Gardenia and szechwan chinaberry fruit composition and application thereof in liver protection

Abstract

The invention discloses a fructus gardeniae and szechwan chinaberry fruit composition and application thereof in liver protection, and belongs to the technical field of biological medicines. The invention provides a cape jasmine fruit and szechwan chinaberry fruit composition, and the proportion of cape jasmine fruit to szechwan chinaberry fruit is (1-2): (1-6), optimally 2: 3. The cape jasmine and szechwan chinaberry fruit composition can be used as an activating agent of DBP; experiments prove that after overexpression of the DBP, the expression quantities of metabolic genes Cyp2a4, ALB, G6PC and Sult1c1 are remarkably increased, which indicates that overexpression of the DBP can promote liver functions. The fructus gardeniae and szechwan chinaberry fruit composition or overexpression DBP provided by the invention can play the role of resisting oxidation and protecting the liver by adjusting an antioxidant function gene, a new thought and strategy are provided for treatment of liver diseases, and an important scientific basis is provided for development of a natural small-molecule DBP agonist.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a composition of gardenia and Sichuan pepper and its application in liver protection. Background Technology

[0002] Liver injury is a serious disease caused by a variety of factors, including chemicals, drugs, viruses, and trauma. These external factors lead to oxidative stress, inflammatory responses, hepatocyte necrosis, apoptosis, and fatty degeneration within the liver, ultimately resulting in abnormalities in liver structure and function. Oxidative stress plays a central role in the pathogenesis of liver injury. Oxidative stress refers to the disruption of the balance between the oxidative and antioxidant systems when the body is subjected to various stimuli, leading to the excessive production of reactive oxygen species (ROS) and free radicals. The liver is an organ particularly susceptible to ROS, which are produced not only during metabolism but also during the biotransformation of foreign organisms. Disruption of the redox balance leads to oxidative stress, which in turn affects liver function, regulates inflammatory pathways, and causes disease. Therefore, oxidative stress is associated with the pathogenesis of acute liver injury and common infectious or metabolic chronic liver diseases, such as viral hepatitis B or C, alcoholic fatty liver disease, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH). Furthermore, oxidative stress plays a crucial role in the progression of liver disease to liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC).

[0003] Given the crucial role of oxidative stress in liver disease, antioxidants are naturally considered a good treatment strategy. To date, research results remain unclear and controversial; however, the therapeutic effects of some antioxidants have been confirmed. Multiple studies have shown that curcumin can protect DNA from ROS damage and support hepatocytes during injury and cirrhosis. Research in the field of chronic hepatitis C virus (HCV) has shown improvement after antiviral treatment with silymarin, which protects glutathione (GSH) within hepatocytes. Ascorbic acid, lipoic acid, quercetin, and mitochondrial quinones are also beneficial for patients with chronic HCV infection. Resveratrol's antioxidant properties can reduce hepatic lipid peroxidation, increase GSH levels in the liver, and scavenge ROS during ethanol-induced liver injury, thus playing a hepatoprotective role. Furthermore, ebuselenium (a glutathione peroxidase analog) is a therapeutic agent for early alcoholic liver injury. Studies have shown that vitamin E can inhibit hepatitis B virus (HBV) replication and suppress transforming growth factor-β (TGF-β) gene expression in a NASH rat model, thus preventing the progression of NAFLD. Currently, the best clinical evidence for successful antioxidant therapy in liver diseases is vitamin E treatment for NASH. There is an urgent need to develop a hepatoprotective composition. Summary of the Invention

[0004] The purpose of this invention is to provide a composition of gardenia and Sichuan pepper and its application in liver protection, in order to solve the problems existing in the prior art. The gardenia and Sichuan pepper composition or overexpression of DBP provided by this invention can exert an antioxidant and liver-protective effect by regulating antioxidant functional genes, providing new ideas and strategies for the treatment of liver diseases, and providing important scientific basis for the development of natural small molecule DBP agonists.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] The present invention provides a composition of gardenia and Sichuan pepper, characterized in that the ratio of gardenia to Sichuan pepper is (1-2):(1-6).

[0007] Preferably, the ratio of gardenia to Sichuan pepper is 2:3.

