Application of emodin and quercetin in preparation of compound preparation medicine for targeted inhibition of TNF-alpha
By optimizing the ratio of emodin and quercetin, a compound preparation targeted inhibition of TNF-α was prepared, which solved the problems of unclear targets and hepatotoxicity when used alone, and achieved the effect of efficiently inhibiting TNF-α activity and reducing toxicity.
Patent Information
- Application Number
- CN202510160445.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-13
- Publication Date
- 2025-05-30
AI Technical Summary
Edhain and quercetin are poorly effective in inhibiting TNF-α activity when used alone due to unclear targets, and emodin is hepatotoxic, which limits its in-depth development.
By optimizing the ratio of emodin and quercetin, with a specific ratio of 3.5:6.5, a compound preparation targeted inhibition of TNF-α was prepared, and the optimal ratio was screened using orthogonal experimental design method, and its efficacy was verified through cell and animal models.
The targeting of emodin and quercetin and inhibiting the activity of TNF-α was achieved, which significantly reduced the TNF-α-mediated inflammatory response, which was much higher than that of using alone, and reduced the toxicity of emodin alone.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of medicine, and particularly relates to the application of emodin and quercetin in the preparation of a compound preparation drug for targeted inhibition of TNF-α. Background Art
[0002] Emodin and quercetin are respectively common bioactive compounds in natural plants and have various biological functions such as anti-inflammatory and antioxidant effects. Although they show certain effects in inhibiting inflammatory reactions when used alone, due to their unclear targets and lack of symptomatic medication, the effects are not satisfactory. Moreover, emodin also has certain hepatotoxicity, which also limits its further development. Emodin and quercetin are widely present in nature. If they cannot be rationally utilized, it will undoubtedly be a great waste of resources. Therefore, how to reduce the toxicity and increase the efficacy of emodin and quercetin is the current key research direction.
[0003] TNF-α (tumor necrosis factor α) is a pro-inflammatory cytokine that participates in various immune-mediated inflammatory reactions and plays an important role in the occurrence and development of inflammatory diseases and is a therapeutic target for various inflammatory diseases. At present, there is no report on the targeting (i.e., directly binding to TNF-α rather than regulating its protein expression) and inhibition of the activity of TNF-α by emodin and quercetin. Whether they can also target and inhibit the activity of TNF-α when used in combination, and whether there is a synergistic effect when used in combination, still lacks sufficient research. Summary of the Invention
[0004] In order to solve the above problems, the present invention provides the application of emodin and quercetin in the preparation of a compound preparation drug for targeted inhibition of TNF-α.
[0005] Specifically, it is achieved through the following technical solutions:
[0006] The application of quercetin and emodin in the preparation of a compound preparation drug for inhibiting TNF-α, and the combination of quercetin and emodin plays a role in reducing toxicity and increasing efficacy.
[0007] Further, the compound preparation drug for inhibiting TNF-α is a drug for targeted inhibition of TNF-α.
[0008] The application of emodin and quercetin in the preparation of a compound preparation drug for inhibiting TNF-α.
[0009] Further, the compound preparation drug for inhibiting TNF-α is a drug for targeted inhibition of TNF-α.
[0010] Further, quercetin and emodin are formulated in a ratio of (3 - 4):(6 - 7). Preferably, the ratio is 3.5:6.5.
[0011] A drug for targeted inhibition of TNF-α, containing quercetin and emodin.
[0012] Furthermore, the ratio of quercetin to emodin is (3 - 4):(6 - 7). The preferred ratio is 3.5:6.5.
[0013] Furthermore, it also contains other drugs for preventing and treating liver injury, as well as pharmaceutical excipients or carriers, and can be prepared into any pharmaceutical preparation suitable for clinical use, mainly including liquid preparations, granules, tablets, infusion granules, soft capsules, hard capsules, dripping pills, sustained-release preparations or injections.
[0014] The beneficial effects of the present invention are as follows:
[0015] The present invention discovers and discloses for the first time that emodin and quercetin can target and inhibit the activity of TNF-α, and can be used as TNF-α inhibitors in the treatment of immune diseases; when emodin and quercetin are used in combination, their effects are much higher than those of emodin and quercetin used alone, and even superior to the methotrexate control group; and when emodin and quercetin are used in combination, they can also significantly reduce the toxicity of emodin used alone.
