Method for increasing content of active ingredients with edible value in radix achyranthis bidentatae

Abstract

The invention relates to the technical field of plant breeding, and particularly provides a method for increasing the content of active ingredients with edible value in radix achyranthis bidentatae. By accurately regulating and controlling the irradiation dose of the heavy ion beam, the content of core active ingredients such as photosynthetic pigments, soluble sugar, total flavonoids and total polysaccharides in the radix achyranthis bidentatae is remarkably increased, the quality of the radix achyranthis bidentatae is effectively improved, and a high-quality raw material is provided for developing high-value-added functional foods and traditional Chinese medicine preparations. According to the method, various antioxidant physiological indexes are comprehensively monitored, so that the mutagenic achyranthes bidentata plants have good growth vigor and stress resistance, and the balance of quality improvement and plant vitality is realized. A new way is provided for resource utilization of the overground stem and leaf parts of the achyranthes bidentata, comprehensive development and utilization of whole-plant resources of the achyranthes bidentata are promoted, resource waste is reduced, and a solid foundation is laid for creating high-yield and high-quality new germplasm of the achyranthes bidentata and promoting modern development of the traditional Chinese medicine industry.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, and in particular to a method for increasing the content of edible active ingredients in Achyranthes bidentata. Background Technology

[0002] Achyranthes bidentata Blume, a perennial herb belonging to the genus Achyranthes in the family Amaranthaceae, is a renowned traditional Chinese medicine, with its dried root used in both traditional Chinese and Mongolian medicine. Modern pharmacological studies have shown that Achyranthes bidentata is rich in various bioactive components, possessing anti-osteoporosis, anti-inflammatory, analgesic, and immunomodulatory effects. The edible value of Achyranthes bidentata is closely related to its various active ingredients: photosynthetic pigments impart excellent sensory qualities and reflect the plant's growth vigor; soluble sugars form the basis of flavor quality and provide energy for the synthesis of other components; total flavonoids and total phenols are important antioxidant functional components; and total polysaccharides are the core active ingredients for immune regulation and anti-aging, serving as a key indicator for evaluating the edible quality of Achyranthes bidentata. With the rise of the health industry, the market demand for Achyranthes bidentata continues to grow.

[0003] However, the current development of the Achyranthes bidentata industry faces severe challenges, with artificial cultivation becoming the main source. However, long-term continuous cropping has led to severe mixing of germplasm resources, significant varietal degeneration, weakened plant resistance, and frequent outbreaks of pests and diseases. Simultaneously, the quality of Achyranthes bidentata medicinal materials is greatly affected by the planting environment and the genetic background of the germplasm. The accumulation of key active ingredients, such as total polysaccharides, is extremely unstable, and their content is significantly affected by cultivation practices such as fertilization levels, planting density, and harvesting time, resulting in inconsistent raw material quality in the market and difficulty in meeting the needs of standardized production. Furthermore, the development and utilization of Achyranthes bidentata currently mainly focuses on the underground roots, while the above-ground stems and leaves are often treated as waste, causing resource waste.

[0004] Heavy ion beams possess high energy transfer linear density and high relative biological effects, enabling them to induce high-frequency, broad-spectrum mutations in plants with minimal damage and easily stabilized mutant traits. However, a systematic dose-response model and mechanism analysis are still lacking regarding how to precisely control irradiation parameters to achieve targeted enhancement of specific active components in Achyranthes bidentata. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides a method for increasing the content of edible active ingredients in Achyranthes bidentata.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for increasing the content of edible active ingredients in Achyranthes bidentata, wherein the edible active ingredients are selected from any one or more of photosynthetic pigments, soluble sugars, total flavonoids and total polysaccharides; the method includes: irradiating Achyranthes bidentata seeds with a heavy ion beam at a dose of 40 Gy to 150 Gy; wherein the heavy ion beam includes a carbon ion beam.

[0007] According to the present invention, a method for increasing the content of edible active ingredients in Achyranthes bidentata is provided, wherein preferably, the photosynthetic pigments are selected from any one or more of chlorophyll a, chlorophyll b, and carotenoids.

[0008] According to the present invention, a method for increasing the content of edible active ingredients in Achyranthes bidentata is provided. Preferably, the edible active ingredients are selected from any one of the following compositional compositions (1) to (2): compositional composition (1) is composed of photosynthetic pigments and soluble sugars; compositional composition (2) is composed of total flavonoids and total polysaccharides.

[0009] More preferably, if the active ingredient with edible value is a component composition (1), then the irradiation dose is 40 Gy to 100 Gy.

[0010] More preferably, if the active ingredient with edible value is a component composition (1), then the irradiation dose is 40 Gy to 80 Gy.

[0011] In some specific embodiments, the active ingredient with edible value is a component composition (1), and the irradiation dose is 40 Gy, 50 Gy, 60 Gy, 70 Gy or 80 Gy.

[0012] More preferably, if the active ingredient with edible value is a component composition (1), then the irradiation dose is 80 Gy.

[0013] More preferably, if the active ingredient with edible value is a component composition (2), then the irradiation dose is 40 Gy or 120 Gy to 150 Gy.

[0014] In some specific embodiments, the active ingredient with edible value is a component composition (2), and the irradiation dose is 120 Gy, 130 Gy, 140 Gy or 150 Gy.

[0015] More preferably, if the active ingredient with edible value is a component composition (2), then the irradiation dose is 120 Gy or 150 Gy.

[0016] According to the present invention, a method for increasing the content of edible active ingredients in Achyranthes bidentata is provided. Preferably, the edible active ingredient content is chlorophyll a and / or chlorophyll b, and the irradiation dose is 40 Gy to 120 Gy.

