related to Qi deficiency and blood deficiency, and using Venn diagrams (Venny 2.1.0) to plot the effective ingredient targets of Yuling Ointment and the target genes for Qi and blood deficiency. The intersection genes obtained from the Venn diagrams were the drug-disease intersection target genes. ② Importing the intersection target genes obtained in the previous step into the interaction gene search tool (STRING) to construct a PPI network diagram and downloading the TSV format file. The PPI network diagram was visualized using Cytoscape software, and its topology was analyzed using Cytoscape, Network Analyzer, and CytoNCA to obtain 45 core target proteins. A "Yuling Ointment-effective ingredient-effective target" network diagram was constructed and its topology analyzed using Cytoscape software, and the top 6 effective ingredients were identified as the material basis.

[0008] Specifically, molecular docking analysis involves verifying the molecular docking of the top six basic components and the top six core targets in the "Yu Ling Gao - Active Ingredients - Key Targets - Disease" network degree value. This includes downloading the 3D structures of key target proteins from the PDB database and the MOL2 file of the main active ingredient from the TCMSP platform. PyMOL software is then used to perform dehydration and hydrogenation operations on the key target proteins. Molecular docking is then performed using Auto Dock Tools 1.5.6 and Auto Dock Vina software, and the binding energy is recorded. Finally, Pymol 2.5.0 is used to visualize compounds with lower molecular docking energies and stable conformations.

[0009] This invention also provides an optimized method for preparing Yuling Ointment, the method comprising the following steps: (1) A series of different Yuling ointment samples were obtained by using different preparation processes; (2) The contents of 6-Paradol (gingerol), Eupafolin (zeolite) and Chrysophanic Acid (chrysophanol) in different Yuling Ointment samples were obtained by high performance liquid chromatography-mass spectrometry analysis. (3) Select the preparation process parameters of Yuling Ointment with high content of potential substances for replenishing qi and nourishing blood as the final preparation process parameters.

[0010] Furthermore, the different preparation processes refer to different preparation steps and / or different process conditions and / or different Yu Ling Gao (Jade Spirit Paste) formulas. The content of potential qi-tonifying and blood-nourishing substances in Yu Ling Gao obtained from different preparation processes varies. By detecting the content of these potential qi-tonifying and blood-nourishing substances, the preparation process with the highest content of these substances is selected as the optimal process.

[0011] The present invention also provides a preferred method for preparing Jade Spirit Ointment, the method comprising the following steps: S1. Soak the longan pulp in water; S2. Chop the soaked longan pulp, mix it with American ginseng powder and white sugar in a mass ratio of 10:1:1, place it in a porcelain bowl, cover it with gauze, and steam it in stages with controlled temperature. Specifically, steam it at a low temperature of 78℃ for 10-14 hours, and then steam it at a high temperature of 95℃ for 6-10 hours. S3. After steaming, proceed with drying; S4. After drying, let it cool to room temperature, then package it to obtain the finished Yu Ling Ointment.

[0012] Further, in step S1, the soaking treatment refers to soaking the dried longan pulp in rice water (the water used to rinse rice). The soaking temperature is controlled between 30-50℃, such as any value or range between 30℃, 35℃, 40℃, 45℃, and 50℃. The soaking time is 2-4 hours, such as 2 hours, 2.5 hours, 3 hours, 3.5 hours, and 4 hours. Rice water soaking is used because rice water has medicinal value such as strengthening the spleen and stomach, and longan pulp itself also has the effect of nourishing the heart and spleen. Soaking in rice water can enhance the medicinal effect of longan pulp to a certain extent, allowing it to better exert its effects of nourishing qi and blood, calming the mind, etc. At the same time, longan pulp is warm in nature, and direct steaming may cause some people to experience symptoms such as internal heat after consumption. Rice water is relatively mild, and soaking longan pulp in it can alleviate its warming nature to a certain extent.

[0013] Furthermore, in step S2, temperature-controlled segmented steaming refers to a steaming process that employs a low-temperature preheating stage followed by a high-temperature steaming stage. During steaming, a low-temperature steaming at 78℃ for 10-14 hours is followed by a high-temperature steaming at 95℃ for 6-10 hours. This temperature-controlled segmented steaming allows the internal cell structure of the longan pulp and American ginseng to remain relatively stable during the low-temperature steaming process, while the subsequent short-duration high-temperature steaming helps the Yuling paste achieve a paste-like texture, minimizing the loss of heat-sensitive active ingredients. Preferably, the total steaming time is 20 hours.

[0014] Preferably, in step S2, the food is first steamed at a low temperature of 78°C for 12 hours, and then steamed at a high temperature of 95°C for 8 hours.

