CN112155208A - Application of garlic organic sulfide in preparing liver-protecting food, health-care product or medicine - Google Patents

Application of garlic organic sulfide in preparing liver-protecting food, health-care product or medicine Download PDF

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CN112155208A
CN112155208A CN202010472152.7A CN202010472152A CN112155208A CN 112155208 A CN112155208 A CN 112155208A CN 202010472152 A CN202010472152 A CN 202010472152A CN 112155208 A CN112155208 A CN 112155208A
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覃思
刘嘉祺
吕承豪
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Hunan Agricultural University
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • A61K31/105—Persulfides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Abstract

The invention belongs to the technical field of pharmacy, and particularly relates to application of garlic organic sulfide in preparation of liver-protecting food, health-care products or medicines. The invention discovers that the garlic organic sulfide can cause the differential expression of liver cancer cell genes after being treated, the DTS has the largest influence on the molecular function of the liver cancer cells after acting on the liver cancer cells, and the garlic sulfide mixture is used for intervening inflammatory cells to carry out protein immunoblotting detection on the expression of inflammation target protein COX-2, and the concentration of the garlic sulfide mixture in a certain range is found to be positively correlated with the anti-inflammatory capability. The garlic organic sulfide can achieve the purpose of protecting the liver by influencing the metabolic pathway and the stress signal pathway of liver cells, is used as a food functional component, has small toxic and side effects, can be used for preparing liver-protecting foods, health-care products or medicines, and develops a new application for the application of the garlic organic sulfide.

Description

Application of garlic organic sulfide in preparing liver-protecting food, health-care product or medicine
Technical Field
The invention belongs to the technical field of pharmacy, and particularly relates to application of garlic organic sulfide in preparation of liver-protecting food, health-care products or medicines.
Background
The liver is the largest digestive gland in the digestive system of the human body and is also the main organ for synthesizing and metabolizing urea. The liver has the main functions of secreting bile, storing glycogen, regulating metabolism of protein, fat and carbohydrate, and the like, and also has the functions of detoxification, hematopoiesis and blood coagulation. Meanwhile, the liver is the largest detoxification organ in the human body, and poisons and wastes generated in the body, ingested poisons, medicines damaging the liver and the like also need to be detoxified by the liver. The liver also breaks down toxic substances absorbed by the intestine or produced by other parts of the body, and then excreted as harmless substances into the bile or blood for subsequent discharge outside the body. When the liver function is abnormal, the metabolic decomposition ability, detoxification ability and bile secretion ability of the human body are reduced.
The main bioactive substance of garlic is garlic organic sulfide (OSCS), which has pungent and spicy taste. Can be divided into two main categories of fat-soluble organic sulfide and water-soluble organic sulfide. The fat-soluble organic sulfur compounds are classified into monosulfur compounds, disulfide compounds, trisulfide compounds and tetrasulfide compounds according to the number of S atoms contained therein. The monosulfide is mainly diallyl monosulfide (DAS). Disulfide compounds are mainly diallyl disulfide (DADS), E, Z-ajoene (ajoene). The trisulfide compounds are mainly diallyl trisulfide (DATS). The tetrasulfide compound is mainly diallyl tetrasulfide compound (DTS). Diallyl thiosulfinate (i.e., allicin) is also a fat-soluble organic sulfide. The water-soluble organosulfur compounds are mainly S-allyl-L-cysteine (SAC) and S-allylmercapto-L-cysteine (SAMC).
At present, the research on the physiological effects of garlic organic sulfide comprises antioxidant activity, anti-inflammatory activity, diabetes inhibition activity, anticancer activity and the like, but the influence of the garlic organic sulfide on the biological function of human liver cells is not researched and reported.
Disclosure of Invention
In view of the above technical problems, the present invention aims to provide an application of garlic organosulfur compounds in preparing liver-protecting foods, health products or medicines.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an application of garlic organic sulfide in preparing liver-protecting food, health-care products or medicines, wherein the garlic organic sulfide comprises one or more of diallyl disulfide, diallyl trisulfide and diallyl tetrasulfide;
the diallyl disulfide has the structural formula:
Figure BDA0002514642630000021
the structural formula of the diallyl trisulfide is as follows:
Figure BDA0002514642630000022
the diallyl tetrasulfide has the structural formula:
Figure BDA0002514642630000023
further, the food, health product or medicament comprises organic sulfide of garlic as an active ingredient.
Further, the mass ratio of diallyl disulfide, diallyl trisulfide and diallyl tetrasulfide in the garlic organic sulfide in the food, the health product or the medicine is 1: 1: 1.
further, the food or the health care product also comprises food additives or food raw materials.
Further, the food additive is one or more of a preservative, a coloring agent, a sweetening agent, a flavoring agent, a leavening agent, a thickening agent, an emulsifying agent and an antifoaming agent.
Furthermore, the medicine also comprises pharmaceutically acceptable auxiliary materials.
Further, the auxiliary materials are one or more of fillers, disintegrants, lubricants, suspending agents, adhesives, sweeteners, flavoring agents, preservatives and matrixes.
Furthermore, the dosage form of the medicine is one of capsules, tablets, granules, powder, oral liquid, pills and injections.