[0008] Optionally, the preparation method of the gardenia and Sichuan pepper composition includes the following steps:

[0009] Gardenia and Sichuan pepper were extracted separately by reflux with methanol for 1 h, filtered, and the first filtrate and the first residue were obtained. The first residue was extracted by reflux with methanol for 1 h, filtered, and the second filtrate and the second residue were obtained. The second residue was extracted by reflux with 50% methanol for 1 h. After reflux extraction with 50% methanol for 3 times, all filtrates were combined, filtered under reduced pressure, evaporated, and the extracts were combined. The extracts were suspended in water to obtain a suspension, which was extracted sequentially with petroleum ether, ethyl acetate, n-butanol and water, and freeze-dried to obtain the gardenia and Sichuan pepper extracts, which were then mixed according to the specified ratio.

[0010] Optionally, the volume ratio of petroleum ether, ethyl acetate, n-butanol, and water to the suspension is 1:1;

[0011] The petroleum ether, ethyl acetate, n-butanol, and water were each extracted three times.

[0012] The present invention also provides the use of the aforementioned gardenia and Sichuan pepper composition in the preparation of DBP activators.

[0013] The present invention also provides a DBP activator, the active ingredient of which is the aforementioned combination of gardenia and Sichuan pepper.

[0014] The present invention also provides the use of the above-described gardenia and Sichuan pepper composition in the preparation of hepatoprotective drugs.

[0015] The present invention also provides a liver-protecting drug, the active ingredient of which is the aforementioned combination of Gardenia jasminoides and Melia toosendan.

[0016] The present invention also provides the use of the aforementioned gardenia and Sichuan pepper composition in the preparation of antioxidant drugs.

[0017] The present invention also provides an antioxidant drug, the active ingredient of which is the aforementioned combination of gardenia and Sichuan pepper.

[0018] The present invention discloses the following technical effects:

[0019] This invention provides a composition of Gardenia jasminoides and Melia toosendan, with a ratio of (1-2):(1-6), optimally 2:3. This composition can act as an activator of DBP. Experimental verification shows that overexpression of DBP significantly upregulates the expression levels of metabolic genes Cyp2a4, ALB, G6PC, and Sult1c1, indicating that DBP overexpression can promote liver function. The Gardenia jasminoides and Melia toosendan composition or DBP overexpression provided by this invention can exert antioxidant and hepatoprotective effects by regulating antioxidant functional genes, providing new ideas and strategies for the treatment of liver diseases and offering important scientific evidence for the development of natural small molecule DBP agonists. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The effects of different ratios of Gardenia jasminoides and Melia toosendan on DBP expression;

[0022] Figure 2 The effect of a 10-fold ratio on the expression of liver function genes ALB, G6PC, CYP2B10, and SUIL1C1;

[0023] Figure 3 The ratio 10 is used to assess the effect of antioxidant-related gene expression.

[0024] Figure 4 The in vitro hepatoprotective effect is achieved with a ratio of 10.

[0025] Figure 5 The expression level of DBP in the DBP overexpression cell model;

[0026] Figure 6 The effect of DBP overexpression on FL83B cell viability;

[0027] Figure 7 To investigate the effect of DBP overexpression on the proliferation of FL83B cells;

[0028] Figure 8 The effect of DBP overexpression on the migration rate of FL83B cells;

[0029] Figure 9 The effect of DBP overexpression on the FL83B cell cycle;

[0030] Figure 10 To investigate the effects of DBP overexpression on metabolism-related genes;

[0031] Figure 11 To knock down the expression level of DBP in the DBP cell model;

[0032] Figure 12 To reduce the effect of DBP on FL83B cell viability;

[0033] Figure 13 To reduce the effect of DBP on the proliferation of FL83B cells;

[0034] Figure 14 The effect of knocking down DBP on the migration rate of FL83B cells;

[0035] Figure 15 To reduce the effect of DBP on the FL83B cell cycle;

[0036] Figure 16 To reduce the impact of DBP on metabolism-related genes;

[0037] Figure 17 To investigate the effects of DBP overexpression on antioxidant genes;

[0038] Figure 18 To reduce the impact of DBP on antioxidant genes. Detailed Implementation

[0039] Example 1

[0040] Ratio: Twelve different ratios of gardenia and Sichuan pepper were set, in the following order: 6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:6, 2:5, 2:4, 2:3, 2:2, 2:1, named ratio 1-12.