[0016] The present invention provides a pharmaceutical composition containing emodin and quercetin, which can target and inhibit the activity of TNF-α and reduce the inflammatory response mediated by TNF-α. The present invention optimizes the ratio of emodin to quercetin by using the orthogonal experimental design method, verifies its curative effect through cell models and animal models, and finds that the drug combination can reduce the toxicity of emodin used alone. This drug combination provides a new treatment option for the treatment of TNF-α-related immune and inflammatory diseases. Description of the Drawings
[0017] Figure 1 It is the DARTS detection of the binding effect between emodin and TNF-α.
[0018] Figure 2 It is the DARTS detection of the binding effect between quercetin and TNF-α.
[0019] Figure 3 It is the CETSA detection of the binding effect between emodin and TNF-α.
[0020] Figure 4 It is the CETSA detection of the binding effect between quercetin and TNF-α.
[0021] Figure 5 It is the SPR detection of the binding effect between emodin and TNF-α.
[0022] Figure 6 It is the KD value of emodin and TNF-α calculated by fitting after SPR detection.
[0023] Figure 7 It is to detect the binding effect of quercetin and TNF-α by SPR and fit the KD value.
[0024] Figure 8 It is the effect of emodin on the viability of L929 cells induced by TNF-α or doxorubicin. A. The effect of emodin on the survival rate of L929 cells induced by TNF-α; B. The effect of emodin on the survival rate of L929 cells induced by doxorubicin.
[0025] Figure 9 It is the effect of quercetin on the viability of L929 cells induced by TNF-α or doxorubicin. A. The effect of quercetin on the survival rate of L929 cells induced by TNF-α; B. The effect of quercetin on the survival rate of L929 cells induced by doxorubicin.
[0026] Figure 10 It is to detect the effect of emodin targeting TNF-α on inhibiting the activation of NF-κB signaling pathway by luciferase reporter gene system. ### p < 0.001 compared with the blank group (Control); *** p < 0.001 compared with the model group (Model).
[0027] Figure 11 It is to detect the effect of quercetin targeting TNF-α on inhibiting the activation of NF-κB signaling pathway by luciferase reporter gene system. *** p < 0.001 compared with the blank group (Control); ### p < 0.001 compared with the model group (TNF-α).
[0028] Figure 12 It is the mRNA expression levels of IL-6, IL-1β and COX2 in emodin-inhibited MH7A cells induced by TNF-α. A. IL-6 level in MH7A cells. B. IL-1β level in MH7A cells. C. COX2 level in MH7A cells. ### p < 0.001 compared with the blank group (Control); * p < 0.05, *** p < 0.001 compared with the model group (Model).
[0029] Figure 13 It is the mRNA expression levels of IL-6, IL-1β and COX2 in quercetin-inhibited MH7A cells induced by TNF-α. A. IL-6 level in MH7A cells. B. IL-1β level in MH7A cells. C. COX2 level in MH7A cells. *** p < 0.001 compared with the blank group (Control); ###p < 0.001 compared with the model group (TNF-α).
[0030] Figure 14 Emodin inhibits the phosphorylation levels of p65 and IκBα in TNF-α-induced MH7A cells. ## p < 0.01, ### p < 0.001 compared with the blank group (Control); ** p < 0.01, *** p < 0.001 compared with the model group (Model).
[0031] Figure 15 Quercetin inhibits the phosphorylation levels of p65 and IκBα in TNF-α-induced MH7A cells. *** p < 0.001 compared with the blank group (Control); ### p < 0.001 compared with the model group (TNF-α).
[0032] Figure 16 Emodin inhibits p65 nuclear translocation in TNF-α-induced MH7A cells (400×).
[0033] Figure 17 Quercetin inhibits p65 nuclear translocation in TNF-α-induced MH7A cells (400×).
[0034] Figure 18 Emodin improves the RA response in CIA rats. A. Toe volume of rats. B. RF level in rat serum. C. TNF-α level in rat serum. D. IL-6 level in rat serum. E. IL-1β level in rat serum. # p < 0.05, ## p < 0.01, ### p < 0.001 compared with the blank group (Control), * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the model group (Model).
[0035] Figure 19 These are the results of quercetin improving foot swelling in CIA rats.
[0036] Figure 20 These are the effects of quercetin on serum inflammatory factors in CIA rats. Relative mRNA expression levels of TNF-α (A), IL-6 (B), IL-1β (C), and RF (D). ** p < 0.01, *** p < 0.001 compared with the blank group (Control), ## p < 0.01,### p < 0.001, compared with the Model group.
