Anti-glycation sprouts and methods of making the same

Abstract

The embodiment of the present application provides a kind of anti-saccharification seedling and its preparation method.The anti-saccharification seedling preparation method includes the following steps: cultivating treatment is carried out to safflower seedling, and anti-saccharification seedling is obtained.The cultivation treatment process is added with inducer, and the inducer includes at least one of sodium chloride, monosaccharide, oligosaccharide and polysaccharide.The preparation method is by adding inducer in the process of safflower seedling growth and development, and the synthesis and accumulation of anti-saccharification component in safflower seedling are effectively promoted, so as to improve the content of anti-saccharification component in the anti-saccharification seedling cultivated.

Description

Technical Field

[0001] This application belongs to the field of vegetable cultivation technology, and in particular relates to a glycation-resistant seedling and its preparation method. Background Technology

[0002] Microgreens are tender vegetables harvested immediately after the cotyledons have fully developed or a set of true leaves have emerged. Cultivation is simple, including hydroponics, and requires no pesticides, making it convenient for home gardening. Common microgreens include radishes, peas, and cauliflower. Compared to mature vegetables, microgreens are not only more tender and delicious but also possess unique nutritional value, acting as enhancers of appearance, taste, and texture. For example, microgreens of Asteraceae vegetables such as red clover contain anti-glycation components that can inhibit the formation of advanced glycation end products (AGEs).

[0003] Advanced Glycation End Products (AGEs) are formed from glucose and protein molecules in the blood, tissue fluid, and cells through a series of complex chemical reactions. AGEs are associated with the pathogenesis of various chronic diseases such as diabetes and its complications, atherosclerosis, osteoporosis, and Alzheimer's disease, and are also closely related to human aging. The earliest AGEs discovered in humans are glycated hemoglobin in diabetic patients, which is considered a biomarker of diabetes progression. Glycated collagen is another AGE closely related to aging. Glycated collagen can generally be observed from age 20 and accumulates at a rate of about 3.7% per year, reaching a 30% to 50% increase by age 80. Therefore, inhibiting the formation of AGEs can effectively control the progression of chronic diseases such as diabetes and delay aging.

[0004] Although the seedlings of Asteraceae vegetables such as safflower seedlings contain anti-glycation components, and consuming these seedlings can have a certain anti-glycation effect, the content of anti-glycation components in safflower seedlings cultivated by existing cultivation methods is relatively low, and the anti-glycation effect is limited. Summary of the Invention

[0005] The purpose of this application is to provide a method for preparing anti-glycation seedlings and anti-glycation seedlings, so as to solve the technical problem of low content of anti-glycation components in safflower seedlings in the prior art.

[0006] In a first aspect, embodiments of this application provide a method for preparing anti-glycation seedlings. The method for preparing anti-glycation seedlings according to embodiments of this application includes the following steps:

[0007] By cultivating safflower seedlings, glycosylation-resistant seedlings were obtained;

[0008] The cultivation process involves the addition of an inducing agent, which includes at least one of sodium chloride, monosaccharides, oligosaccharides, or polysaccharides.

[0009] The method for preparing anti-glycation seedlings in this application uses at least one of sodium chloride, monosaccharides, oligosaccharides, and polysaccharides as an inducer. These inducers are added during the cultivation of safflower seedlings. The inducers provide micro-stimulation to the growth and development of safflower seedlings, effectively promoting the synthesis and accumulation of anti-glycation components in the seedlings and increasing their content. This results in a higher content of anti-glycation components in the cultivated anti-glycation seedlings. Under the same dosage of anti-glycation seedlings, the anti-glycation seedlings prepared by the method in this application have a better anti-glycation effect.

[0010] Secondly, this application provides an anti-glycation seedling. The anti-glycation seedling of this application is cultivated using the anti-glycation seedling preparation method described above, and contains luteolin-7-glucoside.

[0011] The anti-glycation seedlings in this embodiment were cultivated using the preparation method described above. Therefore, the anti-glycation seedlings in this embodiment have high content of anti-glycation components such as luteolin-7-glucoside and good anti-glycation effect. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0013] Figure 1 A schematic diagram showing the anti-glycation test results of extracts obtained from safflower seedlings using different solvents;

[0014] Figure 2 This is a schematic diagram showing the anti-glycation detection results of different eluted fractions obtained by separating 95% ethanol extract in a silica gel column;

[0015] Figure 3 This is a schematic diagram showing the anti-glycation detection results of different eluted components obtained by separating eluted component A15 in a dextran gel column;

[0016] Figure 4 This is a schematic diagram showing the HPLC-UV analysis results of the 95% ethanol extract, eluent fraction B7, and L7G standards. Figure 4 In the figure, A represents the liquid chromatogram of the 95% ethanol extract, dextran fraction B7, and L7G standard. Figure 4In the diagram, B represents the UV spectral scanning results of component B7 separated by the dextran gel column. Figure 4 In the diagram, C represents the ultraviolet spectral scanning results of the L7G standard.

