Method for improving content of dehydrocavidine in corydalis saxicola bunting under drought stress and detection method
By employing scientifically controlled drought stress treatment and high-performance liquid chromatography (HPLC) detection, the problem of fluctuating dehydrocarbeverine content in the artificial cultivation of Coptis chinensis was solved, resulting in a stable improvement in the quality of the medicinal material.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGXI BOTANICAL GARDEN OF MEDICINAL PLANTS
- Filing Date
- 2026-01-21
- Publication Date
- 2026-06-02
AI Technical Summary
The lack of scientifically controllable drought stress treatment methods in current technology leads to large fluctuations in the dehydrocarbeverine content in artificially cultivated Coptis chinensis, affecting the stability of the medicinal material's quality.
Through scientifically controlled drought stress treatment, robust Coptis chinensis plants were selected, and watering was stopped for 6-18 days to induce natural water shortage and drought stress. Combined with cultivation under specific substrate and environmental conditions, leaves and roots were harvested, and the content of dehydrocarbeverine was detected by methanol ultrasonic extraction and high performance liquid chromatography.
It significantly improved the accumulation of dehydrocarbeverine in the leaves and roots of Coptis chinensis, ensuring the improvement of medicinal quality and providing a quantifiable and repeatable technical solution, avoiding irreversible damage to plant growth.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of medicinal plant cultivation and quality control technology. More specifically, this invention relates to a method and detection method for increasing the content of dehydrocarbaverine in Coptis chinensis under drought stress. Background Technology
[0002] Corydalissaxicola Bunting is a traditional medicinal plant unique to the karst regions of my country, possessing properties such as clearing heat and detoxifying, promoting diuresis and relieving pain. Modern pharmacological studies have shown that its main active ingredient, dehydrocarvedin, exhibits significant efficacy in treating hepatitis, cirrhosis, and other diseases, and is considered one of the core active substances responsible for its hepatoprotective effects. Therefore, the content of dehydrocarvedin in the medicinal material is a key indicator for evaluating the quality of Corydalissaxicola Bunting.
[0003] With the intensification of global climate change, drought stress has become one of the major abiotic stresses affecting the growth, development, and accumulation of secondary metabolites in medicinal plants. For Coptis chinensis, drought not only reduces biomass but may also disrupt its secondary metabolic pathways, thereby affecting the synthesis and accumulation of key active ingredients such as dehydrocarbeverine, ultimately leading to unstable quality of the medicinal material.
[0004] Currently, research on *Coptis chinensis* mainly focuses on the isolation of its chemical components and the evaluation of its pharmacological activities. However, systematic and in-depth research is lacking on how drought stress specifically regulates the biosynthesis and accumulation of its core component, dehydrocarbeverine. Particularly in artificial cultivation, water management relies heavily on experience, lacking scientific guidance based on physiological responses and pharmacodynamic component accumulation models. This is a significant reason for the large fluctuations in yield and quality, especially the dehydrocarbeverine content, of artificially cultivated *Coptis chinensis*.
[0005] Therefore, how to maximize the stimulation of secondary metabolic potential and directionally increase the content of dehydrocarbeverine in the medicinal material by scientifically and controllably applying drought stress treatment to maintain the basic survival of Coptis chinensis plants has become a key technical problem that urgently needs to be solved in order to improve the quality and industrial benefits of artificially cultivated Coptis chinensis. Summary of the Invention
[0006] One object of the present invention is to address at least the aforementioned deficiencies and to provide at least the advantages that will be described later.
[0007] Another objective of this invention is to provide a method for increasing the content of dehydrocarbeverine in Coptis chinensis under drought stress. This method can induce and significantly increase the accumulation of its secondary metabolite, dehydrocarbeverine, in leaf and root tissues through scientifically controlled drought stress treatment, while minimizing irreversible damage to the growth of Coptis chinensis plants. This provides a clear, quantifiable, and repeatable technical solution for precisely controlling and improving the quality of Coptis chinensis through water management in artificial cultivation.
[0008] This invention provides a method for increasing the content of dehydrocarbaverine in Coptis chinensis under drought stress, which includes the following steps: (1) Select healthy and vigorous *Coptis chinensis* plants; (2) Stop watering the selected Coptis chinensis plants and subject them to natural drought stress treatment for 6-18 days; (3) Harvest the Coptis chinensis plants when the drought stress ends.
[0009] Preferably, the duration of drought stress in step (2) is 12-18 days.
[0010] Preferably, the robust *Coptis chinensis* plants described in step (1) are obtained through the following method: The seeds of Coptis chinensis were sown in a substrate of peat moss and vermiculite in a volume ratio of 3:1. After culturing for 3 weeks at a temperature of 23±2℃, a relative humidity of 37%-41%, a light duration of 15h / d, and a light intensity of 2000lx, the seeds were transplanted. After transplanting, the seeds were cultured under the same conditions for another 2 months before selection.
