CN115429810B - Application of isoflavone compound in preparing medicament for preventing or treating alcoholic liver injury or dispelling alcohol effect and protecting liver - Google Patents

Application of isoflavone compound in preparing medicament for preventing or treating alcoholic liver injury or dispelling alcohol effect and protecting liver Download PDF

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CN115429810B
CN115429810B CN202110629311.4A CN202110629311A CN115429810B CN 115429810 B CN115429810 B CN 115429810B CN 202110629311 A CN202110629311 A CN 202110629311A CN 115429810 B CN115429810 B CN 115429810B
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alcoholic
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liver injury
isoflavone
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夏增华
张小芸
逄龙
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Suzhou Heyan Biotechnology Co ltd
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Abstract

The invention provides application of isoflavone compounds in preparing medicines for preventing or treating alcoholic liver injury or relieving alcohol and protecting liver. The invention provides application of 6, 7-position ring-formed isoflavone compounds shown in a formula (I) in preparation of medicines for preventing or treating alcoholic liver injury or relieving alcohol and protecting liver for the first time. The activity of 6, 7-position ring-forming isoflavone compounds (compounds 1-15) for treating alcoholic liver injury and relieving alcoholic liver injury is verified through animal experiments, and compared with tectorigenin, the 6, 7-position ring-forming isoflavone compounds have better repairing effect on alcoholic liver injury, and the 6, 7-position ring-forming isoflavone compounds have obvious anti-alcoholic effect through observing the behaviors of alcohol rats before and after administration. Therefore, the invention expands the application field of the isoflavone compound with the 6, 7-position ring.

Description

Application of isoflavone compound in preparing medicament for preventing or treating alcoholic liver injury or dispelling alcohol effect and protecting liver
Technical Field
The invention relates to the field of medicines, in particular to application of an isoflavone compound in preparing a medicine for preventing or treating alcoholic liver injury or relieving alcohol and protecting liver.
Background
Alcoholic liver injury, i.e., alcoholic liver disease (alcoholic liver disease, ALD), is a disease that causes toxic damage to the liver and related health problems after long-term consumption of alcohol, and the incidence of ALD has been on an increasing trend year by year worldwide, and is therefore of great concern. ALD is clinically classified into alcoholic fatty liver (alcoholic fatty liver, AFL), alcoholic hepatitis (alcoholic hepatitis, AH), alcoholic liver fibrosis (alcoholic liver fibrosis, ALF) and alcoholic cirrhosis (alcoholic cirrhosis, AC).
Alcohol is a neurotropic substance that can cause irreversible damage to central and peripheral nerves, and the burden of drinking on public health is 5.1% of the burden of global disease. Alcohol use disorders, including alcohol abuse and alcohol dependence, are one of the major risk factors for premature death, and can cause over 200 diseases, including neuropsychiatric diseases, chronic diseases, cancer, and disabling trauma. Alcohol use disorders are the most common cause of alcoholic liver injury, and can cause a range of liver diseases such as fatty liver, steatohepatitis, liver fibrosis, cirrhosis, liver cancer, and the like.
Various flavonoid compounds are reported to have certain anti-hangover and liver-protecting effects, such as flavonoids derived from chrysanthemum, flavonoids derived from mung beans, flavonoids derived from red bayberry, and the like. Studies have shown that isoflavones derived from kudzuvine flower, such as tectoridin and tectorigenin (tectorigenin), have good liver protecting activity, and that the ethanol dehydrogenase activation effect of tectorigenin is superior to that of tectorigenin.
Chinese patent document (CN 1986556A) discloses a6, 7-position ring-forming isoflavone compound (5, 3',5' -trimethoxy-6, 7-methylenedioxy isoflavone-4 ' -O-beta-D-glucose), and discloses application of the isoflavone compound in preventing and treating estrogen low level and female climacteric syndrome. Regarding the 6, 7-position ring-formed isoflavone compound, no report on the aspects of treating alcoholic liver injury or dispelling alcohol and protecting liver is currently seen.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a new application of the isoflavone compound with 6, 7-position ring formation.