[0015] Further, in step S3, after steaming, the product is moderately dried in an air-source heat pump dryer. During drying, the drying temperature is generally controlled between 40℃ and 60℃, for example, any value or range between 40℃, 45℃, 50℃, and 60℃. The drying time is controlled between 1 and 3 hours, for example, any value or range between 1 hour, 1.5 hours, 2 hours, 2.5 hours, and 3 hours. This drying process reduces the surface moisture content of the Yu Ling Gao after steaming, inhibits the growth and metabolism of microorganisms and enzymes, and thus extends the shelf life of the Yu Ling Gao.

[0016] Compared with the prior art, the present invention has the following advantages: 1. This invention utilizes network pharmacology technology and molecular docking to analyze the potential substances, targets, and pathways by which Yuling Ointment improves Qi and blood deficiency. It analyzes its multi-component, multi-target, and multi-pathway mechanism of action. Active ingredients and their potential targets are obtained using the PubCard and Swiss Target databases. Targets for Qi and blood deficiency are obtained based on GeneCard and OMIM databases. A "Yuling Ointment-Active Ingredient-Key Target-Disease" network is established based on active ingredient and intersection target data. A protein-protein interaction (PPI) network is constructed to screen core targets. Results show that the key components (potential substances) of Yuling Ointment for replenishing Qi and blood include 6-Paradol (gingerol), Eupafolin, and Chrysophanic Acid. Key targets mainly include GAPDH, AKT1, STAT3, and ALB. The binding energies of key components and their target proteins are all less than -5 kJ / mol, indicating stable binding. The method of this invention provides a new perspective on elucidating the molecular mechanism by which Yuling Ointment improves qi and blood deficiency, lays the foundation for further research and development of innovative formulations of Yuling Ointment, and provides support for future research on the molecular mechanisms of traditional Chinese medicine and the development of new therapeutic targets.

[0017] 2. This invention proposes for the first time a quality control method for Yuling Ointment based on effective ingredients, which can provide a certain reference for further improving its quality evaluation standards. By controlling the quality through potential substances that replenish qi and nourish blood, the quality of Yuling Ointment can be controlled in a more standardized manner in industrial production, providing a favorable guarantee for the batch product quality stability in the industrial production of Yuling Ointment.

[0018] 3. This invention optimizes the preparation process of Yuling Paste based on the content of potential substances for replenishing qi and nourishing blood, resulting in a preferred preparation process. This process is simple and easy to operate. It alleviates the warming nature of longan pulp through initial soaking, enhancing its medicinal efficacy, and extends the shelf life of Yuling Paste through subsequent drying. It also promotes the synergistic effect of longan pulp and American ginseng, making it particularly suitable for those who need to replenish qi and blood. By adopting the optimized process, the qi-replenishing and blood-nourishing effects of Yuling Paste are effectively enhanced. Attached Figure Description

[0019] Figure 1 Images of samples of Yuling Paste at five different steaming times; Figure 2 This is a classification diagram of the total metabolites of Yuling Ointment identified under positive and negative ion modes; Figure 3 The images show the differential metabolite volcano diagrams of the Yuling Ointment samples, where (A) represents Y5 vs. Y0, (B) represents Y10 vs. Y0, (C) represents Y20 vs. Y0, and (D) represents Y30 vs. Y0. Figure 4 Venn diagram showing the differences in metabolites between the control group and each steaming group; Figure 5 The diagram shows the intersection of Yuling Ointment and diseases, where (A) shows the intersection of Yuling Ointment and the disease of "Qi deficiency", and (B) shows the intersection of Yuling Ointment and the disease of "blood deficiency". Figure 6 The diagram shows the "drug-ingredient-target" network diagram of Yuling Ointment, where (A) is the "drug-ingredient-target" network diagram for "Qi deficiency" and (B) is the "drug-ingredient-target" network diagram for "blood deficiency". Figure 7 The PPI network diagrams for Yuling Ointment are shown below. (A) is the PPI network diagram for "Qi deficiency" and (B) is the PPI network diagram for "blood deficiency". Figure 8 Heatmaps showing the docking scores of 6 core targets with 6 basic molecules. Figure 9The diagrams show the binding patterns between the six basic material components with the highest binding energy and the core target sites. (A) shows the binding of Eupafolin with ALB, (B) shows the binding of Eupafolin with GAPDH, (C) shows the binding of Chrysophanic Acid with STAT3, (D) shows the binding of Eupafolin with STAT3, (E) shows the binding of 6-Paradol with STAT3, and (F) shows the binding of 6-Paradol with AKT1. Figure 10 Heatmap showing the relative content changes of gingerol, eupatorium, and chrysophanol in each group of Yuling ointment samples. Detailed Implementation

[0020] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Unless otherwise specified, all concentrations mentioned in the following examples and comparative examples are mass percentage concentrations.