[0041] Preparation method: Gardenia jasminoides and Melia toosendan were extracted separately by reflux with methanol. A 5000 mL round-bottom flask was filled 1 / 3 full with the extract, methanol was added to 1 / 2, and the mixture was soaked overnight. The mixture was then refluxed for 1 h, filtered through gauze, yielding the first filtrate and the first residue. The first residue was added to methanol, refluxed for 1 h, and filtered through gauze to obtain the second filtrate and the second residue. The second residue was added to 50% methanol (volume fraction), refluxed for 1 h (the residue was extracted three times with 50% methanol, 1 h each time). All filtrates were combined and subjected to vacuum filtration. Then, the mixture was rotary evaporated at 55℃ to recover methanol, and the extracts were combined. The resulting extract was suspended in water to form a suspension, which was then extracted sequentially with petroleum ether, ethyl acetate, n-butanol, and water (volume ratio of petroleum ether, ethyl acetate, n-butanol, and water to the suspension was 1:1, and each component was extracted three times). The extracted extracts were freeze-dried to obtain the Gardenia jasminoides and Melia toosendan extracts. Weigh the net weight and mix them according to the specified proportions to obtain gardenia and Sichuan pepper compositions in various proportions.

[0042] The effects of different ratios of Gardenia jasminoides and Melia toosendan on DBP expression were investigated using the following methods.

[0043] The mouse normal liver immortalized cell line (FL83B) was purchased from the Beina Chuanglian Biotechnology Research Institute. After the FL83B cells were cultured and passaged to a stable state, they were seeded in 6-well plates. After the cells adhered, gardenia and Sichuan pepper combination (50 μg / mL) were added in different proportions by changing the medium, and the cells were cultured in an incubator for another 6 h. RNA was then extracted for later use.

[0044] Reverse transcription PCR:

[0045] Place a 200 μL enzyme-free EP tube on an ice box and thaw the RNA and all reagents used in the reverse transcription process on the ice box. Mix all the reagents and mix the thawed RNA with a single-channel pipette.

[0046] The reverse transcription system was prepared as shown in Table 1.

[0047] Table 1 Reverse transcription system

[0048] reagents Dosage 5× FastKing-RT SuperMix 4 μL Total RNA 1 μg <![CDATA[RNase Free ddH2O]]> Add to 20 μL

[0049] After shaking and centrifuging the prepared system, proceed with the reaction according to the reaction procedure shown in Table 2.

[0050] Table 2 Reaction Procedure

[0051] reaction temperature reaction time 95℃ 3 min 42℃ 15 min

[0052] Dilute the reverse transcription product cDNA and store it in a -20°C freezer.

[0053] Real-time quantitative PCR:

[0054] Place a 200 μL enzyme-free EP tube on an ice box. Thaw the cDNA and reagents required for real-time quantitative PCR on the ice box. Dilute the cDNA 3-fold and the primers 10-fold before use. Vortex each solution separately to mix. DBP upstream primer: 5'-CTGGCCCGAGTCTTTTTGC-3' (SEQ ID NO.1), DBP downstream primer: 5'-CCAGGTCCACGTATTCCACG-3' (SEQ ID NO.2), with β-actin as the internal reference gene (upstream primers: 5'-GGCTGTATTCCCCTCCATCG-3' (SEQ ID NO.3), 5'-CCAGTTGGTAACAATGCCATGT-3' (SEQ ID NO.4)).

[0055] During the operation, the system should be kept away from light and enzymes, and a 20 μL PCR system should be prepared as shown in Table 3.

