Melatonin preparation for improving salt stress resistance of carya illinoensis and preparation method of melatonin preparation

Abstract

The invention discloses a melatonin preparation for improving the salt stress resistance of carya illinoensis and a preparation method of the melatonin preparation, and relates to the technical field of biology. The melatonin preparation comprises a first active component, a second active component and auxiliary materials, the first active component is melatonin; and the second active component is tuberostemonine or dioscin. According to the invention, tuberostemonine and dioscin are introduced to respectively form a synergistic effect with melatonin, so that the salt stress resistance of the carya illinoensis can be remarkably improved. The tuberostemonine or the dioscin is combined with the melatonin, so that the activity of antioxidant enzymes such as superoxide dismutase and peroxidase can be activated, active oxygen can be effectively removed, and the content of malondialdehyde can be reduced, so that the membrane lipid peroxidation damage can be alleviated, and the cell membrane stability can be maintained; meanwhile, chlorophyll synthesis can be promoted, inhibition of salt stress on photosynthesis is relieved, seedling biomass accumulation is enhanced, the fresh weight is remarkably improved compared with that of salt stress control, and the comprehensive resistance to salt stress is shown.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a melatonin preparation for enhancing the salt stress tolerance of thin-shelled pecans and its preparation method. Background Technology

[0002] Pecan (Carya illinoinensis), an important woody oilseed and ecologically valuable tree species, boasts nutritious fruit and excellent timber, making it highly valuable in both economic and ecological fields. However, soil salinization is increasingly threatening the normal growth and yield of pecans, and improving their salt stress tolerance has become one of the key issues in current forestry research.

[0003] In existing technologies, melatonin, an indole hormone widely found in organisms, has been explored for its application in improving the salt tolerance of pecans due to its significant role in regulating plant growth and development and enhancing plant stress resistance. Studies have shown that melatonin can alleviate the damage caused by salt stress to some extent by regulating the plant's antioxidant enzyme system, ion balance, and hormone signal transduction pathways. However, practical applications have revealed that simply treating pecans with melatonin has a limited effect on improving their salt tolerance, making it difficult to meet the needs of production practice for improving the survival rate and growth quality of pecans in saline soils.

[0004] In recent years, researchers in this field have gradually realized that the effects of a single substance are often limited, and that the combined use of multiple natural substances can achieve synergistic effects. However, the technology of combining natural substances to address salt stress in thin-shelled pecans is still in the exploratory stage, and a mature and efficient solution has not yet been formed. The existing combined formulations have shown inconsistent results in thin-shelled pecans, failing to fully meet the urgent needs of the thin-shelled pecan industry.

[0005] Therefore, there is an urgent need to develop a new melatonin formulation. By introducing other natural substances with synergistic effects, a more scientific and efficient formulation system can be constructed to further enhance the tolerance of thin-shelled pecans to salt stress and provide strong technical support for the promotion and industrial development of thin-shelled pecans in saline-alkali areas. Summary of the Invention

[0006] The purpose of this invention is to provide a melatonin formulation and its preparation method for enhancing the salt stress tolerance of thin-shelled pecans, thereby solving the problems existing in the prior art. This melatonin formulation can significantly enhance the salt stress tolerance of thin-shelled pecans.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a melatonin formulation for enhancing the salt stress tolerance of thin-shelled pecans, comprising a first active ingredient, a second active ingredient, and excipients; the first active ingredient is melatonin; the second active ingredient is stemona alkaloid or diosgenin.

[0009] Furthermore, the concentrations of the first active ingredient and the second active ingredient are 100 μmol / L and 50 μmol / L, respectively.

[0010] Furthermore, the excipients include stabilizers and preservatives.

[0011] Furthermore, the stabilizer is sodium carboxymethyl cellulose.

[0012] Furthermore, the preservative is sodium benzoate.

[0013] Furthermore, the content of the stabilizer is 2-5 g / L; the content of the preservative is 0.3-0.8 g / L.

