Application of hesperidin in improving cold resistance of citrus

Abstract

The application discloses application of hesperetin in improving cold resistance of citrus and belongs to the field of agricultural biotechnology. Exogenous spraying of a medicine containing hesperetin on citrus plants can effectively relieve physiological damage caused by low-temperature stress, and the performance is that high PSII photochemical efficiency is maintained, malondialdehyde content and electrolyte leakage rate are reduced, and antioxidant enzyme activity is enhanced. The application provides a new technical scheme with clear components, efficient action and environment-friendly for cold resistance of citrus, and has important significance for guaranteeing safe production of the citrus industry.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, and relates to the application of hesperidin in improving the cold resistance of citrus, and a method for improving the cold resistance of citrus by exogenously spraying hesperidin. Background Technology

[0002] Citrus, as my country's largest economic fruit tree, is predominantly warm-climate and intolerant of cold, making most varieties extremely sensitive to low-temperature stress. Historical observation data shows that major citrus-producing areas in my country experience a large-scale freezing disaster approximately every 8-10 years. In recent years, due to the rapid expansion of citrus planting areas and the frequent occurrence of extreme low temperatures, short-term freezing damage events caused by the accumulation of cold air have become increasingly common in some areas, making the upgrading of citrus cold-resistant breeding and cultivation techniques particularly urgent. Unlike annual crops, frost damage to citrus not only leads to reduced yields in the current year but also affects tree vigor and yields in subsequent years, and in severe cases, can even cause the death of the entire tree, resulting in long-term economic losses and seriously hindering the sustainable development of the industry. Therefore, exploring cold-resistant germplasm and developing efficient cold-resistant technologies are crucial to ensuring the safety of the industry.

[0003] Currently, exogenous chemical spraying has become an important technical approach to improve the cold resistance of citrus. Discovering key cold-resistant substances from superior cold-resistant germplasm and developing regulatory technologies that mimic their physiological functions based on this has become a more promising new strategy. For example, Xiao et al. (2024) found that the cold-resistant citrus variety Yichang orange accumulated higher levels of sphingosine and chlorogenic acid than the non-cold-resistant HB pomelo, and that spraying sphingosine and chlorogenic acid could enhance the cold resistance of lemon seedlings. Currently, the national plan for accelerating the construction of a strong agricultural nation (2024-2035) is vigorously promoting the "South-to-North Seed Industry Revitalization" initiative, aiming to breed southern varieties adapted to the northern environment and develop supporting regulatory technologies. Against this backdrop, identifying key cold-resistant metabolites in citrus and developing corresponding exogenous regulatory technologies can not only reveal the cold resistance mechanism of citrus but also provide key technical support for improving the cold resistance of varieties.

[0004] This invention, based on metabolomics technology, successfully screened hesperidin, a cold-resistant variety of Yichang orange, by systematically comparing its metabolic profiles from different regions. The hesperidin is specifically highly accumulated in this cold-resistant variety in northern China. Further exogenous spraying experiments confirmed that hesperidin effectively enhances the cold resistance of cold-sensitive citrus varieties. These findings not only provide crucial material evidence for revealing the cold resistance of Yichang orange, but also lay a solid theoretical and technical foundation for developing novel citrus cold-resistant agents with hesperidin as the core component and for cultivating new cold-resistant varieties. This provides a method with a clearly defined, highly effective, and environmentally friendly approach to solving the problem of citrus chilling injury. Summary of the Invention

[0005] The purpose of this invention is to provide a new application of hesperidin in improving the cold resistance of citrus, as well as an efficient, convenient, and environmentally friendly method for cold resistance of citrus.

[0006] To achieve the above objectives, this invention, based on metabolomics technology, successfully screened hesperidin, a substance that accumulates in high amounts in the "Shaanxi-1" Yichang orange variety from northern China, by systematically comparing the metabolic profiles of Yichang oranges from different regions. Further exogenous spraying experiments confirmed that this substance can effectively enhance the cold resistance of cold-sensitive citrus varieties such as lemons.

[0007] The specific technical solution is as follows: This invention provides an application of hesperidin in improving the cold resistance of citrus. Specifically, an agent containing hesperidin is sprayed exogenously onto citrus plants to alleviate the physiological damage caused by low temperature stress to citrus.

