Standardized planting technology and quality control method of traditional Chinese medicinal materials in imitated wild environment

Abstract

The application discloses a kind of traditional Chinese medicinal materials wild environment simulation standardization planting technology and quality control method. Including the following steps: in the rhizome of polygonatum cryodendron of period, artificial control water treatment is carried out to planting area to create drought stress;During stress, the physiological index of polygonatum cryodendron is monitored;When the physiological index reaches preset first threshold value, it is determined that stress has taken effect, and effective stress duration is started to be calculated;When the physiological index falls to preset second threshold value, stress is terminated and water supply is restored;After harvesting, polygonatum cryodendron is detected with chemical potency index and biological potency index.The traditional Chinese medicinal materials wild environment simulation standardization planting technology and quality control method of the embodiment of the application, by with the change of plant physiological index as the criterion for stress initiation and termination, the application of drought stress is matched with the actual physiological response of plant, improves the accuracy and repeatability of stress treatment, and stably promotes the improvement of diosgenin content and in vitro antioxidant potency in polygonatum cryodendron.

Description

Technical Field

[0001] This application relates to the technical field of medicinal plant cultivation, and in particular to a standardized cultivation technique and quality control method for Chinese medicinal herbs in a simulated wild environment. Background Technology

[0002] Polygonatum rhizome is the dried rhizome of Polygonatum yunnanensis, Polygonatum sibiricum, or Polygonatum multiflorum, all belonging to the Liliaceae family. It is a commonly used traditional Chinese medicine for tonifying qi and nourishing yin. The quality of Polygonatum rhizome is highly dependent on the shady, damp, and humus-rich natural environment under the forest canopy. With the gradual depletion of wild resources, semi-wild cultivation under the forest canopy has become the main way to ensure the quality of Polygonatum rhizome.

[0003] However, the timing, intensity, and duration of natural stress factors (such as periodic drought) in semi-wild cultivation environments vary depending on the year and plot, making them difficult to control artificially. This results in inconsistent accumulation levels of medicinal components in different batches of Polygonatum. While existing technologies include artificially controlling water to induce drought stress and enhance the production of secondary metabolites in Polygonatum, the initiation and termination of stress usually rely on a preset fixed number of days, lacking real-time feedback on the plant's physiological state. This can easily lead to limited effectiveness due to insufficient stress or yield loss due to excessive stress. In terms of quality control, current standards mostly use indicators such as polysaccharides for evaluation, which are insufficient to effectively distinguish the quality differences of Polygonatum grown using different methods and at different ages. The quality advantages of semi-wild cultivation products lack quantifiable evaluation methods, making it difficult for high-quality products to gain corresponding market recognition. Summary of the Invention

[0004] This application aims to at least partially address one of the technical problems in the related art.

[0005] Therefore, one objective of this application is to provide a standardized cultivation technology and quality control method for Chinese medicinal materials in a simulated wild environment. After harvesting, the chemical potency index and biological potency index are used for dual testing, and the lower one is used for grading, to ensure that the product meets the corresponding grade requirements in both the content of components and the biological activity, thus providing a quantifiable evaluation basis for the high quality and high price of simulated wild Polygonatum.

[0006] To achieve the above objectives, the first aspect of this application proposes a standardized cultivation technique and quality control method for Chinese medicinal herbs in a simulated wild environment, comprising the following steps: During the rhizome enlargement period of Polygonatum, artificial water control was implemented in the planting area to create drought stress; during the stress period, the physiological indicators of Polygonatum were monitored. When the physiological indicators reach a preset first threshold, it is determined that the stress has taken effect, and the effective stress duration is calculated. When the physiological indicators drop back to a preset second threshold, the stress is terminated and the water supply is restored. Chemical and biological efficacy indicators were tested on the harvested Polygonatum sibiricum. Based on the test results of the two indicators, Polygonatum is divided into at least two quality grades, with the grade determined by the lower of the two indicators.

[0007] The standardized cultivation technology and quality control method for Chinese medicinal materials in a simulated wild environment of this application use changes in the plant's own physiological indicators as the criteria for stress initiation and termination, so that the application of drought stress matches the actual physiological response of the plant, thereby improving the accuracy and repeatability of stress treatment and steadily promoting the increase of diosgenin content and in vitro antioxidant efficacy in Polygonatum sibiricum.

[0008] In addition, the standardized cultivation technology and quality control method for simulated wild environment of Chinese medicinal materials proposed in this application may also have the following additional technical features: In one embodiment of this application, the chemical potency index is the diosgenin content, and the biological potency index is the in vitro antioxidant potency.

[0009] In one embodiment of this application, the quality grade is divided into three levels: premium, first grade, and qualified. Among them, if the diosgenin content is greater than or equal to 0.50 mg / g and the ORAC value is greater than or equal to 80 μmol TE / g, it is classified as extra grade; If the diosgenin content is between 0.30 mg / g and 0.49 mg / g and the ORAC value is between 50 μmol TE / g and 79 μmol TE / g, it is classified as Grade 1. If the diosgenin content is between 0.15 mg / g and 0.29 mg / g and the ORAC value is between 25 μmol TE / g and 49 μmol TE / g, then it is considered qualified.

[0010] In one embodiment of this application, the monitoring of physiological indicators of Polygonatum sibiricum includes the following monitoring methods: Stem micro-change monitoring: The stem diameter change is continuously monitored by a stem micro-change sensor, and the maximum daily shrinkage of the stem exceeding a preset threshold is used as the criterion for stress effectiveness. Canopy temperature difference monitoring uses infrared temperature sensors to monitor the air temperature difference in the canopy, with a sustained positive temperature difference serving as the criterion for the effectiveness of stress. Sampling and testing are conducted by periodically sampling to detect the relative water content or proline content of the leaves, and the detection value reaching a preset threshold is used as the criterion for the stress to take effect.

