Preparation method and anti-oxidation application of sweet osmanthus seed extract

Abstract

The invention relates to the technical field of medicines, and discloses a preparation method and antioxidant application of an osmanthus fragrans seed extract. An innovative antioxidant source is shown, an innovative protection strategy can be provided for animal intestinal health by utilizing the osmanthus fragrans seed extract, and particularly, a remarkable effect is shown in the aspect of preventing and relieving piglet diarrhea. Osmanthus fragrans seed extract has the characteristics of oxidation resistance, inflammation resistance and cell protection, so that cell apoptosis and inflammatory reaction can be relieved, and meanwhile, the resistance of cells to oxidative stress can be improved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for preparing an osmanthus seed extract and an antioxidant application thereof. Background Art

[0002] Osmanthus fragrans, belonging to the genus Osmanthus of the family Oleaceae, is a warm plant. Its seeds are oval in shape, and the outer skin turns purple-black when mature. In the field of traditional medicine, osmanthus occupies a pivotal position. Osmanthus extract is rich in a variety of bioactive ingredients, including flavonoids, terpenes, phenols, etc. These ingredients give osmanthus extract a variety of pharmacological effects, such as antioxidant, anti-inflammatory, and antibacterial.

[0003] At present, the development of osmanthus mainly focuses on the flower itself, while the utilization of osmanthus seeds is relatively small. There is also a lack of research on the antioxidant and antibacterial activities of the chemical components in osmanthus seeds, which leads to waste of resources and potential environmental pollution. As for whether osmanthus seed extract can play an antioxidant role in the pig intestine, there is currently a lack of scientific evidence to support it. Summary of the invention

[0004] In view of this, the present invention proposes a method for preparing an osmanthus seed extract and its antioxidant application, aiming to effectively utilize osmanthus seed resources and explore its potential application in animal intestinal health.

[0005] The present invention provides an application of osmanthus fragrans seeds, wherein the application comprises at least any one of the following:

[0006] A1) Use of the osmanthus seeds in preparing a product for treating piglet diarrhea;

[0007] A2) Use of the sweet osmanthus seeds in preparing a product for preventing diarrhea in piglets;

[0008] A3) Application of the sweet osmanthus seeds in inhibiting cell apoptosis;

[0009] A4) Use of the osmanthus seeds in preparing a product for inhibiting cell apoptosis;

[0010] A5) Application of the sweet osmanthus seeds in resisting oxidative stress;

[0011] A6) Use of the osmanthus seeds in preparing products for resisting oxidative stress;

[0012] A7) Use of the osmanthus seeds in inhibiting cellular inflammation;

[0013] A8) Use of the osmanthus seeds in preparing a product for inhibiting cell inflammation;

[0014] A9) Use of the osmanthus seeds in alleviating peroxide-induced cell death;

[0015] A10) Use of the osmanthus seeds in preparing a product for alleviating peroxide-induced cell death.

[0016] The present invention also provides an application of an osmanthus seed extract, which includes at least any one of the following:

[0017] A1) Use of the osmanthus seeds in preparing a product for treating piglet diarrhea;

[0018] A2) Use of the sweet osmanthus seeds in preparing a product for preventing diarrhea in piglets;

[0019] A3) Application of the sweet osmanthus seeds in inhibiting cell apoptosis;

[0020] A4) Use of the osmanthus seeds in preparing a product for inhibiting cell apoptosis;

[0021] A5) Application of the sweet osmanthus seeds in resisting oxidative stress;

[0022] A6) Use of the osmanthus seeds in preparing products for resisting oxidative stress;

[0023] A7) Use of the osmanthus seeds in inhibiting cellular inflammation;

[0024] A8) Use of the osmanthus seeds in preparing a product for inhibiting cell inflammation;

[0025] A9) Use of the osmanthus seeds in alleviating peroxide-induced cell death;

[0026] A10) Use of the osmanthus seeds in preparing a product for alleviating peroxide-induced cell death.

[0027] The present invention also provides a method for inhibiting cell apoptosis, comprising:

[0028] The cells were cultured in a medium containing Osmanthus fragrans seed extract.

[0029] The present invention also provides a method for alleviating peroxide-induced cell death, comprising:

[0030] The peroxide-induced cells were cultured in a medium containing Osmanthus fragrans seed extract.

[0031] The present invention also provides a method for improving the anti-oxidative stress ability of cells, comprising:

[0032] The cells were cultured in a medium containing Osmanthus fragrans seed extract.

