Method for improving medicinal components of astragalus membranaceus and optimizing tissue structure by utilizing low-temperature quantitative regulation and control in wintering period

Abstract

The invention discloses a method for improving medicinal components of astragalus mongolicus and optimizing a tissue structure by utilizing wintering period low-temperature quantitative regulation and control. The method comprises the following steps: 1, calculating the wintering period low-temperature amount at a position 40cm underground; 2, setting a gradient treatment group and an untreated group; 3, burying the root system of the astragalus membranaceus at a position which is 40cm away from the ground; 4, detecting the percentage contents of astragaloside, miscanin and calycosin-7-glucoside and the content of crude polysaccharide of the astragalus membranaceus according to the set low-temperature amount gradient, and observing the root tissue structure of the astragalus membranaceus; 5, calculating a comprehensive score through a membership function to obtain an optimal low-temperature quantity; 6, wrapping the root system in a woven bag or a breathable container with a sandy soil mixture, and placing the woven bag or the breathable container in a temperature control cellar or a refrigeration house; and 7, treating and setting temperature and time according to the target low-temperature quantity, and performing staged temperature control. According to the method, based on low-temperature quantity gradient regulation and control, the medicinal components and the tissue structure are synergistically improved by optimizing low-temperature treatment parameters below zero.

Description

Technical Field

[0001] The present invention belongs to the technical field of Chinese herbal medicine cultivation, and relates to a method for improving the quality of Astragalus membranaceus, in particular to a method for increasing the content of medicinal components in the roots of Astragalus membranaceus and improving the tissue structure by quantifying the low temperature amount during the overwintering period. Background Art

[0002] As a traditional Chinese herbal medicine, the quality of Astragalus membranaceus depends on the accumulation of secondary metabolites (such as astragaloside IV, polysaccharides, flavonoid glycosides). Existing research shows that the quality of Astragalus membranaceus in the cold regions of the north is excellent, and low temperature in environmental factors can induce the expression of key genes for secondary metabolism and promote the synthesis of medicinal components. However, the existing technology has the following limitations:

[0003] 1. The low temperature amount is not quantified: Most studies only qualitatively describe the influence of low temperature, lacking the quantitative relationship between low temperature intensity and duration, resulting in blind regulation in production practice;

[0004] 2. Lack of research on sub-zero low temperature: Existing technologies mostly focus on above-zero low temperature, and there is insufficient systematic research on long-term sub-zero low temperature;

[0005] 3. Risk of tissue damage: The critical low temperature threshold is not clear, which easily leads to root frostbite or metabolic inhibition. Summary of the Invention

[0006] In view of the above problems existing in the prior art, in order to effectively increase the content of medicinal components in Astragalus membranaceus, the present invention provides a method for improving the medicinal components and optimizing the tissue structure of Astragalus membranaceus by quantitatively regulating the low temperature during the overwintering period. This method is based on the gradient regulation of low temperature amount, and by optimizing the parameters of sub-zero low temperature treatment, the coordinated improvement of medicinal components and tissue structure is achieved.

[0007] The object of the present invention is achieved through the following technical solutions:

[0008] A method for improving the medicinal components and optimizing the tissue structure of Astragalus membranaceus by quantitatively regulating the low temperature during the overwintering period, comprising the following steps:

[0009] Step 1: According to the meteorological data of the target area, taking the root length of two-year-old Astragalus membranaceus as a reference, calculate the low temperature amount during the overwintering period (November to March of the following year) at 40 cm underground:

[0010] Low temperature amount (°C·d) = Σ(monthly average temperature × number of days in that month);

[0011] Step 2: Set gradient treatment groups and a control group according to the low temperature amount during the entire overwintering period from low to high;

[0012] Step 3: When the ground temperature is 0 - 1.5 °C and the soil is not frozen, dig out the roots of Astragalus membranaceus, spray water on the sandy soil and wrap the roots of Astragalus membranaceus, and bury them at 40 cm underground;

