Method for evaluating anti-fatigue effect of acanthopanax tea

Abstract

The invention provides a method for evaluating the anti-fatigue effect of acanthopanax senticosus, which comprises the following steps of: establishing an evaluation system taking the content of syringin and the IC50 value of DPPH free radical scavenging rate as double-effect indexes, and determining a clear judgment critical value of the anti-fatigue effect of acanthopanax senticosus tea: when and only when a sample simultaneously meets the conditions that the IC50 value of DPPH is less than or equal to 35mu g / mL and the content of syringin is more than or equal to 5mg / g, judging the anti-fatigue effect of acanthopanax senticosus tea; the obvious anti-fatigue effect is judged. Experiments prove that the double-index system and the threshold value have significant correlation with the anti-fatigue effect, the prediction deviation is small, the judgment is clear, the result is visual, and a standardized scheme is provided for rapid and low-cost effect evaluation of the acanthopanax tea product.

Description

Technical Field

[0001] This invention belongs to the field of tea quality evaluation technology, specifically relating to a method for evaluating the anti-fatigue effects of Acanthopanax senticosus tea. Background Technology

[0002] In the field of health food function evaluation, anti-fatigue is an important and popular functional claim area. As a traditional Chinese medicine for anti-fatigue, Acanthopanax senticosus has long faced the following industry-level technical challenges in verifying the efficacy of related products. Currently, the gold standard for evaluating anti-fatigue efficacy recognized by international and domestic authoritative institutions is animal testing, primarily the mouse weight-bearing swimming test. While this method can directly reflect overall physiological effects, it suffers from fatal drawbacks such as a lengthy process, high cost, complex operation, and the need for ethical approval. This makes it unsuitable for rapid screening, production process optimization, and product quality control in enterprise product development.

[0003] Existing in vitro detection methods, such as measuring total phenol content and single component content, lack a direct and clear dose-response relationship between these indicators and the final anti-fatigue efficacy. A high total phenol content in a product does not necessarily mean it has a good anti-fatigue effect; it only reflects the product's chemical composition, not its final functional activity. High component content does not directly equate to good efficacy because it involves complex issues such as bioavailability, metabolic transformation, and multi-target synergistic effects. This results in a weak correlation between in vitro indicators and in vivo anti-fatigue effects, poor predictive accuracy, and inability to serve as a reliable basis for efficacy determination. Current quality control of Acanthopanax senticosus tea products mainly focuses on sensory indicators, moisture content, microbial limits, and single marker content. Relying on the measurement of single chemical component content cannot comprehensively reflect the product's overall efficacy. The anti-fatigue effect of Acanthopanax senticosus is the result of the synergistic effect of multiple components such as syringin, eleutherosin, and polysaccharides; a single indicator is incomplete and lacks scientific rigor. High chemical component content does not equate to strong biological activity, nor can it represent the final anti-fatigue efficacy, because it involves complex issues such as bioavailability, metabolic transformation, and target synergy. This renders the indicator completely unusable for efficacy prediction or quality control.

[0004] There are also attempts to combine component content and antioxidant activity as an evaluation of anti-fatigue effect, but this only provides two parallel indicators and fails to establish a clear, explicit and scientifically validated judgment standard through systematic efficacy correlation research. This makes it impossible for enterprises and testing institutions to draw a clear "yes" or "no" conclusion even if they obtain these two test data. Due to the lack of such an authoritative bridge and decision-making basis for the correlation and transformation between in vitro indicators and in vivo efficacy, these in vitro indicators cannot be truly applied to the efficacy prediction and quality control of products. Their evaluation results are neither accepted by regulatory authorities nor recognized by the market. In summary, the current monitoring and evaluation methods for the quality of Acanthopanax senticosus tea have the following defects: (1) The indicators are single and cannot fully reflect the comprehensive efficacy of the product; (2) A quantitative and predictable correlation model between in vitro indicators and in vivo anti-fatigue efficacy has not been established; (3) It has not been established as an authoritative, reliable and exclusive evaluation standard that can be used to replace animal experiments.

[0005] Therefore, the industry urgently needs to establish a new in vitro evaluation method and standard that is strongly correlated with in vivo anti-fatigue efficacy, is fast and low-cost, and is suitable for industrial quality control, in order to fill the huge gap between chemical composition and functional claims. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a method for evaluating the anti-fatigue efficacy of Acanthopanax senticosus tea, and for the first time establishes an IC50 method for the relationship between syringin content and DPPH free radical scavenging rate of Acanthopanax senticosus tea. 50 This dual-effect index linkage evaluation system, verified by extensive experimental data, demonstrates a strong correlation between its efficacy and anti-fatigue effects. Ultimately, it forms a rapid, low-cost, highly accurate, and specific in vitro efficacy evaluation method, directly addressing all the gaps and weaknesses in existing technologies. One objective of this invention is to provide a method for evaluating the anti-fatigue effects of Acanthopanax senticosus tea; another objective is to provide a model for evaluating the anti-fatigue effects of Acanthopanax senticosus tea; and a third objective is to provide an application of dual-effect indexes in evaluating the anti-fatigue effects of Acanthopanax senticosus tea.