[0056] Table 3 PCR System

[0057] reagents Dosage (μL) 2×FastFire Qpcr PreMix 10 μL Forward primers 0.6 μL Reverse primers 0.6 μL cDNA 1 μL <![CDATA[ddH2O]]> 7.8 μL

[0058] After mixing the prepared system by shaking, add it to an 8-tube array and perform the PCR reaction according to the following reaction program as required by the instructions: 95℃ for 2 min; 95℃ for 5 s, 52℃ for 10 s, 72℃ for 15 s, for 40 cycles.

[0059] After the reaction was complete, the transcription level was determined using a threshold comparison method. The results are as follows: Figure 1 As shown, different ratios of Gardenia and Chinaberry combinations can activate DBP expression, with ratio 10 (2:3) showing the most significant effect. Ratio 10 was selected for subsequent experiments for verification. Example 2

[0060] 1. Modeling and grouping of drugs for administration

[0061] Forty-five male C57BL-6 mice (6-8 weeks, 20-22 g) were housed separately according to similar weights under a temperature of 25±2℃, relative humidity of 60-70%, and a 12-hour light / dark cycle. Body weights were carefully recorded for each group. During the experiment, all samples were divided into three groups of 15 mice each: a control group (saline group), an injury group (CCl4 treatment), and an experimental group (10:10 gavage + CCl4 treatment). After a 7-day acclimatization period, the first two groups were gavage with saline once daily for 7 days; the experimental group was gavage with 10:10 gavage once daily for 7 days. Four hours after gavage on day 7, the blank control group received an intraperitoneal injection of olive oil (10 mL / kg), while the latter two groups received an injection of a CCl4-ol mixture (CCl4 to olive oil volume ratio of 1:4). Samples were taken at 2, 5, 7, and 14 days after injury. Blood was collected from the ophthalmic vein. Mice were euthanized by cervical dislocation. The same lobe of the liver was taken from each mouse and fixed in 4% paraformaldehyde. The remaining liver samples from the same location were then divided into equal parts and flash-frozen in liquid nitrogen for preservation.

[0062] 2. Effects on the expression of liver functional genes ALB, G6PC, CYP2B10, and SUIL1C1

[0063] The expression levels of ALB, G6PC, CYP2B10, and SUIL1C1 in the liver were detected using real-time quantitative PCR, as follows:

[0064] RNA was extracted from the liver using standard procedures and subjected to reverse transcription and real-time quantitative PCR as described in Example 1. Primer sequences are shown in Table 4.

[0065] Table 4 Primer Sequences

[0066]

[0067] The results are as follows Figure 2 As shown, a ratio of 10 can promote the expression of liver function genes ALB, G6PC, CYP2B10, and SUIL1C1.

[0068] 3. Effects on the expression of antioxidant-related genes

[0069] The expression levels of antioxidant-related genes GSR, SOD, PPAR, CAT, NOX, KEAP1, HMOX, CYP3A1, and PON in the liver were detected using the same method as in Example 1, and the primer sequences used are shown in Table 5.

[0070] Table 5 Primer Sequences

[0071]

[0072] 4. In vitro liver protection effect

[0073] Liver tissues obtained from different groups were stained with HE and Masson staining. The staining results are as follows: Figure 4 As shown.

[0074] HE: Control Group: The tissue structure was intact, cells were neatly arranged, and there were no obvious pathological changes. No abnormalities such as inflammatory cell infiltration, necrosis, or damage were observed. Model Group: Typical pathological damage was observed, including cell edema and disordered arrangement; inflammatory cell infiltration; areas of necrosis or apoptosis; and fibrous tissue proliferation, indicating successful model construction. The 10-group control group showed significantly reduced pathological damage compared to the model group, manifested as: reduced inflammatory infiltration; a decreased ratio of cell necrosis / apoptosis; partial restoration of tissue structure; and more neat arrangement of hepatic cords than the model group.

[0075] Masson: In the control group, collagen fibers appeared pale blue, sparsely distributed yet regularly arranged, mainly confined to normal tissue structures. The proportion of collagen fibers in the stroma was low. No abnormal fiber proliferation or tissue destruction was observed. In the model group, collagen fiber deposition was significantly increased, appearing as dense blue areas with disordered arrangement. Significant damage and lesions were observed. In the formulation 10, collagen fiber deposition was reduced compared to the model group, the blue area was smaller, and the arrangement tended to be more regular. The degree of damage was reduced, and inflammatory infiltration decreased. These results suggest that formulation 10 may have a hepatoprotective effect.

