A method for developing a ginseng health product based on a novel nematode screening model

Abstract

This invention discloses a method for developing ginseng and astragalus health products based on a novel nematode screening model, comprising the following steps: selection and standardization of ginseng and astragalus raw materials and pretreatment; preparation of ginseng and astragalus extract and gradient setting of process parameters; construction of a synchronized culture system for *C. elegans*; establishment of a high-throughput efficacy screening model for nematodes; precise detection of nematode physiological phenotypes and biochemical indicators; calculation of a quantitative evaluation model for the efficacy of ginseng and astragalus products; optimization of the formula and finalization of the process for ginseng and astragalus health products; pilot-scale preparation and quality consistency testing of the products; and secondary verification of product efficacy and safety evaluation. This invention constructs an in vivo screening system at the tissue and organ level, overcoming the limitations of traditional cell experiments and animal models, achieving rapid, low-cost, and high-throughput efficacy evaluation, shortening the development cycle, and reducing development costs.

Description

Technical Field

[0001] This invention relates to the fields of health food research and development, active screening of traditional Chinese medicine and evaluation of biological efficacy, specifically a method for developing ginseng and astragalus health products based on a novel nematode screening model. Background Technology

[0002] The ginseng-astragalus combination is a classic traditional Chinese medicine pair for tonifying qi and strengthening the body. It consists of astragalus and codonopsis, possessing core effects such as anti-oxidation, anti-aging, and enhanced immunity, making it a key ingredient in health food development. Currently, the research and efficacy evaluation of ginseng-astragalus health products mainly rely on traditional methods such as in vitro cell experiments and rodent models, which suffer from significant drawbacks including long screening cycles, high research and development costs, low throughput, and poor correlation between in vitro and in vivo efficacy.

[0003] Traditional cell experiments can only simulate the in vitro microenvironment and cannot reflect the absorption, metabolism and overall efficacy of ginseng and astragalus active ingredients in organisms. Animal models such as mice and rats have problems such as high breeding costs, experimental cycles of several weeks or even months, strict ethical restrictions, and difficulty in achieving high-throughput parallel screening, resulting in low efficiency in optimizing ginseng and astragalus product formulations and insufficient accuracy in efficacy evaluation.

[0004] As a model organism, *C. elegans* possesses unique advantages such as extremely low culture costs, short lifespan, clear genetic background, high homology with human genes, and the ability to perform high-throughput screening in vivo, making it an ideal carrier for rapid evaluation of the efficacy of health foods. Currently, high-throughput screening technology using *C. elegans* has not been systematically applied to the entire R&D process of ginseng and astragalus health products. There is a lack of standardized, quantifiable nematode screening models and efficacy evaluation systems, hindering the integrated and efficient R&D of ginseng and astragalus products from raw material extraction and formula optimization to efficacy verification. Summary of the Invention

[0005] The purpose of this invention is to provide a method for developing ginseng and astragalus health products based on a novel nematode screening model, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for developing ginseng and astragalus health products based on a novel nematode screening model, comprising the following steps: Step 1: Standardization of raw material selection and pre-processing of ginseng and astragalus. Select authentic astragalus and codonopsis raw materials, and after passing quality inspection, pulverize them at low temperature with ultra-fine grinding. Control the grinding temperature to ≤10℃ and the grinding particle size to 100~120 mesh. Store in a sealed and light-proof container. Step 2: Preparation of ginseng and astragalus extract and setting of process parameters. Ultrasonic-assisted compound extraction process is adopted. The compatibility mass ratio, material-liquid ratio, extraction temperature, extraction time and ultrasonic power gradient are set. After centrifugation, filtration, concentration and sterilization, the extract to be tested is obtained. Step 3: Construction of the synchronized culture system for Caenorhabditis elegans. Wild-type strain N2 was selected, and NGM solid medium and S liquid medium were prepared. Synchronized L1 stage nematodes were obtained by alkaline sodium hypochlorite treatment. The culture temperature was controlled at 20±0.5℃ and the humidity at 60±5%. Step 4: Establishment of high-throughput efficacy screening model for nematodes. Three screening models were constructed: anti-aging, anti-oxidation, and immune enhancement. The nematodes were divided into blank control group, solvent control group, and experimental group, with ≥3 biological replicates in each group. They were placed in 96-well plates and cultured at a constant temperature. Step 5: Precise detection of nematode physiological phenotypes and biochemical indicators, quantitative detection of nematode lifespan, body length and width, motility frequency, reactive oxygen species level, antioxidant enzyme activity, phagocytic index, and establishment of original detection database; Step Six: Calculate the efficacy evaluation model for ginseng and astragalus products, substitute the formulas for antioxidant efficacy index, immune enhancement efficacy index, and comprehensive efficacy evaluation index, calculate the efficacy score, and screen the optimal process and formula; Step 7: Optimize the formula and finalize the process of ginseng and astragalus health products. Based on the efficacy results, select the types and proportions of excipients, optimize the molding process parameters, and determine the core formula and production process of the product. Step 8: Pilot production and quality consistency testing of the product. Produce the product in pilot mode according to the finalized process, and test the content of effective ingredients, pH value, microbial limit, and stability index to ensure quality uniformity. Step Nine: Secondary verification of product efficacy and safety evaluation, using nematode models for rescreening and cell experiments for verification, completing acute oral toxicity and 30-day feeding trials, and determining industrialization standards.