[0011] Preferably, the transplanted plants are cultured under the same conditions for another two months, specifically: Seedlings were transplanted into a substrate with a peat moss to vermiculite volume ratio of 3:1 and cultured under environmental conditions of 23±2℃, relative humidity of 37%-41%, light duration of 15h / d, and light intensity of 2000lx.
[0012] Preferably, the natural water shortage drought stress treatment described in step (2) is carried out under environmental conditions of temperature of 23±2℃, relative humidity of air of 37%-41%, light duration of 15h / d, and light intensity of 2000lx.
[0013] Preferably, the natural drought stress treatment described in step (2) further includes: thoroughly watering the plants before starting the treatment, and then not watering them again until the stress ends.
[0014] Preferably, the harvesting of Coptis chinensis plants in step (3) includes harvesting its leaves and / or roots.
[0015] Preferably, the dehydrocarbaverine content in the leaves of *Coptis chinensis* reached its peak on the 18th day of drought stress.
[0016] The present invention also provides a method for detecting the content of dehydrocarbaverine in the above-obtained Coptis chinensis, comprising the following steps: S1. Preparation of test solution: Take the dried powder of leaves or roots of Coptis chinensis plants subjected to drought stress, add methanol for ultrasonic extraction, filter, and take the filtrate as the test solution; S2. Chromatographic detection: The test solution was injected into a high-performance liquid chromatograph and detected under the following chromatographic conditions: the column was a C18 column packed with octadecylsilane-bonded silica gel; mobile phase A was acetonitrile, and mobile phase B was an aqueous solution of 0.1% phosphoric acid and 0.5% triethylamine; gradient elution was used, with the following program: 0–35 min, the volume percentage of mobile phase A linearly increased from 15% to 50%; 35–45 min, the volume percentage of mobile phase A linearly decreased from 50% to 15%; the detection wavelength was 345–349 nm; the flow rate was 0.9–1.1 mL / min; and the column temperature was 28–32 °C.
[0017] Preferably, in step S1, the ultrasonic extraction time is 45 min; in step S2, the detection wavelength is 347 nm, the flow rate is 1.0 mL / min, and the column temperature is 30℃. The present invention has at least the following beneficial effects: 1. This invention obtains robust *Coptis chinensis* plants and subjects them to a natural water shortage treatment lasting 6 to 18 days, i.e., keeping the plants in a state of continuous water deficit without any watering. This process stimulates the inherent drought stress physiological and metabolic regulatory network of *Coptis chinensis*, adjusting its internal water use strategy and redistributing metabolic resources, thereby promoting the biosynthesis and accumulation of the target active ingredient dehydrocarvedin in its secondary metabolic pathways. Finally, at the end of the stress period, the plants are harvested to obtain medicinal materials with a high content of dehydrocarvedin.
[0018] 2. This invention optimizes the drought stress duration to be between 12 and 18 days, based on a consideration of the balance between stress intensity and secondary metabolic response. Compared to a shorter 6-day treatment, this period can more fully stimulate the physiological and biochemical processes related to drought resistance and secondary metabolism in *Coptis chinensis*, leading to more significant activation and accumulation of dehydrocarbeverine synthesis pathways. Simultaneously, this duration avoids the risk of severe decline in plant viability or metabolic failure that may result from stress exceeding 18 days, thus achieving a more reliable and optimized practical window between effectively increasing the content of target components and maintaining basic plant activity.
[0019] 3. This invention provides a stable and suitable environment for seed germination and early seedling growth by sowing Coptis chinensis seeds in a specific ratio of peat moss and vermiculite substrate and cultivating them for three weeks under strictly controlled temperature, humidity, and light conditions. Following this initial cultivation, transplanting allows seedlings to transition from a relatively dense nursery state to a cultivation stage with ample individual growth space, and they continue to be cultivated for two months under identical environmental parameters. This complete cultivation process aims to select robust and vigorous plants in terms of physiological state and developmental stage through standardized substrate, environmental, and time management, laying the necessary material foundation for subsequent reliable drought stress treatment.
[0020] 4. This invention ensures that the environmental conditions are suitable for the entire growth cycle of the plant, from seedling to stress treatment, by transplanting the seedlings into a mixed substrate of peat moss and vermiculite that is exactly the same as that used in the sowing stage, and continuing to cultivate them for two months under strictly consistent temperature, humidity and light environmental parameters.
[0021] 5. This invention maintains the plants of Coptis chinensis in temperature, humidity and light conditions that are completely compatible with the early seedling stage during the entire natural water shortage and drought stress treatment, which is conducive to the physiological and metabolic response of the plants to drought, especially the accumulation and changes of dehydrocarbeverine.
[0022] 6. This invention involves thoroughly watering the plants before initiating drought stress treatment. This facilitates the physiological activation and storage of *Coptis chinensis* before stress treatment, reducing interference with subsequent stress responses. Subsequently, all forms of external water supplementation are strictly prohibited throughout the entire stress cycle, allowing the plants to rely entirely on their own water reserves and undergo a natural process from sufficient to gradually deficient water. This controlled water deprivation effectively and stably induces drought stress signal transduction within the plant, initiating a series of adaptive metabolic responses, including dehydrocarvedin synthesis.
