Preparation method of oyster peptide and application of oyster peptide in improvement of male reproductive function

Abstract

The invention relates to the technical field of oyster peptides, in particular to a preparation method of oyster peptides and application of the oyster peptides in improvement of male reproductive functions. Oyster peptide is used as a raw material, oleanolic acid type saponin grafting is carried out on the oyster peptide, and the oyster peptide is compounded with chitosan to prepare the ternary complex emulsion. The emulsion can form stable microscopic particles and has stable rheological properties. Simulated digestion experiments show that the emulsion has a slow release effect on digestion of oyster peptide, and at least can reduce the digestion proportion of the oyster peptide in the oral cavity. Besides, when the emulsion and the oyster peptide are used for intervening the rats with the nephrotic syndrome, the nephrotic syndrome can be effectively relieved, meanwhile, the effect of promoting the male sexual function of the rats is achieved, and the oyster peptide has the application prospect of modifying and improving the male reproductive function.

Description

Technical Field

[0001] The present application relates to the field of oyster peptide technology, and specifically to a method for preparing oyster peptides and their application in improving male reproductive function. Background Art

[0002] Oysters are known as "sea milk". They are fresh and smooth, and contain a lot of protein. They are warm-water bivalve molluscs belonging to the class Bivalve. They are one of the four major shellfishes cultured in my country and the first batch of seafood approved by the former Ministry of Health of China that can be used as both food and medicine with health effects. The completeness and quality ratio of essential amino acids in oysters are higher than those in milk and human milk. They also contain taurine, ornithine and many non-essential amino acids with extremely high biological value. They are high-quality raw materials for the development of functional peptides.

[0003] Scientific research results in recent years have shown that oysters contain a large amount of amino acids, B vitamins, polysaccharides, functional peptides, and trace elements and minerals such as Fe, Zn, and Se. The protein content in oysters is 39.1% to 53.1%, which is an excellent source of protein. At the same time, oyster protein can be enzymatically cleaved into a large number of polypeptides with high biological activity.

[0004] Since oyster protein active peptides (also known as oyster peptides) are naturally extracted small molecule protein peptides, they are easy for the human body to absorb and store, and have stable and diverse active ingredients. Many studies have shown that oyster peptides have physiological effects such as inhibiting tumors, anti-oxidation, lowering blood sugar, slowing down aging, and reducing angiotensin converting enzyme activity. Summary of the invention

[0005] The present application uses oyster peptide as a raw material, grafts oleanolic acid saponin, and prepares a ternary complex emulsion with chitosan. The emulsion can form stable microscopic particles and has stable rheological properties. Through simulated digestion experiments, it was found that the emulsion has a sustained release effect on the digestion of oyster peptides, at least reducing its digestion ratio in the mouth. In addition, the use of the emulsion and the oyster peptide to intervene in nephrotic syndrome rats can effectively alleviate their nephrotic syndrome, and at the same time promote the male sexual function of rats, and has the application prospect of modifying and improving male reproductive function.

[0006] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0007] In the first aspect, the embodiment discloses a method for preparing a complex of oyster peptide and oleanolic acid saponin, comprising: subjecting the oyster peptide shown in SEQ ID NO:1 to low-temperature plasma treatment; configuring the oyster peptide after low-temperature plasma treatment into an aqueous solution and keeping it at 4°C overnight; reacting the oyster peptide aqueous solution overnight with the oleanolic acid saponin solution for 24 hours; dialyzing the reaction solution against running water at 4°C for 48 hours, and freeze-drying it in a vacuum.

[0008] In the second aspect, the embodiment discloses a method for preparing an emulsion of a ternary complex, comprising: mixing the complex obtained in the first aspect, chitosan, and lemon essential oil in deionized water; after stirring, high-speed shearing and high-pressure homogenization are performed to obtain an emulsion.

[0009] In a third aspect, the embodiment discloses the application of the emulsion of the ternary complex. The application is selected from: promoting the secretion or expression of gonadotropin-releasing hormone; promoting the secretion or expression of serum testosterone; improving the impaired function of the hypothalamus-pituitary-gonadal axis; reducing the secretion or expression of serum urea nitrogen; reducing the secretion or expression of blood creatinine; reducing the total cholesterol content; reducing the triglyceride content; increasing plasma albumin; improving nephrotic syndrome; and improving sexual function. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Emulsion micrograph of the ternary complex provided for the example.