Compared with the prior art, the invention has the following beneficial effects:
the invention utilizes the gene chip technology to research the influence rule of organic sulfide (di, tri and tetra sulfur) of garlic on the whole genome expression and typical signal channel of human liver cells, and utilizes various analysis methods (GO analysis and IPA analysis) to research the interaction between the channels, finds that the garlic organic sulfide treatment can cause the differential expression of liver cancer cell genes, the DTS has the greatest influence on the molecular functions of liver cancer cells after the DTS acts on the liver cancer cells, the signal channels of Glutamate Metabolism, acid Phase Response Signaling, N RF2-mediated Oxidative Stress Response and the like related to NRF2-mediated Oxidative Stress are obviously changed after being acted by DTS, the molecular functions with the highest change scores under the influence of DTS are cancer, cell apoptosis and cell proliferation, through the analysis of the molecular network with the largest DTS influence, the molecular functions related to the first 3 molecular networks are obtained to correspond to the signal channels and diseases analyzed in the previous step. A cell inflammation model is constructed by using LPS to induce HepG2 cells, and intervention is carried out by using garlic sulfide mixture MIX (DAS: DADS: DATS mixed according to the mass ratio of 1: 1: 1) with different concentrations. The expression of inflammation target protein COX-2 is detected, and the results of the Western blot detection show that: the concentration of the garlic organic sulfide mixture is positively correlated with the anti-inflammatory capability within a certain range. The garlic organic sulfide can achieve the purpose of protecting the liver by influencing the metabolic pathway and the stress signal pathway of liver cells, is used as a food functional component, has small toxic and side effects, can be used for preparing liver-protecting foods, health-care products or medicines, and develops a new application for the application of the garlic organic sulfide.
Drawings
FIG. 1 shows the changes in gene expression after DADS treatment.
FIG. 2 shows the changes in gene expression after DATS treatment.
FIG. 3 shows the change in gene expression after DTS treatment.
FIG. 4 shows the real-time fluorescent quantitative PCR analysis of typical genes after garlic organosulfur treatment of HepG2 cells.
FIG. 5 shows a 10-large typical cell pathway affected after DTS treatment.
FIG. 6 shows the affected 10-mer functions after DTS treatment.
Fig. 7 is a molecular network with a first significance ranking affected by DTS.
Fig. 8 is a molecular network with significance rank second affected by DTS.
Figure 9 is a molecular network with third significance ranking affected by DTS.
FIG. 10 shows the result of the Western blot analysis of COX-2 protein of garlic sulfide mixture in the intervention of inflammatory cells.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The materials used in the following examples are all commercially available from conventional sources.
Application example
The application example provides application of garlic organic sulfide in preparing liver-protecting food, health-care products or medicines, wherein the garlic organic sulfide comprises one or more of diallyl disulfide, diallyl trisulfide and diallyl tetrasulfide.
The food or the health-care product also comprises food additives or food raw materials, wherein the food additives are one or more of preservatives, coloring agents, sweetening agents, flavoring agents, leavening agents, thickening agents, emulsifying agents and defoaming agents.
The medicine also comprises pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials are one or more of a filling agent, a disintegrating agent, a lubricating agent, a suspending agent, an adhesive, a sweetening agent, a flavoring agent, a preservative and a matrix; the dosage form of the medicine can be capsules, tablets, granules, powder, oral liquid, pills, injection and the like.
EXAMPLE 1 cell culture
(1) Cell resuscitation
Human hepatoblastoma HepG2 cell line (American ATCC model cell center) is taken for recovery, and a fast-melting method is adopted: the frozen tube was taken out of the liquid nitrogen and immediately placed in a 37 ℃ water bath to rapidly melt within 1 min. Then the cells are transferred into a culture vessel for culture.
(2) Cell culture
Culturing in DMEM medium containing 10% Fetal Bovine Serum (FBS) under constant temperature of 37 deg.C and stable 5% CO2A gaseous environment.
(3) Cell passage
1. Will be 25cm2Discarding old culture solution in the culture dish, adding 1ml of 0.01M PBS into a disposable pipette tip by using a pipette tip, slightly shaking, washing the cell surface, and washing away residual FBS to prevent influence on digestion of pancreatic enzymes;
2. sucking PBS with the tip of the gun, adding 1ml of 0.05% pancreatin solution to cover the bottom of the bottle, timing for 1min accurately, discarding most pancreatin, and placing in a cell culture box at 37 ℃ for 2 min;
3. observing the digestion condition of the cells by using an inverted microscope, wherein the cells gradually shrink and become round, the cell connection disappears, the space is increased, a few cells fall off from the bottle wall, at the moment, 2ml of DMEM/F12 culture medium (containing 10% FBS) for complete growth is rapidly added, and the cells are lightly blown by using a disposable sterilizing pipette to form a cell suspension;
4. transferring the digested cells into a centrifuge tube, and centrifuging for 5min at the rotating speed of 1000rpm or 800 rpm;
5. sucking out the supernatant with a suction tube, collecting the cell precipitate, adding 3-4ml DMEM/F12 culture medium containing 10% FBS to fully suspend the cells, adding antibiotics (common penicillin) according to the proportion of 1:1000 when necessary, and then respectively placing into 3-4 culture dishes to make the total amount of liquid in each bottle reach 5 ml;
6. the well-mixed cell culture dish was carefully placed under conditions of 37 ℃ and 5% C02The cells in the cell culture box are continuously cultured.