[0023] 7. This invention explicitly targets the leaves and / or roots of *Coptis chinensis* for harvesting, based on experimental findings that dehydrocarbeverine accumulation in different organs of the plant is effectively activated under drought stress. This design allows for targeted harvesting of parts with significantly enriched active ingredients, adhering to the traditional practice of using the whole plant for medicinal purposes while maximizing the utilization of metabolites induced by stress treatment. Furthermore, considering the preference for specific parts (such as leaves or roots) or the differences in component content among different parts in practical applications, this method provides flexible and specific harvesting options, ensuring that the technical solution for improving the quality of medicinal materials through drought stress has clear operational guidance and practical application value.
[0024] 8. The detection method of this invention involves preparing a dried powder from specific parts of drought-stressed *Coptis chinensis* plants, followed by standardized ultrasonic extraction and filtration with methanol to efficiently and stably obtain the dehydrocarbeverine component, thus preparing the test solution. Furthermore, by using an aqueous solution containing acetonitrile and phosphoric acid and triethylamine in a specific ratio as the mobile phase, and executing a specific linear gradient elution program from start to finish, combined with selective detection at wavelengths of 345 to 349 nm, a set of chromatographic separation and quantitative analysis conditions specifically designed for dehydrocarbeverine in the *Coptis chinensis* matrix was constructed. This optimized combination of chromatographic parameters, including precisely controlled flow rate and column temperature, ensures highly selective separation, clear peak shapes, and stable retention behavior of the target component from the complex plant extract. This provides crucial technical support for accurate, reliable, and reproducible quantitative analysis of the changes in dehydrocarbeverine content in *Coptis chinensis* after different drought stress treatments, allowing the evaluation of the effects of the aforementioned stress treatment methods to be based on accurate data.
[0025] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description
[0026] Figure 1 Chromatograms of the reference standard and the sample are shown. (A) Dehydrocarbendazim reference solution; (B) Test solution of leaves treated with Coptis chinensis D-12; 1 is dehydrocarbendazim.
[0027] Figure 2 The samples were Coptis chinensis from different drought treatment groups and a control group.
[0028] Figure 3 and Figure 4 The effect of drought stress on the permeable material of Coptis chinensis; Note: * indicates a significant difference between the drought treatment group and its corresponding control group, *P<0.05; **P<0.01; ***P<0.001.
[0029] Figure 5 The effect of drought stress on antioxidant enzymes in Coptis chinensis; Note: * indicates a significant difference between the drought treatment group and its corresponding control group, *P<0.05; **P<0.01; ***P<0.001.
[0030] Figure 6 The effect of drought stress on photosynthetic pigments in *Rhizophora stylosa*. Note: * indicates a significant difference between the drought treatment group and its corresponding control group, *P<0.05; **P<0.01; ***P<0.001.
[0031] Figure 7The effect of drought stress on the dehydrocarbendazim content in Coptis chinensis; Note: * indicates a significant difference between the drought treatment group and its corresponding control group, *P<0.05; **P<0.01; ***P<0.001. Detailed Implementation
[0032] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.
[0033] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.
[0034] Example 1 A method for increasing the content of dehydrocarbeverine in Coptis chinensis under drought stress, specifically including the following steps: (1) Select healthy and vigorous *Coptis chinensis* plants; The robust *Coptis chinensis* plants were obtained through the following methods: Seeds of Coptis chinensis were sown in a substrate of peat moss and vermiculite in a volume ratio of 3:1. After culturing for 3 weeks at a temperature of 23℃, a relative humidity of 37%-41%, a light duration of 15h / d, and a light intensity of 2000lx, seedlings with uniform growth were selected for transplanting. Seedlings were transplanted into a substrate with a peat moss to vermiculite volume ratio of 3:1 and cultured for 2 months under environmental conditions of 23℃, relative humidity of 37%-41%, light duration of 15h / d, and light intensity of 2000lx. Afterward, robust *Coptis chinensis* plants were selected from the cultured plants for stress treatment. Robust *Coptis chinensis* plants were defined as those cultivated through the above process of "sowing in a 3:1 peat moss + vermiculite substrate, culturing at 23±2℃ for 3 weeks, followed by 2 months of cultivation under the same conditions after transplanting," meeting the following criteria: intact leaves without yellowing, free from pests and diseases, root system without rot, and uniform growth within the same batch (no significant differences in height or vigor).
[0035] (2) Stop watering the selected Coptis chinensis plants and subject them to natural drought stress treatment for 6-18 days; The natural water shortage and drought stress treatment was carried out under environmental conditions of 23℃ temperature, 37%-41% relative humidity, 15h / d light duration, and 2000lx light intensity. The specific procedures include: thoroughly watering the selected *Coptis chinensis* plants before the drought stress treatment begins; then stopping watering until the stress ends. Thorough watering means watering the cultivation substrate by spraying until the substrate is completely saturated (water penetrates to the bottom of the substrate), and water droplets continuously seep out from the bottom of the cultivation container for 3-5 seconds, with no water accumulation on the substrate surface.
