Fruit retention plant source regulator for pawpaw

Abstract

The invention provides a fruit retention plant source regulator. The fruit retention plant source regulator comprises a papaya leaf water extract. The papaya leaf water extract is prepared by the following steps: crushing papaya leaves, adding water, carrying out water bath extraction at 80-90 DEG C for 1-2 hours, and concentrating the obtained filtrate. According to the technical scheme provided by the invention, based on the thinking of cyclic utilization, the papaya leaves are not easy to fall off except drought influence and contain anti-falling components.

Description

Technical Field

[0001] The applicant provides a papaya fruit-preserving plant-derived regulator, which belongs to the field of biopharmaceutical technology. Background Art

[0002] Malus scabra Chaenomeles speciosa (Sweet) Nakai's nearly mature fruit. Although papaya has a large number of flowers, its fruit set rate is only 1-5%, and it suffers from severe flower and fruit drop. Fruit drop is categorized as physiological fruit drop and exogenous infection fruit drop. Physiological fruit drop is caused by malnutrition and insufficient endogenous hormone secretion, while exogenous infection fruit drop is caused by disease, insects, birds, and other pests. To address the causes of papaya fruit drop, appropriate measures such as pruning, early application of base fertilizer, hormone supplementation, and strengthened pest and disease control are implemented to preserve flowers and fruits. The new GAP imposes higher requirements on the production of traditional Chinese medicines. The use of growth regulators such as Zhuanggenling and Wendasu to regulate the growth of harvested organs of traditional Chinese medicines is prohibited. This places higher demands on flower and fruit preservation technologies in papaya production. Therefore, in response to the needs of ecological papaya cultivation, the present invention retains pest-free leaves after harvesting papayas and uses them to preserve papaya fruit the following year.

[0003] Papaya leaves contain a variety of bioactive compounds such as polyphenols and flavonoids. The content of chlorogenic acid is 0.1%-0.8%. As a natural phenolic acid compound, chlorogenic acid plays an important role in plant growth and development, disease resistance, insect resistance, and resistance to external stress environments. Chlorogenic acid has good antibacterial effects and has a good inhibitory effect on Bacillus, Weissella, Enterobacter, Pantoea, Clostridium butyricum, etc. Chlorogenic acid also has insecticidal functions. Studies have shown that applying exogenous chlorogenic acid to tomato fruits can promote the coloring of the fruit, reduce the hardness of the fruit, increase the weight of the fruit, and promote the ripening of the fruit, and its quality and nutritional value are significantly improved. Therefore, the present invention uses chlorogenic acid as the content index component of the papaya leaf water extract. Summary of the Invention

[0004] The present invention provides a fruit-preserving plant-derived regulator, which comprises a papaya leaf water extract.

[0005] The papaya leaf water extract is prepared by adding water to crushed papaya leaves and extracting them in a water bath at 80-90°C for 1-2 hours. The resulting filtrate is concentrated to obtain the papaya leaf water extract. The papaya leaf water extract is a liquid, 1 ml of which is equivalent to 0.2 g of papaya leaves and can be stored at -20°C.

[0006] The fruit-preserving plant-derived regulator further comprises an excipient, and the excipient comprises a combination of one or more of water, corn starch, microcrystalline cellulose, lactose, sucrose, hydroxypropyl cellulose, alginic acid, gelatin, polyethylene glycol, stearic acid, and magnesium stearate.

[0007] Among the above-mentioned different excipients, the papaya leaf water extract can be formed into any one of soluble drugs, gel-type drugs and film-type drugs according to the different excipients.

[0008] The soluble medicine is sprayed onto the fruit or the root of the fruit in a spraying manner.

[0009] The gel medicine is adhered to the root of the fruit or the branches near the fruit in a form of certain viscosity.

[0010] The film-type medicine is adhered to the root of the fruit or the branches near the fruit in the form of forming a certain film layer.

[0011] A papaya fruit-preserving plant-derived regulator comprises the aforementioned fruit-preserving plant-derived regulator, i.e., the aforementioned fruit-preserving drug is used for preserving the fruit of papaya trees during the young fruit stage.

[0012] The dosage of the papaya leaf water extract is 0.05-0.2 g / mL.

[0013] The preferred dosage of papaya leaf water extract is 0.1-0.2 g / mL.

[0014] The chlorogenic acid content in the papaya leaf water extract is 0.05-0.3 g / L, preferably 0.1-0.25 g / L.

[0015] In some preferred cases, the papaya fruit-preserving drug is sprayed with water to achieve the papaya fruit-preserving effect.

[0016] The papaya fruit-preserving effect is achieved by spraying the papaya fruit-preserving drug on the young fruits of the papaya, spraying once every 7-15 days, and spraying 2-4 times.

