Application of clinacanthus nutans extract in preparation of medicine for preventing and treating hepatotoxicity caused by nonyl phenol based on MAPK-Nrf2 pathway

Abstract

The invention discloses an application of clinacanthus nutans extract in preparation of a medicine for preventing and treating hepatotoxicity caused by nonyl phenol based on an MAPK-Nrf2 pathway, nonyl phenol as a widely distributed environmental endocrine disrupter enters a food chain through environmental pollution, and hepatotoxicity can be caused. Researches prove that the clinacanthus nutans leaf and leaf ethanol extract can up-regulate Nrf2 and HO-1 protein expression by inhibiting activation of P38 and ERK in an MAPK-Nrf2 pathway, and recover catalase CAT activity, glutathione GSH content and adenosine triphosphate ATP content in cells, so that the toxic effect of nonyl phenol on HepG2 cells is inhibited. The key effective components in the clinacanthus nutans extract are determined, a reliable mechanism verification model is established, a natural plant source protection strategy is provided for development of liver injury caused by environmental toxicants, and the clinacanthus nutans extract has good application prospects and industrial conversion value.

Description

Technical Field

[0001] The invention relates to the technical field of natural products, and in particular to an application of an alligator mouth flower extract in preparing a drug for preventing and treating hepatotoxicity caused by nonylphenol based on a MAPK-Nrf2 pathway. Background Art

[0002] Nonylphenol (NP) is a widely distributed environmental endocrine disruptor, mainly found in plastic products, detergents and textile printing and dyeing auxiliaries. The industrialization process and the large-scale use of related products have caused NP to enter the environmental circulation system through industrial wastewater discharge, landfill leachate and sewage treatment plant effluent. Due to its high lipid solubility, NP is commonly detected in fish, crops and drinking water systems, and is gradually enriched through the food chain. Therefore, the human body is mainly exposed to NP through three pathways: respiratory inhalation, skin contact and bioaccumulation in the food chain. As the main metabolic and detoxification organ of the body, the liver is the first to be affected by the toxic effects of NP, making it a key target organ for assessing the health risks of NP and exploring natural product intervention strategies.

[0003] The mechanism of hepatotoxicity caused by nonylphenol (NP) is different from traditional hepatotoxicity and has unique pathogenic characteristics. Unlike alcoholic liver disease, which is mainly caused by long-term alcohol intake directly damaging liver cells, non-alcoholic fatty liver disease (NAFLD) originates from an imbalance in energy metabolism, leading to excessive accumulation of lipids in the liver. It is usually associated with metabolic syndromes such as obesity and insulin resistance. The main pathological feature is fat change in hepatocytes, and the inflammatory response develops in an insidious and progressive manner. Drug-induced liver injury, on the other hand, is caused by drugs and their metabolites directly producing hepatotoxicity or inducing immune-mediated injury. The injury patterns are diverse and closely related to individual differences and abnormalities in the drug metabolism enzyme system.

[0004] The specificity of NP hepatotoxicity is mainly manifested as persistent damage mediated by oxidative stress. The specific mechanisms include: NP directly inhibits the activity of key antioxidant enzyme systems (SOD, GSH, CAT), leading to a large accumulation of reactive oxygen species (ROS); NP interferes with the Nrf2 signaling pathway and hinders its nuclear translocation function. Although short-term low-dose NP exposure can activate Nrf2 antioxidant defense, long-term low-dose exposure eventually leads to the collapse of the antioxidant system; NP indirectly activates the MAPK signaling pathway through ROS-mediated oxidative stress, causing mitochondrial dysfunction and triggering apoptosis cascade reactions. More importantly, NP continuously accumulates in organisms through the food chain, and the oxidative damage caused is long-term and persistent, rather than transient. Given the prevalence of environmental exposure to nonylphenol, the entire population faces the potential threat of NP hepatotoxicity, and this toxic effect is hidden and cumulative. Therefore, the development of preventive intervention strategies applicable to a wide range of people is crucial to defend against NP hepatotoxicity.

[0005] At present, the means of detoxifying the hepatotoxicity caused by nonylphenol (NP) are relatively limited. Although traditional antioxidants (such as vitamin C) can scavenge free radicals to a certain extent and reduce the level of reactive oxygen species (ROS), their effect is limited in the hepatotoxicity caused by NP. In particular, they cannot effectively increase the activity of the key antioxidant enzyme system (SOD, GSH, CAT), resulting in poor detoxification effect. Although existing hepatoprotective drugs can improve liver function indicators to a certain extent, they generally have limitations such as insufficient specificity and single mechanism of action, and long-term use may cause adverse reactions and side effects. This situation highlights the urgent need to develop new intervention strategies with specific mechanisms and higher safety for NP hepatotoxicity.

[0006] Clinacanthus nutans (Burm.f.) Lindau is a medicinal and edible plant widely distributed in Southeast Asia, commonly known as Youduncao, Liqing, Qianlizhui, etc. in China. Its traditional eating methods are diverse, including making soup as a herbal tea, fresh juice or directly eating the leaves. In traditional medicine in countries such as Thailand, Indonesia and Malaysia, Clinacanthus nutans is widely used to treat symptoms such as snake bites, herpes infections, skin diseases and burns, showing significant medicinal value.

[0007] Phytochemical studies have shown that Clinacanthus nutans is rich in various bioactive components, especially phenolic and flavonoid compounds, and also contains proteins, vitamin C, various amino acids and important mineral elements such as potassium, calcium, iron, zinc and selenium. Among them, phenolic and flavonoid substances, as the main active components, have significant antioxidant and anti-inflammatory properties, which specifically counteract the oxidative stress mechanism of the hepatotoxicity caused by nonylphenol (NP).