[0011] In one embodiment of this application, during the duration of the effective stress, the aboveground parts of Polygonatum are subjected to mild mechanical trauma treatment to form a combined stress of drought stress and trauma stress.

[0012] In one embodiment of this application, the mild mechanical trauma treatment is performed on the 3rd to 5th day after the drought stress enters its effective period; The trauma method involves gently pressing the surface of the Polygonatum leaves with a roller, or using a row of needles to lightly prick the functional leaves, so that the damaged area of ​​a single leaf does not exceed 10% of the total leaf area.

[0013] In one embodiment of this application, before the drought stress regulation, a seed source screening step is further included: pre-detecting the diosgenin content and in vitro antioxidant potency of candidate Polygonatum seed sources, and screening seed sources that reach the preset threshold for both indicators as seedlings; The preset thresholds are: diosgenin content greater than or equal to 0.30 mg / g, and in vitro antioxidant efficacy ORAC value greater than or equal to 50 μmol TE / g.

[0014] In one embodiment of this application, before harvesting, a potency-guided harvesting step is also included: samples from the planting plots are collected for rapid detection of antioxidant potency, and after confirming that the potency has reached the preset target range, large-scale harvesting is carried out.

[0015] In one embodiment of this application, a process traceability step is also included: uploading the coercion initiation time, the moment when the physiological indicators reach a preset first threshold, the data on changes in physiological indicators during the coercion period, the coercion termination time, and the total effective coercion duration to a blockchain platform to generate an unalterable digital identity card.

[0016] In one embodiment of this application, a valence feedback optimization step is also included: the valence data obtained from the terminal quality evaluation is correlated with the stress regulation parameters to identify the optimal combination of physiological index thresholds and the effective stress duration parameter, and the parameter is applied to the stress regulation of the next round of planting.

[0017] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0018] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 This is a flowchart illustrating the standardized cultivation techniques and quality control methods for medicinal herbs in a simulated wild environment, based on the present application. Figure 2 A bar chart comparing the diosgenin content of different treatment groups based on the standardized cultivation techniques and quality control methods of the simulated wild environment for Chinese medicinal materials in this application; Figure 3This is a bar chart comparing the ORAC antioxidant potency of different treatment groups based on the standardized cultivation techniques and quality control methods of the simulated wild environment for Chinese medicinal materials in this application; Figure 4 This is a daily variation curve of relative water content of leaves during stress, based on the standardized cultivation techniques and quality control methods for medicinal herbs in a simulated wild environment according to this application. Figure 5 This is a daily variation curve of proline content during the stress period of medicinal materials in the standardized cultivation technology and quality control method of simulated wild environment according to this application; Figure 6 Images of Polygonatum sibiricum grown as a blank control in the standardized cultivation techniques and quality control methods for medicinal materials in a simulated wild environment according to this application; Figure 7 Images of Polygonatum sibiricum growing after trauma, based on the standardized cultivation techniques and quality control methods for medicinal materials in a simulated wild environment according to this application; Figure 8 Images of Polygonatum sibiricum grown under drought stress in accordance with the standardized cultivation techniques and quality control methods for medicinal materials in a simulated wild environment as described in this application; Figure 9 Images of Polygonatum sibiricum grown under trauma and drought stress, according to the standardized cultivation techniques and quality control methods for medicinal materials in a simulated wild environment as described in this application. Detailed Implementation

[0019] Embodiments of this application are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application. Rather, embodiments of this application include all variations, modifications, and equivalents falling within the spirit and scope of the appended claims.

[0020] like Figure 1-8 As shown in the embodiments of this application, the standardized cultivation technology and quality control method for Chinese medicinal materials in a simulated wild environment include the following steps: During the rhizome enlargement period of Polygonatum, artificial water control was implemented in the planting area to create drought stress; during the stress period, the physiological indicators of Polygonatum were monitored. When the physiological indicators reach the preset first threshold, it is determined that the stress has taken effect and the effective stress duration is calculated. When the physiological indicators drop back to the preset second threshold, the stress is terminated and the water supply is restored. Chemical and biological efficacy indicators were tested on the harvested Polygonatum sibiricum. Based on the test results of the two indicators, Polygonatum is divided into at least two quality grades, with the grade determined by the lower of the two indicators.

[0021] Specifically, in actual operation: 1.1. Initiation of drought stress.

[0022] During the rapid expansion period of the rhizomes of Polygonatum (usually from July to September each year), retractable rain shelters are used to block natural rainfall in the planting area, causing the soil moisture content to gradually decrease, thereby creating drought stress conditions. The rain shelters are only deployed during rainfall and retracted on sunny days to maintain normal ventilation and light.

[0023] 1.2. Monitoring of physiological indicators.

[0024] During periods of stress, the physiological state of the Polygonatum plants is monitored. Monitoring methods can be selected based on the conditions of the base: where resources permit, stem micro-change sensors can be installed to continuously record changes in stem diameter; or infrared temperature sensors can be used to monitor the temperature difference between the canopy and the air; where resources are limited, technicians can collect leaf samples periodically to determine the relative water content or proline content of the leaves.

[0025] 1.3. Determination of the validity of coercion.

