Preparation method of hippocampus oil and application of hippocampus oil in uric acid reducing products

Abstract

The application discloses a preparation method of hippocampus oil, which comprises the following steps: 1) adding hippocampus dry powder into dichloromethane-methanol mixed solution and stirring uniformly, and then leaching for 24 hours; 2) after filtering the leaching solution, adding water and mixing fully, and then standing for 12 hours, and then collecting the lower organic phase; and 3) performing rotary evaporation and drying on the organic phase. The application further discloses the use of the hippocampus oil in the preparation of a product for reducing uric acid. The hippocampus oil is rich in omega-3 polyunsaturated fatty acids (EPA and DHA), shows moderate down-regulation on xanthine oxidase activity and inhibition on the release of inflammatory factors, and achieves the comprehensive effect of 'inhibiting generation and relieving inflammation'.

Description

Technical Field

[0001] This invention relates to the field of deep processing technology of seafood products, specifically to a method for preparing seahorse oil and its applications. Background Technology

[0002] Hyperuricemia is a metabolic disorder syndrome caused by elevated serum uric acid levels, leading to diseases such as gout and kidney stones. With changes in lifestyle, the prevalence of hyperuricemia is gradually increasing. Currently, clinically used uric acid-lowering drugs are divided into two categories: xanthine oxidase inhibitors and uric acid excretion drugs.

[0003] Xanthine oxidase inhibitors have a stronger effect in lowering uric acid, but adverse reactions and drug interactions limit their use. Uric acid excretion drugs are dependent on kidney function, and some patients experience ineffectiveness or intolerance. Moreover, long-term use of xanthine oxidase inhibitors and uric acid excretion drugs may cause side effects such as liver and kidney damage, skin rashes, and gastrointestinal discomfort.

[0004] This has prompted people to try to discover new and effective uric acid-lowering active substances from safe natural products and to find new safe intervention pathways to improve hyperuricemia and gout.

[0005] Most research on seahorses, a marine product, focuses on peptides or polysaccharides, but there are no reports on the active mechanisms of seahorse oil components. Summary of the Invention

[0006] This invention discloses a method for preparing seahorse oil, comprising the following steps:

[0007] 1) Add the dried seahorse powder to a dichloromethane-methanol mixture and stir well, then extract for 24 hours;

[0008] 2) After filtering the extract, add water and mix thoroughly. After standing for 12 hours, collect the lower organic phase.

[0009] 3) Dry the organic phase by rotary evaporation.

[0010] Furthermore, the dried seahorse powder in step 1) can be made from freeze-dried fresh bloated seahorse powder.

[0011] Furthermore, the conditions for freeze-drying fresh bloated seahorses were -50°C and a vacuum degree of <10 kPa for 48 hours.

[0012] Furthermore, the above-mentioned dichloromethane-methanol mixture can be a dichloromethane-methanol mixture with a volume ratio of 2:1.

[0013] Furthermore, the above-mentioned dry seahorse powder can be added to a dichloromethane-methanol mixture at a material-to-liquid ratio of 1:10.

[0014] Furthermore, the conditions for the rotary evaporation drying described above can be: vacuum degree 0.06~0.08MPa, rotation speed 30~60rpm, cooling temperature -10℃, and water bath temperature 40~50℃.

[0015] The present invention also discloses seahorse oil prepared according to the above method.

[0016] This invention also discloses the use of seahorse oil in the preparation of uric acid-lowering products.

[0017] Furthermore, seahorse oil can be used orally.

[0018] Furthermore, the oral dosage of seahorse oil can be 200–400 mg / kg body weight / day.

[0019] Specifically, the present invention is as follows.

[0020] 1. A method for preparing seahorse oil, comprising the following steps:

[0021] 1) Add the dried seahorse powder to a dichloromethane-methanol mixture and stir well, then extract for 24 hours;

[0022] 2) After filtering the extract, add water and mix thoroughly. After standing for 12 hours, collect the lower organic phase.

