Application of trigonella foetida and extract of trigonella foetida in preparation of medicine for treating liver function impairment

Abstract

The invention relates to the technical field of medicines, in particular to application of creeping woodbetony herb and an extract thereof in preparation of a medicine for treating liver function impairment. The invention provides the following two applications: (1) preventing / treating cell injury and tissue injury caused by oxidation and inflammation; and (2) preventing / treating acute and chronic liver injury. It is fully verified that the creeper creeper extract has an obvious liver protection effect and dose dependence, and liver injury can be obviously protected. In addition, the creeper creeper and the specific creeper creeper extract disclosed in the invention can also reduce the release of alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP), significantly reduce the degree of necrosis of liver cells and reduce the degree of inflammation infiltration.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and specifically to the application of Tetrastigma delavayi Franch. and its extracts in the preparation of drugs for treating liver function impairment. Background Art

[0002] The liver is one of the main organs regulating various physiologies in the human body. Traditional Chinese medicine believes that the liver is mainly responsible for storing blood and regulating qi flow. The role of the liver in the human body is self-evident. Nowadays, liver diseases have become common diseases endangering human physical and mental health and are diseases with a high global mortality rate, and liver injury is an important factor causing liver diseases. Liver injury can be roughly divided into acute liver injury and chronic liver injury. If the formation of liver injury is not controlled, it can further deteriorate into diseases such as liver fibrosis, cirrhosis, and liver cancer. Clinically, there is no drug with a significant therapeutic effect on protecting the liver. Therefore, the research on drugs for liver injury is an important topic and also has good research prospects.

[0003] The Miao medicine Tetrastigma delavayi Franch., also known as Ruiluoqie, is the dried stem, leaf or whole plant of the Araliaceae plant Hedera nepalensis K.Koch var. sinensis (Tobl.) Rehd. Tetrastigma delavayi Franch. belongs to the liver, spleen and lung meridians and is commonly used in clinical practice to treat rheumatic arthralgia, hepatitis, rheumatic sores and toxins, fractures and other diseases. The root of Tetrastigma delavayi Franch. is rich in chemical components, mainly phenolic acids mainly based on chlorogenic acid derivatives, saponins mainly based on hederasaponin derivatives, and flavonoids represented by rutin. Modern pharmacological studies have shown that the crude extract of Tetrastigma delavayi Franch. has anti-diabetic activity, and its ethanol extract has significant antibacterial activity, but its anti-liver injury activity has not been reported. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides Tetrastigma delavayi Franch. and its extracts with the effect of alleviating liver injury, providing an effective drug for treating liver injury, specifically as follows:

[0005] The application of Tetrastigma delavayi Franch. and its extracts in the preparation of drugs for treating liver function impairment.

[0006] Further, the Tetrastigma delavayi Franch. is the dried stem, leaf or whole plant of the Araliaceae plant Hedera nepalensis K.Koch var. sinensis (Tobl.) Rehd.

[0007] Further, the extract of Tetrastigma delavayi Franch. is the extract of the root of Tetrastigma delavayi Franch., especially the ethyl acetate extract of the root of Tetrastigma delavayi Franch.

[0008] Further, the ethyl acetate extract of the root of Tetrastigma delavayi Franch. is prepared by the following method:

[0009] Take 1 kg of the medicinal material of *Tetrastigma delavayi* Franch., dry and pulverize it, reflux extract it twice with 30 times the amount of 95% ethanol for 2 hours each time, and reflux extract it once with 75% ethanol; after concentrating the filtrate in vacuo, an ethanol extract is obtained; then, disperse the ethanol extract with distilled water and extract it with ethyl acetate, and concentrate the filtrate to obtain an ethyl acetate extract.

[0010] Furthermore, the drug for treating impaired liver function is a drug for preventing or treating liver injury.

[0011] Furthermore, one or more pharmaceutically acceptable carriers or excipients are added to the drug for treating impaired liver function. The carriers or excipients include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field. It can be in various dosage forms, such as tablets, powders, injections, granules, powders, capsules or pills, etc.