[0014] The present invention also provides a method for preparing the above-mentioned melatonin preparation, comprising the following steps:

[0015] The first active ingredient and the second active ingredient are dissolved in a solvent to obtain a mixed solution. Then, the stabilizer is added, and after stirring and reacting, the preservative is added. After adjusting the pH to 6.0-6.5, the melatonin preparation is obtained after filtration and packaging.

[0016] Furthermore, the solvent is an aqueous solution of ethanol with a volume fraction of 1-5%.

[0017] The present invention also provides the application of the above-mentioned melatonin preparation in improving the salt stress tolerance of thin-shelled pecans.

[0018] The present invention also provides a method for improving the salt stress tolerance of pecans, including the step of applying the above-mentioned melatonin preparation to pecan plants.

[0019] The present invention discloses the following technical effects:

[0020] This invention introduces stemona alkaloids and diosgenin, which synergistically enhance melatonin, significantly improving the salt stress tolerance of thin-shelled pecans. The combined use of stemona alkaloids or diosgenin with melatonin activates the activity of antioxidant enzymes such as superoxide dismutase (SOD) and peroxidase (POD), effectively scavenging reactive oxygen species and reducing malondialdehyde (MDA) content, thereby mitigating membrane lipid peroxidation damage and maintaining cell membrane stability. Simultaneously, it promotes chlorophyll synthesis, alleviates the inhibition of photosynthesis by salt stress, enhances seedling biomass accumulation, and significantly increases fresh weight compared to the salt-stressed control, demonstrating a comprehensive resistance to salt stress.

[0021] This invention further develops a melatonin formulation for enhancing the salt stress tolerance of pecans. The addition of excipients ensures component homogeneity, duration of action, and formulation safety. This formulation can efficiently induce salt stress tolerance in pecan seedlings, providing a scientifically sound and practical solution for forestry cultivation in saline-alkali areas. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Melatonin (MT) is a newly emerging plant hormone with the following structural formula:

[0028]

[0029] Compared to the five traditional classes of plant hormones—auxin (IAA), gibberellin (GA), cytokinin (CTK), ethylene (ETH), and abscisic acid (ABA)—melatonin identification has only been around for about 20 years. Chemically, MT is a tryptophan derivative, specifically N-acetyl-5-methoxytryptamine, a small indoleamine widely found in plants and animals. In 1958, Lerner et al. isolated and extracted this substance from the pineal gland of cattle, considering it a neurohormone that regulates animal physiological activities. Subsequently, in 1993, van Tassel et al. detected MT in morning glory (Ipomoea leucantha L.) and tomato (Solanum lycopersicum L.) using radioimmunoassay and gas chromatography-mass spectrometry. Then, in 1995, using radioimmunoassay, scientists isolated MT from 24 edible plant species and discovered that MT concentration is species- and organ-specific. Physiologically, MT is similar to other plant hormones; it not only regulates seed germination and root architecture but also modulates diurnal rhythms, photoperiod, and tolerance to biotic and abiotic stresses. Further research revealed that under normal physiological conditions, MT levels in plants are very low, generally ranging from pmol / g fresh weight to nmol / g fresh weight. However, under abiotic stresses such as high temperature, low temperature, drought, salinity, heavy metals, ultraviolet radiation, and waterlogging, MT levels can increase hundreds of times, thereby triggering MT signaling and enhancing the plant's resistance to these abiotic stresses.

[0030] Phloretin, also known as 2,4,6-trihydroxy-3-(4-hydroxyphenyl)phenylacetone, has the following structural formula:

[0031]

[0032] Phlorizin is a plant polyphenol with a dihydrochalcone structure, named for its high concentration in the root bark of apple trees. Due to its unique structure, phlorizin possesses a variety of biological activities, making it a hot research topic in recent years. Studies have found that phlorizin has various pharmacological effects, including antioxidant, antitumor, anti-inflammatory, immunosuppressive, and bone-forming effects. Furthermore, it also has various cosmetic benefits such as delaying aging, whitening skin, reducing inflammation, and removing acne.