[0017] Figure 5 This is a schematic diagram of the LC-MS analysis results of dextran gel column separation of component B7, where... Figure 5 In the figure, A represents the total ion chromatogram of component B7 separated by dextran gel column chromatography. Figure 5 B in the figure represents the negative ion extraction chromatogram with a retention time of 12.43 min. Figure 5 C in the figure represents the positive ion extraction chromatogram with a retention time of 12.41 min;

[0018] Figure 6 A schematic diagram showing the results of the average L7G content determination in the whole plant, leaves, stems and roots of each red clover seedling;

[0019] Figure 7 This is a schematic diagram illustrating the detection results of weight, total length, shoot length, and root length of glycation-resistant seedlings in Examples A1 to A3 and Comparative Examples A1 to A3. Figure 7 In the diagram, A represents the result of the weight measurement. Figure 7 B in the diagram represents the total length measurement results; Figure 7 C in the diagram represents the results of the bud length measurement. Figure 7 D in the diagram represents the root length measurement results;

[0020] Figure 8 This is a schematic diagram showing the detection results of weight, total length, shoot length, and root length of the glycation-resistant seedlings in Examples B1 to B4 and Comparative Example B1. Figure 8 In the diagram, A represents the result of the weight measurement. Figure 8 B in the diagram represents the total length measurement results; Figure 8 C in the diagram represents the results of the bud length measurement. Figure 8 D in the diagram represents the root length measurement results;

[0021] Figure 9 This is a schematic diagram showing the detection results of weight, total length, shoot length, and root length of the glycated seedlings in Examples C1 to C3 and Comparative Examples C1 to C4. Figure 9 In the diagram, A represents the result of the weight measurement. Figure 9 B in the diagram represents the total length measurement results; Figure 9 C in the diagram represents the results of the bud length measurement. Figure 9 D in the diagram represents the root length measurement results;

[0022] Figure 10These are schematic diagrams illustrating the results of the determination of anti-saccharification seedling content in Examples A1 to A3 and Comparative Examples A1 to A3. Figure 10 In this context, A represents the L7G concentration in the anti-glycation seedlings; Figure 10 B in the diagram represents the results of determining the total L7G content in the whole plant of anti-glycation seedlings;

[0023] Figure 11 This is a schematic diagram showing the results of the determination of anti-saccharification seedling content in Examples B1 to B4 and Comparative Example B1. Figure 11 In this context, A represents the L7G concentration in the anti-glycation seedlings; Figure 11 B in the diagram represents the results of determining the total L7G content in the whole plant of anti-glycation seedlings;

[0024] Figure 12 This is a schematic diagram showing the results of the determination of anti-saccharification seedling content in Examples C1 to C3 and Comparative Examples C1 to C4. Figure 12 In this context, A represents the L7G concentration in the anti-glycation seedlings; Figure 12 B in the diagram represents the results of the determination of the total L7G content in the whole plant of anti-glycation seedlings. Detailed Implementation

[0025] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0026] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0027] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0028] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0029] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0030] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0031] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0032] To address the technical problem of low content of anti-glycation components in seedlings of Asteraceae vegetables in existing technologies, this application proposes the following technical solution.

[0033] In a first aspect, embodiments of this application provide a method for preparing anti-saccharification seedlings. The method for preparing saccharification-resistant seedlings according to embodiments of this application includes the following steps:

[0034] By cultivating safflower seedlings, glycosylation-resistant seedlings were obtained;

[0035] The cultivation process involves the addition of an inducer, which includes at least one of sodium chloride, monosaccharides, oligosaccharides, and polysaccharides.

[0036] The method for preparing anti-glycation seedlings in this application involves cultivating safflower seedlings and adding an inducer during the cultivation process. This inducer effectively promotes the synthesis and accumulation of anti-glycation components in the safflower seedlings, increasing their content and resulting in seedlings with high anti-glycation component content and good anti-glycation effect. The anti-glycation components in the seedlings may include, but are not limited to, luteolin-7-glucoside (L7G), which have anti-glycation effects and can inhibit the formation of AGEs (Advanced Glycation End Products). The method in this application uses an inducer to micro-stimulate the growth and development of safflower seedlings, effectively increasing the content of anti-glycation components in the resulting seedlings and improving the anti-glycation effect with the same dosage.

[0037] safflower( Carthamus tinctorius L.) belongs to the genus *Carthamus* of the family Compositae (Asteraceae). Carthamus Safflower (Carthamus tinctorius) is an annual or biennial herbaceous plant, also known as safflower, safflower grass, safflower, safflower, golden safflower, red safflower grass, and safflower vegetable. Safflower is a plant resource used for both food and medicine; its dried flowers are used medicinally and are believed to have the effects of promoting blood circulation, regulating menstruation, dispersing blood stasis, and relieving pain. Therefore, most research on safflower focuses on its flowers. Safflower seedlings contain various nutrients and have a long history of consumption in my country. They can be eaten raw in salads, stir-fried, steamed, or used in soups. They are tender, delicious, and easy to digest, possessing high health-promoting value and are considered a special health vegetable. Hydroponically grown safflower seedlings contain a certain amount of anti-glycation components. The preparation method in this application uses safflower seedlings to cultivate anti-glycation seedlings, which not only gives the resulting anti-glycation seedlings the rich nutrients of safflower seedlings but also the tender, delicious, and easily digestible characteristics of safflower seedlings. In addition, by adding an inducer during the cultivation process to slightly stimulate the growth of safflower seedlings, the synthesis and accumulation of anti-glycation components in the safflower seedlings are further promoted, effectively increasing the content of anti-glycation components in the anti-glycation seedlings obtained through cultivation.

[0038] In some embodiments, safflower seedlings can be obtained by germinating safflower seeds. The safflower seedlings may include safflower buds that germinate from safflower seeds after germination treatment. When the growth cycle of anti-glycation seedlings is the same, for example, 8 days, using newly germinated safflower buds for cultivation treatment to prepare anti-glycation seedlings can prolong the duration of the inducer's effect on the safflower seedlings, further promoting the accumulation of anti-glycation components in the plant and increasing the content of anti-glycation components in the anti-glycation seedlings.