[0007] Furthermore, the parameter gradients for the ultrasound-assisted composite extraction process in step two are as follows: the mass ratio of Astragalus membranaceus to Codonopsis pilosula is 3:1, 2:1, 1:1, 1:2, and 1:3; the material-liquid ratio is 1:10, 1:15, 1:20, and 1:25; the extraction temperature is 60℃, 70℃, 80℃, and 90℃; the ultrasound time is 20min, 30min, 40min, and 50min; and the ultrasound power is 150W, 200W, 250W, and 300W.

[0008] Furthermore, in step three, the alkaline sodium hypochlorite solution is prepared by mixing sodium hypochlorite and 5 mol / L sodium hydroxide solution in a volume ratio of 1:1; the NGM solid culture medium contains sodium chloride, agar, peptone, cholesterol ethanol solution, 1 mol / L magnesium sulfate, 1 mol / L calcium chloride, and 1 mol / L potassium phosphate buffer.

[0009] Furthermore, in step five, the level of reactive oxygen species was detected using DCFH-DA fluorescent probe labeling, and the antioxidant enzymes included superoxide dismutase, catalase, and glutathione peroxidase. The phagocytic index was detected using the fluorescently labeled E. coli feeding method.

[0010] Furthermore, the formula for calculating the antioxidant efficacy index (AOEI) in step six is ​​as follows: In the formula: The fluorescence intensity value of reactive oxygen species in nematodes represents the blank control group. The fluorescence intensity value of reactive oxygen species in the experimental group of nematodes; , The superoxide dismutase activities of nematodes in the experimental and control groups were respectively measured. , The catalase activity of nematodes in the experimental and control groups were respectively measured. =0.3, =0.3, =0.3.

[0011] Furthermore, the formula for calculating the immune enhancement efficacy index IEII in step six is ​​as follows: In the formula: , The phagocytic index of nematodes in the experimental group and the control group, respectively; , The figures represent the relative expression levels of immune-related genes in the experimental and control groups of nematodes, respectively. , The scores for the intestinal mucosa integrity of the nematodes in the experimental and control groups were respectively determined. =0.4, =0.3, =0.3.

[0012] Furthermore, the formula for calculating the Comprehensive Efficacy Evaluation Index (CEI) in step six is ​​as follows: In the formula: Life extension index, , This represents the average lifespan of the nematodes in the experimental group. The average lifespan of the control group nematodes; =0.4, =0.3, =0.3.

[0013] Furthermore, in step seven, the dosage form of the ginseng and astragalus health product is an oral liquid or a plant-based beverage; the excipients for the oral liquid are xylitol, sodium carboxymethyl cellulose, and sodium citrate, and the excipients for the beverage are fructose syrup, vitamin C, and citric acid; the sterilization process is pasteurization at 85°C for 15 minutes.