[0007] To achieve the above objectives, the present invention employs the following technical solution: On the one hand, this invention provides a method for evaluating the anti-fatigue efficacy of Acanthopanax senticosus tea, wherein the evaluation indicators of the method include syringin content and DPPH free radical scavenging rate IC50. 50 value.

[0008] This invention, through extensive experimental research, proposes an IC50 method based on syringin content and DPPH free radical scavenging rate. 50A dual-effect index evaluation system was constructed to evaluate the anti-fatigue efficacy of Acanthopanax senticosus tea. Data verification showed that the evaluation system possessed high accuracy and effectiveness with minimal predictive bias. Firstly, the various pharmacological activities of Acanthopanax senticosus are inseparable from its unique glycoside components, with syringin being one of its characteristic active ingredients. Syringin works synergistically through multiple signaling pathways to regulate the cellular antioxidant defense system, exerting antioxidant, anti-inflammatory, and tissue-protective effects. DPPH free radical scavenging rate is an internationally recognized gold standard method for quantifying the overall in vitro antioxidant capacity of a sample. A product's DPPH IC50... 50 The lower the value, the stronger its ability to scavenge free radicals and resist oxidative stress. Therefore, it possesses strong antioxidant capacity (i.e., DPPH IC). 50 Acanthopanax senticosus tea (with low oxidative stress) can more effectively alleviate exercise-induced oxidative damage, thereby delaying the onset of fatigue.

[0009] However, the research results of this invention found that evaluating the anti-fatigue efficacy of Acanthopanax senticosus tea using changes in any single indicator lacks reliability and certainty. This is because an effective anti-fatigue product must simultaneously meet two conditions: strong functional activity and sufficient active ingredients. Therefore, the content of syringin and the DPPH free radical scavenging rate IC50 are important indicators. 50 The two factors mutually restrict and synergistically influence each other. Only when the syringin content is ≥5 mg / g and the DPPH free radical scavenging rate IC50 is the optimal concentration required for effective treatment. 50 Only Acanthopanax senticosus tea products with a DPPH free radical scavenging rate ≤35 μg / mL possess anti-fatigue effects. In the synergistic effect defined in this invention, the overall functional activity represented by DPPH free radical scavenging rate and the specific material basis represented by syringin content complement and corroborate each other in predicting anti-fatigue efficacy. The accuracy and reliability of the prediction provided by their combination far exceed that of any single indicator. Syringin is the core material basis for anti-fatigue, providing functions such as antioxidation and protection of muscle cells; DPPH reflects overall antioxidant activity, ensuring that the components can effectively scavenge free radicals. If the DPPH value is within the acceptable range but syringin is insufficient, the antioxidant activity may be contributed by other components and is unrelated to the characteristics of Acanthopanax senticosus; conversely, if the syringin value is within the acceptable range but the DPPH value is too high, it indicates that the component may be inactivated or have low bioavailability. Both are synergistic and indispensable, together constituting the necessary and sufficient condition for anti-fatigue efficacy.

[0010] Furthermore, the criterion for the method is: when the syringin content is ≥5 mg / g and the DPPH free radical scavenging rate is IC50... 50 When the concentration of Acanthopanax senticosus tea is ≤35 μg / mL, it is determined that Acanthopanax senticosus tea has anti-fatigue effects; when the concentration of syringin is ≥5 mg / g and the DPPH free radical scavenging rate IC50 is not met, the anti-fatigue effect is determined. 50 When the concentration is ≤35 μg / mL, Acanthopanax senticosus tea is determined to have no anti-fatigue effect.

[0011] In this invention, the threshold for determining the significant anti-fatigue potential of Acanthopanax senticosus tea is defined as follows: syringin content ≥ 5 mg / g and DPPH free radical scavenging rate IC50. 50 ≤35 μg / mL. This threshold was determined based on the following: First, the threshold of syringin (5 mg / g) is more than twice the content in traditional Acanthopanax senticosus leaf tea, representing a critical point for ensuring a significant leap in efficacy; second, the formulation and process research data of this invention indicate that this content is a technical critical point for achieving high-value utilization of the fruit and generating a synergistic effect. DPPH IC 50 The threshold value (35 μg / mL) is based on activity benchmarking against classic antioxidants (VC, Trolox) to ensure the product possesses a potent antioxidant level. Simultaneously, correlation analysis shows that this value is a reliable critical point ensuring a high correlation between in vitro antioxidant activity and in vivo anti-fatigue effect. The combined use of these two thresholds constitutes a mutually reinforcing and indispensable evaluation system. It simultaneously ensures the specificity of the product's active ingredients and the intensity of its functional activity, avoiding the limitations of single-indicator evaluation, thus achieving accuracy, specificity, and reliability in prediction. Samples meeting this condition all showed good anti-fatigue effects in subsequent biological validation.

