Application of radix actinidiae chinensis total flavonoids in preparation of medicine for treating acute alcoholic liver injury

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to an application of Chinese actinidia root total flavonoids in preparation of a medicine for treating acute alcoholic liver injury, and the use dosage of the Chinese actinidia root total flavonoids is 0.09 g / 20g to 0.18 g / 20g according to the use amount of mice. The Chinese actinidia root general flavone extract is taken as a medicinal component, and by establishing an animal alcoholic liver injury model, the Chinese actinidia root general flavone extract is found to have a certain effect of inhibiting the contents of AST and ALT in serum and liver homogenate on mouse acute liver injury caused by alcohol within a certain dosage range, and has a protective effect on acute alcoholic liver injury.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to an application of total flavonoids from a root of Pyrus truncatula in preparing a medicine for treating acute alcoholic liver injury. Background Art

[0002] The root of Actinidia chinensis is the root of the plant Actinidia chinensis, also known as kiwi root. It tastes bitter and is cold in nature. Its functions include clearing away heat and dampness, dispersing blood stasis and dredging meridians, detoxifying and reducing swelling. It is used for common cancers, damp-heat jaundice, dysentery, edema and oliguria, rheumatoid arthritis, and traumatic injuries.

[0003] Total flavonoids from the root of Pyrus truncatula is a general term for flavonoids extracted from the root of Pyrus truncatula. Existing studies have shown that total flavonoids from the root of Pyrus truncatula have antioxidant capacity on rat liver tissue both in vivo and in vitro, and the antioxidant activity is related to the upregulation of SOD1, SOD2, and GPX1 mRNA expression in rat liver tissue. However, there are few reports on the effects of total flavonoids from the root of Pyrus truncatula on acute liver injury, especially on alcohol-induced liver injury. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the present invention is to provide an application of total flavonoids from the root of Pyrus truncatula in the preparation of a drug for the treatment of acute alcoholic liver injury. The total flavonoids extract from the root of Pyrus truncatula is used as a drug ingredient. By establishing an animal alcoholic liver injury model, it is found that the total flavonoids extract from the root of Pyrus truncatula has a certain inhibitory effect on the AST and ALT contents in serum and liver homogenate on acute liver injury in mice caused by alcohol within a certain dosage range, and has a protective effect on the degeneration of some hepatocytes around the central vein of the hepatic lobule in acute alcoholic liver injury.

[0005] The present invention provides an application of total flavonoids of radix pyrifolia in preparing a drug for treating acute alcoholic liver injury. The dosage of the total flavonoids of radix pyrifolia is 0.09g / 20g-0.18g / 20g according to the dosage for mice. According to the conversion of the traditional prescription of radix pyrifolia for treating acute hepatitis recorded in Jiangxi Herbal Medicine, the clinical dosage can be 30g / 60kg-60g / 60kg.

[0006] Furthermore, in the above technical solution, the preparation method of the total flavonoids extract from the root of Pyrus truncatula comprises the following steps: (1) Extraction: Take the root of Pyrus tiliaceus and extract it with 10 times the amount of 55% ethanol under reflux for 3 times, 1.5 h / times. Combine the filtrates and recover the ethanol to obtain the extract. (2) Extraction: The concentrated extract is extracted with petroleum ether and ethyl acetate, respectively, and recovered to obtain a primary extract paste; (3) Purification: The initially extracted dry paste is subjected to column chromatography using a polyamide column, and eluted with water, 20% ethanol, and 80% ethanol in sequence. The eluates are collected, combined, and evaporated to dryness.

[0007] Furthermore, in step (2) of the above technical solution, when petroleum ether is used for extraction, the temperature is 60°C-90°C, and the extraction is performed by shaking for 3 times, 1500 mL / time.

