Application of Rhizoma Coptidis in the preparation of fluorosis drugs

Abstract

The present invention relates to the field of medical technology, specifically to the application of Caulis Agrimoniae in the preparation of fluorosis drugs. Through research, the present invention has found that some components in Caulis Agrimoniae can antagonize oxidative damage and cell apoptosis induced by fluorine poisoning, and further alleviate the effect of liver damage caused by fluorine poisoning, indicating that Caulis Agrimoniae (especially the Caulis Agrimoniae extract extracted according to the specific method in this application) can be used to prepare drugs for fluorosis liver damage, providing technical inspiration for the preparation of drugs for fluorosis liver damage. In addition, the preparation method of the present invention is simple and easy, and Caulis Agrimoniae or Caulis Agrimoniae components prepared by the specific method in this application can be considered to be further developed into safe and effective anti-fluorosis natural medicines. In view of its advantages such as low price and easy preparation, it can be quickly promoted in remote areas, providing an effective and low-cost means for the prevention and treatment of fluorosis in remote areas.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to application of Caulis Aconiti Lateralis Preparata in preparing fluorosis medicine. Background Art

[0002] Fluorine is widely distributed in nature in the form of compounds. Endemic fluorosis (EF), also known as endemic fluorosis, is a serious threat to human health. Fluoride-induced diseases have been reported in many countries around the world, and the prevalence is increasing year by year, posing a serious threat to public safety. Fluoride poisoning is a chronic systemic disease that damages multiple organs and tissues throughout the body. The liver, as the body's primary metabolic organ, is sensitive to fluoride poisoning. Previous studies have shown that fluoride can cause abnormalities in liver tissue structure and function.

[0003] Botanicals are an important source for the search and development of fluorosis treatments. The Miao medicine, Periploca forrestii Schltr., is the dried root or whole plant of the Asclepiadaceae plant. It is listed in the "Quality Standards for Traditional Chinese and Ethnic Medicinal Materials of Guizhou Province" (2003 edition), "Miao Medicine," and "Chinese Materia Medica" (Miao Medicine Volume). Periploca forrestii has the properties of stimulating menstruation, dispelling wind and dampness, promoting blood circulation, and detoxifying. It is used for rheumatic joint pain, injuries from falls, and other conditions. However, there are currently no reports of its use for fluorosis, particularly for fluorosis-induced liver damage. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides the use of Caulis Aconiti Lateralis Preparata in the preparation of fluorosis drugs, which can be used as an effective therapeutic drug for fluorosis, as follows:

[0005] Application of Rhizoma Coptidis in the preparation of fluorosis drugs

[0006] Furthermore, the fluorosis drug is a drug for treating liver damage caused by fluorosis.

[0007] Furthermore, the drug for treating liver damage caused by fluorine poisoning is a drug that alleviates apoptosis of liver damaged cells caused by fluorine poisoning.

[0008] Furthermore, the black bone vine is the dried root or whole plant of Periploca forrestii of the Apocynaceae family.

[0009] Furthermore, the blackthorn vine is an ethanol extract of the blackthorn vine.

[0010] Furthermore, the ethanol extract of Caulis Aconiti Lateralis Preparata is an ethanol extract of Caulis Aconiti Lateralis Preparata.

[0011] Furthermore, the ethanol extract of Caulis Nigra is an ethanol reflux extract of Caulis Nigra.

[0012] Furthermore, the Caulis Aconiti Lateralis Preparata extract is prepared by the following method:

[0013] Dried crude powder of Achyranthes australis was extracted with 70% ethanol under reflux to obtain a 70% ethanol crude extract, Fr.A. Fr.A was dissolved in water and the pH was adjusted to 6 to obtain a water-insoluble fraction, the precipitate fraction Fr.B, and a water-soluble fraction. The water-soluble fraction was adsorbed on D101 macroporous resin and eluted sequentially with water and 85% ethanol. The water and 85% ethanol fractions were collected, recovered, and dried to obtain a water fraction Fr.C and an 85% ethanol fraction Fr.D. The 85% ethanol fraction Fr.D was the final product.