[0008] Clarifying the protective effect and mechanism of action of the ethanol extract of Clinacanthus nutans leaves on NP hepatotoxicity is of great significance for developing targeted liver protection strategies. Applying it to the development of drugs, health products or functional foods can not only provide an effective intervention plan for oxidative stress-related liver injury, but also promote the scientific development and sustainable utilization of this valuable medicinal plant resource, realizing a good example of the modern application of traditional medicinal plants. Summary of the Invention

[0009] To solve the above technical problems, the present invention provides an application of Clinacanthus nutans extract in the preparation of a drug for preventing and treating hepatotoxicity caused by nonylphenol based on the MAPK-Nrf2 pathway.

[0010] The solution of the present invention to the above technical problems is as follows:

[0011] The application of Clinacanthus nutans extract in the preparation of a drug for preventing and treating hepatotoxicity caused by nonylphenol based on the MAPK-Nrf2 pathway, wherein the Clinacanthus nutans extract is an ethanol extract of Clinacanthus nutans leaves, and the extract contains total phenols and total flavonoids.

[0012] Furthermore, after the HepG2 cells were exposed to nonylphenol, the MAPK pathway of the HepG2 cells was activated and the Nrf2 pathway was inhibited. The Clinacanthus nutans extract could inhibit the MAPK pathway and activate the Nrf2 pathway.

[0013] Furthermore, the Clinacanthus nutans extract up-regulated the expression of Nrf2 and HO-1 proteins by inhibiting the activation of P38 and ERK in the MAPK-Nrf2 pathway, thereby inhibiting the activation of the MAPK pathway and the inhibition of the Nrf2 pathway in HepG2 cells after exposure to nonylphenol.

[0014] Furthermore, the Clinacanthus nutans extract up-regulated the expression of Nrf2 and HO-1 proteins by inhibiting the activation of P38 and ERK in the MAPK-Nrf2 pathway, restored the activities of catalase (CAT), the content of glutathione (GSH), and the content of adenosine triphosphate (ATP), thereby inhibiting the activation of the MAPK pathway and the inhibition of the Nrf2 pathway in HepG2 cells after exposure to nonylphenol.

[0015] Furthermore, the Clinacanthus nutans extract inhibited the activation of P38 in the MAPK-Nrf2 pathway, and the specific inhibitor for verifying this target was SB202190. The Clinacanthus nutans extract inhibited the activation of ERK in the MAPK-Nrf2 pathway, and the specific inhibitor for verifying this target was PD98059. The Clinacanthus nutans extract activated Nrf2 and its downstream protein HO-1 in the MAPK-Nrf2 pathway, and the specific inhibitor for verifying this target was ML385.

[0016] Furthermore, the preparation method of the Clinacanthus nutans extract comprises the following steps:

[0017] (1) Freeze-dry fresh Clinacanthus nutans leaves, crush them, pass through a 60-mesh sieve, and collect the material under the sieve;

[0018] (2) Ultrasonically extract the material under the sieve with 70% ethanol at a liquid-to-material ratio of 1:15 to obtain an extract;

[0019] (3) Centrifuge the extract, perform vacuum filtration, concentrate the filtrate under reduced pressure, and remove ethanol to obtain a concentrated solution

[0020] (4) Freeze-dry the concentrated extract to obtain the Clinacanthus nutans extract.

[0021] Furthermore, the conditions for ultrasonic extraction are: ultrasonic power 100 W, ultrasonic temperature 45 °C, extraction time 90 min, and the conditions for concentration under reduced pressure are: rotary evaporation under reduced pressure at 40 - 50 °C.

[0022] Furthermore, the alligator mouth flower extract contains a total phenol content of 57.37 mg / g and a total flavonoid content of 41.56 mg / g.

[0023] Another aspect of the present invention provides a drug for preventing and treating hepatotoxicity caused by nonylphenol based on the MAPK-Nrf2 pathway, comprising the ethanol extract of the leaves of the alligator flower.

[0024] The third aspect of the present invention provides a pharmaceutical preparation for preventing and treating hepatotoxicity caused by nonylphenol based on the MAPK-Nrf2 pathway, comprising the ethanol extract of the above-mentioned alligator flower leaves, and the pharmaceutical preparation for preventing and treating hepatotoxicity caused by nonylphenol based on the MAPK-Nrf2 pathway is in the form of one of a lyophilized powder injection, an injection solution, a tablet or a granule.

[0025] As mentioned above, nonylphenol (NP) is a widely distributed environmental endocrine disruptor, mainly present in plastic products, detergents and textile printing and dyeing auxiliaries, can be detected in water, soil and air, and eventually enter the food chain. The present invention focuses on the application of ethanol extract of alligator mouth flower leaves in the prevention and treatment of hepatotoxicity caused by nonylphenol, and the inventors first discovered that the ethanol extract of alligator mouth flower leaves prevents and treats hepatotoxicity caused by nonylphenol through the MAPK-Nrf2 pathway. Among them, nonylphenol hepatotoxicity is essentially different from hepatotoxicity caused by fatty liver disease and drugs (e.g., acetaminophen APAP) in terms of mechanism.

[0026] Nonylphenol hepatotoxicity and fatty liver disease are fundamentally different in several aspects:

[0027] 1. In terms of triggering factors, nonylphenol hepatotoxicity originates from acute exposure to exogenous environmental toxins (nonylphenol in plastic products and detergents), while fatty liver disease originates from the long-term accumulation of endogenous metabolic imbalance (sitting for long periods of time, high-calorie diet).