[0023] 3) Dry the organic phase by rotary evaporation.

[0024] 2. The method for preparing seahorse oil as described in item 1, characterized in that: the dried seahorse powder in step 1) is made from freeze-dried fresh bloated seahorse powder.

[0025] 3. The method for preparing seahorse oil as described in item 2, characterized in that: the conditions for freeze-drying fresh bloated seahorses are -50℃ and vacuum degree <10kPa for 48h.

[0026] 4. The method for preparing seahorse oil according to any one of items 1 to 3, characterized in that: the dichloromethane-methanol mixture in step 1) is a dichloromethane-methanol mixture with a volume ratio of 2:1.

[0027] 5. The method for preparing seahorse oil as described in item 1, characterized in that: the dry seahorse powder in step 1) is added to a dichloromethane-methanol mixture at a material-liquid ratio of 1:10.

[0028] 6. The method for preparing seahorse oil as described in item 1, characterized in that: the conditions for rotary evaporation drying in step 3) are: vacuum degree 0.06~0.08MPa, rotation speed 30~60rpm, cooling temperature -10℃, and water bath temperature 40~50℃.

[0029] 7. Seahorse oil prepared according to any one of items 1 to 6.

[0030] 8. Uses of seahorse oil in the preparation of uric acid-lowering products.

[0031] 9. The use of seahorse oil as described in item 8 in the preparation of uric acid-lowering products, characterized in that: the seahorse oil is used orally.

[0032] 10. The use of seahorse oil as described in item 9 in the preparation of uric acid-lowering products, characterized in that: the oral dose of the seahorse oil is 200-400 mg / kg body weight / day.

[0033] This invention provides a method for preparing seahorse oil and investigates the effects of seahorse oil on hyperuricemic mice and on an in vitro inflammatory hyperuricemia cell model.

[0034] Seahorse oil is rich in ω-3 polyunsaturated fatty acids (EPA, DHA), which exhibit moderate downregulation of xanthine oxidase activity and inhibition of inflammatory factor release, achieving a comprehensive effect of "inhibiting production + alleviating inflammation." Systematic validation based on in vivo mouse models and in vitro cell models has demonstrated that seahorse oil can significantly reduce serum uric acid, inhibit uric acid synthesis, and improve inflammatory responses within a safe and acceptable dosage range. Attached Figure Description

[0035] Figure 1 This indicates the effect of seahorse oil on serum biochemical indicators in mice with high uric acid.

[0036] Figure 2 This indicates the effect of seahorse oil on uric acid synthesis.

[0037] Figure 3 This indicates the effect of seahorse oil on cell viability in hyperuricemia-induced cellular inflammation.

[0038] Figure 4 This study indicates the effect of seahorse oil on the release of lactate dehydrogenase, which induces cell inflammation in hyperuricemia.

[0039] Figure 5 This indicates the effect of seahorse oil on IL-1β release. Detailed Implementation

[0040] To better understand this invention, the following embodiments are provided in conjunction with the accompanying drawings. It should be understood that the embodiments of this invention are for illustrative purposes only and not for limiting the invention; the scope of protection of this invention is defined solely by the claims. The embodiments provided are merely preferred embodiments and are not intended to limit the invention in any way. Those skilled in the art can make changes, equivalent substitutions, or modifications based on the content of this invention to form different implementations. However, any changes and modifications, and any equivalent substitutions made to the method of this invention without departing from the inventive concept are within the scope of protection of this invention.

[0041] Example 1: Preparation of Seahorse Oil

[0042] Fresh, bloated seahorses were freeze-dried at -50°C and <10 kPa for 48 hours. After freeze-drying, the seahorse powder was pulverized. The seahorse powder was added to a dichloromethane-methanol mixture (2:1, volume ratio) at a material-to-liquid ratio of 1:10 (m / v) and stirred until homogeneous, then extracted for 24 hours. After filtering the extract, 1 / 4 volume of ultrapure water was added and thoroughly mixed. After standing for 12 hours, the lower organic phase was collected. The organic phase was then rotary evaporated. The rotary evaporation conditions were: vacuum 0.06–0.08 MPa, rotation speed 30–60 rpm, cooling temperature -10°C, and water bath temperature 40–50°C. Seahorse oil was obtained after rotary evaporation.