[0036] (3) When the drought stress ends, harvest the leaves and roots of the Coptis chinensis plant.
[0037] Example 2 This embodiment provides a high-performance liquid chromatography (HPLC) method for detecting the content of dehydrocarbeverine in Coptis chinensis obtained from Example 1 or other drought stress treatment methods.
[0038] S1. Preparation of reference solution Accurately weigh 1.16 mg of dehydrocarvitine reference standard (batch number 111667-200401, National Institutes for Food and Drug Control, China), place it in a 5 mL volumetric flask, dissolve and dilute to the mark with methanol, shake well, and prepare a reference standard stock solution with a concentration of 0.232 mg / mL.
[0039] S2. Preparation of the test solution Leaves or roots of *Coptis chinensis* harvested after different periods of drought stress treatment as described in Example 1 were pulverized, sieved, and dried into powder. Approximately 15 mg of the powder was accurately weighed and placed in a 10 mL stoppered centrifuge tube. 2 mL of methanol was accurately added, the tube was sealed, and the weight was determined. The tube was ultrasonically treated (250 W, 40 kHz) for 45 minutes, removed, cooled to room temperature, and weighed again. The lost weight was replenished with methanol, and the tube was shaken well. The solution was filtered through a 0.22 μm microporous membrane, and the subsequent filtrate was collected to obtain the test solution.
[0040] S3. Chromatographic detection Chromatograph: High performance liquid chromatograph (equipped with DAD detector); Chromatographic column: Shim-pack Scepter C18-120 column (4.6mm×250mm, 5μm), or equivalent C18 column; Mobile phase: A is acetonitrile, B is an aqueous solution of 0.1% phosphoric acid and 0.5% triethylamine; Gradient elution program: 0~35 min, phase A increases linearly from 15% to 50%, phase B decreases accordingly; 35~45 min, phase A decreases linearly from 50% to 15%, phase B increases accordingly.
[0041] Detection wavelength: 347nm; Flow rate: 1.0 mL / min; Column temperature: 30℃; Injection volume: 10 μL.
[0042] S4. Determination Method Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the content under the chromatographic conditions described above. Record the chromatograms. Use the retention time of the dehydrocarbeverine reference peak for qualitative analysis, and use the external standard method to calculate the content of dehydrocarbeverine in the test solution based on the peak area.
[0043] S5. Methodological Validation Verify using the method described above: Linearity: With stepwise dilution of the reference stock solution, within the concentration range of 7.25–116.00 μg / mL, the peak area of dehydrocarbeverine showed a good linear relationship with concentration, with the regression equation being y = 43.691x - 91.054, R0. 2 =0.999.
[0044] Precision: The RSD of the peak area of dehydrocarbeverine was 0.85% (n=6) after six consecutive injections of the same test solution, indicating that the instrument has good precision.
[0045] Repeatability: Six parallel samples of the same batch of Coptis chinensis were prepared and tested. The average content of dehydrocarbendazim was 1.10 mg / g, and the RSD was 1.79% (n=6), indicating that the method has good repeatability.
[0046] Stability: The same test solution was injected and measured at room temperature at 0, 2, 4, 6, 12 and 24 h. The RSD of the dehydrocarbeverine peak area was 1.23% (n=6), indicating that the test solution was stable within 24 h.
[0047] Recovery rate: Take 6 portions of Coptis chinensis powder with known content, add a certain amount of dehydrocarbendazim reference solution, prepare and determine according to the above method. The average recovery rate is 103.67%, and the RSD is 1.20% (n=6).
[0048] Test content 1.1 Test Materials The seeds of *Corydalis yanhuanglian* were collected from a *Corydalis yanhuanglian* planting base in Wuzhuan Town, Donglan County, Hechi City, and identified by the Medicinal Botanical Garden as seeds of *Corydalis yanhuanglian*, a plant belonging to the genus *Corydalis* of the Papaveraceae family. Mature and plump *Corydalis yanhuanglian* seeds were selected, impurities were removed, and they were sown in a substrate of peat moss and vermiculite in a 3:1 ratio. The cultivation conditions were: temperature 23℃, humidity 37%–41%, light duration 15 h / d, and light intensity 2000 lx. After three weeks of growth, seedlings with uniform growth were selected for transplanting. After transplanting, the seedlings were further cultivated for two months, and robust, uniformly growing *Corydalis yanhuanglian* were selected as experimental materials.
[0049] Experimental Design This experiment used *Coptis chinensis* plants that were transplanted and cultured for 2 months as experimental materials. Drought stress groups were set up, including drought stress for 6 days (D-6), 12 days (D-12), and 18 days (D-18). A normal watering control group was set up, corresponding to normal watering for 6 days (CK-6), 12 days (CK-12), and 18 days (CK-18). A stress-free day (CK) was also set up as the baseline control for the entire experiment. There were a total of 7 treatment groups, with 7 *Coptis chinensis* plants in each treatment group.