[0037] Figure 21 It is the result graph of screening the best drug combination using the luciferase reporter gene system. ## p < 0.01, compared with the Control group, ** p < 0.01, compared with the Model group, && p < 0.01, compared with the emodin, quercetin, 3:7, and 4:6 groups.
[0038] Figure 22 It is to detect the effects of emodin and quercetin alone or in combination on the mRNA expression levels of inflammatory factors such as IL-6, IL-1β, and COX2. ## p < 0.01, compared with the Control group, ** p < 0.01, compared with the Model group, && p < 0.01, compared with the emodin and quercetin groups.
[0039] Figure 23 It is to detect the improvement effects of emodin and quercetin alone or in combination on the symptoms of rheumatoid arthritis in CIA rats. ## p < 0.01, compared with the Control group, ** p < 0.01, compared with the Model group, && p < 0.01, compared with the emodin, quercetin, and MTX groups.
[0040] Figure 24 It is the result graph of the cell experiment on the compound preparation reducing the toxicity of emodin used alone. ## p < 0.01, compared with the Control group, ** p < 0.01, compared with the Model group.
[0041] Figure 25 It is the result graph of the animal experiment on the compound preparation reducing the toxicity of emodin used alone. ## p < 0.01, compared with the Control group, ** p < 0.01, compared with the Model group. Specific Embodiments
[0042] The following further elaborates on the specific embodiments of the present invention. However, the present invention is not limited to these embodiments. Any improvement or substitution based on the basic spirit of this embodiment still falls within the scope protected by the claims of the present invention.
[0043] 1. Target Binding Experiment
[0044] To verify the binding ability of emodin and quercetin to TNF-α respectively, the following techniques are used in this invention for verification:
[0045] 1.1 Target stability experiment (DARTS)
[0046] Using the DARTS technique, the binding ability of emodin to TNF-α and quercetin to TNF-α are detected respectively. By evaluating the protein stability of TNF-α in the presence of emodin or quercetin, its binding characteristics are further confirmed.
[0047] Experimental method:
[0048] RAW264.7 cells (1.5×10 5 cells / mL) were seeded in 96-well plates and cultured for 24 h, then 1 μg / mL LPS was added and incubated for 24 h. The supernatant was aspirated, and the cells were lysed with RIPA lysis buffer. Centrifuged at 14000 g for 20 min at 4 °C, and the supernatant was collected to obtain the lysate. The lysate was incubated with 200 μM emodin at room temperature for 30 min. At the same time, the lysate was incubated with DMSO as the blank control group. Then it was mixed with protease at different ratios (1:0, 1:100, 1:300, 1:1000, 1:3000, 1:10000). Incubated at room temperature for 30 min, 2 μL of 20× protease inhibitor was added and mixed well. Finally, 6× Loading buffer was added and denatured at 100 °C for 10 min, and Western blot was performed to investigate the effects of emodin and quercetin on the enzyme stability of TNF-α respectively.
[0049] Experimental results:
[0050] The DARTS results showed that compared with the solvent DMSO group, emodin (p<0.001, Figure 1 ) and quercetin (p<0.001, Figure 2 ) significantly reduced the hydrolysis of TNF-α by pronase, indicating that emodin and quercetin can bind to TNF-α, thus enhancing the resistance of TNF-α to protease hydrolysis, suggesting emodin and quercetin.
[0051] 1.2 Cellular thermal shift assay (CETSA)
[0052] Using the CETSA technique, the binding of emodin and quercetin to TNF-α in cells is verified respectively. After treating the cells, the binding effects of emodin and quercetin to TNF-α are evaluated by using the thermal stability changes under different temperature conditions.
[0053] Experimental method:
[0054] Incubate the lysate with 1000 μM emodin at room temperature for 30 min. At the same time, incubate the lysate with DMSO as the blank control group. Aliquot the above incubation solutions into 7 EP tubes in equal volumes and incubate them at room temperature, 60, 64, 68, 72, 76, and 80 °C for 3 min, centrifuge at 14000 g for 10 min at 4 °C, take the supernatant, add 6× Loading buffer and mix well, denature at 100 °C for 10 min, and perform Western blot detection to investigate the effects of emodin and quercetin on the thermal stability of TNF-α.
