Application of rutin composition in preparation of medicine for relieving toxicity of porcine hepatocytes

Abstract

The invention discloses application of a rutin composition in preparation of a medicine for relieving toxicity of porcine hepatocytes, and belongs to the technical field of medicines. The rutin composition comprises rutin, vitamin C, vitamin E, tanshinone IIA, puerarin and licochalcone B. The invention further discloses a preparation method of the rutin composition. According to verification of the embodiment of the invention, it can be obtained that the rutin composition can relieve porcine liver cell toxicity by improving IPLs cell viability, restoring zearalenone to inhibit IPLs cell proliferation and restoring zearalenone to improve ROS yield in IPLs cells.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to application of a rutin composition in preparing a medicine for alleviating the toxicity of pig liver cells. Background Art

[0002] In modern animal husbandry, the scale of pig farming continues to expand, and the resulting disease problems are becoming increasingly prominent. Among them, hepatotoxicity is one of the important factors affecting pig health. When liver cells are damaged, it will not only affect the growth and development and production performance of pigs, but may also lead to death in severe cases, causing economic losses to farmers. Therefore, the development of effective methods to alleviate or treat pig hepatotoxicity has become one of the key research directions. There are currently some products on the market for animal liver protection, but most of them have limited effects or high costs, which limits their widespread use in practical applications.

[0003] In recent years, with the progress of natural product chemistry and pharmacology research, people have found that many plant extracts have good biological activities, including antioxidant, anti-inflammatory and hepatoprotective effects. Rutin is a flavonoid compound widely found in many plants, which has been shown to have strong free radical scavenging and anti-lipid peroxidation abilities. Based on these properties, scientists have begun to explore the possible role of rutin and its derivatives as potential drug ingredients in preventing and treating liver damage caused by various reasons. However, there have been no reports on the use of rutin in the form of a composition to prepare drugs specifically for alleviating pig hepatocyte toxicity. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a rutin composition in the preparation of a drug for alleviating the toxicity of pig liver cells, so as to solve the problems existing in the above-mentioned prior art. The rutin composition provided by the present invention can alleviate the toxicity of pig liver cells by improving the viability of IPLs cells.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] Technical solution 1: Use of a rutin composition in the preparation of a drug for alleviating pig liver cell toxicity, wherein the rutin composition comprises rutin, vitamin C, vitamin E, tanshinone IIA, puerarin and licorice chalcone B.

[0007] Furthermore, the concentration of rutin is 60 μmol / L, the concentration of vitamin C is 2 μmol / L, the concentration of vitamin E is 20 μmol / L, the concentration of tanshinone IIA is 10 μmol / L, the concentration of puerarin is 40 μmol / L and the concentration of licorice chalcone B is 20 μmol / L.

[0008] Furthermore, the porcine liver cell toxicity is caused by zearalenone.

[0009] Furthermore, the mitigation of porcine liver cell toxicity includes increasing IPLs cell viability.

[0010] Furthermore, the mitigation of porcine liver cell toxicity includes restoring zearalenone to inhibit IPLs cell proliferation.

[0011] Furthermore, the mitigation of porcine liver cell toxicity includes restoring zearalenone to increase ROS production in IPLs cells.

[0012] Technical Solution 2: A drug capable of alleviating the toxicity of pig liver cells, the active ingredients of which include a rutin composition; the rutin composition includes rutin, vitamin C, vitamin E, tanshinone IIA, puerarin and licorice chalcone B.

[0013] Furthermore, the concentration of rutin is 60 μmol / L, the concentration of vitamin C is 2 μmol / L, the concentration of vitamin E is 20 μmol / L, the concentration of tanshinone IIA is 10 μmol / L, the concentration of puerarin is 40 μmol / L and the concentration of licorice chalcone B is 20 μmol / L.

[0014] The present invention discloses the following technical effects:

[0015] The present invention discloses an application of a rutin composition in preparing a drug for alleviating pig liver cell toxicity. The rutin composition comprises rutin, vitamin C, vitamin E, tanshinone IIA, puerarin and licorice chalcone B. According to verification according to the embodiments of the present invention, it can be obtained that the rutin composition of the present invention can alleviate pig liver cell toxicity by improving IPLs cell viability, restoring zearalenone to inhibit IPLs cell proliferation, and restoring zearalenone to increase ROS production in IPLs cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1Effects of zearalenone, vitamin C (A), vitamin E (B), tanshinone IIA (C), puerarin (D), licorice chalcone B (E) and rutin (F) on the viability of porcine hepatocytes (n=6); CON: control; ZEA: zearalenone; VC: vitamin C; VE: vitamin E; Tan IIA: tanshinone IIA; PUE: puerarin; LCB: licorice chalcone B; RUT: rutin; ZEA×VC: zearalenone×vitamin C; ZEA×VE: zearalenone×vitamin E; ZEA×Tan IIA: zearalenone×tanshinone IIA; ZEA×PUE: zearalenone×puerarin; ZEA×LCB: zearalenone×licorice chalcone B; ZEA×RUT: zearalenone×rutin; a, b, c indicate significant differences (P<0.05);