[0022] Example 1 S1. Soak rice in 10 times its weight of water for 2 hours, then filter to obtain rice water. Soak longan pulp in the rice water, controlling the soaking temperature at 40℃ and the soaking time at 3 hours. S2. Chop the soaked longan pulp, mix it with American ginseng powder and white sugar in a mass ratio of 10:1:1, place it in a porcelain bowl, cover it with gauze, and steam it at a high temperature of 95℃ for 30 hours.

[0023] S3. After steaming, the product is moderately dried in an air source heat pump dryer at a temperature of 45°C for 2 hours. S4. After drying, let it cool to room temperature, then package it to obtain the finished Yu Ling Ointment.

[0024] Samples were taken at 0h, 5h, 10h, 20h, and 30h of steaming, and the resulting Yuling ointment samples were numbered Y0, Y5, Y10, Y20, and Y30, respectively (see...). Figure 1 ).

[0025] The above samples were analyzed as follows: 1. Metabolomics analysis A metabolomics analysis method based on ultra-high performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HMS) was employed. This method combines a high-quality mzCloud database constructed from standards with the mzVault and MassList databases to match and identify molecular characteristic peaks. First, simple screening was performed using parameters such as retention time and mass-to-charge ratio. Peak alignment was then performed on different samples to improve accuracy. Subsequently, peak extraction was performed based on set ppm and adduct ion information, and peak area was quantified. Finally, a search was conducted using the HMS secondary spectral databases mzCloud and mzVault, as well as the MassList primary database, to identify metabolites. Figure 2 The metabolites identified in both positive and negative ion modes were classified and statistically analyzed, including lipids and lipid molecules (35.43%), phenylpropanoids and polyketides (14.38%), organic heterocyclic compounds (12.96%), organic acids and derivatives (11.15%), organic oxygen compounds (8.64%), benzene compounds (8.09%), nucleosides, nucleotides and analogues (4.24%), alkaloids and derivatives (2.12%), organic nitrogen compounds (1.34%), lignans, neolignans and related compounds (1.26%), homogeneous nonmetallic compounds, organic sulfur compounds, hydrocarbons, mixed metal / nonmetallic compounds, and some unclassified metabolites.

[0026] To specifically compare the effects of different steaming times on metabolites and further explore the potential differential metabolites of Yuling ointment at each time point, a volcano plot analysis was performed on the metabolites of the original group and the processed group (Y5 vs Y0, Y10 vs Y0, Y20 vs Y0, Y30 vs Y0). Figure 3 As shown in the figure, the number of differential metabolites is 400, 570, 714 and 785 respectively, indicating that the number of differential metabolites gradually increases with the extension of steaming time.

[0027] like Figure 4 Venn diagrams showed a total of 249 differentially expressed metabolites between the original and processed groups. In the processed groups (Y5 vs Y0, Y10 vs Y0, Y20 vs Y0, Y30 vs Y0), 50, 44, 41, and 172 differentially expressed metabolites, respectively, were found that distinguished them from the other groups (see...). Figure 4 It can be seen that the content and types of metabolites in Yuling Paste with different steaming time periods are significantly different.

[0028] 2. Network pharmacology analysis: First, the active ingredients in longan pulp and American ginseng were screened using the Traditional Chinese Medicine Systemic Pharmacology Database and Analysis Platform (TCMSP), the Traditional Chinese Medicine and Western Medicine Integrated Pharmacology Research Platform (TCMIP v2.0), and the Encyclopedia of Traditional Chinese Medicine. Only ingredients meeting the criteria of oral bioavailability ≥30% and drug-likeness ≥0.18 were selected. By comparing and screening all obtained active ingredients with all overlapping differential metabolites, 76 dominant ingredients were identified (see Table 1). The SwissTargetPrediction database was used to predict the targets of these ingredients, identifying a total of 3253 targets. After removing duplicates, 911 unique targets remained. Screening in the OMIM and GeneCards databases identified 2195 targets for Qi deficiency and 1648 targets for anemia. Venn diagram analysis showed 286 targets between Yuling Ointment and Qi deficiency, and 178 targets between Yuling Ointment and anemia (see Table 1). Figure 5 To explore the relationship between qi-tonifying and blood-nourishing components and their targets, a "drug-metabolite-target" network was constructed (see...). Figure 6 The network shows that there are many-to-many interactions between the qi-tonifying and blood-nourishing components and the qi-tonifying and blood-nourishing targets, indicating that the qi-tonifying and blood-nourishing effects are due to the synergistic effect of these compounds.