[0011] Figure 2 This is a curve showing the change in apparent viscosity of the emulsion of the ternary composite provided in the example.

[0012] Figure 3 The creaming index of the emulsion of the ternary complex provided in the examples.

[0013] Figure 4 The digestibility of oyster peptides in the oral cavity, stomach and intestinal simulated digestion provided in the examples.

[0014] Figure 5 The FSH results in the blood of each group of rats provided in the examples.

[0015] Figure 6 The LH results in the blood of each group of rats provided in the examples.

[0016] Figure 7 The results of GnRH in the blood of each group of rats provided in the examples.

[0017] Figure 8 The results of T in the blood of each group of rats provided in the examples.

[0018] Fig. 9 The BUN results in the blood of each group of rats provided in the examples.

[0019] Fig.10 The SCr results in the blood of each group of rats provided in the examples.

[0020] Fig.11 The TC results in the blood of each group of rats provided in the examples.

[0021] Fig.12 The TG results in the blood of each group of rats provided in the examples.

[0022] Fig.13 The UTP results in the blood of each group of rats provided in the examples.

[0023] Fig.14 The results of Alb in the blood of each group of rats provided in the examples. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail in conjunction with the examples below. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0025] The present application uses oyster peptide as a raw material, grafts oleanolic acid saponin, and prepares a ternary complex emulsion with chitosan. The emulsion can form stable microscopic particles and has stable rheological properties. Through simulated digestion experiments, it was found that the emulsion has a sustained release effect on the digestion of oyster peptides, at least reducing its digestion ratio in the mouth. In addition, the use of the emulsion and the oyster peptide to intervene in nephrotic syndrome rats can effectively alleviate their nephrotic syndrome, and at the same time promote the male sexual function of rats, and has the application prospect of modifying and improving male reproductive function.

[0026] The embodiment discloses a method for preparing a complex of oyster peptide and oleanolic acid type saponin. The method comprises: subjecting the oyster peptide shown in SEQ ID NO: 1 to low temperature plasma treatment; configuring the oyster peptide treated with low temperature plasma into an aqueous solution and keeping it at 4°C overnight; reacting the oyster peptide aqueous solution overnight with the oleanolic acid type saponin solution for 24 hours; dialyzing the reaction solution with 4°C running water for 48 hours, and vacuum freeze drying.

[0027] In some embodiments, the conditions of the low temperature plasma treatment include: 350w treatment for 5min, and an air intake volume of 120mL / min.

[0028] In some embodiments, the aqueous solution concentration of the oyster peptide is 0.2 g / mL.

[0029] In some embodiments, the dialysis has a molecular weight cut-off of 1000 Da.

[0030] The embodiment discloses a method for preparing an emulsion of a ternary complex, which comprises: mixing the complex obtained in the above embodiment, chitosan, and lemon essential oil in deionized water; stirring, high-speed shearing, and high-pressure homogenization to obtain an emulsion.

[0031] In some embodiments, the compound is added at a ratio of 1.5% (w / v), and the lemon essential oil is added at a ratio of 2.3% (w / v).

[0032] The addition ratio of the chitosan is 1% (w / v).

[0033] In some embodiments, the homogenization pressure is 45 MPa.

[0034] The embodiment discloses the application of the emulsion obtained in the above embodiment, and the application is selected from: promoting the secretion or expression of gonadotropin-releasing hormone; promoting the secretion or expression of serum testosterone; improving the impaired function of the hypothalamus-pituitary-gonadal axis; reducing the secretion or expression of serum urea nitrogen; reducing the secretion or expression of blood creatinine; reducing the total cholesterol content; reducing the triglyceride content; increasing plasma albumin; improving nephrotic syndrome; and improving at least one of sexual function.

[0035] The present application will be described in more detail below in conjunction with more specific embodiments, but this does not constitute a limitation to the present application.

[0036] Example 1: Complex of oyster peptide and oleanolic acid saponin

[0037] 1. Preparation of the complex of oyster peptide and oleanolic acid saponin

[0038] After spreading the oyster peptide (HLRQEEKEEVTVGSLK, 1735.99, synthesized by Shanghai Taopu Technology Co., Ltd., purity 99%) on a culture dish, the culture dish was placed in a low-temperature plasma treatment instrument (SY-DT02S, Suzhou Ops Plasma Technology Co., Ltd.) and treated at 350w for 5min with an air intake of 120mL / min.