[0033] Chlorogenic acid is an organic compound with the chemical formula C2. 16 H 18 O9, the structural formula is as follows:

[0034]

[0035] Chlorogenic acid is one of the main antibacterial and antiviral active pharmacological components of honeysuckle. It is an important bioactive substance with antibacterial, antiviral, white blood cell-boosting, hepatoprotective, choleretic, antitumor, blood pressure-lowering, blood lipid-lowering, free radical scavenging, and central nervous system-stimulating effects. Chlorogenic acid has strong antibacterial and anti-inflammatory effects. It is an effective phenolic antioxidant, with antioxidant capacity stronger than caffeic acid, p-hydroxybenzoic acid, ferulic acid, syringic acid, butylated hydroxyanisole (BHA), and tocopherol. The antioxidant effect of chlorogenic acid is due to its R-OH group, which can form hydrogen free radicals with antioxidant properties, eliminating the activity of hydroxyl free radicals and superoxide anions, thereby protecting tissues from oxidative damage. Chlorogenic acid and its derivatives have stronger free radical scavenging effects than ascorbic acid, caffeic acid, and tocopherol (vitamin E), effectively scavenging DPPH free radicals, hydroxyl free radicals, and superoxide anion free radicals, and also inhibiting the oxidation of low-density lipoprotein.

[0036] tuberostemonine is the main characteristic component of Stemona japonica, with the molecular formula C0.05. 22 H 33 NO4, the structural formula is as follows:

[0037]

[0038] Modern pharmacological studies have shown that stemonasaline has antitussive, paralyzing worm motility, and inhibitory effects on neuromuscular junction excitation in crayfish. A study has disclosed a method for extracting stemonasaline, comprising the following steps: (1) taking 2 kg of Stemona japonica herb and physically pulverizing it; (2) refluxing with 4 liters of 95% ethanol solution for 3 hours, extracting twice; (3) concentrating the extract to dryness; (4) eluting the concentrate using a D101 large-pore Chemicalbook resin column (3 kg), 12 cm in diameter, with 3 liters of 40% methanol solution; (5) collecting the portion containing stemonasaline and concentrating it in 200 mL of anhydrous ethanol, adding 3 mL of hydroiodic acid to form a salt, cooling at 0 degrees Celsius for crystallization overnight, then filtering, drying, and obtaining 7 g of stemonasaline extract.

[0039] Eugenol, scientifically known as 4-allyl-2-methoxyphenol, has the molecular formula C2. 10 H 12 O2, the structural formula is as follows:

[0040]

[0041] Eugenol is naturally found in essential oils such as clove oil, clove basil oil, and cinnamon oil. It is a colorless to pale yellow viscous oily liquid with a strong clove and spicy aroma. Currently, industrial production of eugenol mostly involves isolating eugenol-rich essential oils through alkali treatment. Typically, sodium hydroxide solution is added to the oil to be separated, and after heating and stirring, the non-phenolic oily portion floating on the surface is extracted with a solvent or distilled off with steam. Then, the sodium salt is acidified to obtain crude eugenol, which is washed with water until neutral, and then purified by vacuum distillation.

[0042] Diosgenin, mainly found in the rhizomes of plants in the Dioscoreaceae family (such as Dioscorea nipponica and Dioscorea peltatum), is an important steroidal saponin component in Dioscorea plants. Its molecular formula is C0. 45 H 72 O 16 The structural formula is as follows:

[0043]

[0044] Example 1

[0045] I. Experimental Materials and Methods

[0046] 1.1 Experimental Materials

[0047] Plant materials: Select healthy, uniformly growing two-year-old thin-shelled pecan seedlings and plant them in nutrient pots with a diameter of 15cm and a height of 30cm. The substrate weight of each pot is 1kg. The bottom of the pot is perforated and a tray is placed underneath to ensure drainage.

[0048] Reagents: melatonin, phloretin, chlorogenic acid, stemona alkaloid, eugenol, diosgenin.