[0008] The present invention also provides a method for improving the cold resistance of citrus, comprising spraying an agent containing hesperidin onto citrus plants exogenously.

[0009] The concentration of hesperidin in the agent can be adjusted within a certain range according to factors such as citrus variety, tree age and stress intensity. Preliminary experiments have confirmed that significant cold resistance can be obtained in the range of 0.8-1.5 mg / mL, with 1.1 mg / mL being the preferred concentration.

[0010] The timing of the spraying includes before or during low-temperature stress, preferably both before and during low-temperature stress.

[0011] Preferably, the agent is an aqueous solution of crude extract of Yichang orange leaves containing hesperidin.

[0012] More preferably, the Yichang orange is a cold-resistant variety from a high-latitude, high-altitude region.

[0013] Preferably, the agent further comprises an agriculturally acceptable adjuvant, such as Tween 20.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to demonstrate that hesperidin can significantly improve the cold resistance of citrus, providing a novel active ingredient for the development of citrus cold-resistant agents. Experiments have shown that exogenous spraying of hesperidin can significantly reduce the damage of low temperatures to citrus leaves, manifested in maintaining a tender, green, and normal morphology, and maintaining a high PSII photochemical efficiency. Fv / Fm ), reduce malondialdehyde (MDA) content, and reduce membrane lipid peroxidation (H2O2 and O2). -This invention provides benefits such as protecting cell membrane integrity (reducing electrolyte leakage). The raw material hesperidin used in this invention is naturally derived, offering environmental advantages. Its crude extract preparation process is simple and cost-effective, making it suitable for agricultural application and crucial for ensuring safe production in the citrus industry. Attached Figure Description

[0015] Figure 1 HPLC chromatograms of leaves from 16 representative Yichang orange trees from different regions show that the "Shaanxi-1" and "Shaanxi-2" samples have a high specific accumulation of hesperidin at a retention time of 76.8 min.

[0016] Figure 2 Effects of hesperidin on leaf phenotype and chlorophyll fluorescence under cold stress in lemons.

[0017] Figure 3 : The maximum photochemical efficiency of hesperidin on PSII in lemon leaves under cold stress ( Fv / Fm The impact of ).

[0018] Figure 4 Effects of hesperidin on electrolyte leakage (EL) rate in lemon leaves under cold stress.

[0019] Figure 5 Effects of hesperidin on malondialdehyde (MDA) content in lemon leaves under cold stress.

[0020] Figure 6 Hesperidin's effect on H2O2 and O2 in lemon leaves under cold stress - Effects of content (DAB and NBT staining).

[0021] Figure 7 The effects of hesperidin and crude extracts from hesperidin on the cold resistance of lemon branches under natural low-temperature conditions. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0023] Example 1. Materials and Methods 1.1 Material Preparation From 2024 to 2025, this study conducted a systematic survey and collection of wild Yichang orange resources in 16 locations across seven provinces (autonomous regions and municipalities): Shaanxi, Hubei, Hunan, Sichuan, Chongqing, Guizhou, and Guangxi, along the Qinling Mountains (southern foothills), Daba Mountains, Wuling Mountains, and Mao'er Mountains. A total of 309 leaf samples were obtained (Table 1). The Shaanxi samples are located at the northernmost point of the distribution area, approximately 33.18°N; the Guangxi samples are located at the southernmost point, approximately 25.89°N; the maximum latitude span of the Yichang orange samples is 7.5°. All materials were immediately frozen in liquid nitrogen after processing and stored in an ultra-low temperature freezer at -80°C for later use.

[0024] Table 1. Information on wild Yichang oranges from 16 regions

[0025] Artificial simulation of chilling injury material: 'Eureka Lemon' seedlings were used as the material. Seeds were treated with 20% NaOH to remove pectin, washed with water, and then germinated in seedling trays. After germination, the seeds were transplanted into a mixed potting mix (peat:perlite:vermiculite = 2:1:1) and cultured for three months in a growth chamber under conditions of 16 h light / 8 h darkness, light intensity of 20000 lx, temperature of 26℃, and relative humidity of 75%. Plants with uniform growth were then selected for further experiments.

[0026] Natural chilling injury test materials: New shoots of coarse lemon trees (about 13-15cm long) with uniform vigor from the Citrus Germplasm Resource Base of Huazhong Agricultural University were used to evaluate the physiological response under natural low temperature conditions.