[0039] The oyster peptide treated with low-temperature plasma was prepared into a 0.2 g / mL solution and incubated at 4°C overnight.

[0040] On the second day, 0.04 g / mL oleanolic acid saponin solution (oleanolic acid 3-O-β-D-glucopyranosyl (1→2)-α-L-arabinopyranoside, 750.955, Suzhou Yihua Biopharmaceutical Technology Co., Ltd.) was added. After reacting for 24 hours, the mixture was dialyzed in running water at 4°C for 48 hours using a dialysis bag with a molecular weight cutoff of 1000 Da to ensure that there was no free oleanolic acid saponin in the final dialysate. The solution was vacuum freeze-dried to obtain a complex powder of oyster peptide and oleanolic acid saponin, which was stored at 25°C in the dark for later use.

[0041] 2. Determination of grafting rate

[0042] Add 2.5 mL of 10% (v / v) Folin phenol reagent to 0.5 mL of pre-dialysis solution or post-dialysis solution, mix well and react for 5 min in dark conditions, then add 2 mL of 7.5% (w / v) Na2CO3 solution, react for 2 h in dark conditions and measure the absorbance at 760 nm. Prepare a standard curve with gallic acid to calculate the free oleanolic acid saponin content in the pre-dialysis solution or post-dialysis solution. Grafting efficiency (%) = 100% × C / C0; wherein: C is the polyphenol content in the post-dialysis solution; C0 is the polyphenol content in the pre-dialysis solution. The results show that the grafting rate is 62%.

[0043] Example 2: Emulsion of ternary complex

[0044] 1. Emulsion of ternary complex

[0045] 0.8 g of a complex of oyster peptide and oleanolic acid saponin, 1% (w / v) final concentration of chitosan (Aladdin, deamidation degree 85%), and lemon essential oil (Shanghai McLean) were dissolved and mixed in proportion and added to deionized water to prepare a 100 mL solution. After magnetic stirring at 25°C for 30 min, the solution was sheared using a high-speed shearing machine and then homogenized using a high-pressure homogenizer to obtain an emulsion.

[0046] The optimal conditions for preparing a stable emulsion are 1.5% (w / v) of the complex of oyster peptide and oleanolic acid saponin, 2.3% (w / v) of lemon essential oil, 5 homogenizations, and a homogenization pressure of 45 MPa. Under these conditions, the prepared emulsion has the smallest particle size and the largest absolute value of Zeta potential, exhibits good stability and storage properties, and has no stratification phenomenon, exhibits moderate pseudoplastic fluid characteristics, and is suitable for further applications.

[0047] 2. Optical microscope observation

[0048] The microstructure of the Pickering emulsion stabilized by WPI-EGCG was observed using an optical microscope. The ambient temperature was 25°C, the magnification of the microscope eyepiece was 10 times, and the magnification of the objective lens was 40 times. Figure 1As shown, the droplets are uniform in size and regular in shape.

[0049] 3. Determination of particle size and Zeta potential

[0050] The particle size and Zeta potential of the emulsion system were measured using a Malvern Nano ZS laser particle size analyzer. The average value was taken after three parallel measurements.

[0051] like Figure 1 As shown, the droplet size reaches a minimum value of 172.91±6.54nm, and the absolute value of the Zeta potential reaches a maximum value of 26.32mV.

[0052] 4. Determination of rheological properties of emulsion

[0053] The rheological properties of the Pickering emulsion stabilized by WPI-EGCG were determined using a DHR-1 rheometer with a disc fixture width of 40 mm, a thickness of 1 mm, and a set temperature of 25 ° C. Static shear flow tests were performed to measure the change curve of the apparent viscosity (η) in the range of 0.01 to 1000 s-1.

[0054] like Figure 2 As shown in the figure, under different homogenization pressures, the apparent viscosity of the emulsion of the ternary complex shows a downward trend, indicating that the sample has shear thinning behavior and the emulsion structure is damaged by shear. However, at low shear rate, the apparent viscosity of the emulsion with a homogenization pressure of 45MPa is higher than that of other groups, which shows that this homogenization pressure is most conducive to the formation of an emulsion of a ternary complex with a stable emulsion structure, and makes the particle size of the emulsion smallest and the stability enhanced.