[0012] This invention also verified the effectiveness of the evaluation method provided by this invention using several representative Acanthopanax senticosus tea samples, and used Pearson correlation analysis to analyze the relationship between syringin content and DPPH free radical scavenging rate IC50. 50 The correlation of the values ​​showed that r = -0.92. p < 0.001) indicates a strong negative correlation between the two. This demonstrates their good synergistic effect, forming the core of the evaluation system. Higher syringin content leads to higher DPPH IC50. 50 The lower the value, the stronger the overall antioxidant activity when the core components are sufficient. Therefore, it is necessary to simultaneously meet the requirements of syringin content ≥5 mg / g and DPPH IC50. 50 Samples with a concentration ≤35 μg / mL meet the standards for both basic composition and functional activity, thus synergistically constituting a sufficient condition for anti-fatigue efficacy. The dual-effect index threshold system provided by this invention offers clear judgment and intuitive results, providing a standardized solution for rapid and low-cost efficacy evaluation of Acanthopanax senticosus tea products.

[0013] Furthermore, the method includes the following steps: (1) Processing and preparation of Acanthopanax senticosus tea samples; (2) Syringin content and DPPH free radical scavenging rate IC 50 Value determination; (3) Efficacy determination.

[0014] Furthermore, the Acanthopanax senticosus tea comprises Acanthopanax senticosus fruit and / or leaves.

[0015] In some embodiments, this invention also provides an optimal preparation method for Acanthopanax senticosus fruit and leaf compound tea. The method employs a low-temperature (≤40℃) cell-wall breaking treatment of Acanthopanax senticosus fruit to directionally degrade tannins, achieving a deastringency effect from the root cause, while avoiding the decomposition of heat-sensitive components such as syringin in the fruit. Simultaneously, a segmented roasting process for Acanthopanax senticosus leaves is designed, involving instantaneous fixation at 180℃ and slow roasting at 120℃. This process precisely controls the roasting temperature and time to achieve instantaneous enzyme inactivation and glycoside preservation, while fully enhancing aroma at low temperatures, resulting in a high retention rate of health-promoting components such as syringin and total flavonoids. The Acanthopanax senticosus tea prepared using the process of this invention has a syringin content ≥5 mg / g and a DPPH free radical scavenging rate of IC50. 50 The value is ≤35 μg / mL, which meets the threshold standard for anti-fatigue effect set by this invention. This enables the product to achieve the characteristics of high retention of active ingredients and strong antioxidant activity. The efficacy is significant and predictable, and the anti-fatigue and other health care functions have a solid material basis and have strong market competitiveness.

[0016] On the other hand, this invention provides a model for evaluating the anti-fatigue efficacy of Acanthopanax senticosus tea, including syringin content and DPPH free radical scavenging rate IC50. 50 It has two evaluation parameters.

[0017] Furthermore, the judgment rule for the model is: when the syringin content is ≥5 mg / g and the DPPH free radical scavenging rate IC50 is ≥5 mg / g. 50 When the concentration of Acanthopanax senticosus tea is ≤35 μg / mL, it is determined that Acanthopanax senticosus tea has anti-fatigue effects; when the concentration of syringin is ≥5 mg / g and the DPPH free radical scavenging rate IC50 is not met, the anti-fatigue effect is determined. 50 When the concentration is ≤35 μg / mL, Acanthopanax senticosus tea is determined to have no anti-fatigue effect.

[0018] Furthermore, this invention provides a model for evaluating the anti-fatigue effects of Acanthopanax senticosus tea, the model including syringin content and DPPH free radical scavenging rate IC50. 50 It has two evaluation parameters.

[0019] Furthermore, the judgment rule for the model is: when the syringin content is ≥5 mg / g and the DPPH free radical scavenging rate IC50 is ≥5 mg / g. 50 When the concentration of Acanthopanax senticosus tea is ≤35 μg / mL, it is determined that Acanthopanax senticosus tea has anti-fatigue effects; when the concentration of syringin is ≥5 mg / g and the DPPH free radical scavenging rate IC50 is not met, the anti-fatigue effect is determined. 50 When the concentration is ≤35 μg / mL, Acanthopanax senticosus tea is determined to have no anti-fatigue effect.