[0017] Papaya suffers from serious fruit drop. Both physiological fruit drop and fruit drop caused by exogenous infection seriously affect the yield and profit of papaya. The new version of GAP has put forward higher requirements for the production of Chinese medicinal materials. It is prohibited to use growth regulators such as Zhuanggenling and Gedunculus to regulate the growth of harvested organs of Chinese medicinal materials. The flower and fruit preservation drugs currently used mostly contain growth regulators such as Gedunculus. Therefore, the development of new flower and fruit preservation preparations is an urgent need for papaya ecological cultivation. Based on the idea of ​​recycling, it was found that except for the influence of drought, papaya leaves are not easy to fall off and contain anti-falling ingredients. After papaya leaves fall off naturally in late autumn, substances such as chlorogenic acid degrade quickly. Therefore, retaining the pest-free leaves after harvesting papayas and using them for papaya fruit preservation the following year has achieved good results. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the standard curve of chlorogenic acid.

[0019] Figure 2 Gibberellic acid standard curve.

[0020] Figure 3 Abscisic acid standard curve.

[0021] Figure 4 Fruit retention of papaya after treatment with CK (A) and medium concentration of papaya leaf aqueous extract (B).

[0022] Figure 5 Total fruit drop of 6 papaya trees after treatment with CK (A) and medium concentration (B) of papaya leaf aqueous extract.

[0023] Figure 6 Fruit drop on papaya trees after treatment with CK (A) and medium concentration of papaya leaf aqueous extract (B). DETAILED DESCRIPTION

[0024] Example 1 Experimental Materials Papaya leaves were purchased in Langping Town, Changyang Tujia Autonomous County in August 2024 and identified by Professor Wang Yubing of China Three Gorges University as Malus officinalis, a plant of the Rosaceae family. Chaenomeles speciosa( Sweet ） Nakai leaves. Elisa kits were purchased from Shanghai Hengyuan Biotechnology Co., Ltd.; PBS buffer was from Thermo Fisher Scientific. Chlorogenic acid was produced by Peptide Beauty Biomaterials Factory.

[0025] Experimental instruments and equipment The main experimental instruments used in the experiment are shown in Table 1.

[0026] Table 1 Main experimental instruments

[0027] Experimental methods Establishment of chlorogenic acid standard curve Preparation of Chlorogenic Acid Reference Solution: Accurately weigh 0.00718 g of Chlorogenic Acid Reference Solution into a 25 mL brown volumetric flask. Add 75% methanol to make a stock solution containing 287.2 μg of Chlorogenic Acid Reference Solution per mL, with a purity of 98%.

[0028] Chromatographic conditions: Column: C18 (COSMOSIL 5C18-MS-II Packed Column, 4.6 mm × 250 mm, 5 μm); Mobile phase A: 0.4% phosphoric acid in water, Mobile phase B: acetonitrile, Isocratic elution: 90% 0.4% phosphoric acid in water, 10% acetonitrile, 25 min; Flow rate: 0.8 mL / min; Detection wavelength: 325 nm; Injection volume: 10 μL.

[0029] The chlorogenic acid reference stock solution was diluted with 75% methanol to obtain a series of chlorogenic acid standard solutions with concentrations of 14.36 μg / ml, 28.72 μg / ml, 57.44 μg / ml, 114.88 μg / ml, and 172.32 μg / mL, respectively. The diluted chlorogenic acid standard solutions at varying concentrations were aspirated with a needle, filtered through an organic phase filter, and injected into injection vials. Stored in the dark until ready for use, the chlorogenic acid solution at varying concentrations was injected into the chromatograph using a syringe. The chromatographic peaks were measured and integrated to obtain the peak areas. A standard curve was plotted with the chlorogenic acid solution concentration as the abscissa and the peak area as the ordinate. The regression line equation was y = 35366x + 42770; R² = 0.9999. Preparation of test solution Take 5 ml of papaya leaf water extract, place it in a stoppered conical flask, remove the stopper, dry it at 60-80℃, accurately add 25 ml of methanol, seal it tightly, weigh it, treat it with ultrasound for 30 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain it.

[0030] Preparation of papaya leaf aqueous extract Weigh an appropriate amount of dried papaya leaves, grind them into a fine powder, and pass them through a 20-mesh sieve. Add 10 times the amount of water to the powder and extract in an 80°C water bath for 1 hour with constant stirring. After extraction, filter and collect the filtrate. Repeat the extraction with 10 times the amount of water on the residue. Combine the two filtrates and concentrate to a volume equivalent to 0.2 g of papaya leaves per 1 ml. This aqueous extract of papaya leaves should be stored at -20°C until further use. The chlorogenic acid content is 0.25 g / L as determined by HPLC.