[0049] 1.2 Experimental Methods

[0050] 1.2.1 Experimental Design

[0051] This embodiment sets up 13 treatment groups, each group is repeated 3 times, and each repeat contains 20 seedlings. The specific grouping is as follows:

[0052] CK: Solvent control; the solvent used in each treatment group was a 1% (v / v) aqueous solution of ethanol;

[0053] NaCl: 0.5% NaCl solution (salt stress control);

[0054] Melatonin + NaCl: 0.5% NaCl + 100 μmol / L melatonin;

[0055] Phloretin + NaCl: 0.5% NaCl + 50 μmol / L phloretin;

[0056] Chlorogenic acid + NaCl: 0.5% NaCl + 50 μmol / L chlorogenic acid;

[0057] Stemona alkaloids + NaCl: 0.5% NaCl + 50 μmol / L Stemona alkaloids;

[0058] Eugenol + NaCl: 0.5% NaCl + 50 μmol / L eugenol;

[0059] Diosgenin + NaCl: 0.5% NaCl + 50 μmol / L diosgenin;

[0060] Phloretin + Melatonin + NaCl: 0.5% NaCl + 100 μmol / L Melatonin + 50 μmol / L Phloretin;

[0061] Chlorogenic acid + melatonin + NaCl: 0.5% NaCl + 100 μmol / L melatonin + 50 μmol / L chlorogenic acid;

[0062] Stemona japonica alkaloids + melatonin + NaCl: 0.5% NaCl + 100 μmol / L melatonin + 50 μmol / L Stemona japonica alkaloids;

[0063] Eugenol + Melatonin + NaCl: 0.5% NaCl + 100 μmol / L Melatonin + 50 μmol / L Eugenol.

[0064] Diosgenin + melatonin + NaCl: 0.5% NaCl + 100 μmol / L melatonin + 50 μmol / L diosgenin.

[0065] Treatment method: Irrigate with 200 mL of the corresponding solution every 7 days, for a total of 2 treatments. Maintain salt stress continuously with 0.5% NaCl solution, keeping the substrate moist. Thirty days after the first treatment, collect mature leaves from the middle of the branches to measure physiological indicators.

[0066] 1.2.2 Measurement of physiological indicators

[0067] Plant biomass determination: Ten intact seedlings were randomly selected, cleaned, and weighed.

[0068] Chlorophyll content determination: The chlorophyll content in the leaves was determined by acetone-ethanol extraction method.

[0069] Antioxidant enzyme activity assay: Superoxide dismutase (SOD) activity was determined using the NBT method, while peroxidase (POD) and catalase (CAT) activities were determined according to the method of Chen Jianxun et al.

[0070] Malondialdehyde (MDA) content determination: The MDA content was determined by the thiobarbituric acid method.

[0071] II. Experimental Results

[0072] 2.1 Biomass Measurement Results

[0073] The biomass measurement results for each treatment group are shown in Table 1. Compared with the NaCl treatment group, the fresh weight of seedlings in the Stemona japonica + melatonin + NaCl treatment group and the diosgenin + melatonin + NaCl treatment group was significantly increased, by 49.36% and 48.00% respectively, approaching the level of the water control; other compound treatments (root bark extract, chlorogenic acid, eugenol) combined with melatonin did not significantly increase biomass or were lower than the single reagent treatment groups.

[0074] Table 1. Biomass measurement results for each treatment group

[0075] Processing group Fresh weight (g) CK 65.35 NaCl 40.42 Melatonin + NaCl 51.68 Phloretin + NaCl 46.19 Chlorogenic acid + NaCl 41.25 p-Stemona alkaloids + NaCl 41.69 Eugenol + NaCl 44.48 diosgenin + NaCl 46.25 Phloretin + Melatonin + NaCl 53.40 Chlorogenic acid + melatonin + NaCl 52.19 p-Stemona alkaloids + melatonin + NaCl 60.37 Eugenol + Melatonin + NaCl 51.08 Diosgenin + melatonin + NaCl 59.82

[0076] 2.2 Chlorophyll content determination results

[0077] The chlorophyll content of each treatment group is shown in Table 2. The results showed that NaCl stress led to a significant decrease in total chlorophyll. However, the chlorophyll content of the treatment group (stemona root extract + melatonin + NaCl) and the treatment group (diosgenin + melatonin + NaCl) increased by 24.44% and 21.06% respectively compared with the NaCl control, approaching the non-stress level. The chlorophyll content increase of other compound treatments was less than 10%.