[0028] 2. In terms of pathogenic mechanism, nonylphenol directly attacks mitochondria, leading to membrane potential collapse and oxidative stress, while fatty liver disease causes lipotoxicity and chronic inflammation through abnormal lipid accumulation.

[0029] 3. In the core pathway, nonylphenol hepatotoxicity mainly activates the MAPK-Nrf2 antioxidant pathway to respond to exogenous toxins, while fatty liver disease involves the TLR4 / MyD88 / NF-κB pro-inflammatory pathway.

[0030] 4. In terms of research markers, nonylphenol hepatotoxicity mainly measures the activity of the antioxidant enzyme catalase (CAT) and the content of glutathione (GSH) to reflect the degree of oxidative stress, while fatty liver disease focuses on the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) to evaluate liver cell damage.

[0031] 5. In terms of the protection mechanism, the Clinacanthus nutans extract against nonylphenol hepatotoxicity mainly exerts its effect by protecting mitochondrial function and maintaining membrane potential, while the treatment of fatty liver disease focuses on regulating lipid metabolism and reducing the level of triglyceride (TG).

[0032] Differences between nonylphenol hepatotoxicity and hepatotoxicity caused by drugs (such as acetaminophen APAP):

[0033] 1. Different sources: Nonylphenol: An environmental endocrine disruptor, present in plastic products, detergents, and textile printing and dyeing auxiliaries, and enters the food chain through environmental pollutants. Acetaminophen: A commonly used antipyretic and analgesic drug, safe at normal doses (0.9 - 2 g / day), and overdose (>4 g / day) can cause drug-induced liver injury.

[0034] 2. Toxic activation mechanism: Nonylphenol: Does not require metabolic activation, directly attacks hepatocyte mitochondria, leading to the collapse of membrane potential. Acetaminophen: Needs to be metabolically activated by CYP2E1 enzyme into the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). At normal doses, NAPQI binds to GSH for detoxification, and when in excess, GSH depletion causes liver injury.

[0035] 3. Key signaling pathways: Nonylphenol: Mainly involves the MAPK-Nrf2 antioxidant pathway, inhibits the expression of antioxidant proteins such as Nrf2, HO-1, and NQO1, and activates the p-ERK / ERK and p-P38 / P38 signal transduction. Acetaminophen: Mainly activates the JNK and NF-κB signaling pathways. NAPQI causes mitochondrial dysfunction and ROS production, secondarily activating the inflammatory pathway.

[0036] 4. Changes in biochemical markers: Nonylphenol: The content of GSH and the activity of CAT decrease significantly with the increase of dose (a passive consumption caused by ROS accumulation), the content of ATP decreases severely, the expression of antioxidant proteins (Nrf2, HO-1, NQO1) is inhibited, and the mitochondrial membrane potential collapses, directly reflecting its toxic mechanism of damaging mitochondrial function and inhibiting the antioxidant defense system. Acetaminophen: Serum transaminases (ALT / AST) increase significantly, GSH is consumed due to direct binding to NAPQI for detoxification, the activity of SOD decreases, the level of MDA increases, the pro-apoptotic protein Bax increases accompanied by the decrease of the anti-apoptotic protein Bcl-2, and the pro-inflammatory factors (TNF-α, IL-1β) increase while the anti-inflammatory factor (IL-10) decreases, constituting the typical biochemical characteristics of acute drug-induced liver injury.

[0037] 5. Differences in protection mechanisms: Nonylphenol: Focuses on maintaining mitochondrial function and repairing the antioxidant system. It mainly upregulates the expression of Nrf2 and HO-1 proteins by inhibiting the activation of P38 and ERK in the MAPK-Nrf2 pathway, and restores the activities of catalase CAT, glutathione GSH, and adenosine triphosphate ATP. Paracetamol: Mainly targets the metabolic activation link, inhibits the activity of CYP2E1 to block the generation of NAPQI, promotes the non-toxic metabolic pathway of APAP via SULT1A1 and UGT1A1, supplements N-acetylcysteine (NAC) as a GSH precursor to detoxify the already generated NAPQI, and reduces the inflammatory response by inhibiting the JNK and NF-κB signaling pathways.

[0038] The present invention has the following advantages over the prior art:

[0039] 1. It is determined that total phenols and total flavonoids in Clinacanthus nutans extracts are the key effective components for preventing and treating nonylphenol-induced hepatotoxicity.

[0040] 2. The action targets of Clinacanthus nutans extracts are verified by specific inhibitors, and it is confirmed that its hepatoprotective effect depends on the activation of the MAPK-Nrf2 signaling pathway, establishing a reliable mechanism verification model.

[0041] 3. It is revealed that Clinacanthus nutans extracts can upregulate the expression of key antioxidant proteins (Nrf2, HO-1) and restore the activities of antioxidant enzymes (GSH, CAT), providing an effective measure for solving the antioxidant system damage caused by NP.

[0042] 4. It is confirmed that Clinacanthus nutans extracts can repair the mitochondrial ATP synthesis disorder caused by nonylphenol, solving the problem of energy metabolism disorder caused by environmental toxins.

[0043] 5. It provides a natural plant source for preparing drugs, health products, or functional foods against nonylphenol hepatotoxicity, and has good application prospects and industrial transformation value. Description of the Drawings

[0044] Figure 1 For detecting the effect of NP-exposed HepG2 cells on Nrf2 pathway-related proteins by western blot in Example 2.

[0045] Figure 2 For detecting the effect of NP-exposed HepG2 cells on MAPK pathway-related proteins by western blot in Example 2.

[0046] Figure 3 For detecting the effect of NP on oxidative stress indexes of HepG2 cells by kit in Example 3.