Example 2 cytotoxicity assays
MTT assay to check DSs for cytotoxicity. HepG2 cells were seeded at a density of 104 cells per well in 96-well plates and then preincubated at 37 ℃ for 24 h; then adding the specified dose (0, 1. mu.M, 10. mu.M and 20. mu.M) of garlic organosulfur compounds, and incubating for 24 h; MTT was then added to the plate (final concentration 0.5mg/ml) and incubated for 4 h; acidic isopropanol (HCl in isopropanol of 0.04-0.10N) was added to dissolve the formazan crystals and the Optical Density (OD) was measured at 575nm using a microplate reading spectrophotometer (Thermo Scientific, USA). Cell viability was determined by comparing DSs the OD of the treated cells with the OD of the untreated cells.
Example 3RNA extraction and Gene chip detection
HepG2 cells were pre-cultured in petri dishes for 24h and then treated with 10. mu.M concentration of DADS, DATS, DTS dissolved in 0.1% DMSO for 10 h. Total RNA was extracted using the Isogen RNA kit (Nippon Gene co., tokyo, japan) according to the instructions and RNA quality was evaluated by automated capillary gel electrophoresis on an agilent bioanalyzer 2100(Palo Alto, CA, usa). These total RNA samples were labeled according to the standard single cycle amplification and labeling protocol developed by Affymatrix (Santa Clara, Calif., USA), and the cRNA was labeled for 2h at 40 ℃.
(1) RNA extraction
The RNA extraction needs to be carefully operated, and the whole process is carried out in a fume hood by wearing disposable gloves and a mask, and the RNA extraction method in the using instruction of the Isogen RNA kit is referred to as the following steps:
1. adding 0.8mL of Isogen into each hole of a 6-hole plate subjected to one-time sterilization, fully blowing and beating for about 2min to fully crack the liquid, blowing and transferring the liquid into a 1.5mL EP tube by using a gun head after the liquid is changed into a state with better fluidity from a viscous state, blowing and beating for 2 times, uniformly mixing, and standing for 5 min;
2. phase separation: adding 200uL chloroform into an EP tube, violently mixing uniformly for 30s, standing for 30s, and violently mixing uniformly for 30 s;
RNA precipitation: the colorless aqueous phase was aspirated, about 200mL, transferred to a new EP tube (without aspiration of the intermediate and organic layers), then equal volume of isopropanol was added, vortexed and mixed for 30s, left to stand at room temperature for 10min, and centrifuged at 12000rpm for 5min at room temperature.
RNA washing: the white pellet at the bottom of the EP tube was RNA, the supernatant was carefully aspirated and discarded, 0.7mL of 75% ethanol was added to the pellet, and the RNA pellet was washed using vortex shaking. Then centrifuged at 12000rpm for 2min and the washing was repeated twice.
5. RNA dissolution: carefully sucking and discarding supernatant, and naturally drying the RNA precipitate in a clean bench for 10-20 min. Dissolving RNA in 20uL DEPC treated water, measuring RNA concentration by ultraviolet method, adjusting concentration, subpackaging RNA samples, and storing in an ultra-low temperature refrigerator at-80 ℃.
(2) Reverse transcription of RNA
Reverse transcribing the RNA extracted in step (1) into more stable cDNA by reference to the instructions of Promega First-Strand Synthesis of cDNA:
1. the whole procedure was performed on ice, adding to the PCR tube: 2ug total RNA, 1uL OLIGO (dT)18, add DEPC treated water to 12uL, mix well and centrifuge slightly briefly;
heating at 70 ℃ for 5min in a PCR instrument, taking out, and immediately cooling on ice for 30 s;
3. after a short centrifugation, on ice: add 5uL of 5 × Reaction Buffer, 1uL of rnase inhibitor, 1.25uL of 40mM Dntp mix, luL M-MuLV reverse transcriptase, DEPC water (final volume 25uL), mix well and centrifuge briefly;
4. put into a PCR instrument and programmed as follows: taking out at 37 deg.C for 60min, and cooling on ice;
5. the reverse transcribed cDNA was split and stored at-80 ℃.
6. Gene chip detection
Following amplification and labeling, GeneChip assays were performed using the Affimatrix Gene Chip Human U133plus 2.0Array system, Affymatrix GeneChip U133plus 2.0 chips containing oligonucleotides of more than 44K, and the hybridization fluorescence was scanned in an Affymatrix scanner. And obtaining a preliminary experiment result.
Gene chip results were first classified according to the gene ontology ID (GO ID) (http:// www.geneontology.org /), and then analyzed by the Ingeneity Path Analysis System (IPA) (http:// www.ingenuity.com).
Example 4 Gene chip expression profiling
According to the results of the preliminary experiments, the experimental group of HepG2 cells was treated with 10. mu. mol, while the control group was not treated with DADS, DATS, DTS. Under these conditions, HepG2 cells showed no cytotoxicity for treatment with DADS, DATS, DTS. Cellular mRNA was prepared and processed for hybridization to human oligonucleotide DNA chips as described in example 3.