[0027] 1.2 Reagents and Instruments Main reagents used in the experiment: hesperidin standard (Shanghai Yuanye Biotechnology Co., Ltd.); methanol and acetonitrile (Fisher Scientific, USA); formic acid (Aladdin Reagent Co., Ltd.); Tween-20 (Sinopharm Chemical Reagent Co., Ltd.); propylene glycol detection kit (Nanjing Jiancheng Biotechnology Co., Ltd.); DAB and NBT staining agents (Beijing Cooler Master Technology Co., Ltd.).

[0028] Experimental instruments used: High-performance liquid chromatograph (HPLC, Waters 1525 system, USA); Fluorescence imaging system (Mini-IMAGING-PAM, WALZ, Germany); Ultraviolet spectrophotometer (UV-1800, Shimadzu, Japan); Conductivity meter (SG23, Mettler Toledo, China); High-speed benchtop refrigerated centrifuge (H1850R, Hunan Xiangyi); LED intelligent light incubator (Wuhan Ruihua Instruments); Sample grinder (A11, IKA, Germany); Ultrasonic cleaner (DTC-15J, Dingtai Biochemical Technology); MilliQ ULTRA ultrapure water system (Millipore, USA); Rotary evaporator (Dalong Xingchuang, China).

[0029] 1.3 Experimental Methods 1.3.1 Extraction and Detection of Citrus Metabolites The extraction and detection of citrus metabolites were carried out in accordance with the methods described in the literature (Chen JJ, Zhang HY, Pang YB, Cheng YJ, Deng XX, Xu J. Comparative study of flavonoid production in lycopene-accumulated and blonde-flesh sweet oranges (Citrus sinensis) during fruit development[J]. Food Chem, 2015, 184: 238-246.).

[0030] Citrus metabolite extraction: Weigh 0.1 g of freeze-dried powder from Yichang orange leaves, add 3 mL of 80% methanol aqueous solution for extraction, and use an FS60 ultrasonic cleaner to break up the sample with ultrasound for 1 h at a temperature of 40℃. Invert the sample every 20 min to mix it. Finally, centrifuge the extract at 3500 rpm for 10 min, collect the supernatant, carefully aspirate the supernatant with a syringe, filter it through a 0.22 μm microporous membrane into a 2 mL sample vial for HPLC metabolic detection.

[0031] Crude extract extraction method: Combining the experimental method for extracting citrus metabolites, the filtered supernatant was poured into a 10 ml centrifuge tube and concentrated by rotary evaporation under reduced pressure at 45℃ to obtain crude extract solid.

[0032] Metabolic assay methods and procedures: Qualitative and quantitative analysis was performed using an HPLC system (1525 binary gradient pump, 2998 photodiode array detector, and 2707 autosampler) (Waters Co., Milford, MA, USA) combined with a standard curve prepared using commercial hesperidin standards. Flavonoids were separated using a C18 Hypersil GOLD column (4.6 × 250 mm, 5 μm; Thermo Scientific, Waltham, MA). The gradient elution system and instrument conditions were set as follows: solvent system: water (A, 0.15% formic acid), acetonitrile (B, 0.15% formic acid); gradient program: 0-10 min 10-15% B, 10-20 min 15-20% B, 20-35 min 20-25% B, 35-45 min 25-30% B, 45-55 min 30-35% B, 55-60 min 35-45% B, 60-65 min 45-55% B, 65-70 min 55-75% B, 70-75 min 75% B, 75-78 min 75-10% B, 78-80 min 10% B. The flow rate was 1 mL / min, the injection volume was 10 μL, and the column temperature was 25 ℃. Chromatographic data were analyzed using a Waters Breeze 2 HPLC system.

[0033] 1.3.2 Artificial Climate Chamber Simulation of Cold Damage Experiment Experimental designs in 1.3.2 and 1.3.3 were based on the methods described in the literature (Xiao P, Qu J, Wang Y, Fang T, XiaoW, Wang YL, Zhang Y, Khan M, Chen QY, Xu XY, Li CL, Liu JH. Transcriptome and metabolome atlas reveals contributions of sphingosine and chlorogenic acid tocold tolerance in Citrus[J]. Plant Physiol. 2024, 96(1): 634-650.).