[0033] The invention also provides a medicine for preventing and / or treating piglet diarrhea, which comprises an osmanthus seed extract.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention reveals an innovative source of antioxidants, paving the way for the development of new antioxidant products. Using osmanthus seed extract, we are able to provide an innovative protective strategy for animal intestinal health, especially showing significant effects in the treatment and prevention of piglet diarrhea. The antioxidant, anti-inflammatory and cytoprotective properties of osmanthus seed extract help to reduce cell apoptosis and inflammatory responses, while enhancing the resistance of cells to oxidative stress. In addition, the extraction method of the present invention is simple and efficient, and can be easily implemented on an industrial scale, indicating good application prospects and market potential. Overall, the present invention not only provides a new perspective for the comprehensive utilization of osmanthus seeds, but also provides a new way for the innovative development of animal intestinal health and antioxidants, and has important social and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0037] Figure 1 The model was constructed for the effects of H2O2 concentration and osmanthus seed extract on IPEC-J2 cell viability; A was the effect of different concentrations of H2O2 treatment on IPEC-J2 cell survival rate; B was the effect of different concentrations of osmanthus seed extract on IPEC-J2 cell survival rate; NC was negative control; OS was Osmanthus fragrans'seeds;

[0038] Figure 2 Different concentrations of osmanthus seed extracts alleviated the oxidative stress of IPEC-J2 cells induced by H2O2; A is the experimental setting flow chart; B is the results of different concentrations of osmanthus seed extracts on the oxidative stress of IPEC-J2 cells induced by H2O2; NC is the negative control; OS is Osmanthus fragrans'seeds;

[0039] Figure 3 The pathway diagram of the Osmanthus fragrans seed extract on the expression of genes related to H2O2-induced cell anti-oxidative stress and inflammation;

[0040] Figure 4The effect of osmanthus fragrans seed extract on the expression of genes related to the cell antioxidant stress pathway induced by H2O2; AF is the expression of cell antioxidant stress related genes Keap1, Nrf2, SOD1, SOD2, CAT, GSH in different treatment groups detected by fluorescence quantitative PCR technology; NC is negative control; OS is Osmanthus fragrans' seeds;

[0041] Figure 5 The effect of osmanthus seed extract on the expression of genes related to cell inflammation induced by H2O2; AD is the expression of cell inflammation-related genes NF-κB, TNF-α, IL-18, and IL-1β in different treatment groups detected by fluorescence quantitative PCR technology; NC is the negative control; OS is Osmanthus fragrans' seeds. DETAILED DESCRIPTION

[0042] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features described in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0043] Example 1

[0044] 1. Materials and Methods

[0045] 1. Main experimental reagents

[0046] Contains 0.25% Trypsin-EDTA (1×) (Gibco, Catalog No. 25200-056); PBS (1×) (Bioss, Catalog No. C01-01001); DMEM high glucose medium (Gibco, Catalog No. C11995500BT); Penicillin-Streptomycin (10000U / mL) (Gibco, Catalog No. 15140122); Fetal bovine serum (Gibco, Catalog No. 10100147); SYBR qPCR Master Mix (Accurate Biology, Catalog No.: AG11701), and cck8 detection kit (APE×BIO, Catalog No.: K1018311). The primers used in the experiment were designed and synthesized by Guangzhou Huada Biotechnology Co., Ltd. The primer sequences are shown in Table 1.

[0047] Table 1

[0048]

[0049]

[0050] 2. Test consumables

[0051] Cells: Porcine jejunal IPEC-J2 cell line was used.

[0052] Instruments and equipment: including fluorescence quantitative PCR instrument LCR480Ⅱ, incubator, inverted microscope Axio Vert A1, cell culture dishes, 75% alcohol cotton balls, 1mL pipettes, sterilized pipette tips, sealing film, 60mm cell culture dishes, 96-well plates, 12-well plates, etc.

[0053] 3. Cell culture

[0054] IPEC-J2 cells were cultured in a cell culture dish containing DMEM complete medium (supplemented with 10% fetal bovine serum) at 37°C and 5% CO2. When the cells grew to a confluence of about 80%, they were washed with 2 mL of PBS to remove non-adherent cells, and then 1 mL of trypsin was added for 2 minutes for digestion and passage.

[0055] 4. Preparation method of osmanthus seed extract

[0056] Take 100 grams of mature osmanthus seeds, crush them with a grinder and pass them through a 40-mesh sieve. Then, add 95% ethanol for ultrasonic extraction according to a solid-liquid ratio of 1:10, set the ultrasonic power to 280W, extract for 30 minutes each time, and extract 3 times in total. After combining the three extracts, recover them under reduced pressure to obtain an osmanthus seed ethanol extract.