(1) DADS processing
As shown in FIG. 1, in the DADS-treated gene chip-derived data, the expression of 3 genes was changed by 5-fold or more, and the expression of all 3 genes was up-regulated. The fold change in expression of 2 genes was between 4-fold and 5-fold, and the expression of all 2 genes was up-regulated. The fold change in expression of 13 genes was between 3-fold and 4-fold, with all 13 genes up-regulated. The expression of 110 genes varied between 2-fold and 3-fold, with 85 genes up-regulated and 25 genes down-regulated. In general, the fold change in expression of 128 genes (0.23% by weight, 128: 54675) among the 54675 genes was higher than 2 fold in the experimental group-derived data treated with DADS.
Genes whose expression was changed by 5-fold or more by treating HepG2 cells with DADS were F2RL2 and EGR 1. The gene whose expression varied between 4-fold and 5-fold was NCF 2. The genes with expression change fold between 3-fold and 4-fold include PALLD, SLC6A6, SLC7A11, FOS, SPP1, AGPAT9, LTB4DH, EID3, etc. From gene chip gene data, it is known that all genes whose gene expression changes by 3 times or more are up-regulated. There are 110 genes whose gene expression changes between 2-fold and 3-fold, and 85 genes whose expression is up-regulated, such as IL11, TLR6, TRIB1, PLEKHA3, FGL2, GCLC, TUSC3, JAG1, OSGIN1, and the like. The expression gene is down-regulated by 15 genes such as SEMA3C, SLC2A2, TncRNA, KRTAP1-3, FOXA1, MALAT1 and the like.
(2) DATS processing
As shown in fig. 2, in the gene chip source data after the DATS treatment, the expression of 4 genes was changed by 5 or more times, the expression of 3 genes was up-regulated, and the expression of 1 gene was down-regulated. The fold change in expression of 7 genes was between 4-fold and 5-fold, 6 genes were up-regulated, and 1 gene was down-regulated. The fold change in expression of 32 genes was between 3-fold and 4-fold, 18 genes were up-regulated and 14 genes were down-regulated. The expression of 201 genes varied between 2-fold and 3-fold, with 99 genes up-regulated and 102 genes down-regulated. Overall, the fold change in the expression of 244 of the 54675 genes (0.45% by weight, 244: 54675) was greater than 2 fold in the experimental group-derived data treated with DATS.
After the treatment of HepG2 cells with DATS, 4 genes with the fold change of gene expression greater than or equal to 5 fold, EGR1 and F2RL2 for up-regulation of gene expression, and DKK1 for down-regulation of gene expression were present. The expression of the gene is up-regulated by 4-fold and 5-fold, and the expression of the gene is up-regulated by 7 genes, such as HSPA1A, SLC7A11, EID3, EGR1 and the like, and the expression of the gene is down-regulated by NTN 4. The expression of the gene with the fold change of 3 times and 4 times is up-regulated by FOS, HSPA1A, SLC7A11, JAG1, NCF2, AGPAT9 and the like, and the expression of the gene is down-regulated by FOXA1, ANKRD1, SLC2A2, PROX1 and the like. The genes with the up-regulated gene expression change between 2-fold and 3-fold are MCL1, PALLD, TRIB1, HSPA1A, ABCC1, GCLC, FOSL1, IL11, TLR6, TUSC3 and the like, and the genes with the down-regulated gene expression include TNFAIP3, TncRNA, LGR4, TOX3, PKP2, ARL4C and the like.
(3) DTS processing
As shown in FIG. 3, in the gene chip source data after DTS treatment, the expression of 4 genes was changed by 5-fold or more, and the expression of 4 genes was all up-regulated. The fold change in expression of 8 genes was between 4-fold and 5-fold, 7 genes were up-regulated, and 1 gene was down-regulated. The fold change in expression of 29 genes was between 3-fold and 4-fold, 21 genes were up-regulated and 8 genes were down-regulated. The expression of 203 genes varied between 2-fold and 3-fold, with 115 genes up-regulated and 88 genes down-regulated. In general, the fold change in the expression of 244 genes (0.45% by weight, 244: 54675) among the 54675 genes was higher than 2 fold in the experimental group-derived data treated with DTS.
After the HepG2 cells are treated by DTS, EGR1, F2RL2 and EID3 are used for up-regulation of gene expression with the gene expression change multiple more than or equal to 5 times. The fold change of gene expression is between 4-fold and 5-fold, SLC7A11, JAG1, FOS, TRIB1 and the like are up-regulated, and DKK1 is down-regulated. The gene expression fold change is 3-fold and 4-fold, the gene expression is up-regulated by HSPA1A, SLC7A11, NCF2, AGPAT9, LTB4DH, RIT1, ARL6IP2 and the like, and the gene expression is down-regulated by FOXA1, KRTAP1-3, SYT15 and the like. The fold of gene change is between 2-fold and 3-fold, and the gene expression is up-regulated by PALLD, EGR1, EGR3, GCLC, FOSL1, TLR6, JAG1, TncRNA, HSPA1A and the like. The gene expression is reduced by ARL4C, TNFAIP3, FSTL3, GAD1, PROX1, LOC643401 and the like.
