A method for rejuvenating and restoring wild Corydalis understory populations

Abstract

This invention provides a method for the reintroduction and rejuvenation of wild Corydalis rhizome under forest populations. The method includes selecting a reintroduction habitat, sowing Corydalis rhizome tubers in the understory of the reintroduction habitat, and planting under controlled conditions of canopy closure, light intensity, and removal of understory litter until harvest. This invention selects key ecological factors that restrict the growth of wild Corydalis rhizome in Chinese fir forests, such as canopy closure and understory litter, to effectively maintain the content of the main active ingredient, corydaline B, while significantly increasing the content of corydaline A.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicinal herb cultivation technology, and in particular to a method for the restoration and rejuvenation of wild Corydalis yanhusuo understory populations. Background Technology

[0002] Corydalis yanhusuo is a traditional Chinese medicine, first recorded in Li Zhongli's *Ben Cao Yuan Shi* (Original Materia Medica) during the Qing Dynasty. It has a long history of cultivation, with its tubers used medicinally for their blood-activating, qi-regulating, and pain-relieving effects. The wild Corydalis yanhusuo herb growing in Dapan Mountain, Pan'an County, Zhejiang Province, is one of the few large-scale distribution areas of this herb in China and a major production area. The area has a subtropical monsoon climate with an average annual temperature of 17.3℃, a maximum temperature (July) of 35℃, a minimum temperature (January) of 2℃, an average annual rainfall of 1427.7mm, a relative humidity of 77%, and 1822.8 hours of sunshine annually. The altitude ranges from 580.6m to 712.5m, and the soil type is sandy loam.

[0003] Corydalis is an early spring plant. From February to April, before most plants have sprouted, it takes full advantage of sunlight to grow first, which is conducive to the formation of a dominant community. In early summer, the shade provided by tree leaves prevents strong direct sunlight, allowing the tubers to develop fully. However, due to the ban on logging in nature reserves, the regeneration of Chinese fir forests is insufficient. High canopy density leads to insufficient sunlight, and the allelopathic effect of the thick litter layer under the forest floor restricts the release of soil nutrients. These problems severely restrict the germination of Corydalis seeds and the growth of seedlings. As a result, in recent years, the wild Corydalis community in the Dapan Mountain Nature Reserve has shown a sharp decline in population size, failure to flower and bear fruit, leading to a severe decline in the wild Corydalis population.

[0004] Current Chinese herbal medicine cultivation techniques mainly focus on artificial cultivation, domestication, and planting, lacking effective measures for the rejuvenation of wild Chinese herbal medicines and understory planting techniques. Because Corydalis rhizome only roots under the mother bulb, its underdeveloped root system results in poor water absorption, insufficient to cope with transpiration caused by excessive sunlight, while insufficient sunlight cannot meet its energy needs for growth. Therefore, it has high requirements for its growing environment.

[0005] Therefore, it is necessary to provide a method for the reintroduction and rejuvenation of wild Corydalis understory populations to solve the aforementioned technical problems. Summary of the Invention

[0006] This invention provides a method for the restoration and rejuvenation of wild Corydalis understory populations. This method selects a Chinese fir forest area and selects ecological factors that restrict the growth of wild Corydalis, such as canopy closure and understory litter, to effectively preserve the content of alkaloids in wild Corydalis tubers, especially the content of corydaline A.

[0007] The specific technical solution is as follows:

[0008] A method for rejuvenating and reintroducing wild Corydalis rhizome under forest cover includes: selecting a reintroduction habitat; sowing wild Corydalis rhizome tubers under the forest cover of the reintroduction habitat; and rejuvenating the wild Corydalis rhizome under controlled canopy closure, light intensity regulation, and removal of understory litter until harvest; wherein the canopy closure is 0.7; and the light intensity is 50–100 W / m². 2 .

[0009] Furthermore, the reintroduced habitat is a flat, sandy soil area of ​​Chinese fir forest with an altitude of 600–700m, a slope of 5°–15°, and a windward orientation.

[0010] Furthermore, the Chinese fir forest area is mainly composed of middle-aged trees with a height of over 6m and few shrubs; the humus layer of the soil in the forest area is over 3cm, and the soil is loose and fertile.