[0050] Before the experiment, all treatment groups of *Coptis chinensis* were thoroughly watered. Subsequently, the stress groups (D-6, D-12, D-18) underwent natural drought treatment, while the control groups (CK-6, CK-12, CK-18) were watered every 2 days until the substrate surface was slightly moist and no excess water seeped through. Samples were collected on day 0 (CK), day 6 (CK-6 and D-6), day 12 (CK-12 and D-12), and day 18 (CK-18 and D-18) of stress.
[0051] Test items and methods 1.3.1 Biomass After the treatment was completed, the *Coptis chinensis* plants from each treatment group were collected. The root substrate of the *Coptis chinensis* plants was washed with clean water and the moisture was absorbed with paper towels. The fresh weight of the whole plant and the biomass of the aboveground parts were measured.
[0052] 1.3.2 Relative water content of leaves Mature leaves from each treatment group of *Coptis chinensis* were collected, and 1g of fresh weight was taken. The leaves were then soaked in water until fully saturated, and their saturated weight was measured. The saturated leaves were then dried in an oven at 75℃ until constant weight, and their dry weight was measured. The relative water content of the leaves was calculated. The calculation formula is as follows: Relative water content of leaves (%) = [(fresh weight of leaves - dry weight of leaves) / (saturated weight of leaves - dry weight of leaves)] × 100; 1.3.3 Physiological Indicators The photosynthetic pigment content was determined by organic solvent extraction. organic solvent extraction ) determination, proline ( proline, Pro The content was determined by the ninhydrin colorimetric method. ninhydrin colorimetric ) determination, soluble protein ( soluble protein The content was determined by Coomassie brilliant blue staining method ( ) Coomassie Brilliant Blue staining ) determination; soluble sugar ( soluble sugar The anthrone colorimetric method was used. anthrone colorimetric ) was determined; the WST-8 method was used respectively ( water-soluble tetrazolium salt-8 ), Guaiacol method ( guaiacol colorimetric ) Determination of superoxide dismutase (S uperoxide dismutase SOD), peroxidase ( peroxidase POD); using the thiobarbituric acid method ( Thiobarbituric Acid ) [8] Determination of malondialdehyde (MDA) malonaldehyde The content of MDA was determined. All procedures and calculations were performed in accordance with the kit instructions provided by Suzhou Mengxi Biomedical Technology Co., Ltd.
[0053] 1.3.4 Determination of the content of active pharmaceutical ingredients Preparation of reference solution: Accurately weigh 1.16 mg of dehydrocarbeverine reference standard and dissolve it in 5 mL of methanol to prepare a stock solution with a concentration of 0.232 mg / mL. Dehydrocarbeverine reference standard (batch number 111667-200401, mass fraction 100%) was purchased from the National Institutes for Food and Drug Control, China.
[0054] Preparation of test solution: Take leaves and roots of each treated Coptis chinensis, prepare dried powder, take 15 mg of each, dissolve in 2 mL of methanol, sonicate for 45 min, filter through a 0.22 μm microporous membrane, and test.
[0055] HPLC chromatographic conditions: A Shim-pack Scepter C18-120 column (4.6 × 250 mm, 5 μm) was used, with acetonitrile as mobile phase A and 0.1% phosphoric acid-0.5% triethylamine as mobile phase B. Gradient elution conditions: 0–35 min, 15%–50% A; 35–45 min, 50%–15% A; detection wavelength: 347 nm; flow rate: 1.0 mL / min; column temperature: 30 °C; injection volume: 10 μL. Under these chromatographic conditions, the theoretical plate number calculated based on the dehydrocarvedin peak was not less than 50,000, and the resolution was greater than 1.5. The chromatograms were recorded as follows. Figure 1 .
[0056] Linearity assessment: The prepared dehydrocarbeverine stock solution was diluted 2, 4, 8, 16, 20, and 32 times, respectively. The concentration of the reference standard dehydrocarbeverine was plotted on the x-axis. x Its peak area is the vertical axis ( y Plot a standard curve. Based on the analysis of the standard curve, obtain the regression equation. y =43.691 x -91.054, R 2 =0.999. The results indicate that dehydrocarvedin exhibits good linearity in the range of 7.25–116.00 μg / mL.
[0057] Precision test: Accurately pipette an appropriate amount of the test solution prepared as described above, and inject it continuously for 6 determinations under the chromatographic conditions described above, recording the peak area. Measure the peak area of dehydrocarvedin. RSD =0.85% n =6), indicating that the instrument has good precision.
[0058] Repeatability test: Accurately weigh 6 portions of Coptis chinensis sample, and prepare 6 parallel test solutions of a certain concentration according to the above method. Inject each solution separately and record the peak area. The average content of dehydrocarbeverine was calculated to be 1.10 mg / g. RSD =1.79% ( n =6), indicating that the method has good repeatability.