[0043] Example 2: Preparation of Hippocampal Peptides

[0044] A certain mass of dried hippocampal powder was added to distilled water and 3% compound protease (120 U / mg, Shanghai Yuanye Biotechnology Co., Ltd., product number: S10155) at a material-to-liquid ratio of 1:10 (m / v) and stirred until homogeneous. The pH was adjusted to 7.5 with 1 mol / L NaOH and 1 mol / L HCl, and enzymatic hydrolysis was carried out at 50℃ for 5 h. The hydrolysate was centrifuged at 7500 rpm for 15 min, and the supernatant was filtered through a 0.45 μm microporous membrane. The filtered enzymatically hydrolyzed peptide solution was freeze-dried at -50℃ and a vacuum degree <10 kPa for 48 h to obtain hippocampal peptide powder.

[0045] Example 3, Animal Experiment

[0046] Thirty-six male C57BL / 6 mice, 6–8 weeks old, 18–20 g, SPF grade, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0047] After one week of acclimatization, animals were randomly divided into four groups according to body weight: a normal control group, a model group, a low-dose seahorse oil group (200 mg / kg body weight / day), a high-dose seahorse oil group (400 mg / kg body weight / day), a seahorse peptide group (200 mg / kg body weight / day), a seahorse powder group (200 mg / kg body weight / day), and an allopurinol group (allopurinol 25 mg / kg body weight / day, positive control), with six animals in each group. Using 5% sodium carboxymethyl cellulose (CMC-Na) as the suspension medium, each group was administered the corresponding test substance by gavage at 4:00 PM daily. The normal control group and the model group received an equal volume of 5% sodium carboxymethyl cellulose solution for 14 consecutive days.

[0048] Fourteen days after gavage administration of the test substance, adenine and potassium oxonate were prepared into a suspension (using 5% sodium carboxymethyl cellulose as the suspension medium) and administered orally at 200 μL at 10:00 AM daily. Except for the control group, all other groups received adenine 120 mg / kg body weight / day and potassium oxonate 1800 mg / kg body weight / day. The control group received an equal volume of 5% sodium carboxymethyl cellulose solution. This was continued for two weeks to establish a hyperuricemia model.

[0049] After the final gavage experiment, mice were anesthetized after a 12-hour fast, and urine and blood samples were collected. After sacrifice, kidney and liver tissues were rapidly separated, weighed, and stored at -80°C. Blood samples were centrifuged at 4500 rpm and 4°C for 15 minutes to obtain serum.

[0050] Example 4: Determination of serum uric acid, serum creatinine and serum urea nitrogen

[0051] The uric acid (UA) test kit was purchased from Nanjing Jiancheng Bioengineering Institute, serial number C012-2-1. The creatinine (Cr) assay kit (sarcosine oxidase method) was purchased from Nanjing Jiancheng Bioengineering Institute, serial number C011-2-1. The blood urea nitrogen (BUN) test kit was purchased from Nanjing Jiancheng Bioengineering Institute, serial number C013-2-1.

[0052] Follow the instructions in the kit to measure serum uric acid (SUA), serum creatinine (sCr), and serum blood urea nitrogen (BUN) levels.

[0053] Serum uric acid, serum urea nitrogen, and serum creatinine levels are important indicators for assessing whether uric acid metabolism is impaired. Measurement results are shown below. Figure 1 .