[0039] In some embodiments, the safflower seedlings used for cultivation to prepare saccharification-resistant seedlings may further include tender safflower seedlings. The tender safflower seedlings are those obtained by germinating safflower seeds after germination treatment and allowing them to grow for a period of time. In a further embodiment, the tender safflower seedlings can be seedlings with a growth cycle of less than 14 days after germination treatment. In the exemplary example, the growth cycle of the safflower seedlings used for cultivation treatment after germination treatment can be typical but not limiting times such as 0.5 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, and 14 days, or any time between any two numerical ranges. By using tender safflower seedlings for cultivation treatment to prepare saccharification-resistant seedlings, the cultivation treatment time with added inducing agents can be shortened while maintaining the same total growth cycle of the saccharification-resistant seedlings, thus improving the production efficiency of the saccharification-resistant seedlings. For example, when the total growth cycle of anti-saccharification seedlings after safflower seed germination is 8 days, if safflower seedlings that have grown for another 3 days after germination are used for cultivation treatment with an inducer, the cultivation treatment time with the inducer is 5 days, which effectively shortens the cultivation treatment time with the inducer and reduces the consumption of the inducer.

[0040] In some embodiments, safflower seeds may be disinfected before germination treatment. When safflower seedlings are obtained by germinating disinfected safflower seeds, the disinfection method may include, but is not limited to, ultraviolet disinfection, heat disinfection, and disinfectant disinfection. In exemplary cases, safflower seeds can be disinfected by sun exposure; alternatively, they can be disinfected by soaking them in a disinfectant solution (such as sodium hypochlorite solution). These disinfection methods are economical, convenient, and easy to implement in home cultivation. Disinfecting safflower seeds effectively reduces the viruses and bacteria they carry, improves germination rate, enhances seedling growth, and further promotes the synthesis and accumulation of anti-glycation components in the seedlings during cultivation, thereby increasing the content of anti-glycation components in the seedlings.

[0041] In some embodiments, the germination treatment can be carried out in the dark, with a temperature of 20–28 °C, a humidity of 60%–85%, and a duration of 36–48 h. In exemplary embodiments, the germination treatment temperature can be typical but not limiting temperatures such as 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, and 28 °C, or any temperature between any two temperature ranges. The germination treatment humidity can be typical but not limiting humidity such as 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, and 85%, or any humidity between any two numerical ranges. The germination treatment time can be typical but not limiting time such as 36 h, 38 h, 40 h, 42 h, 44 h, 46 h, and 48 h, or any time between any two numerical ranges. By controlling the germination conditions, such as temperature, humidity, and time, within a specified range, the germination rate of safflower seeds is further improved, as are the quality and growth activity of safflower seedlings. This further assists the inducer in promoting the synthesis and accumulation of nutrients and anti-glycation components during the growth and development of safflower seedlings. In a further embodiment, an inducer can be added during the germination process. By adding the inducer during germination, the safflower buds can come into contact with the inducer, thereby allowing the inducer to promote the synthesis of anti-glycation components in the plant as early as possible in the growth and development of safflower seedlings. This further promotes the synthesis and accumulation of anti-glycation components during the cultivation process, increasing the content of anti-glycation components in the anti-glycation seedlings.

[0042] In some embodiments, monosaccharides may include glucose, oligosaccharides may include at least one of sucrose and trehalose, and polysaccharides may include β-cyclodextrin. The addition of these inducers can effectively promote the synthesis and accumulation of anti-glycation components in safflower seedlings, increasing the content of anti-glycation components in the seedlings. It should be noted that the inducer added during the cultivation process can be one, two, or more, and is not specifically limited. In the example, when there is only one inducer, the inducer can be sucrose; when there are two or more inducers, the inducer can include sucrose and sodium chloride.

[0043] In some embodiments, prior to the cultivation treatment, the preparation method of this application may further include: preparing an inducer solution from an inducer, and then adding the inducer solution to the cultivation treatment system. In further embodiments, the concentration of the inducer solution may be 0.1‰ to 10‰, optionally 3‰ to 6‰ or 0.5‰ to 2.5‰. In exemplary examples, the concentration of the inducer solution may be typical but not limiting concentrations such as 0.1‰, 0.5‰, 1.0‰, 1.5‰, 2.0‰, 2.5‰, 3.0‰, 3.5‰, 4.0‰, 4.5‰, 5.0‰, 6.0‰, 7.0‰, 8.0‰, 9.0‰, and 10.0‰, or any concentration between any two concentration ranges. By controlling the concentration of the inducer solution within a certain range, the inducer solution achieves a suitable osmotic pressure, further promoting the absorption of the inducer by safflower seedlings. Simultaneously, it further promotes the synthesis of anti-saccharification components in the safflower seedlings induced by the inducer, promoting the accumulation of anti-saccharification components and further increasing the content of anti-saccharification components in the seedlings. It should be noted that the concentration of the inducer solution in this application specification is a mass concentration.

[0044] In some embodiments, when the inducing agent solution includes a sucrose solution, the concentration of the sucrose solution can be 0.1‰ to 10‰, optionally 0.5‰ to 5‰ or 0.5‰ to 2.5‰. In exemplary examples, the concentration of the sucrose solution can be typical but not limiting concentrations such as 0.1‰, 0.5‰, 1.0‰, 1.5‰, 2.0‰, 2.5‰, 3.0‰, 3.5‰, 4.0‰, 4.5‰, 5.0‰, 6.0‰, 7.0‰, 8.0‰, 9.0‰, and 10.0‰, or any concentration between any two concentration ranges. By controlling the concentration of the sucrose solution within this range, the osmotic pressure in the induction treatment system is adjusted, promoting the absorption of sucrose by the safflower seedlings, thereby promoting the effect of sucrose as an inducing agent, further inducing the synthesis of anti-saccharification components in the safflower seedlings, promoting the accumulation of anti-saccharification components, and further increasing the content of anti-saccharification components in the anti-saccharification seedlings.