[0055] 5. Storage stability study

[0056] Creaming index is an indicator of the degree of droplet aggregation in the emulsion, and the creaming index is positively correlated with the degree of droplet aggregation. Take 30mL of freshly prepared emulsion in a glass bottle, seal it and store it at 4℃ and 25℃ for one month. Observe the changes in the emulsion every 5 days and calculate the creaming index (Creaming Index, CI). CI = 100% × hs / ht, where hs is the volume of the clear liquid / cm3, and ht is the total volume of the emulsion / cm 3 .

[0057] like Figure 3 As shown, the milking index was greater than zero only on the 20th day, which further indicates that the emulsion storage stability of the ternary complex provided by the present invention is better.

[0058] Example 3: Simulated digestion experiment

[0059] 1. Prepare sample solution

[0060] The emulsion of the ternary complex provided in the above examples, and the aqueous solution containing 0.2 g / mL oyster peptide.

[0061] 2. Simulate oral stage

[0062] 3.5 mL of simulated saliva (SSF, CZ0241, Regen Biotech) was mixed with 5 mL of the emulsion sample and shaken at 120 rpm in a 37°C water bath for 5 min to simulate the oral digestion stage. The resulting solution was reserved for the next stage.

[0063] 3. Simulated gastric stage

[0064] 7.5 mL of simulated gastric fluid (SGF, R30388, source leaf) was mixed with 10 mL of the emulsion after oral digestion, and the mixture was shaken at 120 rpm in a 37°C water bath for 2 h. The resulting solution was reserved for the next stage.

[0065] 4. Simulated intestinal stage

[0066] Take 20 mL of simulated gastric digestive fluid and 11 mL of simulated intestinal fluid (SIF, PH1841, PHYGENE), and shake them continuously at 120 rpm in a 37°C water bath for 2 h.

[0067] 5. Detection of oyster peptide consumption rate in consumption fluid at different stages

[0068] Oral digestive juice, gastric digestive juice and intestinal digestive juice were collected separately, centrifuged at 3000rpm and 4℃ for 10min, the supernatant was concentrated by ultrafiltration using a 3kD ultrafiltration centrifuge tube, the filtrate was collected, and the oyster peptide content was determined by BCA protein assay. The digestibility of oyster peptides at each stage was calculated based on the volume. The digestibility was the percentage of the mass difference of oyster peptides before and after digestion to the mass of oyster peptides before consumption.

[0069] like Figure 4 As shown, the digestibility of oyster peptides in the sample liquid of the experimental group in the oral stage was significantly lower than that in the control group, and digestion only began in the gastrointestinal consumption stage, indicating that the emulsion of the ternary complex provided by the present invention can improve the digestion of oyster peptides in the gastrointestinal tract and is beneficial to biological absorption.

[0070] Embodiment 4:

[0071] Nephrotic syndrome is a disease characterized by glomerular lesions. It may be caused by a variety of causes, including primary glomerular diseases and secondary diseases. Traditional Chinese medicine believes that the kidney is the innate foundation of the human body and an important organ of the reproductive system. The kidney is responsible for reproduction, growth, and development. The kidney includes two aspects: kidney yang and kidney yin. Kidney yang represents the properties of yang, warmth, and activity, while kidney yin represents the properties of yin essence, nourishment, and moisture. The normal function of the reproductive system is closely related to the health of the kidneys.

[0072] Serum urea nitrogen, serum creatinine, total cholesterol, triglycerides, plasma albumin and 24h urine protein quantification will change accordingly in patients with nephrotic syndrome. Serum urea nitrogen and serum creatinine are commonly used blood renal function indicators. In nephrotic syndrome, the glomerular filtration membrane is damaged, resulting in decreased renal filtration function, and serum urea nitrogen and serum creatinine levels usually increase, reflecting a decrease in glomerular filtration rate. The kidneys of patients with nephrotic syndrome have a weakened ability to handle and excrete lipids, resulting in increased lipid content in the blood, often accompanied by abnormal lipid metabolism, manifested as increased total cholesterol and triglycerides. Due to the damage of the glomerular filtration membrane, large molecular proteins such as albumin will be lost from the urine, resulting in a decrease in the albumin level in the plasma. 24h urine protein quantification is an indicator for assessing renal protein loss. Due to the damage of the glomerular filtration membrane, a large amount of protein will be lost from the urine, resulting in an increase in 24h urine protein quantification. Changes in these biochemical indicators can provide important information about renal function and the severity of nephrotic syndrome.