[0057] 5. Grouping and treatment of antioxidant test

[0058] The experimental grouping is shown in Table 2. IPEC-J2 cells were evenly spread in a 12-well plate and divided into the following four groups: control group, H2O2 treatment group, H2O2 combined with osmanthus seed extract treatment group, each group containing three replicates. After 4 hours of H2O2 treatment, osmanthus seed extract was added to each group and cultured for another 24 hours. Afterwards, cell samples were collected for subsequent detection and analysis.

[0059] Table 2

[0060]

[0061] 7. Data Analysis

[0062] In subsequent experiments, SPSS26.0 software will be used to perform t test and one-way ANOVA to evaluate the differences between different groups. In order to perform multiple comparison tests between groups, the LSD method will be applied. The preparation of the chart will be completed with the help of GraphPad Prism 8.0.1 software. Statistically, when the P value is less than 0.05, the difference is considered significant.

[0063] 2. Test methods and results

[0064] 1. To investigate the effects of model concentration of H2O2 and osmanthus seed extract on IPEC-J2 cell viability

[0065] First, IPEC-J2 cells were evenly seeded in a 96-well plate with a cell concentration of 1×10 4 After one night of culture, H2O2 and osmanthus seed extract were diluted to different concentrations for 4 hours. Then the cell samples were collected and the absorbance (OD value) was measured at 450nm using CCK-8 kit to evaluate the survival status of the cells. Figure 1 shown.

[0066] Figure 1 A revealed a dose-dependent relationship between H2O2 treatment concentration and IPEC-J2 cell survival rate. As the H2O2 concentration increased, the cell survival rate decreased significantly. When the H2O2 concentration reached 350 μmol / L, the cell survival rate dropped to about 50%, so this concentration was selected as the condition for constructing an in vitro IPEC-J2 cell oxidative stress model. In addition, the study found that different concentrations of osmanthus seed extract (5, 10, 20, 50, 100 μg / mL) had no significant effect on the survival rate of IPEC-J2 cells ( Figure 1 B).

[0067] 2. Effects of Osmanthus fragrans seed extract on alleviating H2O2-induced oxidative stress

[0068] The cell concentration was adjusted to approximately 1 × 10 4 IPEC-J2 cells were evenly distributed in a 96-well plate, and a control group, a 350 μmol / L H2O2 treatment group, and an experimental group treated with 350 μmol / L H2O2 and different concentrations of osmanthus seed extract were set up (see Figure 2 A). After 4 hours of H2O2 treatment, the cells were cultured with osmanthus seed extract for 24 hours. The cells were then collected and the absorbance (OD value) was measured at 450 nm using a CCK-8 kit to evaluate cell survival. Figure 2 shown.

[0069] like Figure 2 As shown in Figure B, compared with the control group, treatment with 350 μmol / L H2O2 for 4 hours significantly reduced the survival rate of IPEC-J2 cells. However, after treatment with 350 μmol / L H2O2, the cells were cultured with different concentrations of osmanthus seed extract solutions (50, 100, 200 μg / mL) for 24 hours, and it was found that both 100 and 200 μg / mL of extracts could significantly alleviate cell death caused by oxidative stress induced by H2O2 and significantly improve cell survival rate.

[0070] 3. Study on the molecular mechanism of osmanthus seed extract in alleviating H2O2-induced oxidative stress

[0071] The Keap1 / Nrf2 signaling pathway is a classic pathway for cells to respond to anti-oxidative stress. In order to further study the molecular mechanism of how Osmanthus fragrans seed extract alleviates H2O2-induced oxidative stress in IPEC-J2 cells and prevents cell death, the present invention used RT-qPCR technology to quantitatively analyze the genes involved in the cell oxidative stress and inflammatory response pathways.

[0072] 3.1 Osmanthus fragrans seed extract alleviates H2O2-induced oxidative stress

[0073] 3.1.1 Osmanthus fragrans seed extract regulates the expression of antioxidant-related genes induced by H2O2

[0074] Experimental Results Figure 3 and Figure 4 The results showed that compared with the untreated group, H2O2 treatment of IPEC-J2 cells significantly increased the expression of Keap1 and decreased the expression of Nrf2, SOD1, SOD2, CAT, and GSH ( Figure 4 A-4F). This indicates that H2O2 treatment of IPEC-J2 cells mainly leads to cell death by activating the Keap1 / Nrf2 / SOD / GSH antioxidant stress pathway. However, when 200 μg / mL of Osmanthus fragrans seed extract was used to treat IPEC-J2 cells, compared with the H2O2 treatment group, Osmanthus fragrans seed extract significantly reduced the H2O2-induced high expression of Keap1 ( Figure 4 A), and Nrf2 levels were significantly increased ( Figure 4 B). In addition, the addition of osmanthus seed extract changed the expression levels of downstream target genes related to the Keap1 / Nrf2 signaling pathway ( Figure 4 C-4F), indicating that Osmanthus fragrans seed extract activated this pathway. These data revealed that Osmanthus fragrans seed extract effectively alleviated H2O2-induced IPEC-J2 cell death by activating the Keap1 / Nrf2 / SOD / GSH antioxidant stress pathway.