[0011] Furthermore, the wild Corydalis tubers are disease-free, newly grown in the current year, with a neat surface and a diameter of 1-1.5cm. The Corydalis tubers are uniformly rinsed with clean water and stored in a 4℃ constant temperature refrigerator.

[0012] Furthermore, the sowing time is from late September to early October.

[0013] Furthermore, the sowing method is as follows: the land is prepared into raised beds, and fallen leaves and weeds on the soil surface are removed. The raised beds are 10cm deep and the row spacing is 15cm. Sowing is completed with a plant spacing of 20cm×5cm and the sowing amount is 2 seeds per hole.

[0014] Furthermore, the canopy closure is 0.7, and the light intensity is 50 W / m². 2 .

[0015] This invention demonstrates that adjusting the forest stand structure can improve the light intensity under the forest canopy, thereby promoting the recovery and growth of understory herbaceous plants.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention selects ecological factors that restrict the growth of wild Corydalis rhizome, such as canopy closure and understory litter, in Chinese fir forest areas, and significantly increases the content of alkaloids in wild Corydalis rhizome tubers, especially the content of corydaline A. Attached Figure Description

[0018] Figure 1 Distribution of various components of Corydalis alkaloids under different planting methods;

[0019] Among them, "field" means planting Corydalis in a field; "understory" means the planting scheme provided in Example 1; and "high canopy closure to remove litter" means the -H planting scheme provided in Example 1. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments. The following are only specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto.

[0021] Example 1

[0022] The specific steps for restoring and rejuvenating wild Corydalis yanhusuo understory populations are as follows:

[0023] (1) Habitat selection

[0024] This case study takes the terrain and climate conditions of Dapan Mountain Nature Reserve in Pan'an County as an example. The reintroduction habitat for Corydalis yanhusuo is selected under Chinese fir forests at an altitude of 600-700m, where wild Corydalis yanhusuo can grow. The terrain is selected with sandy soil with a slope of 5°-15° and the windward side. Under natural conditions, the soil moisture holding capacity and the humidity of the air in the forest stand are kept relatively stable to avoid the problem of limited growth of Corydalis yanhusuo due to poor soil permeability. At the same time, it is convenient to manage the growth of medicinal materials and the reintroduction base.

[0025] The adjacent forests in the reintroduction area should be free from pests and diseases, with middle-aged forests with a tree height of 6m or more being preferred. The habitat should have few shrubs to avoid nutrient competition. Corydalis prefers a warm and humid climate. The average annual precipitation in the reintroduction habitat is 1409.8-1527.8 mm, the humus layer of the forest soil is more than 3 cm, and the soil is loose and fertile.

[0026] The canopy closure and light intensity of the sample plots were controlled by thinning the canopy of Chinese fir forests. Three gradients of canopy closure (light intensity) were set up, and litter removal was combined with the process to minimize interference from other conditions.

[0027] The method for measuring canopy closure is as follows: the canopy closure of the regression plot is measured visually along the diagonal; a sample point is set at 1m intervals along the diagonal, and the sample point is observed vertically upwards to see whether it is covered by the tree canopy as the counting standard, thus obtaining the canopy closure of the forest stand, that is: canopy closure = number of sample points covered by the tree canopy / total number of sample points.

[0028] The method for measuring light intensity is as follows: using a total solar radiation sensor, on a sunny day between 10:00 and 11:00, a sampling point is set at 1m intervals along the diagonal of the selected regression habitat plot. The measurement point is 30cm above the ground and the measurement data is recorded. The average value of the results is taken.

[0029] The following table shows the processing steps:

[0030] Table 1 Experimental schemes for different light intensities under the removal of litter by Corydalis yanhusuo in forests

[0031]

[0032] (2) Seed selection and cultivation

[0033] To rejuvenate the tubers, select healthy, disease-free, newly grown Corydalis rhizomes from the current year. Medium-sized tubers with a diameter of 1-1.5 cm and a uniform surface are preferred. Rinse the Corydalis tubers uniformly with clean water, dry them in the sun, and store them in a refrigerator at a constant temperature of 4℃.

[0034] The best time for sowing and cultivation is from late September to early October. Remove surface debris and weeds. Planting pits should be 8-13 cm deep and 5 cm in diameter, with a plant spacing of 10 cm × 25 cm. Sow 3-5 seeds per hole, taking extra care to avoid damaging the Corydalis tubers during sowing.