[0020] Furthermore, this invention provides the use of syringin in the preparation of a model for evaluating the anti-fatigue efficacy of Acanthopanax senticosus tea, wherein the model is evaluated based on syringin content and DPPH free radical scavenging rate IC50. 50 The value is used to determine the anti-fatigue effect of Acanthopanax senticosus tea.

[0021] In another aspect, the present invention provides a kit for carrying out the method described above, the kit comprising reagents for determining DPPH free radical scavenging rate and reagents for determining syringin content.

[0022] The present invention has the following beneficial effects: 1. Creatively linking syringin content with DPPH free radical scavenging rate IC50 50 By synergistically correlating these two different dimensions of indicators, a more comprehensive and scientific evaluation system is formed. This method requires only conventional biochemical testing equipment, and the testing process can be completed within a few hours, without the need for animal husbandry or complex in vivo experiments. Therefore, this invention achieves a revolutionary reduction in evaluation costs and an exponential increase in evaluation efficiency, enabling companies to afford efficacy monitoring for every batch of products.

[0023] 2. A quantitative and predictable efficacy assessment threshold was established. Through extensive experimental data verification, the DPPH IC was clearly defined. 50 The clear critical standard of ≤35 μg / mL and syringin content ≥5 mg / g overcomes the limitations of a single indicator and establishes a more scientific and accurate dual-effect indicator evaluation system that is strongly correlated with anti-fatigue efficacy. The prediction bias is significantly lower than that of traditional in vitro methods, enabling the evaluation results to leap from possible qualitative inferences to binary quantitative judgments of "yes or no", achieving a fundamental transformation from fuzzy to precise.

[0024] 3. This invention enables the seamless integration of anti-fatigue efficacy evaluation into the production quality control process for the first time, realizing the transformation from post-production testing to process control. It can be used for real-time monitoring, raw material screening, and process optimization, greatly improving the level of industrial technology. Attached Figure Description

[0025] Figure 1 Four-quadrant analysis diagram for the in vitro evaluation of the anti-fatigue efficacy of Acanthopanax senticosus tea. Detailed Implementation

[0026] To make the inventive objectives, technical solutions, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description.

[0027] The following examples are provided to facilitate a better understanding of the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0028] Example 1: The optimal preparation process of the Acanthopanax senticosus fruit and leaf compound tea provided by the present invention This embodiment takes the production of 1 kg of Acanthopanax senticosus fruit and leaf compound tea bags as an example. The specific preparation steps are as follows: (1) Raw material preparation: Select 5 kg of mature, unrotten Acanthopanax senticosus fruit, wash them thoroughly, and drain off the surface moisture; select 3.5 kg of tender, disease-free Acanthopanax senticosus leaves, wash them thoroughly, and drain off the surface moisture.

[0029] (2) Raw material processing 1. Segmented temperature-controlled roasting of leaf processing a) Put the drained Acanthopanax senticosus leaves into the intelligent electric heating blower tea frying machine.

[0030] b) First stage (high temperature blanching): Quickly raise the temperature of the wok to 182°C, add the leaves and stir-fry quickly for 28 seconds. Once the leaves soften and the aroma is released, remove them from the wok immediately.

[0031] c) Second stage (medium-low temperature slow roasting): Quickly transfer the withered leaves to another tea frying machine set at 118℃ and stir-fry slowly for 16 minutes until the leaves feel crisp to the touch and have a rich aroma.

[0032] d) After the stir-fried leaves cooled to room temperature, samples were taken. The retention rate of syringin was determined to be 92.6% by HPLC.

[0033] 2. Low-temperature cell wall breaking and astringency removal treatment of fruits: a) The residual heat from the blanching of the leaves is introduced into the fruit processing area through the control system to pre-dry the Acanthopanax senticosus fruits. After drying, the fruits are sent into an ultra-micro low-temperature cell-wall breaking machine. b) Set the core parameters of the equipment: temperature control is set to 38℃, spindle speed is set to 28000 rpm, and processing time is set to 6 minutes; c) After treatment, samples were taken for testing. The tannin content was determined using the Folin-Ciocalteu method to be 2.1 mg / g, which met the expected standard (≤2.5 mg / g).

[0034] (3) Compound 1. Weigh 400 g of processed Acanthopanax senticosus pulp powder and 300 g of stir-fried Acanthopanax senticosus leaves in a weight ratio of 40:30; to adjust the flavor, add 200 g of dried hawthorn powder and 100 g of crushed chrysanthemum as auxiliary ingredients.

[0035] 2. Place the above raw materials in a three-dimensional mixer and mix for 15 minutes until homogeneous to obtain Acanthopanax senticosus fruit and leaf compound tea bag.