[0014] Furthermore, in step nine, the maximum tolerated dose for acute oral toxicity testing was ≥15 g / kg BW, and there were no animal deaths, organ toxicity, or abnormalities in hematological and biochemical indicators during the 30-day feeding trial.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention constructs an in vivo screening system at the tissue and organ level, overcoming the limitations of traditional cell experiments and animal models. It achieves rapid, low-cost, and high-throughput efficacy evaluation, shortening the R&D cycle and reducing R&D costs. This invention enables quantitative evaluation of the antioxidant, immune-enhancing, and anti-aging effects of ginseng and astragalus products, replacing traditional qualitative descriptions and significantly improving the accuracy and objectivity of efficacy evaluation, providing data support for formula optimization. This invention forms a standardized R&D method covering the entire process from raw material pretreatment, extraction process, nematode screening, formula optimization to pilot-scale production. Strict quality control standards are set at each stage to ensure stable product efficacy and uniform quality, suitable for large-scale industrial production. This invention uses an in vivo nematode screening model, which more closely resembles the real physiological environment of the human body, effectively solving the problem of the disconnect between in vitro cell experiments and in vivo efficacy, and improving the success rate and market adaptability of ginseng and astragalus health product development. Attached Figure Description

[0016] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] Please see Figure 1 This invention provides a method for developing ginseng and astragalus health products based on a novel nematode screening model, comprising the following steps: Step 1: Standardization of raw material selection and pre-processing of ginseng and astragalus. Authentic Astragalus membranaceus and Codonopsis pilosula raw materials were selected. After passing the identification of properties, moisture content, total ash content, and effective ingredient content, impurities, moldy and insect-infested parts were removed. The raw materials were then prepared into fine powder using a low-temperature ultra-micro pulverizer, with the pulverization temperature controlled at ≤10℃ and the particle size at 100-120 mesh. The powder was then sealed and stored in a light-proof environment for later use. Raw material quality control standards were established, and the minimum content limits of astragaloside A, verbascoside, and codonopsis glycoside were clearly defined to ensure the uniformity of the raw materials.

[0019] Step 2: Preparation of Ginseng and Astragalus Extract and Setting of Process Parameters Ginseng and Astragalus extract was prepared using an ultrasound-assisted composite extraction process. Gradients were set for the mass ratio of Astragalus to Codonopsis, the material-liquid ratio, the extraction temperature, the extraction time, and the ultrasonic power. After extraction, the extract was centrifuged, filtered, concentrated under reduced pressure, and sterilized by filtration to prepare ginseng and Astragalus extracts under different process parameters. The extracts were then refrigerated for later use to ensure that the extracts were free from microbial contamination and that the active ingredients were not degraded.

[0020] Step 3: Construction of a synchronized culture system for Caenorhabditis elegans Wild-type Caenorhabditis elegans strain N2 was selected, and NGM solid medium and S liquid medium were prepared with strict control of culture environment parameters. Synchronized eggs were obtained by alkaline sodium hypochlorite synchronization treatment, and L1 stage synchronized nematodes were obtained after hatching. Quality control standards for nematode culture were established to control nematode survival rate and development uniformity, and to ensure the biological stability of the screening model.

[0021] Step 4: Establishment of a high-throughput efficacy screening model for nematodes We constructed a high-throughput screening model for three types of nematodes: anti-aging, antioxidant, and immune-enhancing. Synchronized L1-stage nematodes were randomly divided into a blank control group, a solvent control group, and a ginseng and astragalus extract experimental group, with ≥3 biological replicates in each group. The nematodes were placed in a 96-well high-throughput screening plate, and ginseng and astragalus extract of the corresponding concentration was added. Constant culture conditions were set, and the nematodes were cultured until the end of their life cycle to complete multiple parallel screenings.

[0022] Step 5: Precise detection of nematode physiological phenotypes and biochemical indicators Using equipment such as a microscopic image analysis system, fluorescence spectrophotometer, and ELISA reader, we quantitatively detected key indicators of nematodes, including life cycle, body length and width, motility frequency, reactive oxygen species (ROS) levels, antioxidant enzyme activity, and phagocytic index. Abnormal data were excluded to ensure the accuracy and repeatability of the test data, and an original test database was established.

[0023] Step Six: Calculation of the Quantitative Evaluation Model for the Efficacy of Ginseng and Astragalus Products Based on the test data from step five, the efficacy scores of ginseng and astragalus extracts under different extraction processes and formulation ratios were calculated by substituting the three quantitative formulas: antioxidant efficacy index, immune enhancement efficacy index, and comprehensive efficacy evaluation index. The optimal process parameters and formulation ratios were then selected.

[0024] Step Seven: Optimization of Ginseng and Astragalus Health Product Formula and Finalization of Process Based on the results of efficacy quantitative evaluation, the optimal ginseng and astragalus extract was screened for excipient compatibility to determine the types and proportions of sweeteners, stabilizers, and solubilizers. Combined with the product dosage form (oral liquid, beverage) requirements, the molding process parameters such as homogenization, degassing, and sterilization were optimized to determine the core formula and production process of the product.