As described above, the genes whose gene expression was changed by 4-fold or more after DADS treatment were EGR1, NCF2 and F2RL 2. The genes with the gene expression change fold more than 4 times after the DATS treatment are HSPA1A///HSPA1B, EGR1, DKK1, SLC7A11, NTN4 and F2RL 2. The genes with the fold change of gene expression more than 4 times after the DTS treatment include EGR1, DKK1, SLC7A11, JAG1, FOS, F2RL2, EID3 and TRIB 1. In the three experimental groups, the gene expression varied by more than 4 times, and EGR1, NCF2 and F2RL2 were observed. The EGR1 (early growth factor-1) gene is an important nuclear transcription factor, is an important upstream molecule for cell proliferation and differentiation, and is an anti-cancer gene. Mutations in the NCF2 (neutrophil factor-2) gene can lead to chronic granulomatous disease. The F2RL2 (coagulation factor II receptor 2) gene is a member of the protease activated receptor family and is activated by thrombin, and F2RL2 is a cofactor for thrombin-activated F2RL3, which mediates thrombin-induced phosphoinositide hydrolysis and is expressed in various tissues.
Example 5 expression control and Real-Time RT-PCR validation of DADS, DATS, DTS on liver drug metabolizing enzyme, inflammation-related gene, glycolipid metabolism-related gene, cell proliferation and apoptosis-related gene
(1) Expression regulation of liver drug metabolizing enzymes by DADS, DATS and DTS
The phase I enzyme is mainly responsible for binding heterologous substances with carriers on the cell surface in the drug metabolism process, the phase II enzyme is used for enhancing the water solubility of the phase I enzyme or exposing specific binding sites of the phase II enzyme so as to facilitate the binding with transport proteins, and the transport enzyme is used for transporting the exogenous substances out of cells and discharging the exogenous substances out of the body through blood circulation. The gene expression changes of 35 drug metabolizing enzymes and transporters were observed in this experiment. Coenzyme I related enzymes such as classical phase I enzymes represent FMO5 and NCF 2; the related value of the II-phase coenzyme is noted that the differentially expressed II-phase coenzyme also comprises antioxidant enzymes such as GCLC, GCLM, SQSTM1 and the like; there are 10 genes associated with transporters (e.g., ATP and SLC family).
In the DTS treatment group, NCF2 is a gene with up-regulated expression more than 2 times in the phase I coenzyme; genes with up-regulated expression of more than 2-fold in phase II coenzymes or antioxidant proteins include GCLC, GCLM, SQSTM1 and HMOX 1; genes with up-regulated expression in the transporter by more than 2-fold are SLC6A6 and SLC2A 2.
TABLE 1 Gene expression changes of drug metabolizing enzymes and transporters in1 DSs-treated cells
Figure BDA0002514642630000091
Figure BDA0002514642630000101
(2) Modulation of expression of inflammation-associated genes by DADS, DATS, DTS
DSs are involved in NF-. kappa.B signaling, IL-6signaling and IL-2 signaling. According to the data of the gene chip, the gene expression changes by more than 2 times, and genes related to inflammatory cell pathways of the gene chip comprise TLR6, FOS, SOCS1, BCL10, TNFAIP3 and the like. DSs has the greatest influence on the expression change of the FOS gene, the expression change of the FOS gene by the DTS treatment group is more than 4 times, and the expression change of the DADS experiment group and the expression change of the DATS experiment group are both more than 3 times.
TABLE 2 DSs Regulation of expression of inflammation-associated genes
Figure BDA0002514642630000102
(3) Expression regulation of glycolipid metabolism-related genes by DADS, DATS and DTS
Among the genes involved in glycolipid metabolism, the expression of genes treated with DSs changed by more than 2-fold and was GCLC, GCLM, SLC2A2, GCKR. Wherein, the expression of the SLC2A2 gene is changed by more than 3 times by DATS and DTS experimental groups and is down-regulated.
TABLE 3 DSs expression control of glycolipid metabolism-related genes
Figure BDA0002514642630000111
(4) Expression regulation of cell proliferation and apoptosis-related genes by DADS, DATS and DTS
DSs, F2RL2, HSPA1A///HSPA1B, GCLC, GCLM, SLC2A2, EGR1, FOS, BCL10, TLR6, NCF2, etc., show more than 2-fold change in expression of genes related to apoptosis. It is known from the data in the gene chip that DSs can promote the expression of apoptosis genes, especially F2RL2 gene, HSPA1A// HSPA1B gene, FOS gene and NCF2 gene, the fold change of gene expression is more than 3 times, and the fold expression of F2RL2 gene is even more than 14 times. DSs also down-regulate the expression of the SLC2A2 gene. F2RL1, SOCS1, FOS, FOSL1, HSPA1A, JAG1, SPP1 and the like show more than 2-fold change of expression of genes related to cell proliferation. From the data on the gene chip, the most typical gene affecting cell proliferation was F2RL2 gene.