The invention provides an application of isoflavone compound shown in a formula (I) and pharmaceutically acceptable salt thereof in preparing medicines for preventing or treating alcoholic liver injury or relieving alcohol and protecting liver,
wherein:
R 1 selected from hydrogen, C 1 -C 8 Alkyl or C of (2) 3 -C 10 Cycloalkyl of (c);
R 2 、R 3 independently of one another selected from hydrogen, C 1 -C 8 Alkyl, C of (2) 3 -C 10 Cycloalkyl, C 4 -C 10 Aryl or C of (2) 3 -C 10 Heteroaryl of (a);
R 4 、R 7 、R 8 independently of each other selected from hydrogen, hydroxy, C 1 -C 8 Alkyl, C of (2) 3 -C 10 Cycloalkyl, C 1 -C 8 Alkoxy or C of (2) 4 -C 20 Is a glycosyl residue of (a);
R 5 、R 6 independently of each other selected from hydrogen, hydroxy, C 1 -C 8 Alkyl, C of (2) 3 -C 10 Cycloalkyl, C 1 -C 8 Alkoxy, C 4 -C 20 Is a glycosyl residue, -OC (O) NHR, or R 5 And R is 6 Are connected to each other to form a ring a,
the ring A is selected from C 3 -C 7 Carbocycles of C 3 -C 7 Heterocyclic ring of C 4 -C 10 Aryl, C of (2) 3 -C 10 Heteroaryl of (a);
r is selected from C 1 -C 10 Is a hydrocarbon group.
Further, the method comprises the steps of,
R 1 selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl;
R 2 、R 3 independently of each other, selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl;
R 4 、R 7 、R 8 independently of each other selected from hydrogen, hydroxy, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methoxy, ethoxy, n-propoxy, isopropoxy or from At least one formed glycosyl residue;
R 5 、R 6 independently of each other selected from hydrogen, hydroxy, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methoxy, ethoxy, n-propoxy, isopropoxy, -OC (O) NHR, or from At least one formed glycosyl residue, or R 5 And R is 6 Are connected with each other to form 1, 3-dioxolane,
r is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.
Further, the method comprises the steps of,
R 1 selected from hydrogen or methyl;
R 2 、R 3 independently of each other selected from hydrogen or phenyl;
R 4 selected from hydrogen, hydroxy, methoxy or fromAt least one formed glycosyl residue;
R 5 、R 6 independently of one another, selected from hydrogen, hydroxy, methoxy, -OC (O) NHR, or R 5 And R is 6 Are connected with each other to form 1, 3-dioxolane;
R 7 、R 8 independently of each other selected from hydrogen, hydroxy, methoxy, or from At least one formed glycosyl residue;
r is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.
Further, the method comprises the steps of,
R 1 selected from hydrogen or methyl;
R 2 、R 3 independently of each other selected from hydrogen or phenyl;
R 4 selected from hydrogen, hydroxy or methoxy;
R 5 selected from hydrogen, hydroxy or-OC (O) NHR;
R 6 selected from hydrogen, hydroxy or methoxy;
alternatively, R 5 And R is 6 Are connected with each other to form 1, 3-dioxolane;
R 7 selected from hydrogen, hydroxy or methoxy;
R 8 selected from hydrogen, hydroxy, methoxy, or fromA glycosyl residue formed by one of (a) and (b);
r is selected from n-propyl.
Further, the method comprises the steps of,
the isoflavone compound has a structure shown in a formula (a), (b) or (c):
further, the method comprises the steps of,
the isoflavone compound has a structure shown as follows:
further, the medicament also comprises a pharmaceutically acceptable carrier.
Further, the medicine is added with conventional auxiliary materials and is prepared into clinically acceptable tablets, capsules, oral liquid, powder, dripping pills, granules or injections according to a conventional process.
Further, the alcoholic liver injury includes: alcoholic fatty liver, alcoholic liver fibrosis, alcoholic hepatitis, and alcoholic cirrhosis.
