CN113546070B - Use of oligomeric stilbene monomer compound Isohop in the preparation of products for increasing animal fat deposition - Google Patents
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Abstract
The invention discloses application of an oligomeric stilbene monomer compound Isohop in preparation of products for improving animal fat deposition, and through activity research on the oligomeric stilbene monomer compound Isohop, the application of the oligomeric stilbene monomer compound Isohop in preparation of products for improving animal fat deposition proves that the oligomeric stilbene monomer compound Isohop can promote accumulation of lipid in C2C12 cells and improve expression levels of transcription factors PPARgamma and FABP 4.
Description
Technical Field
The invention belongs to the field of development of pharmaceutical active compounds in plants, and particularly relates to application of an oligomeric stilbene monomer compound Isohop in preparation of products for improving animal fat deposition.
Background
Stilbene compounds are a generic term for a class of compounds having a 1, 2-stilbene skeleton and polymers of their composition. Is mainly present in xylem of plant, and is found in Rheum officinale of Polygonaceae at the earliest, and comprises stilbene, diphenylethyl, phenanthrene monomers and polymers thereof. The oligomeric stilbenes are non-flavone polyphenols and are present in gentiaceae, paeoniaceae, gnetaceae, rosaceae, cyperaceae, leguminosae, moraceae, papilionaceae, iridaceae, vitaceae, etc. In recent years, research reports that stilbene compounds have various biological activities, such as neuroprotection, antivirus, antibacterial, anti-inflammatory, anti-tumor, antioxidant, anti-AIDS and liver protection effects.
Iris lacteal pall.var.chinensis (Fisch.) Koidz.) and the like, are perennial herb perennial root plants of Iris genus (Iris) of the phylum Iridaceae (Angiospermae) and the like. Iris is a traditional Chinese herbal medicine, and is a whole herb of plant Iris lactea according to Chinese herbal medicine, and has bitter and slightly sweet taste and slightly cold nature. It enters kidney, bladder and liver meridians. It has effects of clearing heat and detoxicating, promoting urination and treating stranguria, promoting blood circulation and detumescence, and can be used for treating pharyngitis, stranguria with turbid urine, arthralgia, carbuncle and malignant sore etc., and leaf, flower, root and seed of Iris lactea can also be used as medicines. The Chinese iris can be used as a medicine after being dried in the sun, has salty, sour, bitter and slightly cool taste, has the functions of clearing heat and cooling blood, and is used for treating symptoms such as sore throat, hematemesis and the like; the Chinese iris root has flat nature and sweet taste, has the effects of clearing heat and detoxicating, and is used for treating symptoms such as toothache, acute infectious hepatitis, pharyngitis, and the like; the seeds have the effects of clearing heat and detoxicating, promoting diuresis and stopping bleeding, and can be used for treating jaundice, leucorrhea, diarrhea, hematemesis and other diseases. Modern pharmacology shows that Iris lactea has various pharmacology effects, such as radiosensitization, antibiosis, immunity enhancement, anticancer, glycolipid metabolism improvement and the like.
Disclosure of Invention
In livestock production, intramuscular fat deposition affects the cross-sectional marble score of meat, closely related to the quality of meat (e.g., pork, beef, etc.). Muscle fat mainly includes intramuscular fat and intramuscular fat. Intermuscular fat is the fat between muscle fiber bundles; intramuscular fat is present on the adventitia, fasciculi, and even on the endometrium, and fat is present on capillaries of the myofibrillar membrane of livestock with good nutritional status. The intramuscular fat content is closely related to the organoleptic quality and the edible quality of meat, and directly influences the flavor, juiciness, tenderness, color and the like of the meat, and finally influences the meat quality and the economic value. Therefore, the research of regulating and controlling fat deposition in meat is of great significance to the healthy development of animal husbandry and the satisfaction of consumers for high-quality meat.
In Bohai ocean and the like (influence of ademetionine on adipogenic differentiation and fat deposition of myoblasts, 2012), the influence of methionine metabolite ademetionine (SAM) on adipogenic differentiation and fat deposition of muscle-derived pluripotent stem cells is studied by taking mouse myoblasts (C2C 12) as a model, and the C2C12 cells treated by SAM show morphological characteristics of fat cells; intracellular fat deposition levels increased and exhibited dose-dependent effects with increasing SAM treatment concentrations; the expression level of mRNA and protein of fat specific genes PPARgamma and C/EBP alpha of cells is obviously increased, which shows that SAM can promote the adipogenic differentiation of C2C12 cells and the deposition of fat in cells, thereby increasing the content of intramuscular fat in animal muscles and obviously improving the quality of meat products.