[0076] Example 3: Effects of DBP on the biological characteristics of FL83B cells

[0077] The immortalized mouse liver cell line (FL83B) was purchased from the BeiNa ChuangLian Biotechnology Research Institute. This embodiment first investigates whether DBP can inhibit the tumor biological characteristics of FL83B cells and promote their differentiation, thus better reflecting the characteristics of hepatocytes, by examining the tumor biological behavior of FL83B cells. Further investigation is needed to explore how DBP regulates the expression of antioxidant genes.

[0078] 1. Construction of a cell model overexpressing DBP

[0079] Construction method: One day before transfection, cells in the logarithmic growth phase were digested with trypsin, resuspended, and plated at an appropriate density to achieve 70%-90% confluence at transfection. Lipofectamine 2000 and the DBP overexpression vector were transfected into FL83B cells using liposome transfection. DBP expression was validated at both mRNA and protein levels.

[0080] To reveal the effects of DBP on the biological characteristics of FL83B cells, the expression of mRNA in FL83B cells overexpressing DBP was examined using RT-qPCR: cellular RNA was extracted, reverse transcribed, and then subjected to real-time quantitative PCR, following the same methods as in Example 1. The results are as follows: Figure 5 As shown, compared with the control group, DBP expression was significantly upregulated (P<0.01), indicating that the DBP overexpression cell model was successfully constructed.

[0081] 2. Effects of DBP overexpression on cell proliferation

[0082] like Figure 6 As shown, cell proliferation was detected by CCK-8 assay. Compared with the control group, the viability of FL83B cells was significantly reduced after DBP overexpression (P<0.01).

[0083] The detection method is as follows:

[0084] (1) Preparation of cell suspension: 2000 cells per well;

[0085] (2) Seeding in 96-well plates: 100 μL of cell suspension per well (repeated three times) according to the number of cells to be seeded.

[0086] (3) Incubate at 37℃ for 24 h;

[0087] (4) Add 10 μL of CCK-8 enhancement solution to each well and mix the CCK-8 enhancement solution with the culture medium (v:v=1:9). A crucial point to emphasize in this step is to avoid air bubbles forming in the wells, as this can negatively impact the final test results.

[0088] (5) Incubate in an incubator for 2 h and measure the absorbance at 450 nm;

[0089] (6) Calculate the cell viability according to the formula in the instruction manual.

[0090] 3. Effects of DBP overexpression on cell colony formation

[0091] To further clarify the effect of DBP overexpression on the proliferation ability of FL83B cells, cell colony formation assays were used to detect cell proliferation. The results are as follows: Figure 7As shown, compared with the control group, the number of clones formed by cells after DBP overexpression was significantly reduced (P<0.01).

[0092] The detection method is as follows:

[0093] (1) After 24 h of transfection of different groups in 6-well plates, cells were collected by trypsin digestion and centrifugation. The cells were resuspended in 1 mL of complete culture medium, 5 μL of which was transferred to an EP tube and 45 μL of PBS was added and mixed. 5 μL of the mixture was then transferred to a hemocytometer and counted under a microscope. The required number of cells was transferred to complete culture medium and mixed. 700 cells per well were then seeded into a new 6-well plate. Three replicates were set up for the control group and the experimental group.

[0094] (2) Observe the cell condition and change the medium as needed;

[0095] (3) Take it out at 14 days, aspirate the culture medium, stain with 1% crystal violet solution in the dark for 1 h, discard the crystal solution and wash it, and place it on white paper for 48 h in the dark for taking pictures.

[0096] 4. The effect of DBP overexpression on migration

[0097] like Figure 8 As shown, compared with the control group, the migration rate of FL83B cells overexpressing DBP was significantly decreased (P < 0.05).

[0098] The detection method is as follows:

[0099] (1) After transfecting different groups of cells in 6-well plates for 24 h, trypsin digestion was performed, and the cells were collected by centrifugation and 1 mL of complete culture medium was added.