[0013] Step 4: According to the set low-temperature quantity gradient, detect the percentage contents of astragaloside IV, ononin, and calycosin in astragalus at 60 d, 80 d, 100 d, and 180 d respectively (by HPLC method) and detect the crude polysaccharide content (by sulfuric acid-phenol colorimetric method), and simultaneously observe the root tissue structure of astragalus (by paraffin section method);

[0014] Step 5: Calculate the comprehensive score through the membership function to obtain the optimal low-temperature quantity;

[0015] Step 6: Use the optimal low-temperature quantity for artificial regulation. Wrap the roots with a sand-soil mixture (sand: soil = 1:1, water content 10-15%) in a woven bag or a breathable container and place them in a temperature-controlled cellar or cold storage;

[0016] Step 7: Set the temperature and time according to the target low-temperature quantity for treatment, simulate natural fluctuations, and perform staged temperature control. For example, -280 °C·d requires -8×35 days. Staged temperature control (simulating natural fluctuations): Stage 1: -5 °C (simulating early winter), lasting for 10 days (low-temperature quantity = -50); Stage 2: -7 °C (simulating deep winter), lasting for 30 days (low-temperature quantity = -210); Stage 3: -4 °C (simulating early spring), lasting for 5 days (low-temperature quantity = -20), until the cumulative low-temperature quantity = -280.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1. Compared with the previous low-temperature treatment methods, the low-temperature treatment method adopted by the present invention precisely quantifies and gradient designs the sub-zero low-temperature quantity, proposes a calculation model of "low-temperature quantity (°C·d) = Σ(monthly average soil temperature × number of days)", and for the first time quantifies the sub-zero low-temperature intensity and duration into adjustable parameters; determines that the optimal low-temperature quantity is -280 (°C·d), which increases the contents of astragaloside IV, crude polysaccharide, and ononin by 2.99 times, 6.11 times, and 23.9 times respectively; the synergistic optimization mechanism of tissue structure and medicinal components is found, and it is found that low temperature induces an increase in the width of phloem cells in the root system (up to 82.83 μm, 49% higher than the control) and sparsification of vessels, promoting the accumulation of secondary metabolites; and this treatment method will not cause frost damage to the roots of astragalus, enabling the roots of astragalus to still be active and not affecting the subsequent slicing treatment of fresh samples. Therefore, in the later stage, this result characteristic can be utilized in the production regulation and initial processing in the place of origin of astragalus.

[0019] 2. The method of the present invention is applicable to temperate and cold temperate regions with an annual average temperature ≤ 10 °C and the soil temperature during the overwintering period (from November to March of the following year) can drop to about -10 °C, including but not limited to Northeast China, Northwest China, and cold and cool regions at high altitudes in China.

[0020] 3. By quantifying the low-temperature quantity (low-temperature intensity × duration), the present invention can be flexibly adapted to different climate zones to meet the demand for continuous low-temperature accumulation during the overwintering period. Description of the Drawings

[0021] Figure 1 Effect of different low temperature amounts on the content of astragaloside IV, the main medicinal ingredient in the roots of Astragalus membranaceus

[0022] Figure 2 Effect of different low temperature amounts on the content of crude polysaccharide, the main medicinal ingredient in the roots of Astragalus membranaceus

[0023] Figure 3 Effect of different low temperature amounts on the content of formononetin glucoside, the main medicinal ingredient in the roots of Astragalus membranaceus

[0024] Figure 4 Effect of different low temperature amounts on the content of calycosin-7-O-β-D-glucoside, the main medicinal ingredient in the roots of Astragalus membranaceus

[0025] Figure 5 Anatomical structure of the roots of Astragalus membranaceus under different low temperature amounts, A - DW0, B - DW1, C - DW2, D - DW3, E - DW4; P - periderm, F - fiber, Cw - cell wall, Ve - vessel, Xr - xylem ray, Is - intercellular space

[0026] Figure 6 Verification of indoor low temperature regulation Detailed Embodiment

[0027] The technical solution of the present invention will be further described below in conjunction with the drawings, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention, without departing from the spirit and scope of the technical solution of the present invention, shall be covered by the protection scope of the present invention.