[0029] Table 1. Key ingredients of Yuling Paste that contribute to its qi-tonifying and blood-nourishing effects. To evaluate the key components and core targets of longan pulp and American ginseng in replenishing qi and nourishing blood, we imported the obtained active ingredients into Cytoscape 3.10.1 software and used the Network Analyzer algorithm tool to analyze the imported data. After comparison and screening, a total of 6 components were identified as the material basis for Yuling Ointment's qi-replenishing and blood-nourishing effects. These 6 components are: 6-Paradol (gingerol), Eupafolin (eupatorin), Chrysophanic Acid (chrysophanol), Poricoic acid B (poriolic acid B), Atractylenolide III (atractylenolide III), and Ziyuglycoside (sanguisorbin).

[0030] Further protein-protein interaction network analysis was performed on 286 and 178 common potential targets using the STRING data platform, specifically targeting the species “Homo sapiens”. The results were imported into Cytoscape 3.10.1 software, and topology parameter analysis was performed using its CytoNCA plugin. Core targets of the protein network were selected based on degree ranking, with the top 5 targets chosen as core targets (see [link to relevant documentation]). Figure 7 The core targets for Yuling Ointment's Qi-tonifying effects are Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), AKT serine / threonine kinase1 (AKT1), Albumin (ALB), Interleukin-6 (IL6), and Tumor necrosis factor (TNF). The core targets for its blood-nourishing effects are GAPDH, AKT1, IL6, TNF, and Signal transducer and activator of transcription 3 (STAT3). Both groups contain four shared targets and two unique targets; therefore, six targets were selected as the core targets. The high similarity of the core targets for both Qi-tonifying and blood-nourishing effects indicates that, at the molecular level, these pathways likely function through the same or closely related targets, further demonstrating the complexity of biological networks and the multifunctionality of targets.

[0031] 3. Molecular docking analysis: Molecular docking technology was used to analyze the affinity between key components of Yuling Ointment and its core target proteins. Six target sites with high degree values ​​in the PPI network were selected as the core targets in this study: GAPDH, AKT1, IL6, TNF, STAT3, and ALB. The basic substances 6-Paradol, Eupafolin, Chrysophanic Acid, Poricoic Acid B, Ziyuglycoside I, and Atractylenolide III were paired with these core targets for docking. The binding stability between the basic substances and the core targets was evaluated. Based on the binding energy, the lower the docking binding energy, the higher the binding interaction and matching degree between the target protein and the compound. Molecular docking scores are shown below. Figure 8 The results showed that the binding energies of the 12 docking groups were less than -5.0 kcal·mol⁻¹. -1 This indicates that these compounds have good binding ability with the core target site; the binding energy is less than -7.0 kcal·mol⁻¹. -1Two groups showed positive binding activity, indicating strong binding activity. This suggests that Yuling Ointment improves Qi deficiency and anemia through multiple pathways and targets, a result that is largely consistent with previous network pharmacology findings.

[0032] Simultaneously, the six docking results with the lowest binding energy will be visualized to obtain the binding pattern diagrams of each compound with the core target protein, see [link to visualization]. Figure 9 The components identified are, respectively, *Gynostemma pentaphyllum*-ALB, *Gynostemma pentaphyllum*-GAPDH, *Emodin*-STAT3, *Gynostemma pentaphyllum*-STAT3, *Gingerol*-STAT3, and *Gingerol*-AKT1. As shown in the figure, besides some surrounding hydrophobic interactions, *Gynostemma pentaphyllum* forms hydrogen bonds with Lys212, Glu79, Arg80, Glu24, and Asp158; *Emodin* with Asp97 and Glu96; and 6-Paradol with Glu96, Met99, and Ala230. These interactions achieve strong binding to the core targets, effectively promoting the formation of stable complexes between proteins and small molecules, and exhibiting a strong correlation with the targets. Therefore, it can be inferred that *Gingerol*, *Gynostemma pentaphyllum*, and *Emodin* are the key components for the efficacy of Yuling Ointment, and ALB, GAPDH, STAT3, and AKT1 are the key targets for the efficacy of Yuling Ointment.

[0033] To further ensure that Yuling Paste exerts its effects of replenishing qi and nourishing blood, and to ensure the stability of the finished product quality, the appropriate steaming time for Yuling Paste is estimated by combining the results of non-targeted metabolomics and network pharmacology. Figure 10 The graph shows the changes in six key components during the steaming process. It indicates that the levels of gingerol, zedoaria, and rhein consistently increased in groups Y0, Y5, Y10, and Y20, reaching their maximum in group Y20, and then decreased in group Y30. Therefore, it can be inferred that the optimal steaming time for Yuling Paste should be controlled at 20 hours, at which point its qi-tonifying and blood-nourishing effects are best.

[0034] Example 2 S1. Soak rice in 10 times its weight of water for 2 hours, then filter to obtain rice water. Soak longan pulp in the rice water, controlling the soaking temperature at 40℃ and the soaking time at 3 hours. S2. Chop the soaked longan pulp, mix it with American ginseng powder and white sugar in a mass ratio of 10:1:1, place it in a porcelain bowl, cover it with gauze, and steam it in stages with controlled temperature. Steam it at a low temperature of 78℃ for 12 hours, and then steam it at a high temperature of 95℃ for 8 hours.