[0031] The extract was diluted with water during use to produce a high-concentration papaya leaf extract group (1 ml equivalent to 0.2 g of papaya leaf, with a measured chlorogenic acid concentration of 0.25 g / L), a medium-concentration papaya leaf extract group (1 ml equivalent to 0.1 g of papaya leaf, with a chlorogenic acid concentration of 0.125 g / L), and a low-concentration papaya leaf extract group (1 ml equivalent to 0.05 g of papaya leaf, with a chlorogenic acid concentration of 0.0625 g / L). These three concentration gradients constituted the experimental papaya leaf extract group, with the chlorogenic acid group having a concentration of 0.125 g / L. Pure water served as the control group. Six papaya trees of identical age, size, growth, and flowering period were randomly selected as groups, for a total of 30 trees. These trees were sprayed on young fruiting papayas.

[0032] Determination of papaya fruit retention by papaya leaf water extract The papaya is the nearly mature fruit of the Malus serrata, a plant in the Rosaceae family. Malus serrata is a deciduous shrub, about 1.5-3 meters tall, with dark brown bark and prickles. Its twigs are coarse, yellowish-brown and glossy. Its leaves are leathery, emerald green when young and dark green when older. They are narrowly elliptical, with sharply serrated margins, 4-9 cm long and 1.3-5 cm wide, acute at the apex and narrowly cuneate at the base. It has 8-11 pairs of lateral veins, with the primary vein concave and prominently raised on the dorsal surface. The petioles are 0.8-1.5 cm long. The flowers are fragranceless, borne in clusters, and flowering and leafing occur simultaneously. The buds are ellipsoidal, with five sepals. The tepals are five simple, predominantly pink, with rarer variations of red and white. Stamens are apetalous, and the anthers are pale yellow. The petals are flat, obovate, rounded at the apex and broadly cuneate at the base. The calyx tube is cylindrical, and the pedicel is short. The fruit is ovoid, 3-7 cm in diameter, with grooved fruiting spots and no clearly developed seeds. Flowering occurs from March to April. Young papaya fruits generally refer to the early stages of fruit development, from the time the flowers have fertilized and formed into fruit and are initially set, until the fruit reaches maturity. These fruits are called young fruits. The fruit forms and grows for one to two months after the flowers have fallen, from mid-April to early June. Young fruits are initially small, round or oval, with a smooth surface and a firm texture. They are light green in color, gradually turning dark green as they grow. The average number of young fruits per tree is approximately 120 ± 30.

[0033] Count the number of fruit remaining on the sprayed papaya trees. Spray twice, 10-15 days after the flowers have faded. Count the number of young fruit on each tree and perform the first spraying. Spray again 15 days later, spray again. Use 1000 ml per tree for each spray (e.g., dissolve the high-dose formulation (0.2 g / 1 ml) in 1000 ml and spray. This method applies to other dosages and comparative examples). Adjust the dosage based on tree size. Fifteen days after the second spraying, count the number of fruit remaining on the experimental and control papaya trees again to calculate the fruit retention rate.

[0034] Fruit retention rate = number of fruits remaining after 1 month / initial number of fruits remaining × 100%.

[0035] Determination of phytohormone content in papaya fruit The content of phytohormones in papaya fruit was determined by double-antibody one-step sandwich enzyme-linked immunosorbent assay.

[0036] Wash and dry the papaya, carefully cut the middle part of the papaya with a sterilized planer, and accurately weigh 1g of the fresh weight of the papaya samples from the experimental group and the blank group respectively. To reduce experimental errors, one sample should include papayas from different strains at the same concentration; grind the papaya sample evenly in a mortar and pestle, with a homogenate ratio of 10%, that is, 1g of tissue + 9mL of homogenate (pH 7.2-7.4, concentration of 0.01mol / L PBS), and the grinding process must be kept in an ice bath throughout; centrifuge at 3000rpm for 20min, take the supernatant for testing, and freeze the rest for later use; use an enzyme-immune system plant hormone ELISA detection kit to determine its content.

[0037] ELISA kit for determination of plant hormones Sample Addition: Set up blank wells, standard wells, and test sample wells. Accurately add 50 μL of the standard sample to the ELISA-coated plate. First, add 40 μL of sample diluent to the test sample wells, followed by 10 μL of the test sample. Add the sample to the bottom of the plate well, avoiding contact with the well walls. Gently shake to mix. Follow the instructions.

[0038] Draw a standard curve: With the horizontal axis as the standard concentration and the horizontal axis as the OD value, perform linear regression and draw a standard curve as shown below: Figure 2 、 Figure 3 , Gibberellin regression equation Y=0.0023x+0.0125, R 2 =0.9991, abscisic acid regression equation Y=0.0031x+0.0024, R 2 =0.9994.