[0026] When a significant change in physiological indicators is detected and reaches a pre-set first threshold, the plant is determined to have entered a state of stress, and the effective duration of stress is then calculated. For example, a decrease in the relative water content of leaves from the normal 85% or above to below 75%, or a significant accumulation of proline content to more than twice the pre-stress level, can be used as indicators of effective stress.

[0027] 1.4. The maintenance and termination of coercion.

[0028] After the stress takes effect, continue to maintain controlled watering. Continue to monitor physiological indicators daily. When physiological indicators gradually recover and fall back to the pre-set second threshold, it indicates that the plant has adapted to the current stress intensity or that the stress has been sufficiently effective. At this point, terminate the stress, remove the rain shelter, and restore normal water supply. If physiological indicators do not fall back due to weather or other reasons, set a maximum stress duration as a safety boundary, and forcibly terminate the stress upon expiration.

[0029] 1.5. Post-harvest potency testing.

[0030] After harvesting Polygonatum, samples are taken for two types of testing: one is chemical potency index testing, which determines the content of specific medicinal components; the other is biological potency index testing, which determines the in vitro biological activity of the medicinal material (such as antioxidant capacity).

[0031] 1.6. Classification of quality grades.

[0032] Based on the test results of chemical and biological potency indicators, Polygonatum is classified into at least two quality grades, such as superior and qualified. The grading rule is: the lower of the grades corresponding to the two indicators is taken as the final grade. For example, if a sample meets the superior standard for chemical potency but only the qualified standard for biological potency, then the sample is ultimately classified as qualified. This rule ensures that the product must reach the corresponding level in both types of indicators to obtain the corresponding grade.

[0033] In one embodiment of this application, the chemical potency index is the content of diosgenin, and the biological potency index is the in vitro antioxidant potency.

[0034] Specifically, in actual operation: 2.1. Detection of diosgenin content.

[0035] Diosgenin is a hydrolysis product of steroidal saponins in Polygonatum sibiricum, and its content can reflect the total amount of saponins in Polygonatum sibiricum to a certain extent.

[0036] In practice, the Polygonatum sibiricum sample is first dried and pulverized, then extracted with ethanol using ultrasound. After acid hydrolysis, the extract is further extracted with an organic solvent to separate diosgenin from other impurities. High-performance liquid chromatography (HPLC) is then used for determination, typically employing an evaporative light scattering detector (EVS). The content of diosgenin in the sample is calculated by comparing its peak area with that of a diosgenin reference standard, and the result is expressed in milligrams per gram of dried medicinal material.

[0037] 2.2. In vitro antioxidant potency detection.

[0038] In vitro antioxidant potency is used to measure the bioactivity of Polygonatum sibiricum in scavenging free radicals and resisting oxidative damage.

[0039] In practice, the oxygen radical absorbance capacity (ORAC) method can be used for determination. First, the Polygonatum sibiricum sample is extracted with a suitable solvent. The extract is then diluted and added to a reaction system containing sodium fluorescein and a free radical initiator. As free radicals damage fluorescein, the fluorescence signal gradually decays. The presence of antioxidants slows down this decay process. Changes in fluorescence intensity are continuously recorded using a fluorescence microplate reader. The area under the curve (AUC) protecting fluorescein is calculated and compared with the protective effect of the standard antioxidant Trolox. The results are expressed as the antioxidant capacity equivalent to micromoles of Trolox per gram of dried medicinal material.

[0040] 2.3. Considerations for selecting the two indicators.

[0041] Diosgenin was chosen as the chemical potency indicator because it has certain specificity in Polygonatum sibiricum, the reference standard is readily available, the detection method is mature, and the results are reproducible. In vitro antioxidant potency was chosen as the biological potency indicator because this indicator has a certain biological correlation with the traditional effects of Polygonatum sibiricum, such as "delaying aging," and the detection method is widely used in related fields and is relatively simple to operate.

[0042] Furthermore, the quality grades are divided into three levels: Special Grade, Grade 1, and Qualified. Among them, if the diosgenin content is greater than or equal to 0.50 mg / g and the ORAC value is greater than or equal to 80 μmol TE / g, it is classified as extra grade; If the diosgenin content is between 0.30 mg / g and 0.49 mg / g and the ORAC value is between 50 μmol TE / g and 79 μmol TE / g, it is classified as Grade 1. If the diosgenin content is between 0.15 mg / g and 0.29 mg / g and the ORAC value is between 25 μmol TE / g and 49 μmol TE / g, then it is considered qualified.

[0043] The specific numerical thresholds for the three grades of premium, first grade, and qualified were initially set based on the data distribution patterns after batch determination of diosgenin content and ORAC antioxidant efficacy of Polygonatum samples with different planting methods and different growth years.

[0044] In practice, samples of Polygonatum were collected from different sources, including those grown in semi-wild forests for more than six years, those grown in semi-wild forests for three to five years, and those grown in ordinary fields for three years. The diosgenin content and ORAC value were measured. The results showed that the two indicators of the samples from different planting methods exhibited a relatively obvious stratified distribution, and the above three grade intervals were divided accordingly.

[0045] Among them, the premium grade corresponds to samples with both indicators at a high level, with diosgenin content reaching 0.50 mg / g or higher and ORAC value reaching 80 μmol TE / g or higher. These samples are usually from forest-grown semi-wild planting plots with a long growth period and sufficient natural environmental stress. The first grade corresponds to samples with both indicators at a medium-to-high level, with the indicator range between premium and qualified. The qualified grade corresponds to samples with both indicators reaching the entry level of this grading system, with diosgenin content not less than 0.15 mg / g and ORAC value not less than 25 μmol TE / g. Polygonatum with a value lower than this threshold is not included in this grading system.