Guo Gongfang et al (Kruppel-Like Factor 3 (KLF 3) gene effect on beef fat deposition, 2019) disclose that beef quality is mainly affected by fat deposition, fat deposition is mainly closely related to fat cell differentiation and fatty acid metabolism, and how to increase intramuscular fat content to meet the needs of consumers on beef tenderness and flavor is always a hot spot of beef fat deposition biomolecule regulation research, so that the document provides a certain theoretical basis for further defining the effect of KLF3 gene in regulating beef fat deposition by determining the effect of KLF3 gene in beef fat cell differentiation and fatty acid metabolism.
The patent CN110590881 separates and identifies various compounds from Iris lactea, and finds that stilbene compounds in Iris lactea have the effect of inhibiting lipid accumulation. Surprisingly, however, the present inventors have found that oligomeric stilbene based monomer compounds Isohop, which have lipid-elevating effects, promote intramuscular cellular lipid accumulation and can enhance muscle fat deposition in animals.
In the experiment, the mouse myoblasts C2C12 cells are adopted for the experiment, and C2C12 is the representative of the pluripotent stem cells of muscle sources and is the in vitro model most commonly used for researching muscle development.
In the application of the oligomeric stilbene monomer compound Isohop provided by the invention, the chemical structural formula of the oligomeric stilbene monomer compound is as follows:
the products of the present invention include, but are not limited to, pharmaceuticals, feeds, etc.
Further, the product is a product that promotes intramuscular fat deposition in an animal.
Further, the product is a product that promotes accumulation of cellular lipids.
Lipid droplets are currently believed to form on the Endoplasmic Reticulum (ER), where small molecules including triglycerides, glycerol and free fatty acids are transferred into the cell, where they are synthesized and secreted into the cell matrix in the form of lipid droplets.
Further, the product is a pparγ agonist.
The PPARgamma agonist is a product for increasing the expression level of PPARgamma.
Further, the product is a FABP4 agonist.
The FABP4 agonist is a product for increasing the expression level of FABP 4.
Further, the product is a Caveolin-1 agonist.
The Caveolin-1 agonist is a product for improving the expression level of Caveolin-1.
Further, the animal is selected from the group consisting of pigs and cattle.
Further, the product is a product for preventing and/or treating a muscle lipid metabolism disorder.
The invention provides an application of the oligomeric stilbene monomer compound VitD in preparing products for improving animal meat quality.
The invention also provides application of the oligomeric stilbene monomer compound in preparing any one of PPARgamma agonist, FABP4 agonist and Caveolin-1 agonist.
The beneficial effects of the invention are as follows:
(1) The oligomeric stilbene monomer compound provided by the invention can promote accumulation of lipid drops in C2C12 cells and increase the content of triglyceride, and can improve the expression level of transcription factors PPARgamma and FABP4, promote accumulation of lipid and remarkably improve intramuscular fat deposition.
(2) The invention more comprehensively digs the medicinal value of the iris, expands the application of the iris, and has important significance in the life science field, the medical science field, the animal husbandry field and the like.
Drawings
FIG. 1 effect of oligomeric stilbene tetramer compounds on lipid accumulation of C2C12 cells;
FIG. 2 effect of oligomeric stilbene tetramer compounds on triglyceride content of C2C12 cells;
FIG. 3 effect of oligomeric stilbene tetramer compounds on the expression levels of C2C12 cell transcription factors.
Detailed Description
The use of the oligomeric stilbene monomer compounds for ameliorating disorders associated with lipid metabolism is further illustrated by the following examples and experiments.
Materials and reagents
The test compound is an oligomeric stilbene monomer compound separated from Iris pallium seed, and is epsilon-Viniferin (epsilon-vin), vinisinA-13 b-o-glucoside (VitAOG), vinisinA (VitA), vinisinD (VitD), hopeaphenol (Hop) and Isohopeaphenol (Isohop) respectively. The chemical structural formula of all the compounds is shown as follows:
C2C12 mouse myoblasts were purchased from the Shanghai cell bank of the national academy of sciences.