[0017] More preferably, if the content of the edible active ingredient is chlorophyll a and / or chlorophyll b, then the irradiation dose is 80 Gy to 100 Gy.

[0018] In some specific embodiments, if the content of the edible active ingredient is chlorophyll a and / or chlorophyll b, then the irradiation dose is 80 Gy, 85 Gy, 90 Gy, 95 Gy or 100 Gy.

[0019] More preferably, if the content of the edible active ingredient is chlorophyll a and / or chlorophyll b, then the irradiation dose is 80 Gy.

[0020] According to the present invention, a method for increasing the content of edible active ingredients in Achyranthes bidentata is provided. Preferably, the edible active ingredient is carotenoid, and the irradiation dose is 40 Gy to 100 Gy.

[0021] More preferably, if the content of the edible active ingredient is carotenoid, then the irradiation dose is 80 Gy to 100 Gy.

[0022] In some specific implementations, if the content of the edible active ingredient is carotenoid, then the irradiation dose is 80 Gy, 85 Gy, 90 Gy, 95 Gy, or 100 Gy.

[0023] More preferably, if the edible active ingredient is carotenoid, then the irradiation dose is 80 Gy.

[0024] According to the present invention, a method for increasing the content of edible active ingredients in Achyranthes bidentata is provided. Preferably, the edible active ingredient content is soluble sugar, and the irradiation dose is 40 Gy to 120 Gy.

[0025] More preferably, if the content of the edible active ingredient is soluble sugar, then the irradiation dose is 40 Gy to 80 Gy or 120 Gy.

[0026] In some specific implementations, if the content of the edible active ingredient is soluble sugar, then the irradiation dose is 40 Gy, 50 Gy, 60 Gy, 70 Gy, 80 Gy, or 120 Gy.

[0027] More preferably, if the edible active ingredient is soluble sugar, then the irradiation dose is 80 Gy.

[0028] According to the present invention, a method for increasing the content of edible active ingredients in Achyranthes bidentata is provided. Preferably, the edible active ingredient content is total flavonoids, and the irradiation dose is 40 Gy or 100 Gy to 150 Gy.

[0029] More preferably, if the content of the edible active ingredient is total flavonoids, then the irradiation dose is 40 Gy or 120 Gy to 150 Gy.

[0030] In some specific implementations, the content of the edible active ingredient is total flavonoids, and the irradiation dose is 40 Gy, 120 Gy, 130 Gy, 140 Gy or 150 Gy.

[0031] More preferably, if the content of the edible active ingredient is total flavonoids, then the irradiation dose is 150 Gy.

[0032] According to the present invention, a method for increasing the content of edible active ingredients in Achyranthes bidentata is provided. Preferably, the edible active ingredient content is total polysaccharides, and the irradiation dose is 40 Gy or 120 Gy to 150 Gy.

[0033] More preferably, if the content of the edible active ingredient is total polysaccharide, then the irradiation dose is 40 Gy, 120 Gy, 130 Gy, 140 Gy or 150 Gy.

[0034] More preferably, if the content of the edible active ingredient is total polysaccharide, then the irradiation dose is 40 Gy.

[0035] According to the present invention, a method for increasing the content of edible active ingredients in Achyranthes bidentata is provided, wherein preferably, the dose rate of heavy ion beam irradiation is 70 Gy / min to 90 Gy / min.

[0036] More preferably, the dose rate of heavy ion beam irradiation is 75 Gy / min to 85 Gy / min.

[0037] In some specific implementations, the dose rate of heavy ion beam irradiation is 75 Gy / min, 76 Gy / min, 77 Gy / min, 78 Gy / min, 79 Gy / min, 80 Gy / min, 81 Gy / min, 82 Gy / min, 83 Gy / min, 84 Gy / min or 85 Gy / min.

[0038] More preferably, the dose rate of the heavy ion beam irradiation is 80 Gy / min.

[0039] According to the present invention, a method for increasing the content of edible active ingredients in Achyranthes bidentata is provided, wherein preferably, the total energy of the heavy ion beam is 960 MeV to 970 MeV.

[0040] More preferably, the total energy of the heavy ion beam is 962 MeV to 968 MeV.

[0041] In some specific implementations, the total energy of the heavy ion beam is 962 MeV, 963 MeV, 964 MeV, 965 MeV, 966 MeV, 967 MeV, or 968 MeV.

[0042] More preferably, the total energy of the heavy ion beam is 967 MeV.

[0043] According to the present invention, a method for increasing the content of edible active ingredients in Achyranthes bidentata is preferably provided in which the average energy transfer linear density of the heavy ion beam is 30 keV / μm to 40 keV / μm.

[0044] More preferably, the average energy transfer linear density of the heavy ion beam is 32 keV / μm to 38 keV / μm.

[0045] In some specific implementations, the average energy transfer linear density of the heavy ion beam is 32 keV / μm, 33 keV / μm, 34 keV / μm, 35 keV / μm, 36 keV / μm, 37 keV / μm, or 38 keV / μm.

[0046] More preferably, the average energy transfer linear density of the heavy ion beam is 34 keV / μm.

[0047] According to the present invention, a method for increasing the content of edible active ingredients in Achyranthes bidentata is preferably further comprising: cultivating Achyranthes bidentata seeds irradiated with heavy ion beams to obtain Achyranthes bidentata plants.

[0048] More preferably, the Achyranthes bidentata plant is an Achyranthes bidentata seedling.