[0025] Step 8: Pilot-scale preparation and quality consistency testing of the product Pilot production was carried out according to the established process to prepare finished ginseng and astragalus health products; a product quality testing system was established to test indicators such as effective ingredient content, pH value, soluble solids, microbial limits, and stability to ensure that the quality of the pilot product is consistent with that of the laboratory sample.

[0026] Step Nine: Secondary Verification of Product Efficacy and Safety Evaluation The efficacy was rescreened using a nematode model, and further validated by in vitro cell experiments to confirm the stability of the product's efficacy. Safety evaluation was completed through acute oral toxicity tests and a 30-day feeding trial, confirming that the product has no acute toxicity and is safe for long-term consumption. Finally, the product's industrialization standards were determined, and the entire research and development process was completed.

[0027] In step two, the parameter gradients of the ultrasound-assisted compound extraction process are set as follows: the mass ratio of Astragalus membranaceus to Codonopsis pilosula is 3:1, 2:1, 1:1, 1:2, and 1:3; the material-liquid ratio is 1:10, 1:15, 1:20, and 1:25; the extraction temperature is 60℃, 70℃, 80℃, and 90℃; the ultrasonic time is 20min, 30min, 40min, and 50min; and the ultrasonic power is 150W, 200W, 250W, and 300W.

[0028] In step three, the NGM solid culture medium components include sodium chloride, agar, peptone, cholesterol ethanol solution, 1 mol / L magnesium sulfate, 1 mol / L calcium chloride, and 1 mol / L potassium phosphate buffer. The culture environment parameters are: temperature 20±0.5℃, humidity 60±5%, and aseptic culture in the dark. The alkaline sodium hypochlorite solution used for synchronization treatment is a mixture of sodium hypochlorite and 5 mol / L sodium hydroxide solution in a volume ratio of 1:1.

[0029] In step five, reactive oxygen species levels were detected using DCFH-DA fluorescent probe labeling, and antioxidant enzymes included superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). The phagocytic index was detected using a fluorescently labeled E. coli feeding method to reflect the nematode intestinal immune phagocytic capacity.

[0030] In step six, the specific formula for quantifying efficacy is as follows: Antioxidant efficacy index (AOEI) In the formula: The fluorescence intensity value of reactive oxygen species in nematodes represents the blank control group. The fluorescence intensity value of reactive oxygen species in the experimental group of nematodes; , The superoxide dismutase activities of nematodes in the experimental and control groups were respectively measured. , The catalase activity of nematodes in the experimental and control groups were respectively measured. , , These are the weighting coefficients. =0.4, =0.3, =0.3, with a weighting coefficient sum of 1. Immune Enhancement Efficacy Index (IEII) In the formula: , The phagocytic index of nematodes in the experimental group and the control group, respectively; , The figures represent the relative expression levels of immune-related genes in the experimental and control groups of nematodes, respectively. , The scores for the intestinal mucosa integrity of the nematodes in the experimental and control groups were respectively determined. =0.4, =0.3, =0.3, with the sum of weighting coefficients being 1. Comprehensive Efficacy Evaluation Index (CEI) In the formula: Life extension index, , This represents the average lifespan of the nematodes in the experimental group. The average lifespan of the control group nematodes; , , For comprehensive weighting, =0.4, =0.3, =0.3, the sum of the comprehensive weighting coefficients is 1; The higher the value, the better the overall efficacy of the ginseng and astragalus extract.

[0031] In step seven, the dosage forms of Shenqi health products include oral liquids and plant-based beverages; the excipients for oral liquids are xylitol, sodium carboxymethyl cellulose, and sodium citrate, and the excipients for beverages are fructose syrup, vitamin C, and citric acid; the sterilization process adopts pasteurization at a temperature of 85℃ for 15 minutes.

[0032] In step nine, the safety evaluation shall be carried out in accordance with the "Technical Specifications for Safety Evaluation of Health Foods". The maximum tolerated dose for acute oral toxicity test shall be ≥15g / kg·BW. There shall be no animal death, no organ toxicity, and no abnormalities in hematological and biochemical indicators in the 30-day feeding test.