[0028] The present invention provides a method for improving the quality of Astragalus membranaceus, and the method includes the following steps:

[0029] In late October, when the soil was not frozen, the roots of Astragalus membranaceus were dug out, trying to keep the roots of Astragalus membranaceus intact. After mixing with 1:1 sand and spraying a small amount of water, the roots of Astragalus membranaceus were wrapped and buried 40 cm underground (referring to the root length of two-year-old Astragalus membranaceus var. mongholicus. After the low temperature during the overwintering period, the medicinal components of two-year-old Astragalus membranaceus increased significantly. Calculating the low temperature at this depth is crucial. The low temperature calculated based on the soil temperature at 40 cm deep can be used as a basis for artificially regulating the quality of Astragalus membranaceus in the future, which has practical production significance. Moreover, the roots of Astragalus membranaceus will not be frozen at this depth. Because the fresh roots of Astragalus membranaceus have a large water content after harvest, if the depth is inappropriate, the roots of Astragalus membranaceus will be damaged by freezing and lose their activity). Both the upper and lower parts were covered with woven bags for convenient winter sample collection. According to the soil temperature at 40 cm underground provided by the Daqing Meteorological Bureau in 2022, the total low temperature during the local winter from November to April of the following year was estimated. The total low temperature = the sum of the low temperature per month (average soil temperature × number of days). Different levels of low temperature were set, with a total of 5 gradients, -140 (DW1), -280 (DW2), -420 (DW3), -600 (DW4), and the untreated one was CK (DW0). Samples were taken at 60 d, 80 d, 100 d, and 180 d, with 3 replicates. The calculation basis of the low temperature is shown in Table 1:

[0030] Table 1 Soil Temperature at 40 cm Depth Underground in Daqing City in 2022

[0031] [[ID=?]]

[0032] Note: The data was provided by the Daqing Meteorological Bureau

[0033] Test results:

[0034] 1. Influence of low temperature on medicinal components of Astragalus membranaceus roots

[0035] As [[ID=1?]] Figures 1 to 4 shown, the four main medicinal components of Astragalus membranaceus showed a trend of first increasing and then decreasing with the increase of low temperature. As Figure 1 known, the content of astragaloside Ⅳ increased significantly during the overwintering period. Among them, it was the highest when the low temperature was -420 (DW3), with a content as high as 0.37%, which was 4.23 times higher than that of CK (DW0). As Figure 2 known, the crude polysaccharide increased significantly when the low temperature was between -280 and -600. Among them, it was the highest when the low temperature was -280 (DW2), up to 4.30%, which was 6.14 times higher than that of CK. Figure 3 As known, formononetin increased significantly. Among them, it was the highest when the low temperature was -280 (DW2), up to 1.23%, which was 23.57 times higher than that of CK. Figure 4It can be seen that the content of calycosin-7-O-β-D-glucoside increases significantly when the low temperature is between -280 and -600. There is no significant difference in these two treatment times, which are 0.114% and 0.118% respectively. Compared with CK, they increase by 26% and 32% respectively.

[0036] To obtain the optimal low temperature for promoting the accumulation of medicinal components in Astragalus membranaceus, the membership function analysis was carried out on the main medicinal components at low temperatures of 0, -140, -240, -480 and -600 respectively. As shown in Table 2, when the low temperature is -280 (DW2), the membership function values of crude polysaccharide and ononin are the highest; when the low temperature is -420 (DW3), the membership function values of astragaloside IV and calycosin-7-O-β-D-glucoside are the highest; among them, when the low temperature is -280 (DW2), the average membership function score of the four medicinal components is the highest, which is 0.87. To sum up, low temperature is helpful for the accumulation of medicinal components in the roots of Astragalus membranaceus. Except for calycosin-7-O-β-D-glucoside, the increase in astragaloside IV, crude polysaccharide and ononin is obvious. At low temperatures of -280 (DW2) and -420 (DW3), the comprehensive accumulation of medicinal components is significantly higher than that of other treatments.

