Mass marker for preventing and treating kidney-yang deficiency of medicinal and edible formula composition as well as preparation method and application of mass marker

Abstract

The invention relates to the technical field of traditional Chinese medicine research, in particular to a quality marker for preventing and treating kidney-yang deficiency of a medicinal and edible formula composition as well as a preparation method and application of the quality marker. The medicinal and edible formula composition is prepared from the following raw materials in parts by weight: 1-2 parts of cortex cinnamomi, 6-8 parts of fructus momordicae, 2-4 parts of pericarpium citri reticulatae, 2-4 parts of rhizoma zingiberis, 8-10 parts of radix ginseng, 29-31 parts of radix astragali, 4-6 parts of radix glycyrrhizae preparata and 14-16 parts of poria cocos. The mass markers comprise [6]-isoshogaol, 3, 5, 6, 7, 8, 3 ', 4'-heptamethoxyl flavone, 1-dehydrogenation-[8]-zingiberendione, 6-methyl zingiberendiol, nobiletin, peterolide and the like. The ginseng formula for regulating the middle warmer is reasonably added and subtracted, so that the traditional Chinese medicine composition is applied to prevention and treatment of the kidney-yang deficiency syndrome, and a new application prospect is given to the classic famous formula. Meanwhile, on the basis of spectrum-effect association research, a quality marker group associated with the kidney-yang deficiency effect is screened and established for the first time, and scientific, accurate, stable and controllable product quality of the medicinal and edible formula composition can be achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine research, and particularly to a quality marker for preventing and treating kidney yang deficiency by a medicine and food homologous formula composition, and its preparation method and application. Background Art

[0002] Kidney yang deficiency syndrome is one of the common syndromes in traditional Chinese medicine, and its clinical manifestations include soreness and weakness of the waist and knees, fear of cold and cold limbs, mental listlessness, sexual function decline, clear and long urine, etc. With the acceleration of the modern life rhythm, the increase in pressure and bad living habits, the prevalence rate of kidney yang deficiency syndrome shows an upward trend, seriously affecting the quality of life. At present, for the prevention and treatment of kidney yang deficiency, Western medicine mainly uses hormone replacement therapy, but there are certain side effects and risks in its long-term application. Traditional Chinese medicine has accumulated rich experience in the prevention and treatment of kidney yang deficiency. Among them, warming and tonifying kidney yang is the fundamental treatment method, and classic prescriptions such as Jinkui Shenqi Pills and Yougui Pills are widely used with definite curative effects. Renshen Lizhong Decoction originated from a traditional Chinese medicine decoction in "Wa Tai" Volume 6, which cited "Shan Fan Fang". It is mainly composed of ingredients such as ginseng, dried ginger, licorice, poria, cinnamon, tangerine peel, astragalus membranaceus, etc. Its core efficacy lies in warming the middle-jiao and dispelling cold, replenishing qi and strengthening the spleen. Traditionally, it is mainly used to treat cholera with continuous diarrhea and the abdominal discomfort caused by kidney qi deficiency. According to the theory of traditional Chinese medicine, "the spleen is the foundation of acquired constitution, and the kidney is the foundation of congenital constitution". They support each other physiologically and affect each other pathologically. Insufficient spleen yang can, over time, affect kidney yang, leading to spleen-kidney yang deficiency; conversely, warming and tonifying kidney yang also helps to strengthen the transportation and transformation of spleen yang. Therefore, based on the theories of "tonifying fire to generate earth" and "strengthening earth to control water", the representative prescription of warming the middle-jiao and strengthening the spleen, Renshen Lizhong Decoction, is modified and applied to the prevention and treatment of spleen-kidney yang deficiency syndrome, especially kidney yang deficiency syndrome, which has theoretical basis and development potential and good application prospects.

[0003] Developing traditional Chinese medicine prescriptions into medicine and food homologous products suitable for modern people to use, with clear quality standards and substance bases of efficacy, faces many challenges: the quality markers (Q-Markers) are not clear. At present, the quality control of prescriptions such as Renshen Lizhong Decoction is mostly limited to the determination of the content of individual index components (such as ginsenosides and glycyrrhizic acid) in single herbs, lacking a comprehensive quality marker group of multiple components and multiple targets that can overall reflect the efficacy characteristics of this compound prescription in "warming and tonifying the spleen and kidney (especially emphasizing kidney yang)". This makes it difficult to scientifically guarantee the product quality uniformity, stability and efficacy correlation, restricting its industrialization and modernization development. The adaptability of medicine and food homology is insufficient. The taste of traditional decoctions is not good, and direct application may affect its acceptance as a daily product. It is necessary to scientifically optimize the formula according to the attribute of "medicine and food homology" to improve its palatability while maintaining the core efficacy.

[0004] While traditional Chinese medicine theory supports the use of Renshen Lizhong Decoction for treating kidney yang deficiency, systematic modern pharmacological studies are lacking to elucidate its specific mechanism of action and the spectrum-effect relationship between key quality markers and its efficacy in preventing and treating kidney yang deficiency. Therefore, there is an urgent need to rationally modify and combine this classic formula, Renshen Lizhong Decoction, following the guidelines of the food-medicine homology catalogue, to identify its core quality markers for preventing and treating kidney yang deficiency, ultimately forming a reliable and effective food-medicine homology formula. Summary of the Invention

[0005] To address the above shortcomings, this invention provides a quality marker for the effect of a food-medicine homology formula in preventing and treating kidney yang deficiency. It modifies Renshen Lizhong Tang, a representative formula for warming the middle and strengthening the spleen, and identifies its core quality marker for preventing and treating kidney yang deficiency. This formula shows promising application prospects in the prevention and treatment of kidney yang deficiency. The specific technical solution is as follows: A quality marker for the effect of a food-medicine homology formula composition in preventing and treating kidney yang deficiency, wherein the food-medicine homology formula composition is composed of the following raw materials in parts by weight: 1-2 parts cinnamon, 6-8 parts monk fruit, 2-4 parts dried tangerine peel, 2-4 parts dried ginger, 8-10 parts ginseng, 29-31 parts astragalus, 4-6 parts prepared licorice root, and 14-16 parts poria cocos; the quality marker includes: [6]-isoshogaol, 3,5,6,7,8,3′,4′-heptamethoxy Flavonoids, 1-dehydro-[8]-gingerdione, 6-methylgingeradiol, hesperidin, perolin, 1-(3,4-dimethoxyphenyl)-5-hydroxydecane-3-one, dihydro-[6]-gingerol, isotrichol, isoglycyrrhizin, 3-hydroxy-9,10-dimethoxystigmine, astragalus isoflavone, hesperidin, naringenin, glycyrrhizin P, glycyrrhizin R, glycyrrhizin, pine resin, Ceylon cinnamon, 8-dehydroshogaol.