[0008] Furthermore, in step (2) of the above technical solution, when ethyl acetate is used for extraction, the aqueous solution after petroleum ether extraction is first placed on a water bath to evaporate the petroleum ether, cooled, and then transferred to a separatory funnel and extracted with ethyl acetate by shaking for 4 times, 1500 mL / time, the ethyl acetate liquids are combined, the ethyl acetate is recovered, the residue is completely dissolved with methanol, transferred to an evaporating dish, and evaporated to dryness.

[0009] Furthermore, in step (3) of the above technical solution, the specifications of the polyamide column are: pore size of 30 mesh-60 mesh, column height of 50 cm, and radius of 2.5 cm.

[0010] Furthermore, in step (3) of the above technical solution, the primary dry paste is first mixed with water and polyamide powder and evaporated to dryness, then transferred to a polyamide column and eluted with water, 20% ethanol and 80% ethanol in sequence; wherein the water and 20% ethanol eluates are combined and evaporated to dryness; the 80% ethanol eluate is evaporated to dryness and then dissolved with methanol, then centrifuged, the centrifuged liquid is evaporated to dryness and then dissolved with ethyl acetate in small amounts several times, evaporated to dryness, and finally the above evaporated materials are combined to obtain a dry paste for later use.

[0011] Furthermore, in step (3) of the above technical solution, the mass of the water and 20% ethanol used is 70 to 75 times the mass of the initial dry paste, respectively, and the mass of the 80% ethanol used is 140 to 150 times the mass of the initial dry paste.

[0012] Advantages compared to the prior art: The invention adopts ethanol as extraction solvent for the root of Actinidia chinensis, extracts it with petroleum ether and ethyl acetate, and then purifies it with a polyamide column to obtain the ethyl acetate extraction part of the alcohol extract of Actinidia chinensis root, which is the total flavonoids extract of kiwifruit root. The method has simple process, low cost and no toxic side effects.

[0013] The present invention establishes an animal alcoholic liver injury model and finds that the total flavonoids extract of the root of Pyrus truncatula has a certain inhibitory effect on the AST and ALT contents in serum and liver homogenate on the acute liver injury in mice caused by alcohol within a certain dosage range, and has a protective effect on the degeneration of some hepatocytes around the central vein of the liver lobule of the mice with alcoholic acute liver injury. The extract has good medicinal value in the preparation of drugs for treating acute alcoholic liver injury, filling the gap in the research on drugs for acute alcohol-induced liver injury using the total flavonoids of the root of Pyrus truncatula. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a diagram of liver tissue sections of mice in the Normal group in Example 3 of the present invention; Figure 2This is a diagram of liver tissue sections of mice in the Model group in Example 3 of the present invention; Figure 3 This is a liver tissue section of mice in the T1 group in Example 3 of the present invention; Figure 4 This is a diagram of liver tissue sections of mice in the T2 group in Example 3 of the present invention. DETAILED DESCRIPTION

[0015] The above-mentioned technical features of the present invention and the technical features specifically described below (such as implementation cases) can be combined with each other to form new or preferred technical solutions, but the present invention is not limited to these embodiments, and these embodiments do not limit the present invention in any way.

[0016] The experimental methods in the following examples are conventional methods unless otherwise specified. The preparations involved in the following examples are common commercial products unless otherwise specified and can be purchased from the market.

[0017] Experimental instruments and reagents: 1. Instrument Centrifuge, Shanghai Medical Instrument (Group) Co., Ltd. Surgical Instrument Factory; UV spectrophotometer, Japan Shimadzu UV-2401pc / 2450; HH-4 digital display constant temperature water bath, Jintan Jiangnan Instrument Factory.

[0018] 2. Drug reagents The root of Pear Tree was purchased from the Chinese medicine pharmacy of Jiangxi Provincial Hospital of Traditional Chinese Medicine (Thermosensitive Moxibustion Branch); ALT test kit, Shanghai Rongsheng Bio-Pharmaceutical Co., Ltd.; AST test kit, Shanghai Rongsheng Bio-Pharmaceutical Co., Ltd.; Ganlixin capsule, Jiangsu Zhengda Tianqing Pharmaceutical Co., Ltd.; ethanol, Guangdong Chemical Reagent Engineering Technology Research and Development Center.