[0046] When two indicators of a sample fall into different grade ranges, the lower grade shall prevail. For example, if a sample has a diosgenin content that meets the premium standard, but its ORAC value only meets the first grade standard, then the sample shall be classified as first grade. This rule, to a certain extent, ensures that products that obtain a certain grade meet the corresponding levels in both chemical and biological potency.

[0047] It should be noted that the specific numerical thresholds mentioned above are initially set based on the current sample data and detection conditions. As the sample size accumulates, the detection methods are optimized, and the research on the pharmacodynamic material basis of Polygonatum is deepened, these thresholds can be adjusted and optimized in subsequent practice.

[0048] In one embodiment of this application, physiological indicators of Polygonatum are monitored, and the monitoring method includes: Stem micro-change monitoring: The stem diameter change is continuously monitored by a stem micro-change sensor, and the maximum daily shrinkage of the stem exceeding a preset threshold is used as the criterion for stress effectiveness. Canopy temperature difference monitoring uses infrared temperature sensors to monitor the air temperature difference in the canopy, with a sustained positive temperature difference serving as the criterion for the effectiveness of stress. Sampling and testing are conducted by periodically sampling to detect the relative water content or proline content of the leaves, and the detection value reaching a preset threshold is used as the criterion for the stress to take effect.

[0049] In actual planting bases, one of the three monitoring methods mentioned above can be selected for implementation based on the available equipment.

[0050] If using stem micro-change monitoring, the stem micro-change sensor should be installed at the base of the pseudostem of the Polygonatum plant before the onset of stress. The clamping force of the sensor should be adjusted to ensure a tight fit to the stem surface without causing mechanical damage. The sensor is connected to a field data logger via a data cable, which records changes in stem diameter at preset time intervals. Under normal water supply conditions, the stem diameter of Polygonatum exhibits a regular diurnal contraction and diurnal expansion fluctuation throughout the day. After entering drought stress, the daytime contraction amplitude gradually increases, while the nighttime recovery amplitude decreases, and the maximum daily contraction gradually rises. When the monitoring data shows that the maximum daily stem contraction continuously exceeds the preset threshold, the stress is considered to have taken effect, and timing begins. The recovery of stem diameter is then continuously monitored as a reference for whether to terminate the stress.

[0051] If canopy temperature difference monitoring is used, infrared temperature sensors need to be installed within the planting area, ensuring their field of view covers the entire canopy area of ​​the Polygonatum odoratum. Simultaneously, an air temperature sensor should be installed in the same area as a reference. The sensors synchronously collect canopy surface temperature and air temperature at regular time intervals, calculating the difference between the two. Under normal water supply conditions, Polygonatum odoratum dissipates heat through transpiration, and the canopy temperature is usually slightly lower than or close to the air temperature. As drought stress develops, stomata gradually close, transpiration cooling weakens, and the canopy temperature gradually rises. When the canopy-air temperature difference changes from a negative or zero value to a sustained positive value, and several consecutive readings remain positive, the stress is considered to have taken effect. Thereafter, temperature difference changes are continuously monitored, and when the temperature difference returns to the pre-stress level, it can be used as a reference for terminating the stress.

[0052] If sampling is used for testing, several fixed sampling points should be selected within the planting area, and functional leaf samples should be collected at fixed times each day. After collection, the samples should be placed in moisturizing bags and transferred to a simple workbench at the field edge for processing as soon as possible. The testing indicators can be one or both of the following: relative leaf water content or proline content. The method for determining the relative leaf water content is as follows: first, weigh the fresh weight of the leaves; then, immerse the leaves in clean water until they are fully saturated and weigh them again; finally, dry them and weigh them again. The relative water content of the leaves is then calculated using a formula.

[0053] Proline content can be determined using a kit method. Leaves are chopped and extracted with a specific solution. After a colorimetric reaction, the absorbance is read using a colorimeter or portable spectrophotometer, and the proline concentration is calculated. When the detected value reaches a preset threshold, such as when the relative water content of the leaves drops below 75% or the proline content rises to more than twice the pre-stress level, stress is considered to have taken effect. Sampling and testing continue daily thereafter. When the detected value falls back to the preset termination threshold, stress can be terminated and water supply restored.

[0054] In one embodiment of this application, during the effective stress period, the aboveground parts of Polygonatum are subjected to mild mechanical trauma treatment to form a combined stress of drought stress and trauma stress.

[0055] Furthermore, treatment for mild mechanical trauma was administered on days 3 to 5 after the drought stress entered its effective period; The trauma method involves gently pressing the surface of the Polygonatum leaves with a roller, or using a row of needles to lightly prick the functional leaves, so that the damaged area of ​​a single leaf does not exceed 10% of the total leaf area.

[0056] Three to five days after the drought stress enters its effective period, the plant is already in a confirmed stress state. At this time, a mild mechanical wound is applied to the above-ground parts of Polygonatum to introduce a wound signal, so that drought stress and wound stress form a compound stress effect.

[0057] Specifically, the wound treatment is carried out as follows: Take a lightweight roller, 30cm wide and weighing approximately 250g / cm width, and slowly and evenly push it along the top of the plant canopy between the rows of Polygonatum plants, so that the roller lightly presses on the leaf surface. During the pushing process, the roller uses its own weight to generate slight pressure on the leaf, causing local indentations on the leaf surface without breaking or falling off.