[0078] Table 2. Results of chlorophyll content measurement for each treatment group

[0079]

[0080]

[0081] 2.3 Results of Antioxidant Enzyme Activity Assay

[0082] POD, SOD, and CAT are important components of the enzymatic defense mechanism in plants under stress conditions. They can scavenge excess reactive oxygen species and free radicals in plants, thereby improving their stress resistance. Among them, SOD converts excess free radicals into H2O2, while POD and CAT work together to scavenge H2O2. The results of the antioxidant enzyme activity assays for each treatment group are shown in Table 3. The results showed that in the *Stemona japonica* + melatonin + NaCl treatment group, the activities of SOD, POD, and CAT increased by 42.32%, 19.91%, and 21.12% respectively compared with the NaCl control. In the diosgenin + melatonin + NaCl treatment group, the activities of SOD, POD, and CAT increased by 42.46%, 19.45%, and 17.78% respectively compared with the NaCl control, all significantly higher than other combined treatments. The increase in enzyme activity was relatively low in the single melatonin treatment, indicating that the combined use of *Stemona japonica* or diosgenin with melatonin can synergistically enhance the antioxidant system.

[0083] Table 3 Results of antioxidant enzyme activity assays for each treatment group

[0084] Processing group SOD activity (U / g) POD activity (U / g) CAT activity (U / g) CK 305.1 286.9 89.2 NaCl 348.3 324.5 150.6 Melatonin + NaCl 454.0 362.1 167.5 Phloretin + NaCl 426.2 356.4 164.3 Chlorogenic acid + NaCl 407.8 347.5 161.2 p-Stemona alkaloids + NaCl 418.5 340.9 157.0 Eugenol + NaCl 445.4 354.7 159.7 diosgenin + NaCl 431.9 350.8 156.2 Phloretin + Melatonin + NaCl 456.2 370.8 168.2 Chlorogenic acid + melatonin + NaCl 432.8 365.9 169.5 p-Stemona alkaloids + melatonin + NaCl 495.7 389.1 182.4 Eugenol + Melatonin + NaCl 434.9 367.5 160.8 Diosgenin + melatonin + NaCl 496.2 387.6 180.4

[0085] 2.4 MDA content determination results

[0086] When plants are subjected to abiotic stress, their cells undergo membrane lipid peroxidation, and MDA is one of the indicators for measuring the degree of oxidative stress. Higher MDA content indicates a higher degree of membrane lipid peroxidation in the plant, and more severe damage to the cell membrane. The MDA content measurements for each treatment group are shown in Table 4. The results showed that the MDA content of the *Stemona japonica* + melatonin + NaCl treatment group and the *diosgenin* + melatonin + NaCl treatment group was reduced by 33.03% and 30.62% respectively compared to the NaCl control, significantly lower than other combined treatment groups, indicating that their cell membrane stability was the best. The MDA content of the eugenol + melatonin combination treatment was even higher than that of the melatonin-only group, which may have exacerbated membrane lipid peroxidation.

[0087] Table 4. Results of MDA content determination in each treatment group

[0088] Processing group MDA content (mmol / g) CK 11.02 NaCl 19.53 Melatonin + NaCl 17.31 Phloretin + NaCl 18.20 Chlorogenic acid + NaCl 17.69 p-Stemona alkaloids + NaCl 18.54 Eugenol + NaCl 18.18 diosgenin + NaCl 18.67 Phloretin + Melatonin + NaCl 16.59 Chlorogenic acid + melatonin + NaCl 17.03 p-Stemona alkaloids + melatonin + NaCl 13.08 Eugenol + Melatonin + NaCl 18.88 Diosgenin + melatonin + NaCl 13.55

[0089] In summary, this invention confirms that the combination of stemona alkaloids or diosgenin with melatonin can significantly enhance the salt stress tolerance of pecan seedlings. The mechanism may involve synergistically enhancing the activity of antioxidant enzymes (SOD, POD, CAT), promoting chlorophyll synthesis, and reducing membrane lipid peroxidation. The combined use of phloretin, chlorogenic acid, eugenol, and melatonin did not significantly improve the salt tolerance of pecan; some treatments even showed antagonistic effects (e.g., eugenol).