[0035] (3) Maintenance and management of plants in forests

[0036] During the restoration and rejuvenation of wild Corydalis rhizome, fertilization, water management, and weeding should be avoided as much as possible. Weeds should be cleared the following spring depending on their growth to prevent them from growing too tall, shading the plants, and competing for resources; surface debris should also be removed. Entry by humans and livestock is prohibited to prevent trampling of seedlings and theft of medicinal materials and seeds.

[0037] (4) Forest harvesting

[0038] In May of the following year, Corydalis rhizome and soil samples were collected from the reintroduced forest plots. The relevant data were measured, compiled, and organized.

[0039] (5) Farmland harvesting

[0040] In Pan'an County, Zhejiang Province, the production area of ​​Corydalis yanhusuo, five farmlands that have been growing Corydalis yanhusuo for many years were randomly selected to harvest Corydalis yanhusuo from farmlands including Shanghu, Mataoshan, Dashanxia, ​​Mazhai, and Jinyun. The content of alkaloid medicinal components in Corydalis yanhusuo grown in local farmlands was measured.

[0041] Experiments such as sample collection were conducted during the growth of Corydalis yanhusuo, with the yield of a single plant and the content of seven alkaloids in the medicinal material as the main analytical objects.

[0042] The test results are shown in the table below:

[0043] Table 2 Results of component analysis in Example 1

[0044]

[0045] Note: Values ​​are mean ± standard deviation. Different lowercase letters indicate significant differences (P < 0.05) between different treatments.

[0046] According to the test results in Table 2, increasing canopy closure and reducing direct sunlight intensity effectively increased the content of protopine, berberine hydrochloride, palmatine hydrochloride, tetrahydrocorydaline, corydaline A, dehydrocorydaline, and corydaline B among the alkaloids in Corydalis yanhusuo during the reintroduction of wild Corydalis yanhusuo populations in the forest. The content of the key alkaloid corydaline A was significantly increased by 38% in the forest environment compared to the field, and corydaline B was increased by 6%. This indicates that the method used in this study for reintroducing wild Corydalis yanhusuo populations in the forest can effectively maintain the medicinal efficacy of the plant, thus providing a stable growth environment for wild Corydalis yanhusuo and contributing to the protection of wild germplasm resources.

[0047] Comparative Example 1

[0048] This comparative example is basically the same as Example 1, except that the method of handling fallen objects is to retain the fallen objects.

[0049] Table 3 Experimental schemes for different light intensities under the condition of retaining litter from Corydalis yanhusuo in the forest.

[0050]

[0051] Experiments such as sample collection were conducted during the growth of Corydalis yanhusuo, with the yield of a single plant and the content of seven alkaloids in the medicinal material as the main analytical objects.

[0052] Table 4. Component detection results of Comparative Example 1

[0053]

[0054]

[0055] According to the test results in Table 4, under the experimental treatment of retaining litter, the alkaloids of wild Corydalis rhizomes in the forest maintained the same trend as the litter removal treatment group under different canopy closure (light intensity) treatments.

[0056] Compared with Example 1, it can be found that the content of medicinal components decreased after retaining the fir litter, with a decrease of 5% to 10% as measured. This indicates that the treatment method of removing litter can effectively increase the content of medicinal components.

[0057] To better understand the medicinal value of alkaloids in wild-grown Corydalis rhizomes under forest cover, we compared the average alkaloid content of wild-grown Corydalis rhizomes with that of Corydalis rhizomes grown in five different farmlands in the area. (See attached table.) Figure 1It was found that planting Corydalis under forest cover effectively increased the content of the key alkaloid corydaline in the tubers. The unique habitat and climate under forest cover altered the alkaloid synthesis characteristics of Corydalis. Compared with farmland cultivation, the proportion of corydaline A in the total alkaloids increased from 22.19% to 41.69%, and the proportion of corydaline B in the total alkaloids increased from 10.53% to 13.19%. The plant devoted more nutrients to the synthesis of corydaline A and corydaline B.