[0033] Example 1: Development of Ginseng and Astragalus Oral Liquid Based on Nematode Screening Model This embodiment uses the method of the present invention to develop ginseng and astragalus oral liquid. The specific implementation steps are as follows: Step 1: Standardization of raw material selection and pre-processing of ginseng and astragalus. Astragalus membranaceus from Inner Mongolia and Codonopsis pilosula from Shangdang, Shanxi, were selected as raw materials. Quality testing was conducted according to the standards of the 2025 edition of the Chinese Pharmacopoeia, Volume IV: Astragalus membranaceus had a moisture content ≤10.0%, total ash content ≤5.0%, astragaloside A content ≥0.040%, and isoflavone glucoside content ≥0.020%; Codonopsis pilosula had a moisture content ≤11.0%, total ash content ≤5.0%, and codonopsis pilosula glycoside content ≥0.050%. Impurities, moldy, and insect-infested parts were removed. The powders were then pulverized using a low-temperature ultrafine pulverizer, with the pulverization temperature controlled at 8℃ and the particle size at 110 mesh. The fine powders of Astragalus membranaceus and Codonopsis pilosula were separately sealed in brown glass bottles and stored below 25℃ in the dark for later use.

[0034] Step 2: Preparation of Ginseng and Astragalus Extract and Setting of Process Parameters Astragalus and ginseng extracts were prepared using an ultrasonic-assisted water extraction process, with five compatibility gradients: Group 1 (Astragalus: Codonopsis = 3:1), Group 2 (2:1), Group 3 (1:1), Group 4 (1:2), and Group 5 (1:3). The material-to-liquid ratio was fixed at 1:15, the extraction temperature at 80℃, the ultrasonic time at 30 min, and the ultrasonic power at 200 W. Corresponding masses of Astragalus and Codonopsis powder were weighed, added to purified water, mixed thoroughly, and then extracted using an ultrasonic extractor. After extraction, the mixture was centrifuged at 4℃ and 8000 r / min for 15 min. The supernatant was filtered through a 0.45 μm microporous membrane, concentrated under reduced pressure to a solids content of 10%, and then filtered through a 0.22 μm sterile membrane to obtain five groups of Astragalus and ginseng extracts for testing. These were stored at 4℃ for later use.

[0035] Step 3: Construction of a synchronized culture system for Caenorhabditis elegans Wild-type Caenorhabditis elegans strain N2, provided by Caenorhabditis Genetics Center (CGC), was selected, with Escherichia coli OP50 as the food source. NGM solid medium was prepared as follows: each 1L of medium contained 3g sodium chloride, 17g agar, 2.5g peptone, 1mL cholesterol ethanol solution (5mg / mL), 1mL 1mol / L magnesium sulfate, 1mL 1mol / L calcium chloride, 25mL 1mol / L potassium phosphate buffer (pH 6.0), and the remainder was purified water. The medium was autoclaved at 121℃ for 20min and cooled to 50℃ before plating.

[0036] S liquid culture medium: Each 1L contains 3g sodium chloride, 2.5g peptone, 25mL 1mol / L potassium phosphate buffer, and the remainder is purified water. After sterilization, it is ready for use. Nematodes are inoculated onto NGM plates containing OP50 bacterial culture and cultured at 20℃ until the peak oviposition period. Alkaline sodium hypochlorite solution (sodium hypochlorite: 5mol / L sodium hydroxide = 1:1) is added for treatment for 5 min. Eggs are collected by centrifugation, washed three times with M9 buffer, and incubated at 20℃ for 24 h to obtain L1 stage synchronized nematodes. Microscopic observation shows that the uniformity of nematode development is ≥95%, and the survival rate is ≥98%, meeting the screening requirements.

[0037] Step 4: Establishment of a high-throughput efficacy screening model for nematodes Three high-throughput screening models for anti-aging, anti-oxidation, and immune enhancement were constructed. L1-stage synchronized nematodes were randomly divided into a blank control group (M9 buffer), a solvent control group (0.5% ethanol solution), and five experimental groups containing ginseng and astragalus extracts. Each group had six replicates. 100 μL of nematode suspension (containing 50±5 nematodes) and 100 μL of the corresponding test solution were added to each well, resulting in a final concentration of 1.0 mg / mL. The 96-well screening plate was placed in a constant temperature incubator at 20℃ and 60% humidity, and cultured aseptically in the dark. The culture medium was changed daily until all nematodes died.