[0049] More preferably, the Achyranthes bidentata seedlings are 6 to 10 weeks old.

[0050] In some specific implementations, the Achyranthes bidentata seedlings are 6, 7, 8, 9, or 10 weeks old.

[0051] More preferably, the Achyranthes bidentata seedlings are 8 weeks old.

[0052] More preferably, the seeds of Achyranthes bidentata irradiated with heavy ion beams are soaked before cultivation.

[0053] More preferably, the seeds are soaked in water or water containing indoleacetic acid.

[0054] According to the present invention, a method for increasing the content of edible active ingredients in Achyranthes bidentata is provided, wherein the edible active ingredients are located in the aerial part of Achyranthes bidentata.

[0055] More preferably, the edible active ingredient is located in the leaves of Achyranthes bidentata.

[0056] Secondly, the present invention also provides a method for preparing an extract of Achyranthes bidentata, comprising: extracting the extract from an Achyranthes bidentata plant; wherein the Achyranthes bidentata plant is treated by any of the methods described above for increasing the content of edible active ingredients in Achyranthes bidentata.

[0057] According to the present invention, a method for preparing Achyranthes bidentata extract is preferably provided in which the extraction method includes any one or more of the following: alcohol extraction, water extraction, organic solvent extraction, enzymatic hydrolysis, buffer extraction, homogenization extraction, and ultrasound-assisted extraction.

[0058] According to a method for preparing an Achyranthes bidentata extract provided by the present invention, preferably, the extract includes any one or more of photosynthetic pigments, soluble sugars, total flavonoids, and total polysaccharides.

[0059] According to a method for preparing Achyranthes bidentata extract provided by the present invention, preferably, the extract is obtained from the above-ground parts of the Achyranthes bidentata plant.

[0060] More preferably, the extract is obtained from the leaves of the Achyranthes bidentata plant.

[0061] According to the present invention, a method for preparing Achyranthes bidentata extract is provided, wherein the Achyranthes bidentata plant is an Achyranthes bidentata seedling.

[0062] More preferably, the Achyranthes bidentata seedlings are 6 to 10 weeks old.

[0063] In some specific implementations, the Achyranthes bidentata seedlings are 6, 7, 8, 9, or 10 weeks old.

[0064] More preferably, the Achyranthes bidentata seedlings are 8 weeks old.

[0065] Thirdly, the present invention also provides the application of Achyranthes bidentata extract in the preparation of food or pharmaceuticals, wherein the Achyranthes bidentata extract is prepared by the preparation method described above.

[0066] According to the present invention, the application of Achyranthes bidentata extract in the preparation of food or medicine is preferably provided in that the active ingredient of the food, medicine or health product includes the Achyranthes bidentata extract.

[0067] According to the application of the Achyranthes bidentata extract provided by the present invention in the preparation of food or pharmaceuticals, preferably, the food further includes food science-acceptable excipients.

[0068] According to the application of the Achyranthes bidentata extract provided by the present invention in the preparation of food or pharmaceuticals, preferably, the pharmaceuticals further include pharmaceutically acceptable excipients.

[0069] The application of the Achyranthes bidentata extract provided by the present invention in the preparation of food or pharmaceuticals, preferably, the food also includes health products.

[0070] More preferably, the health product also includes excipients that are acceptable in health product science.

[0071] In some specific implementations, the excipients include any one or more of the following: flavoring agents, nutrients, diluents, excipients, stabilizers, and thickeners.

[0072] Compared with the prior art, the present invention has the following beneficial effects: This invention, through precise control of heavy ion beam irradiation dosage, significantly increases the content of core active ingredients in Achyranthes bidentata, such as photosynthetic pigments, soluble sugars, total flavonoids, and total polysaccharides, effectively improving the quality of Achyranthes bidentata and providing high-quality raw materials for the development of high-value-added functional foods and traditional Chinese medicine preparations. This invention also comprehensively monitors multiple antioxidant physiological indicators, enabling the mutant Achyranthes bidentata plants to exhibit good growth vigor and stress resistance, achieving a balance between quality improvement and plant vitality. This invention provides a new approach for the resource utilization of the above-ground stems and leaves of Achyranthes bidentata, helping to promote the comprehensive development and utilization of the entire plant resource, reducing resource waste, and laying a solid foundation for creating new high-yield and high-quality Achyranthes bidentata germplasm and promoting the modernization of the traditional Chinese medicine industry. Attached Figure Description

[0073] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0074] Figure 1 This invention provides the test examples of the effects of heavy ion beam irradiation mutagenesis at different irradiation doses on the emergence and growth of Achyranthes bidentata.

[0075] Figure 2 This invention provides the effect of heavy ion beam irradiation mutagenesis at different irradiation doses on the survival rate of Achyranthes bidentata seedlings (8 weeks).

[0076] Figure 3 This invention provides the effect of heavy ion beam irradiation mutagenesis at different irradiation doses on the plant height of Achyranthes bidentata seedlings (8 weeks).

[0077] Figure 4This invention provides the effect of heavy ion beam irradiation mutagenesis at different irradiation doses on the root length of Achyranthes bidentata seedlings (8 weeks). Detailed Implementation

[0078] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0079] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0080] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0081] Example 1: A method for irradiating Achyranthes bidentata with a heavy ion beam This embodiment provides a method for irradiating Achyranthes bidentata with a heavy ion beam, the specific steps of which are as follows: Plump Achyranthes bidentata seeds were selected and placed in a heavy ion beam irradiation device for irradiation. The heavy ion beam parameters were set as follows: carbon ion type, total energy of 967 MeV, average energy transfer linear density of 34 keV / μm, dose rate of 80 Gy / min, and irradiation dose of 40 Gy.