[0045] In some embodiments, when the inducing agent solution includes a sodium chloride solution, the concentration of the sodium chloride solution can be 3‰ to 6‰. In exemplary examples, the concentration of the sodium chloride solution can be a typical but not limiting concentration such as 3‰, 4‰, 5‰, or 6‰, or any concentration between any two concentration ranges. Controlling the concentration of the sodium chloride solution within this range adjusts the osmotic pressure in the induction treatment system, increases the absorption of sodium chloride by safflower seedlings, further promotes the effect of sodium chloride as an inducing agent, enhances the synthesis and accumulation of anti-saccharification components in safflower seedlings, and further increases the content of anti-saccharification components in anti-saccharification seedlings.

[0046] In some embodiments, the cultivation treatment may include at least one of hydroponics, aeroponics, and traditional soil cultivation, without specific limitations. These cultivation methods, especially hydroponics, can improve the effectiveness of the induction treatment and also have the advantage of being suitable for home cultivation. When using hydroponics, the inducer can be added to the hydroponic nutrient solution, and the plant roots can be placed above or immersed in the nutrient solution; alternatively, the inducer can be dissolved and added by spraying. When using aeroponics, the inducer can be dissolved and added by spraying. When using traditional soil cultivation, the inducer can be added by watering with the solution, spraying the inducer onto the plant leaves, or mixing the inducer into the soil, without specific limitations.

[0047] It should be noted that, in addition to the inducer, other nutrients required for plant growth, such as N, P, and K, can be added during the cultivation process, depending on actual needs. There are no specific restrictions.

[0048] In some embodiments, the temperature of the cultivation treatment can be 20–28 °C, and the humidity can be 60%–85%. In exemplary cases, the temperature of the cultivation treatment can be typical but not limiting temperatures such as 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, and 28 °C, or any temperature between any two temperature ranges; the humidity of the cultivation treatment can be typical but not limiting humidity such as 60%, 65%, 70%, 75%, 80%, and 85%, or any humidity between any two numerical ranges. Controlling the temperature and humidity of the cultivation treatment within this range further promotes the growth and development of safflower seedlings, promotes the accumulation of nutrients in the safflower seedlings, and promotes the synthesis and accumulation of anti-glycation components, thereby increasing the content of anti-glycation components in the anti-glycation seedlings.

[0049] In some embodiments, the light intensity for the cultivation treatment can be 2000-5000 Lux, and the light duration can be 8-12 hours / day. In exemplary cases, the light intensity for the cultivation treatment can be typical but not limiting light intensities such as 2000 Lux, 2500 Lux, 3000 Lux, 3500 Lux, 4000 Lux, 4500 Lux, and 5000 Lux, or any light intensity between any two numerical ranges; the light duration for the cultivation treatment can be typical but not limiting light durations such as 8 hours / day, 8.5 hours / day, 9.5 hours / day, 10 hours / day, 10.5 hours / day, 11 hours / day, 11.5 hours / day, and 12 hours / day, or any light duration between any two numerical ranges. By controlling the light intensity and duration of the cultivation treatment within this range, the photosynthesis of the plants is further promoted, as well as the growth and development of the plants and the synthesis and accumulation of nutrients. This increases the content of nutrients in the glycosylation-resistant seedlings and further promotes the synthesis and accumulation of anti-glycosylation components, thus increasing the content of anti-glycosylation components in the glycosylation-resistant seedlings.

[0050] In some embodiments, the cultivation treatment period can be 6 to 14 days. In exemplary cases, the cultivation treatment period can be a typical but not limiting time such as 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days, 10 days, 11 days, 12 days, 13 days, or 14 days, or any time between any two numerical ranges. Controlling the cultivation treatment time within this range ensures that the anti-glycation seedlings have a tender texture while increasing the total accumulation of other nutrients and anti-glycation components in the anti-glycation seedlings.

[0051] In some embodiments, when the inducer is sucrose, the method for preparing anti-glycation seedlings according to this application may include the following steps:

[0052] Step S01: Wash the safflower seeds with water to remove impurities from the surface of the safflower seeds. Then, soak the seeds in a 7g / L sodium hypochlorite solution for 5 minutes for disinfection. After disinfection, wash the seeds with water for 1 minute. Repeat three times to remove the residual sodium hypochlorite solution.

[0053] Step S02: Take an appropriate amount of sucrose solution with a concentration of 0.1‰~10‰, immerse half of the seeds, and germinate them in the dark for 36~48 hours under the conditions of temperature 20~28℃ and humidity 60~85%.

[0054] Step S03: Germinated seeds after dark germination treatment are cultivated under the conditions of temperature 20~28 ℃, humidity 60~85%, light intensity 2000~5000 Lux, and light duration 8~12 hours / day. The sucrose solution is changed every 2 days during the cultivation period. Harvest after 6~14 days to obtain sugar-resistant seedlings.

[0055] After germinating safflower seeds with sucrose solution and cultivating them in sucrose solution, the growth and development of safflower seedlings were effectively promoted by controlling the cultivation conditions. This promoted the synthesis and accumulation of nutrients in the safflower seedlings and effectively induced the synthesis of anti-glycation components in the safflower seedlings, thus increasing the content of anti-glycation components in the anti-glycation seedlings.

[0056] Secondly, this application provides an anti-glycation seedling prepared by the method described above. The anti-glycation seedling in this application contains L7G.

[0057] The anti-glycation seedlings in this embodiment were cultivated using the preparation method described above. Therefore, the anti-glycation seedlings in this embodiment have a high content of anti-glycation components such as L7G and a good anti-glycation effect.