[0047] Figure 4To detect the effect of CE on the viability of HepG2 cells by the CCK-8 method in Example 4.

[0048] Figure 5 To detect the effect of CE on the viability of NP-exposed HepG2 cells by the CCK-8 method in Example 5.

[0049] Figure 6 To detect the effect of CE on the oxidative stress indexes of NP-exposed HepG2 cells by the kit in Example 6.

[0050] Figure 7 To detect the effect of CE on the Nrf2 pathway-related proteins in NP-exposed HepG2 cells by western blot in Example 7.

[0051] Figure 8 To detect the effect of CE on the ERK / P38 pathway-related proteins in NP-exposed HepG2 cells by western blot in Example 7.

[0052] Figure 9 To detect the effect of P38 inhibitor on the Nrf2 and HO-1 proteins after CE intervention in NP-exposed HepG2 cells by western blot in Example 8.

[0053] Figure 10 To detect the effect of ERK inhibitor on the Nrf2 and HO-1 proteins after CE intervention in NP-exposed HepG2 cells by western blot in Example 8.

[0054] Figure 11 To detect the effect of P38 inhibitor on the CAT activity after CE intervention in NP-exposed HepG2 cells by the kit in Example 9.

[0055] Figure 12 To detect the effect of ERK inhibitor on the CAT activity after CE intervention in NP-exposed HepG2 cells by the kit in Example 9.

[0056] Figure 13 To detect the effect of Nrf2 inhibitor on the Nrf2 and HO-1 proteins after CE intervention in NP-exposed HepG2 cells by western blot in Example 10.

[0057] Figure 14 To detect the effect of Nrf2 inhibitor on the upstream ERK pathway proteins after CE intervention in NP-exposed HepG2 cells by western blot in Example 10.

[0058] Figure 15To detect the effect of Nrf2 inhibitor on the upstream P38 pathway protein after CE intervened in NP-exposed HepG2 cells in Example 10 by western blot.

[0059] Figure 16 To detect the effect of Nrf2 inhibitor on the CAT activity after CE intervened in NP-exposed HepG2 cells in Example 11 by kit. Detailed implementation manners

[0060] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0061] Example 1:

[0062] This example is about the extraction method of ethanol extract from Andrographis paniculata Nees leaves.

[0063] 1. The specific operation steps of the ethanol extract from Andrographis paniculata Nees leaves are as follows:

[0064] Step 1. Freeze-dry the fresh Andrographis paniculata Nees leaves, crush them through a 60-mesh sieve, and collect the material under the sieve.

[0065] Step 2. Mix the material under the sieve obtained in Step 1 with 70% ethanol according to a solid-liquid ratio of 1:15, and perform ultrasonic extraction. The extraction conditions are: ultrasonic power 100W, ultrasonic temperature 45°C, and extraction time 90 min. Centrifuge the Andrographis paniculata Nees extract at 3000 rpm for 10 min. Repeat the ultrasonic extraction for the Andrographis paniculata Nees leaf residue, combine the extracts and then centrifuge, and perform vacuum filtration. Concentrate the filtrate by removing ethanol in a rotary evaporator under reduced pressure at 40 - 50°C to obtain a concentrated extract.

[0066] Step 3. Freeze-dry the concentrated extract obtained in Step 2 to obtain the ethanol extract from Andrographis paniculata Nees leaves (CE), with a yield of 25.6%, and store it in the dark at -20°C for standby. The formula for calculating the CE yield is as follows:

[0067] CE yield (%) = (m2 / m1) × 100% (Formula 1)

[0068] In Formula 1, CE is the ethanol extract from Andrographis paniculata Nees leaves, m1 is the mass (g) of dry Andrographis paniculata Nees leaves, and m2 is the mass (g) of the ethanol extract from Andrographis paniculata Nees leaves.

[0069] 2. The total phenol and total flavonoid contents in CE were measured by the Folin-Ciocalteu method and the sodium nitrite-aluminum chloride method respectively. The results showed that the total phenol content in CE was 57.37 mg / g, and the total flavonoid content was 41.56 mg / g.

[0070] Example 2:

[0071] This example is about verifying the inhibitory effect of nonylphenol (NP)-exposed human hepatocellular carcinoma (HepG2) cells on the Nrf2 and MAPK pathways.

[0072] (1) The HepG2 cells were obtained from Wuhan Pusai Biotechnology Co., Ltd. The cell culture medium was MEM complete medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine. After resuscitating the cryopreserved HepG2 cells, they were resuspended and mixed well with the complete culture medium and placed in a sterile culture dish, and then cultured in an incubator at 37°C and 5% CO 2 . When the cells were in the logarithmic growth phase, they were subcultured or plated in a timely manner for further experiments.

[0073] (2) Cells in the logarithmic growth phase were seeded at 3×10 5 cells / well in a 6-well plate for 24 h. Then, different concentrations (0, 40, 50, 60, and 70 μM) of NP were added and the cells were treated for 24 h.

[0074] (3) Total cellular protein extraction: The cells were lysed on ice with RIPA lysis buffer for 30 min, centrifuged (4°C, 12,000 rpm, 5 min), and the supernatant was collected. The protein concentration was determined using a BCA protein assay kit. The protein samples were boiled in a boiling water bath for 7 min and stored in aliquots at -80°C.

[0075] (4) 15 μg of protein sample was added to each well and protein electrophoresis was performed using 10% SDS-PAGE. Then, the proteins were transferred onto a PVDF membrane and blocked with 5% non-fat milk for 1.5 h. Subsequently, the protein samples were incubated overnight at 4°C with primary antibodies against p-ERK, ERK, p-P38, P38, p-JNK, JNK, HO-1, Nrf2, and NQO1. Horseradish peroxidase-conjugated secondary antibody was added and incubated at room temperature at a ratio of 1:6000 for 1 h. The gray values of the samples were analyzed using Image J. The results were expressed as the gray value of the target protein divided by the gray value of the internal reference protein.