[0054] In all the figures of this invention, the results are expressed as Mean ± SEM. Significance analysis was performed using one-way ANOVA, followed by Tukey's test. "##" above the data bar indicates a highly significant difference (p < 0.01) between the model group and the normal group; "*" above the data bar indicates a significant difference (p < 0.1) between the corresponding experimental group and the model group; "**" above the data bar indicates a highly significant difference (p < 0.01) between the corresponding experimental group and the model group.

[0055] like Figure 1 As shown, after drug intervention, the serum urea nitrogen and serum uric acid levels in the model group were significantly higher than those in the normal group, indicating that adenine and potassium oxonate successfully induced a mouse model of hyperuricemia. Compared with the model group, intervention with high-dose or low-dose seahorse oil significantly reduced serum uric acid, serum urea nitrogen, and serum creatinine levels, and the degree of improvement showed a dose-related relationship; the higher the intervention dose, the more significant the improvement effect. Compared with the model group, high-dose seahorse oil intervention reduced serum uric acid, urea nitrogen, and creatinine levels by 37.93%, 53.88%, and 50.21%, respectively, showing better uric acid-lowering activity compared with seahorse peptides and seahorse powder, and was superior to the allopurinol group.

[0056] Example 5: Detection of xanthine oxidase (XOD) and adenosine deaminase (ADA) activities

[0057] The mouse xanthine oxidase (XOD) ELISA research kit was purchased from Jiangsu Jingmei Biotechnology Co., Ltd., catalog number JM-11523M1. The mouse adenosine deaminase (ADA) ELISA research kit was purchased from Jiangsu Jingmei Biotechnology Co., Ltd., catalog number JM-11527M1.

[0058] Take 0.1g of liver tissue obtained in Example 3 from each group and place it in a 2mL grinding tube. Add 900μl of physiological saline containing a mixture of 1% benzyl sulfonyl fluoride (PMSF) and a protease phosphatase inhibitor (general type, 50X, Shanghai Beyotime Biotechnology Co., Ltd., catalog number P1045) and two 5mm stainless steel grinding beads. Set the grinding parameters as follows: frequency 60Hz, grinding time 30 seconds, pause time 15s, number of cycles 2, and temperature 4℃. After grinding, centrifuge at 12000rpm and 4℃ for 5min. Take the supernatant and strictly determine the xanthine oxidase and adenosine deaminase activities according to the kit instructions.

[0059] Xanthine oxidase and adenosine deaminase are enzymes that play key roles in regulating uric acid production. The measurement results are as follows: Figure 2As shown in the figure, compared with the model group, the activities of xanthine oxidase and adenosine deaminase in liver tissue were significantly reduced in the allopurinol group, the high-dose seahorse oil group, and the low-dose seahorse oil group, demonstrating superior inhibitory activity compared with the same dose of seahorse peptide. Furthermore, seahorse oil showed a dose-dependent effect; compared with the model group, high-dose seahorse oil intervention reduced xanthine oxidase and adenosine deaminase activities by 38.14% and 29.55%, respectively, with activities comparable to allopurinol. Seahorse powder intervention reduced xanthine oxidase and adenosine deaminase activities, but the reduction was not significant. These results indicate that seahorse oil has good anti-hyperuricemia activity.

[0060] Example 6: Cell culture and cell viability assay, cell damage detection

[0061] RAW264.7 mouse mononuclear macrophages (STR identification correct) were purchased from Guangzhou Saiku Biotechnology Co., Ltd., catalog number CC9001. The CCK-8 cell culture proliferation and toxicity assay kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number CA1210. A trace amount of the L-lactate dehydrogenase (L-LDH) activity assay kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., product model BC0685-100T / 48S.

[0062] RAW264.7 mouse mononuclear macrophages were seeded in 96-well plates and cultured in DMEM high-glucose medium (containing GlutaPlus, 1.5 g / L sodium bicarbonate, Wuhan Saiwell Biotechnology Co., Ltd., catalog number G4524-500ML) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin at 37°C in a 5% CO2 incubator. When the cell confluence reached 70%–80%, 500 ng / ml lipopolysaccharide (LPS) (LPS derived from Escherichia coli O55:B5, Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number L2880) was added for pretreatment.