[0055] Experimental results:
[0056] The CETSA results showed that with the increase of temperature, the protein abundance of TNF-α in the solvent DMSO group decreased significantly (p < 0.01, Figure 3 , Figure 4 ), while the protein abundances of TNF-α in the emodin group and quercetin group were significantly more stable than those in the DMSO group (p > 0.05, Figure 3 , Figure 4 ), indicating that emodin and quercetin can bind to the TNF-α protein, thereby enhancing the thermal stability of TNF-α
[0057] 1.3 Surface Plasmon Resonance (SPR)
[0058] Experimental method:
[0059] Dissolve the drugs in PBS-Surfactant P20 solution containing 5% DMSO (PBS-P) at concentrations of 3.125, 6.25, 12.5, 25, and 50 μM. Use Biacore T200 (GE Healthcare, Boston, MA, USA) for binding kinetics evaluation. Couple the recombinant TNF-α protein to the BIAcore S series CM5 chip according to the kit instructions. The final immobilization level is 8700 response units. In the PBS-P immobilization buffer containing 5% DMSO and milli-Q water, contact for 100 s at a flow rate of 20 μL / min and dissociate for 150 s. At the same time, set the PBS-P solvent correction curve containing 5% DMSO. Use the Biacore T200 evaluation software for kinetic fitting.
[0060] Experimental results:
[0061] The results showed that emodin and quercetin could bind to TNF-α, generating response signals ( Figure 5 , Figure 7 ), the KD of emodin was approximately 26.49 μM ( Figure 6 ), and the KD value of quercetin was 2.664 μmol / L ( Figure 7) It indicates that emodin and quercetin can target TNF-α and have good binding ability with TNF-α.
[0062] 2. Verification of anti-TNF-α activity
[0063] Next, the inhibitory effects of emodin and quercetin on TNF-α activity were verified by the following experimental methods respectively:
[0064] 2.1 L929 cell model
[0065] Using the L929 cell death model induced by TNF-α, the optimal ratio of emodin and quercetin was screened out, and its anti-TNF-α effect was further verified by the luciferase reporter gene system and qRT-PCR.
[0066] Experimental method:
[0067] Use The luminescent cell viability assay kit to detect cell viability. L929 cells (1.5×10 5 cells / mL) were seeded in 96-well plates and cultured for 24 h. Act D (1 μg / mL) and TNF-α (7.5 ng / mL) were mixed with 0, 5, 10, and 15 μM drugs (emodin or quercetin) respectively, and Act D (1 μg / mL) and 0.5 μM doxorubicin were mixed with 0, 5, 10, and 15 μM drugs (emodin or quercetin) respectively. After co-incubation at 37 °C for 30 min, the cells were treated for 12 h respectively. The supernatant was aspirated, and 100 μL of reagent was added to each well, shaken for 4 min, incubated at room temperature for 8 min, and the chemiluminescence signal was detected at 1 s / well.
[0068] Experimental results:
[0069] The results showed that compared with the Model group, emodin and quercetin significantly increased the survival rate and cell viability of L929 cells induced by TNF-α (p<0.001, Figure 8 A, Figure 9 A), and had no significant effect on the viability of L929 cells induced by doxorubicin (p>0.05, Figure 8 B, Figure 9 B). It is suggested that emodin and quercetin can antagonize TNF-α activity and inhibit L929 cell damage.
[0070] 2.2 Luciferase reporter gene system
[0071] The luciferase reporter gene system was used to evaluate whether emodin and quercetin have inhibitory effects on the activity induced by TNF-α at different concentrations. By measuring the fluorescence signal intensity, its ability to inhibit TNF-α activity was determined.
[0072] Experimental method:
[0073] HEK293T cells (1.5×10 5 cells / mL) were seeded in 96-well plates and cultured for 12 h. The transfection mixture (10 μL per well) was added, and the micro oscillator was used for 30 s (the transfection mixture was composed of FUGENE 6 transfection reagent and pNL3.2.NF-κB-RE[NlucP / NF-κB-RE / Hygro] at a ratio of 3:1). After 24 h, 100 μL of pre-incubated TNF-α and drugs at different concentrations (emodin or quercetin) were added to the cells. At the same time, pre-incubated TNF-α and 10 μM UCB-9260 were used as positive controls, and the cells were treated for 1 h. Then the cells were placed in an incubator and cultured for another 5 h. The culture plates were equilibrated at room temperature for 10 min, and 100 μL fluorescence detection reagent (substrate: buffer = 1:50) was added. After reacting for 3 min, the chemiluminescence signal was detected.