1. Induced differentiation of C2C12 cells
The C2C12 cells are induced to differentiate by adopting a cocktail method, and the induced differentiation method is as follows: inoculating cells with good cell state on culture plate, and plating with density of 5×10 4 Each/mL was incubated with 10% FBS-containing high-sugar DMEM mediumCulturing until cell density reaches 85% -90%, removing the complete culture solution after two days of contact inhibition, adding induced liquid (0 d when induced liquid is added), culturing cells 2d, then changing to induced liquid II, culturing cells 2d, changing to normal culture solution, culturing for 4d, and changing liquid every other day. At 8d of induction, the appearance of a large number of lipid droplets in the cell was observed.
C2C12 cell induced I culture medium: 10 μg/mL Insulin, 0.5mM IBMX, 1 μM Dex, and 10 μM Rosi;
C2C12 cell-induced II culture broth: 10. Mu.g/mL Insulin and 10. Mu.M Rosi.
2. SRB method for measuring cell viability
Sulforhodamine B (SRB) colorimetry is a method for detecting cell proliferation. The method comprises the following specific steps: (1) Cells were grown in 1X 10 cells 5 Is inoculated in 96-well plates, cells are cultured in high-sugar DMEM medium containing 10% FBS, and an oligomeric stilbene extract is added for 48 hours. (2) After the incubation, the stock solution was discarded, 100. Mu.L of fresh DMEM was added to each well, followed by 25. Mu.L of 50% TCA (protected from light), left at room temperature for 5min, and left at 4℃for 1h. (3) The stock solution was discarded, each well was rinsed 5 times with flowing ultra pure water, and the wells were forcibly thrown out after each rinsing. And (5) inverted on an ultra-clean workbench for 15min to blow dry (high wind). (4) 70 μl of 0.4% SRB was added to each well and shaken for 30min (protected from light). (5) The SRB dye solution was discarded, and the fixed cells were rinsed 4 times with 1% glacial acetic acid per well, and forced out after each rinsing. And (5) inverted on an ultra-clean workbench for 15min to blow dry (high wind). (6) mu.L of 100mM Tribase was added to each well and the mixture was incubated at 37℃for 20min with shaking. (7) detecting the OD value at 540nm wavelength by using an enzyme-labeled instrument. Cell viability was calculated according to the formula.
Cell viability (%) = (drug group OD/blank group OD) ×100%
Example 1 Effect of oligomeric stilbene monomer Compounds on the relevant index of C2C12 cells
Different induction times C2C12 intracellular lipid droplet accumulation conditions
1. Oil red O dyeing
Cells with good cell status were inoculated on 6-well plates with plating densities of 5×10 4 individual/mL, waiting for cell densityAnd (3) performing induction differentiation when the degree reaches about 85% -90%, wherein the induction process is as described in 2.1, fixing the cells for 30min by using 4% neutral formaldehyde after the induction is finished, adding the oil red O working solution prepared in advance to dye the surfaces of the cells after the cell fixing is finished, and standing for 60min in a dark place. After the dyeing is finished, washing the cells with 70% ethanol, discarding redundant dye, washing with ultrapure water for 3-4 times, and finally observing and photographing under a microscope. The results are shown in FIG. 1.
Determination of TG content
The cells were grown at 5X 10 4 Inoculating the cell density of the cell to a 6-hole plate, and carrying out induced differentiation when the cell density reaches about 85% -90%, and carrying out TG content determination on the 8 th induced day, wherein the specific method is as follows: (1) cell pretreatment: at the 8 th day of induction, the cell culture solution was aspirated away, and cells were digested by adding pancreatin digest after washing twice with cold PBS. (2) cell collection: after cell digestion, cells were resuspended in PBS and centrifuged at 1000g for 5min to collect cell pellet. (3) ultrasonic disruption: an appropriate amount of PBS was added to the collected pellet, followed by ultrasonication (3 min). (4) measurement: mu.L of cell disruption suspension was added to each well of a 96-well plate, 2. Mu.L of distilled water was added to a blank well, 2. Mu.L of standard substance was added to a standard well, 200. Mu.L of assay solution was added to each well, and after incubation at 37℃for 10min, absorbance was read at 510 nm. And measuring the protein concentration in the sample to be measured by means of the BCA method, correcting, and finally calculating the content of TG according to a formula. The results are shown in FIG. 2.