[0036] Example 2: Evaluation method for the anti-fatigue effect of Acanthopanax senticosus tea provided by the present invention The process of the evaluation method for the anti-fatigue effect of Acanthopanax senticosus tea provided by this invention is as follows: Figure 1 As shown, the process includes testing reagents, sample processing and index determination, and efficacy assessment, as detailed below: I. Test Reagents (1) Sample testing a) Extraction solvent: 70% (v / v) aqueous ethanol solution.

[0037] b) Equipment: analytical balance, ultrasonic cleaner, constant temperature water bath, centrifuge, volumetric flasks, pipettes, etc.

[0038] (2) DPPH free radical scavenging activity detection a) Reagent: DPPH (1,1-diphenyl-2-trinitrophenylhydrazine), purity ≥ 97%, prepared into an ethanol stock solution with a concentration of 0.1 mmol / L, and stored in the dark and refrigerated.

[0039] b) Instrument: Ultraviolet-visible spectrophotometer.

[0040] (3) Detection of syringin content a) Core reagent: Syringin standard (purity ≥ 98%), used to prepare standard curve.

[0041] b) Chromatography system: High performance liquid chromatograph, equipped with a C18 column and ultraviolet detector.

[0042] c) Mobile phase: methanol and ultrapure water, using a gradient elution program.

[0043] II. Sample processing and index detection (1) Sample processing ① Sampling: After uniformly pulverizing the Acanthopanax senticosus tea product, pass it through an 80-mesh sieve. Accurately weigh 1000 g of sample powder and place it in a 50 mL stoppered conical flask; ② Extraction: Accurately add 25.00 mL of 70% (v / v) ethanol solution to the conical flask; ③Ultrasonic extraction: Place the conical flask in an ultrasonic cleaner and extract ultrasonically for 30 minutes at 60±2°C; ④ Volume adjustment and filtration: After the extract has cooled to room temperature, replenish the lost mass with 70% ethanol and shake well. Take an appropriate amount of extract into a centrifuge tube, centrifuge at 12000 rpm for 10 minutes, and filter the supernatant through a 0.45 μm microporous membrane. The resulting filtrate is the sample solution to be tested and is ready for use.

[0044] (2) DPPH free radical scavenging rate IC 50 Value determination ① Solution preparation: Weigh 20 mg of DPPH reagent into a beaker, dissolve it in anhydrous ethanol, and then dilute to a volumetric flask of 250 mL. Store in the dark.

[0045] ② Sample addition and reaction: Pipette 2 mL of the diluted solution into 2 mL of DPPH-ethanol solution in a test tube, and record this as A. i Following the method described above, 2 mL of diluted fermentation broth and 2 mL of DPPH-ethanol solution were replaced with 2 mL of anhydrous ethanol, and the absorbance was measured and recorded as A0 and A1. r (Blank control). The experiment was repeated 3 times.

[0046] ③Incubation: Let the reaction stand in the dark at room temperature for 30 min.

[0047] ④ Measurement: Immediately use a UV spectrophotometer to measure the absorbance value of each group at a wavelength of 517 nm.

[0048] ⑤ Calculation of DPPH free radical scavenging rate: K (%) = [1 - (A i -A r ) / A0]×100 ⑥ DPPH IC 50 Value calculation: With sample concentration as the X-axis and DPPH free radical scavenging rate percentage as the Y-axis, nonlinear regression fitting was performed using analysis software. 50 The value is the concentration value on the X-axis derived from the dose-response curve fitted by the software when Y=50%.

[0049] (3) Determination of syringin content ①Chromatographic conditions: Column: C18 reversed-phase column (250 mm × 4.6 mm, 5 μm); Column temperature: 30°C; Mobile phase: Phase A is methanol, Phase B is 0.1% phosphoric acid aqueous solution; Flow rate: 1.0 mL / min; Detection wavelength: 220 nm; Injection volume: 10 μL; Gradient elution program is shown in the table below: Table 1 Gradient elution conditions for syringin

[0050] ② Construction of standard curves Accurately weigh syringin standard, prepare a series of standard solutions with methanol, and inject them for determination under the chromatographic conditions described above. Plot a standard curve with peak area on the ordinate and standard concentration on the abscissa.

[0051] ③ Sample determination The prepared sample solution was injected and analyzed under the above chromatographic conditions. The peak area of ​​the syringin chromatographic peak was recorded, and the syringin content (mg / g) in the sample was calculated by substituting it into the standard curve equation.

[0052] III. Efficacy Determination The DPPH IC measured from the sample 50 The value and syringin content are compared with the judgment criteria established in this invention: if both DPPH IC are satisfied... 50 If the value is ≤35 μg / mL and the syringin content is ≥5 mg / g, the Acanthopanax senticosus tea sample is judged to have significant anti-fatigue effects; if any one or both indicators fail to meet the threshold standard, its anti-fatigue effects are judged to be insignificant.