[0035] S3. After steaming, the product is moderately dried in an air source heat pump dryer at a temperature of 45°C for 2 hours. S4. After drying, let it cool to room temperature, then package it to obtain the finished Yu Ling Ointment.

[0036] Example 3 The Yuling ointment was prepared according to the method of Example 2, except that in step S1, when soaking the longan pulp, the soaking temperature was controlled at 30°C and the soaking time was 2 hours.

[0037] Example 4 The Yuling ointment was prepared according to the method of Example 2, except that in step S2, the segmented steaming method was as follows: first, it was steamed at a low temperature of 78°C for 18 hours, and then steamed at a high temperature of 95°C for 2 hours.

[0038] Example 5 The Yuling ointment was prepared according to the method of Example 2, except that in step S2, the segmented steaming method was as follows: first, it was steamed at a low temperature of 78°C for 2 hours, and then steamed at a high temperature of 95°C for 18 hours.

[0039] Example 6 The Yuling ointment was prepared according to the method of Example 2, except that in step S3, the drying temperature was controlled at 60°C and the drying time was controlled at 1 hour.

[0040] Comparative Example 1 The Yuling paste was prepared according to the method of Example 2, except that step S1 was omitted, the longan pulp was not soaked, and step S2 was performed directly.

[0041] Comparative Example 2 The Yuling ointment was prepared according to the method of Example 2, except that in step S2, the steaming process did not use segmented temperature control, but was steamed at 95°C for 20 hours.

[0042] Comparative Example 3 The Jade Spirit Cream was prepared according to the method in Example 2, except that step S3 was omitted, and the product was directly cooled to room temperature and packaged after steaming.

[0043] Component testing Yuling Paste uses longan pulp and American ginseng as its main raw materials. Although the Chinese Pharmacopoeia does not explicitly specify its indicator components, relevant literature indicates that total polysaccharides can serve as an indicator component for longan pulp. Measuring the total polysaccharide content helps assess the quality of longan pulp and ensure the overall quality of Yuling Paste. The main quality indicator components for American ginseng are saponins; measuring the total saponin content can effectively assess the quality of American ginseng, thus reflecting the quality of Yuling Paste. Simultaneously, through network pharmacology and molecular docking technology, three components—6-Paradol, Eupadol, and Chrysophanic Acid—were screened. These components play a potentially important role in Yuling Paste's qi-tonifying and blood-nourishing effects. Therefore, measuring the contents of these five components—total saponins, total polysaccharides, 6-Paradol, Eupadol, and Chrysophanic Acid—can not only control the quality of Yuling Paste's raw materials but also provide multi-dimensional evidence for its quality evaluation and efficacy mechanism research.

[0044] 1. Determination of total saponin content Preparation of reference solution: Accurately weigh 5 mg of ginsenoside Rb1 reference standard, place it in a 5 mL volumetric flask, dissolve it in methanol and dilute to volume to obtain a reference solution with a mass concentration of 1 mg / mL.

[0045] Preparation of the standard curve: Accurately pipette 100 μL, 300 μL, 500 μL, 700 μL, 900 μL, and 1100 μL of the reference solution into 10 mL stoppered test tubes, evaporate the solvent at low temperature, add 0.2 mL of 5% vanillin-glacial acetic acid solution, then add 0.8 mL of perchloric acid, shake well, and place in a 60 ℃ water bath for 30 min. Remove and immediately cool in cold water, accurately add 5.0 mL of glacial acetic acid, shake well, and let stand for 30 min. Use the corresponding reagents (vanillin-glacial acetic acid solution and perchloric acid solution) as blank controls, and measure the absorbance at a wavelength of 540 nm. Plot the standard curve with absorbance as the ordinate (Y) and mass concentration as the abscissa (X).

[0046] Determination of sample content: Accurately measure 0.1 mL of methanol into a 10 mL test tube, add 1 g of each Yuling ointment sample, sonicate for 30 min, cool to room temperature, and then dilute to the mark with methanol and shake well. Filter through a 0.45 µm microporous membrane, take 0.1 mL of the filtrate into a 10 mL test tube, evaporate the solvent at 80℃, add 0.2 mL of 5% vanillin-glacial acetic acid solution, then add 0.8 mL of perchloric acid, shake well, and place in a 60℃ water bath for 30 min. Remove and immediately cool in cold water, accurately add 5.0 mL of glacial acetic acid, shake well, and let stand for 30 min. Measure the absorbance at 540 nm. Calculate the total saponin content according to the standard curve.