[0014] Furthermore, the fluorosis medicine is a medicine in various dosage forms made from Rhizoma Coptidis as raw material.

[0015] Compared with the prior art, the technical effects created by the present invention are embodied in:

[0016] The present invention provides that some components of Rhizoma Coptidis can antagonize oxidative damage and cell apoptosis induced by fluorine poisoning, further alleviate the effect of liver damage caused by fluorine poisoning, and illustrate that Rhizoma Coptidis (especially the Rhizoma Coptidis extract extracted according to the specific method of this application) can be used to prepare drugs for liver damage caused by fluorine poisoning, providing technical inspiration for the preparation of drugs for liver damage caused by fluorine poisoning. Fluorine poisoning is common in economically underdeveloped remote areas. Rhizoma Coptidis is a commonly used folk medicine that is easy to obtain and inexpensive, and the preparation method of the present invention is simple and easy. If the prepared Rhizoma Coptidis components are further developed into safe and effective natural anti-fluorine poisoning medicines, given their advantages such as low price and easy preparation, they can be quickly promoted in remote areas, providing an effective and inexpensive means for the prevention and treatment of fluorine poisoning in remote areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are the pathological sections of the liver tissues of rats in each group (×400). Note: A. control group; B. fluoride-exposed group; C. Fr.D low-dose group; D. Fr.D medium-dose group; E. Fr.D high-dose group.

[0018] Figure 2 This is the effect of Fr.D on cell apoptosis in each group. Note: A. control group; B. fluoride-exposed group; C. Fr.D low-dose group; D. Fr.D medium-dose group; E. Fr.D high-dose group.

[0019] Figure 3 is the expression of apoptosis-related proteins in the cells of each group. Note: A. control group; B. fluoride-exposed group; C. Fr.D low-dose group; D. Fr.D medium-dose group; E. Fr.D high-dose group.

[0020] Figure 4These are the pathological sections of rat liver tissues in the control group, Fr.A crude extract group, and Fr.D component group. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0022] In this study, fluoride-induced L-02 cells, primary hepatocytes and rats were used to construct in vitro and in vivo fluoride exposure models. Cell Counting Kit-8 (CCK-8) was used to detect cell viability. The levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and ROS were detected by the kit. Annexin V-FITC / PI cell apoptosis kit was used to detect cell apoptosis rate. Western blotting was used to detect cell apoptosis. The expression levels of B lymphocytoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), caspase-3, cleaved caspase-3, caspase-9 and cleaved caspase-9 proteins in cells were detected by blot method, and the protective effect and mechanism of the Fr.D component of the black bone vine prepared by the present invention on liver damage caused by fluorine poisoning were explored to provide a theoretical basis for its clinical application.

[0023] 1. Materials

[0024] 1.1 Experimental Animals

[0025] SPF male Sprague-Dawley rats, weighing 120 ± 10 g, were provided by the Experimental Animal Center of Guizhou Medical University (Certificate Number: SCXK(Guizhou)2018-0001). The rats were maintained under environmental conditions of 24 ± 1°C, 45–55% relative humidity, a 12:12 h light / dark cycle, and free access to food. The animal experiments were reviewed and approved by the Experimental Animal Ethics Committee of Guizhou Medical University. Animal handling during the experiments complied with the "Guiding Opinions on the Ethical Treatment of Laboratory Animals" issued by the Ministry of Science and Technology of China.

[0026] 1.2 Experimental cells

[0027] Normal human hepatocytes (L-02) cell line was purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.

[0028] Primary rat hepatocytes were extracted from SPF-grade SD rats.