[0006] On the other hand, the present invention also provides a method for preparing a quality marker for the effect of the above-mentioned food-medicine homology formula composition in preventing and treating kidney yang deficiency, comprising the following steps: (1) Take the raw medicinal materials, add water and decoct 2-3 times, each time for 1-2 hours. After decoction, concentrate to obtain concentrated liquid; (2) Separate the concentrate by passing it through a macroporous resin column, wash it with water until the eluent is nearly colorless, and then elute it with gradients of ethanol-water solutions with concentrations of 25-35%, 45-55%, and 70-80%, respectively, and collect the ethanol eluents of different concentrations in segments. (3) The eluent obtained in step (2) is subjected to high-resolution liquid chromatography-mass spectrometry for component identification. The eluents that can identify 20 chemical components, including [6]-isoshogaol, 3,5,6,7,8,3′,4′-heptamethoxyflavone, 1-dehydro-[8]-gingerdione, 6-methylgingeradiol, noriheptamethrin, peroroliline, 1-(3,4-dimethoxyphenyl)-5-hydroxydecane-3-one, dihydro-[6]-gingerol, isotrichol, isoglycyrrhizin, 3-hydroxy-9,10-dimethoxysphingane, astragalus isoflavone, hesperidin, naringenin, glycyrrhizin P, glycyrrhizin R, glycyrrhizin, pinocembrin, Ceylon cinnamon, and 8-dehydroshogaol, are concentrated and dried to obtain the quality marker of the formulation composition for preventing and treating kidney yang deficiency.

[0007] Preferably, in the preparation method of the quality marker of the effect of the above-mentioned food-medicine homology formula composition in preventing and treating kidney yang deficiency, in step (3), take 100 μL of the eluent, put it in a 1.5 mL centrifuge tube, add 300 μL of methanol, vortex and mix for 1 min; centrifuge at 4℃ and 12000 rpm for 5-10 min; take 100 μL of the solution, add 100 μL of ultrapure water, mix well, and put it in a sample bottle for testing.

[0008] Preferably, in the method for preparing the quality marker of the effect of the above-mentioned food-medicine homology formula composition in preventing and treating kidney yang deficiency, in step (3), the chromatographic conditions are: ACQUITY UPLC HSS T3 column, column temperature is 40°C, injection volume is 5.0-8.0 µL, flow rate is 0.3 mL / min; the mobile phase consists of the following components: phase A: water + 0.1% formic acid, phase B: acetonitrile; the elution gradient of the column is: 0-1 min, phase B volume fraction is 2%; 1-14 min, phase B volume fraction is 2-30%; 14-25 min, phase B volume fraction is 30-100%; 25-28 min, phase B volume fraction is 100%; 28-28.1 min, phase B volume fraction is 100-2%; 28.1-29.5 min, phase B volume fraction is 2%.

[0009] Preferably, in the method for preparing the quality marker of the effect of the above-mentioned food-medicine homology formula composition in preventing and treating kidney yang deficiency, the mass spectrometry conditions are as follows: the ion source voltages for positive and negative ions are 3.7 kV and 3.5 kV, respectively; the capillary heating temperature is 320°C; the sheath gas pressure is 30 psi, and the auxiliary gas pressure is 10 psi; the solvent heating and evaporation temperature is 300°C; both the sheath gas and the auxiliary gas are nitrogen; the collision gas is nitrogen, and the pressure is 1.5 mTorr; and Full scan / dd-MS is used. 2 Data acquisition mode, Fullscan parameters: resolution 70000, automatic gain control target 1×10⁻⁶.6 Maximum isolation time 50 ms, mass-to-charge ratio scan range 100-1500; dd-MS 2 Parameters: Resolution 17500, Automatic Gain Control target 1×10 5 The maximum isolation time is 50 ms.

[0010] Preferably, in the preparation method of the quality marker of the effect of the above-mentioned food-medicine homology formula composition in preventing and treating kidney yang deficiency, in step (2), the mixture is eluted by gradient elution with ethanol-water solution of mass concentrations of 30%, 50%, and 75%, respectively.

[0011] On the other hand, the present invention also provides an analytical method for the quality markers of the above-mentioned food-medicine homology formula composition for preventing and treating kidney yang deficiency, comprising the following steps: S1. The effective components of the food-medicine homology formula composition were analyzed using the traditional Chinese medicine systems pharmacology database and analysis platform. The effective targets of the food-medicine homology formula composition were then sequentially imported into the SwissTargetPrediction database and screened using Probability>0.1 as the screening condition. S2. Using the GeneCards database with "kidney function; endocrine disorder; metabolic syndrome" as search keywords, relevant targets for kidney yang deficiency-related diseases were obtained; Venn was used to draw a Venn diagram of the effective components of the food-medicine homology formula composition and the targets related to kidney yang deficiency diseases. The intersection targets obtained are the target genes of the effective components of the food-medicine homology formula composition for kidney yang deficiency diseases. S3. Upload the genes that act on the common target of the drug and the disease to the STRING database to draw the PPI network diagram, and perform topological parameter analysis in Cytoscape 3.10 software; obtain the "traditional Chinese medicine-component-disease-target" interaction network diagram between the drug-food homology formula composition and kidney yang deficiency-related diseases, and obtain the top 20 effective components; S4. The collected co-acting gene targets were analyzed using the DAVAD database. With P < 0.05, GO function and KEGG signaling pathway enrichment analysis were performed. The top-ranked data were selected and visualized on the MicroBioinformatics platform.

[0012] On the other hand, the present invention also provides the application of the quality markers of the above-mentioned food-medicine homology formula composition for preventing and treating kidney yang deficiency in the preparation of drugs for preventing and treating kidney yang deficiency.

[0013] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention rationally modifies the traditional "Ginseng Lizhong Decoction," primarily used for warming the middle jiao, dispelling cold, tonifying qi, and strengthening the spleen, expanding its application to the prevention and treatment of kidney yang deficiency syndrome, thus giving this classic formula new application prospects. Simultaneously, for medicinal and edible homologous formulations with kidney yang deficiency prevention and treatment effects, based on "spectrum-efficacy" correlation studies, a group of 20 chemical components constituting kidney yang deficiency efficacy quality markers was screened and identified for the first time. A comprehensive quality marker system precisely correlated with efficacy was established, which is conducive to achieving scientific, precise, stable, and controllable product quality of medicinal and edible homologous formulations.

[0014] 2. In the preparation method of the quality marker of the medicinal and edible homology formula composition for preventing and treating kidney yang deficiency of the present invention, by optimizing the chromatographic conditions and high-resolution mass spectrometry parameters in the component identification method, 20 chemical components can be identified simultaneously to obtain the quality marker of the medicinal and edible homology formula composition.