TG content= (OD sample-OD blank)/(OD proof-OD blank). Times.calibrator concentration (mM)/protein concentration of sample to be measured (gprot/L)
3. Effect of oligomeric stilbene monomer Compounds on the expression level of C2C12 cell lipid-forming transcription factors
C2C12 cells with good growth state were cultured at 5×10 4 Inoculating the cell sample on a 6-hole plate at the density of each mL, performing induced differentiation when the cell density reaches about 85% -90%, adding a tested compound for treatment during induction, collecting cells after the induction is finished, and extracting protein from the cell sample obtained by treatment, wherein the specific operation method comprises the following steps:
(1) The RIPA lysate and PMSF are mixed uniformly in a ratio of 100:1 (e.g., phosphatase inhibitor is added when analyzing phosphorylated proteins) to prepare a cell lysate. Cell sample: cells in 6-well plates were washed 3 times with cold PBS, 100. Mu.L of cell lysate was added to each well, lysed on ice for 5min, then scraped with a cell scraper, collected into a 1.5mL centrifuge tube, and again placed in a refrigerator at 4℃for full lysis for 30min. Finally, the liver tissue homogenate and the cell lysate are centrifuged at 12000g at 4℃for 15min, and the supernatant protein is taken out in a new EP tube.
(2) BCA assay for protein content: taking out an appropriate amount of protein sample, diluting the protein standard with PBS buffer solution to 0.5mg/mL according to the requirements of the BCA protein quantitative kit instruction, and mixing the reagent A in a ratio of 50:1: and (3) uniformly mixing the reagent B to prepare a working solution. Standards and PBS were added to 96-well plates as shown in table 1, with 20 μl per well, and typically 3 multiplex wells per concentration, to draw standard curves. 1 μl of protein sample was added to a 96-well plate and 20 μl of PBS was added. 200 mu L of BCA working solution is added into each hole, the mixture is fully and uniformly mixed, the samples are subjected to oscillation incubation for 30min at 37 ℃ by a microplate incubation oscillator, absorbance values are measured at 562nm of an enzyme label instrument, and the protein concentration of the samples is calculated according to a standard curve. The prepared protein sample is heated at 100deg.C for 10min to completely denature the protein. And before protein loading, the sample is centrifugally and uniformly mixed for use.
TABLE 1BCA method protein quantification Table
(3) SDS-PAGE electrophoresis: selecting proper concentration of separating gel according to molecular weight of target protein, preparing proper solution system (lower gel), standing the lower gel at room temperature for more than 40min until the lower gel is completely coagulated, preparing 5% concentrated gel (upper gel), and inserting into comb with proper size. After the concentrated gel is solidified, placing the gel plate into an electrophoresis tank, filling electrophoresis liquid between the two plates, pulling out a comb, and running the gel after a protein sample and a marker are added. Electrophoresis conditions: s1, 80V for 30min; and S2, 120V, about 60-90 min, and ending electrophoresis until bromophenol blue in the buffer solution runs to the lowest end of the separation gel.
(4) Transferring: and immediately transferring the membrane after the protein electrophoresis is finished, and cutting the position of the target protein according to the molecular weight according to the marker band. And taking out the PVDF film, cutting the PVDF film into the same size as the glue, placing the cut film into a methanol solution for activating for 1min, and then placing the cut film into a film transferring solution. And (3) clamping the electric transfer clamp according to the sequence of the transfer clamp blackboard, the filter paper, the adhesive PVDF, the filter paper and the transfer clamp whiteboard, placing the blackboard opposite to the black surface of the groove, placing the electrophoresis tank, filling the transfer membrane liquid into the whole groove, and placing the transfer membrane instrument into an ice-water mixture after electrifying. The film transfer condition is constant current 250mA for 90min.
(5) Closing: after the transfer was completed, the PVDF membrane was quickly gently removed from the transfer plate with forceps, rinsed in TBST buffer for 5min, and immediately thereafter blocked with TBST containing 5% nonfat milk powder on a room temperature shaker for 1h.