[0058] Press down once per row, without repeated rolling. After treatment, randomly select 10 plants to check the damage. Estimate the damaged leaf area of ​​a single plant to be about 5% to 8% of the total leaf area of ​​that plant, and keep it below 10%.

[0059] Alternatively, a row of needles can be used for wound treatment: Take a row of multi-needle needles spaced 1cm apart, select 3 to 5 fully unfolded functional leaves from the middle of each Polygonatum plant, and evenly prick 8 to 10 small holes on each leaf surface, with a hole diameter of about 0.3mm, just enough to pierce the epidermis but not penetrate the leaf. Either method can be chosen based on the site conditions and operating habits; both methods are equally effective.

[0060] After the wound treatment is completed, the plant continues to be under drought stress without additional watering until the monitoring signal indicates that the stress has ended.

[0061] To verify the effects of combined stress, the following four sets of comparative experiments were set up on the same plot of land at the base: Group A (drought stress only): Drought stress was applied using a dynamic feedback method. No trauma treatment was applied during the stress period, and normal water supply was restored after the stress ended.

[0062] Group B (Combined Treatment of Drought Stress and Trauma): On the 4th day after the drought stress entered the effective period, the above-mentioned roller trauma treatment was applied once, and the rest of the operation was the same as Group A.

[0063] Group C (Trauma Treatment Only): Under normal water supply conditions, the same roller trauma treatment was applied once at the same time as Group B, without any drought stress.

[0064] Group D (blank control): Normal water management, no drought stress treatment, and no trauma treatment. Natural rainfall combined with routine forest soil moisturizing served as the baseline reference for the other three groups.

[0065] Each treatment group consisted of 30 plants, with isolation rows at least 2 meters wide between groups to prevent lateral soil moisture infiltration. After harvesting, the diosgenin content and ORAC antioxidant potency of the rhizomes of each group were measured.

[0066] The average values ​​of diosgenin content and ORAC value for each group were as follows: Group D (blank control): diosgenin content was 0.22 mg / g, ORAC value was 35 μmol TE / g; Group A (drought stress only): diosgenin content was 0.46 mg / g, ORAC value was 72 μmol TE / g; Group C (wound treatment only): diosgenin content was 0.28 mg / g, ORAC value was 42 μmol TE / g; Group B (drought stress + wound treatment): diosgenin content was 0.58 mg / g, ORAC value was 92 μmol TE / g.

[0067] The test results show that both drought stress and trauma treatments have a certain effect on increasing the content of diosgenin and antioxidant potency of diosgenin. However, when the two are applied in combination at an appropriate time, the increase in both indicators is greater than the sum of the increases of the individual treatments, showing a synergistic effect. Group B's comprehensive grade assessment reached the top grade standard, Group A's comprehensive grade was Grade 1, and Groups C and D's comprehensive grades were both qualified.

[0068] It should be noted that the above data are based on the conditions of this experiment, and the specific values ​​may fluctuate due to differences in planting environment, variety, and operating parameters.

[0069] In one embodiment of this application, before drought stress regulation, a seed source screening step is also included: pre-detecting the diosgenin content and in vitro antioxidant potency of candidate Polygonatum seed sources, and screening seed sources that meet the preset threshold for both indicators as seedlings; The preset thresholds are: diosgenin content greater than or equal to 0.30 mg / g, and in vitro antioxidant efficacy ORAC value greater than or equal to 50 μmol TE / g.

[0070] In practice, a small amount of tissue samples were cut from the rhizomes of the selected Polygonatum plants, dried, pulverized, and then tested. The content of diosgenin was determined by high-performance liquid chromatography (HPLC), and the in vitro antioxidant potency was determined by the oxygen free radical absorbance assay.

[0071] The test results were compared with preset thresholds. Seed sources were selected when the diosgenin content was greater than or equal to 0.30 mg / g and the ORAC value was greater than or equal to 50 μmol TE / g. Seed sources that did not meet these thresholds were not included in the subsequent planting process. The selected seed source rhizomes were cut into pieces, the cut surfaces were disinfected, and then used for seedling cultivation using conventional methods or directly as seed tubers.

[0072] The practical basis for setting the above threshold lies in the accumulation of data from various sources of Polygonatum sibiricum germplasm from this base and surrounding production areas over many years. This data revealed that germplasm with a diosgenin content below 0.30 mg / g or an ORAC value below 50 μmol TE / g, even under the same semi-wild cultivation conditions and after multiple rounds of stress regulation, are unlikely to achieve the potency level of the final product reaching Grade 1 or premium grade standards. Conversely, germplasm that meets this threshold exhibits a more significant potency response under stress conditions in its offspring. Therefore, using this threshold as a reference benchmark for germplasm selection allows for the exclusion of individuals with low potency potential at the beginning of the planting process, providing a germplasm-level guarantee for improving the overall grade ratio of the final product.

[0073] In one embodiment of this application, before harvesting, a potency-guided harvesting step is also included: samples from the planting plots are collected for rapid detection of antioxidant potency, and after confirming that the potency has reached the preset target range, large-scale harvesting is carried out.

[0074] Specifically, about 15 days before the scheduled harvest, select 5 to 10 sampling points in a diagonal or quincunx pattern from the planting area. Collect a portion of the rhizome from one Polygonatum plant at each point as a test sample. Take care not to damage the integrity of the entire rhizome during sampling so that plants that do not meet the standards can continue to grow in the field. Perform rapid antioxidant potency testing as soon as possible after sample collection. Rapid testing methods can include the DPPH test strip method or a portable near-infrared spectroscopy method.