[0018] Figure 2 The effects of each treatment group on the viability of pig hepatocytes (n=6); CON: control; ZEA: zearalenone; VC: vitamin C; VE: vitamin E; RUT: rutin; VC×VE×ZEA: vitamin C×vitamin E×zearalenone; VC×RUT×ZEA: vitamin C×rutin×zearalenone; VE×RUT×ZEA: vitamin E×rutin×zearalenone; VC×VE×RUT×ZEA: vitamin C×vitamin E×rutin×zearalenone; VC×VE×Tan IIA×ZEA: vitamin C×vitamin E×tanshinone IIA×zearalenone; VC×VE×PUE×ZEA: vitamin C×vitamin E×puerarin×zearalenone; VC×VE×LCB×ZEA: vitamin C×vitamin E×licylocazone B×zearalenone; VC×VE×Tan IIA×PUE×LCB×ZEA: Vitamin C×Vitamin E×Tanshinone IIA×Puerin×Glycyrrhizic chalcone B×Zea zearalenone; VC×VE×RUT×Tan IIA×ZEA: Vitamin C×Vitamin E×Rutin×Tanshinone IIA×Zea zearalenone; VC×VE×RUT×PUE×ZEA: Vitamin C×Vitamin E×Rutin×Puerin×Zea zearalenone; VC×VE×RUT×LCB×ZEA: Vitamin C×Vitamin E×Rutin×Glycyrrhizic chalcone B×Zea zearalenone; VC×VE×RUT×Tan IIA×PUE×LCB×ZEA: Vitamin C×Vitamin E×Rutin×Zea zearalenone×Tanshinone IIA×Puerin×Glycyrrhizic chalcone B; a, b, c indicate significant differences (P<0.05);

[0019] Figure 3The effects of each treatment group on the proliferation of pig hepatocytes under fluorescence microscope (n=6), where CON: control; ZEA: zearalenone; VC×VE×RUT×Tan IIA×PUE×LCB×ZEA: vitamin C×vitamin E×rutin×zearalenone×tanshinone IIA×puerarin×licoricechalcone B; a, b, c indicate significant differences (P<0.05), and the magnification ratio is 200μm;

[0020] Figure 4 The statistical results of the effects of zearalenone and six drugs on the proliferation of pig hepatocytes (n=6), where CON: control; ZEA: zearalenone; VC×VE×RUT×Tan IIA×PUE×LCB×ZEA: vitamin C×vitamin E×rutin×zearalenone×tanshinone IIA×puerarin×licoricechalcone B; a, b, c indicate significant differences (P<0.05);

[0021] Figure 5 The effect of zearalenone and six drugs on the production of active oxygen in pig hepatocytes under fluorescence microscope (n=6), where CON: control; ZEA: zearalenone; VC×VE×RUT×Tan IIA×PUE×LCB×ZEA: vitamin C×vitamin E×rutin×zearalenone×tanshinone IIA×puerarin×licoricechalcone B; a, b, c indicate significant differences (P<0.05), magnification ratio is 200μm;

[0022] Figure 6 The statistical results of the effects of zearalenone and six drugs synergistically treated groups on the production of reactive oxygen species in pig hepatocytes (n=6), where CON: control; ZEA: zearalenone; VC×VE×RUT×Tan IIA×PUE×LCB×ZEA: vitamin C×vitamin E×rutin×zearalenone×tanshinone IIA×puerarin×licoricechalcone B; a, b, c indicate significant differences (P<0.05). DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Example 1

[0029] 1 Test method

[0030] 1.1 Cell recovery

[0031] IPLs cells were donated by the College of Animal Science and Technology (Veterinary College) of Shandong Agricultural University. Take out the frozen cell cryotube from the liquid nitrogen tank, place it in a 37°C water bath, and take it out after thawing. Take a 15mL centrifuge tube and add 5mL of complete cell culture medium (89% Dulbecco's Modified Eagle's Medium, DMEM)-high glucose medium (SH30022.01, Hyclone, USA) + 10% fetal bovine serum (FBS, 10099141, Gibco, USA) + 1% penicillin-streptomycin mixture (PS, 15140122, Gibco, USA). Transfer the thawed cells to a centrifuge tube using a pipette, gently blow and mix, centrifuge at 1000r / min for 3min, discard the supernatant, add 1mL of complete cell culture medium, gently blow and mix 40-50 times to resuspend the cells, take a 100mm culture dish, use a Pasteur pipette to add 9mL of complete cell culture medium, evenly inoculate the mixed cell suspension into the culture dish, gently shake it crosswise on the flat surface, observe the cell status under a microscope, and culture it in a 37℃, 5% CO2 cell culture incubator if there is no abnormality.