[0015] 3. The quality markers of the medicinal and edible homologous formulation composition of this invention for preventing and treating kidney yang deficiency significantly improved typical symptoms such as cold intolerance, lethargy, and brittle hair in a hydrocortisone-induced kidney yang deficiency mouse model; effectively increased body weight and exercise endurance (significantly prolonged fall time and distance in the rotarod test); and regulated abnormal organ coefficients, protecting the kidneys, spleen, thymus, and adrenal glands from damage. It significantly increased the decreased levels of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) in the serum of kidney yang deficiency mice, alleviated pathological damage to adrenal tissue, and effectively inhibited excessive apoptosis of adrenal cells, demonstrating a protective effect on endocrine glands at the organ and cellular levels. Simultaneously, it improved the survival rate of TM3 testicular interstitial cells and promoted testosterone secretion; exhibiting good preventive and therapeutic effects against kidney yang deficiency and anti-fatigue efficacy. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a Venn diagram of the effective target of the food-medicine homology formula composition in Example 2 of the present invention - the target related to kidney yang deficiency; Figure 2This is a network diagram of the "Chinese medicine-ingredient-disease-target" of the Chinese medicine-food homology formula composition in Example 2 of the present invention; the orange octagons represent kidney yang deficiency and related diseases, the green squares represent the constituent drugs of the modified formula of Renshenli, the blue circles represent the effective ingredients of the modified formula of Renshenli, the pink represents the target, and the small circle composed of the blue circles on the right is the top 10 effective ingredients with the highest Degree value. Figure 3 The effect of the food-medicine homology formula composition in Example 2 of this invention on kidney yang deficiency: (a) bubble chart of GO functional enrichment analysis, (b) bubble chart of KEGG pathway enrichment analysis; Figure 4 The detection peak diagrams of the quality markers of the food-medicine homology formula composition in Example 3 of the present invention are as follows: (a) detection peak diagram in positive ion mode; (b) detection peak diagram in negative ion mode. Figure 5 This describes the effects of each treatment group on the body weight of mice with kidney-yang deficiency in Experiment 1 of this invention. Figure 6 The following are the pathological changes in the kidney tissue of mice with kidney yang deficiency in each treatment group in Experiment 1 of this invention: (a) blank control group; (b) model group; (c) Jin Kui Shen Qi Wan group; (d) medium dose group of the composition; (e) medium dose group of quality markers; Figure 7 The pathological changes of spleen tissue in mice with kidney-yang deficiency in each treatment group in Experiment 1 of this invention are as follows: (a) blank control group; (b) model group; (c) Jin Kui Shen Qi Wan group; (d) medium dose group of the composition; (e) medium dose group of quality markers; Figure 8 The pathological changes of ovarian tissue in mice with kidney-yang deficiency in each treatment group in Experiment 1 of this invention are as follows: (a) blank control group; (b) model group; (c) Jin Kui Shen Qi Wan group; (d) medium dose group of the composition; (e) medium dose group of quality markers; Figure 9 The pathological changes of testicular tissue in mice with kidney-yang deficiency in each treatment group in Experiment 1 of this invention are as follows: (a) blank control group; (b) model group; (c) Jin Kui Shen Qi Wan group; (d) medium dose group of the composition; (e) medium dose group of quality marker.

[0018] Figure 4Legend in the figure: 1 is 8-dehydroshogaol, 2 is peroroliline, 3 is naringenin, 4 is hesperidin, 5 is glycyrrhizin, 6 is Ceylon cinnamyl, 7 isotrifolin, 8 isoglycyrrhizin, 9 is 3-hydroxy-9,10-dimethoxysphingane, 10 is astragalus isoflavone, 11 is 6-methylshogaol, 12 is 3,5,6,7,8,3′,4′-heptamethoxyflavone, 13 is norihesperidin, 14 is 1-(3,4-dimethoxyphenyl)-5-hydroxydecane-3-one, 15 is [6]-shogaol, 16 is dihydro-[6]-shogaol, 17 is glycyrrhizin R, 18 is glycyrrhizin P, 19 is pine resin, 20 is 1-dehydro-[8]-shogaol. Detailed Implementation

[0019] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods.

[0020] The raw medicinal materials used in the following examples and comparative examples are as follows: Cinnamon is the bark of the cinnamon tree (Cinnamomum cassia Presl), a plant belonging to the Lauraceae family.

[0021] Monk fruit is the fruit of the cucurbitaceous plant *Siraitia grosvenorii* (Swingle) C. Jeffrey ex AM Lu et ZY Zhang.

[0022] Dried tangerine peel is the peel of Citrus reticulata Blanco and its cultivated varieties, belonging to the Rutaceae family.

[0023] Dried ginger is the rhizome of the ginger plant Zingiber officinale Rosc. (family Zingiberaceae).

[0024] Ginseng refers to the root and rhizome of Panax ginseng CA Mey., a plant belonging to the Araliaceae family.

[0025] Astragalus is the root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge., both belonging to the Fabaceae family.

[0026] Licorice is the root and rhizome of Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., or Glycyrrhiza glabra L., all belonging to the legume family.

[0027] Poria cocos is the sclerotium of the fungus Poria cocos (Schw.) Wolf, belonging to the Polyporaceae family.

[0028] All the above-mentioned Chinese medicinal materials were purchased from Guangxi Xianzhu Chinese Medicine Co., Ltd.

[0029] Example 1 A medicinal and edible formula composition, also known as the modified Renshen Lizhong Decoction, is a medicinal and edible formula composition with the effects of preventing and treating kidney yang deficiency and anti-fatigue, obtained by adding or subtracting ingredients based on the formula of Renshen Lizhong Decoction. It consists of the following ingredients: 1.5 parts cinnamon, 7 parts monk fruit, 3 parts dried tangerine peel, 3 parts dried ginger, 9 parts ginseng, 30 parts astragalus, 5 parts prepared licorice root, and 15 parts poria cocos.

[0030] A method for preparing a medicinal and edible formula composition includes: taking medicinal materials according to the formula ratio, adding 8 times the mass of water, decocting 3 times, 2 hours each time, filtering after decoction, concentrating the filtrate into a paste, drying, and obtaining powder, the weight of the powder being 15% of the weight of the original medicinal materials.

[0031] The above compositions can be used (but are not limited to) in the production of tea drinks, ointments, powders, tablets, granules, oral liquids, milk tea ingredient packets, candy tablets, energy bars and other product forms.

[0032] Example 2 A method for analyzing the quality markers of the effect of a medicinal and edible homologous formula composition (modified Renshen Lizhong formula) in preventing and treating kidney yang deficiency, wherein the formula of the medicinal and edible homologous formula composition is the same as in Example 1, and includes the following steps: S1. Acquisition of Active Ingredients and Targets: The active ingredients of the medicinal and edible formulation were finally determined by consulting the Pubchem, Reaxys, and TCMSP databases and literature. They were sequentially imported into the SwissTargetPrediction (http: / / www.swisstargetprediction.ch / ) database and screened for effective targets of the active ingredients with a probability > 0.1. The obtained effective targets were corrected by the Uniprot (https: / / www.uniprot.org / ) database and displayed as GeneSymbols. Finally, 92 active pharmaceutical ingredients were identified and 812 targets were predicted. S2. Acquisition of targets for kidney-yang deficiency: Using the GeneCards database (https: / / www.genecards.org / ), with "kidney function; endocrine disorder; metabolic sydrome" as search keywords, genes related to kidney-yang deficiency were searched and collected. After removing duplicates, a total of 2590 related disease targets were obtained. A Venn diagram was constructed using the Venn diagram tool (http: / / www.bioinformatics.com.cn / ) to plot the effective ingredient targets of the food-medicine homology formula composition against the targets related to kidney-yang deficiency (see...). Figure 1 The intersection targets obtained are 379 common targets of the effective components of the modified Ginseng Formula for Kidney Yang Deficiency. S3. Network Model Construction and Analysis: Using Cytoscape 3.10 software, a network diagram of the interaction between the mass spectrometry results of the modified Ginseng Lizhong formula and kidney-yang deficiency-related diseases was drawn (see...). Figure 2Then, the genes targeting the common effects of the drug and the disease were uploaded to the STRING database (https: / / stringdb.org / ) to draw a PPI network diagram. Topological parameter analysis was performed in Cytoscape 3.10 software. The results were sorted by the target gene degree value, and the core genes with the highest degree ranking were selected. The larger the degree value, the more important the role of the effective active ingredients of the ginseng compound in treating diseases related to kidney yang deficiency. The network is a complex network composed of protein-protein interactions, where the edges represent the interaction between the effective ingredients of the ginseng compound and kidney yang deficiency. The denser the lines, the deeper the association. The nodes represent proteins. The larger the node, the darker the color. The top 10 core targets were selected by the node degree value, specifically including PIK3CA, PIK3R1, PIK3CB, STAT3, SRC, HRAS, PIK3CD, AKT1, GRB2, and HSP90AA1, and the top 20 effective ingredients were obtained (see Table 1). Table 1. Top 20 active ingredients in food-medicine homology formulations (by Degree value)