[0082] Example 2: A method for irradiating Achyranthes bidentata with a heavy ion beam This embodiment provides a method for irradiating Achyranthes bidentata with a heavy ion beam, the specific steps of which are as follows: Plump Achyranthes bidentata seeds were selected and placed in a heavy ion beam irradiation device for irradiation. The parameters of the heavy ion beam were set as follows: ion type carbon ion, total energy 967 MeV, average energy transfer linear density 34 keV / μm, dose rate 80 Gy / min, and irradiation dose 80 Gy.

[0083] Example 3: A method for irradiating Achyranthes bidentata with a heavy ion beam This embodiment provides a method for irradiating Achyranthes bidentata with a heavy ion beam, the specific steps of which are as follows: Plump Achyranthes bidentata seeds were selected and placed in a heavy ion beam irradiation device for irradiation. The heavy ion beam parameters were set as follows: carbon ion type, total energy of 967 MeV, average energy transfer linear density of 34 keV / μm, dose rate of 80 Gy / min, and irradiation dose of 100 Gy.

[0084] Example 4: A method for irradiating Achyranthes bidentata with a heavy ion beam This embodiment provides a method for irradiating Achyranthes bidentata with a heavy ion beam, the specific steps of which are as follows: Plump Achyranthes bidentata seeds were selected and placed in a heavy ion beam irradiation device for irradiation. The parameters of the heavy ion beam were set as follows: ion type: carbon ions, total energy: 967 MeV, average energy transfer linear density: 34 keV / μm, dose rate: 80 Gy / min, and irradiation dose: 120 Gy.

[0085] Example 5: A method for irradiating Achyranthes bidentata using a heavy ion beam. This embodiment provides a method for irradiating Achyranthes bidentata with a heavy ion beam, the specific steps of which are as follows: Plump Achyranthes bidentata seeds were selected and placed in a heavy ion beam irradiation device for irradiation. The heavy ion beam parameters were set as follows: carbon ion type, total energy of 967 MeV, average energy transfer linear density of 34 keV / μm, dose rate of 80 Gy / min, and irradiation dose of 150 Gy.

[0086] Example 6: A method for irradiating Achyranthes bidentata using a heavy ion beam. This embodiment provides a method for irradiating Achyranthes bidentata with a heavy ion beam, the specific steps of which are as follows: Plump Achyranthes bidentata seeds were selected and placed in a heavy ion beam irradiation device for irradiation. The parameters of the heavy ion beam were set as follows: ion type carbon ions, total energy 967 MeV, average energy transfer linear density 34 keV / μm, dose rate 80 Gy / min, and irradiation dose 200 Gy.

[0087] Example 7: A method for irradiating Achyranthes bidentata with a heavy ion beam This embodiment provides a method for irradiating Achyranthes bidentata with a heavy ion beam, the specific steps of which are as follows: Plump Achyranthes bidentata seeds were selected and placed in a heavy ion beam irradiation device for irradiation. The parameters of the heavy ion beam were set as follows: ion type carbon ions, total energy 967 MeV, average energy transfer linear density 34 keV / μm, dose rate 80 Gy / min, and irradiation dose 250 Gy.

[0088] Example 8: A method for irradiating Achyranthes bidentata with a heavy ion beam This embodiment provides a method for irradiating Achyranthes bidentata with a heavy ion beam, the specific steps of which are as follows: Plump Achyranthes bidentata seeds were selected and placed in a heavy ion beam irradiation device for irradiation. The heavy ion beam parameters were set as follows: carbon ion type, total energy of 967 MeV, average energy transfer linear density of 34 keV / μm, dose rate of 80 Gy / min, and irradiation dose of 300 Gy.

[0089] The effect of heavy ion beam irradiation on the morphological and physiological and biochemical indicators of Achyranthes bidentata. 1. Testing Method (1) Seed irradiation and cultivation The dried seeds of Achyranthes bidentata (with plump, uniform size and a thousand-seed weight of 3.8611g, sourced from the Niujiayingzi medicinal herb planting base in Kalaqin Banner, Chifeng City, and available to the public from the applicant) were randomly divided into 10 portions of 150 seeds each. Nine portions were treated according to the methods in Examples 1 to 8, and the remaining portion served as a control without heavy ion beam irradiation. Each method was repeated three times.

[0090] The planting soil consists of nutrient soil, native soil and sand in a mass ratio of 2:1:1. It is disinfected and mixed before sowing.

[0091] The treated Achyranthes bidentata seeds and control seeds were soaked in distilled water at room temperature for 24 hours, and then sown in experimental planting pots containing planting soil, with 30 plants per pot and 5 pots for each replicate of each method.

[0092] When grown indoors, water regularly. The growing period is 8 weeks (56 days). During the growing period, maintain constant light and temperature conditions, with a temperature of 25±1℃ and a photoperiod of 12 hours of light and 12 hours of darkness.

[0093] (2) Morphological index determination After the growing season, the survival rates of Achyranthes bidentata irradiated by the methods in Examples 1-8 and the control Achyranthes bidentata were statistically analyzed. Ten Achyranthes bidentata plants were randomly sampled for each method to measure plant height and root length.