[0039] Experimental results Compared with the blank control, papaya leaf extract at all concentrations demonstrated a fruit-retaining effect. The medium and low concentration groups significantly increased the fruit retention rate of medicinal papaya, demonstrating a significant fruit-retaining effect, with the medium concentration group showing the best effect. Chlorogenic acid also exhibited a fruit-retaining effect, but this effect was lower than that observed in the medium concentration group of the same chlorogenic acid content. This suggests that papaya leaf extract contains multiple components with fruit-retaining properties.

[0040] Table 2 Effects of different concentrations of papaya leaf water extract on papaya fruit retention rate ( ±s, n=6)

[0041] Note: The data in the table are mean ± SD; * represents P < 0.05 compared with the blank group; ** represents P < 0.01 compared with the blank group, the same below.

[0042] Fruit drop Because the medium concentration has a good effect on fruit retention, the fruit drop of the medium concentration group and the blank group were also investigated. All fallen fruits were randomly collected from 6 papaya trees and weighed. The results showed that the medium concentration can significantly reduce papaya fruit drop compared with the blank group.

[0043] Table 3 Effect of concentration of papaya leaf water extract on papaya fruit drop ( ±s, n=6)

[0044] The fruit drop weight refers to the following: all the fallen fruits under a group of 6 papaya trees were weighed separately, and the average was obtained for statistical analysis.

[0045] like Figure 5 、 Figure 6 As shown in the results, the fruits on papaya trees sprayed with the aqueous extract of papaya leaves were significantly denser. The number of fruits dropped from papaya trees was significantly reduced. The number of fruits dropped from the harvested trees was significantly less than that in the control group.

[0046] Effects on endogenous hormones Compared with the blank control, different concentrations of papaya leaf extract significantly reduced the endogenous abscisic acid (GA) content in papaya and significantly increased the gibberellin (ABA) content, with the medium concentration having the greatest effect on endogenous GA and ABA. The fruit-preserving effect of papaya leaf extract may be related to its ability to reduce endogenous GA and increase gibberellin.

[0047] Abscisic acid is found in high concentrations in wilting leaves and in organs and tissues that are about to fall off. Abscisic acid can promote fruit ripening. However, high levels of abscisic acid can also specifically induce senescence, accelerating fruit and leaf drop.

[0048] Gibberellic acid (GA) plays a significant role in regulating seed germination, stem elongation, flowering induction, stress response, and fruit development. Increasing endogenous GA concentrations promotes fruit development and avoids the need for exogenous GA. This promotes fruit growth and development, enhances disease and pest resistance, and promotes fruit preservation.

[0049] Table 4 Effects of different concentrations of papaya leaf water extract on endogenous ABA and GA in papaya ( ±s, n=6) .

Claims

1. A fruit-preserving plant-derived regulator, characterized in that: The fruit-protecting plant-derived regulator includes papaya leaf water extract.

2. The fruit-protecting plant-derived regulator according to claim 1, characterized in that The papaya leaf water extract is prepared by adding water to crushed papaya leaves and extracting them in a water bath at 80-90° C. for 1-2 hours. The obtained filtrate is concentrated to obtain the papaya leaf water extract.

3. The fruit-protecting plant-derived regulator according to claim 2, characterized in that The papaya leaf water extract includes chlorogenic acid.

4. The fruit-protecting plant-derived regulator according to claim 3, characterized in that The fruit-preserving medicine further comprises an excipient, which comprises a combination of one or more of water, corn starch, microcrystalline cellulose, lactose, sucrose, hydroxypropyl cellulose, alginic acid, gelatin, polyethylene glycol, stearic acid, and magnesium stearate.

5. A papaya fruit-preserving plant-derived regulator, characterized in that: The invention comprises the fruit-protecting plant-derived regulator according to any one of claims 1 to 4.

6. The papaya fruit-preserving plant-derived regulator according to claim 5, characterized in that The dosage of papaya leaf water extract is 0.05-0.2g / mL.

7. The papaya fruit-preserving plant-derived regulator according to claim 6, characterized in that The dosage of papaya leaf water extract is 0.1-0.2 g / mL.

8. The papaya fruit-preserving plant-derived regulator according to claim 5, characterized in that The chlorogenic acid content in the papaya leaf water extract is 0.0625-0.3 g / L, preferably 0.125-0.25 g / L.

9. The papaya fruit-preserving plant-derived regulator according to any one of claims 5 to 8, characterized in that The papaya fruit-preserving plant-derived regulator is sprayed after adding water to achieve the papaya fruit-preserving effect.

10. The papaya fruit-preserving plant-derived regulator according to claim 9, characterized in that The papaya fruit preservation effect is to spray papaya fruit preservation drugs on the young fruits of papaya, once every 7-15 days, and spray 2-4 times.

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