[0012] A drug for treating impaired liver function contains *Tetrastigma delavayi* Franch. or its extract. It can be a drug prepared by combining *Tetrastigma delavayi* Franch. or its extract with other drugs, excipients, etc.

[0013] Use of *Tetrastigma delavayi* Franch. and its extract in the preparation of a drug for preventing / treating cell damage and tissue damage caused by oxidation and inflammation.

[0014] Compared with the prior art, the technical effects of the present invention are reflected in:

[0015] The present invention provides the following two uses of *Tetrastigma delavayi* Franch. and its extract: (1) for preventing / treating cell damage and tissue damage caused by oxidation and inflammation; (2) preventing / treating acute and chronic liver injury. It is fully verified that the extract of *Tetrastigma delavayi* Franch. has an obvious liver protection effect and dose dependence, and can significantly protect liver injury. In this application, it is also disclosed that *Tetrastigma delavayi* Franch. and a specific extract of *Tetrastigma delavayi* Franch. can also reduce the release of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), significantly reduce the degree of hepatocyte necrosis, and reduce the degree of inflammatory infiltration. Description of the Drawings

[0016] Figure 1 HPLC chromatogram (270 nm) of the ethyl acetate extract of the Miao medicine *Tetrastigma delavayi* Franch.

[0017] Figure 2 Protective activity of the ethyl acetate extract of *Tetrastigma delavayi* Franch. against acute injury of HepG2 cells caused by paracetamol.

[0018] Figure 3 Effect of the ethyl acetate fraction of *Tetrastigma delavayi* Franch. on acute liver injury in mice caused by paracetamol. Detailed Embodiments

[0019] The technical solution of the present invention will be further limited in combination with specific implementation manners below, but the scope of protection claimed is not limited only to the description made.

[0020] Example 1 Preparation of ethyl acetate fraction of *Tetrastigma obtectum*

[0021] Extract preparation: 1 kg of *Tetrastigma obtectum* powder was refluxed and extracted twice with 95% ethanol and once with 75% ethanol, each time for 2 - 3 hours. After the filtrates were concentrated and combined, an ethanol extract paste of *Tetrastigma obtectum* was obtained. Then, this paste was dispersed with distilled water and extracted three times with ethyl acetate. The filtrates were concentrated and combined, and then freeze-dried under vacuum to obtain a powder of ethyl acetate extract of *Tetrastigma obtectum*, which was placed in a desiccator for standby.

[0022] Extract detection: Weigh 2 mg of the ethyl acetate extract of *Tetrastigma obtectum*, dissolve it in 1 mL of methanol, filter it through a 0.22 μm microporous filter membrane, and inject the sample.

[0023] Chromatographic conditions:

[0024] Instrument: Agilent 1260 high performance liquid chromatograph (Agilent Technologies);

[0025] Chromatographic column: Aglient Zorbax Extend-C18 (5 μm, 4.6×250 mm, Aglient Technologies., Tnc., CA, USA);

[0026] Mobile phase: acetonitrile (A) - 0.1% formic acid (B);

[0027] Elution program: 0 min, 5% acetonitrile - formic acid water; 10 min, 15% acetonitrile - formic acid water; 20 min, 35% acetonitrile - formic acid water; 25 min, 50% acetonitrile - formic acid water; 35 min, 70% acetonitrile - formic acid water; 40 min, 100% acetonitrile.

[0028] Detection wavelength: 270 nm.

[0029] In this experiment, using information such as peak elution time and accurate molecular weight, each chromatographic peak was matched with chemical components and reference substances in a known database to obtain the identification results;

[0030] 2. Experimental results.

[0031] The HPLC chromatogram of the ethyl acetate extract of *Tetrastigma obtectum* is shown in Figure 1。The 11 compounds in the figure (numbered 1-11) are: protocatechuic acid (1), p-hydroxybenzoic acid (2), 3-pyridinol, 4-methoxy-(9CI) (3), caffeic acid (4), homoeriodictyol-7-O-glucoside (5), resveratrol-3-O-glucoside (6), isochlorogenic acid A (7), emodin-1-glucoside (8), 4,5-Di-O-caffeoylquinic acid (9), 3,4-Di-O-caffeoylquinic acid methyl ester (10), ethyl caffeate (11).