[0032] 1.2 Cell passaging

[0033] When the cells occupy about 80% of the entire culture dish area, they can be passaged. Use a Pasteur pipette to suck out the culture medium in the culture dish and discard it, add 2mL PBS to rinse with a pipette, suck out and discard it, add 2mL 0.25% trypsin (15400054, Gibco, USA), gently shake it to make it evenly distributed, put it in a 37℃, 5% CO2 cell culture incubator for digestion for 2min, observe the cell state under a microscope, and after most cells become round and free, add 4mL of complete cell culture medium to stop digestion, gently blow and mix, transfer the cells to a 15mL centrifuge tube, centrifuge at 1,000r / min for 3min, discard the supernatant, add an appropriate amount of complete cell culture medium, gently blow and mix 40-50 times, resuspend the cells, and evenly inoculate them into a culture dish containing complete cell culture medium. Observe the cell state under a microscope. If there is no abnormality, put it in a 37℃, 5% CO2 cell culture incubator for culture.

[0034] 1.3 Cell cryopreservation

[0035] When the cells occupy about 80% of the entire culture dish area, the cell collection steps are the same as 1.2, and the cell freezing solution (80% complete cell culture medium + 10% FBS + 10% dimethyl sulfoxide (DMSO) (VWRC0231, Amresco, USA) is added, and the cells are gently pipetted and mixed for 40-50 times to resuspend the cells, and the cells are dispensed into sterile cryopreservation tubes (labeled with the generation number, date, name and cell name), placed at 4°C for 0.5h, transferred to -20°C for 0.5h, transferred to -80°C for 24h, and then transferred to a liquid nitrogen tank for long-term storage.

[0036] 1.4 Cell treatment

[0037] 1.4.1 Toxin and drug configuration

[0038] ZEA: Zearalenone; VC: Vitamin C; VE: Vitamin E; Tan IIA: Tanshinone IIA; PUE: Puerarin; LCB: Licorice Chalcone B; RUT: Rutin. Dissolve ZEA (Fermentek, Israel, purity ≥ 98%) powder with DMSO and prepare a stock solution with a concentration of 20mmol / L, store it at -20°C in the dark, and use it to prepare ZEA with a suitable concentration. The previous study of the present invention found that the suitable concentration of ZEA acting on IPLs is 60μmol / L for 24h. Therefore, the present invention also uses the same concentration and treatment time. Dissolve Vitamin C (VC, Solarbio, China, purity ≥ 98%) powder with DMSO and prepare it with a stock solution with a concentration of 20mmol / L, store it at 4°C, and use it to prepare VC with different concentrations. Vitamin E (VE, Solarbio, China, purity ≥ 98%) powder was dissolved in diethyl pyrocarbonate (DEPC) water (R0021, Beyotime, China) and prepared into a stock solution with a concentration of 20 mmol / L, which was stored at 4°C for the preparation of different concentrations of VE. Cocrystal rutin (RUT, Eutectic Technology, China, 75% rutin, the remaining 25% L-proline) powder was dissolved in DMSO and prepared into a stock solution with a concentration of 20 mmol / L, which was stored at 4°C for the preparation of different concentrations of RUT. Tanshinone IIA (Tan IIA, Huacui Biotechnology, China, purity ≥ 99%) powder was dissolved in DMSO and prepared into a stock solution with a concentration of 20 mmol / L, which was stored at 4°C for the preparation of different concentrations of Tan IIA. Puerarin (PUE, Anhui Medical University, China, purity ≥ 98%) powder was dissolved in DMSO and prepared into a stock solution with a concentration of 20 mmol / L, stored at 4°C, and used to prepare different concentrations of PUE. Licorice chalcone B (LCB, Herbest Biotech, China, purity ≥ 98%) powder was dissolved in DMSO and prepared into a stock solution with a concentration of 20 mmol / L, stored at 4°C, and used to prepare different concentrations of LCB.