[0076] The effects of NP on Nrf2 pathway-related proteins in HepG2 cells are shown as Figure 1 follows. Compared with the control group, the protein levels of Nrf2, HO-1, and NQO1 were downregulated in a dose-dependent manner, and this downregulation was statistically significant (P < 0.05 or P < 0.01). These results suggest that NP affects the expression of Nrf2 pathway-related proteins.

[0077] The effects of NP on MAPK pathway-related proteins in HepG2 cells are shown as Figure 2 follows. At 60 μM, NP upregulated the ratios of p-ERK / ERK and p-P38 / P38 (P < 0.01); while the ratio of p-JNK / JNK was upregulated at 70 μM (P < 0.01).

[0078] Example 3:

[0079] This example is about the effect of NP on the oxidative stress of HepG2 cells. The indicators include the content of glutathione (GSH), the activity of catalase (CAT), and the content of adenosine triphosphate (ATP).

[0080] (1) When measuring GSH and CAT, cells in the logarithmic growth phase were seeded into 24-well plates at a density of 2×10 5 cells / well; when measuring ATP, cells in the logarithmic growth phase were seeded into 24-well plates at a density of 2×10 5 cells / well. After 24 h, cells were treated with different concentrations (0, 40, 50, 60, and 70 μM) of NP for 24 h; the culture medium was discarded, and the cells were washed twice with PBS. The culture plates were placed on ice, and the cells were lysed with Western and IP lysis buffer. 100 μL of lysis buffer was added to each well and lysed for 30 min; during the lysis process, the cells were pipetted with a pipette tip to ensure sufficient cell lysis. The treated cells were collected in EP tubes, centrifuged (4 °C, 12,000 rpm, 5 min), and the supernatant was taken for use; according to the instructions, the supernatant was taken for corresponding detection, and the protein concentration was detected with a BCA kit at the same time. The final results were normalized according to the protein content. Four parallels were set for each concentration. The GSH content was expressed as μmol per gram of protein, the CAT activity was expressed as enzyme activity units per milligram of protein, and the ATP content was expressed as μmol per milligram of protein.

[0081] The effect of NP on the oxidative stress of HepG2 cells is as Figure 3 shown. After treating HepG2 cells with different concentrations of NP for 24 h, the GSH content in the cells decreased in a dose-dependent manner, and this decrease was statistically significant at 50 μM - 70 μM (P < 0.01). The CAT activity showed a trend of first decreasing and then increasing, and the activity decreased to the lowest at 60 μM (P < 0.01), but there was no significant difference in the CAT activity between the 60 μM group and the 70 μM group (P > 0.05). When NP acted on HepG2 cells for 24 h, the ATP content showed a trend of first increasing and then decreasing, and at 60 μM and 70 μM, compared with the control group, this decrease was statistically significant (P < 0.05 or P < 0.01). Based on the above results, 60 μM was used as the NP poisoning concentration for subsequent experiments.

[0082] Example 4:

[0083] This example is about the cytotoxicity of CE on HepG2 cells.

[0084] (1) Inoculate cells in the logarithmic growth phase into a 96-well plate at a density of 3000 cells / well. After 24 h, add CE at different concentrations (0, 0.125, 0.25, 0.5, 1, 2, 4, 8, and 16 mg / mL), with 6 parallels for each concentration. After incubation for 24 h, discard the culture medium, wash with PBS, and then add 10 μL of CCK-8 reagent to each well. Incubate in the dark for 2 h, measure the absorbance at 450 nm, and calculate the cell viability.

[0085] The effect of CE on the viability of HepG2 cells is as Figure 4 shown. CE had an activating effect on the viability of HepG2 cells at a low concentration (0.125 mg / mL) (P < 0.05), and showed a dose-dependent inhibitory effect (P < 0.01), that is, cytotoxicity, at high concentrations (4 mg / mL - 16 mg / mL).

[0086] Example 5:

[0087] This example is about the effect of CE on the viability of HepG2 cells poisoned with NP.

[0088] (1) Inoculate cells in the logarithmic growth phase into a 96-well plate at a density of 3000 cells / well. After 24 h, add 60 μM NP and CE at different concentrations (0, 0.25, 0.5, 1, and 2 mg / mL), with 6 parallels for each concentration. After incubation for 24 h, discard the culture medium, wash with PBS, and then add 10 μL of CCK-8 reagent to each well. Incubate in the dark for 2 h, measure the absorbance at 450 nm, and calculate the cell viability.

[0089] The improvement effect of CE on hepatocyte injury caused by NP is as Figure 5 shown. Compared with the control group, the cell viability of the NP-poisoned group decreased to 88.51% (P < 0.01), indicating that the NP cell injury model was successfully constructed. Compared with the NP-poisoned group, the cell viability was increased after intervention with different concentrations of CE (P < 0.01). These results suggest that CE has a protective effect on NP-poisoned cells.

[0090] Example 6:

[0091] This example is about the effect of CE on the oxidative stress of HepG2 cells poisoned with NP. The indicators include GSH content, CAT activity, and ATP content.