[0090] Example 2

[0091] A melatonin preparation for enhancing the salt stress tolerance of thin-shelled pecans comprises 100 μmol / L melatonin, 50 μmol / L stemona alkaloid, 3 g / L stabilizer, 0.6 g / L preservative, and the balance being solvent. The preparation method is as follows:

[0092] (1) Add solvent, i.e., 1% (v / v) aqueous ethanol solution, to the reaction vessel, heat to 45°C, set the stirring speed to 200 r / min, then add melatonin and p-stemonine, and continue stirring until completely dissolved to obtain a mixed solution.

[0093] (2) Slowly add the stabilizer sodium carboxymethyl cellulose, increase the stirring speed to 400 r / min, stir for 20 min to form a uniform colloid, cool to room temperature, add the preservative sodium benzoate, and stir for 10 min to fully disperse it.

[0094] (3) Adjust the pH to 6.5, filter through a 0.45μm filter membrane, fill into a light-proof plastic bottle, seal and store in a cool and dry place.

[0095] Example 3

[0096] A melatonin preparation for enhancing the salt stress tolerance of thin-shelled pecans comprises 100 μmol / L melatonin, 50 μmol / L stemona alkaloid, 2 g / L stabilizer, 0.8 g / L preservative, and the balance being solvent. The preparation method is as follows:

[0097] (1) Add solvent, namely 2% (v / v) aqueous ethanol solution, to the reaction vessel, heat to 40°C, set the stirring speed to 180 r / min, then add melatonin and p-stemonine, and continue stirring until completely dissolved to obtain a mixed solution.

[0098] (2) Slowly add the stabilizer sodium carboxymethyl cellulose, increase the stirring speed to 350 r / min, stir for 25 min to form a uniform colloid, cool to room temperature, add the preservative sodium benzoate, and stir for 8 min to fully disperse it.

[0099] (3) Adjust the pH to 6.0, filter through a 0.45μm filter membrane, fill into a light-proof plastic bottle, seal and store in a cool and dry place.

[0100] Example 4

[0101] A melatonin preparation for enhancing the salt stress tolerance of thin-shelled pecans comprises 100 μmol / L melatonin, 50 μmol / L stemona alkaloid, 5 g / L stabilizer, 0.3 g / L preservative, and the balance being solvent. The preparation method is as follows:

[0102] (1) Add solvent, namely 5% (v / v) aqueous ethanol solution, to the reaction vessel, heat to 50°C, set the stirring speed to 250 r / min, then add melatonin and p-stemonine, and continue stirring until completely dissolved to obtain a mixed solution.

[0103] (2) Slowly add the stabilizer sodium carboxymethyl cellulose, increase the stirring speed to 450 r / min, stir for 15 min to form a uniform colloid, cool to room temperature, add the preservative sodium benzoate, and stir for 12 min to fully disperse it.

[0104] (3) Adjust the pH to 6.3, filter through a 0.45μm filter membrane, fill into a light-proof plastic bottle, seal and store in a cool and dry place.

[0105] Example 5

[0106] A melatonin preparation for enhancing the salt stress tolerance of thin-shelled pecans comprises 100 μmol / L melatonin, 50 μmol / L diosgenin, 3 g / L stabilizer, 0.6 g / L preservative, and the balance being solvent. The preparation method is as follows:

[0107] (1) Add solvent, i.e., 1% (v / v) ethanol aqueous solution, to the reaction vessel, heat to 45°C, set the stirring speed to 200 r / min, then add melatonin and diosgenin, and continue stirring until completely dissolved to obtain a mixed solution.