TABLE 4 DSs Regulation of expression of cell proliferation and apoptosis-related genes
Figure BDA0002514642630000112
Figure BDA0002514642630000121
(5) Real-Time RT-PCR validation
After HepG2 cells are treated by samples with the same concentration, total RNA is extracted and then is reversely transcribed into cDNA, finally, a fluorescent reagent bag and a real-time fluorescent quantitative PCR instrument are used for detecting and statistically analyzing target genes, 6 typical genes in drug metabolizing enzyme and antioxidant protein are selected for RNA transcription level verification, the selected genes are NCF2, SQSTM1, HMOX1, GCLC, GCLM and SLC6A6 which belong to genes corresponding to I/II phase antioxidant protein and III phase transport protein respectively, and the research result is shown in figure 4. Real-time PCR was performed using SYBR Green 2-step qRT-PCR Kit. Results are expressed as relative expression levels. Each value represents the mean ± SD of three independent experiments, # p <0.05vs.
The selected gene shows similar expression results between the DNA chip and RT-PCR data, and the expressed gene differential expression trends are the same, wherein the expression level of the typical inflammation suppressor IL11 is increased by 4.21 times in the real-time PCR and increased by 3.33 times in the DNA chip, and the reason for the difference of the expression multiples is that the RT-PCR result is obtained by real-time fluorescence measurement, and the more tiny and accurate gene differential expression can be recorded in real time, so the measured difference multiple value is larger and more accurate. These data indicate that DSs treatment of HepG2 cells did enhance their antioxidant gene expression, reduce inflammatory responses, and increase metabolic regulation-related gene expression.
Example 6 IPA analysis (taking DTS treatment as an example)
(1) DTS handles the main typically affected signal paths
Typical pathways affected by DTS processing can help locate the signal channel for which it is primarily responsible. Fig. 5 shows ten typical signal paths that are significantly altered by DTA. The most affected signal channel in the DTS-treated group was Glutamate Metabolism (Glutamate Metabolism), with a negative log of p-value of 3.47 at the maximum; the second affected channel is the Acute Phase Response Signaling (Acute Phase Response signal).
The p-value of the DTS activated NRF2-mediated Oxidative Stress Response takes the negative logarithm of 3.08. Is a very important and complex signal transmission path. The nuclear factor Nrf2 (also known as Nfe212) is a transcription factor that regulates the redox state of cells. Nrf2 has the ability to promote the expression of various protective genomes of cells in response to various stimuli, including redox signals, inflammation, growth factors, and changes in energy supply, making it a key role in cellular adaptation. In addition, other Signaling pathways that can also be significantly activated in the DTS-treated group are NF-. kappa.B Signaling, IL-6Signaling, and IL-2 Signaling. All three cellular pathways are inflammatory cellular pathways. Therefore, DTS may also activate inflammatory cell pathways, indicating its anti-inflammatory potential.
(2) Ten-macromolecule function altered by DTS treatment
As shown in FIG. 6, the ten macromolecular functions affected by DTS are Cancer, Cell Death, Cellular Growth and Proliferation, Tissue Development, Cellular Development, Reproductive System Disease, Cycle, hematology System Development and Function, and Cell Morphology, respectively. The DTS has expression change of more than 2 times to F2RL1, SLC2A2, FOS, PALLD, TRIB1 and other genes, and the DTS can obviously influence cell proliferation and apoptosis and cell cycle block. Furthermore, the expression of DTS on cancer-related genes such as FOSL1, JAG1, FLRT2, BCL10 was also changed by 2 times or more, suggesting that DTS may have an anticancer effect.
(3) Molecular networks and signal channel analysis
The most significant ten-macromolecule networks after DTS treatment are shown in table 5. Among them, the most affected by DTS is Gene Expression, Protein Synthesis, Cell Death (score: 49 points). Three main biological functions are involved in this network: gene expression, protein synthesis and apoptosis. Next, is the Cardiovascular System Development and Function, Tissue Morphology, Amino Acid Metabolism (score: 39 points).
TABLE 5 Ten-macromolecule networks most pronounced after DTS treatment
Figure BDA0002514642630000141
Figure BDA0002514642630000151
The present example used IPA biological software to perform typical signal channel and network analysis on the up, down and comprehensive total data sets, and constructed a biological network of comparable differential metabolites for the DTS-treated group versus the control group without DTS treatment. The classification attributes of the molecules with different shapes are illustrated in the drawing, wherein the red marked metabolic products are up-regulated metabolic products, the green marked metabolic products are down-regulated metabolic products, the solid line represents the direct action relationship between the two molecules, and the dotted line represents the indirect action relationship between the two molecules. Networks and signaling pathways describe functional relationships between gene products based on known interactions reported in the literature.