[0092] (1) When measuring GSH and CAT, inoculate cells in the logarithmic growth phase into a 24-well plate at a density of 2×10 5 cells / well; when measuring ATP, inoculate cells in the logarithmic growth phase into a 24-well plate at a density of 2×10 5Cells were seeded at a density of

[0093] The effect of CE on improving the cell state of NP-exposed HepG2 cells is shown as Figure 6 follows. Compared with the control group, the contents of GSH, CAT activity and ATP content in the NP-exposed group decreased (P<0.05 or P<0.01); after CE intervention, the contents of GSH, CAT activity and ATP content all increased in a dose-dependent manner (P<0.05 or P<0.01). Among them, the contents of GSH and ATP showed obvious improvement effects at a low dose (0.25 mg / mL) (P<0.05 or P<0.01), while the improvement effect of CAT activity began at medium and high doses (1 mg / mL) (P<0.01). The above results suggest that CE has an intervention effect on the oxidative stress damage of cells caused by NP, and the effects on the contents of GSH and ATP are more obvious than those on CAT activity.

[0094] Example 7:

[0095] This example is about the effect of CE on the Nrf2 and ERK / P38 pathways of NP-exposed HepG2 cells.

[0096] (1) Cells in the logarithmic growth phase were seeded at a density of 3×10 5 cells / well in a 6-well plate for 24 h. Then, 60 μM NP and CE at different concentrations (0, 0.25, 0.5, 1 and 2 mg / mL) were added and the cells were treated for 24 h.

[0097] (2) Total cell protein extraction: Cells were lysed on ice with RIPA lysis buffer for 30 min, centrifuged (4℃, 12,000 rpm, 5 min), and the supernatant was collected. The protein concentration was measured using a BCA protein assay kit. The protein samples were boiled in a boiling water bath for 7 min and stored in aliquots at -80℃.

[0098] (3) Add 15 μg of protein sample to each well and perform protein electrophoresis using 10% SDS-PAGE. Then transfer it to a PVDF membrane and block it with 5% non-fat milk for 1.5 h. Subsequently, incubate the protein sample with primary antibodies against p-ERK, ERK, p-P38, P38, HO-1, and Nrf2 overnight at 4°C. Add a horseradish peroxidase-labeled secondary antibody and incubate it at a ratio of 1:6000 at room temperature for 1 h. Analyze the gray value of the sample by ImageJ. The result is expressed as the gray value of the target protein divided by the gray value of the internal reference protein.

[0099] The expression levels of proteins related to the Nrf2 pathway in CE-treated NP-exposed HepG2 cells are as Figure 7 shown. Compared with the control group, Nrf2 and HO-1 were both downregulated in the NP-exposed group (P<0.05). After CE intervention, the content of Nrf2 first increased and then decreased, and the expression of its downstream pathway protein HO-1 was upregulated. This indicates that CE has a regulatory effect on the expression of proteins related to the Nrf2 pathway in NP-exposed HepG2 cells.

[0100] The expression levels of proteins related to the ERK / P38 pathway in CE-treated NP-exposed HepG2 cells are as Figure 8 shown. CE at doses of 0.25 mg / mL - 1 mg / mL dose-dependently downregulates the ratios of p-ERK / ERK and p-P38 / P38 induced by NP, while the combination of NP and 2 mg / mL CE upregulates the ratios of p-ERK / ERK and p-P38 / P38 again, suggesting that CE at doses of 0.25 mg / mL - 1 mg / mL has an improving effect on the activation of the ERK and P38 pathways induced by NP.

[0101] Example 8:

[0102] This example is to verify the effect of CE on the expression of Nrf2 and HO-1 in NP-exposed HepG2 cells through the P38 pathway and ERK pathway using P38 inhibitor and ERK inhibitor.

[0103] (1) Take cells with intact adherent and normal growth state, discard the cell culture medium, wash twice with PBS, then add P38 inhibitor or ERK inhibitor to pretreat the cells for 2 h in the culture plate; discard the medium, wash twice with PBS, use pure medium, co-culture with 60 μM NP and different concentrations (0 and 1 mg / mL) of CE, co-culture with 1 mg / mL CE and different concentrations of inhibitors, and treat the cells with pure inhibitor, and place them in an incubator (37°C, 5% CO2) for 24 h, with 4 parallels set for each concentration.

[0104] (2) The operation steps of western blot are the same as those in (2)(3) of Example 7.

[0105] The expression levels of Nrf2 and HO-1 in CE-treated NP-exposed HepG2 cells with different concentrations of P38 inhibitor (SB202190) were as Figure 9 shown. Different concentrations of SB202190 could all down-regulate the ratio of p-P38 / P38 in NP-exposed HepG2 cells (P<0.01), and up-regulate the expression of downstream pathway proteins Nrf2 and HO-1 (P<0.05 or P<0.01).

[0106] The expression levels of Nrf2 and HO-1 in CE-treated NP-exposed HepG2 cells with different concentrations of ERK inhibitor (PD98059) were as Figure 10 shown. PD98059 could dose-dependently down-regulate the ratio of p-ERK / ERK in NP-exposed HepG2 cells (P<0.05 or P<0.01), and dose-dependently up-regulate the expression of downstream pathway proteins Nrf2 and HO-1 (P<0.05).

[0107] Example 9:

[0108] This example was to verify the effect of CE on the CAT activity in NP-exposed HepG2 cells via the P38 pathway and ERK pathway by using P38 inhibitor and ERK inhibitor.

[0109] (1) Take cells with intact adherent state and normal growth, discard the cell culture medium, wash twice with PBS, then add P38 inhibitor or ERK inhibitor to pretreat the cells for 2 h in the culture plate; discard the medium, wash twice with PBS, use pure medium, co-culture with 60 μM NP and different concentrations (0 and 1 mg / mL) of CE, co-culture with 1 mg / mL CE and different concentrations of inhibitor, and treat the cells with pure inhibitor, and place them in an incubator (37 °C, 5% CO2) for 24 h.