[0029] 1.3 Reagents

[0030] Sodium fluoride (NaF) (Shanghai Aladdin Biochemical Technology Co., Ltd.); DMEM culture medium, trypsin, FBS (Gibco, USA); CCK-8 (GlpBio, USA); phosphate buffered saline (PBS), glycine, sodium dodecyl sulfate (SDS), bovine serum albumin (BSA), tris (Tris), Percoll cell separation medium, BCA protein concentration determination kit (Beijing Solaibao Technology Co., Ltd.); SDS-PAGE gel preparation kit ( (Sangon Biotech); phenylmethylsulfonyl fluoride (PMSF), protein lysis buffer (RIPA) (Dalian Meilun Biotechnology Co., Ltd.); SDS-protein pre-staining Maker (Shanghai Aibixin Biotechnology Co., Ltd.); PVDF membrane (Millipore, Germany); ECL developer (Shanghai Biyuntian Biotechnology Co., Ltd.); LDH, MDA, ALT, AST, ROS, SOD, GSH-Px and CAT detection kits (Nanjing Jiancheng Bioengineering Institute); Annexin V-FITC apoptosis detection kit (BD Biosciences, USA); GAPDH, Bcl-2, Bax, Caspase-3, Caspase-9 (abcam, USA); cleaved Caspase-3 and cleaved Caspase-9 (Wanlei Biotechnology Co., Ltd.), goat anti-rabbit Ig G secondary antibody (abcam, USA); the black bone vine medicinal material was purchased from Wandong Chinese medicinal material market in Guiyang City and identified by Associate Professor Liu Chunhua of the Department of Medicinal Botany and Pharmacognosy of Guizhou Medical University as the dried root or whole plant of Periploca forrestii of the Apocynaceae family.

[0031] 1.4 Instruments

[0032] JJ-CJ-2ND II clean bench (Beijing Donglian Har Instrument Manufacturing Co., Ltd.), fresco17 refrigerated high-speed centrifuge, 311 CO2 saturated humidity incubator and multifunctional microplate reader (Thermo Scientific, USA), PowerPac Basic electrophoresis instrument (Bio-Rad, USA), Trans-Blot Turbo semi-dry transfer apparatus (Bio-Rad, USA), TS100 fluorescence inverted microscope (Nikon, Japan), G:BOX Chemi XL1.4 gel imaging system (Syngene, UK), YXQ-LS-50SII high-pressure steam sterilizer (Shanghai Boxun Industrial Co., Ltd. Medical Instrument Factory), C6plus flow cytometer (BD Biosciences, USA).

[0033] 2 Experimental methods

[0034] 2.1 Preparation of Caulis Aconiti Lateralis Preparata components

[0035] 10 kg of dried crude powder of Achyranthes australis was extracted with 70% ethanol under reflux four times, at 7, 6, 6, and 4 times the volume, for 2 hours each time. The extracts were filtered, the filtrates combined, and the ethanol recovered to yield 1254 g of a 70% ethanol crude extract, Fr.A. Fr.A was dissolved in water, and the pH was adjusted to 6 to yield a water-insoluble fraction, the precipitate fraction Fr.B (179.5 g), and a water-soluble fraction. The water-soluble fraction was adsorbed on D101 macroporous resin and eluted sequentially with water and 85% ethanol. The water and 85% ethanol fractions were collected, recovered, and dried to yield 402.6 g of the water fraction Fr.C and 403.5 g of the 85% ethanol fraction Fr.D. Fr.A, Fr.B, Fr.C, and Fr.D were prepared with DMSO to a concentration of 100 mg / mL. -1 The mother solution was stored at -20℃ for future use and diluted with culture medium to the required concentration before use.