[0058] This case study integrates the data from Example 1 and Comparative Example 1 and performs a two-factor test. The results are shown in Table 5. Individual treatments of litter and canopy closure significantly affect the medicinal properties of the alkaloid corydaline. Changes in canopy closure alter light intensity, thus affecting plant photosynthesis, and consequently influencing the alkaloid content in the tubers by regulating the photosynthetic and transpiration rates.

[0059] Table 5. Two-way ANOVA of Corydaline

[0060]

[0061] Note: For significant differences between treatments, * indicates P < 0.05, ** indicates P < 0.01.

[0062] In addition, we conducted a correlation analysis on forest light intensity and Corydalis alkaloid content, and the results are shown in Table 6.

[0063] Table 6. Correlation between forest light intensity and Corydalis alkaloid content

[0064]

[0065] Note: * indicates a significant correlation (<0.05); ** indicates a significant correlation (<0.01).

[0066] Pearson correlation analysis showed a highly significant negative correlation between forest canopy light intensity and Corydalis alkaloids, indicating that reducing light intensity can effectively increase the alkaloid content of Corydalis under forest canopy. This suggests that adjusting forest canopy closure can effectively improve the alkaloid content of wild Corydalis tubers under forest canopy, and increase the content of alkaloids such as corydaline A and B.

[0067] Example 2

[0068] Soil samples were collected within 1 meter of the site where Corydalis yanhusuo was harvested in May, and the physicochemical properties of the soil were determined. Soil pH was measured using a composite electrode method with a soil-to-water ratio of 2.5:1. Soil organic carbon (SOC) content was determined using the potassium dichromate oil bath external heating method. Total nitrogen (TN) content was determined using a Kjeldahl nitrogen analyzer-distillation method. Total phosphorus (TP) content was determined using the acid-molybdenum antimony colorimetric method. Available phosphorus (AVP) content was determined using the hydrochloric acid-ammonium fluoride method. The DOC and DON content were determined as follows: 5g of fresh soil was extracted with 25ml of ultrapure water (soil-to-water ratio 5:1), shaken at 170rpm for 0.5h at 25℃, centrifuged for 20min, and then filtered through a 0.45μm filter membrane into a plastic bottle for TOC analysis. Soil microbial carbon (MBC), nitrogen (MBN), and phosphorus (MBP) were determined using the chloroform fumigation extraction method, with the above indicators measured according to the "Soil Agrochemical Analysis" standard. The collected physicochemical properties of the soil were analyzed using IBM SPSS 22.0 software. Multiple comparisons of different litter with the same canopy closure were performed using independent paired t-tests (P<0.05).

[0069] Table 7. Soil physicochemical properties at the maturity stage of wild Corydalis yanhusuo

[0070]

[0071] Note: Values ​​are mean ± standard deviation. Different capital letters indicate significant differences between different litter treatments at the same canopy closure (P < 0.05).

[0072] Different lowercase letters indicate significant differences in the same litter treatment under different canopy closure levels (P < 0.05).

[0073] Soil samples were collected in May under six different treatment conditions, and relevant physicochemical properties were measured. pH, organic carbon, total nitrogen, total phosphorus, and soil moisture content were the main analytical parameters. According to the results in Table 7, there were no significant differences in the main nutrients in the soil under different canopy closure treatments with the same litter. Under the same canopy closure, there were no significant differences in soil pH, phosphorus content, and soil moisture between litter retention and litter removal treatments. Soil organic carbon content showed a decreasing trend with increasing canopy closure under different litter treatments; however, there was no significant difference between different litter treatments under the high canopy closure treatment used in this scheme. Soil nitrogen content did not show an overall changing trend, suggesting that litter treatment may not be the main influencing factor. Using the method described in this scheme, litter removal treatment under short-term reintroduction of wild Corydalis yanhusuo populations in forests will not affect the continuous input of soil nutrients other than nitrogen; appropriate nitrogen supplementation treatment can ensure the supply and cycling of soil nutrients.