[0032] Example 2. Protective effect of the extract of Tetrastigma obtectum on acetaminophen-induced HepG2 hepatocyte injury.

[0033] Preparation of acetaminophen stock solution: Accurately weigh 151.16 mg of acetaminophen powder, add it to 1 ml of cell-grade DMSO, and dissolve it completely by ultrasound. The concentration of the stock solution is 1 M, and it is stored at -20 °C. When used, it is diluted to 10 mM with serum-free medium.

[0034] HepG2 cells in the logarithmic growth phase were digested with trypsin and then subjected to viable cell counting (the survival rate was verified by trypan blue staining method to be ≥ 95%). A cell suspension was prepared at a density of 5×10 4 cells / mL. 100 μL of the cell suspension (containing 1×10 3 cells / well) was inoculated into a 96-well plate and incubated in an incubator at 37 °C and 5% CO2 for 24 hours to ensure that the cells were completely adherent. After discarding the medium, 100 μL of drug-containing medium was added to each well (the final concentration of the drug was 10 μM, and 3 parallel wells were set for each concentration. The blank control group was added with an equal amount of complete medium without the drug).

[0035] Blank control group: Blank DMEM medium without acetaminophen (added to blank wells without cells);

[0036] Model group: After pre - treating with serum - free DME medium for 3 h, add 10 mM paracetamol and culture for 24 h;

[0037] Positive drug group: After pre - treating with serum - free DMEM medium containing 25 μg / mL silybin for 3 h, add 10 mM paracetamol and co - culture for 24 h;

[0038] Group administered with ethyl acetate fraction of Tetrastigma delavayi: Add serum - free DMEM medium containing 100, 50, 25, 12.5, 6.25 μg / mL of the ethyl acetate fraction of Tetrastigma delavayi respectively, pre - treat for 3 h, then add 10 mM paracetamol and co - culture for 24 h;

[0039] Continue to culture the 96 - well plate for 24 h. 30 minutes before the end of the experiment, add 10 μL of CCK - 8 reagent to each well and incubate in the dark for 1 - 2 h. Measure the absorbance (OD value) of each well at a wavelength of 450 nm using an enzyme - linked immunosorbent assay reader. After correcting with the blank control group, calculate the cell survival rate according to the formula (OD value of the experimental group / OD value of the blank control)×100%. Use GraphPad Prism for data analysis and draw the dose - response curve.

[0040] 2. Experimental results.

[0041] As Figure 2 shown, the ethyl acetate (EtOAc) fraction of Tetrastigma delavayi showed a significant hepatoprotective effect in the concentration range of ≥50 μg / mL, and its effect intensity was comparable to that of the classic liver - protecting drug silybin (Silibinin). It has significant activity in protecting acute liver injury and can be used for preparing drugs for protecting acute hepatitis liver injury.

[0042] Example 3. Protective effect of the ethyl acetate extract (EtOAc) of Tetrastigma delavayi on acute liver injury in mice caused by paracetamol.

[0043] 1. Experimental materials and methods.

[0044] 6 - week - old SPF - level C57BL male mice were purchased from the Experimental Animal Center of Guizhou Medical University, given 12 h of light and 12 h of darkness, at a constant temperature, and fed with normal feed and water. After adapting in a sterile environment for 4 days, the experiment was carried out.

[0045] Animals were randomly divided into 5 groups (n = 8). 1) The control group and 2) the model group of animals were gavaged with 0.5% sodium carboxymethylcellulose solution every day. 3) The positive drug group was gavaged with 50 mg / mL silybin suspension (mixed in 0.5% sodium carboxymethylcellulose solution) at 10 mL / kg every day. 4) The low-dose protection group of the extract of *Tetrastigma obtectum* and 5) the high-dose protection group of the extract of *Tetrastigma obtectum* were respectively gavaged with 50 mg / mL and 100 mg / mL suspensions of the extract of *Tetrastigma obtectum* (the administration doses were 50 mg / kg and 100 mg / kg) at 10 mL / kg every day. After continuous administration for 7 days, 6 hours after gavage on the 7th day, 1) the control group was intraperitoneally injected with normal saline (10 mL / kg), and 2) - 5) groups were intraperitoneally injected with acetaminophen for modeling (300 mg / kg, dissolved in normal saline). After 24 hours, blood was collected by eye socket puncture, and liver tissue was taken after cardiac perfusion. After the blood samples were left standing at room temperature for 2 hours, serum was obtained by centrifugation at 8000 g for 15 minutes at 4°C.