[0033] S4. The collected co-acting gene targets were analyzed using the DAVAD database. For each target, P < 0.05, GO function and KEGG signaling pathway enrichment analysis were performed. The top 20 data points were selected and visualized on the MicroBio platform (see [link to analysis]). Figure 4 GO enrichment analysis showed that the active ingredients of the modified Ginseng Formula participate in the biological processes (BP) regulating kidney yang deficiency, mainly including protein phosphorylation, response to exogenous stimuli, negative regulation of apoptosis, and the insulin-like growth factor receptor signaling pathway. Cellular components (CC) involved in regulating kidney yang deficiency mainly include the plasma membrane, cytoplasmic matrix, and membrane rafts. Molecular functions (MF) involved in regulating kidney yang deficiency mainly include protein kinase activity, ATP binding, protein tyrosine kinase activity, and protein serine / threonine kinase activity. KEGG pathway enrichment analysis revealed signaling pathways including PI3K / AKT, MAPK, and endocrine resistance.

[0034] Example 3 A quality marker for the effect of a food-medicine homology formula composition in preventing and treating kidney yang deficiency, the food-medicine homology formula composition is composed of the following raw materials in parts by weight: 1.5 parts cinnamon, 7 parts monk fruit, 3 parts dried tangerine peel, 3 parts dried ginger, 9 parts ginseng, 30 parts astragalus, 5 parts prepared licorice, 15 parts poria cocos; the quality marker includes: [6]-isoshogaol, 3,5,6,7,8,3′,4′-heptamethoxyflavone, 1-de- Hydrogen-[8]-gingerone, 6-methylgingerol, hesperidin, perolin, 1-(3,4-dimethoxyphenyl)-5-hydroxydecane-3-one, dihydro-[6]-gingerol, isotrichol, isoglycyrrhizin, 3-hydroxy-9,10-dimethoxystigmine, astragalus isoflavone, hesperidin, naringenin, glycyrrhizin P, glycyrrhizin R, glycyrrhizin, pine resin, Ceylon cinnamon, 8-dehydroshogaol.

[0035] A method for preparing a quality marker for the effect of a food-medicine homology formula composition in preventing and treating kidney yang deficiency includes the following steps: (1) Weigh the medicinal materials according to the ratio, add 8 times the amount of water and decoct 3 times, 2 hours each time. After decoction, concentrate to a concentrated liquid with a mass ratio of 3 times that of the original medicinal materials. (2) Separate the concentrate by passing it through a D101 macroporous resin column, wash it with water until the eluent is nearly colorless, and then elute it with an ethanol-water gradient with a mass ratio of 30:70, 50:50 and 75:25 respectively. The amount of ethanol-water used is 5 times the mass of the original drug material. Collect the ethanol eluent of different concentrations in segments. (3) The eluent obtained in step (2) was subjected to high-resolution liquid chromatography-mass spectrometry for component identification. The eluents that could be identified were [6]-isoshogaol, 3,5,6,7,8,3′,4′-heptamethoxyflavone, 1-dehydro-[8]-gingerdione, 6-methylgingeradiol, noriheptamethrin, peroroliline, 1-(3,4-dimethoxyphenyl)-5-hydroxydecane-3-one, and dihydro-[6]- The eluent of 20 chemical components, including gingerol, isotrichol, isoglycyrrhizin, 3-hydroxy-9,10-dimethoxystigmine, astragalus isoflavone, hesperidin, naringenin, glycyrrhizin P, glycyrrhizin R, glycyrrhizin, pinocembrin, Ceylon cinnamyl oleate, and 8-dehydroshogaol, was concentrated and dried to obtain a dry powder, which is the quality marker for the composition's effect in preventing and treating kidney yang deficiency. The weight of the dry powder is 4.8% of the weight of the original medicinal material.

[0036] The high-resolution liquid chromatography-mass spectrometry (HPLC-MS) method for component identification includes the following steps: Take 100 μL of eluent, place it in a 1.5 mL centrifuge tube, add 300 μL of methanol, and vortex to mix for 1 min; centrifuge at 4 °C and 12000 rpm for 10 min, take 100 μL of the solution, add 100 μL of ultrapure water, mix well, place it in a sample vial, and perform high-resolution liquid chromatography-mass spectrometry (HPLC-MS) for detection; Chromatographic conditions: Chromatographic separation was performed using a Vanquish Flex UHPLC (Thermo Fisher Scientific Inc., Waltham, MA, USA) ultra-high performance liquid chromatography system equipped with an ACQUITY UPLC HSS T3 column (2.1 mm (inner diameter) × 100 mm (length), 1.8 μm (particle size)) (Waters Corp., Milford, MA, USA). The mobile phase consisted of phase A (water + 0.1% formic acid) and phase B (acetonitrile), with the elution gradient shown in Table 2. The flow rate was 0.3 mL / min, the column temperature was 40°C, and the injection volume was 6.0 µL.

[0037] Table 2 Elution gradient table

[0038] Mass spectrometry conditions: Mass spectrometry analysis was performed using a quadrupole orbital ion trap mass spectrometer (Q Exactive, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a thermospray ion source. The ion source voltages for positive and negative ions were 3.7 kV and 3.5 kV, respectively; the capillary heating temperature was 320°C; the sheath gas pressure was 30 psi, and the auxiliary gas pressure was 10 psi; the solvent evaporation temperature was 300°C; both the sheath gas and auxiliary gas were nitrogen; the collision gas was nitrogen at a pressure of 1.5 mTorr. Full scan / dd-MS was used. 2 Data acquisition mode, Full scan parameters: resolution 70000, automatic gain control target 1×10⁻⁶. 6 Maximum isolation time 50 ms, mass-to-charge ratio scan range 100 – 1500; dd-MS 2 Parameters: Resolution 17500, Automatic Gain Control target 1×10 5 Maximum isolation time 50 ms, maximum scanning of secondary debris of 10 ions (dynamic exclusion), mass separation window 2, collision energy 10 V, 30 V, 60 V, intensity limit 1×10 5 .