[0073] The present application constructs nephrotic syndrome rats, intervenes with the ternary complex emulsion prepared in the above example, and uses an oyster peptide aqueous solution as a control to evaluate the technical advantages of the ternary complex emulsion prepared by the present application over the oyster peptide.

[0074] 1. Prepare sample solution

[0075] The emulsion of the ternary complex provided in the above examples, and the aqueous solution containing 0.2 g / mL oyster peptide.

[0076] 2. Group experiment

[0077] Male SD rats were kept in a clean-grade environment with humidity maintained at 50%, temperature controlled at 23°C, 12h alternating lighting system, free access to food and water.

[0078] 3. Modeling and grouping

[0079] All male SD rats that had undergone 1 week of adaptive feeding were randomly divided into:

[0080] Blank group: Rats received 1 mL of distilled water by gavage daily.

[0081] Model group: On the first and eighth days of the experiment, rats received tail vein injection of doxorubicin at a dose of 4.0 mg / kg and 3.5 mg / kg, respectively. To ensure that the nephrotic syndrome model established in SD rats has a low mortality rate and relative stability, and received oral gavage of 1 mL of distilled water daily, with 10 rats in each group. Doxorubicin was selected because of its high toxicity, narrow safety window, and wide range of side effects.

[0082] Experimental group: On the first and eighth days of the experiment, rats were injected with doxorubicin via the tail vein at doses of 4 mg / kg and 3.5 mg / kg, respectively, to construct a nephrotic syndrome model, and simultaneously received oral gavage of 40 mg / kg of the sample solution (emulsion of the ternary complex) at intervals daily.

[0083] Control group: On the first and eighth days of the experiment, rats were injected with doxorubicin via the tail vein at doses of 4 mg / kg and 3.5 mg / kg, respectively, to construct a nephrotic syndrome model, and at the same time received oral gavage of 40 mg / kg sample solution (containing 0.2 g / mL oyster peptide aqueous solution) at daily intervals.

[0084] 4. Sample collection

[0085] After the experiment was completed, all rats were sampled after 30 days of intervention. Before sampling, the rats were placed in metabolic cages for fasting, but water was retained, and their 24-hour urine was collected at the same time, and the urine volume was recorded in detail. The collected urine specimens will be used for quantitative detection of 24-hour urine protein.

[0086] After the urine specimens are collected, 4 to 8 mL of blood is drawn through the abdominal aorta. The drawn blood is left at room temperature for 1 hour. After the blood is completely coagulated, it is centrifuged at 4000 r / min for 10 minutes. The supernatant is divided into EP tubes and stored in a -80°C refrigerator. These blood samples will be used for subsequent ELISA and biochemical tests.

[0087] Subsequently, 0.9% saline was injected through the left ventricle to flush the organs. After the kidneys became lighter in color, the kidney tissue was removed and the wet weight was measured. The obtained kidney tissue was fixed in 4% paraformaldehyde for subsequent HE staining and Masson staining. In addition, the testicles were removed and fixed in 4% paraformaldehyde for HE staining.

[0088] 5. Determination of the levels of sex hormones FSH, LH, GnRH, and T

[0089] Immunohistochemistry was used to detect the levels of FSH (serum follicle-stimulating hormone), LH (luteinizing hormone), GnRH (gonadotropin-releasing hormone), and T (serum testosterone) in the abdominal aorta blood of rats in each experimental group. In the male reproductive system, hormones such as FSH, LH, GnRH, and T interact with each other to form a fine regulatory mechanism to ensure the normal operation of male reproductive function. When the function of the hypothalamus-pituitary-gonadal axis is impaired, the release of GnRH will be reduced, which in turn affects the release of LH and FSH. The previously frozen serum was thawed, and then the ELISA kit was used to accurately determine the concentration levels of FSH, LH, GnRH, and T in the serum according to the strict operating procedures of the relevant instructions.

[0090] like Figures 5 to 8 As shown in the figure, the concentration levels of FSH and LH in the serum of rats in the model group were significantly higher than those in the blank group, and the concentration levels of GnRH and T were significantly lower than those in the blank group, indicating that the nephrotic syndrome model was successfully established.