[0094] (3) Measurement of physiological and biochemical indicators After the growing season, leaf samples of *Achyranthes bidentata* irradiated by the methods in Examples 1-8 and the control *Achyranthes bidentata* were collected. The contents of chlorophyll, POD, SOD, CAT, MDA, proline, soluble sugars, total flavonoids, total polysaccharides, and total phenols were determined by ultraviolet spectrophotometry. The determination methods are as follows: 1) Chlorophyll content determination 0.10 g of leaf sample was weighed and extracted with ethanol to obtain extract 1. After being stored in the dark for 24 h, sample solution 1 was obtained. The absorbance of sample solution 1 was measured by spectrophotometry at 663 nm, 645 nm and 470 nm (D, respectively). 663 D 645 and D 470 ), repeat three times.

[0095] The concentrations of chlorophyll a (Ca), chlorophyll b (Cb), total carotenoids (Cx.c), and total chlorophyll content (CT) are calculated using the following formulas (1) to (4): Formula (1): Ca = 13.95D 663 -6.88D 645 ; Formula (2): Cb=24.96D 645 -7.32D 663 ; Formula (3): Cx.c = (1000D) 470 -2.05Ca-114.8Cb) / 245; Formula (4): CT = Ca + Cb = 18.16D 645 +6.63D 663 .

[0096] 2) POD content determination Weigh 0.25g of leaf sample, add 0.75ml of phosphate buffer, grind, and centrifuge at 3000r / min for 10min to obtain grinding buffer 1. Add 2.9ml of phosphate buffer, 0.95ml of guaiacol solution, and 1ml of 2% H2O2 solution to a test tube, then immediately add 0.05ml of grinding buffer 1 and mix well to obtain sample solution 2. Perform a POD enzymatic reaction for 1min, and measure the absorbance of sample solution 2 at 470nm using spectrophotometry. Repeat three times.

[0097] The POD content is calculated according to the following formula (5): Formula (5): POD=(ΔA470×Vt1) / (W1×Vs1×0.01×t1); Where ΔA470 is the difference in absorbance at 470 nm before and after the POD enzymatic reaction; Vt1 is the total volume of sample solution 2; W1 is the fresh weight of the sample for determining POD content, 0.25 g; Vs1 is the volume of grinding solution 1 used during the POD enzymatic reaction, 0.05 ml; and t1 is the POD enzymatic reaction time, 1 min.

[0098] 3) SOD content determination Weigh 0.25g of leaf sample, add 0.75ml of phosphate buffer, grind, and centrifuge at 4000r / min for 10min at 4℃ to obtain grinding buffer 2. Take 0.1ml of grinding buffer 2, add 0.05mol / L phosphate buffer, 130nmol / L methionine solution, 750μmol / L nitroblue tetrazolium solution, 100μmol / L disodium ethylenediaminetetraacetate solution, 20μmol / L riboflavin solution, and distilled water respectively, mix well to obtain sample solution 3. After incubation for 10min, measure the absorbance of sample solution 3 at 560nm using spectrophotometry. Repeat three times, and set up a light control tube (25℃, under light) and a dark control tube (25℃, in the dark), where the dark control tube is used for zeroing.

[0099] The SOD content is calculated using the following formula (6): Formula (6): SOD=((A0-As)×Vt2) / A0×0.5×Fw1×V1; Where A0 is the absorbance of the control tube at 560 nm; As is the absorbance of the sample tube at 560 nm; Vt2 is the total volume of sample solution 3; Fw1 is the fresh weight of the sample for determining SOD content, 0.25 g; and V1 is the volume of grinding solution 2, 0.1 ml.

[0100] 4) CAT content determination Weigh 0.10 g of leaf sample, add 1 ml of phosphate buffer solution, grind in an ice bath, place in a centrifuge tube, and centrifuge at 12000 r / min for 20 min at 4℃ to obtain grinding solution 3. The CAT content was determined using the hydrogen peroxide decomposition method. Mix 200 ml of phosphate buffer solution with 0.3092 ml of 30% H2O2 to obtain a reaction solution. Mix 3 ml of the reaction solution with 0.1 ml of grinding solution 3 to obtain sample solution 4, and perform a CAT enzymatic reaction. The absorbance of sample solution 4 was then measured spectrophotometrically at 240 nm, repeated three times.

[0101] The CAT content is calculated using the following formula (7): Formula (7): CAT = (ΔA240 × Vt3) / (W2 × Vs2 × 0.01 × t2); Where △A240 is the difference in absorbance at 240nm before and after the CAT enzymatic reaction; Vt3 is the total volume of sample solution 4; W2 is the fresh weight of the sample for determining CAT content, 0.10g; Vs2 is the volume of grinding solution 3 used in the CAT enzymatic reaction, 0.1ml; and t2 is the CAT enzymatic reaction time.

[0102] 5) MDA content determination Weigh 0.25g of leaf sample, add 5ml of 10% trichloroacetic acid solution and grind in an ice bath. Transfer to a centrifuge tube and centrifuge at 4000r / min for 10min. Take 2ml of supernatant, add 2ml of 0.6% thiobarbituric acid solution, mix well, and heat in a boiling water bath for 30min. After cooling, centrifuge again and take the supernatant to obtain sample solution 5. Measure the absorbance of sample solution 5 at 600nm, 532nm, and 450nm (D5, D6, D7, D8, D9 ... 600 D 532 and D 450 ), repeat three times.

[0103] The MDA content is calculated using the following formulas (8) and (9): Formula (8): C1 = 6.45 (D 532 -D 600 -0.56D 450 ; Formula (9): MDA=C1×Vt4 / Fw2; Where C1 is the concentration of MDA in sample solution 5; Vt4 is the volume of sample solution 5; and Fw2 is the fresh weight of the sample for determining MDA content, 0.25g.