[0108] (2) Slowly add the stabilizer sodium carboxymethyl cellulose, increase the stirring speed to 400 r / min, stir for 20 min to form a uniform colloid, cool to room temperature, add the preservative sodium benzoate, and stir for 10 min to fully disperse it.

[0109] (3) Adjust the pH to 6.5, filter through a 0.45μm filter membrane, fill into a light-proof plastic bottle, seal and store in a cool and dry place.

[0110] Example 6

[0111] A melatonin preparation for enhancing the salt stress tolerance of thin-shelled pecans comprises 100 μmol / L melatonin, 50 μmol / L diosgenin, 2 g / L stabilizer, 0.8 g / L preservative, and the balance being solvent. The preparation method is as follows:

[0112] (1) Add solvent, namely 2% (v / v) aqueous ethanol solution, to the reaction vessel, heat to 40°C, set the stirring speed to 180 r / min, then add melatonin and diosgenin, and continue stirring until completely dissolved to obtain a mixed solution.

[0113] (2) Slowly add the stabilizer sodium carboxymethyl cellulose, increase the stirring speed to 350 r / min, stir for 25 min to form a uniform colloid, cool to room temperature, add the preservative sodium benzoate, and stir for 8 min to fully disperse it.

[0114] (3) Adjust the pH to 6.0, filter through a 0.45μm filter membrane, fill into a light-proof plastic bottle, seal and store in a cool and dry place.

[0115] Example 7

[0116] A melatonin preparation for enhancing the salt stress tolerance of thin-shelled pecans comprises 100 μmol / L melatonin, 50 μmol / L diosgenin, 5 g / L stabilizer, 0.3 g / L preservative, and the balance being solvent. The preparation method is as follows:

[0117] (1) Add solvent, i.e. 5% (v / v) ethanol aqueous solution, to the reaction vessel, heat to 50°C, set the stirring speed to 250 r / min, then add melatonin and diosgenin, and continue stirring until completely dissolved to obtain a mixed solution.

[0118] (2) Slowly add the stabilizer sodium carboxymethyl cellulose, increase the stirring speed to 450 r / min, stir for 15 min to form a uniform colloid, cool to room temperature, add the preservative sodium benzoate, and stir for 12 min to fully disperse it.

[0119] (3) Adjust the pH to 6.3, filter through a 0.45μm filter membrane, fill into a light-proof plastic bottle, seal and store in a cool and dry place.

[0120] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A melatonin preparation for enhancing the salt stress tolerance of thin-shelled pecans, characterized in that, It includes a first active ingredient, a second active ingredient, and excipients; the first active ingredient is melatonin; the second active ingredient is stemona alkaloid or diosgenin.

2. The melatonin formulation according to claim 1, characterized in that, The concentrations of the first active ingredient and the second active ingredient are 100 μmol / L and 50 μmol / L, respectively.

3. The melatonin formulation according to claim 1, characterized in that, The excipients include stabilizers and preservatives.

4. The melatonin formulation according to claim 3, characterized in that, The stabilizer is sodium carboxymethyl cellulose.

5. The melatonin formulation according to claim 3, characterized in that, The preservative is sodium benzoate.

6. The melatonin formulation according to claim 3, characterized in that, The stabilizer content is 2-5 g / L; the preservative content is 0.3-0.8 g / L.

7. A method for preparing a melatonin formulation as described in any one of claims 1-6, characterized in that, Includes the following steps: The first active ingredient and the second active ingredient are dissolved in a solvent to obtain a mixed solution. Then, the stabilizer is added, and after stirring and reacting, the preservative is added. After adjusting the pH to 6.0-6.5, the melatonin preparation is obtained after filtration and packaging.

8. The preparation method according to claim 7, characterized in that, The solvent is an aqueous solution of ethanol with a volume fraction of 1-5%.

9. The use of a melatonin preparation as described in any one of claims 1-6 in enhancing the salt stress tolerance of thin-shelled pecans.

10. A method for improving the salt stress tolerance of thin-shelled pecans, characterized in that, The step includes applying the melatonin preparation of any one of claims 1-6 to pecan plants.

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