[0019] 3. Experimental Animals Kunming mice, half male and half female, weighing 18g-22g, were provided by the Animal Experiment Center of Jiangxi University of Traditional Chinese Medicine.

[0020] The present invention is described in further detail below in conjunction with embodiments: Example 1: Preparation of total flavonoids extract from Pyrus truncatula root The specific steps include: (1) Extraction: Take 1 kg of Pyrus tiliaceus root and extract it with 10 times the amount of 55% ethanol under reflux for 3 times, 1.5 h / times. Combine the filtrates, recover the ethanol and concentrate to obtain 1500 mL of extract. (2) Extraction: ①Petroleum ether extraction: Place the extract in a separatory funnel and shake with petroleum ether (60℃-90℃) for 3 times, 1500mL / time, discard the petroleum ether liquid, and obtain the aqueous liquid part; ② Ethyl acetate extraction: Place the aqueous portion after petroleum ether extraction on a water bath to evaporate the petroleum ether, cool, and then transfer to a separatory funnel, shake and extract with ethyl acetate 4 times, 1500 mL / time, combine the ethyl acetate liquid, recover the ethyl acetate, dissolve the residue with methanol, and transfer to an evaporating dish to obtain 14 g of primary dry paste solid; (3) Purification Take 14 g of the above-mentioned primary dry paste, 50 mL of water, mix with 100 g of polyamide powder, evaporate to nearly dryness, transfer to a polyamide column (30 mesh-60 mesh, column height 50 cm, radius 2 cm), first elute with 1000 mL of water, collect the eluate; then elute with 1000 mL of 20% ethanol, collect the eluate; finally elute with 2000 mL of 80% ethanol, collect the eluate.

[0021] Combine the water and 20% ethanol elutions, evaporate to dryness, and obtain 2.9 g of dry paste; The 80% ethanol elution part was evaporated to dryness, dissolved with methanol, and then centrifuged. The centrifugal liquid was evaporated to dryness and then dissolved with ethyl acetate in small amounts several times, and evaporated to dryness to obtain 2.5 g of dry paste.

[0022] Combine the dry paste obtained above and set aside.

[0023] Example 2: Acute toxicity test of total flavonoids extract from Pyrus truncatula root on mice 1. Experimental Materials 1.1 Experimental animals Ordinary grade Kunming mice weigh 18g-22g, half male and half female.

[0024] 1.2 Experimental equipment 1mL syringe, gavage needle, mouse cage, balance, etc.

[0025] 2. Preliminary Experiment 2.1 Dose estimation According to the maximum daily dosage of the root of Pyrola paniculata for humans (60 kg), 4 liang = 200 g of raw medicine / 60 kg, which is converted into a daily dosage of 0.09 g / 20 g for mice (20 g). This dosage is used as the minimum dosage for the acute toxicity experiment in mice, and four dosage groups are set with a ratio of 1:0.5 between the two adjacent high and low dosages. The total flavonoid extract of the root of Pyrola paniculata is the dry paste obtained by the method in Example 1, and CMC-NA (sodium carboxymethyl cellulose) is used to assist dissolution.

[0026] 2.2 Drug preparation Dissolve the dry paste in the solvent to make up the volume, place the prepared liquid in a brown bottle and prepare it before use.

[0027]

[0028] 2.3 Animal Preparation Sixteen mice were taken, half of which were male and half were female, and randomly divided into four groups, with four mice in each group, half of which were male and half were female.

[0029] 2.4 Experimental methods The drug was administered by oral gavage at a rate of 0.2 mL / 10 g. After administration, the mice were given water and feed according to normal feeding conditions and observed for 14 days.

[0030] 3. Formal Experiment Five doses were determined based on the preliminary experimental results. The administered doses should range from the dose where no toxicity was observed to the dose where severe toxicity (life-threatening) occurred.