[0074] Experimental results:
[0075] The results showed that compared with the Model group, emodin and quercetin significantly reduced the chemiluminescence signal of the NlucP-NF-κB reporter gene induced by TNF-α (p<0.001, Figure 10 , Figure 11 ). The above results indicated that emodin and quercetin could inhibit the activation of the NF-κB signaling pathway through TNF-α.
[0076] 3. Anti-inflammatory effects of emodin and quercetin by inhibiting TNF-α activity
[0077] 3.1 qRT-PCR
[0078] Using qRT-PCR technology, the mRNA expression levels of inflammatory factors such as IL-6, IL-1β, and COX2 induced by TNF-α were detected after treatment with emodin and quercetin, respectively, to evaluate their anti-inflammatory effects.
[0079] Experimental method:
[0080] MH7A cells (1.5×105 cells / mL) were seeded in 6-well plates and cultured for 24 h. Pre-incubated TNF-α and drugs at different concentrations were added, and after treating the cells for 24 h, they were rinsed twice with PBS. Total RNA was extracted according to the instructions of the total RNA extraction kit (Promega), and reverse transcribed into cDNA using the PrimeScript RT kit (TaKaRa). The primer sequences are shown in Table 1. The PCR program was 95°C for 15 s, 60°C for 15 s, and 72°C for 30 s. Forty PCR cycles were performed, and the relative gene expression (fold change) was calculated using the 2-ΔΔCT method. The data were normalized to the GAPDH level.
[0081] Table 1 Primers for target genes
[0082]
[0083] Experimental results:
[0084] Compared with the Model group, emodin significantly decreased the mRNA expression levels of IL-6, IL-1β, and COX2 in TNF-α-induced MH7A cells (p<0.001, Figure 12 ), and quercetin could significantly reduce the relative expression levels of IL-6, IL-1β, and COX-2 mRNA ( Figure 13 (A-C), P<0.001). The results showed that emodin and quercetin could inhibit the expression of inflammatory factors induced by TNF-α.
[0085] 3.2 Western blot
[0086] The Western blot technique was used to detect the effects of emodin and quercetin on downstream signaling molecules (such as p65, p-p65, IκB, p-IκB) induced by TNF-α, and to analyze their inhibitory effects on the NF-κB signaling pathway.
[0087] Experimental method:
[0088] MH7A cells (1.5×10 5Cells (at a density of [X] cells / mL) were seeded in a culture dish and cultured for 24 h. Pre-incubated TNF-α and drugs at different concentrations were added, and after treating the cells for 24 h, RIPA lysis buffer containing 1% PMSF was added to each sample to lyse the cells. The cell lysates were collected and centrifuged at 12,000 g for 20 min at 4 °C. The supernatant was collected, and the protein concentration was determined using the BCA method. 6× loading buffer was mixed with the total protein and boiled for 10 min. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked in a blocking buffer (5% BSA) at 4 °C for 3 h, and then incubated overnight at 4 °C with p65 (1:1000), p-p65 (1:1000), IκB (1:1000), p-IκB (1:1000), or GAPDH (1:5000). It was washed 5 times with TBST (5 min each time), and then incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. It was washed 5 times with TBST (5 min each time), and the membrane was photographed using a gel imaging system (Thermo Fisher Scientific). The scanned images were analyzed, and the gray value ratio of the target protein to GAPDH in each lane was analyzed separately to calculate the relative protein expression level.
[0089] Experimental results:
[0090] The results showed that compared with the Model group, emodin and quercetin significantly decreased the phosphorylation levels of IκBα and p65 in MH7A cells induced by TNF-α (p < 0.001, Figure 14 , Figure 15 ).
[0091] 3.3 Cellular immunofluorescence
[0092] The inhibitory effects of emodin and quercetin on the nuclear translocation of p65 protein were observed through cellular immunofluorescence experiments to further verify their anti-inflammatory mechanisms.