[0047] 2. Determination of total polysaccharide content Preparation of reference solutions: Preparation of 6% phenol solution: Accurately weigh an appropriate amount of crystalline phenol, add a certain proportion of distilled water and mix thoroughly to prepare a 6% phenol solution; accurately weigh 100 mg of anhydrous glucose dried to constant weight, add water to a final volume of 100 mL to prepare a 1 mg / mL standard glucose solution. Take 0 μL, 10 μL, 20 μL, 40 μL, 60 μL, 80 μL, and 100 μL of the 1 mg / mL glucose standard solution respectively, add water to a final volume of 1000 μL, mix well, and obtain glucose solutions of 0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL.

[0048] Preparation of the standard curve: Pipette an appropriate amount of glucose standard solution into a 10 mL stoppered test tube, add 0.5 mL of 6% phenol solution, and shake the mixture thoroughly to allow it to react completely. Pipette 2.5 mL of concentrated sulfuric acid (let it flow naturally), mix quickly, and allow to stand in a fume hood for 30 min. Measure the absorbance at 490 nm. Plot a standard curve of glucose concentration versus absorbance with OD490 as the ordinate and concentration as the abscissa.

[0049] Determination of test sample content: Accurately pipette 1 g of each Yuling ointment sample into a 100 mL volumetric flask, dissolve and filter with distilled water to obtain the test solution. Take 1 mL of the test solution, mix with 0.5 mL of 6% phenol solution, add 2.5 mL of concentrated sulfuric acid and react thoroughly. After standing for 30 min, measure the absorbance at 490 nm. Calculate the total polysaccharide content according to the standard curve.

[0050] 3. Determination of gingerol content Preparation of reference solutions: Accurately weigh 10 mg of gingerol standard into a 10 mL volumetric flask, dissolve in methanol and dilute to the mark, then shake well to obtain a 1 mg / mL standard solution. Accurately measure 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the standard solution into 10 mL volumetric flasks, dilute to the mark with methanol, and shake well to obtain reference solutions with concentrations of 0.01 mg / mL, 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, and 0.1 mg / mL, respectively. Preparation of test solution: Weigh 0.5 g (accurate to 0.0001 g) of each Yuling ointment sample and place it in a 50 mL stoppered centrifuge tube. Add 20 mL of methanol, stopper tightly, weigh, and record the initial weight. Place the centrifuge tube in an ultrasonic cleaner for ultrasonic extraction for 30 min. Remove, cool, weigh again, replenish the lost weight with methanol, shake well, centrifuge at 5000 r / min for 10 min, collect the supernatant, filter through a 0.45 μm microporous membrane, and use the filtrate as the test solution.

[0051] Chromatographic conditions: Column: C18 column (250 mm × 4.6 mm, 5 μm); Mobile phase: methanol-water (65:35, V / V); Flow rate: 1.0 mL / min; Column temperature: 30 ℃; Injection volume: 10 μL; Detection wavelength: 280 nm.

[0052] Determination: Reference solutions of different concentrations were injected into the chromatographic column for analysis. A standard curve was plotted with the concentration of the reference solution on the x-axis and the peak area on the y-axis. The test solution was injected into the chromatographic column for analysis, and the peak area was recorded. Based on the peak area of ​​the test solution, the content of gingerol in the sample was calculated from the standard curve.

[0053] 4. Determination of quercetin content Preparation of reference solution: Accurately weigh an appropriate amount of quercetin reference standard and place it in a 10 mL volumetric flask. Dissolve and dilute to the mark with acetonitrile-water (80:20, V / V), and shake well to prepare a standard solution with a concentration of 1 mg / mL. Accurately pipette 0.05 mL, 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, and 0.8 mL of the standard solution into separate 10 mL volumetric flasks, dilute to the mark with acetonitrile-water (80:20, V / V), and shake well to obtain a series of reference solutions with concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, and 80 μg / mL, respectively.

[0054] Preparation of test solution: Weigh approximately 100 mg of each Yuling ointment sample and place it in a 50 mL volumetric flask. Add an appropriate amount of methanol, sonicate for 30 min, cool to room temperature, and then dilute to the mark. Shake well. Filter through a 0.45 µm microporous membrane and collect the filtrate for testing.

[0055] Chromatographic conditions: Column: C18 column (4.6 mm × 250 mm, 5 µm); Mobile phase: acetonitrile-0.1% formic acid solution (40:60, V / V); Flow rate: 1.0 mL / min; Detection wavelength: 350 nm; Column temperature: 25℃; Injection volume: 10 µL.

[0056] Determination: Reference solutions of different concentrations were injected into the chromatographic column for analysis. A standard curve was plotted with the concentration of the reference solution on the x-axis and the peak area on the y-axis. The test solution was injected into the chromatographic column for analysis, and the peak area was recorded. Based on the peak area of ​​the test solution, the content of quercetin in the sample was calculated from the standard curve.