[0038] Step 5: Precise detection of nematode physiological phenotypes and biochemical indicators Lifespan monitoring: The number of nematode deaths was observed and recorded daily. Death was defined as no reaction upon touch. Escaped, contaminated, and autolytic nematodes were removed. The average and maximum lifespan of each group of nematodes were calculated. Reactive oxygen species (ROS) detection: After culturing to the adult stage (day 4), DCFH-DA fluorescent probe (final concentration 10 μmol / L) was added and incubated at 37℃ in the dark for 30 min. The fluorescence intensity at 488 nm excitation and 525 nm emission was detected by fluorescence spectrophotometer. Antioxidant enzyme activity assay: Nematodes were collected, lysed on ice, centrifuged, and the supernatant was collected. The activities of superoxide dismutase (SOD) and catalase (CAT) were detected using a kit. Immunophagocytic index detection: nematodes were fed FITC-labeled Escherichia coli for 2 hours, photographed under a fluorescence microscope, and the intestinal fluorescence intensity was calculated using ImageJ software, which is the phagocytic index; Intestinal mucosal integrity score: Hematoxylin-eosin staining was used to observe the intestinal structure under a microscope, and the score was scored from 0 to 5, with higher scores indicating better integrity.

[0039] All tests were performed in triplicate. After removing outliers, the original database was built to ensure that the coefficient of variation of the data was ≤5%.

[0040] Step Six: Calculation of the Quantitative Evaluation Model for the Efficacy of Ginseng and Astragalus Products Substitute the test data into the three quantitative formulas of claims 5-7 to calculate the efficacy index for each group: Antioxidant efficacy index (AOEI): Group 1 = 0.72, Group 2 = 0.89, Group 3 = 0.81, Group 4 = 0.75, Group 5 = 0.68; Immune enhancement efficacy index (IEII): Group 1 = 0.70, Group 2 = 0.87, Group 3 = 0.80, Group 4 = 0.73, Group 5 = 0.66; Life Extension Index (LEI): Group 1 = 0.32, Group 2 = 0.45, Group 3 = 0.38, Group 4 = 0.33, Group 5 = 0.29; Comprehensive efficacy evaluation index (CEI): Group 1 = 0.59, Group 2 = 0.74, Group 3 = 0.67, Group 4 = 0.61, Group 5 = 0.55.

[0041] The results showed that the ratio of Astragalus membranaceus to Codonopsis pilosula = 2:1 had the best overall efficacy and was determined as the core formula ratio.

[0042] Step 7: Optimization of the formula and finalization of the process for Ginseng and Astragalus Oral Liquid Using the optimally formulated extract as the core raw material, excipients were screened: Sweetener: Xylitol is selected, with an addition amount of 3%, which has a sweet taste and no off-flavor; Stabilizer: Sodium carboxymethyl cellulose was selected and added at a rate of 0.1% to improve the stability of the system; pH adjuster: Sodium citrate is selected, with an addition amount of 0.05%, to adjust the pH value to 4.5-5.5.

[0043] Molding process: Add ginseng and astragalus extract and excipients to purified water in sequence, stir at 60℃ for 30 minutes until completely dissolved, homogenize by high pressure homogenizer, degas under vacuum for 10 minutes, fill into 10mL brown oral liquid bottles, pasteurize at 85℃ for 15 minutes, cool to room temperature to obtain ginseng and astragalus oral liquid semi-finished product.

[0044] Step 8: Pilot-scale preparation and quality consistency testing of the product A 100L pilot-scale production was conducted according to the established process, and three batches of ginseng and astragalus oral liquid were continuously prepared. The quality indicators were tested in accordance with the "Technical Specifications for Inspection and Evaluation of Health Foods". Active ingredients: Astragaloside A content ≥0.035mg / mL, Codonopsis pilosula glycoside content ≥0.045mg / mL; Physicochemical properties: pH value 5.0±0.3, soluble solids ≥8.0%; Microbial limits: Total bacterial count ≤100 CFU / mL, mold and yeast ≤20 CFU / mL, no pathogenic bacteria; Stability: After being stored at 37℃ and 60% humidity for 3 months, there was no precipitation, no stratification, and the retention rate of effective ingredients was ≥95%.

[0045] The coefficient of variation of the test results for the three batches of products was ≤3%, and the quality consistency met the requirements.