[0034] Experimental groups and treatments: As shown in Table 2, the normal temperature treatment group was sprayed with a 10% Tween 20 aqueous solution; the low temperature treatment group was sprayed with a 10% Tween 20 aqueous solution; hesperidin treatment 1: 1.1 mg / mL hesperidin was sprayed before the cold damage, and a 10% Tween 20 aqueous solution was sprayed during the cold damage; hesperidin treatment 2: 1.1 mg / mL hesperidin was sprayed; no external spraying was performed after the cold damage. Hesperidin was dissolved in a 10% Tween 20 aqueous solution and prepared to a concentration of 1.1 mg / mL for spraying treatment.

[0035] Table 2. Reagent settings for each group in the artificial climate chamber simulation of cold damage experiment.

[0036] Table 3 Temperature settings for each group in the artificial climate chamber simulation of cold damage experiment

[0037] Spraying method: Spray twice daily, until the leaves are wet to the point of dripping. As shown in Table 3, the normal temperature group was kept in the artificial climate chamber at 26℃ for 6 days; the other three treatments were kept in the artificial climate chamber at 26℃ for 3 days before the cold injury, and then transferred to the LED intelligent light incubator at 4℃ for 3 days during the cold injury; the light conditions were kept consistent with the artificial climate chamber; after the cold injury, during the sample collection period, no external spraying was applied, and each group was transferred to the normal temperature condition of 26℃ for sample collection.

[0038] Physiological index determination: (1) After the leaves were dark adapted for 30 min, the maximum photochemical efficiency of PSII was determined using a modulated fluorescence imaging system. Fv / Fm) (2) Take leaves and break them into leaf discs. Place them in deionized water and shake slowly at room temperature for 45 min. Measure the conductivity (C1). After cooling in a boiling water bath for 10 min, measure the conductivity (C2). EL (%) = (C1-CK1) / (C2-CK2)×100% to complete the detection of electrolyte leakage (EL) in lemon leaves after stress. (3) Measure the malondialdehyde (MDA) content according to the instructions of Nanjing Jiancheng reagent kit. (4) Immerse the leaves in freshly prepared DAB (1 mg / mL, pH 3.8) or NBT (1 mg / mL) staining solution and incubate at room temperature in the dark until color development. Then decolorize with 80% ethanol in a 65℃ water bath and take photos to record the results. That is, measure the H2O2 and O2 content. • Histochemical staining.

[0039] 1.3.3 Natural Low Temperature Test Experimental groups and treatments: As shown in Table 4, the blank group received no external treatment; the solvent spraying group received a 10% Tween 20 aqueous solution; the hesperidin spraying group received 1.1 mg / mL hesperidin; and the crude extract spraying group received 220 mg / mL of crude extract from 'SX1' Yichang orange leaves (at this concentration, the crude extract contained 1.1 mg / mL of hesperidin). Both hesperidin and the crude extract from 'SX1' Yichang orange leaves were dissolved in a 10% Tween 20 aqueous solution to prepare the concentrations set for the experimental groups, namely 1.1 mg / mL and 220 mg / mL, respectively, for spraying.

[0040] Table 4. Reagent settings for each group in the natural low temperature test.

[0041] Table 5 Temperature settings for each group in the natural low temperature test

[0042] Spraying method: Spray twice daily, ensuring the leaves are moistened to the point of dripping. Since the freezing weather was accompanied by rain and snow, the spraying treatment was conducted before the freezing damage occurred. As shown in Table 5, before the freezing damage, January 12–14 were sunny with outdoor temperatures ranging from 10 to 20℃; during the freezing damage, January 15–17 were rainy and snowy, with outdoor temperatures dropping to -5 to -1℃, accompanied by freezing rain and heavy snow; after the freezing damage, January 18–20 were cloudy to overcast with outdoor temperatures rising to 5 to 8℃. The experiment lasted a total of 9 days, with the low-temperature stress phase (-5 to -1℃) lasting for 3 days, meeting the requirements for natural low-temperature cold damage stress experiments.

[0043] Photographic observation: Morphological observations were conducted daily for each experimental group, especially after the low-temperature freezing injury treatment was completed, until significant phenotypic differences appeared between the groups; phenotypic images of each group were photographed and recorded daily, and it was ensured that the sample number corresponded one-to-one with the image label, and that the results were consistent.