[0058] In some embodiments, the L7G content in the resistant seedlings can be 76.85-231.13 μg / g, based on the whole plant wet weight. In exemplary cases, the L7G content in the resistant seedlings can be typical but not limiting values ​​such as 80 μg / g, 90 μg / g, 100 μg / g, 110 μg / g, 120 μg / g, 130 μg / g, 140 μg / g, 150 μg / g, 160 μg / g, 170 μg / g, 180 μg / g, 190 μg / g, 200 μg / g, 210 μg / g, 220 μg / g, and 230 μg / g, or any value between any two ranges.

[0059] In some embodiments, since the anti-glycation seedlings described above contain a high content of anti-glycation components, they can be used not only as vegetables for consumption but also for extracting anti-glycation components. In a further embodiment, the anti-glycation seedlings can be extracted using ethanol and water to obtain an anti-glycation extract, wherein the concentration of ethanol and water is 95%, and the anti-glycation seedlings are those described above.

[0060] Extraction of anti-glycation components from anti-glycation seedlings using ethanol and water effectively extracts the anti-glycation components, resulting in a high extraction yield and good anti-glycation effect.

[0061] In some embodiments, the anti-saccharification seedlings can be dried and / or crushed before extraction. The drying process can be freeze-drying, which quickly and effectively removes moisture from the seedlings, reducing the impact of moisture on the extraction process and minimizing the destruction of the anti-saccharification components during drying. Crushing further increases the contact area between the anti-saccharification seedlings and the ethanol-water mixture, thereby improving the extraction rate.

[0062] In some embodiments, ultrasonic treatment may be performed during the ethanol-water extraction process. In some embodiments, after ethanol-water extraction, the extract may be further separated and purified using a silica gel column and / or a dextran gel column.

[0063] In the exemplary example, the method for extracting anti-glycation extracts from anti-glycation seedlings according to the embodiments of this application may include the following steps:

[0064] Step G10: After freeze-drying the anti-glycation seedlings, break the cell walls and grind them, then extract them with ethanol and water to obtain an ethanol and water extract.

[0065] Step G20: The ethanol-water extract is subjected to a first purification treatment using a silica gel column to obtain a crude extract;

[0066] Step G30: The crude extract is subjected to a second purification treatment using a dextran gel column to obtain an anti-glycation extract.

[0067] The elution solvent for the first purification process may include at least one of n-hexane, a mixture of n-hexane and ethyl acetate, ethyl acetate, a mixture of dichloromethane and methanol, methanol, and acetone, and the elution solvent for the second purification process may include methanol.

[0068] The ethanol-water extract was separated and purified by silica gel column and dextran gel column, which further improved the content and purity of the anti-glycation components in the anti-glycation extract and enhanced its anti-glycation effect.

[0069] In some embodiments, the silica gel column in step G20 can be 100-200 mesh, and the first purification treatment can be eluted sequentially with hexane, a mixture of hexane and ethyl acetate in a ratio of (4:1) to (1:4), ethyl acetate, a mixture of dichloromethane and methanol in a ratio of (4:1) to (1:1), and methanol and acetone. In some embodiments, the dextran gel column can be Sephadex LH-20, and the elution solvent for the second purification treatment can be methanol. By controlling the conditions of the first purification and the second purification treatment, the purity of the anti-glycation component in the anti-glycation extract can be further improved.

[0070] In some embodiments, liquid chromatography can be used to determine the anti-glycation components in anti-glycation seedlings. In further embodiments, the determination method may include the following steps:

[0071] Step S10: Provide the anti-glycation seedlings from the above-described embodiments of this application, and perform crushing and methanol extraction to obtain a sample solution;

[0072] Step S20: The anti-glycation component in the sample solution is determined by liquid chromatography, wherein the anti-glycation component includes L7G.

[0073] In some embodiments, the chromatographic conditions for liquid chromatography may include: performance on a Waters Arc™ HPLC system (Waters, Milford, MA) equipped with a Waters 2998 PAD detector (Waters, Milford, MA). The column is a Waters XBridge C18 (4.6 mm × 250 mm, 5 μm). The mobile phase is 0.3% acetic acid solution and acetonitrile. Gradient elution is used with a range of 15% to 90%, an elution time of 45 min, an injection volume of 10 μL, and a flow rate of 1.0 mL / min.

[0074] The above-described liquid chromatography method was used to detect the content of anti-glycation components, including L7G, with high accuracy and good repeatability. Preparing sample solutions by crushing and extracting anti-glycation seedlings with methanol effectively extracts anti-glycation components such as L7G from the seedlings into the sample solution, further improving the accuracy of the detection results.

[0075] To enable those skilled in the art to clearly understand the above-described implementation details and operations of this application, and to demonstrate the significant improvement in the performance of the anti-glycation seedling preparation method and the anti-glycation seedlings in the embodiments of this application, the above technical solutions are illustrated below through multiple embodiments.

[0076] 1. Identification of anti-glycation components in safflower seedlings

[0077] 1.1 Extraction of anti-glycation components from red clover

[0078] Take safflower seeds and germinate them in the dark for two days. Then, cultivate the germinated safflower seeds at a temperature of 25 ℃, a humidity of 75%, and a light intensity of 2500 Lux. Keep the daily light and dark time fixed at 12 hours and change the water every two days. Harvest the safflower seedlings on the 8th day.