[0039] Data analysis: The acquired mass spectrometry data were processed using Progenesis QI 3.0 software (Waters Corp., MA, USA). The steps were as follows: importing raw data, peak extraction, and deconvolution. Data on 20 quality marker compounds, including [6]-isoshogaol, 3,5,6,7,8,3′,4′-heptamethoxyflavone, 1-dehydro-[8]-gingerdione, 6-methylgingeradiol, noriheptacortine, peroroliline, 1-(3,4-dimethoxyphenyl)-5-hydroxydecane-3-one, dihydro-[6]-gingerol, isotrichol, isoglycyrrhizin, 3-hydroxy-9,10-dimethoxystigmine, astragalus isoflavone, hesperidin, naringenin, glycyrrhizin P, glycyrrhizin R, glycyrrhizin, pine resin, Ceylon cinnamon, and 8-dehydroshogaol, were compiled (constructed from literature, public databases, etc.). The identification results were analyzed by multidimensional comprehensive judgment based on the parent ion mass error, secondary fragment matching, isotope distribution, peak intensity, etc., to obtain the final results.

[0040] Figure 4 Table 3 shows the detection peaks of the 20 quality markers in positive ion mode and negative ion mode, respectively.

[0041] Table 3 shows the identification results of 20 quality indicators in this embodiment.

[0042] The results show that the high-resolution liquid chromatography-mass spectrometry method in this embodiment can identify 20 quality markers: (1) 8-dehydroshogaol, (2) peroroliline, (3) naringenin, (4) hesperidin, (5) glycyrrhizin, (6) Ceylon cinnamicin, (7) isotrichol, (8) isoglycyrrhizin lactone, (9) 3-hydroxy-9,10-dimethoxysphingane, (10) astragaloside, (11) 6 -Methyl gingerol, (12)3,5,6,7,8,3′,4′-heptamethoxyflavone, (13)hesperidin, (14)1-(3,4-dimethoxyphenyl)-5-hydroxydecane-3-one, (15)[6]-isoshogaol, (16)dihydro-[6]-gingerol, (17) glycyrrhizin R, (18) glycyrrhizin P, (19) pine resin, (20)1-dehydro-[8]-gingerdione.

[0043] Comparative Example 1 This comparative example provides a high-resolution liquid chromatography-mass spectrometry (LC-MS) method for component identification, which differs from the high-performance liquid chromatography (HPLC) method for component identification in Example 3 in that the mobile phase consists of the following components: Phase A: water, Phase B: methanol, and the elution gradient is shown in Table 4.

[0044] Table 4 Elution gradient of Comparative Example 1

[0045] The component identification method of this comparative example can only identify 12 quality markers: (1) 8-dehydrogingerenol, (3) naringenin, (4) hesperetin, (5) liquiritin, (6) cinnamtannin B1, (10) astragaloisoflavan, (11) 6-methylgingediol, (12) 3,5,6,7,8,3′,4′-heptamethoxyflavone, (13) nobiletin, (15) [6]-isogingerenol, (16) dihydro-[6]-gingerol, (19) pachymic acid.

[0046] Comparative Example 2 This comparative example provides a method for identifying components by high-resolution liquid chromatography-mass spectrometry. The difference from the method for identifying components by high-performance liquid chromatography in Example 3 is that the mobile phase consists of the following: Phase A: water + 0.1% formic acid, Phase B: methanol, and the elution gradient is shown in Table 5.

[0047] Table 5 Elution Gradient of Comparative Example 2

[0048] The component identification method of this comparative example can only identify 15 quality markers: (1) 8-dehydrogingerenol, (2) perlolyrine, (3) naringenin, (5) liquiritin, (6) cinnamtannin B1, (7) isotrifolin, (9) 3-hydroxy-9,10-dimethoxypterocarpan, (10) astragaloisoflavan, (11) 6-methylgingediol, (12) 3,5,6,7,8,3′,4′-heptamethoxyflavone, (13) nobiletin, (14) 1-(3,4-dimethoxyphenyl)-5-hydroxydecane-3-one, (18) glycyrrhetol P, (19) pachymic acid, (20) 1-dehydro-[8]-gingerdione.

[0049] Experimental Example 1 Animal Pharmacological Verification Test on the Prevention and Treatment of Kidney Yang Deficiency by Quality Markers of the Medicine-Food Homology Formula Composition 1. Materials 1.1 Animals SPF Kunming white mice, half male and half female, 6 - 8 weeks old, purchased from Guangxi Medical University, with the animal production license number SCXK Gui 2020-0003, and raised in the animal experiment barrier system of Guangxi Academy of Chinese Medicine. The experimental unit's usage license number is SYXK Gui 2022-0001. The laboratory temperature is 23 ± 1°C, and the humidity is 60 ± 3%. The animal experiment was approved by the Experimental Animal Management and Ethics Committee of Guangxi Academy of Chinese Medicine (approval number 2025062570).

[0050] 1.2 Drugs and Reagents The food-medicine homology formula composition was prepared in Example 1, and the quality marker for the effect of the food-medicine homology formula composition in preventing and treating kidney yang deficiency was prepared in Example 3. Jin Kui Shen Qi Wan was purchased from Beijing Tongrentang Technology Development Co., Ltd. Pharmaceutical Factory (batch number Z11020054); hydrocortisone injection was purchased from Shanxi Ruicheng Kelong Veterinary Drug Co., Ltd. (batch number 040191269); Cyclic adenosine monophosphate (cAMP) assay kit (batch number WK102LHLN5095), Cyclic guanosine monophosphate (cGMP) assay kit (batch number WA03H8VVL4921), mouse corticotropin-releasing hormone (CRH) assay kit (batch numbers WP078840P6313, WK09PX0682280), and mouse adrenocorticotropic hormone (CRH) assay kit were also prepared. Hormone (ACTH) detection kit (batch number WK0708JD68684) was purchased from Wuhan Yilairuit Biotechnology Co., Ltd.; 4% paraformaldehyde (batch number 2500050007) and 10% neutral buffered formalin fixative (batch number 2500030001) were purchased from Beijing Solarbio Science & Technology Co., Ltd.; one-step TUNEL apoptosis detection kit (batch numbers A259D250702 and A259B250715) were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; and anti-fluorescence quenching mounting medium (containing DAPI) (batch number 2500050003) was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0051] 1.3 Instruments Electronic balance (model FA2004B, YP20001) Shanghai Yueping Scientific Instruments (Suzhou) Manufacturing Co., Ltd.; Small animal rotundus fatigue tester (model XR-6D rat rotundus fatigue tester) Shanghai Xinruan Information Technology Co., Ltd.; Biological microscope (model ECLIPSE Ci-L plus) Nanjing Nikon Jiangnan Optical Instrument Co., Ltd.; Full-wavelength microplate reader (model Multiskan SkyHigh) Singapore Life Technology Holdings Pte Ltd.; Infrared oven (model W2002-Z) Airmate Electric Shenzhen Co., Ltd.; Electric ceramic stove (model LC-EA3S) Guangdong Shunde Zhongchen Electric Co., Ltd.