[0110] (2) Discard the medium, wash the cells twice with PBS, place the culture plate on ice, lyse the cells with Western and IP lysis buffer, add 100 μL of lysis buffer to each well and lyse for 30 min; pipette the cells during lysis to ensure sufficient cell lysis. Collect the treated cells in EP tubes, centrifuge (4 °C, 12,000 rpm, 5 min), and take the supernatant for use; perform corresponding detections on the supernatant according to the instructions, and simultaneously detect the protein concentration with a BCA kit, and finally normalize the results according to the protein content. Set 4 parallels for each concentration, and express the CAT activity in enzyme activity units per milligram of protein.

[0111] The effect of different concentrations of P38 inhibitor (SB202190) on the CAT activity in CE-treated NP-exposed HepG2 cells was as Figure 11As shown in the figure. Compared with the control group, the CAT activity in the NP poisoning group decreased (P<0.01). After the intervention of CE or SB202190, the CAT activity recovered (P<0.01). The results of the CE intervention were the same as those of the SB202190 intervention, suggesting that CE restored the CAT activity through the P38-Nrf2 pathway and finally played an intervention role.

[0112] The effects of different concentrations of ERK inhibitor (PD98059) on the CAT activity after CE intervened in NP-poisoned HepG2 cells are as Figure 12 shown in the figure. Compared with the control group, the CAT activity in the NP poisoning group decreased (P<0.01). After the intervention of CE or PD98059, the CAT activity recovered (P<0.01). The results of the CE intervention were the same as those of the PD98059 intervention, suggesting that CE restored the CAT activity through the ERK-Nrf2 pathway and finally played an intervention role.

[0113] Example 10:

[0114] This example is to verify the detoxification effect of CE on NP-poisoned HepG2 cells mediated by the Nrf2 pathway by using an Nrf2 inhibitor.

[0115] (1) Take the cells with intact adherent and normal growth state, discard the cell culture medium, wash twice with PBS, then add Nrf2 inhibitor to pretreat the cells in the culture plate for 2 hours; discard the culture medium, wash twice with PBS, use pure culture medium, co-culture with 60 μM NP and different concentrations (0 and 1 mg / mL) of CE, co-culture with 60 μM NP, 1 mg / mL CE and different concentrations of inhibitor, and treat the cells with pure inhibitor, and place them in an incubator (37 °C, 5% CO2) for 24 hours. Set 4 parallels for each concentration.

[0116] (2) The operation steps of western blot are the same as those in (2) and (3) of Example 7.

[0117] The expression levels of Nrf2 and HO-1 after CE intervened in NP-poisoned HepG2 cells with different concentrations of Nrf2 inhibitor (ML385) are as Figure 13 shown in the figure. Compared with the control group, the expression of Nrf2 and HO-1 in the NP poisoning group was significantly down-regulated (P<0.01), and the expression of Nrf2 and HO-1 increased after the intervention of CE (P<0.01); after further adding the Nrf2 inhibitor (ML385), compared with the CE intervention group, the expression of Nrf2 and HO-1 decreased in a dose-dependent manner (P<0.01), and the activation of CE on Nrf2 and HO-1 was inhibited again. It is suggested that CE may play a detoxification role through the Nrf2 pathway.

[0118] The effects of Nrf2 inhibitor (ML385) at different concentrations on the expression levels of upstream ERK pathway proteins in CE-treated NP-exposed HepG2 cells are as follows Figure 14 shown. Compared with the control group, the ratio of p-ERK / ERK was upregulated in the NP-exposed group (P<0.01), and the ratio of p-ERK / ERK was significantly decreased by CE treatment (P<0.01); after further addition of Nrf2 inhibitor (ML385), the ratio of p-ERK / ERK was dose-dependently upregulated compared with the CE treatment group (P<0.01), and the ERK pathway was activated again. It is suggested that CE can affect the ERK pathway through Nrf2 in turn.

[0119] The effects of Nrf2 inhibitor (ML385) at different concentrations on the expression levels of upstream P38 pathway proteins in CE-treated NP-exposed HepG2 cells are as follows Figure 15 shown. Compared with the control group, the ratio of p-P38 / P38 was increased in the NP-exposed group (P<0.01), and the ratio of p-P38 / P38 was significantly decreased by CE treatment (P<0.01); after further addition of Nrf2 inhibitor (ML385), the ratio of p-P38 / P38 was dose-dependently upregulated compared with the CE treatment group (P<0.01), and the P38 pathway was activated again. It is suggested that CE can affect the P38 pathway through Nrf2 in turn.

[0120] Example 11:

[0121] This example is to verify the effect of CE on the CAT activity of NP-exposed HepG2 cells through the Nrf2 pathway by Nrf2 inhibitor.

[0122] (1) Take the cells with intact adherent and normal growth state, discard the cell culture medium, wash twice with PBS, then add Nrf2 inhibitor to pretreat the cells for 2 h in the culture plate; discard the medium, wash twice with PBS, and use pure medium, 60 μM NP and CE at different concentrations (0 and 1 mg / mL) for co-culture, 1 mg / mL CE and different concentrations of inhibitor for co-culture, and pure inhibitor to treat the cells, and place them in an incubator (37 °C, 5% CO2) for 24 h.

[0123] (2) Discard the culture medium, wash the cells twice with PBS, place the culture plate on ice, and lyse the cells with Western and IP lysis buffer. Add 100 μL of lysis buffer to each well and lyse for 30 min. During the lysis process, pipette the cells to ensure complete lysis. Collect the processed cells in an EP tube, centrifuge (4°C, 12,000 rpm, 5 min), and take the supernatant for use. According to the instructions, take the supernatant for corresponding detection, and at the same time, detect the protein concentration with a BCA kit. The final results are normalized according to the protein content. Set 4 parallels for each concentration, and the CAT activity is expressed as enzyme activity units per milligram of protein.