[0075] When using the DPPH test strip method, a small amount of fresh cross-section is cut from the rhizome of Polygonatum odoratum. The detection end of the test strip is then placed in contact with the sap of the cross-section. The colorimetric reaction is allowed to proceed according to the time specified in the kit instructions. The colorimetric results are then compared with a standard colorimetric card to determine the corresponding antioxidant activity range. When using a portable near-infrared spectrometer, the rhizome of Polygonatum odoratum is sliced ​​or pulverized and placed in the detection window. The ORAC estimated efficacy value is directly read using the instrument's built-in prediction model.

[0076] When rapid testing results show that over 80% of the sample points have reached the preset target potency range, large-scale harvesting can begin. If the potency has not yet reached the target range, harvesting should be appropriately delayed, with sampling and retesting conducted 7 to 10 days later, until the potency meets the standard before harvesting. This step provides a reference for determining the harvesting timing in addition to growth years and appearance characteristics, helping to improve the overall potency compliance rate of the harvested batches.

[0077] In one embodiment of this application, a process traceability step is also included: uploading the coercion initiation time, the moment when the physiological indicators reach a preset first threshold, the data on changes in physiological indicators during the coercion period, the coercion termination time, and the total effective coercion duration to a blockchain platform to generate an unalterable digital identity card.

[0078] Specifically, during the implementation of coercion, the following data will be recorded in real time and uploaded to the blockchain platform through field data collection terminals or mobile devices: the specific date and time of coercion initiation, the specific moment when the monitored physiological indicators reach the preset first threshold, the daily monitoring values ​​of physiological indicators and their changing trends during the coercion period, the specific date and time of coercion termination, and the total effective coercion duration from the coercion taking effect to the coercion termination.

[0079] The aforementioned data is automatically timestamped and plot location information is added upon generation at the data collection terminal. After encryption, it is uploaded to the blockchain platform, where a unique digital identity is generated for each batch of Polygonatum. This identity is assigned to the product packaging in the form of a QR code or digital code. Consumers or downstream stakeholders can scan the code to access a complete record of the stress regulation experienced by this batch of Polygonatum during cultivation. Once uploaded, the record cannot be tampered with. This step ensures the complete preservation and traceability of key process parameters related to stress regulation, providing process data support for the efficacy evaluation of the final product during the cultivation process.

[0080] In one embodiment of this application, a valence feedback optimization step is also included: the valence data obtained from the terminal quality evaluation is correlated with the stress regulation parameters to identify the optimal combination of physiological index thresholds and the effective stress duration parameter, and the parameter is applied to the stress regulation of the next round of planting.

[0081] In practice, after a planting cycle, the final quality evaluation data of this batch of Polygonatum (including the measured values ​​of diosgenin content and ORAC potency) is summarized and compared with the stress regulation parameters recorded during the planting process (including the types of physiological indicators used, the specific settings of the first and second thresholds, the effective stress duration, and the daily variation curves of physiological indicators during the stress period). By comparing the differences in potency data between different batches, the corresponding differences in stress parameters are traced, and the combinations of physiological indicator thresholds and the range of effective stress duration that can achieve higher potency levels under the conditions of this plot are gradually identified.

[0082] For example, if comparative analysis reveals that batches using relative leaf water content as a monitoring indicator, with a first threshold set at 75%, and effective stress lasting 6 to 7 days, exhibit higher overall terminal potency than batches using other parameter combinations, then this parameter combination is determined as the preferred scheme for the next planting round. In the stress control of the next planting round, this preferred parameter combination is directly used for setting and operation. In this way, after each planting cycle, the stress parameters can be calibrated and optimized based on the potency data from the previous round, gradually bringing the stress control scheme closer to the optimal configuration under the conditions of this plot and variety.

[0083] Specifically, this application discloses a standardized cultivation technique and quality control method for Chinese medicinal herbs in a simulated wild environment. This method integrates stress control during cultivation with post-harvest potency testing and grading, forming a complete technical process from field management to final evaluation. The following section uses a four-year-old Polygonatum multiflorum plant from a simulated wild cultivation base in a certain region as an example to explain each step of the method in detail.

[0084] 1. Seed source screening.

[0085] Before planting commenced, candidate Polygonatum sibiricum germplasm sources underwent preliminary testing for diosgenin content and in vitro antioxidant potency. Small tissue samples were taken from the rhizomes of the candidates, dried, and pulverized. The diosgenin content was determined by high-performance liquid chromatography (HPLC), and the ORAC value was determined by oxygen radical absorbance capacity assay (ORAC). The screening criteria were a diosgenin content greater than or equal to 0.30 mg / g and an ORAC value greater than or equal to 50 μmol TE / g. Germplasm sources that did not meet these criteria were not included in the subsequent planting process.

[0086] The selected seed stems contained 0.35 mg / g of diosgenin and had an ORAC value of 55 μmol TE / g. After being cut and disinfected, the seed stems were transplanted in November 2019 using conventional methods, with a plant spacing of 28 cm and a row spacing of 38 cm. They were planted on gentle slopes under broad-leaved forests at an altitude of approximately 800 meters, with a canopy closure of 0.5 to 0.6. The soil was humus-rich sandy loam with a pH of approximately 5.8. No chemical pesticides or fertilizers were used throughout the planting process. Pest and disease control was achieved through yellow sticky traps and Bacillus subtilis spraying.

[0087] 2. Dynamic feedback of drought stress.

[0088] In July 2023, the rhizome of Polygonatum entered its fourth growth year and began to be regulated under drought stress.

[0089] 2.1 Coercive initiation.