FIG. 7 shows the molecular network with the first significance of the change after up-and down-regulation of the gene by DTS (score: 49). 35 genes are involved in the construction of the network, of which 25 are differentially expressed. NF-kB is used as the central gene of the molecular network and controls the transcription activity of a plurality of inflammatory factors and the expression of antioxidant genes. The number of the upregulated genes is 12, and the gene with the most obvious change of the upregulated fold is HSPA 1A. The number of the down-regulated genes is 13, and the gene with the most obvious change of the down-regulation fold is BIRC 3. In the molecular network, the up-regulation expression change multiple of HSPA1A is the largest after the indirect action of NF-kB, and is 3.8. It promotes the correct folding of newly translated and misfolded proteins, as well as stabilizing or degrading mutant proteins. Is helpful for DNA repair. Its function contributes to biological processes including signal transduction, apoptosis, protein homeostasis, cell growth and differentiation. It is associated with a number of cancers, neurodegenerative diseases, cellular senescence and aging, and inflammatory diseases such as diabetes, type 2 and rheumatoid arthritis. In the molecular network, NCF2 (neutrophil cytoplasmic factor 2) is up-regulated and expressed after being indirectly acted by NF-kB, NCF2 is also called NADPH oxidase, is a multi-protein compound with 67 kilodalton molecular subunits and is related to chronic diseases, particularly chronic inflammation, Alu repeated induction deletion in NCF2 gene is a new mechanism for causing p 67-hypoxic Chronic Granulomatosis (CGD), and the chronic granulomatosis is related to life-threatening infection and dysregulated granulomatous inflammation. BIRC3 has the maximum expression change factor of 3.35 under the indirect action of NF-kappa B. BIRC3 is a member of the apoptotic family and inhibits apoptosis by interfering with Caspase activation. Comprising three BIR domains, a UBA domain, a CARD domain and a loop finger domain.
FIG. 8 shows the molecular network with the second most significant change after up-and down-regulation of the gene by DTS (score: 39). 35 genes are involved in the construction of the network, and 21 genes are differentially expressed. ERK, as a central gene in this molecular network, controls the transcriptional activity of a number of inflammatory factors and the expression of antioxidant genes. The number of the up-regulated genes is 16, and the gene with the most obvious change of the up-regulation fold is EGR 1. The number of down-regulated genes was 5, and the gene with the most significant fold change was NTN 4.
FIG. 9 shows the third molecular network (score: 34) with significance of changes following up-and down-regulation of the gene by DTS. 35 genes are involved in the construction of the network, of which 19 are differentially expressed. HNF4A is used as the central gene of the molecular network and controls the transcription activity of a plurality of inflammatory factors and the expression of antioxidant genes. The number of upregulated genes was 9, and the gene with the most significant fold change was F2RL 2. There were 10 genes downregulated, the gene with the most significant fold change being CLDN 1.
Example 7 intervention of Garlic sulfide mixture on inflammatory cells
(1) LPS (lipopolysaccharide) constructed cell inflammation model
Lipopolysaccharide (LPS) is the outer membrane component of gram-negative bacteria and is also the most widely used substance with a promoting inflammatory production today, and LPS endotoxin has been used for over a century as a powerful agent for the evaluation of anti-inflammatory agents. A series of studies by Greisman et al, in the early 60's of the 20 th century, injected subjects with LPS preparations and examined the human response to LPS endotoxins systematically. The detection result shows that the intravenous injection of lipopolysaccharide causes symptoms similar to gram-negative sepsis, such as the rise of body temperature, the release of neutrophils, CRP and the rapid rise of some inflammatory factors, namely IL-8, IL-6, TNF-alpha and the like. The in vivo action of LPS is mainly recognized by Toll-like receptors, the recognition involves the combination of LPS and binding protein thereof and the combination of LPS and CD14, and then the LPS receptor complex with TLR4 as the center generates strong immune response, so that the expression of some inflammatory factors is increased.
This example used HepG2 cells treated in example 1 and induced with LPS to construct a cellular inflammation model.
(2) Western blotting
Western blotting is a relatively common test method in which proteins are separated by polyacrylamide gel electrophoresis (PAGE) according to their molecular weight, transferred and immobilized on a membrane carrier, and selectively detected with specific antibodies, during which the proteins are colored. The expression of the target protein to be detected in the living body can be known by analyzing the coloration. Briefly, the sample is denatured by proteins and then subjected to gel electrophoresis. Soaking and incubating in a synthetic or animal derived antibody (called primary antibody), the primary antibody can recognize and combine with a specific target protein, washing the residual antibody, and adding a secondary antibody, which can recognize and combine with the primary antibody, wherein the secondary antibody can be visually displayed by various methods such as staining, immunofluorescence and radioactivity, so that the specific target protein can be indirectly detected. This technique is commonly used to qualitatively identify a specific protein from a complex biological sample and provide information on its molecular weight.
2.1 protein extraction
The extraction of protein is a process of crushing cells and then placing the cells under certain conditions to fully extract the protein. Adding protease inhibitor into the cell treated by the medicine, crushing on ice by using an ultrasonic crusher, centrifuging for 5min at a low temperature of 4 ℃, taking the supernatant, using Dithiothreitol (DTT) or 2-mercaptoethanol to expand the secondary structure and the tertiary structure of the protein, finally placing the supernatant in a protein boiling instrument for 5min, further leading the protein to lose the secondary structure in the process, cooling the protein to room temperature after denaturation, and carrying out vortex oscillation for several times, thus extracting the target protein.
2.2 SDS-PAGE electrophoresis
Polyacrylamide gels have limited protein loading capacity to enable separation of proteins at different molecular weights. Firstly, pouring glue and loading, preparing 10% separation glue according to a proportion, sucking liquid glue by a gun head to discharge slowly, adding a layer of water on the glue, and horizontally placing for 1 h. After the separation glue is solidified, preparing 4% of concentrated glue according to the proportion, immediately shaking up, slowly and vertically inserting the comb into the glue after adding (preventing the unsolidified glue solution from being sprayed out as much as possible), and vertically pulling out the comb after the glue is solidified and checking the adhesion condition of each sample hole. Putting the prepared rubber plate into an electrophoresis tank, taking out a sample to be loaded into a centrifuge tube, adding a sample loading buffer solution, pouring the prepared electrophoresis solution into the electrophoresis tank, sucking the sample by using a microsyringe, and inserting a needle into a sample loading hole to slowly add the sample.