[0079] Safflower seedlings were freeze-dried and then ground to obtain safflower seedling powder. The powder was then extracted sequentially with petroleum ether, n-hexane, ethyl acetate, 95% ethanol, and water at a 1:10 ratio using ultrasonic extraction at room temperature for 30 min. After each extraction, the mixture was filtered, and the filtrate was used for anti-glycation testing in a fructose-bovine serum albumin (BSA) model. The residue was then used for extraction with the next solvent. The anti-glycation test results of the filtrates obtained from each solvent extraction are shown below. Figure 1As shown, the filtrate obtained from the 95% ethanol extraction exhibited the best anti-glycation effect. It should be noted that the data in the embodiments of this application were tested using Tukey's test, with different letter examples indicating statistically significant differences between groups. p <0.05), the presence of the same letter indicates that the difference between groups is not statistically significant. For example Figure 2 The letter 'a' represents group A15 and the letter 'b' represents group A16. These are different letters, indicating that the difference between groups A15 and A16 is statistically significant. p <0.05), Figure 2 Both A12 and A13 contain the letter 'c', indicating that there is no statistically significant difference between the groups A12 and A13.

[0080] The filtrate obtained from the 95% ethanol extraction was evaporated to dryness by rotary evaporation. The evaporated product was dissolved in a small amount of methanol and coarsely separated using a 100-200 mesh silica gel column (60 mm × 50 cm). The elution solvents were used in the following order: n-hexane, n-hexane:ethyl acetate = 4:1, n-hexane:ethyl acetate = 1:4, ethyl acetate, dichloromethane:methanol = 4:1, dichloromethane:methanol = 1:1, dichloromethane:methanol = 1:4, methanol, and acetone, with 200 mL of each solvent. A total of 21 eluent fractions were collected and labeled A1 to A21. Anti-glycation assays were performed on eluent fractions A1 to A21, and the results are as follows: Figure 2 As shown, the elution fraction A15 exhibited the best anti-glycation effect.

[0081] Elution fraction A15 was evaporated to dryness by rotary evaporation and further separated using a Sephadex LH-20 dextran gel column (30 mm × 35 cm) with methanol as the elution solvent. A total of nine elution fractions were collected and labeled B1 to B9. Anti-glycation assays were performed on elution fractions B1 to B9, and the results are as follows: Figure 3 As shown, the elution fraction B7 exhibits the best anti-glycation effect.

[0082] The method for detecting anti-glycation in the fructose-bovine serum albumin (BSA) model was as follows: 45.05 g fructose and 5 g bovine serum albumin (BSA) were added to 100 mL PBS to obtain a BSA mixture with a fructose concentration of 2.5 M and a BSA concentration of 50 mg / mL. For each BSA model, 1 mL of the BSA mixture was mixed with 50 μL of the extraction filtrate or elution fraction as the experimental group, and 1 mL of the BSA mixture was mixed with 50 μL of methanol as the control group. After incubation at 37 ℃ for 7 days, 200 μL of each mixture was collected for detection. The fluorescence intensity of the formed AGEs was measured using a microplate reader (BioTek, Winooski, USA) at excitation / emission wavelengths of 370 / 440 nm. The AGEs inhibition rate of the experimental group was calculated using the following formula:

[0083] The inhibition rate of AGEs (%) = (1-Trt / Ctl)×100%, where Trt is the fluorescence intensity of the experimental group and Ctl is the fluorescence intensity of the control group.

[0084] 1.2 Structural Assessment

[0085] The 95% ethanol extract and eluent B7 were analyzed by HPLC-UV and LC-MS, and the chromatographic information was compared with that of the L7G standard. The HPLC-UV chromatographic conditions were the same as those used in the subsequent determination of the anti-glycation component content, with a scanning wavelength of 200–400 nm. The HPLC-UV analysis results of the 95% ethanol extract, eluent B7, and L7G standard are shown below. Figure 4 As shown, the LC-MS analysis of elution fraction B7 is as follows: Figure 5 As shown. Figure 4 As shown, the retention time of eluent B7 is consistent with that of the L7G standard, and the maximum absorption wavelength of the main peak of eluent B7 is the same as that of the main peak of L7G in the UV spectrum. Figure 4 and Figure 5 It can be seen that the main component of elution fraction B7 is L7G.

[0086] LC-MS Method: LC-MS analysis was performed on a Waters ACQUITY UPLC H-stage chromatographic system (Waters, Milford, MA) and an XEVO TQ-XS tandem triple quadrupole mass spectrometer (Waters, Milford, MA). The column was a Waters ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm), and the column temperature was 35 ℃. The mobile phase was 0.1% formic acid solution and acetonitrile, with a gradient elution rate of 5%–90%, an elution time of 35 min, an injection volume of 1 μL, and a flow rate of 0.2 mL / min. In addition, the operating conditions for ESI-MS were set as follows: ionization mode, positive (ESI+) and negative (ESI-); ion source temperature, 120 °C; desolvation temperature, 500 °C; capillary voltage, 2.5 kV; cone gas flow rate, 150 L / hr; dissolving gas flow rate, 1000 L / h; collision gas flow rate, 0.15 mL / min. The complete MS scan range was 50–1500 m / z. Total ion chromatograms (TIC) and extracted ion chromatograms (XIC) at specific m / z were recorded.

[0087] 1.3 Determination of L7G content in different parts of *Safflower Sprouts*

[0088] Take the same *Corydalis camara* as described in section 1.1 above, separate the leaves, stems, and roots, and determine the L7G content in the whole plant. Simultaneously, determine the L7G content in the leaves, stems, and roots separately. The method for determining the L7G content in the whole plant, leaves, stems, and roots is the same as the method described in section 3.2 below. The results are as follows: Figure 6 As shown. Figure 6 As shown, L7G in *Sedum aizoon* is mainly concentrated in the leaves, with a small amount distributed in the stems, while L7G was not detected in the roots of *Sedum aizoon*.