[0039] 1.4.2 Screening of toxin and drug concentrations in IPLs

[0040] Add 60 μL of 1 mg / mL poly-lysine solution to each well of the 96-well plate, place in the incubator for 10 min, aspirate and discard; add 100 μL of PBS to each well, place in the incubator for 10 min, aspirate and discard, wash twice with PBS, and dry. When the cells occupy about 80% of the entire culture dish area, resuspend the cells with trypsin, follow the same steps as 1.2, count the cells using a counting plate, dilute the cells with complete cell culture medium, and inoculate 10 cells per well. 4IPLs of cells were placed in 96-well plates. When the cells grew to 70% of the bottom area of ​​the culture dish, the IPLs cells were treated with 0, ZEA 60μmol / L, ZEA 60μmol / L×VC (0.5, 1, 2, 4 and 6μmol / L) for 24 h, the cell viability was detected, and the final concentration of ZEN×VC for treating IPLs cells was screened out; the IPLs cells were treated with 0, ZEA 60μmol / L, ZEA 60μmol / L×VE (15, 20, 25, 30 and 35μmol / L) for 24 h, the cell viability was detected, and the final concentration of ZEN×VE for treating IPLs cells was screened out; the IPLs cells were treated with 0, ZEA 60μmol / L, ZEA 60μmol / L×RUT (20, 40, 50, 60, 80μmol / L) for 24 h, the cell viability was detected, and the final concentration of ZEN×RUT for treating IPLs cells was screened out; IPLs cells were treated with 0, ZEA 60μmol / L, ZEA 60μmol / L×PUE (10, 20, 40, 60, 80μmol / L) for 24h, the cell viability was detected, and the final concentration of ZEN×PUE for IPLs cells was screened out; IPLs cells were treated with 0, ZEA 60μmol / L, ZEA 60μmol / L×LCB (10, 20, 40, 60, 80μmol / L) for 24h, the cell viability was detected, and the final concentration of ZEN×LCB for IPLs cells was screened out; 0, ZEA 60μmol / L, ZEA 60μmol / L×LCB were used to prepare the above-screened VC, VE, Tan IIA, PUE, LCB and RUT concentrations. Twelve drug treatment groups, including 60 μmol / L, VC×VE×ZEA, VC×RUT×ZEA, VE×RUT×ZEA, VC×VE×RUT×ZEA, VC×VE×Tan IIA×ZEA, VC×VE×PUE×ZEA, VC×VE×LCB×ZEA, VC×VE×Tan IIA×PUE×LCB×ZEA, VC×VE×RUT×Tan IIA×ZEA, VC×VE×RUT×PUE×ZEA, VC×VE×RUT×LCB×ZEA and VC×VE×RUT×Tan IIA×PUE×LCB×ZEA, were used to detect cell viability and screen the optimal treatment group.

[0041] 1.4.5 Cell viability assay

[0042] Cell inoculation and toxin and drug treatment were the same as in 1.4.2. CCK-8 kit (C0038, Beyotime, Shanghai, China) was used to detect IPLs cell viability. 10 μL CCK reagent was added to each well and placed in a cell culture incubator for 2 h. The absorbance value was measured at a wavelength of 450 nm on a microplate reader to calculate the cell viability.

[0043] 1.4.6 Cell proliferation assay

[0044] Cell inoculation and toxin and drug treatment were the same as in 1.4.2. The EdU cell proliferation detection kit (C10310-1, Beyotime, Shanghai, China) was used to detect the proliferation of IPLs cells. After the cells were treated with toxins and drugs for 22 h, 100 μL of complete cell culture medium containing 50 μmol / L EdU was added to each well and incubated in a cell culture incubator for 2 h. The cells were aspirated and discarded. The cells were washed twice with PBS, and 50 μL of cell fixative (PBS containing 4% paraformaldehyde) was added and incubated at room temperature for 30 min. The cells were aspirated and discarded. 50 μL of 2mg / L glycine, incubate for 5min, aspirate and discard, wash with PBS, add 100μL permeabilization agent (PBS containing 0.5% TritonX-100), incubate for 10min, aspirate and discard, wash with PBS, add 100μL Apollo staining reaction solution, incubate at room temperature for 30min in dark, aspirate and discard, add 100μL Hoechst33342 reaction solution, incubate at room temperature for 30min in dark, aspirate and discard, wash with PBS and observe under 40x fluorescence microscope.