[0091] The concentration levels of FSH and LH in the serum of rats in the experimental group and the control group were significantly lower than those in the model group, and the concentration levels of GnRH and T were significantly higher than those in the model group, indicating that the sample solutions provided by the experimental group and the control group respectively had an intervention effect on rats with nephrotic syndrome. However, the concentration levels of FSH and LH in the serum of rats in the experimental group were lower than those in the control group, and the concentration levels of GnRH and T were significantly higher than those in the control group, indicating that the intervention effect of the ternary complex emulsion provided by the experimental group on the various sex hormones in the serum of rats with nephrotic syndrome was stronger than that of the aqueous solution containing 0.2g / mL oyster peptide.

[0092] 6. Blood biochemical index detection

[0093] Serum and urine samples were collected separately and placed in a fully automatic biochemical instrument. Serum urea nitrogen (BUN), serum creatinine (SCr), total cholesterol (TC), triglycerides (TG), plasma albumin (Alb) and 24-hour urine protein (UTP) were detected strictly according to the operating instructions.

[0094] like Figures 9 to 14 As shown in the figure, the concentration levels of BUN, SCr, TC, TG and UTP in the serum of the model group rats were significantly higher than those in the blank group, and the concentration level of Alb was significantly lower than that in the blank group, indicating that the nephrotic syndrome model was successfully established. The concentration levels of BUN, SCr, TC, TG and UTP in the serum of the experimental group rats were significantly lower than those in the model group, and the concentration level of Alb was significantly higher than that in the blank group, indicating that the sample solution provided by the experimental group had an intervention effect on nephrotic syndrome rats. However, only the concentration levels of BUN and Alb in the serum of the control group rats were significantly different from those in the model group, and there were no significant differences in other biochemical indicators, which also indicated that the sample solution provided by the control group had limited intervention effect on the serum biochemistry of nephrotic syndrome rats.

[0095] In summary, the present application obtains a complex by grafting oleanolic acid saponin with oyster peptides, and then prepares an emulsion of a ternary complex with chitosan, which can not only improve the sex hormone level of rats with nephrotic syndrome, but also improve the application prospects of male reproductive function. In addition, the emulsion of the ternary complex can also promote the improvement of serum biochemical indicators of rats with nephrotic syndrome and improve their kidney disease symptoms. Moreover, the emulsion of the ternary complex has a better effect on improving the sex hormone levels and serum biochemical indicators of rats with nephrotic syndrome than general oyster peptides.

[0096] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A method for preparing a complex of oyster peptide and oleanolic acid saponin, comprising: The oyster peptide shown in SEQ ID NO: 1 is subjected to low-temperature plasma treatment; The oyster peptide treated with low-temperature plasma was prepared into an aqueous solution and kept at 4°C overnight; The oyster peptide aqueous solution was reacted with the oleanolic acid type saponin solution overnight for 24 hours; The reaction solution was dialyzed against running water at 4°C for 48 h and then freeze-dried in vacuum.

2. The preparation method according to claim 1, wherein the conditions of the low temperature plasma treatment include: 350w treatment for 5min, air intake volume 120mL / min.

3. The preparation method according to claim 1, wherein the concentration of the aqueous solution of the oyster peptide is 0.2 g / mL.

4. The preparation method according to claim 1, wherein the molecular weight cut-off of the dialysis is 1000Da.

5. A method for preparing a ternary complex emulsion, comprising: Mixing the composite obtained according to any one of claims 1 to 4, chitosan and lemon essential oil in deionized water; After stirring, high-speed shearing and high-pressure homogenization, an emulsion is obtained.

6. The preparation method according to claim 5, wherein the addition ratio of the complex is 1.5% (w / v), the addition ratio of the lemon essential oil is 2.3% (w / v), and the addition ratio of the chitosan is 1% (w / v).

7. The preparation method according to claim 5, wherein the homogenization pressure is 45 MPa.

8. The use of the emulsion of the ternary complex according to any one of claims 5 to 7, wherein the use is selected from at least one of the following: Promote the secretion or expression of gonadotropin-releasing hormone; Promote serum testosterone secretion or expression; Improve the impaired function of the hypothalamus-pituitary-gonadal axis; Reduce serum urea nitrogen secretion or expression; Reduces serum creatinine secretion or expression; Lower total cholesterol levels; Lower triglyceride levels; Increase plasma albumin; Improvement of nephrotic syndrome; and Improve at least one of the sexual functions.

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