[0124] The effects of different concentrations of Nrf2 inhibitor (ML385) on the CAT activity of CE-treated NP-exposed HepG2 cells are shown as Figure 16 follows. Compared with the control group, the CAT activity decreased in the NP-exposed group (P < 0.01). After CE intervention, the CAT activity recovered (P < 0.01). After further adding the Nrf2 inhibitor (ML385), the CAT activity decreased again. It is suggested that CE may feedback-regulate ERK and P38 signals through the Nrf2 pathway, thereby restoring the CAT activity and finally exerting an intervention effect.

[0125] The above is a preferred embodiment of the present invention. However, the embodiments of the present invention are not limited to the above content. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of an extract of Alligator Mouth Flower in the preparation of a drug for preventing and treating hepatotoxicity caused by nonylphenol based on the MAPK-Nrf2 pathway, characterized in that: The alligator mouth flower extract is an ethanol extract of alligator mouth flower leaves, and the extract contains total phenols and total flavonoids.

2. The use of the crocodile mouth flower extract according to claim 1 in the preparation of a drug for preventing and treating hepatotoxicity caused by nonylphenol based on the MAPK-Nrf2 pathway, characterized in that: After the nonylphenol infects the HepG2 cells, the MAPK pathway of the HepG2 cells is activated and the Nrf2 pathway is inhibited. The alligator mouth flower extract can inhibit the MAPK pathway and activate the Nrf2 pathway.

3. Use of the crocodile mouth flower extract according to claim 2 in the preparation of a drug for preventing and treating hepatotoxicity caused by nonylphenol based on the MAPK-Nrf2 pathway, characterized in that: The alligator mouth flower extract upregulates the expression of Nrf2 and HO-1 proteins by inhibiting the activation of P38 and ERK in the MAPK-Nrf2 pathway, thereby inhibiting the activation of the MAPK pathway and the inhibition of the Nrf2 pathway in HepG2 cells after nonylphenol poisoning HepG2 cells.

4. Use of the crocodile mouth flower extract according to claim 3 in the preparation of a drug for preventing and treating hepatotoxicity caused by nonylphenol based on the MAPK-Nrf2 pathway, characterized in that: The alligator mouth flower extract upregulates the expression of Nrf2 and HO-1 proteins by inhibiting the activation of P38 and ERK in the MAPK-Nrf2 pathway, and restores the activity of catalase CAT, the content of glutathione GSH and the content of adenosine triphosphate ATP, thereby inhibiting the activation of the MAPK pathway and the inhibition of the Nrf2 pathway in HepG2 cells after nonylphenol poisoning HepG2 cells.

5. Use of the crocodile mouth flower extract according to claim 4 in the preparation of a drug for preventing and treating hepatotoxicity caused by nonylphenol based on the MAPK-Nrf2 pathway, characterized in that: The crocodile mouth flower extract inhibits the activation of P38 in the MAPK-Nrf2 pathway, and the verified specific inhibitor of this target is SB202190. The crocodile mouth flower extract inhibits the activation of ERK in the MAPK-Nrf2 pathway, and the verified specific inhibitor of this target is PD98059. The crocodile mouth flower extract activates Nrf2 and its downstream protein HO-1 in the MAPK-Nrf2 pathway, and the verified specific inhibitor of this target is ML385.

6. Use of the crocodile mouth flower extract according to claim 1 in the preparation of a drug for preventing and treating hepatotoxicity caused by nonylphenol based on the MAPK-Nrf2 pathway, characterized in that: The preparation method of the alligator mouth flower extract comprises the following steps: (1) Fresh alligator mouth flower leaves are freeze-dried, crushed, passed through a 60-mesh sieve, and the sieve residue is collected; (2) the sieve material was ultrasonically extracted with 70% ethanol at a liquid-to-solid ratio of 1:15 to obtain an extract; (3) centrifuging the extract, vacuum filtering, concentrating the filtrate under reduced pressure, removing ethanol, and obtaining a concentrated solution; (4) The concentrated extract is freeze-dried to obtain an alligator flower extract.

7. Use of the crocodile mouth flower extract according to claim 6 in the preparation of a drug for preventing and treating hepatotoxicity caused by nonylphenol based on the MAPK-Nrf2 pathway, characterized in that: The conditions of the ultrasonic extraction are: ultrasonic power 100W, ultrasonic temperature 45°C, extraction time 90min, and the reduced pressure concentration conditions are: reduced pressure rotary evaporation at 40-50°C.

8. Use of the crocodile mouth flower extract according to claim 7 in the preparation of a drug for preventing and treating hepatotoxicity caused by nonylphenol based on the MAPK-Nrf2 pathway, characterized in that: The alligator mouth flower extract contains a total phenol content of 57.37 mg / g and a total flavonoid content of 41.56 mg / g.

9. A drug for preventing and treating hepatotoxicity caused by nonylphenol based on the MAPK-Nrf2 pathway, characterized in that: The invention discloses an ethanol extract comprising the leaves of the alligator mouth flower according to claim 1.

10. A pharmaceutical preparation for preventing and treating hepatotoxicity caused by nonylphenol based on the MAPK-Nrf2 pathway, characterized in that: The ethanol extract of alligator mouth flower leaves according to claim 1 is included, and the pharmaceutical preparation for preventing and treating hepatotoxicity caused by nonylphenol based on MAPK-Nrf2 pathway includes one of lyophilized powder injection, injection, tablet or granule.

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