[0053] In this embodiment, 20 samples of Acanthopanax senticosus tea were collected, and the anti-fatigue efficacy of each sample was determined using the evaluation method provided by this invention. Figure 1 As shown, the content of syringin was 5 mg / g and DPPH IC 50 A threshold of 35 μg / mL was used to divide the samples into quadrants, clearly separating the effective zone into the target group and the non-target group. The 10 samples falling into the effective zone were considered to have significant anti-fatigue potential.

[0054] Example 3: Verification of the accuracy of the evaluation method provided by the present invention To verify the accuracy and effectiveness of the evaluation method provided in Example 2 of this invention in determining the anti-fatigue efficacy of Acanthopanax senticosus tea, 15 Acanthopanax senticosus tea samples were randomly collected in this example. The syringin content and DPPH free radical scavenging rate IC50 in the samples were then determined according to the method in Example 2. 50 Value, according to the judgment criteria, syringin content ≥5 mg / g and DPPHIC 50 A value ≤35 μg / mL was used to determine whether the sample had anti-fatigue effects. The results are shown in Table 2.

[0055] Table 2. Determination of the anti-fatigue efficacy of 15 Acanthopanax senticosus tea samples

[0056] This embodiment then further verifies the accuracy of the above method's judgment results for 15 samples using a mouse weighted swimming test (standard method). The experimental steps are as follows: The experiment consisted of one blank control group and 15 experimental groups. Six healthy adult male mice, weighing 14–21g, were selected for each group. The control group was fed a basal diet and cooled boiled water. The experimental groups received a diet supplemented with 3% Acanthopanax senticosus tea and a 0.5% tea infusion. After 30 days of continuous feeding, 30 minutes after the mice's last meal, a lead weight equal to 5% of their body weight was placed on the base of their tails, and they were placed in a swimming tank with water temperature controlled at 25°C ± 0.5°C and a water depth of no less than 30 cm. The time from the start of swimming to the mice's exhaustion and death was recorded. If the swimming time of the experimental group mice was significantly longer than that of the control group (…), the experimental group mice were considered to have… p If the value is less than 0.05, the experiment is considered positive, indicating that the Acanthopanax senticosus tea sample has an anti-fatigue effect.

[0057] Experimental tests showed that mice fed Acanthopanax senticosus tea samples 1, 2, 3, 4, 8, 9, 10, 11, 12, and 13 had significantly longer swimming times than the control group. p <0.05), which is about 35-39% longer than the control group; while the swimming time of mice fed with samples from other experimental groups was only slightly longer than or comparable to the control group. This result indicates that Acanthopanax senticosus tea samples 1-4 and 8-13 have significant anti-fatigue effects, while other samples do not have anti-fatigue effects, which is consistent with the judgment results in Table 2. This also fully proves that the judgment criteria of the evaluation method provided by the present invention are completely effective and the judgment results are accurate, and can be used for the quality judgment of Acanthopanax senticosus tea.

[0058] Example 4: Verification of the universality of the evaluation method provided by the present invention To verify that the evaluation method provided by this invention can be used to evaluate Acanthopanax senticosus tea products from different origins or processes, and has broad applicability, this embodiment collected Acanthopanax senticosus tea samples prepared by the optimal process of this invention, as well as Acanthopanax senticosus tea from different origins and processes for comparative evaluation. To maximize sample diversity, a total of 30 Acanthopanax senticosus tea samples were prepared, and the materials were grouped as follows: Group A (prepared using the optimal process of this invention, n=6): prepared according to the optimal embodiment 1 of this invention; Group B (different origins, n=12): using raw materials from Heilongjiang, Shaanxi and Hebei, prepared according to a uniform but non-optimal basic process to highlight the differences in the raw materials themselves; Group C (different process groups, n=9): Using the same batch of raw materials from Heilongjiang, the leaves were stir-fried in a single temperature zone at three different fixation temperatures of 160℃, 180℃ and 200℃ respectively, and the influence of process parameters was investigated. Group D (control and imbalance group, n=3): included one commercially available Acanthopanax senticosus leaf tea product and two self-made samples with an imbalanced ratio of fruit to leaf (fruit:leaf = 1:0.5, fruit:leaf = 1:1.5).

[0059] The syringin content and DPPH free radical scavenging rate (IC50) of all 30 samples were determined according to the method in Example 2. 50 The value is used to determine whether the sample has anti-fatigue effects based on the judgment criteria.