[0046] Serum samples were subjected to blood biochemical analysis to detect the indexes of ALT, AST, and ALP. The liver tissues were fixed with paraformaldehyde and then embedded in paraffin, sectioned, stained with HE, and photographed.

[0047] 2. Experimental results.

[0048] The measurement data of the experimental data were expressed as mean ± standard deviation. The comparison of two means was analyzed by one-way ANOVA in SPSS 16.0 statistical software. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the model group.

[0049] After hepatocytes are damaged, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) will be released, and the concentrations of these enzymes in serum reflect the degree of liver injury. As Figure 3 (A) shows, the blood biochemical results show that the extract of *Tetrastigma obtectum* has obvious protective effects and dose dependence, and significantly protects against acute liver injury caused by carbon tetrachloride.

[0050] Under the optical microscope, in the liver tissue sections of non-damaged mice, it can be seen that the size and morphology of hepatocytes are normal, there are no liver parenchymal cell lesions, and the central vein and portal area are normal, as Figure 3 (B) shows. In the model group damaged by carbon tetrachloride, the hepatocytes around the central vein in the mouse liver tissue have no normal cell morphology, the hepatocytes are swollen, and some cells show ballooning degeneration, with inflammatory cell infiltration. After administration of the ethyl acetate extract of *Tetrastigma obtectum*, the degree of hepatocyte necrosis is significantly reduced, and the degree of inflammatory infiltration is decreased. In the low-dose administration group, some cells still show ballooning degeneration, and in the high-dose administration group, there is almost no ballooning degeneration. This is consistent with the blood biochemical results, indicating that the ethyl acetate extract of *Tetrastigma obtectum* has obvious effects on protecting liver injury.

[0051] Finally, it should be noted that the above embodiments are only relatively representative examples of the present invention. Obviously, the technical solutions of the present invention are not limited to the above embodiments and there can be many variations. All variations that can be directly derived or associated by those of ordinary skill in the art from the disclosed content of the present invention shall be considered as within the protection scope of the present invention.

Claims

1. Application of delavay ivy and its extract in the preparation of drugs for treating impaired liver function.

2. The application according to claim 1, wherein The delavay ivy is the dry stem, leaf or whole plant of the Chinese ivy (Hedera nepalensis K. Koch var. sinensis (Tobl.) Rehd.), which belongs to the Araliaceae family.

3. The application according to claim 1, wherein The extract of delavay ivy is the extract of the root of delavay ivy.

4. The application according to claim 3, characterized in that, The extract of the root of delavay ivy is the ethyl acetate extract of the root of delavay ivy.

5. The application according to claim 4, wherein The ethyl acetate extract of the root of delavay ivy is prepared by the following method: After drying and pulverizing the delavay ivy root medicinal material, it is soaked and extracted twice with 30 times the amount of 95% ethanol for 2 hours each time, and soaked and extracted once with 75% ethanol for 7 days each time; after the filtrate is concentrated in vacuo, an ethanol extract is obtained; then, the ethanol extract is dispersed with distilled water and extracted with ethyl acetate, and the filtrate is concentrated to obtain an ethyl acetate extract.

6. The application according to claim 1, characterized in that, The drug for treating impaired liver function is a drug for preventing or treating liver injury.

7. The application according to claim 1, characterized in that, The drug for treating impaired liver function also contains a pharmaceutically acceptable carrier or excipient.

8. A drug for treating liver function impairment, characterized in that, It contains delavay ivy or its extract.

9. The drug for treating impaired liver function according to claim 8, wherein, The drug for treating impaired liver function is a drug prepared by combining delavay ivy or its extract with other drugs, excipients, etc.

10. Application of delavay ivy and its extract in the preparation of drugs for preventing / treating cell damage and tissue damage caused by oxidation and inflammation.

Images