[0089] 2. Anti-glycation seedlings and their preparation methods

[0090] Example A1

[0091] This embodiment provides a glycation-resistant seedling, which is obtained by cultivating safflower seedlings. The cultivation process includes the addition of an inducing agent, sucrose. The preparation method of this glycation-resistant seedling is as follows:

[0092] Step S1: Wash the safflower seeds with water for one minute each time, repeating three times to remove impurities from the surface. Then, soak the safflower seeds in a 7g / L sodium hypochlorite solution for 5 minutes for disinfection. After soaking, wash with water for one minute, repeating three times to remove any residual sodium hypochlorite solution.

[0093] Step S2: Take an appropriate amount of sucrose solution with a concentration of 5.0‰, and immerse half of the safflower seeds in the sucrose solution. Germinate the seeds in the dark for 48 hours at 25℃ and 75% humidity to obtain germinated safflower seeds.

[0094] Step S3: Using the germinating safflower seeds from Step S2 as safflower seedlings, the seedlings are cultivated using hydroponics, with the sucrose solution from Step S2 as the hydroponic nutrient solution. The nutrient solution is changed every two days during the cultivation process. The cultivation temperature is 25 ℃, the humidity is 75%, the light intensity is 2500 Lux, and the daily light and dark periods are fixed at 12 hours and 12 hours respectively. Harvesting is carried out after 8 days of cultivation to obtain the saccharification-resistant seedlings of this embodiment.

[0095] Examples A2 to A3

[0096] Examples A2 to A3 each provide a saccharification-resistant seedling. The saccharification-resistant seedlings and their preparation methods in Examples A2 to A3 are basically the same as those in Example A1, except that the nutrient solutions (containing inducers) for the germination and cultivation treatments of the saccharification-resistant seedlings in Examples A2 to A3 are shown in Table 1.

[0097] Comparative Examples A1 to Comparative Examples A3

[0098] Comparative Examples A1 to A3 each provide a saccharification-resistant seedling. The saccharification-resistant seedlings and their preparation methods in Comparative Examples A1 to A3 are basically the same as those in Example A1, except that the nutrient solutions for the germination and cultivation treatments of the saccharification-resistant seedlings in Comparative Examples A1 to A3 are shown in Table 1.

[0099] The saccharification-resistant seedlings of Examples A1 to A3 and Comparative Examples A1 to A3 were prepared by simultaneously germinating and cultivating safflower seeds from the same batch in the same incubator. These safflower seeds were sourced from Aosen Horticulture in Muyang County, Jiangsu Province.

[0100]

[0101] Examples B1 to B4 and Comparative Example B1

[0102] Examples B1 to B4 and Comparative Example B1 each provide a saccharification-resistant seedling. The preparation methods of the saccharification-resistant seedlings in Examples B1 to B4 and Comparative Example B1 are basically the same as those in Example A1, except that the nutrient solutions for the germination and cultivation treatments of the saccharification-resistant seedlings in Examples B1 to B4 and Comparative Example B1 are shown in Table 2. The saccharification-resistant seedlings in Examples B1 to B4 and Comparative Example B1 were prepared by simultaneously germinating and cultivating safflower seeds from the same batch in the same incubator. These safflower seeds were sourced from Aosen Horticulture in Muyang County, Jiangsu Province.

[0103]

[0104] Examples C1 to C3 and Comparative Examples C1 to C4

[0105] Examples C1 to C3 and Comparative Examples C1 to C4 each provide a saccharification-resistant seedling. The preparation methods of the saccharification-resistant seedlings in Examples C1 to C3 and Comparative Examples C1 to C4 are basically the same as those in Example A1, except that the nutrient solutions for the germination and cultivation treatments of the saccharification-resistant seedlings in Examples C1 to C3 and Comparative Examples C1 to C4 are shown in Table 3. The saccharification-resistant seedlings in Examples C1 to C3 and Comparative Examples C1 to C4 were prepared by simultaneously germinating and cultivating safflower seeds from the same batch in the same incubator. These safflower seeds were sourced from Aosen Horticulture in Muyang County, Jiangsu Province.

[0106]

[0107] 3. Quality testing of anti-glycation seedlings

[0108] 3.1 Investigation on the growth of glycogen-resistant seedlings

[0109] The saccharification-resistant seedlings from Examples A1 to A3, Comparative Examples A1 to A3, Examples B1 to B4, Comparative Example B1, Examples C1 to C3, and Comparative Examples C1 to C4 were taken respectively. The saccharification-resistant seedlings were weighed, and their total length, shoot length, and root length were measured. Shoot length refers to the total length of the upper part of the root. The results are as follows: Figures 7 to 9 As shown.

[0110] like Figures 7 to 9As shown, the saccharification-resistant seedlings of Comparative Examples A1, B1, and C1 were cultivated under the same conditions—in water without the addition of any inducing agents. However, the weight and total length of the saccharification-resistant seedlings of Comparative Examples A1, B1, and C1 were not entirely the same. This is because the saccharification-resistant seedlings of Comparative Examples A1, B1, and C1 were cultivated using different batches of safflower seeds, and there are certain differences between different batches of safflower seeds.

[0111] like Figure 7 As shown in A, the addition of sodium hypochlorite significantly inhibited the growth of safflower seedlings, and the weight of the saccharification-resistant seedlings in Comparative Example A2 was significantly lower than that of the saccharification-resistant seedlings in other examples and comparative examples.

[0112] like Figure 8 As shown in A, when sucrose is used as an inducer and the concentration of the sucrose solution is 0.1‰~2.5‰, the total weight of the sucrose-resistant seedlings obtained by cultivation is not significantly different from the total weight of the sucrose-resistant seedlings obtained by water cultivation in Example B1.