[0100] (2) Carefully observe under an optical microscope. When the confluence is found to be about 90%, make a cross with the pipette tip that has been sterilized by high temperature and high pressure.

[0101] (3) Remove the original culture medium and rinse with PBS. One thing to note is that this operation must be done gently and slowly.

[0102] (4) Then place it under a microscope for photography and record the 0 h data;

[0103] (5) After 24 hours, take a picture from the same field of view as in step (4), and make a careful record based on the result.

[0104] (6) The migration rate is equal to the difference in area before and after migration divided by the area before migration × 100%.

[0105] 5. Effects of DBP overexpression on the cell cycle

[0106] Flow cytometry was used to detect the cell cycle of FL83B cells overexpressing DBP. Figure 9 As shown, compared with the control group, the G0 / G1 ratio was significantly increased after DBP overexpression (P<0.01).

[0107] The detection method is as follows:

[0108] (1) After 48 h of transfection of different groups of cells in 6-well plates, the cells were digested with trypsin, centrifuged and collected. The cells of different groups were collected into 15 mL centrifuge tubes.

[0109] (2) Wash the cells with PBS, then centrifuge at 1500 rpm for 5 min to collect them;

[0110] (3) Discard the supernatant, add 500 μL of 70% pre-cooled ethanol to the centrifuge tube to fix for 12 h, and store at 4 ℃;

[0111] (4) Centrifuge before the experiment, remove the supernatant, wash twice with PBS, and discard the PBS after centrifugation;

[0112] (5) Add RNAase A solution to the cell pellet and then resuspend it. Keep it in a water bath at 37 °C for about 30 min.

[0113] (6) On this basis, add 300 μL of PI staining solution, mix gently and incubate at 4°C for about 30 min. One thing to note is that this step must be done in the dark.

[0114] (7) Detect the data using flow cytometry and record the data carefully.

[0115] 6. Effects of DBP overexpression on metabolism-related genes

[0116] The expression of metabolic gene populations in FL83B cells overexpressing DBP was examined using RT-qPCR. Figure 10 As shown, compared with the control group, the expression levels of metabolic genes Cyp2a4, ALB, G6PC, and Sult1c1 were significantly upregulated (P<0.05). This indicates that overexpression of DBP can promote liver function.

[0117] Example 4: Effects of DBP knockdown on the biological characteristics of FL83B cells

[0118] 1. Construction of DBP knockdown cell model

[0119] Construction method: One day before transfection, cells in the logarithmic growth phase were digested with trypsin, resuspended, and plated at an appropriate density to achieve 70%-90% confluence at transfection. Lipofectamine 2000 and the si-DBP plasmid were transfected into FL83B cells using liposome transfection. DBP expression was verified at the mRNA and protein levels.

[0120] RT-qPCR was used to examine mRNA expression in FL83B cells with DBP knockdown. Figure 11 As shown, compared with the control group, DBP expression was significantly downregulated (P<0.01), indicating that the DBP knockdown cell model was successfully constructed.

[0121] 2. Effects of DBP knockdown on cell proliferation

[0122] The CCK-8 assay was used to detect the proliferation of FL83B cells. For example... Figure 12 As shown, compared with the control group, cell viability was significantly increased after DBP knockdown (P<0.01).

[0123] 3. The effect of DBP knockdown on cell colony formation

[0124] Cell colony formation assay was used to detect cell proliferation, and the results were as follows: Figure 13 As shown, compared with the control group, the number of clones formed by FL83B cells with DBP knockdown was significantly increased (P<0.01).

[0125] 4. The impact of knocking down DBP on migration

[0126] Scratch tests are used to examine cell migration ability, such as... Figure 14 As shown, compared with the control group, the migration rate of FL83B cells with DBP knockdown was significantly increased (P < 0.01).

[0127] 5. The effect of DBP knockdown on the cell cycle

[0128] Flow cytometry was used to examine the cell cycle of DBP-knockdown FL83B cells, such as... Figure 15 As shown, compared with the control group, knocking down DBP significantly reduced the proportion of cells in the G2 / M phase (P<0.01).