[0093] Experimental method:
[0094] Cover slips were placed in a 6-well plate with a small amount of culture medium dropped on them. MH7A cells ([X]×10 5Cells were seeded at a density of [[[cell density]]] cells / mL in 6-well plates and cultured for 24 h. Pre-incubated TNF-α and drugs at different concentrations were added, and after treating the cells for 24 h, the cells were rinsed 3 times with PBS (5 min each time). 4% paraformaldehyde was added and the cells were fixed at room temperature for 20 min, then rinsed 3 times with PBS (5 min each time). 0.5% Triton X-100 was added and the cells were incubated at room temperature for 20 min, then rinsed 3 times with PBS (5 min each time). The cells were blocked in blocking buffer (5% BSA) at 4 °C for 30 min, then incubated with p65 (1:50) overnight at 4 °C, rinsed 3 times with PBS (5 min each time), and then incubated with the secondary antibody at room temperature for 2 h. DAPI was added for counterstaining, and the cells were incubated in the dark for 10 min, then rinsed 3 times with PBS (5 min each time). Images were observed and acquired using a Zeiss LSM 900 laser confocal microscope (Carl Zeiss AG, BW, Germany).
[0095] Experimental results:
[0096] The results showed that compared with the Model group, emodin and [[drug name]] could significantly reduce p65 nuclear translocation in TNF-α-induced MH7A cells (p < 0.001, Figure 16 , Figure 17 ).
[0097] 3.3 Animal model
[0098] Animal model experiments were conducted on the selected drug combinations with better effects to further verify their anti-inflammatory effects and safety in vivo.
[0099] Experimental method:
[0100] Female specific pathogen-free Wistar rats (weighing 210±10 g) were purchased from Tianqin Biotechnology Co., Ltd. in Changsha City (SCXK(Xiang) 2022-0011). After 1 week of adaptive feeding, the rats were randomly divided into a control group and a model group. The hair at the root of the rat's tail was shaved off with a hair clipper, and 0.2 mg of collagen emulsion (mixed with incomplete Freund's adjuvant and bovine type II collagen solution, 1 mg / mL) was injected intradermally into the tail of each rat. Seven days after the primary immunization, the same collagen emulsion was injected in the same way to enhance the immunity to prepare a CIA rat model. The normal group was injected with normal saline in the same way. The successfully prepared rats were randomly divided into 5 groups: model group (CIA), methotrexate group (MTX, 0.9 mg / kg), low-dose drug group (20 mg / kg), medium-dose drug group (40 mg / kg), and high-dose drug group (80 mg / kg), with 6 rats in each group. The MTX group was given MTX by gavage 2 times a week, the drug groups were given emodin or quercetin by gavage once a day, and the control group and the model group were given 0.5% sodium carboxymethylcellulose (CMC-Na) solution by gavage every day. It was given continuously for 35 days. The toe volume of the left foot of the rats was measured every week. And 12 h after the last administration, the rats were fasted but not water-deprived, and blood was collected from the abdominal aorta to detect the levels of RF, TNF-α, IL-1β, and IL-6 in the serum according to the ELISA kit.
[0101] Experimental results:
[0102] The results were as Figure 18 shown. Compared with the Model group, emodin and quercetin significantly reduced the toe volume of CIA rats and alleviated the swelling of the left ankle joint of CIA rats (p<0.001, Figure 18 A, Figure 19 ), and reduced the levels of RF, TNF-α, IL-6, and IL-1β in the serum of CIA rats (p<0.05, Figure 18 D-E, Figure 20 ).
[0103] 4. Optimization and screening of drug combinations
[0104] 4.1 Orthogonal experimental design
[0105] The orthogonal experimental design method was used in the present invention to optimize the ratio of emodin to quercetin, and the experimental design was as follows:
[0106] Fix the total drug amount: The total amount of drugs in each group was 10 μM.
[0107] Adjust the ratio: The ratios of emodin to quercetin were different, but the sum of their concentrations should be equal to the preset total drug amount.
[0108] Calculation method: The total drug amount in each group = emodin concentration + quercetin concentration. Therefore, the concentrations of emodin and quercetin were allocated according to the set ratios.
[0109] Select the orthogonal array of L20(2^10) to design 20 groups of different experimental conditions, and screen out the optimal ratio of emodin to quercetin. The ratios are as follows:
[0110] Table 2 Orthogonal array of L20(2^10)
[0111]
[0112] 4.2 L929 cell model
[0113] Use the L929 cell death model induced by TNF-α to screen out the optimal ratio of emodin and quercetin.