[0031] Before the experiment, mice were fasted but not watered for 16 hours. After being weighed the next day, they were randomly divided into 6 groups: a blank control group and 5 dose groups, with 10 mice in each group. Each group of animals was given a single dose by gavage, with a total dose of 20 mL / kg, divided into 2-3 doses within 24 hours (each dose was 4-6 hours apart), and continued to fast for 3-4 hours after administration.

[0032] After administration, the experimental animals should be regularly observed for signs of poisoning and death on the first day, and then carefully observed at least once a day. Record in detail changes in coat and skin, eyes and mucous membranes, respiration, circulation, autonomic and central nervous systems, limb movements and behavior. Pay special attention to whether symptoms such as tremors, convulsions, salivation, diarrhea, lethargy and coma occur. The time when signs of toxicity appear and disappear and the time of death should be recorded.

[0033] The observation period is generally no more than 14 days, depending on the severity of the animal's poisoning reaction, the speed of symptom onset and the length of the recovery period. If there is a delayed death, the observation period can be extended. During the observation period, the living animals are weighed weekly. At the end of the observation period, the surviving animals should be weighed. After being killed, they are subjected to gross anatomical examinations and all gross pathological changes are recorded.

[0034] 4. Experimental Results In the preliminary experiment, the maximum allowable amount of total flavonoids extract from the root of Pyrus truncatula was 0.2 mL / 10 g, and no animal poisoning symptoms or death were observed; in the formal experiment, no animal poisoning or death was observed within 14 days. The above results of acute toxicity experiments on mice show that the total flavonoids extract from the root of Pyrus truncatula is highly safe for oral administration.

[0035] Example 3: Intervention test of total flavonoids extract from Pyrus truncatula root on acute alcoholic liver injury in mice 1. Dosage and grouping The total flavonoids extract from the root of Pyrus truncatula was prepared by the method of Example 1. According to the traditional prescription of the root of rattan pear for treating acute hepatitis recorded in Jiangxi Herbal Medicine, the clinical dosage for human is 4 liang / day (i.e. 200g / 60kg), which is converted into the dosage for mice as 0.6g / 20g; according to the dosage of the root of rattan pear as a compound, the clinical dosage for human is 30g / 60kg, which is converted into the dosage for mice as 0.09g / 20g; therefore, the mouse dosage of the total flavonoid extract of the root of rattan pear is set at T1 as 0.09g / 20g (T0 is 0.045g / 20g, T2 is 0.18g / 20g, T3 is 0.27g / 20g, T4 is 0.36g / 20g, and T5 is 0.45g / 20g), and the drug is prepared with CMC-NA (sodium carboxymethyl cellulose), and sonicator is used to assist dissolution or suspension, and the drug is administered by gavage at a dosage volume of 10mL / kg, and the N group and the M group are given an equal volume of distilled water. The above are all raw drug dosages.

[0036] The mice were divided into normal control group, model control group and total flavonoids from Pyrus truncatula root administration groups (T0, T1, T2, T3, T4 and T5), with 8 mice in each group, half of which were male and half were female.

[0037] 2. Establishment of Alcoholic Liver Injury Model The mice were given 50% ethanol by gavage at a dosage of 12 mL / kg, which induced an acute alcoholic liver injury model in mice, characterized by mild increase in serum ALT and AST and hepatocyte degeneration.

[0038] 3. Experimental Procedure Mice were given preventive medication for 2 days. After the medication on the second day, they were fasted but not watered for 14 hours. On the third day, 50% ethanol (12 mL / kg) was given ig to establish the model. After 3 hours, the mice were given ig. After 1 hour, blood was collected from the eye sockets and allowed to stand at room temperature for more than half an hour. The supernatant was aspirated at 3500 rpm for 5 minutes. The ALT and AST kits were operated according to the instructions, and the absorbance A value was measured on a UV spectrophotometer. The liver tissue was taken and 10% liver tissue homogenate was made. The absorbance A value was measured according to the instructions of the ALT and AST kits. The liver tissue was taken, embedded in paraffin, and sliced ​​for observation.