[0057] 5. Determination of rhein content Preparation of reference solutions: Accurately weigh approximately 10 mg of rhein standard and place it in a 10 mL volumetric flask. Dissolve and dilute to the mark with methanol, then shake well to obtain a 1 mg / mL standard solution. Accurately pipette 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the standard solution into separate 10 mL volumetric flasks, dilute to the mark with methanol, and shake well to obtain reference solutions with concentrations of 0.01 mg / mL, 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, and 0.1 mg / mL.

[0058] Preparation of test solution: Accurately weigh approximately 1.0 g (accurate to 0.0001 g) of each Yuling ointment sample and place it in a 50 mL stoppered conical flask. Accurately add 25 mL of methanol-0.1% phosphoric acid solution (85:15, V / V), weigh the sample, sonicate (power 250 W, frequency 40 kHz) for 30 min, cool, weigh the sample again, replenish the lost weight with methanol-0.1% phosphoric acid solution, shake well, centrifuge at 8000 r / min for 10 min, take the supernatant, filter it through a 0.45 μm microporous membrane, and use the filtrate as the test solution.

[0059] Chromatographic conditions: Column: C18 column (4.6 mm × 250 mm, 5 µm); Mobile phase: methanol-0.1% phosphoric acid solution (85:15, V / V); Flow rate: 1.0 mL / min; Detection wavelength: 254 nm; Column temperature: room temperature; Injection volume: 10 µL.

[0060] Determination: Reference solutions of different concentrations were injected into the chromatographic column for analysis. A standard curve was plotted with the concentration of the reference solution on the x-axis and the peak area on the y-axis. The test solution was injected into the chromatographic column for analysis, and the peak area was recorded. Based on the peak area of ​​the test solution, the content of rhein in the sample was calculated from the standard curve.

[0061] 6. Measurement Results The results of the determination of the content of different components in various samples of Yuling Ointment are shown in Table 2 below: Table 2. Results of the determination of the content of each component in Yuling Ointment The results showed that the content of various components in the samples of Examples 2-6 was generally higher than that in Comparative Examples 1-3. Among them, the content of gingerol, quercetin, and rhein in Example 2 was relatively higher, and the content of total saponins and total polysaccharides in Example 3 was relatively higher. In order to verify whether the three index components screened in this experiment are superior to the traditional index components of the two raw materials, further verification is needed in conjunction with animal experiments. Specifically, Example 2 was used as treatment group A and Example 3 was used as treatment group B for verification.

[0062] Animal experiments verified: 1. Modeling and drug administration: Forty-eight healthy, clean-grade KM mice, approximately 7 weeks old and weighing (18±4) g, were selected, with half males and half females. During the experiment, the room temperature was controlled at (20.00±2.00)℃, with alternating lighting for 12 hours. After 3 days of acclimatization, the 48 mice were randomly divided into four groups of 12 mice each: normal group, model group, treatment group A, and treatment group B. For animal modeling, except for the normal control group, the other groups received subcutaneous injections of 2% acetylphenylhydrazine at 0.2 mg / kg on days 1 and 4. On days 4, 6, and 8 after the first acetylphenylhydrazine administration, 0.25 ml / 10 g of blood was excised from the orbital cavity. On days 5, 7, and 9 after the first acetylphenylhydrazine administration, 0.3 mg / kg of 1% cyclophosphamide was injected intraperitoneally. This treatment was continued for 10 days to establish the model. The normal group received an equal volume of physiological saline intraperitoneally at the same time points. Meanwhile, except for the normal group which was fed daily, the other groups were fasted for two days after a full meal on day 1, and then fed again on day 4 to establish a mouse model of qi and blood deficiency. Starting on day 5, treatment group A was administered the sample from Example 2, and treatment group B was administered the sample from Example 3, diluted with water at a concentration of 1 g / mL, and administered by gavage at 0.02 ml / g once daily for 10 consecutive days. The normal group and model group were administered the corresponding doses of distilled water. The mice's mental state, food and water intake, as well as the smoothness of their fur, and the color of their ears, tails, and claws were monitored daily.

[0063] 2. Measurement of mouse body weight and thymus index: After the last administration (example sample), the weight of the mice was weighed and recorded. The mice were euthanized by dislocation of the neck. The thymus and pancreas of the mice were taken, rinsed with physiological saline, drained with filter paper, weighed with an electronic balance, and the thymus index was calculated.

[0064] 3. Peripheral blood count: The number of red blood cells (RBCs) and hemoglobin (HGBs) in the peripheral blood of mice in each group was detected using a blood analyzer.

[0065] 4. ELISA method for content detection: Serum samples from each group were collected and the levels of ALB, GAPDH, STAT3, and AKT1 in the serum were measured strictly according to the following procedures: ① Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) kit, ② Mouse albumin (ALB) ELISA kit, ③ Mouse signal transduction activator of transcription 3 (STAT3) detection kit, and ④ Mouse AKT1 protein (AKT1) ELISA kit.