Electrophoresis and membrane transfer were carried out next, and the electrophoresis time was set to 1.5 hours and the voltage was set to 75V for this experiment. After the electrophoresis is finished, the membrane is transferred, and 6 filter papers and 1 nitrocellulose membrane are prepared for one membrane. The cut membrane was immersed in methanol for about 5min to activate it. Then cutting the glue according to the molecular weight of the target protein and covering the glue on filter paper, then covering the film on the glue, putting the filter paper and closing the clamp, and carrying out the whole operation in the transfer liquid. The film transfer is carried out for 45 min. After the membrane is transferred, the membrane is washed by TBST, and the membrane is put into coating liquid and is kept at room temperature for being shaken and sealed for about one hour.
And finally, carrying out immunoreaction and development, taking out the membrane from the sealing solution, washing the membrane by TBST, adding primary antibody, incubating for 1-2 h at room temperature, and eluting by TBST for 10min each time. The secondary antibody was prepared and contacted with the membrane as above, incubated overnight at 4 ℃ and then eluted with TBST 3 times the next day on a shaker, and a color-developing solution was added to carry out a chemiluminescent reaction. The results are shown in FIG. 10.
FIG. 10 shows the induction of HepG2 cells with endotoxin LPS to establish a model of cellular inflammatory stress. Meanwhile, the protein immunoblotting is used for detecting COX-2, and the result shows that the concentration of a garlic sulfide mixture MIX (DAS: DADS: DATS mixed according to the mass ratio of 1: 1: 1) in a certain range is in positive correlation with the anti-inflammatory capacity. Wherein M1(MIX-1, concentration: 5 μ M), M2(MIX-2, concentration: 10 μ M), and CTL (control) are used as blank control, and L (LPS) Lipopolysaccharides are used for establishing a cell inflammation stress model.
In conclusion, the invention researches the influence rule of garlic organic sulfide on human hepatocyte gene expression by advanced nutritional genomics and molecular biology methods, finds that the treatment of DADS, DATS and DTS can cause the differential expression of hepatoma cell genes, and the number of genes with the differential expression more than 2 times of that of molecular functions in three categories classified according to GO catalogue is the largest, which indicates that DTS acts on hepatoma cells and has the largest influence on the molecular functions thereof, wherein the function with the largest base factor is protein combination, and indicates that molecular signal channels in cells are obviously influenced by DTS.
Glutamate Metabolism, acid Phase Response Signaling, NRF2-mediated Oxidative Stress Response are the most significantly altered typical signal channels upon DTS, and activation of these signal channels is associated with NRF2-mediated Oxidative Stress, respectively. The molecular functions with the highest score of altered DTS are cancer, apoptosis and cell proliferation, demonstrating that the occurrence or metabolism of these diseases is associated with differential expression of the above-mentioned related genes, activation of signaling pathways. Through the analysis of the molecular network with the largest DTS influence, the molecular functions related to the first 3 molecular networks are obtained to correspond to the signal channels and diseases analyzed in the previous step. In particular, the core of the first molecular network is the inflammatory response. The garlic organic sulfide can achieve the purpose of protecting the liver by influencing the metabolism of liver cancer cells, is used as a food functional component, has small toxic and side effects, and can be used for preparing liver-protecting foods, health-care products or medicines.

Claims (8)

1. The application of garlic organic sulfide in preparing liver-protecting food, health-care products or medicines, wherein the garlic organic sulfide comprises one or more than one of diallyl disulfide, diallyl trisulfide and diallyl tetrasulfide;
the diallyl disulfide has the structural formula:
Figure FDA0002514642620000011
the structural formula of the diallyl trisulfide is as follows:
Figure FDA0002514642620000012
the diallyl tetrasulfide has the structural formula:
Figure FDA0002514642620000013
2. the use according to claim 1, wherein garlic organosulfide is included as an active ingredient in the food, health product or pharmaceutical.
3. The use according to claim 2, wherein the mass ratio of diallyl disulfide, diallyl trisulfide and diallyl tetrasulfide in the organic sulfide of garlic in the food, health product or medicament is 1: 1: 1.
4. the use according to claim 2, wherein the food or health product further comprises a food additive or food material.
5. The use according to claim 4, wherein the food additive is one or more of a preservative, a colorant, a sweetener, a flavoring agent, a leavening agent, a thickener, an emulsifier, and an antifoaming agent.
6. The use of claim 2, wherein the medicament further comprises a pharmaceutically acceptable excipient.
7. The use according to claim 6, wherein the adjuvant is one or more of a filler, a disintegrant, a lubricant, a suspending agent, a binder, a sweetener, a flavoring agent, a preservative, and a matrix.
8. The use of claim 6, wherein the medicament is in the form of one of capsules, tablets, granules, powder, oral liquid, pills and injections.
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