[0039] SPSS software was used to perform T test on the data, p < 0.05 indicated a difference, and p < 0.01 indicated a significant difference.

[0040] 3.1 The effect of the total flavonoids extract of Pyrus truncatula on ALT in the serum and liver homogenate of mice with alcoholic acute liver injury was statistically analyzed, and the effects of different doses of total flavonoids extract of Pyrus truncatula on the ALT index of mice were analyzed. The results are shown in Table 1.

[0041] Table 1 Effects of total flavonoids extract from Pyrus truncatula root on ALT in serum and liver homogenate of mice with alcoholic acute liver injury (x±s, n=8)

[0042] *: Compared with the Normal group, p≤0.05, there is a difference; **: Compared with the Normal group, p≤0.01, there is a significant difference; # : Compared with the Model group, p≤0.05, there is a difference; ## : Compared with the Model group, p≤0.01, there is a significant difference.

[0043] From the experimental results in Table 1, it can be seen that compared with the Normal group, the Model group had significant differences in serum and liver homogenate ALT levels, indicating that the model was successfully established; compared with the Model group, the total flavonoids extract group of Pyrus truncatula root had differences in T1, T2, T3, and T4, among which T1 and T2 had significant differences, and T3 and T4 had differences, indicating that the total flavonoids extract of Pyrus truncatula root had a protective effect on alcoholic acute liver injury in mice.

[0044] 3.2 The effect of the total flavonoids extract of Pyrus truncatula root on AST in the serum and liver homogenate of mice with alcoholic acute liver injury was statistically analyzed, and the effects of different doses of total flavonoids extract of Pyrus truncatula root on the AST index of mice were analyzed. The results are shown in Table 2.

[0045] Table 2 Effects of total flavonoids extract from Pyrus truncatula root on AST in serum and liver homogenate of mice with alcoholic acute liver injury (x±s, n=8)

[0046] *: compared with the Normal group, p≤0.05, there is a difference; **: compared with the Normal group, p≤0.01, there is a significant difference; ***: compared with the Normal group, p≤0.001, there is a significant difference.

[0047] # : Compared with the Model group, p≤0.05, there is a difference; ## :Compared with the Model group, p≤0.01, there is a significant difference; ### : Compared with the Model group, p≤0.001, there is a significant difference.

[0048] From the experimental results in Table 2, it can be seen that compared with the Normal group, the Model group had significant differences in serum and liver homogenate AST levels, indicating that the model was successfully constructed; compared with the Model group, the total flavonoids extract group of Pyrus truncatula root had differences in T1, T2, T3, and T4, with significant differences in T1 and T2, and differences in T3 and T4, indicating that the total flavonoids extract of Pyrus truncatula root had a protective effect on alcoholic acute liver injury in mice.

[0049] 3.3 Analyze the effect of total flavonoids extract from radix pyrifolia on liver tissue sections of mice with acute alcoholic liver injury to understand the protective effect of total flavonoids extract from radix pyrifolia on the specific liver tissue of mice with acute alcoholic liver injury. Figures 1 to 4 As shown: Normal group: The liver tissue structure is basically normal, the liver lobule structure is intact, the nucleolus is clear, and there are almost no fat droplets in the cytoplasm ( Figure 1 ); Model group: The liver lobule structure is incomplete, the hepatic cords are not arranged evenly, the hepatocytes are swollen, the cytoplasm is loose and balloon-like, a large number of fat vacuoles appear, and the cell nuclei are fragmented and dissolved ( Figure 2 ); T1 group of total flavonoids extract from Pyrus truncatula: some hepatocytes around the central vein of the hepatic lobule were swollen, with loose cytoplasm, and cell nuclear fragmentation and dissolution were still observed, but it was improved compared with the Model group ( Figure 3 ); T2 group of total flavonoids extract from Pyrus truncatula: a small number of hepatocytes around the central vein degenerated, the lesion area became smaller, the swelling was reduced, only a small number of fat vacuoles were seen, and the edema and necrosis of hepatocytes were significantly improved ( Figure 4 ).