[0104] 6) Proline content determination Weigh 0.20g of leaf sample, cut it into small pieces and put it into a test tube. Add 5ml of 3% sulfosalicylic acid solution and perform the first extraction in a boiling water bath for 10min. After cooling, centrifuge and collect the supernatant to obtain extract 2. Take 2ml of extract 2, 2ml of glacial acetic acid and 3ml of colorimetric solution, and perform the second extraction in a boiling water bath for 40min to obtain sample 6. Measure the absorbance of sample 6 at 520nm using spectrophotometry. Repeat four times and construct a standard curve of concentration versus absorbance at 520nm using proline standard.

[0105] The proline content is calculated using the following formula (10): Formula (10): Proline content = (C2×V2 / a1) / W3; Where C2 is the proline content of the sample calculated from the standard curve; V2 is the total volume of sample solution 6; a1 is the volume of extract solution 2 taken before the second extraction, 2 ml; W3 is the fresh weight of the sample for determining the proline content, 0.20 g.

[0106] 7) Determination of soluble sugar content The anthrone method was used for determination. 0.10 g of leaf sample was weighed, added to 10 ml of distilled water, and ground to obtain soluble buffer 4. The buffer was placed in a test tube, heated in a boiling water bath for 30 min, cooled, centrifuged, and the supernatant was collected to obtain extract 3. 0.5 ml of extract 3 was taken and mixed with 1.5 ml of distilled water, 1 ml of 9% phenol solution, and 5 ml of concentrated sulfuric acid to obtain sample solution 7. The absorbance of sample solution 7 was then measured at 620 nm. This was repeated three times, and a standard curve was constructed using sucrose standards to compare the concentration with the absorbance at 620 nm.

[0107] The soluble sugar content is calculated according to the following formula (11): Formula (11): Soluble sugar content = (C3×V3 / a2×n) / (W4×10) 3 ); Where C3 is the soluble sugar content of the sample calculated from the standard curve; a2 is the volume of extract 3 taken, 0.5 ml; V3 is the total volume of sample solution 7; n is the dilution factor of sample solution 7, 1; W4 is the fresh weight of the sample for determining soluble sugar content, 0.10 g.

[0108] 8) Determination of total flavonoid content The sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method was used for determination. 0.10 g of leaf sample was weighed, dried at 60℃ to constant weight, ground, and passed through a 40-mesh sieve. 0.1 g of sample powder was accurately weighed, added to 2 ml of 70% ethanol, ground, then sonicated for 60 min, centrifuged for 10 min, and the supernatant was collected. The precipitate was then added to 2 ml of 70% ethanol, sonicated for 60 min, centrifuged for 10 min, and the supernatant was collected. The two supernatants were combined, concentrated at 65℃, and finally diluted to 10 ml to obtain extract 4. 1.6 ml of extract 4 was mixed with 0.2 ml of 5% sodium nitrite solution, 0.2 ml of 10% aluminum nitrate solution, and 2 ml of 4% sodium hydroxide solution. The volume was then increased to 5 ml with 70% ethanol to obtain sample solution 8. The control tube used 70% ethanol instead of extract 4. The absorbance of sample solution 8 was measured spectrophotometrically at 510 nm, repeated three times. A standard curve was constructed using rutin standard to compare concentration with absorbance at 510 nm.

[0109] The total flavonoid content is calculated using the following formula (12): Formula (12): Total flavonoid content = (Y1 × V4 × D) / W5; Where Y1 is the total flavonoid content of the sample calculated from the standard curve; V4 is the volume of sample solution 8, 5 ml; D is the dilution factor of sample solution 8, 1; and W5 is the dry weight of the sample for determining the total flavonoid content.

[0110] 9) Determination of total polysaccharide content Weigh 0.05g of leaf sample and use the reagent kit from Shanghai Keaibo Biotechnology Co., Ltd. to determine the absorbance of sample solution 9 after extracting the leaf sample. The absorbance of sample solution 9 was measured at 490nm, and a standard curve of concentration versus absorbance at 490nm was constructed using the polysaccharide standard in the kit.

[0111] The total polysaccharide content is calculated according to the following formula (13): Formula (13): Total polysaccharide content = 5Y2 ÷ W6; Wherein, Y2 is the total polysaccharide content of the sample calculated from the standard curve; W6 is the fresh weight of the sample for determining the total polysaccharide content, 0.05g.

[0112] 10) Determination of total phenol content Weigh 0.10 g of leaf sample and use the reagent kit from Beijing Solarbio Science & Technology Co., Ltd. After extracting the leaf sample to obtain sample solution 10 according to the kit instructions, measure the absorbance of sample solution 10 at 760 nm, and construct a standard curve of concentration versus absorbance at 760 nm using gallic acid standard in the kit.

[0113] The total phenol content is calculated using the following formula (14): Formula (14): Total phenol content = X × V5 / W7; Where X is the total phenol content of the sample calculated from the standard curve; V5 is the volume of sample solution 10; and W7 is the fresh weight of the sample for determining the total phenol content, 0.10 g.

[0114] (4) Data processing Data was processed using Excel, images were plotted using Origin, and significance of differences was analyzed using SPSS.