[0059] Stability test: Accurately pipette an appropriate amount of the test solution prepared according to the above method, and inject it for detection at 0, 2, 4, 6, 12, and 24 h under the chromatographic conditions described above, recording the peak area. The peak area of dehydrocarvedin was measured. RSD =1.23% ( n =6), the results showed that the test sample was stable within 24 hours.
[0060] Recovery test: Six portions of Coptis chinensis powder with known content, approximately 0.02 g each, were accurately weighed. An appropriate amount of dehydrocarbendazim reference solution was accurately added, and the test solution was prepared according to the above method. The solutions were injected separately, and the peak areas were recorded and the recovery rate was calculated. The average recovery rate of dehydrocarbendazim was found to be 103.67%. RSD =1.20%( n =6).
[0061] Data Analysis Data were processed and analyzed using Microsoft Excel 2019 and IBM SPSS Statistics 27 software, and graphs were created using GraphPad Prism 10.
[0062] Effects of drought stress on the growth and morphological characteristics of Coptis chinensis After drought stress treatment, the *Coptis chinensis* plants in different treatment groups were observed and photographed. As the degree of drought stress increased, the growth status of *Coptis chinensis* showed significant differences. Plants in the normally watered group had extended leaves and well-developed root systems; while plants in the drought-stressed group exhibited drooping and wrinkled leaves and reduced fibrous roots. Furthermore, as the stress intensified, the degree of leaf wilting and the reduction in fibrous roots further increased. Figure 2 ).
[0063] With increasing drought stress, the fresh weight and aboveground biomass of *Coptis chinensis* gradually decreased; both reached their peak reduction after 18 days of drought stress (Table 1). Under drought stress, the relative water content of *Coptis chinensis* leaves initially increased and then decreased; after 6 days of drought stress, the relative water content of *Coptis chinensis* leaves was higher than that of the control group, but decreased with increasing drought stress (Table 1). This indicates that drought stress negatively impacts the growth and development of *Coptis chinensis*.
[0064] Table 1 Fresh weight, aboveground biomass, and relative water content of Coptis chinensis under drought stress Note: * indicates a significant difference between the drought treatment group and its corresponding control group. P <0.05;** P <0.01; *** P <0.001 2.2 Effects of drought stress on the content of permeability regulating substances in Coptis chinensis Depend on Figure 3 and Figure 4 It was found that drought stress significantly affected the contents of proline, soluble sugar, soluble protein, and malondialdehyde in *Coptis chinensis*. Under drought stress, the proline content in both leaves and roots of *Coptis chinensis* showed an increasing trend. Compared with the control group that was normally watered during the same period, at 6 days of drought stress, the increase in proline content in the roots of *Coptis chinensis* was relatively small, at 19.05%, while the increase in proline content in the leaves was significantly larger, increasing by 580.23%. With the extension of stress time, the proline content in the leaves was significantly higher than that in the roots. At 18 days of drought stress, the proline content in the leaves increased by 6585.27% compared with the control group, and the increase in the roots was 1325.78%.
[0065] Under drought stress, the soluble sugar content in both leaves and roots of *Coptis chinensis* showed an increasing trend, with the increase becoming significant after 12 days of drought stress. After 18 days of drought stress, the rate of increase in root soluble sugar content slowed, while the soluble sugar content in leaves still increased significantly. Compared with the control group under normal irrigation, after 6 days of drought stress, the soluble sugar content in leaves showed almost no increase, while the soluble sugar content in roots increased by 83.58%. With the extension of stress duration, after 18 days of stress, the soluble sugar content in leaves increased significantly by 971.08%, while the soluble sugar content in roots increased by only 72.20%.
[0066] Meanwhile, as the severity of drought stress intensified, the soluble protein content in both the leaves and roots of *Coptis chinensis* showed an upward trend. Compared with the normal watering treatment group, 6 days of drought stress had little effect on the soluble protein content of *Coptis chinensis* leaves, increasing by only 4.35%; the largest increase in soluble protein content was observed in the leaves at 18 days of drought stress, reaching 112.76%; the soluble protein content in the roots increased more rapidly, with the largest increase at 12 days of drought stress, reaching 450.31%.
[0067] Furthermore, with the intensification of drought stress, the malondialdehyde (MDA) content in the leaves of *Coptis chinensis* showed a gradual upward trend, while the MDA content in the roots showed a trend of first increasing and then decreasing. Compared with the control at the same time point, the increase in MDA content in leaves was minimal at 6 days of drought stress, only 8.4%; at 12 and 18 days of drought stress, the MDA content in leaves increased significantly compared with the control at the same time point, increasing by 157.81% and 238.66%, respectively; the MDA content in roots increased by 15.18%, 22.28%, and 43.87% at 6, 12, and 18 days of drought stress, respectively. These results indicate that *Coptis chinensis* leaves and roots respond to different levels of drought stress through the accumulation of osmotic regulatory substances and membrane system response mechanisms.