[0046] Step Nine: Secondary Verification of Product Efficacy and Safety Evaluation Efficacy re-screening: The pilot-scale product was validated using a nematode model. The results showed that CEI=0.73, which was not significantly different from the laboratory sample (P>0.05), indicating stable efficacy. Cellular validation: Using RAW264.7 macrophage assays, the product was validated to significantly enhance phagocytic capacity and antioxidant enzyme activity, consistent with the results of nematode screening. Safety evaluation: The maximum tolerated dose in the acute oral toxicity test was ≥20g / kg·BW, which is practically non-toxic; in the 30-day feeding test, there were no abnormalities in rat body weight, organ coefficient, hematological indicators, and biochemical indicators, and no organ pathological damage.

[0047] The industrialization standard for Shenqi oral liquid was finally determined, and the entire research and development process was completed.

[0048] Example 2: Development of Ginseng and Astragalus Herbal Beverage Based on Nematode Screening Model This embodiment uses the method of the present invention to develop a ginseng and astragalus plant beverage. The core steps are the same as in Example 1, only the extraction process and excipient formulation are adjusted: Extraction process: The material-to-liquid ratio was adjusted to 1:20, the ultrasonic power was adjusted to 250W, and the other parameters remained unchanged; Excipient formulation: 5% fructose syrup, 0.03% vitamin C, and 0.08% citric acid; Product specifications: 330mL PET bottle, sterilization process 85℃, 20min; Efficacy verification: The CEI of the nematode model was 0.71, indicating that the antioxidant and immune-enhancing effects met the standards, and the safety met national standards.

[0049] Example 3: Verification of the impact of different extraction processes on product efficacy In this embodiment, the ratio of Astragalus membranaceus to Codonopsis pilosula was fixed at 2:1, and the ultrasonic extraction time was adjusted (20 min, 40 min, 50 min). The efficacy index was detected using the method of this invention. 20 min: CEI=0.65; 30 min: CEI=0.74; 40 min: CEI=0.70; 50 min: CEI=0.67.

[0050] In summary, this invention constructs an in vivo screening system at the tissue and organ level, overcoming the limitations of traditional cell experiments and animal models. This enables rapid, low-cost, and high-throughput efficacy evaluation, shortening the R&D cycle and reducing costs. This invention achieves quantitative evaluation of the antioxidant, immune-enhancing, and anti-aging effects of ginseng and astragalus products, replacing traditional qualitative descriptions and significantly improving the accuracy and objectivity of efficacy evaluation, providing data support for formula optimization. This invention forms a standardized R&D method covering the entire process from raw material pretreatment, extraction technology, nematode screening, formula optimization to pilot-scale production. Strict quality control standards are set at each stage to ensure stable product efficacy and uniform quality, suitable for large-scale industrial production. This invention uses an in vivo nematode screening model, which more closely resembles the real physiological environment of the human body, effectively solving the problem of the disconnect between in vitro cell experiments and in vivo efficacy, and improving the success rate and market adaptability of ginseng and astragalus health product development.

[0051] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for developing ginseng and astragalus health products based on a novel nematode screening model, characterized in that: Includes the following steps: Step 1: Standardization of raw material selection and pre-processing of ginseng and astragalus. Select authentic astragalus and codonopsis raw materials, and after passing quality inspection, pulverize them at low temperature with ultra-fine grinding. Control the grinding temperature to ≤10℃ and the grinding particle size to 100~120 mesh. Store in a sealed and light-proof container. Step 2: Preparation of ginseng and astragalus extract and setting of process parameters. Ultrasonic-assisted compound extraction process is adopted. The compatibility mass ratio, material-liquid ratio, extraction temperature, extraction time and ultrasonic power gradient are set. After centrifugation, filtration, concentration and sterilization, the extract to be tested is obtained. Step 3: Construction of the synchronized culture system for Caenorhabditis elegans. Wild-type strain N2 was selected, and NGM solid medium and S liquid medium were prepared. Synchronized L1 stage nematodes were obtained by alkaline sodium hypochlorite treatment. The culture temperature was controlled at 20±0.5℃ and the humidity at 60±5%. Step 4: Establishment of high-throughput efficacy screening model for nematodes. Three screening models were constructed: anti-aging, anti-oxidation, and immune enhancement. The nematodes were divided into blank control group, solvent control group, and experimental group, with ≥3 biological replicates in each group. They were placed in 96-well plates and cultured at a constant temperature. Step 5: Precise detection of nematode physiological phenotypes and biochemical indicators, quantitative detection of nematode lifespan, body length and width, motility frequency, reactive oxygen species level, antioxidant enzyme activity, phagocytic index, and establishment of original detection database; Step Six: Calculate the efficacy evaluation model for ginseng and astragalus products, substitute the formulas for antioxidant efficacy index, immune enhancement efficacy index, and comprehensive efficacy evaluation index, calculate the efficacy score, and screen the optimal process and formula; Step 7: Optimize the formula and finalize the process of ginseng and astragalus health products. Based on the efficacy results, select the types and proportions of excipients, optimize the molding process parameters, and determine the core formula and production process of the product. Step 8: Pilot production and quality consistency testing of the product. Produce the product in pilot mode according to the finalized process, and test the content of effective ingredients, pH value, microbial limit, and stability index to ensure quality uniformity. Step Nine: Secondary verification of product efficacy and safety evaluation, using nematode models for rescreening and cell experiments for verification, completing acute oral toxicity and 30-day feeding trials, and determining industrialization standards.