[0115] 2. Test Results (1) Effects of different heavy ion beam irradiation doses on the morphological parameters of Achyranthes bidentata Table 1. Morphological Indications of Achyranthes bidentata

[0116] like Figure 1As shown, different heavy ion beam irradiation doses significantly affected the growth of Achyranthes bidentata. With the continuous increase of radiation dose, the growth inhibition and damage of Achyranthes bidentata seedlings became more and more severe: at low doses (≤80 Gy), the emergence rate and growth of seedlings were basically unaffected, and the surviving seedlings were still all alive and growing normally until 23 weeks later; however, as the dose increased to a medium level (100-150 Gy), the emergence rate began to decline, and the growth momentum of Achyranthes bidentata seedlings weakened slightly. The surviving seedlings were still all alive and growing normally until 23 weeks later; when the dose reached a high level (≥200 Gy), the radiation exhibited a strong lethal effect, resulting in a precipitous decrease in the number of seedlings, and the very few surviving plants were also severely underdeveloped and extremely stunted.

[0117] Regarding survival rates, such as Figure 2 As shown in Table 1, the survival rate of Achyranthes bidentata plants continued to decline with increasing heavy ion beam irradiation dose, with the average survival rate decreasing from 60% at 0 Gy to 6.444% at 300 Gy. When the irradiation dose was not higher than 150 Gy, the average survival rate was above 40%.

[0118] Regarding plant height, such as Figure 3 As shown in Table 1, the plant height of Achyranthes bidentata showed a continuous decreasing trend with the increase of heavy ion beam irradiation dose. The average plant height decreased from 7.33 cm at 0 Gy to 3.52 cm at 300 Gy. When the irradiation dose was not higher than 150 Gy, the average plant height was above 5 cm.

[0119] Regarding root length, such as Figure 4 As shown in Table 1, the root length of Achyranthes bidentata plants exhibited a nonlinear response as the irradiation dose of heavy ion beam increased. Compared with 0 Gy, the root length of Achyranthes bidentata plants increased significantly at an irradiation dose of 40 Gy, reaching a peak of 11.27 cm. Subsequently, the root length showed a continuous downward trend as the irradiation dose increased. When the irradiation dose was 150 Gy, the root length slightly rebounded, but afterwards (irradiation dose ≥200 Gy), the root length was significantly damaged.

[0120] (2) Effects of different heavy ion beam irradiation doses on the physiological and biochemical indicators of Achyranthes bidentata 1) Effects on photosynthetic pigments Table 2. Photosynthetic pigment status of Achyranthes bidentata.

[0121] Note: "-" in the table indicates that the growth of Achyranthes bidentata seedlings under 300Gy irradiation dose was significantly poor, with low survival rate and poor growth. There were too few leaf samples to meet the required sample loading for measurement, so the corresponding index was not measured.

[0122] As shown in Table 2, the contents of chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll all reached their maximum values ​​at an irradiation dose of 80 Gy, namely 2.629±0.002 mg / g, 1.009±0.006 mg / g, 0.562±0.003 mg / g, and 3.638±0.005 mg / g, respectively. When the irradiation dose was 40 Gy to 100 Gy or 250 Gy, the contents of chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll were significantly higher than those of the control Achyranthes bidentata.

[0123] The above results indicate that low to medium doses of heavy ion beam irradiation can significantly enhance the photosynthetic capacity and photoprotective capacity of plants.

[0124] 2) Effect on oxidation reaction Table 3. Antioxidant enzyme system of Achyranthes bidentata.

[0125] Note: "-" in the table indicates that the growth of Achyranthes bidentata seedlings under 300Gy irradiation dose was significantly poor, with low survival rate and poor growth. There were too few leaf samples to meet the required sample loading for measurement, so the corresponding index was not measured.

[0126] As shown in Table 3, compared with the control Achyranthes bidentata, the POD activity of Achyranthes bidentata significantly increased under heavy ion beam irradiation, reaching a peak at 80 Gy (312.6±39.075 U / gFW) and a secondary peak at 150 Gy (208.9±59.831 U / gFW). Under heavy ion beam treatment, the SOD activity of Achyranthes bidentata decreased with increasing dose in the low dose range (0–100 Gy), reaching the lowest level at 100 Gy (178.515±1.206 U / gFW). After that, the SOD activity gradually increased with increasing irradiation dose and basically maintained at the level of 183 U / gFW to 201 U / gFW. The CAT activity fluctuated and increased with increasing irradiation dose, reaching a peak at 100 Gy (218.215±202.743 U / gFW).

[0127] Table 4 Oxidative stress damage of Achyranthes bidentata

[0128] Note: "-" in the table indicates that the growth of Achyranthes bidentata seedlings under 300Gy irradiation dose was significantly poor, with low survival rate and poor growth. There were too few leaf samples to meet the required sample loading for measurement, so the corresponding index was not measured.

[0129] As shown in Table 4, compared with the control Achyranthes bidentata, the MDA content of Achyranthes bidentata was significantly reduced under heavy ion beam treatment at medium and low irradiation doses (40 Gy to 100 Gy), reaching the lowest value at 80 Gy (7.815 ± 0.074 mmol / g FW), rebounding and reaching the maximum value at 120 Gy (34.18 ± 0.074 mmol / g FW), and then the MDA content of Achyranthes bidentata was significantly reduced under high irradiation doses (150 Gy to 250 Gy). As the irradiation dose of heavy ion beam increased, the proline content of Achyranthes bidentata showed a fluctuating trend, reaching the lowest value at 120 Gy, and increasing sharply to 55.313 ± 4.18 μg / g at a high dose of 200 Gy.