[0114] Experimental method:
[0115] Use The luminescent cell viability assay kit to detect cell viability. Seed L929 cells (1.5×10 5 cells / mL) into 96-well plates and culture for 24 h. Mix Act D (1 μg / mL) and TNF-α (7.5 ng / mL) with the drug respectively, incubate at 37 °C for 30 min, then treat the cells for 12 h respectively. Aspirate the supernatant, add 100 μL of reagent to each well, shake for 4 min, incubate at room temperature for 8 min, and detect the chemiluminescence signal at 1 s / well.
[0116] Experimental results:
[0117] It can be seen from the results (Table 3) that compared with experimental group 1 (using quercetin alone) and group 21 (using emodin alone), the ratios of quercetin:emodin of 3:7 (group 7), 3.5:6.5 (group 8) and 4:6 (group 9) have the best effects. We selected these 3 ratios and further verified them using the NlucP-NF-κB reporter gene system.
[0118] Table 3 Cell survival rate
[0119]
[0120] 4.3 Luciferase reporter gene system
[0121] Further verify its anti-TNF-α effect through the luciferase reporter gene system
[0122] Experimental method:
[0123] Seed HEK293T cells (1.5×10 5Cells (at a density of cells / mL) were seeded in a 96-well plate and cultured for 12 h. The transfection mixture (10 μL per well) was added, and the plate was shaken on a microplate shaker for 30 s (the transfection mixture was composed of FUGENE 6 transfection reagent and pNL3.2.NF-κB-RE[NlucP / NF-κB-RE / Hygro] at a ratio of 3:1). After 24 h, 100 μL of pre-incubated TNF-α and different concentrations of the drug were added to the cells. At the same time, pre-incubated TNF-α and 10 μM UCB-9260 were used as a positive control, and the cells were treated for 1 h. Then the cells were placed back in the cell culture incubator and cultured for an additional 5 h. The culture plate was equilibrated at room temperature for 10 min, and 100 μL of
[0124] Experimental results:
[0125] From the results of the L929 and luciferase reporter gene systems ( Figure 21 ), when the ratio of quercetin to emodin was 3.5:6.5, the anti-TNF-α activity of the composition was optimal.
[0126] 5. Anti-inflammatory effect of the compound preparation by inhibiting TNF-α activity
[0127] The anti-inflammatory effect of the compound preparation was further verified at the cellular and animal levels
[0128] 5.1 Verification at the cellular level
[0129] The qRT-PCR technique was used to detect the mRNA expression levels of inflammatory factors such as IL-6, IL-1β, and COX2 in TNF-α-induced MH7A cells after treatment with emodin and quercetin alone or in combination, and to evaluate their anti-inflammatory effects.
[0130] Experimental method:
[0131] MH7A cells (1.5×10 5 cells / mL) were seeded in a 6-well plate and cultured for 24 h. Pre-incubated TNF-α and the drug were added to the cells. After 24 h of treatment, the cells were rinsed twice with PBS, and total RNA was extracted according to the instructions of the total RNA extraction kit (Promega) and reverse transcribed into cDNA using the PrimeScript RT kit (TaKaRa). The primer sequences are shown in Table 1. The PCR program was 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s. Forty PCR cycles were performed, and the relative gene expression (fold change) was calculated using the -ΔΔCT method. The data were normalized to the GAPDH level.
[0132] Table 4 Primers for target genes
[0133]
[0134] Experimental results:
[0135] It can be seen from Figure 22 that when the ratio of quercetin to emodin is 3.5:6.5, the composition has the best anti-TNF-α activity.
[0136] 5.2 Verification at the animal level
[0137] Animal model experiments were carried out on the selected drug combinations with better effects to further verify their anti-inflammatory effects and safety in vivo.
[0138] Experimental method:
[0139] 5.1.1 Female specific pathogen-free Wistar rats (body weight 210±10 g) were purchased from Changsha Tianqin Biotechnology Co., Ltd. (SCXK(Xiang)2022-0011). After 1 week of adaptive feeding, the rats were randomly divided into a control group and a model group. The hair at the root of the rat tail was shaved off with a hair clipper, and 0.2 mg of collagen emulsion (mixed with incomplete Freund's adjuvant and bovine type II collagen solution, 1 mg / mL) was injected intradermally into the tail of each rat. Seven days after the primary immunization, the same collagen emulsion was injected in the same way to enhance the immunity to prepare a CIA rat model. The normal group was injected with normal saline in the same way. The successfully prepared rats were randomly divided into 5 groups: model group (CIA), methotrexate group (MTX, 0.9 mg / kg), emodin group (80 mg / kg), quercetin group (80 mg / kg), and compound preparation group (80 mg / kg), with 6 rats in each group. The MTX group was given MTX by gavage 2 times a week, the emodin group was given emodin by gavage once a day, and the control group and the model group were given 0.5% carboxymethylcellulose sodium (CMC-Na) solution by gavage every day. It was continuously administered for 35 days. After the last administration, the rats were fasted but not water-deprived for 12 h, and blood was collected from the abdominal aorta to detect the levels of RF, TNF-α, IL-1β, and IL-6 in the serum according to the ELISA kit.