The technical scheme of the invention has the following advantages:
the invention provides application of 6, 7-position ring-forming isoflavone compounds shown in a formula (I) in preparation of medicines for preventing or treating alcoholic liver injury or relieving alcohol and protecting liver for the first time. The activity of 6, 7-position ring-forming isoflavone compounds (compounds 1-15) for treating alcoholic liver injury and relieving alcoholic liver injury is verified through animal experiments, an alcoholic rat model is constructed through experimental rat alcohol lavage, and the compounds 1-15 and tectorigenin are respectively used for administration, so that compared with tectorigenin, the 6, 7-position ring-forming isoflavone compounds have better repairing effect on alcoholic liver injury, and the behavior of an alcoholic rat before and after administration is observed, so that the 6, 7-position ring-forming isoflavone compounds have obvious anti-alcoholic effect. Therefore, the invention expands the application field of the isoflavone compound with the 6, 7-position ring.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
Preparing a chromatographic column: c (C) 18 The diameter of the chromatographic column is 2.6cm, the length of the chromatographic column is 20cm, and the particle size of the packing material is 10 mu m.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The materials or instruments used are all conventional products commercially available, including but not limited to those used in the examples of the present application.
The percentages of solutions in the examples and experimental examples represent the volume fraction (v/v).
Example 1 preparation of Compounds 1-6
Weighing 15kg of iris whole herb, cutting, adding 6 times of 70% ethanol, heating, extracting at 65 ℃ for 2 hours each time, combining the extracts, concentrating under reduced pressure until no alcohol smell exists, sequentially extracting the obtained extract (1020 g) with ethyl acetate and n-butanol (3 times of each solvent extraction, 1.5L of each solvent extraction), respectively combining each extract, concentrating under reduced pressure to obtain 640g of ethyl acetate part, 220g of n-butanol part and 160g of pure water part.
Separating the pure water part by silica gel column (column diameter 4 cm. Times.height 50cm, column volume 650 mL), and gradient eluting with chloroform-methanol (50:1, 20:1, 10:1, 1:1) to obtain 4 components Fr.HO1-Fr.HO4. Wherein fr.ho2 was separated by preparative chromatography (60% aqueous methanol, 70% aqueous methanol, 80% aqueous methanol elution), 70% aqueous methanol fraction yielded compound 1 (268 mg) and compound 2 (294 mg).
Separating ethyl acetate part by silica gel column (column diameter 6 cm. Times.60 cm, column volume 1.7L), and gradient eluting with petroleum ether-ethyl acetate (100:0, 50:1, 20:1, 10:1, 5:1, 0:100) to obtain 6 components Fr.EAC1-Fr.EAC6. Wherein fr.eac3 is separated by silica gel column chromatography (petroleum ether-ethyl acetate 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 8:1, 6:1, 4:1, 2:1), and the eluents at the same spot position are combined according to the detection result of Thin Layer Chromatography (TLC), to obtain 15 fractions fr.eac3-1 to fr.eac3-15, fr.eac3-5 are separated by preparative chromatography (elution with 85% aqueous methanol) to obtain compound 3 (520 mg) and compound 5 (266 mg); fr.EAC5 was separated by silica gel column chromatography (20:1, 17:1, 15:1, 12:1, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1) and the eluates at the same spot position were combined according to the Thin Layer Chromatography (TLC) detection result to give 10 fractions Fr.EAC5-1 to Fr.EAC5-10, fr.EAC5-5 was separated by preparative chromatography (75% aqueous methanol elution) to give compound 4 (587 mg).
Separating n-butanol part by silica gel column (column diameter 6 cm. Times.60 cm, column volume 1.7L), and gradient eluting with chloroform-methanol (100:0, 50:1, 20:1, 10:1, 5:1, 0:100) to obtain 6 components Fr.NBA1-Fr.NBA6. Wherein fr.nba2 was separated by silica gel column chromatography (chloroform-methanol 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 5:1), and the eluates at the same spot positions were combined according to the detection result of Thin Layer Chromatography (TLC), to obtain 11 fractions fr.nba2-1 to fr.nba2-11, fr.nba2-8 were separated by preparative chromatography (70% aqueous methanol elution) to obtain compound 6 (614 mg).
Subjecting the above prepared compounds to HPLC-MS respectively 1 H-NMR、 13 C-NMR performs structure confirmation, and the structure confirmation data is referred to as follows:
compounds 1-5: elliger C A, halloin J M.Phenolics induced in Beta vulgaris by rhizoctonia solani infection [ J ]. Phytochemistry (Oxford), 1994,37 (3): 691-693.
Compound 6: choudhary M I, hareem S, siddiqui H, et al A benzil and isoflavone from Iris tenuifolia [ J ]. Phytochectry, 2008,69 (9): 1880-1885.