[0090] A retractable rain shelter was erected above the planting area. The shelter had a bamboo frame and was covered with a transparent plastic film. It was only deployed during rainfall to block natural rainwater and retracted on sunny days to maintain normal ventilation and sunlight. As soil moisture content gradually decreased, drought stress conditions began to develop.

[0091] 2.2 Monitoring of physiological indicators.

[0092] A sampling method was used to collect functional leaf samples from five fixed sampling points within the planting area daily from 8:00 AM to 9:00 AM. The samples were placed in moisturizing bags and transferred to a simple workbench at the field edge, where the relative water content and proline content of the leaves were measured simultaneously. The method for determining the relative water content of the leaves was as follows: first, the fresh weight of the leaves was weighed; then, the leaves were immersed in clean water until fully saturated, and the saturated weight was measured; finally, the leaves were dried in a portable drying oven at 80℃ for 2 hours, and the dry weight was measured. The relative water content of the leaves was calculated using the formula: "(fresh weight - dry weight) / (saturated weight - dry weight) × 100%". The proline content was determined using a commercially available proline detection kit. The leaves were chopped and extracted with sulfosalicylic acid solution. After a ninhydrin colorimetric reaction, the absorbance was read at 520 nm using a portable spectrophotometer, and the proline concentration was calculated by referring to a standard curve.

[0093] Before the stress began, the relative water content of the leaves was measured to be approximately 88%, and the proline content was measured to be approximately 15 μg / g fresh weight.

[0094] 2.3 Determination of the effectiveness of coercion.

[0095] On the fourth day after the water control treatment began, monitoring data showed that the relative water content of the leaves decreased to 73%, and the proline content increased to 35 μg / g fresh weight, more than twice the pre-stress level. Both indicators reached the preset first threshold. Based on this, it was determined that the stress had taken effect, and the effective stress duration was calculated.

[0096] 2.4 Duration and Termination of Coercion.

[0097] Water control continued after the stress took effect, and the aforementioned physiological indicators were monitored daily. On the third day after the stress took effect, under the continued drought, a mild mechanical wounding treatment was applied to the plants. The wounding treatment was performed using a lightweight roller, 30 cm wide, with a weight of approximately 250 g / cm width. The roller was slowly and evenly pushed once along the canopy of the Polygonatum plants between rows, creating localized indentations on the leaf surface without breaking or falling off the leaves. Ten plants were randomly inspected after the treatment, and the estimated damage area per plant was approximately 6% to 8% of the total leaf area. After the wounding treatment, the drought stress condition was maintained.

[0098] By the 7th day after the stress took effect, the relative water content of the leaves had recovered to 82%, and the proline content had fallen back to 25 μg / g fresh weight, approximately 1.6 times the pre-stress level, reaching the preset second threshold. The rain shelter was removed that day, and normal water supply was restored. The total effective stress duration for this batch was 7 days.

[0099] 3. Process traceability.

[0100] During the stress application process, data such as the stress initiation time, the moment when physiological indicators reached the first threshold, daily leaf relative water content and proline content, stress termination time, and total effective stress duration were uploaded in real time to the blockchain platform via field mobile terminals. Timestamps and plot location information were automatically added during data upload, and after encryption, a unique digital ID corresponding to each batch of Polygonatum was generated and assigned to the product packaging in the form of a QR code.

[0101] 4. Valence guides harvesting.

[0102] In late October 2023, 15 days before the scheduled harvest, eight sampling points were selected from the plot in a quincunx pattern. A portion of the rhizome from one Polygonatum plant was collected at each point, and the antioxidant potency was rapidly tested using the DPPH test strip method. The fresh cross-section of the rhizome was placed in contact with the test strip end, and after the specified time for color development, the result was compared with a standard colorimetric card. The results showed that the antioxidant activity of seven out of the eight sampling points reached the preset target range, accounting for over 80%, confirming that large-scale harvesting could commence.

[0103] 5. Terminal valence detection and classification.

[0104] After harvesting, the Polygonatum sibiricum was cleaned, dried, and then sampled for testing of chemical and biological potency indicators. The content of diosgenin was determined using high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD), and the in vitro antioxidant potency was determined using the ORAC method. In this example, the diosgenin content was 0.58 mg / g, and the ORAC value was 92 μmol TE / g. According to the grading standards, both indicators met the requirements for premium grade, and this batch of Polygonatum sibiricum was rated as premium grade.

[0105] 6. Valence feedback optimization.

[0106] At the end of this planting cycle, the terminal potency data and stress regulation parameters were summarized and compared. In this batch, leaf relative water content was used as the main monitoring indicator, the first threshold was approximately 73%, effective stress lasted for 7 days, and wound treatment was applied on the 3rd day after stress took effect, resulting in a terminal potency reaching the highest level. This parameter combination was recorded and archived as the optimal scheme for stress regulation in the next planting cycle. In the next planting cycle, this parameter combination was directly used, and the potency data was compared again after harvest for continuous iterative optimization.

[0107] 7. Comparative verification of the effects of combined stress.