[0036] 2.2 Preparation and culture of primary rat hepatocytes

[0037] SD rats were intraperitoneally injected with 2% sodium pentobarbital and 100 U·kg -1 Heparin was added, and consumables were soaked in 75% ethanol. The fur was disinfected, the abdominal cavity was opened, the portal vein was cannulated, and the liver was irrigated with pre-perfusion fluid at 37°C and pH = 7.2-7.5 for 10 minutes, and then irrigated with collagenase IV for 10 minutes. When the liver became soft, the liver tissue was cut and scraped with a cell scraper. The cell suspension was filtered through a 400-mesh filter and the cell suspension was collected. The cell suspension was centrifuged at 400 r / min. -1 , centrifuge for 3 minutes, centrifuge 3 times, add serum-free medium to resuspend and mix into one tube, add serum-free medium to 6 mL, add 3.42 mL Percoll solution and 0.38 mL 10× PBS in sequence, resuspend again, and rotate at 1300 r·min-1 Centrifuge for 7 minutes, discard the supernatant, and resuspend in complete culture medium. Culture the cells in a 37°C, 5% CO2 incubator.

[0038] 2.3 Establishment of fluoride poisoning model

[0039] The fluorosis model was established according to the literature method. L-02 cells and primary hepatocytes were cultured in vitro, and cells in the logarithmic growth phase were taken and 1.5×10 5 The cells were inoculated into 96-well plates and cultured for 24 h, and then divided into control group and fluoride-treated group (5 mmol★L - 1 The control group was treated with complete culture medium, and the fluoride-treated group was treated with 5 mmol·L -1 After treatment with NaF medium for 12 hours, the supernatant was discarded and medium containing 5% CCK-8 was added and incubated for 2 hours. The optical density (OD) value was measured at a wavelength of 450 nm to calculate the cell survival rate.

[0040] 2.4 In vitro anti-fluorosis activity of different components of Rhizoma Coptidis

[0041] Cells in the logarithmic growth phase were plated in 96-well plates according to 2.3. After culturing for 24 h, the cells were divided into a control group, a fluoride-treated group, and a drug-treated group. Complete medium was added to the control group and the fluoride-treated group, and 200 μg mL -1 After culturing for 12 h, 5 mmol·L -1 After incubation for 48 h in a 37°C, 5% CO2 incubator, discard the supernatant and test cell viability according to 2.3. Treat cells in the same manner, collect the cell supernatant, and test LDH release and MDA content in cells according to the instructions of the LDH and MDA detection kits.

[0042] 2.6 Investigation of the anti-fluorosis activity of Fr.D using animal models

[0043] 2.6.1 Animal modeling and grouping

[0044] Fifty SPF SD rats weighing 120±10 g were randomly divided into a control group, a fluoride-exposed group, a low-dose, a medium-dose, and a high-dose Fr.D group (100 mg·kg -1 , 200mg·kg -1 , 400mg·kg -1 ), 10 in each group. The control group was free to drink tap water (fluoride content less than 0.5 mg·L -1 Sodium fluoride was added to tap water in the fluoride group and the Fr.D low-, medium-, and high-dose groups, with a fluoride concentration of 150 mg·L -1Eight weeks after modeling, rats in all groups, except the control group, were allowed to drink fluoridated tap water freely. The low-, medium-, and high-dose Fr.D groups were gavage-administered once daily for four weeks, and liver tissue and blood were collected for subsequent experiments.

[0045] 2.6.2 Detection of rat serum LDH, MDA, ALT and AST levels

[0046] Blood 4°C, 4000 rpm -1 The cells were centrifuged for 10 min, and serum was collected and the levels of LDH, MDA, ALT, and AST in serum were determined according to the kit instructions.

[0047] 2.6.3 Rat liver pathological sections

[0048] Rat liver tissue was washed with saline, fixed in 10% paraformaldehyde, dehydrated, and embedded in paraffin. The liver was then sectioned into 5 μm slices. Paraffin sections were dewaxed and stained with hematoxylin and eosin (HE). Images of the liver were taken at 20x magnification. Liver histopathology was also photographed.