[0044] 2. Experimental Results 2.1 Hesperidin in "Shaanxi-1" Yichang oranges accumulates at a particularly high level A systematic comparative analysis of HPLC chromatograms of Yichang orange leaves from 16 regions revealed that samples from the two northernmost regions, "Shaanxi-1" and "Shaanxi-2," showed a particularly high accumulation of hesperidin at a retention time of 76.8 min. Figure 1 Among them, the content of hesperidin in single plants 'SX-1' and 'SX-2' was as high as 5.93±0.07 (mg / g DW) and 3.78±0.19 (mg / g DW), respectively, while it was not detected in single plants in other regions, suggesting that hesperidin may be one of the marker metabolites affecting the ecological adaptability of Yichang oranges in the north and south.

[0045] 2.2 Hesperidin can significantly improve the cold resistance of lemons, and its effect is dose-dependent. 2.2.1 Evaluation of the cold resistance of hesperidin in artificially simulated cold damage The aforementioned metabolic analysis indicates that hesperidin accumulation in the Citrus genus exhibits significant germplasm specificity, with particularly high accumulation in the 'Shaanxi-1' Yichang orange variety. The 'Shaanxi-1' Yichang orange was collected from a high-latitude, high-altitude region on the southern slopes of the Qinling Mountains, an area frequently experiencing prolonged sub-zero temperatures in winter. However, this community has managed to survive and propagate stably through seedlings. The high accumulation of hesperidin in the cold-resistant 'Shaanxi-1' germplasm strongly suggests that hesperidin may have participated in the low-temperature adaptation process of this germplasm.

[0046] To investigate the effects of hesperidin on the cold resistance of citrus and its mechanism of action, four treatment groups were set up using Eureka lemon seedlings as material. Samples were taken before (day 3 of the experiment), during (day 5 of the experiment), and after (day 7 of the experiment) cold damage to measure relevant physiological indicators. The results are as follows: Phenotypic observation and chlorophyll fluorescence imaging analysis revealed that the leaves of the room temperature treatment group remained a bright green throughout the experiment. After the cold damage, the leaves of the low temperature treatment group showed severe wilting, and the chlorophyll fluorescence images showed large areas of white spots indicating leakage. The leaves of the orange peel oil treatment group 1 showed slight wilting, and some leakage areas were visible in the fluorescence images. The leaves of the orange peel oil treatment group 2 remained in a normal state, with a bright green color, and the fluorescence images showed no significant difference from those of the room temperature treatment group. Figure 2 ).

[0047] chlorophyll fluorescence parameters Fv / Fm Analysis revealed that before the frost damage, each treatment group... Fv / Fm The values ​​were all between 0.79 and 0.81, with no significant differences between groups. After the frost damage, the low-temperature treatment group... Fv / Fm The mean value decreased to 0.699, while in group 1 treated with hesperidin it decreased to 0.736, and in group 2 it remained at 0.791, significantly higher than the low-temperature treatment group. Three days after the cold injury, the low-temperature treatment group... Fv / Fm The concentration of hesperidin further decreased to 0.428, with 0.645 in group 1 and 0.751 in group 2, significantly higher than that in the low-temperature treatment group. Figure 3 ).

[0048] Analysis of leaf electrolyte leakage rate (EL) revealed that the electrolyte leakage rate in the normal temperature treatment group was 14.81%. After frost damage, the low temperature treatment group had the highest electrolyte leakage rate, reaching 25.83%. The leakage rate in hesperidin treatment 1 was 22.29%, and in hesperidin treatment 2 it was 18.88%. Figure 4 ).

[0049] Data on malondialdehyde (MDA) content after stress showed that the low-temperature treatment group had the highest MDA content, reaching 4.12 nmol / mg prot (FW, the same below); hesperidin group 1 had 2.07 nmol / mg prot; and hesperidin treatment group 2 had 1.16 nmol / mg prot, significantly lower than the low-temperature treatment group, and not significantly different from the normal temperature treatment group (1.02 nmol / mg prot). Figure 5 ).