[0066] 5. Animal experiment results: Table 3. Results of mouse body weight, thymus index, RBC, and HGB measurements in each group. During the experiment, the normal control group mice had shiny, strong fur, agile movements, normal appetite, and normal light red paws and ears. Their food and water intake were normal. The model group mice had messy, dry fur, slow movements, reduced activity, huddled together, and lay down listlessly. Their paws and ears were lighter in color, and their water and food intake were reduced. The treatment group A showed significant improvement in fur color, activity level, appetite, ear and tail color compared to the model group. The treatment group B mice showed significantly better mental state than the model group, their fur became smooth again, their ear and paw colors returned to light red, and their food and water intake gradually returned to normal.

[0067] Compared with the normal group, the model group had significantly lower body weight and thymus index. P <0.05; Compared with the model group, the body weight and thymus index of mice in the treatment group were significantly increased, with the increase in treatment group A being more significant than that in treatment group B. P <0.05; compared with the model group, the number of RBCs and HGBs in the treatment group was significantly increased ( P <0.05 (see Table 3). Similarly, the increase in treatment group A was more significant than that in treatment group B. This indicates that the mouse model of Qi and Blood Deficiency Syndrome was successfully established, and that Yuling Ointment has a certain therapeutic effect on mice with Qi and Blood Deficiency Syndrome. Among them, the therapeutic effect of Yuling Ointment in Example 2 was better than that in Example 3.

[0068] Table 4. Detection results of ALB, GAPDH, STAT3, and AKT1 in each group Compared with the normal control group, the serum levels of ALB, GAPDH, STAT3 and AKT1 in the model group were significantly increased. P <0.05; Compared with the model group, the serum levels of ALB, GAPDH, STAT3 and AKT1 in the treatment group were significantly decreased ( P <0.05), see Table 4, where the decrease was more significant in treatment group A than in treatment group B. This indicates that "Yuling Ointment" may exert its therapeutic effect on mice with qi and blood deficiency by regulating the expression of ALB, GAPDH, STAT3, and AKT1.

[0069] The animal experiments described above clearly show that the sample in Example 2 has a significantly better therapeutic effect on mice with qi and blood deficiency than the sample in Example 3. This indicates that using 6-Paradol (gingerol), Eupafolin (a type of herb), and Chrysophanic Acid (rhein) as quality control components can better enhance the qi-tonifying and blood-nourishing efficacy of Yuling Ointment than using traditional total saponins and total polysaccharides, further confirming the reliability of network pharmacology prediction.

Claims

1. A quality control method for Yuling Ointment, characterized in that: The quality control components are the potential substances for replenishing qi and nourishing blood. The higher the content of the quality control components, the better the quality of Yuling Ointment. The potential substances for replenishing qi and nourishing blood are gingerol, zedoaria oleracea var. truncatum and rhein.

2. An optimized preparation process for Yuling Ointment, characterized in that: Includes the following steps: (1) A series of different Yuling ointment samples were obtained by using different preparation processes; (2) The contents of gingerol, zedoaria and rhein, potential substances for replenishing qi and nourishing blood, in different Yuling ointment samples were obtained by high performance liquid chromatography-mass spectrometry analysis. (3) Select the preparation process parameters of Yuling Ointment with high content of potential substances for replenishing qi and nourishing blood as the final preparation process parameters.

3. The preparation process optimization method according to claim 2, characterized in that: The different preparation processes refer to different preparation steps or / and different preparation process conditions or / and different Yuling Ointment formulas.

4. A method for preparing Jade Spirit Ointment, characterized in that: Includes the following steps: S1. Soak the longan pulp in water; S2. Chop the soaked longan pulp, mix it with American ginseng powder and white sugar in a mass ratio of 10:1:1, place it in a porcelain bowl, cover it with gauze, and steam it in stages with controlled temperature. Specifically, steam it at a low temperature of 78℃ for 10-14 hours, and then steam it at a high temperature of 95℃ for 6-10 hours. S3. After steaming, proceed with drying; S4. After drying, let it cool to room temperature, then package it to obtain the finished Yu Ling Ointment.

5. The preparation method according to claim 4, characterized in that: In step S1, the soaking temperature is 30-50℃ and the soaking time is 2-4 hours.

6. The preparation method according to claim 4, characterized in that: In step S2, the total steaming time is 20 hours.

7. The preparation method according to claim 4, characterized in that: In step S2, the mixture is first steamed at a low temperature of 78℃ for 12 hours, and then steamed at a high temperature of 95℃ for 8 hours.

8. The preparation method according to claim 4, characterized in that: In step S3, the drying temperature is 40℃-60℃ and the drying time is 1-3h.

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