[0075] 3.1.2 Osmanthus fragrans seed extract regulates the expression of H2O2-induced inflammation-related genes

[0076] Experimental Results Figure 3 and Figure 5 It was further shown that compared with the untreated group, the treatment of IPEC-J2 cells with H2O2 alone significantly increased the expression of NF-κB, TNF-α, IL-18, and IL-1β ( Figure 5 A-5D), which indicates that H2O2 treatment of IPEC-J2 cells mainly leads to cell death by activating the expression of NF-κB / TNF-α / IL-18 / IL-1β cell inflammation-related genes. However, after IPEC-J2 cells were treated with 200 μg / mL of osmanthus seed extract, osmanthus seed extract significantly reduced the high expression of NF-κB, IL-18, and IL-1β induced by H2O2 ( Figure 5 A, 5C, 5D), TNF-α expression showed a downward trend, but the difference was not significant ( Figure 5 B). These data indicate that Osmanthus fragrans seed extract effectively alleviated H2O2-induced IPEC-J2 cell death by inhibiting the expression of cellular inflammation-related genes.

[0077] Conclusion

[0078] In summary, the present invention is the first to explore the antioxidant effect of osmanthus seed extract in porcine intestinal IPEC-J2 cells. The study revealed the molecular mechanism by which osmanthus seed extract can alleviate the oxidative stress and inflammatory response of porcine intestinal IPEC-J2 cells caused by H2O2, thereby protecting cell survival. Our findings provide scientific basis and theoretical support for the use of osmanthus seed extract to prevent and alleviate piglet diarrhea caused by intestinal oxidative stress through nutritional regulation strategies in pig farming practice.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. An application of osmanthus fragrans seeds, characterized in that: The application includes at least one of the following: A1) Use of the osmanthus seeds in preparing a product for treating piglet diarrhea; A2) Use of the sweet osmanthus seeds in preparing a product for preventing diarrhea in piglets; A3) Application of the sweet osmanthus seeds in inhibiting cell apoptosis; A4) Use of the osmanthus seeds in preparing a product for inhibiting cell apoptosis; A5) Application of the sweet osmanthus seeds in resisting oxidative stress; A6) Use of the osmanthus seeds in preparing products for resisting oxidative stress; A7) Use of the osmanthus seeds in inhibiting cellular inflammation; A8) Use of the osmanthus seeds in preparing a product for inhibiting cell inflammation; A9) Use of the osmanthus seeds in alleviating peroxide-induced cell death; A10) Use of the osmanthus seeds in preparing a product for alleviating peroxide-induced cell death.

2. An application of an osmanthus seed extract, characterized in that: The application includes at least one of the following: A1) Use of the osmanthus seeds in preparing a product for treating piglet diarrhea; A2) Use of the sweet osmanthus seeds in preparing a product for preventing diarrhea in piglets; A3) Application of the sweet osmanthus seeds in inhibiting cell apoptosis; A4) Use of the osmanthus seeds in preparing a product for inhibiting cell apoptosis; A5) Application of the sweet osmanthus seeds in resisting oxidative stress; A6) Use of the osmanthus seeds in preparing products for resisting oxidative stress; A7) Use of the osmanthus seeds in inhibiting cellular inflammation; A8) Use of the osmanthus seeds in preparing a product for inhibiting cell inflammation; A9) Use of the osmanthus seeds in alleviating peroxide-induced cell death; A10) Use of the osmanthus seeds in preparing a product for alleviating peroxide-induced cell death.

3. A method for inhibiting cell apoptosis, characterized in that: include: The cells were cultured in a medium containing Osmanthus fragrans seed extract.

4. A method for alleviating peroxide-induced cell death, characterized in that: include: The peroxide-induced cells were cultured in a medium containing Osmanthus fragrans seed extract.

5. A method for improving the ability of cells to resist oxidative stress, characterized in that: include: The cells were cultured in a medium containing Osmanthus fragrans seed extract.

6. A drug for preventing and / or treating piglet diarrhea, characterized in that: The medicine comprises osmanthus seed extract.

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