[0074] Example 3

[0075] This embodiment is basically the same as Embodiment 1, except that it measures and statistically analyzes the chlorophyll content, intercellular CO2 concentration, and transpiration rate of Corydalis yanhusuo under forest cover during April, when its growth is most vigorous. The relative chlorophyll content is expressed using SPAD (soil and plant analyzer development) values, measured using a SPAD-502PLUS chlorophyll meter (Konica Minolta, Japan). Measurements were taken at three locations on each leaf: the leaf tip, leaf midsection, and leaf base, and the average value was calculated. On a sunny day between 9:00 and 11:00 AM, a LI-6400XT photosynthesis system (LI-COR, USA) was used to measure the net photosynthetic rate Pn, stomatal conductance Gs, intercellular carbon dioxide concentration Ci, and transpiration rate Tr of Corydalis yanhusuo. The light intensity was set at 900 μmol / m² / s, the leaf chamber flow rate at 200 ml / min, and the temperature was not controlled. Three healthy leaves from the same location were selected from each plant. When the increase or decrease in the net photosynthetic rate of each leaf was less than 0.5, five consecutive values ​​were recorded and the average was taken. This experiment was conducted continuously for two years, from 2021 to 2022, in the Dapan Mountain Nature Reserve. The statistical results are illustrated in the following charts:

[0076] Table 8. Photosynthetic characteristics of wild Corydalis during flowering period

[0077]

[0078]

[0079] Note: Values ​​are mean ± SD. Different lowercase letters indicate significant differences under different canopy closure treatments (P < 0.05), and different uppercase letters indicate significant differences under different litter treatments (p < 0.05). In the two-way analysis of light and canopy closure, NS indicates no significant difference (P > 0.05), * indicates a significant difference (P < 0.05), and ** indicates an extremely significant difference (P < 0.01).

[0080] According to the test results in Table 8, the photosynthetic index of Corydalis yanhusuo in the forest showed an overall trend of decreasing photosynthetic index with decreasing light intensity during the flowering period, which is consistent with the experimental expectations. A comprehensive analysis of the photosynthetic index data of Corydalis yanhusuo during the flowering period of 2021 and 2022 revealed that different litter treatments had little effect on the net photosynthetic rate and stomatal conductance of Corydalis yanhusuo. Litter and canopy closure treatments had significant effects on the intercellular CO2 concentration and transpiration rate of Corydalis yanhusuo, respectively. Meanwhile, the transpiration rate of the litter removal treatment was generally lower than that of the litter retention treatment, which may explain why the alkaloid content in the litter removal treatment was higher than that in the retention treatment group, but the specific mechanism of this effect is unclear.

Claims

1. A method for the reintroduction and rejuvenation of wild Corydalis yanhusuo forest understory populations, characterized in that, include: Select a reintroduction habitat, sow wild Corydalis tubers under the forest canopy in the reintroduction habitat, and rejuvenate the wild Corydalis under controlled conditions of canopy cover, light intensity and removal of litter until harvest; The canopy closure is 0.7; the light intensity is 50 W / m². 2 ; The reintroduced habitat is a flat, sandy soil area of ​​Chinese fir forest with an altitude of 600-700 m, a slope of 5°-15°, and a windward orientation. By controlling the canopy density and light intensity under the forest canopy, as well as removing forest litter, the content of corydaline A and corydaline B in wild Corydalis yanhusuo was increased.

2. The method for rejuvenating and restoring wild Corydalis yanhusuo understory populations as described in claim 1, characterized in that, The Chinese fir forest area is mainly composed of middle-aged trees with a height of 6 m or more and few shrubs; the soil humus layer in the forest area is more than 3 cm, and the soil is loose and fertile.

3. The method for rejuvenating and restoring wild Corydalis yanhusuo understory populations as described in claim 1, characterized in that, The wild Corydalis tubers are disease-free, newly grown in the current year, with a neat surface and a diameter of 1 to 1.5 cm. The Corydalis tubers are uniformly rinsed with clean water and stored in a 4 ℃ constant temperature refrigerator.

4. The method for rejuvenating and restoring wild Corydalis yanhusuo understory populations as described in claim 1, characterized in that, The sowing time is from late September to early October.

5. The method for rejuvenating and restoring wild Corydalis yanhusuo understory populations as described in claim 1, characterized in that, The sowing method is as follows: remove fallen leaves and weeds from the soil surface, make raised beds with a depth of 10 cm and a row spacing of 15 cm, and sow at a plant spacing of 10 cm × 25 cm, with a sowing amount of 2 seeds per hole.

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