[0037] Table 2 Membership function scores

[0038]

[0039] 2. Effects of different low temperatures on the root tissue structure of Astragalus membranaceus

[0040] Under low temperature environment, obvious changes have occurred in the secondary phloem cells of the roots of Astragalus membranaceus. When the roots of Astragalus membranaceus are not treated with low temperature, the phloem parenchyma cells are arranged neatly, with a compact and regular structure, intact and clearly visible, and no cell damage occurs (see Table 3, Figure 5 A). After the low temperature of -140 (DW1), the phloem parenchyma cells of the roots are deformed, the structure begins to be disordered, the cells develop from nearly round to oval, there is a pulling phenomenon in the cells, the cell wall is damaged, cell gaps appear, the xylem parenchyma cells swell and deform, the vessels are sparse and the diameter becomes smaller (see Table 3, Figure 5 B); when the low temperature reaches -280, the phloem parenchyma cells are more slender, the degree of cell structure disorder increases, the cell gaps become significantly larger, the cell wall is severely damaged, and the cell walls of some cells begin to disintegrate, and the structure is blurred and unclear. The xylem parenchyma cells swell and deform, and 2 vessels are clustered, sparse and small in diameter (see Table 3, Figure 5 C); when the low temperature reaches -480, although the ground temperature is still below 0℃, it has begun to rise. The disorder phenomenon of the phloem parenchyma cells has been significantly improved, the cell pulling phenomenon has been alleviated, the cell gaps become smaller, the shape of the xylem parenchyma cells shows signs of recovery, 2 vessels are clustered, with a large number and a large diameter (see Table 3, Figure 5D); When the low-temperature amount is -600, the rising amplitude of the ground temperature becomes larger, and the phloem parenchyma cells and xylem parenchyma cells basically return to the state of normal growth. The cell structure is arranged tightly, the cells change from long oval to nearly round, and the cell gaps basically disappear. Two xylem vessels are aggregated together in groups and have a large diameter (see Table 3, Figure 5 E).

[0041] Table 3 Anatomical structure parameters of Astragalus membranaceus roots under different low-temperature amounts

[0042]

[0043] Note: BPCL length of phloem parenchyma cells, BPCW width of phloem parenchyma cells, BPCC perimeter of phloem parenchyma cells, BPCA area of phloem parenchyma cells, BTCW wall thickness of phloem parenchyma cells, CD diameter of vessels, WPCL length of xylem parenchyma cells, WPCW width of xylem parenchyma cells, WPCT wall thickness of xylem parenchyma cells, WPCC perimeter of xylem parenchyma cells, WPCA area of xylem parenchyma cells. Different lowercase letters indicate significant differences (P<0.05).

[0044] 3. Correlation between the root tissue structure and medicinal components of Astragalus membranaceus under different low-temperature amounts

[0045] According to the results of Pearson correlation analysis, there is a certain correlation between the anatomical structure characteristics of Astragalus membranaceus roots and medicinal components under low-temperature treatment. The width of phloem parenchyma cells (BPCW) has a highly significant positive correlation with crude polysaccharides and ononin, and a significant positive correlation with astragaloside IV. The perimeter of phloem parenchyma cells (BPCC) has a significant positive correlation with astragaloside IV and calycosin-7-O-β-D-glucoside (Table 4). Through the analysis of the root structure of Astragalus membranaceus, it is found that under low temperature, the width and perimeter of the phloem cells of the roots with higher medicinal components of Astragalus membranaceus are also relatively larger.

[0046] Table 4 Correlation between the root tissue structure and medicinal components of Astragalus membranaceus

[0047]

[0048] Note: BPCL length of phloem parenchyma cells, BPCW width of phloem parenchyma cells, BPCC perimeter of phloem parenchyma cells, BPCA area of phloem parenchyma cells, BTCW wall thickness of phloem parenchyma cells, CD diameter of vessels, WPCL length of xylem parenchyma cells, WPCW width of xylem parenchyma cells, WPCT wall thickness of xylem parenchyma cells, WPCC perimeter of xylem parenchyma cells, WPCA area of xylem parenchyma cells. For correlation comparison, *P<0.05, **P<0.01.