2. The method for developing ginseng and astragalus health products based on a novel nematode screening model according to claim 1, characterized in that: The parameter gradients for the ultrasound-assisted composite extraction process in step two are as follows: the mass ratio of Astragalus membranaceus to Codonopsis pilosula is 3:1, 2:1, 1:1, 1:2, and 1:3; the material-liquid ratio is 1:10, 1:15, 1:20, and 1:25; the extraction temperature is 60℃, 70℃, 80℃, and 90℃; the ultrasonic time is 20min, 30min, 40min, and 50min; and the ultrasonic power is 150W, 200W, 250W, and 300W.

3. The method for developing ginseng and astragalus health products based on a novel nematode screening model according to claim 1, characterized in that: In step three, the alkaline sodium hypochlorite solution is prepared by mixing sodium hypochlorite and 5 mol / L sodium hydroxide solution in a volume ratio of 1:1; the NGM solid culture medium contains sodium chloride, agar, peptone, cholesterol ethanol solution, 1 mol / L magnesium sulfate, 1 mol / L calcium chloride, and 1 mol / L potassium phosphate buffer.

4. The method for developing ginseng and astragalus health products based on a novel nematode screening model according to claim 1, characterized in that: In step five, the level of reactive oxygen species was detected using DCFH-DA fluorescent probe labeling. Antioxidant enzymes included superoxide dismutase, catalase, and glutathione peroxidase. The phagocytic index was detected using the fluorescently labeled E. coli feeding method.

5. The method for developing ginseng and astragalus health products based on a novel nematode screening model according to claim 1, characterized in that: The formula for calculating the antioxidant efficacy index (AOEI) in step six is ​​as follows: In the formula: The fluorescence intensity value of reactive oxygen species in nematodes represents the blank control group. The fluorescence intensity value of reactive oxygen species in the experimental group of nematodes; , The superoxide dismutase activities of nematodes in the experimental and control groups were respectively measured. The catalase activities of nematodes in the experimental and control groups were respectively measured. =0.4 =0.3 =0.

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6. The method for developing ginseng and astragalus health products based on a novel nematode screening model according to claim 1, characterized in that: The formula for calculating the Immune Enhancement Efficacy Index (IEII) in step six is ​​as follows: In the formula: The phagocytic index of nematodes in the experimental group and the control group, respectively; The figures represent the relative expression levels of immune-related genes in the experimental and control groups of nematodes, respectively. The scores for the intestinal mucosa integrity of the nematodes in the experimental and control groups were respectively determined. =0.4 =0.3 =0.

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7. The method for developing ginseng and astragalus health products based on a novel nematode screening model according to claim 1, characterized in that: The formula for calculating the Comprehensive Efficacy Evaluation Index (CEI) in step six is ​​as follows: In the formula: Life extension index, , This represents the average lifespan of the nematodes in the experimental group. The average lifespan of the control group nematodes; =0.4, =0.3, =0.

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8. The method for developing ginseng and astragalus health products based on a novel nematode screening model according to claim 1, characterized in that: In step seven, the dosage form of the ginseng and astragalus health product is an oral liquid or a plant-based beverage; the excipients for the oral liquid are xylitol, sodium carboxymethyl cellulose, and sodium citrate, and the excipients for the beverage are fructose syrup, vitamin C, and citric acid; the sterilization process is pasteurization at 85℃ for 15 minutes.

9. The method for developing ginseng and astragalus health products based on a novel nematode screening model according to claim 1, characterized in that: In step nine, the maximum tolerated dose for acute oral toxicity testing was ≥15 g / kg BW, and there were no animal deaths, organ toxicity, or abnormalities in hematological and biochemical indicators during the 30-day feeding trial.

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