[0052] 2. Methods 2.1 Grouping and Modeling and Drug Administration Healthy mice, half male and half female, were acclimatized for 3 days and then randomly divided into a blank control group, a model group, a positive group for Jin Kui Shen Qi Wan (1.55 g / kg), and low, medium, and high doses (2.39, 4.78, and 9.56 g / kg) of a medicinal and food homologous formula, as well as low, medium, and high doses (2.39, 4.78, and 9.56 g / kg) of a quality marker group, with 10 mice in each group. The dosages were based on the weight of the original medicinal materials. Except for the blank control group, the other groups were subcutaneously injected with hydrocortisone 25 mg / kg for 14 consecutive days to establish a kidney-yang deficiency model. Simultaneously with model establishment, each treatment group was administered the corresponding dose of hydrocortisone to distilled water via gavage for 23 consecutive days. The blank and model control groups were given an equal volume of distilled water.

[0053] 2.2 Detection of indicators for kidney yang deficiency syndrome 2.2.1 Morphological observation During the modeling and drug administration period, observe the activity level, mental state, excretion, coat color changes, diet and water intake of mice, and observe whether the model group mice exhibit symptoms similar to those of kidney yang deficiency, such as aversion to cold and preference for warmth, lethargy, messy and dull fur, huddling together and less movement, and emaciation.

[0054] 2.2.2 Weight Observe and record the activity level and mental state of the mice daily. Record the mice's weight every 6 days and plot a weight change curve.

[0055] 2.2.3 Rotary rod motion function experiment On day 22 of drug administration, the rotarod exercise experiment was formally conducted. The time and distance traveled by the mice on the rotarod were measured. The speed of the rotarod was adjusted to 40 revolutions / minute. Each experimental mouse was tested 3 times, and the average value was taken as the result. Before the formal experiment, the mice underwent exercise adaptation training for 20 minutes each day for 1 day at speeds of 10 revolutions / minute, 20 revolutions / minute, and 40 revolutions / minute respectively.

[0056] 2.3 Calculation of organ coefficients Mice were fasted for 12 hours after the last administration of the drug, but allowed free access to water. They were then weighed, sacrificed, and their kidneys, spleen, adrenal glands, and thymus were harvested. The corresponding organ coefficients were calculated as follows: Organ coefficient (%) = Organ weight (g) / Mouse body weight (g) × 100% 2.4 Observe the pathological changes in the kidney, spleen, ovary, and testis tissues. After the last administration, mice were euthanized by blood collection from the orbital cavity. The kidneys, spleen, ovaries, and testes were dissected, fixed, dehydrated, embedded, and sectioned in 10% neutral formalin fixative. Paraffin sections were prepared, stained with hematoxylin and eosin (HE), and the pathological changes of the kidney, spleen, ovary, and testes were observed under a microscope.

[0057] 2.5 Detection of adrenal tissue cell apoptosis Paraffin sections of adrenal gland tissue were prepared and mounted according to the TUNEL kit instructions. After mounting with anti-fluorescence quenching mounting solution, the sections were observed under a fluorescence microscope. The observed fluorescent cells were apoptotic cells, and the proportion of TUNEL-positive cells was counted.

[0058] 2.6 Detection of serum markers After mice were euthanized by blood collection from their orbital fossa, the supernatant serum was collected by centrifugation. The levels of cAMP and cGMP in the serum, as well as the expression levels of ACTH and CRH, were detected by ELISA.

[0059] 2.7 Statistical Analysis Statistical analysis was performed using IBM SPSS Statistics 31.0.0.0 software. All values ​​are expressed as follows: Mean squared (±S) values ​​are represented using Origin 2022 software. One-way ANOVA was used to compare sample means between groups.

[0060] 3. Results 3.1 Effects of the formulation composition on kidney yang deficiency syndrome Compared with the blank control group, the model group mice exhibited symptoms similar to those of kidney yang deficiency, such as arched backs, sparse and messy fur, huddling together with little movement, and lethargy; compared with the model group, the condition of mice in each treatment group was significantly improved. Figure 5 As shown, compared with the blank control group, the body weight of mice in the model group decreased significantly over time; compared with the model group, the body weight of mice in the Jin Kui Shen Qi Wan group and the low-dose group of the formula composition increased, while the body weight of mice in the medium and high-dose groups of the formula composition increased significantly. The body weight growth trend of the quality marker group and the composition group was similar.

[0061] As shown in Table 6, compared with the blank control group, the dwell time and range of mice in the model group on the rotundus were significantly reduced (P < 0.001); compared with the model group, the dwell time and range of mice in each drug administration group were also significantly increased (P < 0.001). Among them, the dwell time and range of mice in the composition group and the quality marker group increased with the increase of dosage. The medium dosage group of the composition group had a similar effect on improving motor function as the Jin Kui Shen Qi Wan group.

[0062] Table 6. Effects of the formulation composition on the motor ability of mice with kidney-yang deficiency.

[0063] 3.2 Effects of the formulation composition on organ coefficients in mice with kidney-yang deficiency As shown in Table 7, compared with the blank control group, the coefficients of kidney, thymus, and adrenal gland in the model group decreased (P < 0.001); compared with the model group, each drug administration group increased the coefficients of kidney, spleen, thymus, and adrenal gland in mice with kidney yang deficiency to varying degrees (P < 0.001), among which the dosage group of the composition was comparable to the effect of Jin Kui Shen Qi Wan.

[0064] Table 7. Effects of the formulation composition on organ coefficients in mice with kidney-yang deficiency.

[0065] 3.3 Effects of the formulation composition on the pathological changes of the kidney, spleen, ovary, and testis tissues in mice with kidney-yang deficiency like Figure 6 As shown, the kidneys of mice in the blank control group were intact, with clear glomerular and tubular structures and no obvious pathological changes. In contrast, the glomeruli of mice in the model group were atrophied, the tubular epithelial cells showed eosinophilic changes, and the tubules were dilated into a sac-like shape. Compared with the model group, the glomerular atrophy symptoms and tubular dilation were reduced in mice in each drug administration group.

[0066] like Figure 7 As shown, the spleen tissue structure of mice in the blank control group was clear, the boundary between the red and white pulp was clear, and the white pulp area was large, with no obvious pathological changes. In contrast, compared with the blank control group, the white pulp area of ​​mice in the model group was smaller, and the boundary between the red and white pulp was blurred. Compared with the model group, the white pulp area of ​​mice in each drug administration group increased to varying degrees, and the boundary between the red and white pulp was also relatively clear and improved.

[0067] like Figure 8 As shown, multiple corpus luteum tissues and follicles at different developmental stages were visible in the ovaries of mice in the blank control group, with no obvious pathological changes. In contrast, compared with the blank control group, the model group mice showed a reduction in oocytes at all stages and a reduction in the granulosa layer of follicular cells. Compared with the model group, the mice in each drug-treated group showed follicles at different developmental stages and an increase in the granulosa layer of follicular cells.

[0068] like Figure 9 As shown, the seminiferous tubules of mice in the blank control group showed intact structures and no obvious pathological changes. In contrast, the spermatogenic cell layers of mice in the model group were reduced and interstitial edema was observed. Compared with the model group, the spermatogenic cell layers of mice in each drug administration group were increased.

[0069] 3.4 Effects of the formulation composition on apoptosis of adrenal tissue cells in mice with kidney-yang deficiency TUNEL staining was performed on adrenal tissue sections of mice in each group to detect cell apoptosis. The results are detailed in Table 8. Compared with the blank control group, the proportion of TUNEL-positive cells in the model group was significantly increased, indicating increased cell apoptosis (P < 0.001). Compared with the model group, the proportion of TUNEL-positive cells in each treatment group was significantly decreased, indicating reduced cell apoptosis (P < 0.001).