[0130] The above results indicate that Achyranthes bidentata exhibits a dynamic and phased adaptive strategy with varying irradiation doses. Specifically, at low irradiation doses (40 Gy–80 Gy), Achyranthes bidentata achieves better membrane protection with less resource input by adjusting the composition of its antioxidant enzyme system (dominated by POD), demonstrating a positive adaptive regulatory capacity. At medium irradiation doses (100 Gy–120 Gy), Achyranthes bidentata's defense system is temporarily dysregulated, with insufficient activity of the core enzyme CAT, leading to a decrease in hydrogen peroxide scavenging capacity and an increase in oxidative damage. At high irradiation doses (>120 Gy), Achyranthes bidentata's core antioxidant capacity is substantially enhanced, with SOD and CAT efficiently and synergistically scavenging free radicals, keeping membrane damage at a low level, accumulating large amounts of proline, and maintaining cell survival in vivo through osmotic regulation and weak antioxidant effects.

[0131] 3) Effects on active ingredients Table 5. Active ingredients of Achyranthes bidentata

[0132] Note: "-" in the table indicates that the growth of Achyranthes bidentata seedlings under 300Gy irradiation dose was significantly poor, with low survival rate and poor growth. There were too few leaf samples to meet the required sample loading for measurement, so the corresponding index was not measured.

[0133] As shown in Table 5, compared with the control Achyranthes bidentata, the soluble sugar content of Achyranthes bidentata significantly increased under heavy ion beam irradiation. With increasing irradiation dose, the soluble sugar content of Achyranthes bidentata first reached a peak at 80 Gy (19.463±0.013 mg / g). Subsequently, it remained basically within the range of 5 mg / g to 10 mg / g at 100 Gy to 250 Gy; at 300 Gy, it experienced an explosive accumulation reaching 20.656±0.013 mg / g, more than 5 times that of the control Achyranthes bidentata. This may be the result of Achyranthes bidentata converting irradiated oligosaccharide fragments into signaling molecules through metabolic reprogramming, thereby activating the emergency protection mechanism.

[0134] At 40 Gy, the total flavonoid content of Achyranthes bidentata was significantly higher than that of the control Achyranthes bidentata. At 80 Gy, the total flavonoid content of Achyranthes bidentata returned to a level comparable to that of the control Achyranthes bidentata. At 100 Gy to 150 Gy, the total flavonoid content of Achyranthes bidentata was significantly higher than that of the control Achyranthes bidentata, reaching a maximum value of 36.729 ± 0.014 mg / g at 150 Gy. At 200 Gy to 250 Gy, the total flavonoid content of Achyranthes bidentata was significantly lower than that of the control Achyranthes bidentata.

[0135] Under heavy ion beam irradiation, the total polysaccharide content of Achyranthes bidentata reached its maximum value (30.991±0.421 mg / g) at 40 Gy. The total polysaccharide content of Achyranthes bidentata decreased significantly and was lower than that of the control at 80 Gy to 100 Gy. The total polysaccharide content of Achyranthes bidentata rebounded again at 120 Gy to 150 Gy and was significantly higher than that of the control. Then, at 200 Gy, it dropped to a level significantly lower than that of the control. At 250 Gy, it rebounded for the third time and was significantly higher than that of the control.

[0136] Under heavy ion beam irradiation, the total phenol content of Achyranthes bidentata was not significantly different from that of the control Achyranthes bidentata. The total phenol content reached a peak at 200 Gy (4.663±0.969 mg / g), indicating that the heavy ion beam irradiation method did not have a direct regulatory effect on the total phenol content of Achyranthes bidentata.

[0137] In another independent experiment, similar results were obtained when Achyranthes bidentata seeds irradiated with heavy ion beams were soaked in distilled water containing 7.5 mg / L indoleacetic acid at room temperature for 24 hours and then subjected to germination tests in an incubator.

[0138] In another independent experiment, the offspring of Achyranthes bidentata obtained by the methods of Examples 1-8 were subjected to indoor planting experiments and showed similar results to their corresponding parents.

[0139] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for increasing the content of edible active ingredients in Achyranthes bidentata, characterized in that, The edible active ingredients are selected from any one or more of photosynthetic pigments, soluble sugars, total flavonoids, and total polysaccharides. The method includes irradiating Achyranthes bidentata seeds with a heavy ion beam at a dose of 40 Gy to 150 Gy; the heavy ion beam includes a carbon ion beam.

2. The method according to claim 1, characterized in that, The photosynthetic pigments are selected from any one or more of chlorophyll a, chlorophyll b, and carotenoids.

3. The method according to claim 1, characterized in that, The edible active ingredient is selected from any one of the following ingredient compositions (1) to (2): The composition (1) consists of photosynthetic pigments and soluble sugars; The composition (2) consists of total flavonoids and total polysaccharides.

4. The method according to claim 3, characterized in that, If the active ingredient with edible value is component composition (1), then the irradiation dose is 40 Gy to 100 Gy.

5. The method according to claim 4, characterized in that, The irradiation dose is 40 Gy to 80 Gy.

6. The method according to claim 3, characterized in that, If the active ingredient with edible value is component composition (2), then the irradiation dose is 40 Gy or 120 Gy to 150 Gy.

7. The method according to any one of claims 1 to 6, characterized in that, The dose rate of heavy ion beam irradiation is 70 Gy / min to 90 Gy / min.

8. The method according to any one of claims 1 to 6, characterized in that, The method also includes: cultivating Achyranthes bidentata seeds irradiated with heavy ion beams to obtain Achyranthes bidentata plants.

9. A method for preparing an extract of Achyranthes bidentata, characterized in that, include: An extract was obtained from the Achyranthes bidentata plant; The Achyranthes bidentata plant was obtained by processing it using the method described in any one of claims 1 to 6.

10. The application of Achyranthes bidentata extract in the preparation of food or medicine, characterized in that, The Achyranthes bidentata extract was prepared by the method described in claim 9.

Images