Example 2 preparation of Compounds 7-9
Weighing 15kg of fresh Ateleia hereunder-smithii Pittier leaves, cutting into small blocks, adding 3 times of diethyl ether for soaking and extracting for 3 times, each time for 24 hours, merging the extracting solutions, concentrating, adding methanol into the concentrated solution, dissolving, mixing the mixture with silica gel, separating by a silica gel column (column diameter is 6cm multiplied by 60cm, column volume is 1.7L), gradient eluting by chloroform-methanol (20:1, 10:1, 2:1), collecting 10:1 parts of each gradient by 3 column volumes, concentrating and drying (10 g), separating by medium pressure chromatography (silica gel column, column diameter is 4.6cm multiplied by 60cm, column volume is 1L), eluting by cyclohexane-ethyl acetate (1:1), merging the eluates (254 nm) at the same spot positions according to the detection result of Thin Layer Chromatography (TLC), and obtaining six fractions: fr.a to fr.f. Wherein fr.c was isolated by preparative chromatography (eluting with 75% aqueous methanol) to give compound 7 (671 mg); fr.D was isolated by preparative chromatography (eluting with 75% aqueous methanol) to give compound 8 (264 mg); fr.E was isolated by preparative chromatography (eluting with 80% aqueous methanol) to give compound 9 (289 mg).
Subjecting the above prepared compounds to HPLC-MS respectively 1 H-NMR、 13 C-NMR performs structure confirmation, and the structure confirmation data is referred to as follows:
compound 7-9 structure: veitch NC, sutton PS, kite GC, ireland HE.Six new isoflavones and a 5-deoxyflavonol glycoside from the leaves of Ateleia herbert-smithii.J Nat prod.2003;66 (2):210-216.
EXAMPLE 3 preparation of Compounds 10-11
Cercis chinensis (Mad)Cutting 35kg of huca latifolia) fruit into small pieces, chopping an aliquot with a stirrer, extracting with 6 times 90% ethanol for 3 times (40 ℃) for 2 hours each time, concentrating the extract into an extract, sequentially extracting with ethyl acetate and n-butanol (3 times each solvent extraction, 1.5L each solvent extraction), combining each extract, and extracting the ethyl acetate extract with anhydrous Na 2 SO 4 The water was removed and concentrated (79 g), and the mixture was separated by a silica gel column (column diameter: 6 cm. Times. Height: 60cm, column volume: 1.7L) and successively purified by petroleum ether, petroleum ether: ethyl acetate (9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1), chloroform-methanol (9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 2:1, 1:1), methanol gradient elution, and combining the eluates at the same spot positions according to the detection result of Thin Layer Chromatography (TLC), to obtain 20 fractions Fr.1-Fr.20. Wherein fr.11 (1.35 g) was separated by a silica gel column, petroleum ether: ethyl acetate (90:1, 50:1, 20:1, 1:1), and combining the eluates at the same spot position according to the detection result of Thin Layer Chromatography (TLC), to obtain 13 fractions Fr.11-1 to Fr.11-13, fr.11-5, and subjecting to preparative chromatographic separation (85% methanol aqueous solution elution) to obtain compound 10 (167 mg); fr.11-8 was subjected to preparative chromatography (70% aqueous methanol elution) to give compound 11 (108 mg).
Subjecting the above prepared compounds to HPLC-MS respectively 1 H-NMR、 13 C-NMR performs structure confirmation, and the structure confirmation data is referred to as follows:
compound 10: siddiqui B, khan S, kardar M N, et al chemical Constituents from the Fruits of Madhuca latifolia [ J ]. Helvetica Chimica Acta,2004,87 (5).
Compound 11: siddiqui, B.S., khan, S., & Nadeem Kardar, M. (2010) A new isoflavone from the fruits ofMadhuca latifolia. Natural Product Research,24 (1), 76-80.