[0070] Table 8. Effects of the formulated composition on adrenal cell apoptosis in mice with kidney-yang deficiency.

[0071] 3.5 Effects of the formulation composition on the expression levels of HPA axis markers in the serum of mice with kidney-yang deficiency The results are detailed in Table 9. Compared with the blank control group, the levels of ACTH and CRH in the serum of mice in the model group were significantly decreased (P < 0.001). Compared with the model group, the levels of ACTH in the serum of mice in each treatment group were significantly increased, and the levels of CRH were also significantly improved in the Jin Kui Shen Qi Wan group and the high-dose group of the combination (P < 0.001).

[0072] Table 9. Effects of the formulation composition on serum ACTH and CRH levels in mice with kidney-yang deficiency.

[0073] 3.6 Effects of the formulation composition on serum cAMP and cGMP levels in mice with kidney-yang deficiency The results are detailed in Table 10. Compared with the blank control group, the serum cAMP level in the model group mice was significantly decreased (P < 0.001); compared with the model group, the serum cAMP level in each treatment group mice was significantly improved (P < 0.001). Meanwhile, the serum cGMP level in the model group mice was significantly increased compared with the blank control group (P < 0.001); the serum cGMP level in each treatment group mice was significantly decreased compared with the model group (P < 0.001).

[0074] Table 10 Effects of the formulation composition on serum cAMP and cGMP levels in mice with kidney-yang deficiency

[0075] The above results indicate that the composition of the present invention has good effects in preventing and treating kidney yang deficiency and anti-fatigue. At the same time, the quality markers are basically consistent with the preventive and treatment effects of the composition, indicating that the main components that exert the comprehensive therapeutic effect in the composition of the present invention are 20 components such as [6]-isoshogaol, 3-hydroxy-9,10-dimethoxysphingane, and astragalus isoflavone.

[0076] Experimental Example 2: The efficacy of quality markers of food-medicine homology formulations on TM3 cell viability and testosterone secretion. Materials and Instruments: Cell Counting Kit-8 (CCK-8) cell proliferation kit and PBS (1X) buffer (Shenzhen Mohong Technology Co., Ltd.); Mouse testicular interstitial cells TM3 and TM3 cell culture medium (Wuhan Pronosai Life Science Co., Ltd.); Mouse testosterone ELISA kit (batch number WL06T86NT5725) (Wuhan Yilairui Biotechnology Co., Ltd.); Full-wavelength microplate reader, Singapore Life Technology Holdings Pte Ltd (Model: Multiskan SkyHigh); Inverted biological microscope, Guangzhou Mingmei Optoelectronic Technology Co., Ltd. (Model: MIX60); CO2 incubator, Wiegand Technology (Beijing) Co., Ltd. (Model: WCI-120); Low-speed centrifuge, Beijing Lanjie Keke Co., Ltd. (Model: L-CM-1001A).

[0077] The formulation composition group is the composition powder prepared in Example 1, and the quality marker group is the quality marker powder prepared in Example 3.

[0078] Experimental methods: Effects of the formulation composition on TM3 cell viability and testosterone secretion Cell Culture: After thawing the frozen TM3 cells, they were cultured in a TM3 cell culture medium at 37°C and 5% CO2 saturated humidity. Microscopic observation was performed to observe cell adhesion. When the cells adhered, the culture medium was aspirated, and the cells were washed three times with PBS until no floating cells remained. Then, TM3 cell culture medium was added again for continued culture. When the cells reached 80%-90% confluence in the culture flask, they were passaged every 2 days. Cells in the logarithmic growth phase were used for experiments.

[0079] CCK-8 assay for cell viability: 6 cells per well 10 3 Cells were seeded in 96-well plates and randomly divided into a blank control group, a model group, and groups containing different concentrations of the formulation composition and quality markers. After 24 h of culture, the cell model groups, groups containing different concentrations of the formulation composition, and quality marker groups were incubated with 5 μmol / L hydrogen peroxide for 4 h to induce cell modeling. The formulation composition groups were then incubated with samples at final concentrations of 62.5, 125, 250, and 500 μg / mL, respectively, while the quality marker groups were incubated with samples at final concentrations of 20, 40, 80, and 160 μg / mL, respectively (the concentration of the quality markers was set to be the same as the amount of the original medicinal material used in the formulation composition). Each group was divided into three replicates. After culturing for another 24 h in an incubator, 10 μL of CCK8 reagent was added to each well, and the cells were incubated at 37°C in the dark for 4 h. The OD values ​​of each group were then measured using a microplate reader at a wavelength of 450 nm. The cell viability of each group was calculated based on the measured absorbance using the following formula.

[0080] Cell viability (%) = ×100% Testosterone content determination: After 24 hours of culture with TM3 cells, the supernatant was collected. Testosterone content was detected using a mouse testosterone ELISA kit, following the manufacturer's instructions, and the OD value was measured at 450 nm.

[0081] (3) Data processing Each experiment was performed three times independently, and the results were averaged. Software was used to perform significance analysis of the data, and the data were calculated as follows: ±S represents the symbol.

[0082] Results and Analysis: Effects of the composition and quality markers on TM3 cell survival: The effects of different concentrations of the composition on TM3 cell survival are shown in Table 11. Compared with the control group, the H2O2-induced TM3 cell model group showed a significant decrease in cell survival (P < 0.001), indicating the successful establishment of the TM3 cell kidney-yang deficiency model. Compared with the model group, the composition group promoted TM3 cell proliferation to varying degrees within the concentration range of 62.5–500 μg / mL, showing a concentration-dependent effect at concentrations of 62.5, 125, and 250 μg / mL. The 250 μg / mL concentration showed the strongest promoting effect, with a survival rate of 76%, which was significantly different from the model group (P < 0.001). The trend of the quality marker group was the same as that of the composition group. However, at concentrations of 62.5 μg / mL for the combined drug and 20 μg / mL for the quality marker, there was no significant difference between the treatment group and the model group (P > 0.05). At high concentrations (500 μg / mL, 160 μg / mL), it is likely related to the toxicity that begins with excessive drug concentration. Although there was a significant difference compared to the model group (P < 0.01), the survival rate was lower than that at lower concentrations (250 μg / mL, 80 μg / mL).

[0083] Table 11 Effect of the formulation composition on TM3 cell viability

[0084] Effects of the formulation and quality markers on testosterone secretion in TM3 cells: At the lowest concentration, the formulation and quality markers had no significant promoting effect on TM3 cells. Considering the potential toxicity of the formulation and quality markers at the highest concentration, testosterone secretion was analyzed at 125 μg / mL and 250 μg / mL formulations, as well as 40 μg / mL formulations. As shown in Table 12, compared with the control group, the testosterone secretion in the model group was significantly reduced (P < 0.001), indicating successful establishment of the TM3 cell kidney-yang deficiency model. Compared with the model group, both treatment groups increased testosterone secretion in TM3 cells to varying degrees. The highest testosterone secretion was achieved at a concentration of 125 μg / mL, at which point the secretion level was 0.15 ng / mL.