[0108] To verify the effect of combined drought and trauma stress, four comparative experiments were set up in the same plot, with 30 plants in each group and a row width of no less than 2 meters. Group A only received the above-mentioned dynamic feedback drought stress without trauma treatment; Group B received a rolling trauma treatment on the 4th day after the drought stress took effect; Group C received the same trauma treatment once under normal water supply conditions without drought stress; Group D served as a blank control, with normal water management and no stress treatment. After harvest, the diosgenin content and ORAC value of each group were measured, and the average values ​​are as follows: Group D: diosgenin content 0.22 mg / g, ORAC value 35 μmolTE / g; Group C: diosgenin content 0.28 mg / g, ORAC value 42 μmolTE / g; Group A: diosgenin content 0.46 mg / g, ORAC value 72 μmolTE / g; Group B: diosgenin content 0.58 mg / g, ORAC value 92 μmolTE / g. The improvement in the two indicators for Group B was greater than the sum of the improvement in Groups A and C, demonstrating a synergistic effect. According to this grading standard, Group B's overall grade is Special Grade, Group A is Grade 1, and Groups C and D are both Qualified.

[0109] In summary, the standardized cultivation technology and quality control method for medicinal herbs in a simulated wild environment, as described in this application, use changes in the plant's own physiological indicators as the criteria for stress initiation and termination. This ensures that the application of drought stress matches the actual physiological response of the plant, improving the accuracy and repeatability of stress treatment and steadily promoting the increase of diosgenin content and in vitro antioxidant potency in Polygonatum sibiricum. Introducing mild mechanical trauma treatment during the effective period of drought stress utilizes the synergistic effect of the two stress signals to further enhance the accumulation of medicinal components. After harvesting, dual testing of chemical and biological potency indicators is used, with the lower indicator used for grading, ensuring that the product meets the corresponding grade requirements in both component content and biological activity dimensions. This provides a quantifiable evaluation basis for the high quality and premium price of simulated wild Polygonatum sibiricum.

[0110] In the description of this specification, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0111] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0112] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A standardized cultivation technique and quality control method for Chinese medicinal herbs in a simulated wild environment, characterized in that, Includes the following steps: During the rhizome enlargement period of Polygonatum, artificial water control was carried out in the planting area to create drought stress; During the period of stress, the physiological indicators of Polygonatum were monitored; When the physiological indicators reach a preset first threshold, it is determined that the stress has taken effect, and the effective stress duration is calculated. When the physiological indicators drop back to a preset second threshold, the stress is terminated and the water supply is restored. Chemical and biological efficacy indicators were tested on the harvested Polygonatum sibiricum. Based on the test results of the two indicators, Polygonatum is divided into at least two quality grades, with the grade determined by the lower of the two indicators.

2. The method according to claim 1, characterized in that, The chemical potency indicator is the content of diosgenin, and the biological potency indicator is the in vitro antioxidant potency.

3. The method according to claim 2, characterized in that, The quality grades are divided into three levels: Special Grade, Grade 1, and Qualified. Among them, if the diosgenin content is greater than or equal to 0.50 mg / g and the ORAC value is greater than or equal to 80 μmol TE / g, it is classified as extra grade; If the diosgenin content is between 0.30 mg / g and 0.49 mg / g and the ORAC value is between 50 μmol TE / g and 79 μmol TE / g, it is classified as Grade 1. If the diosgenin content is between 0.15 mg / g and 0.29 mg / g and the ORAC value is between 25 μmol TE / g and 49 μmol TE / g, then it is considered qualified.

4. The method according to claim 1, characterized in that, The monitoring methods for the physiological indicators of Polygonatum include: Stem micro-change monitoring: The stem diameter change is continuously monitored by a stem micro-change sensor, and the maximum daily shrinkage of the stem exceeding a preset threshold is used as the criterion for stress effectiveness. Canopy temperature difference monitoring uses infrared temperature sensors to monitor the air temperature difference in the canopy, with a sustained positive temperature difference serving as the criterion for the effectiveness of stress. Sampling and testing are conducted by periodically sampling to detect the relative water content or proline content of the leaves, and the detection value reaching a preset threshold is used as the criterion for the stress to take effect.

5. The method according to claim 1, characterized in that, During the duration of the effective stress, mild mechanical trauma treatment is also performed on the aboveground parts of Polygonatum to create a combined stress of drought stress and trauma stress.

6. The method according to claim 5, characterized in that, The mild mechanical trauma treatment was performed on the 3rd to 5th day after the drought stress entered its effective period; The trauma method involves gently pressing the surface of the Polygonatum leaves with a roller, or using a row of needles to lightly prick the functional leaves, so that the damaged area of ​​a single leaf does not exceed 10% of the total leaf area.

7. The method according to claim 1, characterized in that, Before the drought stress regulation, a seed source screening step is also included: the diosgenin content and in vitro antioxidant potency of candidate Polygonatum seed sources are pre-detected, and seed sources that meet the preset threshold for both indicators are selected as seedlings. The preset thresholds are: diosgenin content greater than or equal to 0.30 mg / g, and in vitro antioxidant efficacy ORAC value greater than or equal to 50 μmol TE / g.

8. The method according to claim 1, characterized in that, Before harvesting, the process also includes a potency-guided harvesting procedure: samples are collected from the planting area for rapid testing of antioxidant potency. Once the potency is confirmed to have reached the preset target range, large-scale harvesting can proceed.

9. The method according to claim 1, characterized in that, It also includes process traceability steps: uploading the coercion initiation time, the moment when physiological indicators reach the preset first threshold, the data on changes in physiological indicators during the coercion period, the coercion termination time, and the total effective coercion duration to the blockchain platform to generate an unalterable digital identity card.

10. The method according to claim 1 or 9, characterized in that, It also includes a valence feedback optimization step: the valence data obtained from the terminal quality evaluation is correlated with the stress regulation parameters to identify the optimal combination of physiological indicator thresholds and the effective stress duration parameter, and the parameter is applied to the stress regulation of the next planting.

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