[0050] H2O2 and O2 were detected using POD and SOD staining methods, respectively. • The lighter the leaf color after staining with POD and SOD, the higher the levels of H2O2 and O2. • The lower the content of these substances, the higher the activity of POD and SOD. Both of these indicators can be used to evaluate the degree of leaf damage under stress. It was found that compared to the low-temperature treatment group, the leaves in the two groups treated with hesperidin showed lighter staining, and hesperidin treatment 2 was more effective than hesperidin treatment 1. This indicates that after hesperidin treatment, the levels of H2O2 and O2 in the leaves decreased. • The content is relatively low, meaning that the activity of POD and SOD is stronger, the antioxidant capacity is improved, and the resistance to cold damage is enhanced. Figure 6 ).

[0051] Artificial cold resistance experiments showed that exogenous application of hesperidin significantly reduced the damage caused by low temperature to lemon leaves, manifested in maintaining a tender green morphology, maintaining high PSII photochemical efficiency, low MDA content, and reducing membrane lipid peroxidation (H2O2 and O2). • It also protects cell membrane integrity (EL). Among them, the cold resistance effect of hesperidin treatment 2 was significantly higher than that of hesperidin treatment 1. That is, the cold resistance effect of continuous spraying of hesperidin during the low temperature stress period (full treatment) is better than that of pretreatment only before low temperature stress. It can be seen that hesperidin can improve the physiological state of lemon leaves under cold stress, and the improvement effect is positively correlated with the amount of hesperidin sprayed.

[0052] 2.2.2 Experiment on freezing damage treatment in natural production areas To further investigate the cold resistance effects of hesperidin and crude extracts from 'SX1' Yichang orange leaves under natural low-temperature conditions, a natural low-temperature freezing injury experiment was conducted at the National Citrus Breeding Center of Huazhong Agricultural University from January 12 to 20, 2026.

[0053] like Figure 7The lemon branches were shown as follows: before frost damage (day 3), during frost damage (day 6), and after frost damage (day 9). Results showed no significant differences in leaf morphology among the treatment groups before frost damage. During frost damage, the leaves in the control group and the solvent-sprayed group showed obvious water-soaked appearance; the leaves in the hesperidin-sprayed group and the crude extract-sprayed group showed less water-soaking. After frost damage, the water-soaking in the control group and the solvent-sprayed group persisted and worsened, with leaves showing curling and browning, indicating that low temperature had severely damaged the integrity of the leaf cell membrane system. The hesperidin-sprayed group and the crude extract-sprayed group did not show obvious curling or browning; although some leaves showed slight water-soaking, they remained relatively green.

[0054] Further comparison revealed that the crude extract spraying group exhibited better cold resistance than the hesperidin spraying group. Before frost damage, there was no difference in branch condition among the groups; during frost damage, the former group showed less water immersion in its leaves and remained upright throughout; the latter group showed slight wilting of the two young leaves at the tip during frost damage, but recovered somewhat afterward. These results indicate that exogenous spraying of 1.1 mg / mL hesperidin can alleviate the damage to lemon leaves caused by low temperatures, consistent with the results of artificially simulated cold damage treatment. This experiment also found that the crude extract's cold resistance was superior to that of hesperidin alone, which may be a synergistic effect of hesperidin and other cold-resistant components in the crude extract. This study will provide a scientific basis for the development and utilization of hesperidin and the "Shaanxi-1" Yichang orange in terms of cold resistance.

Claims

1. The application of hesperidin in the preparation of agents for improving the cold resistance of citrus fruits, characterized in that: Exogenous spraying of agents containing hesperidin onto citrus plants can alleviate the physiological damage caused by low-temperature stress to citrus.

2. A method for improving the cold resistance of citrus fruits, characterized in that: The agent containing hesperidin was sprayed exogenously onto the citrus plants.

3. The method according to claim 2, characterized in that: The concentration of hesperidin in the preparation is 0.8-1.5 mg / mL.

4. The method according to claim 2, characterized in that: The spraying should be performed before the onset of low temperature stress.

5. The method according to claim 2, characterized in that: The timing of the spraying is before and during low-temperature stress.

6. The method according to claim 2, characterized in that: The agent is an aqueous solution of crude extract from Yichang orange leaves containing hesperidin.

7. The method according to claim 6, characterized in that: The Yichang oranges mentioned are cold-resistant varieties from high-latitude, high-altitude regions.

8. The method according to claim 2, characterized in that: The agent also contains agriculturally acceptable adjuvants.

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