[0049] Study on the mechanism of 2.7Fr.D's anti-fluorosis effect

[0050] 2.7.1 Cell viability, LDH, MDA, SOD, GSH-Px, CAT, and ROS level detection

[0051] Primary rat hepatocytes were extracted and inoculated into 6-well plates and cultured to the logarithmic growth phase. They were then divided into control group, fluoride-exposed group, low-, medium-, and high-dose Fr.D groups. Complete medium was added to the control group and fluoride-exposed group, while 100 mg·kg Fr.D low-, medium-, and high-dose Fr.D groups were added to the culture medium. -1 , 200mg·kg -1 , 400mg·kg -1 Culture cells in Fr.D medium for 12 hours. NaF was then added and cultured for 48 hours. The cell supernatant was collected and LDH release was determined according to the LDH assay kit instructions. Cells were collected, rinsed twice with PBS, and after sonication, MDA content, SOD, GSH-Px, CAT activity, and ROS levels were determined according to the assay kit instructions.

[0052] 2.7.2 Cell apoptosis rate detection

[0053] The cells were divided into groups and processed according to the method in 2.7.1. The supernatant was discarded, and the cells were rinsed twice with PBS. The cells were treated according to the instructions of the Annexin V-FITC apoptosis kit. The cells were incubated in the dark at room temperature for 15 minutes, and then the cell apoptosis was detected by flow cytometry within 1 hour.

[0054] 2.7.3 Western blot analysis of the effect of Fr.D on the expression of apoptosis-related proteins

[0055] Cells were grouped and treated as in 2.7.1. Total protein was extracted and protein concentration was determined using a BCA kit. Equal amounts of denatured protein (20 μg) were loaded and separated by SDS-PAGE electrophoresis. The membranes were transferred and blocked with 5% BSA for 2 h at room temperature. The expression of GAPDH (1:1500), Bax (1:1500), Bcl-2 (1:1500), Caspase-3 (1:1500), cleaved Caspase-3 (1:1500), Caspase-9 (1:1500), and cleaved Caspase-9 (1:1500) proteins was detected. After incubation, the membranes were washed and incubated with HRP-conjugated goat anti-rabbit IgG (1:10,000) at room temperature for 1 h. Finally, the membranes were developed and exposed using ECLplus developer. Protein signals were measured using the G:BOX Chemi XL1.4 system, and grayscale values ​​were analyzed using Image J software. The ratio of the grayscale value of the target protein to the internal reference GAPDH was used as the relative expression of the target protein.

[0056] 2.8 Differences in hepatotoxicity between crude Fr.A extract and Fr.D fraction

[0057] Thirty healthy clean grade SD rats weighing 120±10g were randomly divided into control group, Fr.A (400mg·kg -1 ) and Fr.D group (400 mg·kg -1 In addition to the control group, Fr.A and Fr.D groups were given oral administration once a day for 4 weeks, and liver tissue and blood were collected for pathological sections and ALT and AST levels.

[0058] 2.9 Statistical Analysis

[0059] SPSS 25.0 software was used to analyze the data. One-way ANOVA and t-test were used to compare the data between groups and between two groups, respectively, and P < 0.05 was considered statistically significant.

[0060] 3 Results

[0061] 3.1 Effects of various components of Achyranthes bidentata on fluoride-induced L-02 cell survival rate, LDH and MDA

[0062] Compared with the control group, the cell survival rate in the fluoride-exposed group was significantly decreased, while LDH and MDA levels were significantly increased (P < 0.05). Compared with the fluoride-exposed group, the cell survival rate in the Fr.D group was significantly increased (P < 0.05). Compared with the fluoride-exposed group, LDH and MDA levels in the Fr.C and Fr.D groups were significantly decreased (P < 0.05). See Table 1.