[0063] Two hours after pretreatment, the seahorse oil group and the allopurinol group were cultured with 10 μg / ml seahorse oil and 200 μg / ml allopurinol, respectively.

[0064] After 1 hour of culture, the model group, seahorse oil group, and allopurinol group were stimulated with 300 ng / μl soluble uric acid (UA) for 3 hours.

[0065] Subsequently, 10 μL of CCK-8 working solution was added to each well, and the cells were incubated for 1 h. Cell viability was assessed by measuring absorbance at 450 nm. Cell culture supernatant was collected simultaneously, and L-lactate dehydrogenase (L-LDH) release levels were detected strictly according to the kit instructions. Furthermore, interleukin-1β levels were detected using a sandwich ELISA method according to the instructions of the mouse interleukin-1β (IL-1β) ELISA research kit (Jiangsu Jingmei Biotechnology Co., Ltd., catalog number JM-02323M2).

[0066] Figure 3 This indicates the effect of seahorse oil on cell viability in cases of hyperuricemia-induced cellular inflammation. For example... Figure 3 As shown, seahorse oil can alleviate hyperuricemia-induced inflammatory death of mononuclear macrophages in RAW264.7 mice. Compared with the model group, the cell survival rate after seahorse oil intervention increased significantly by 21.1% (p<0.01), and the improvement effect was comparable to that of the allopurinol group.

[0067] The amount of lactate dehydrogenase leakage can be used to assess cell growth status and the degree of cell damage. The test results are as follows: Figure 4 As shown in the figure, compared with the normal group, the release of lactate dehydrogenase in the model group cells was significantly increased, which was 4.6 times that of the normal group (p<0.01). Seahorse oil could significantly inhibit the release of lactate dehydrogenase. Although it was slightly less than that of the allopurinol group, it was significantly lower than that of the model group by 22.72% (p<0.01).

[0068] The combined results of cell viability and lactate dehydrogenase leakage indicate that seahorse oil significantly improves cellular inflammation induced by high uric acid.

[0069] Inflammatory factor test results as follows Figure 5 As shown, the inflammatory response plays an important role in the pathogenesis of hyperuricemia. Compared with the normal group, the IL-1β content in the cell culture supernatant of the model group was significantly increased. However, intervention with allopurinol or seahorse oil significantly inhibited the expression of inflammatory factors, and the inhibitory effect of seahorse oil was better than that of allopurinol. Compared with the model group, the inhibition rate of seahorse oil reached 60.92%. These results indicate that seahorse oil can effectively alleviate the inflammatory response of hyperuricemia and exert an anti-inflammatory effect.

Claims

1. The use of seahorse oil in the preparation of uric acid-lowering products, characterized in that, The method of use for the seahorse oil is oral administration, and the oral dosage of the seahorse oil is 400 mg / kg body weight / day; The preparation method of the seahorse oil includes the following steps: 1) The dried seahorse powder is made from freeze-dried fresh bloated seahorse powder. The dried seahorse powder is added to a 2:1 volume ratio of dichloromethane-methanol mixture and stirred evenly for 24 hours. 2) After filtering the extract, add water and mix thoroughly. After standing for 12 hours, collect the lower organic phase. 3) Dry the organic phase by rotary evaporation.

2. The use as described in claim 1, characterized in that: The conditions for freeze-drying fresh bloated seahorses were -50℃ and vacuum degree <10kPa for 48h.

3. The use as described in claim 1 or 2, characterized in that: Step 1) The dried seahorse powder is added to a dichloromethane-methanol mixture at a material-liquid ratio of 1:

10.

4. The use as described in claim 1, characterized in that: The conditions for rotary evaporation drying in step 3) are: vacuum degree 0.06~0.08MPa, rotation speed 30~60rpm, cooling temperature -10℃, and water bath temperature 40~50℃.

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