[0060] Upon testing, out of the 30 samples in this embodiment, 8 samples were deemed to meet the standards, including 6 samples prepared using the optimal process and 2 samples prepared using a blanching temperature of 180℃ from different process groups. A total of 22 samples did not meet the standards. The syringin content and DPPH IC of the meeting and non-meeting groups were compared. 50 The values ​​all showed highly significant differences ( p < 0.001). The two indicators maintained a significant negative correlation across the entire sample range (Pearson r = -0.84, p < 0.001 indicates that the evaluation system has consistent and robust internal logic. All compliant samples are concentrated in the efficacy-significant zone in the upper right corner. All non-compliant samples, regardless of whether the cause is poor raw material origin, improper processing technology, or imbalanced formula ratio, are accurately excluded from the efficacy-significant zone and distributed in the other three quadrants. This fully demonstrates that the dual-indicator thresholds set in this invention can be universally applied to Acanthopanax senticosus products from different sources and processes, and can accurately and effectively identify efficacy potential. The core production area, Heilongjiang, has the best raw material quality and is easy to meet the standards; the second-best production area, Shaanxi, can also meet the standards under reasonable processes; while the inferior production area, Hebei, cannot meet the standards due to insufficient internal components. The roasting temperature has a great impact on DPPH activity (antioxidant capacity). Too low a temperature (160℃) may result in insufficient enzyme inactivation, while too high a temperature (200℃) will directly destroy antioxidant components, causing even if the syringin content is close to the threshold, it will still be judged as ineffective due to insufficient DPPH activity. This highlights the superiority and necessity of dual-indicator evaluation compared to single-indicator evaluation.

[0061] The above results indicate that the method established in this invention, characterized by "syringin content ≥ 5 mg / g and DPPH IC," is effective. 50 The in vitro evaluation method with a threshold of ≤35 μg / mL has broad applicability and high robustness. Its discrimination results are not limited by the specific origin of Acanthopanax senticosus raw materials or specific processing technology, and can serve as a universal and reliable standard for screening and quality control of the anti-fatigue efficacy potential of various Acanthopanax senticosus tea products.

[0062] Example 5: Screening of Dual-Effect Indicators The results of the above examples fully demonstrate that the present invention provides a solution with "syringin content ≥ 5 mg / g and DPPH IC". 50The evaluation system constructed using the dual-effect index of "≤35 μg / mL" is reliable, accurate, and widely applicable when used to evaluate Acanthopanax senticosus tea samples. To verify the significant accuracy and superiority of the dual-effect index provided by this invention compared to other single or combined indicators, this embodiment further employs different indicator evaluation standards to determine the anti-fatigue effect of Acanthopanax senticosus tea, as detailed below: Standard 1: When the content of syringin is ≥5 mg / g, it is determined that Acanthopanax senticosus tea has anti-fatigue effects; Standard 2, DPPH IC 50 When the value is ≤35 μg / mL, it is determined that Acanthopanax senticosus tea has anti-fatigue effects; Standard 3: Syringin content ≥ 5 mg / g and DPPH IC 50 When the value is ≤35 μg / mL, it is determined that Acanthopanax senticosus tea has anti-fatigue effects; Standard 4: When the content of syringin is ≥5 mg / g and the total flavonoid content is ≥40 mg / g, it is determined that Acanthopanax senticosus tea has anti-fatigue effects; Standard 5: Syringin content ≥ 5 mg / g and ABTS IC 50 When the value is ≤40 μg / mL, it is determined that Acanthopanax senticosus tea has anti-fatigue effects; In this embodiment, 10 commercially available Acanthopanax senticosus tea samples were randomly selected, and then the content of syringin and DPPHIC was determined. 50 Value, total flavonoid content and ABTS IC 50 The five criteria were then used to determine whether the Acanthopanax senticosus tea sample had anti-fatigue effects. Finally, a weighted swimming experiment was conducted on mice to verify whether the sample had anti-fatigue effects. The results of the five criteria are shown in Table 3.

[0063] Table 3.5 Judgment Criteria for Determining Whether 10 Acanthopanax senticosus Tea Samples Have Anti-fatigue Effects

[0064] Note: "Yes" means that the standard judges the sample to have anti-fatigue effect, and "No" means that the standard judges the sample to have no anti-fatigue effect.