[0050] Observations of pathological tissue morphology suggest that the total flavonoids extract from the root of Pyrus truncatula has a certain protective effect on the degeneration of some hepatocytes around the central vein of the hepatic lobule in mice with acute alcoholic liver injury.

[0051] In summary, the total flavonoids extract of the root of Pyrus truncatula of the present invention has a certain inhibitory effect on the content of AST and ALT in serum and liver homogenate on acute liver injury caused by alcohol in mice within the dosage range of T1-T4, but the efficacy is not proportional to the increase in dosage. Among them, T1 and T2, the efficacy increases with the increase of dosage, at T3, the efficacy decreases instead, at T4, the efficacy is consistent with T3, T0 and T5 have no effect, indicating that the higher the dosage, the better. At the same time, the total flavonoids extract of the root of Pyrus truncatula can significantly improve the degeneration of some hepatocytes around the central vein of the liver lobule of mice with alcoholic acute liver injury, has a positive effect on preventing alcoholic acute liver injury in mice, and has obvious improvement and protective effects on various biochemical indicators of the liver of alcoholic acute liver injury within a certain dosage range.

[0052] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An application of total flavonoids from the root of Pyrus truncatula in the preparation of a drug for treating acute alcoholic liver injury, characterized in that: Calculated based on the dosage for mice, the dosage of the total flavonoids from the root of Pyrus truncatula is 0.09g / 20g-0.18g / 20g.

2. The use according to claim 1, characterized in that: The preparation method of the total flavonoids extract of the root of Pyrus truncatula comprises the following steps: (1) extraction: taking the root of Pyrus truncatula, refluxing and extracting it with 10 times the amount of 55% ethanol for 3 times, 1.5 hours per time, combining the filtrate, recovering the ethanol, and obtaining an extract; (2) extraction: extracting the concentrated extract with petroleum ether and ethyl acetate in turn, recovering the extract, and obtaining a primary extract paste; (3) purification: performing column chromatography on the primary extract paste using a polyamide column, eluting with water, 20% ethanol, and 80% ethanol in turn, collecting the eluate, combining and evaporating to obtain the extract.

3. The use according to claim 2, characterized in that: In step (2), when petroleum ether is used for extraction, the temperature is 60°C-90°C, and the extraction is performed by shaking for 3 times, 1500 mL / time.

4. The use according to claim 2, characterized in that: In step (2), when ethyl acetate is used for extraction, the aqueous solution after petroleum ether extraction is first placed on a water bath to evaporate the petroleum ether, cooled, and then transferred to a separatory funnel and extracted with ethyl acetate by shaking for 4 times, 1500 mL / time, the ethyl acetate solutions are combined, the ethyl acetate is recovered, the residue is completely dissolved with methanol, transferred to an evaporating dish, and evaporated to dryness.

5. The use according to claim 2, characterized in that: In step (3), the specifications of the polyamide column are: pore size of 30 mesh to 60 mesh, column height of 50 cm, and radius of 2.5 cm.

6. The use according to claim 2, characterized in that: In step (3), the primary dry paste is first mixed with water and polyamide powder and evaporated to dryness, then transferred to a polyamide column and eluted with water, 20% ethanol and 80% ethanol in sequence; wherein the water and 20% ethanol eluates are combined and evaporated to dryness; the 80% ethanol eluate is evaporated to dryness and then dissolved in methanol and then centrifuged; the centrifuge liquid is evaporated to dryness and then dissolved in ethyl acetate in small amounts several times and evaporated to dryness; finally, the above evaporated materials are combined to obtain a dry paste for later use.

7. The use according to claim 2, characterized in that: In step (3), the mass of water and 20% ethanol used is 70 to 75 times the mass of the initial dry paste, respectively, and the mass of 80% ethanol used is 140 to 150 times the mass of the initial dry paste.

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