[0140] Experimental results:
[0141] It can be seen from Figure 23 that the emodin / quercetin combination (3.5:6.5) can significantly reduce the levels of RF, TNF-α, IL-1β, and IL-6 in the serum, and the effect is significantly better than that of emodin or quercetin alone.
[0142] 6. The compound preparation can reduce the toxicity of emodin alone
[0143] Although emodin has good anti-inflammatory effects, it has certain hepatotoxicity, which limits its clinical use. [Citation: Guo, Y.; Song, J.; Liu, Y.; Yuan, M.; Zhong, W.; Guo, Y.; Guo, L. Study on the Hepatotoxicity of Emodin and Its Application in the Treatment of Liver Fibrosis. Molecules 2024, 29, 5122.]. Therefore, we also investigated whether the compound preparation could reduce the hepatotoxicity caused by emodin at the cellular and animal levels.
[0144] Experimental methods:
[0145] 6.1 Cell model
[0146] Experimental methods:
[0147] Use A luminescent cell viability assay kit was used to detect cell viability. Human primary hepatocytes (1.5×10 5 cells / mL) were seeded in 96-well plates and cultured for 24 h. Drugs were added, and after co-incubation at 37 °C for 30 min, the cells were treated for 12 h respectively. The supernatant was aspirated, and 100 μL of reagent was added to each well. The mixture was shaken for 4 min and incubated at room temperature for 8 min, and the chemiluminescence signal was detected at 1 s / well.
[0148] Experimental results:
[0149] From Figure 24 it can be seen that emodin can significantly kill human primary hepatocytes, while the emodin / quercetin composition (3.5:6.5) has no obvious effect on the survival rate of human primary hepatocytes.
[0150] 6.2 Animal model
[0151] Experimental methods:
[0152] Eighteen healthy clean-grade SD rats, weighing 120 ± 10 g. After 1 week of adaptive feeding and observation, they were randomly divided into a control group, an Emodin group (400 mg·kg -1 ), and a compound preparation group (620 mg·kg -1 ), with 6 rats in each group. After continuous administration for 4 weeks, blood was collected for subsequent experiments.
[0153] Experimental results:
[0154] From Figure 25It can be seen that emodin can significantly cause liver injury in rats, while the emodin / quercetin composition (3.5:6.5) does not cause liver injury.
[0155] The above cell and animal experiments show that the compound preparation can reduce the toxicity of emodin used alone.
[0156] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to embrace all changes falling within the equivalent meaning and scope of the claims within the protection scope of the present invention.
Claims
1. The application of quercetin and emodin in the preparation of a compound preparation for inhibiting TNF-α, characterized in that: The combination of quercetin and rhein has the effect of reducing toxicity and increasing efficacy.
2. The use according to claim 1, characterized in that The compound preparation drug for inhibiting TNF-α is a drug for inhibiting TNF-α in a targeted manner.
3. Application of emodin and quercetin in the preparation of compound preparations for inhibiting TNF-α.
4. The use according to claim 3, characterized in that The compound preparation drug for inhibiting TNF-α is a drug for inhibiting the activity of TNF-α in a targeted manner.
5. The use according to claim 3, characterized in that Quercetin and emodin are mixed in the ratio of (3-4):(6-7).
6. The use according to claim 5, characterized in that The ratio of quercetin to rhein is 3.5:6.
5.
7. A drug for targeted inhibition of TNF-α, characterized in that: Contains quercetin and rhein.
8. The drug according to claim 7, characterized in that The ratio of quercetin to rhein is (3-4):(6-7).
9. The drug according to claim 8, characterized in that The ratio of quercetin to emodin is 3.5:6.
5.
10. The drug according to claim 7, characterized in that The invention also contains other drugs for preventing and treating liver damage and drug excipients or carriers.