[0045] 1.4.7 Detection of Cellular Reactive Oxygen Species Production

[0046] Cell inoculation and toxin and drug treatment were the same as in 1.4.2. A positive control well was set up, and the ROS production in IPLs cells was detected using a ROS detection kit (S0033M, Beyotime, Shanghai, China). After the cells were treated with toxins and drugs for 23.5 h, the ROS positive control reagent was added to the positive control well (Rosup, 50 mg / mL), and the cells were placed in the incubator for further culture for 0.5 h, and the reactive oxygen fluorescent probe with a final concentration of 10 μmol / L was added. After incubation in the incubator for 20 min, the cells were washed 3 times, observed under a 40x fluorescence microscope, and the fluorescence intensity was scanned using ImageJ software.

[0047] 2 Results and analysis

[0048] 2.1 Cell viability assay

[0049] The effects of ZEA 60 μmol / L×VC (0.5, 1, 2, 4, and 6 μmol / L), ZEA 60 μmol / L×VE (15, 20, 25, 30, and 35 μmol / L), ZEA 60 μmol / L×Tan IIA (2, 5, 8, 10, and 15 μmol / L), ZEA 60 μmol / L×PUE (10, 20, 40, 60, and 80 μmol / L), ZEA 60 μmol / L×LCB (10, 20, 40, 60, and 80 μmol / L), and ZEA 60 μmol / L×RUT (20, 40, 50, 60, 80, and 100 μmol / L) on the viability of IPLs cells after 24 h of treatment are shown in the table. Figure 1 . As shown in the figure, compared with the control group, 60μmol / LZEA can significantly reduce the cell viability of IPLs (P<0.05); 2μmol / LVC, 20μmol / LVE, 10μmol / L Tan IIA, 40μmol / LPUE, 20μmol / L LCB and 60μmol / L RUT can significantly increase the cell viability of IPLs (P<0.05). Therefore, ZEA 60μmol / L×VC 2μmol / L, ZEA 60μmol / L×VE 20μmol / L, ZEA 60μmol / L×Tan IIA 10μmol / L, ZEA 60μmol / L×PUE 40μmol / L, ZEA 60μmol / L×LCB 40μmol / L and ZEA 60μmol / L×RUT 60μmol / L were selected for subsequent experiments.

[0050] The effects of each treatment group on the viability of IPLs cells after 24 h of treatment were shown in Table 2. Figure 2 .Depend on Figure 2 It can be seen that each treatment group can significantly improve the cell viability of IPLs (P<0.05), among which the cell viability of IPLs in the VC×VE×RUT×Tan IIA×PUE×LCB×ZEA treatment group was most significantly improved. Therefore, this treatment group was selected for subsequent experiments.

[0051] 2.2 Cell proliferation assay

[0052] The effects of ZEA and VC×VE×RUT×Tan IIA×PUE×LCB×ZEA on the proliferation of IPLs cells after 24 h of synergistic treatment are shown in Figure 3 and 4 As can be seen from the figure, compared with the control group, 60 μmol / L ZEA can significantly inhibit the proliferation of IPLs cells (P<0.05); the VC×VE×RUT×Tan IIA×PUE×LCB×ZEA synergistic treatment group can significantly restore the inhibition of IPLs cell proliferation by ZEA (P<0.05).

[0053] 2.3 Detection of cell reactive oxygen species production

[0054] The effects of ZEA and VC×VE×RUT×Tan IIA×PUE×LCB×ZEA on the proliferation of IPLs cells after 24 h of synergistic treatment are shown in Figure 5 and 6 As can be seen from the figure, compared with the control group, 60μmol / LZEA can significantly increase the production of ROS in IPLs cells (P<0.05); the VC×VE×RUT×Tan IIA×PUE×LCB×ZEA synergistic treatment group can significantly restore the situation in which ZEA increases the production of ROS in IPLs cells (P<0.05).

[0055] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of a rutin composition in the preparation of a drug for alleviating the toxicity of pig liver cells, characterized in that: The rutin composition comprises rutin, vitamin C, vitamin E, tanshinone IIA, puerarin and licorice chalcone B.

2. The use according to claim 1, characterized in that: The porcine hepatocyte toxicity was induced by zearalenone.

3. The use according to claim 1, characterized in that: The mitigation of porcine liver cell toxicity includes improving IPLs cell viability.

4. The use according to claim 1, characterized in that: The mitigation of porcine hepatocyte toxicity includes restoring zearalenone to inhibit IPLs cell proliferation.

5. The use according to claim 1, characterized in that: The mitigation of porcine hepatocyte toxicity includes restoring zearalenone to increase ROS production in IPLs cells.

6. A drug capable of alleviating the toxicity of pig liver cells, characterized in that: The effective ingredients include a rutin composition; the rutin composition includes rutin, vitamin C, vitamin E, tanshinone IIA, puerarin and licorice chalcone B.

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