(6) Incubation resistance: after the end of the blocking, the PVDF membrane was placed in the diluted primary antibody solution and incubated overnight at 4℃on a horizontal shaking table with slow shaking.
(7) Secondary antibody incubation: after the primary antibody incubation is finished, the membrane is washed three times for 8min once by TBST. The PVDF membrane was then incubated in solution with HRP-labeled murine or rabbit secondary antibodies for 1h at room temperature with slow shaking.
(8) Developing: after the secondary antibody incubation is finished, the membrane is washed three times by TBST for 8min once. After the film washing is finished, the mixed ECL developing solution is dripped on the PVDF film, and a gel imaging system is used for photographing.
(9) Analysis of results: the gray values of the target protein and the internal reference protein were quantitatively analyzed by Image J software, and the relative expression amount of the protein was the ratio of the target protein to the internal reference protein (ratio of phosphorylated to non-phosphorylated protein).
Statistical analysis
The graph pad Prism7 software is used for mapping and statistical analysis, independent sample t test is adopted for comparison of the average numbers between two groups, one-way ANOVA (one-way ANOVA) is adopted for mutual comparison of the average numbers between multiple groups of data, the statistical data are expressed as mean value + -standard deviation (x + -s), the expression of P <0.05 has statistical significance, and the expression of P <0.01 has very significant statistical significance.
Results
As shown in FIG. 1, there was a large accumulation of lipid droplets in the induced differentiation cell diff group compared to the undifferentiated cell undiff group (P < 0.01). Compounds VitAOG, vitA and Hop significantly reduced accumulation of intracellular lipid droplets (P < 0.05) after treatment, whereas compounds VitD and Isohop significantly increased accumulation of lipid droplets (P < 0.01). After the dyeing is finished, the relative content of lipid drops is analyzed by using the isopropanol dissolution and coloring lipid titration amount, and the experimental result shows that the relative quantitative result of the lipid drops is consistent with the result of oil red O dyeing.
As a result, as shown in fig. 2, the intracellular TG content of the induced differentiated cell diff group was significantly increased (P < 0.01) compared to the undifferentiated cell undiff group. The intracellular TG content showed a different trend of change after tetrameric compound treatment, wherein the compound VitAOG was able to significantly reduce intracellular TG content (P < 0.01), whereas TG content in VitD, isohop treated cells was elevated, wherein TG content in Isohop treated cells was significantly elevated (P < 0.01).
The results are shown in FIG. 3, and the expression levels of the transcription factors PPARgamma and FABP4 in the induced differentiated cell diff group are significantly increased (P < 0.01) compared with the undifferentiated cell undiff group. Tetrameric compounds VitAOG, vitA and Hop significantly reduced pparγ, FABP4 expression levels (P < 0.01), whereas compounds VitD and Isohop significantly increased pparγ, FABP4 expression levels (P < 0.01).
In conclusion, the oligomeric stilbene monomer compounds VitAOG, vitA, hop and epsilon-Vin can inhibit accumulation of lipid droplets and TG content of cells and reduce expression levels of proteins such as PPARgamma and FABP4, while the compounds VitD and Isohop can promote accumulation of lipid droplets in cells and increase expression levels of transcription factors PPARgamma and FABP4, and remarkably increase expression level of Caveolin-1.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.
Claims (4)
1. The use of an oligomeric stilbene monomer compound Isohop for the preparation of a product for increasing the deposition of animal fat; the chemical structural formula of the oligomeric stilbene monomer compound is as follows:
the product is a product for improving animal meat quality.
2. The use according to claim 1, wherein the product is a product that promotes intramuscular fat deposition in an animal.
3. The use according to claim 1, wherein the product is a product that promotes accumulation of cellular lipids.
4. The use according to claim 1, wherein the animal is selected from the group consisting of pigs, cattle.
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| CN110590881B (en) * | 2019-09-23 | 2023-05-16 | 中国科学院西北高原生物研究所 | Oligomeric stilbene compounds in Iris seed kernel, and extraction method and application thereof |
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| 唐炳华，等.《简明生物化学》.中国中医药出版社,2019,第103页. * |
| 铁芳芳，等 .马蔺化学成分及药理作用研究进展.《天然产物研究与开发》.2017,第第29卷卷(第第29卷期),第2152-2156页. * |
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