[0085] Table 12 Effects of the formulation composition on testosterone secretion in TM3 cells

[0086] The above results indicate that the formulation composition and the quality marker can improve the survival rate of TM3 cells and promote testosterone secretion. The formulation composition showed the greatest effect in promoting testosterone secretion at a concentration of 125 μg / mL, while the quality marker showed the greatest effect at a concentration of 80 μg / mL. The formulation composition and quality marker can improve gonadal function by promoting testosterone secretion, thereby indirectly regulating kidney function and exhibiting a certain kidney-tonifying effect.

[0087] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A quality marker for the effect of a food-medicine homology formula composition in preventing and treating kidney yang deficiency, characterized in that, The medicinal and edible formula composition consists of the following raw materials in parts by weight: 1-2 parts cinnamon, 6-8 parts monk fruit, 2-4 parts dried tangerine peel, 2-4 parts dried ginger, 8-10 parts ginseng, 29-31 parts astragalus, 4-6 parts prepared licorice root, and 14-16 parts poria cocos; the quality markers include: [6]-isoshogaol, 3,5,6,7,8,3′,4′-heptamethoxyflavone, 1-dehydro-[8]- Ginger dione, 6-methyl gingerol, hesperidin, perolin, 1-(3,4-dimethoxyphenyl)-5-hydroxydecane-3-one, dihydro-[6]-gingerol, isotrichol, isoglycyrrhizin, 3-hydroxy-9,10-dimethoxystigmine, astragalus isoflavone, hesperidin, naringenin, glycyrrhizin P, glycyrrhizin R, glycyrrhizin, pine resin, Ceylon cinnamon, 8-dehydroshogaol.

2. A method for preparing a quality marker for the effect of the medicinal and edible homology formula composition as described in claim 1 in preventing and treating kidney yang deficiency, characterized in that, Includes the following steps: (1) Take the raw medicinal materials, add water and decoct 2-3 times, each time for 1-2 hours. After decoction, concentrate to obtain concentrated liquid; (2) Separate the concentrate by passing it through a macroporous resin column, wash it with water until the eluent is nearly colorless, and then elute it with gradients of ethanol-water solutions with concentrations of 25-35%, 45-55%, and 70-80%, respectively, and collect the ethanol eluents of different concentrations in segments. (3) The eluent obtained in step (2) is subjected to high-resolution liquid chromatography-mass spectrometry for component identification. The eluents that can identify 20 chemical components, including [6]-isoshogaol, 3,5,6,7,8,3′,4′-heptamethoxyflavone, 1-dehydro-[8]-gingerdione, 6-methylgingeradiol, noriheptacortine, peroroliline, 1-(3,4-dimethoxyphenyl)-5-hydroxydecane-3-one, dihydro-[6]-gingerol, isotrichol, isoglycyrrhizin, 3-hydroxy-9,10-dimethoxysphingane, astragalus isoflavone, hesperidin, naringenin, glycyrrhizin P, glycyrrhizin R, glycyrrhizin, pinocembrin, Ceylon cinnamon, and 8-dehydroshogaol, are concentrated and dried to obtain the quality marker of the formulation composition for preventing and treating kidney yang deficiency.

3. The method for preparing quality markers for the effect of the food-medicine homology formula composition on preventing and treating kidney yang deficiency according to claim 2, characterized in that, In step (3), take 100 μL of the eluent, place it in a 1.5 mL centrifuge tube, add 300 μL of methanol, vortex and mix for 1 min; centrifuge at 4℃ and 12000 rpm for 5-10 min; take 100 μL of the solution, add 100 μL of ultrapure water, mix well, and place it in a sample vial for testing.

4. The method for preparing quality markers of the effect of the food-medicine homology formula composition on preventing and treating kidney yang deficiency according to claim 2, characterized in that, In step (3), the chromatographic conditions are: ACQUITY UPLC HSS T3 column, column temperature 40°C, injection volume 5.0-8.0 µL, flow rate 0.3 mL / min; the mobile phase consists of the following components: phase A: water + 0.1% formic acid, phase B: acetonitrile; The elution gradient of the chromatographic column is: The volume fraction of phase B is 2% from 0 to 1 minute. 1-14 min, B phase volume fraction is 2-30%; 14-25 min, B phase volume fraction is 30-100%; 25-28 min, B phase volume fraction is 100%; 28-28.1 min, B phase volume fraction is 100-2%; 28.1-29.5 min, B phase volume fraction is 2%.

5. The method for preparing the quality marker of the effect of the food-medicine homology formula composition on preventing and treating kidney yang deficiency according to claim 2, characterized in that, The mass spectrometry conditions were as follows: ion source voltages for positive and negative ions were 3.7 kV and 3.5 kV, respectively; capillary heating temperature was 320°C; sheath gas pressure was 30 psi, and auxiliary gas pressure was 10 psi; solvent heating and evaporation temperature was 300°C; both sheath gas and auxiliary gas were nitrogen; the collision gas was nitrogen at a pressure of 1.5 mTorr; and Full scan / dd-MS was used. 2 Data acquisition mode, Full scan parameters: resolution 70000, automatic gain control target 1×10⁻⁶. 6 Maximum isolation time 50 ms, mass-to-charge ratio scan range 100-1500; dd-MS 2 Parameters: Resolution 17500, Automatic Gain Control target 1×10 5 The maximum isolation time is 50 ms.

6. The method for preparing quality markers of the effect of the food-medicine homology formula composition on preventing and treating kidney yang deficiency according to claim 2, wherein in step (2), the mixture is eluted by gradient elution with ethanol-water solutions of concentrations of 30%, 50%, and 75%.

7. A method for analyzing the quality markers of the medicinal and edible homologous formula composition as described in claim 1 for preventing and treating kidney yang deficiency, characterized in that, Includes the following steps: S1. The effective components of the food-medicine homology formula composition were analyzed using the traditional Chinese medicine systems pharmacology database and analysis platform. The effective targets of the food-medicine homology formula composition were then sequentially imported into the SwissTargetPrediction database and screened using Probability>0.1 as the screening condition. S2. Using the GeneCards database with "kidney function; endocrine disorder; metabolic syndrome" as search keywords, relevant targets for kidney yang deficiency-related diseases were obtained; Venn was used to draw a Venn diagram of the effective components of the food-medicine homology formula composition and the targets related to kidney yang deficiency diseases. The intersection targets obtained are the target genes of the effective components of the food-medicine homology formula composition for kidney yang deficiency diseases. S3. Upload the genes that act on the common target of the drug and the disease to the STRING database to draw the PPI network diagram, and perform topological parameter analysis in Cytoscape 3.10 software; obtain the "traditional Chinese medicine-component-disease-target" interaction network diagram between the drug-food homology formula composition and kidney yang deficiency related diseases, and obtain the top 20 effective components; S4. The collected co-acting gene targets were analyzed using the DAVAD database. With P < 0.05, GO function and KEGG signaling pathway enrichment analysis were performed. The top-ranked data were selected and visualized on the MicroBioinformatics platform.

8. The application of the quality marker of the effect of the food-medicine homology formula composition as described in claim 1 in preventing and treating kidney yang deficiency in the preparation of drugs for preventing and treating kidney yang deficiency.

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