[0068] Effects of drought stress on antioxidant enzymes in Coptis chinensis Different antioxidant enzyme activities in Coptis chinensis respond differently to drought stress. Figure 5 The SOD activity in the leaves and roots of *Coptis chinensis* showed different responses to drought stress. In leaves, SOD activity peaked at 6 days of drought stress, increasing by 57.80% compared to the control at the same time point. At 12 days of drought stress, it remained significantly higher than the control, but decreased significantly by 28.53% at 18 days. In roots, SOD activity increased with increasing stress intensity. Compared to the normal irrigation control, SOD activity increased by only 13.92% at 6 days of drought stress, but increased by 123.46% at 18 days.
[0069] Under drought stress, the POD activities in both leaves and roots of *Coptis chinensis* showed an increasing trend, reaching their highest levels at 18 days of drought stress. Leaf POD activity increased continuously with the severity of drought stress; compared to the control at the same time point, leaf POD activity increased by 37.21%, 24.59%, and 20.37% at 6, 12, and 18 days of drought stress, respectively. In the early stages of drought stress, root POD activity increased relatively slowly, but significantly increased at 18 days of drought stress, increasing by 97.52% compared to the control at the same time point. In summary, the response of POD and SOD activities in *Coptis chinensis* to drought stress exhibits organ- and enzyme-specific differences. POD activity increased with increasing drought stress, while the trends in SOD activity varied across different organs.
[0070] Effects of drought stress on photosynthetic pigment content of Coptis chinensis Drought stress significantly affected the content of photosynthetic pigments in *Rhizophora stylosa*. Figure 6Under drought stress, the contents of chlorophyll a, chlorophyll b, carotenoids, and chlorophyll (a+b) in *Coptis chinensis* all showed an increasing trend, with the highest contents observed after 18 days of drought stress; the chlorophyll a / b ratio, however, showed a decreasing trend. Compared with normal irrigation during the same period, drought stress increased chlorophyll a content by 15.94%, 13.86%, and 78.33% after 6, 12, and 18 days, respectively; the increase in chlorophyll b was even greater, at 21.57%, 35.23%, and 146.68%, respectively. Due to the influence of drought stress, the increase in chlorophyll b content was consistently greater than that of chlorophyll a, leading to a decreasing trend in the chlorophyll a / b ratio as the severity of drought stress intensified. Compared with the control group at the same time, after 6, 12, and 18 days of drought stress, the carotenoid content increased by 8.44%, 28.99%, and 102.56%, respectively; the chlorophyll (a+b) content increased by 17.26%, 18.88%, and 94.93%, respectively. It can be seen that Coptis chinensis adapts to drought stress by upregulating the content of photosynthetic pigments.
[0071] Effects of drought stress on the content of active medicinal components in Coptis chinensis Under drought stress, the content of dehydrocarvedin, the active medicinal ingredient in Coptis chinensis, increased significantly. Figure 7 Compared with the control group, the increase in dehydrocarbendazim content in leaves was relatively small in the early stages of drought, increasing by 5.29% after 6 days of drought stress and by 46.19% after 12 days; however, the content increased by 587.93% after 18 days of drought stress. Referring to the control group with normal irrigation, the dehydrocarbendazim content in roots increased by 12.81%, 118.84%, and 24.24% after 6, 12, and 18 days of drought stress, respectively, with the largest increase observed at 12 days of drought stress and the highest content at 18 days. These results suggest that dehydrocarbendazim may play a role in the stress adaptation of *Coptis chinensis*.
[0072] The above experiments show that plant responses to drought stress exhibit significant physiological and biochemical consistency, manifested as the accumulation of osmotic regulators, changes in antioxidant enzyme activity, and an increase in membrane lipid peroxidation products. These responses are prevalent across different species, reflecting a conserved strategy for plants to cope with drought stress. With increasing drought stress, the levels of proline, soluble sugars, and soluble proteins in the plant continuously accumulate, while the activities of SOD and POD in the roots continue to rise, and the MDA content increases synchronously. This result further confirms the conservation of this mechanism in plants. However, *Coptis chinensis* exhibits species-specificity in its drought response. Regarding the utilization of osmotic regulators, at the same drought stress treatment level, the soluble sugar content in the leaves and roots of *Coptis chinensis* is generally higher than that of proline and soluble protein, indicating that *Coptis chinensis* prefers soluble sugars as the main regulator, and the accumulation of soluble sugars in the leaves is more pronounced, similar to that of *Dendrobium nobile*. Dendrobium nobile Lindl.), Dendrobium officinale ( Dendrobium officinale Unlike *Kimuraet Migo*, which uses proline as the main osmotic regulator, this reflects a unique selective preference of *Rhizophora stylosa* for osmotic regulators. Regarding changes in antioxidant enzyme activity, the SOD activity in *Rhizophora stylosa* leaves showed a trend of first increasing and then decreasing, while the SOD and POD activities in roots consistently increased with increasing stress, unlike wheat (…). Triticum aestivum L.), Tangut daphne ( Daphne tangutica Unlike plants such as Maxim, it reflects the species specificity of Coptis chinensis formed during its long-term adaptation to karst habitats.