[0113] like Figure 9 As shown, the addition of glucose and maltose mainly inhibits the growth of roots in glycosylated seedlings, with less inhibition on the growth of the upper part of the roots.

[0114] 3.2 Determination of anti-glycation component content

[0115] Take the anti-glycation seedlings from Examples A1 to A3, Comparative Examples A1 to A3, Examples B1 to B4, Comparative Example B1, Examples C1 to C3, and Comparative Examples C1 to C4, and determine the content of the anti-glycation component L7G. The results are as follows: Figures 10 to 12 As shown. The method for determining the content of the anti-glycation component L7G is as follows:

[0116] Thirty glycation-resistant seedlings were collected and samples were extracted on the day of harvest. The specific extraction steps were as follows: the surface moisture of the red flower seedlings was absorbed and weighed, and then placed in a high-speed blender for thorough cell wall disruption and homogenization. The homogenized sample was then extracted with 30 mL of methanol and ultrasonically extracted at room temperature for 30 min. After filtration, the filtrate was stored at -20 ℃. The concentration of L7G in the filtrate was determined by HPLC, and the concentration and total content of L7G in the glycation-resistant seedlings were calculated. Before HPLC injection, the samples were diluted to an appropriate concentration as needed, and L7G standard solutions of suitable concentrations were prepared and injected into the HPLC system.

[0117] Chromatographic conditions: HPLC analysis was performed on a Waters Arc™ HPLC system (Waters, Milford, MA) equipped with a Waters 2998 PAD detector (Waters, Milford, MA). The column was a Waters XBridge C18 (4.6 mm × 250 mm, 5 μm). The mobile phase was 0.3% acetic acid solution and acetonitrile. Gradient elution was used with a range of 15%–90%, an elution time of 45 min, a flow rate of 1.0 mL / min, a detection wavelength of 254 nm, and a column temperature of 25 °C.

[0118] like Figure 10 As shown, compared to Example A1 without an inducer, although Comparative Examples A2 and A3, using sodium hypochlorite as an inducer, could increase the L7G concentration in the whole wet product of the glycosylated seedlings, the content of L7G in the whole glycosylated seedlings did not increase significantly because sodium hypochlorite significantly inhibited the growth of the seedlings. However, Examples A1 to A4, using sodium chloride and sucrose as inducers, not only increased the concentration of L7G in the glycosylated seedlings (wherein, the L7G concentration in the glycosylated seedlings is based on the whole plant wet weight, the same below), but also significantly increased the total L7G content in the whole glycosylated seedlings. This indicates that using sodium chloride and sucrose as inducers can effectively promote the synthesis and accumulation of L7G and increase the total L7G content in the glycosylated seedlings.

[0119] like Figure 11 As shown, compared with Comparative Example B1 without the addition of an inducer, the anti-glycation seedlings cultivated in Examples B1 to B4 with 0.1‰~2.5‰ sucrose as an inducer not only had a higher concentration of L7G in the anti-glycation seedlings, but also a significantly higher total L7G content in the whole plant. This indicates that different concentrations of inducers can effectively promote the synthesis and accumulation of L7G and increase the total L7G content in the anti-glycation seedlings.

[0120] like Figure 12 As shown, compared to Comparative Example C1 without any inducer, the total L7G content in Comparative Example C2, cultivated with maltose as an inducer, did not increase significantly. Similarly, in Comparative Examples C3 to C4, cultivated with chitosan and xylitol, the L7G concentration and total L7G content did not increase significantly. However, in Examples C1 to C3, cultivated with glucose, trehalose, and cyclodextrin as inducers, both the L7G concentration and total L7G content in the entire plant increased.

[0121] Figures 10 to 12This indicates that the method for preparing anti-glycation seedlings in this application can effectively promote the synthesis and accumulation of L7G in safflower seedlings by adding a specific inducer during the cultivation process, thereby increasing the total L7G content in the anti-glycation seedlings obtained from the cultivation process.

[0122] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing anti-saccharification seedlings, characterized in that, Includes the following steps: By cultivating safflower seedlings, glycosylation-resistant seedlings were obtained; The cultivation process includes the addition of an inducing agent, which is sodium chloride, glucose, sucrose, trehalose, or β-cyclodextrin. Prior to the cultivation treatment, the preparation method further includes: The inducer is prepared into an inducer solution, wherein... When the inducing agent solution is a sucrose solution, the concentration of the sucrose solution is 0.5‰~2.5‰; When the inducing agent solution is a sodium chloride solution, the concentration of the sodium chloride solution is 3‰~6‰; When the inducing agent solution is a glucose solution, the concentration of the glucose solution is 1‰; When the inducing agent solution is a trehalose solution, the concentration of the trehalose solution is 1‰; When the inducing agent solution is a β-cyclodextrin solution, the concentration of the β-cyclodextrin solution is 1‰; The cultivation treatment method is hydroponics, and the nutrient solution for hydroponics is the inducer solution; The safflower seedlings are obtained by adding the inducer solution to safflower seeds and germinating them.

2. The preparation method according to claim 1, characterized in that, The cultivation treatment satisfies at least one of the following (1) to (5): (1) The cultivation treatment time is 6 to 14 days; (2) The light intensity for the cultivation treatment is 2000~5000 Lux; (3) The light exposure time for the cultivation treatment is 8-12 hours / day; (4) The temperature for the cultivation treatment is 20~28 ℃; (5) The humidity of the cultivation treatment is 60%~85%.

3. The preparation method according to claim 1, characterized in that, Prior to the germination treatment, the safflower seeds are also disinfected.

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