[0129] 6. Effects of DBP knockdown on metabolism-related genes

[0130] The expression of metabolic gene populations in DBP-knocked FL83B cells was examined using RT-qPCR. Figure 16As shown, compared with the control group, the expression levels of metabolic genes Cyp2a4, ALB, G6PC, and Sult1c1 were significantly downregulated (P<0.05). This indicates that knocking down DBP can inhibit liver function.

[0131] Example 5: Regulatory role of DBP in antioxidant genes

[0132] This embodiment uses RT-qPCR to detect antioxidant functional genes and analyzes whether DBP can regulate the expression of antioxidant functional genes after inhibiting the biological characteristics of FL83B tumors and promoting hepatocyte differentiation.

[0133] 1. The regulatory role of DBP overexpression in antioxidant genes

[0134] The expression of antioxidant gene clusters mRNA in FL83B cells overexpressing DBP was examined using RT-qPCR, following the same detection method as in Example 2. The results are as follows: Figure 17 As shown, compared with the control group, the expression levels of SOD1 and PPARα were significantly upregulated after DBP overexpression (P<0.05), while the expression levels of NOX4 and HMOX1 were significantly downregulated (P<0.05). These results indicate that DBP overexpression can significantly upregulate the expression of antioxidant genes and significantly inhibit the expression of oxidative stress genes.

[0135] 2. Knocking down the regulatory role of DBP in antioxidant genes

[0136] The expression of antioxidant gene clusters mRNA in DBP-knocked FL83B cells was examined using RT-qPCR, following the same method as in Example 2. The results are as follows: Figure 18 As shown, compared with the control group, DBP knockdown significantly downregulated the expression levels of SOD1 and PPARα (P<0.05), while significantly upregulated the expression levels of NOX4 and HMOX1 (P<0.05). These results indicate that DBP knockdown significantly inhibits the expression of antioxidant genes and significantly upregulates the expression of oxidative stress genes.

[0137] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A combination of Gardenia jasminoides Ellis and Melia toosendan Sieb. et Zucc., characterized in that, The ratio of the Gardeniae and the Xiangai is (1-2):(1-6).

2. The combination of gardenia and xiaozhi according to claim 1, wherein, The ratio of the Gardeniae and the Xiangai is 2:

3.

3. The combination of gardenia and xiaozhi according to claim 1, wherein, The preparation method of the Gardeniae and the Xiangai composition comprises the following steps: The Gardeniae and the Xiangai are extracted by refluxing with methanol for 1 hour, and then filtered to obtain a first filtrate and a first filter residue; the first filter residue is extracted by refluxing with methanol for 1 hour, and then filtered to obtain a second filtrate and a second filter residue; the second filter residue is extracted by refluxing with methanol with a volume fraction of 50% for 1 hour, and then extracted by refluxing with methanol with a volume fraction of 50% for 3 times; all the filtrates are combined, filtered under reduced pressure, evaporated, and then the extract is combined; the extract is suspended with water to obtain a suspension, which is extracted with petroleum ether, ethyl acetate, n-butanol and water in sequence, and then freeze-dried to obtain the Gardeniae and the Xiangai extract; and the Gardeniae and the Xiangai extract are mixed according to the ratio to obtain the Gardeniae and the Xiangai composition.

4. The combination of gardenia and xiaozhi according to claim 1, wherein, The volume ratio of the petroleum ether, the ethyl acetate, the n-butanol and the water to the suspension is 1:

1. The petroleum ether, the ethyl acetate, the n-butanol and the water are repeatedly extracted for 3 times.

5. The Gardeniae and the Xiangai composition of claim 1 is applied to the preparation of a DBP activator.

6. A DBP activator, characterized by, The effective component is the Gardeniae and the Xiangai composition of claim 1.

7. The Gardeniae and the Xiangai composition of claim 1 is applied to the preparation of a liver-protecting drug.

8. A hepatoprotective drug, characterized by comprising, The effective component is the Gardeniae and the Xiangai composition of claim 1.

9. The Gardeniae and the Xiangai composition of claim 1 is applied to the preparation of an antioxidant drug.

10. An antioxidant medicament, characterized by, The effective component is the Gardeniae and the Xiangai composition of claim 1.

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