[0073] In this study, as drought stress intensified, the content of dehydrocarbaverine in *Coptis chinensis* showed a significant upward trend, while the relative water content of leaves decreased and the MDA content continuously increased, suggesting that the accumulation of dehydrocarbaverine may be related to the physiological processes of *Coptis chinensis* in response to drought stress. Considering the characteristics of the native karst habitat of *Coptis chinensis*, this type of environment not only experiences frequent droughts but also generally suffers from nitrogen deficiency in the soil. Alkaloids, as secondary metabolites with both physiological activity and environmental adaptability, do not require the large amounts of nitrogen consumed by soluble proteins during their accumulation. They can perform dual functions of osmotic regulation and antioxidant activity, thus adapting to the dual limitations of karst habitats. Previous studies have found that *Catharanthus roseus* (… Catharanthu sroseus Rauvolfia ( Rauvolfia vomitoria Plants such as *Coptis chinensis* can enhance drought resistance by activating relevant signaling pathways to increase the activity of key enzymes in alkaloid synthesis, thereby increasing the accumulation of terpenoid indole alkaloids and reserpine. Therefore, the accumulation of dehydrocarvedin in *Coptis chinensis* under drought conditions may not be a simple response to the synthesis of medicinal active ingredients, but rather an evolutionary result of karst plants adapting to special habitats over a long period.
[0074] In summary, plants exhibit a certain degree of consistency in their physiological and biochemical responses to drought, but *Coptis chinensis* also demonstrates species specificity in its drought response. The performance of *Coptis chinensis* suggests that karst plants may have evolved mechanisms superior to those in other habitats for coping with drought.
[0075] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.
Claims
1. A method for increasing the content of dehydrocarbaverine in Coptis chinensis under drought stress, characterized in that, Includes the following steps: (1) Select healthy and vigorous *Coptis chinensis* plants; (2) Stop watering the selected Coptis chinensis plants and subject them to natural drought stress treatment for 6-18 days; (3) Harvest the Coptis chinensis plants when the drought stress ends.
2. The method according to claim 1, characterized in that, The duration of drought stress described in step (2) is 12-18 days.
3. The method according to claim 1, characterized in that, The robust *Coptis chinensis* plants mentioned in step (1) were obtained through the following methods: The seeds of Coptis chinensis were sown in a substrate of peat moss and vermiculite in a volume ratio of 3:
1. After culturing for 3 weeks at a temperature of 23±2℃, a relative humidity of 37%-41%, a light duration of 15h / d, and a light intensity of 2000lx, the seeds were transplanted. After transplanting, the seeds were cultured under the same conditions for another 2 months before selection.
4. The method according to claim 3, characterized in that, The phrase "continue cultivation under the same conditions for 2 months after transplanting" specifically refers to: Seedlings were transplanted into a substrate with a peat moss to vermiculite volume ratio of 3:1 and cultured under environmental conditions of 23±2℃, relative humidity of 37%-41%, light duration of 15h / d, and light intensity of 2000lx.
5. The method according to any one of claims 1 to 4, characterized in that, The natural water shortage drought stress treatment described in step (2) was carried out under environmental conditions of temperature 23±2℃, relative humidity of air 37%-41%, light duration of 15h / d, and light intensity of 2000lx.
6. The method according to claim 1, characterized in that, The natural drought stress treatment described in step (2) also includes: watering the plants thoroughly before starting the treatment, and then not watering them again until the stress ends.
7. The method according to any one of claims 1 to 6, characterized in that, The harvesting of Coptis chinensis plants in step (3) includes harvesting its leaves and / or roots.
8. The method according to claim 1, characterized in that, The content of dehydrocarbaverine in the leaves of Coptis chinensis reached its peak on the 18th day of drought stress.
9. A method for detecting the content of dehydrocarbaverine in Coptis chinensis obtained by the method according to any one of claims 1-8, characterized in that, Includes the following steps: S1. Preparation of test solution: Take the dried powder of leaves or roots of Coptis chinensis plants subjected to drought stress, add methanol for ultrasonic extraction, filter, and take the filtrate as the test solution; S2. Chromatographic detection: The test solution was injected into a high-performance liquid chromatograph and detected under the following chromatographic conditions: the column was a C18 column packed with octadecylsilane-bonded silica gel; mobile phase A was acetonitrile, and mobile phase B was an aqueous solution of 0.1% phosphoric acid and 0.5% triethylamine; gradient elution was used, with the following program: 0–35 min, the volume percentage of mobile phase A linearly increased from 15% to 50%; 35–45 min, the volume percentage of mobile phase A linearly decreased from 50% to 15%; the detection wavelength was 345–349 nm; the flow rate was 0.9–1.1 mL / min; and the column temperature was 28–32 °C.
10. The detection method according to claim 9, characterized in that, In step S1, the ultrasonic extraction time is 45 min; in step S2, the detection wavelength is 347 nm, the flow rate is 1.0 mL / min, and the column temperature is 30℃.