[0063] Table 1 Effects of various components of Acanthocereus chinensis on cell viability, LDH and MDA ( n=10)

[0064]

[0065] Note: Compared with the control group 1) P<0.05; compared with the fluoride group 2) P<0.05. (Same as Table 2-7)

[0066] 3.2 Fr.D component can antagonize fluoride poisoning-induced liver damage in rats

[0067] Compared with the control group, the levels of LDH, MDA, ALT and AST in the serum of the fluoride group were significantly increased (P<0.05), and the cell damage in the liver tissue increased. Compared with the fluoride group, the levels of LDH, MDA, ALT and AST in the serum of the rats in the medium and high dose Fr.D groups were significantly decreased (P<0.05), and the degree of cell damage in the liver tissue was significantly improved. See Table 2, Table 3 and Figure 1 .

[0068] Table 2. Effects of Fr.D components on LDH and MDA content ( n=10)

[0069]

[0070] Table 3. Effects of Fr.D components on ALT and AST levels ( n=10)

[0071]

[0072] 3.3 Study on the mechanism of action of Fr.D component against fluorosis

[0073] Compared with the control group, the cell viability, SOD, GSH-Px, and CAT levels in the fluoride-exposed group were significantly decreased, while LDH, MDA, and ROS levels were significantly increased (P < 0.05). Compared with the fluoride-exposed group, the cell viability, SOD, GSH-Px, and CAT levels in the medium- and high-dose Fr.D groups were significantly increased, while LDH, MDA, and ROS levels were significantly decreased (P < 0.05). See Table 4.

[0074] Table 4. Effects of Fr.D components on cell viability, LDH, MDA, SOD, GSH-Px, CAT and ROS contents ( n=10)

[0075]

[0076] 3.4 Apoptosis rate of primary hepatocytes

[0077] A small number of damaged or early apoptotic cells were found in the control group, with an apoptosis rate of 1.59%. Compared with the control group, the apoptosis rate of the fluoride-exposed group was 66.00% (P<0.05). After pretreatment with medium and high concentrations of Fr.D, the apoptosis rates were 47.72% and 29.37%, respectively, which were significantly lower than those in the model group (P<0.05). Figure 2 and Table 5. The results showed that Fr.D could significantly reduce the apoptosis rate of primary hepatocytes induced by fluoride.

[0078] Table 5. Effects of Fr.D components on apoptosis rate of primary hepatocytes ( n=10)

[0079]

[0080] 3.5 Expression levels of apoptosis pathway-related proteins in primary hepatocytes

[0081] Compared with the control group, the ratios of cleaved Caspase-3 / Caspase-3 and cleaved Caspase-9 / Caspase-9 in the fluoride-exposed group were significantly increased, and the expression of Bcl2 / Bax was significantly decreased (P<0.05). Compared with the fluoride-exposed group, the ratios of cleaved Caspase-3 / Caspase-3 and cleaved Caspase-9 / Caspase-9 were significantly decreased, and the expression of Bcl2 / Bax was significantly increased after Fr.D pretreatment (P<0.05). See Table 6 and Figure 3 .

[0082] Table 6. Effects of Fr.D components on the expression of apoptosis proteins in primary hepatocytes ( n=3)

[0083]

[0084] 3.2 This preparation method can greatly reduce the hepatotoxicity of Caulis Aconiti Lateralis Preparata

[0085] Compared with the control group, the serum ALT and AST levels in the Fr.A crude extract group were significantly increased (P<0.05), and the cell damage in the liver tissue increased. Compared with the Fr.A crude extract group, the serum ALT and AST levels in the Fr.D group were significantly lower (P<0.05), and there was no liver tissue cell damage, which was no different from the control group. See Table 7 and Figure 4 .

[0086] Table 7. Effects of Fr.A crude extract and Fr.D fraction on ALT and AST content ( n=10)

[0087]

[0088] 4 Discussions

[0089] Fluorosis is a zoonotic disease widespread in many countries and regions. Long-term exposure to fluorine can cause damage to multiple organs and tissues. Fluoride can damage the liver, a key detoxification organ, and can lead to metabolic abnormalities.