[0049] Through Tables 1 - 4, Figures 1 to 5It can be seen that: with the increase of the low-temperature amount, the contents of medicinal components such as astragaloside IV, crude polysaccharide, formononetin glucoside, and calycosin-7-O-β-D-glucoside show a change of first increasing and then decreasing. Through the membership function, it is obtained that the comprehensive accumulation of medicinal components in the DW2 treatment is significantly higher than that in other treatments. Compared with the CK, the contents of the four medicinal components are increased by 2.99 times, 6.11 times, 23.9 times, and 1.27 times respectively, and the increase amplitude of formononetin glucoside is the largest. The low temperature during the overwintering period changes the root tissue structure of astragalus. The width of phloem cells increases, the perimeter increases, and the vessels become sparse. When the low-temperature amount reaches -600 (DW4), although the low-temperature amount increases, the temperature rises and the tissue structure begins to recover. This kind of change is beneficial to the accumulation of medicinal components in astragalus. Therefore, the low temperature during the overwintering period can improve the content of medicinal components in the roots of astragalus. When the low-temperature amount is -280, the effect on improving the quality of astragalus medicinal materials is the best.

[0050] As Figure 6 shown, according to the low-temperature amount obtained by the present invention, under the indoor low-temperature regulation, the contents of several medicinal components such as astragaloside IV, polysaccharide, calycosin-7-O-β-D-glucoside, and calycosin in the roots of astragalus are greatly increased. The results are close to the contents of medicinal components in astragalus under the optimal low-temperature amount during outdoor overwintering, and there is no damage to the appearance of the astragalus roots.

[0051] What the present invention mainly emphasizes is the low-temperature amount, which is calculated by the product of the low-temperature degree and time adopted. At the same time, it is required that the range of the low-temperature degree is below 0°C but not exceeding -10°C. The low-temperature treatment time is related to the temperature at that time. For example, 280 °C·d requires -8°C × 35 days.

Claims

1. A method for quantitatively regulating and controlling by using low temperature during the overwintering period to improve the medicinal components and optimize the tissue structure of Astragalus membranaceus, characterized in that The method includes the following steps: Step 1: According to the meteorological data of the target area, taking the root length of two-year-old Astragalus membranaceus as a reference, calculate the low-temperature amount at 40 cm underground during the overwintering period; Step 2: Set gradient treatment groups and a control group according to the low-temperature amount during the entire overwintering period from low to high; Step 3: When the ground temperature is 0 - 1.5 °C and the soil is not frozen, dig the roots of Astragalus membranaceus, spray water on the sandy soil and then wrap the roots of Astragalus membranaceus, and bury them at 40 cm underground; Step 4: According to the set low-temperature amount gradient, detect the percentage contents of astragaloside IV, ononin, and calycosin-7-O-β-D-glucoside and the crude polysaccharide content of Astragalus membranaceus at 60 d, 80 d, 100 d, and 180 d respectively, and at the same time observe the root tissue structure of Astragalus membranaceus; Step 5: Calculate the comprehensive score through the membership function to obtain the optimal low-temperature amount; Step 6: Wrap the roots with a sandy soil mixture in a woven bag or a breathable container, and place them in a temperature-controlled cellar or cold storage; Step 7: Set the temperature and time according to the target low-temperature amount treatment, simulate natural fluctuations, and perform staged temperature control.

2. The method for improving the medicinal components and optimizing the tissue structure of Astragalus membranaceus by quantitatively regulating low temperature during the overwintering period according to claim 1, wherein The calculation formula for the low-temperature amount during the overwintering period is: Low-temperature amount (°C·d) = Σ(monthly average temperature × number of days in that month).

3. The method for improving the medicinal components of Astragalus membranaceus and optimizing the tissue structure by quantitatively regulating the low temperature during the overwintering period according to claim 1, characterized in that The roots of Astragalus membranaceus are mixed with sandy soil at a ratio of 1:1, and the water content is 10 - 15%.

4. The method for improving the medicinal components of Astragalus membranaceus and optimizing its tissue structure by quantitatively regulating low temperature during the overwintering period according to claim 1, characterized in that In Step 7, the natural fluctuations include three stages: Stage 1: Early winter; Stage 2: Midwinter; Stage 3: Early spring.

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