[0065] The results of the weighted swimming experiment in mice showed that the swimming time of mice fed samples 1, 2, 5, and 10 was comparable to that of the control group, while the swimming time of mice in the other experimental groups was significantly longer than that of the control group. This indicates that samples 1, 2, 5, and 10 of Acanthopanax senticosus tea do not have significant anti-fatigue effects, while samples 3, 4, and 6-9 have significant anti-fatigue effects. This is consistent with the judgment results of Standard Three, further proving the accuracy of the evaluation system provided by this invention, and also proving that the judgment results using a single indicator or other combined indicators are one-sided. This is because the content of a single chemical component or an antioxidant indicator can only reflect one aspect of the product's efficacy, while anti-fatigue involves the coordination of multiple aspects such as antioxidant, energy metabolism, and neural regulation. Therefore, using only syringin as an indicator cannot reflect the overall functional activity of the product, and may lead to the risk of meeting the ingredient standard but not the efficacy. Using only DPPH value as an indicator cannot distinguish the source of activity, which may lead to the loophole of replacing the core functional ingredients with cheap antioxidants, lacking scientific basis. Using syringin + total flavonoids as a detection indicator is still within the category of component content, lacking direct functional activity characterization; using syringin + ABTS IC 50 The evaluation system for ABTS may lack sufficient scientific basis because its correlation with anti-fatigue oxidative stress pathways may not be as direct as that with DPPH. It is noteworthy that sample 4 was misjudged as invalid according to standard four, demonstrating that even if the component content meets the standard, efficacy cannot be accurately predicted without functional activity characterization, highlighting the necessity of introducing the DPPH activity indicator in this invention. In summary, the present invention provides a method based on "syringin content ≥ 5 mg / g and DPPH IC50". 50 The dual-effect index evaluation system of "≤35 μg / mL" provides a sufficient scientific basis for determining the anti-fatigue effect of Acanthopanax senticosus tea. The judgment is accurate, efficient and suitable for promotion and application.

[0066] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any modifications and alterations made by those skilled in the art without departing from the spirit and scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for evaluating the anti-fatigue efficacy of Acanthopanax senticosus tea, characterized in that, The evaluation indicators of the method include syringin content and DPPH free radical scavenging rate (IC50). 50 value.

2. The method as described in claim 1, characterized in that, The criterion for determining the effectiveness of the method is as follows: when the syringin content is ≥5 mg / g and the DPPH free radical scavenging rate is IC50... 50 When the concentration of Acanthopanax senticosus tea is ≤35 μg / mL, it is determined that Acanthopanax senticosus tea has anti-fatigue effects; when the concentration of syringin is ≥5 mg / g and the DPPH free radical scavenging rate IC50 is not met, the anti-fatigue effect is determined. 50 When the concentration is ≤35 μg / mL, Acanthopanax senticosus tea is determined to have no anti-fatigue effect.

3. The method as described in claim 2, characterized in that, Includes the following steps: (1) Preparation and processing of Acanthopanax senticosus tea samples; (2) Syringin content and DPPH free radical scavenging rate IC 50 Value determination; (3) Efficacy determination.

4. The method as described in claim 3, characterized in that, in, The Acanthopanax tea comprises Acanthopanax fruit and / or leaves.

5. A model for evaluating the anti-fatigue efficacy of Acanthopanax senticosus tea, characterized in that, Including syringin content and DPPH free radical scavenging rate IC 50 Two evaluation parameters are used.

6. The model as described in claim 5, characterized in that, The model's judgment rule is: when the syringin content is ≥5 mg / g and the DPPH free radical scavenging rate is IC50... 50 When the concentration of Acanthopanax senticosus tea is ≤35 μg / mL, it is determined that Acanthopanax senticosus tea has anti-fatigue effects; when the concentration of syringin is ≥5 mg / g and the DPPH free radical scavenging rate IC50 is not met, the anti-fatigue effect is determined. 50 When the concentration is ≤35 μg / mL, Acanthopanax senticosus tea is determined to have no anti-fatigue effect.

7. A model for evaluating the anti-fatigue efficacy of Acanthopanax senticosus tea, characterized in that, The model includes syringin content and DPPH free radical scavenging rate IC. 50 Two evaluation parameters are used.

8. The use as described in claim 7, characterized in that, The model's judgment rule is: when the syringin content is ≥5 mg / g and the DPPH free radical scavenging rate is IC50... 50 When the concentration of Acanthopanax senticosus tea is ≤35 μg / mL, it is determined that Acanthopanax senticosus tea has anti-fatigue effects; when the concentration of syringin is ≥5 mg / g and the DPPH free radical scavenging rate IC50 is not met, the anti-fatigue effect is determined. 50 When the concentration is ≤35 μg / mL, Acanthopanax senticosus tea is determined to have no anti-fatigue effect.

9. The use of syringin in preparing a model for evaluating the anti-fatigue efficacy of Acanthopanax senticosus tea, characterized in that, The model uses syringin content and DPPH free radical scavenging rate IC50 as parameters. 50 The value is used to determine the anti-fatigue effect of Acanthopanax senticosus tea.

10. A kit for carrying out the method as described in any one of claims 1-4, characterized in that, The kit includes reagents for determining DPPH free radical scavenging rate and reagents for determining syringin content.

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