[0090] Under fluoride poisoning conditions, cellular peroxidation levels in the liver significantly increase, leading to liver tissue cell damage and apoptosis, which in turn triggers liver injury. ALT, AST, and LDH serve as important serum biomarkers for assessing liver injury, and their expression levels change dynamically with the progression of liver injury. We found that serum LDH, ALT, and AST levels were elevated in fluoride-exposed rats, indicating liver tissue damage. After treatment with the Fr.D component, these levels decreased, and the extent of liver damage was reversed. These findings were also observed in primary hepatocytes.

[0091] Cell apoptosis caused by oxidative stress also plays a significant role in liver damage. During cell injury in vitro and in vivo, intracellular oxidative stress drives cell damage and apoptosis, with ROS being the primary marker for detection. Simultaneously, SOD removes ROS from the body and cells through dismutation reactions, while GSH-Px catalyzes the reaction of hydrogen peroxide with reduced glutathione to produce water, thereby reducing oxidative stress levels and ultimately reducing the production of the lipid peroxidation product MDA, thereby protecting the body from damage. This study showed that compared with the fluoride-exposed group, treatment with the Fr.D component reduced the levels of the oxidative metabolites MDA and ROS, while increasing the levels of the antioxidant enzymes SOD, GSH-Px, and CAT. This suggests that the extract of Scutellaria baicalensis can exert its anti-tissue damage effect by enhancing its ability to scavenge ROS in cells.

[0092] Oxidative stress is closely linked to cell apoptosis. To further validate this, our laboratory examined changes in the expression of classic apoptotic proteins, including Caspase-3, Caspase-9, Bax, and Bcl-2. Our findings indicate that in the fluoride-exposed L-02 cells, the ratios of Cleaved-Caspase-3 / Caspase-3 and Cleaved-Caspase-9 / Caspase-9 were upregulated, while the ratio of BCL-2 / Bax was downregulated. This suggests that apoptosis is increased in fluorosis-induced hepatocytes and that treatment with the Fr.D component alleviates this apoptosis.

[0093] In summary, the Fr.D component of Achyranthes bidentata may achieve its effect on resisting fluoride poisoning damage to hepatocellular cells by increasing the activities of enzymes such as GSH-Px and SOD and the level of GSH in the liver, reducing the contents of ROS, MDA, and LDH, and inhibiting cell apoptosis.

[0094] In addition, since studies have reported that A. truncatum has some hepatotoxicity, we also investigated the safety of Fr.D. The results showed that the crude extract of A. truncatum, Fr.A, has some hepatotoxicity, but after treatment with the preparation method of the present invention, the obtained Fr.D component has no hepatotoxicity.

[0095] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solutions of the present invention are not limited to the above embodiments and are subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. The use of an ethanol extract of Caulis Aconiti Lateralis Preparata in the preparation of a drug for treating liver damage caused by fluorosis, characterized in that: The ethanol extract of the black bone vine is specifically prepared by the following method: taking dried black bone vine medicinal material crude powder, refluxing and extracting it with 70% ethanol to obtain a 70% ethanol crude extract Fr.A, adding water to dissolve Fr.A, adjusting the pH to 6, and obtaining a water-insoluble part, namely, a precipitated component Fr.B and a water-soluble part. The water-soluble part is adsorbed on D101 macroporous resin, eluted with water and 85% ethanol in sequence, collecting the water and 85% ethanol elution parts, recovering and drying to obtain a water component Fr.C and an 85% ethanol component Fr.D, and the 85% ethanol component Fr.D is the final product, namely the final black bone vine ethanol extract.

2. The use according to claim 1, characterized in that The drug for treating liver damage caused by fluorine poisoning is a drug for alleviating apoptosis of liver damage cells caused by fluorine poisoning.

3. The use according to claim 1, characterized in that The black bone vine is the dried root or the whole plant of Periploca forrestii of the genus Periploca of the Apocynaceae family.

4. The use according to claim 1, characterized in that The medicine for treating liver damage caused by fluorosis is a medicine in various dosage forms made from the ethanol extract of Achyranthes bidentata as a raw material.

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