EXAMPLE 4 preparation of Compounds 12-13
25kg of dried whole grass of herba Salsolae Collinae, cutting, reflux-extracting with 95% ethanol for 3 times each for 2h, mixing the extractive solutions, concentrating under reduced pressure to obtain 1.97kg of extract, dispersing the extract in water, sequentially extracting with petroleum ether, chloroform and n-butanol (each solvent for 3 times and 1.5L each), mixing the extractive solutions, and concentrating under reduced pressure to obtain 650g of petroleum ether, 773g of chloroform and 434g of n-butanol. Separating chloroform part by silica gel column (column diameter 6 cm. Times.60 cm, column volume 1.7L), gradient eluting petroleum ether-ethyl acetate (100:0, 50:1, 20:1, 10:1, 5:1, 0:100) to obtain 6 components Fr.1-Fr.6, wherein Fr3 is separated by silica gel column chromatography (dichloromethane-ethyl acetate 100:1, 70:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 8:1, 6:1, 4:1, 2:1), combining eluents at the same spot position according to Thin Layer Chromatography (TLC) detection result to obtain 13 fractions (Fr 3-1-Fr 3-13), and subjecting Fr3-3 to preparative chromatography (85% methanol aqueous solution elution) to obtain compound 12 (166 mg); fr3-8 was subjected to preparative chromatography (eluting with 75% aqueous methanol) to give compound 13 (357 mg).
Subjecting the above prepared compounds to HPLC-MS respectively 1 H-NMR、 13 C-NMR performs structure confirmation, and the structure confirmation data is referred to as follows:
compounds 12-13: woldu Y, abegaz B.Isoflavonoids from Salsola somalensis [ J ]. Phytochemistry,1990,29 (6): 2013-2015.
EXAMPLE 5 preparation of Compounds 14-15
Step 1: 87.07g of raw material 1 and 8mL of DMF (dimethylformamide) were weighed and added to CCl 4 In (500 mL) solution, after stirring at room temperature for 30min, CCl containing 120g of bis (trichloromethyl) carbonate was added dropwise 4 (200 mL) of the solution, the mixture was slowly refluxed for 6 hours (75 ℃ C.), naturally cooled to room temperature, and the reaction solution was concentrated under reduced pressure, and the crude residue was passed through trueAir distillation purification, collecting distillate, namely iminochloride (intermediate 1);
step 2: 1g of 5,6,7,3'-Tetrahydroxy-4' -methoxofenaset and 0.93g of dichlorodiphenyl methane are weighed and added into 50mL of diphenyl ether, the mixture is heated and stirred at 180 ℃ for reaction for 30 minutes, the mixture is cooled to room temperature, petroleum ether (150 mL) is added dropwise, the solid is filtered, the solid is dissolved by ethyl acetate and then is stirred with silica gel (dry sample), and the mixture is purified by a silica gel column (phi 2cm multiplied by 60 cm) and then the mixture is purified by petroleum ether: ethyl acetate 10:1, 3:1, 1:1 elution, and eluent (3:1 fractions) were collected according to TLC results to afford compound 14 (1.12 g, 73.5% yield) as a yellow solid;
step 3: 500mg of compound 14 was weighed into anhydrous DMF (100 mL), 205mg of potassium carbonate and 52mg of potassium iodide were sequentially added, the mixture was stirred in an ice water bath (0 ℃) for 10min, 265mg of intermediate 1 was further added, the ice water bath (0 ℃) was stirred for 2h, the reaction mixture was taken out, left standing at room temperature for 12 hours, the mixture was dissolved in water (150 mL), extracted 3 times with ethyl acetate, 80mL each time, the organic layer was collected, washed with saturated brine (200 mL), dried over sodium sulfate and concentrated under reduced pressure to give a crude solid, the solid was dissolved with ethyl acetate and then stirred with silica gel (dry loading), and then purified by silica gel column (. Phi.2 cm. Times.50 cm), using petroleum ether: ethyl acetate 10:1, 5:1, 1:1, 0:1 solution was used as eluent, and the eluent was collected according to TLC results (1:1 fraction) to give compound 15 (389 mg, yield 66.1%).
Compound 14:
1 HNMR(300MHz,DMSO-d6):
3.87(s,3H),7.04(s,1H),7.47-7.59(m,10H),7.61(d,J＝6.69Hz,1H),7.65(s,1H),7.74(d,J＝6.69Hz,1H),7.96(s,1H)
13 C-NMR(75MHz,DMSO-d6):
56.23,94.52,103.31,104.95,119.15,124.35,126.21,126.30,126.49(2C),128.79,129.04(4C),129.13(4C),139.27(2C),141.95,145.62,152.76,152.94,154.41,155.77,164.01,182.44
compound 15:
1 HNMR(300MHz,DMSO-d6):
1.12(t,3H),1.59(m,2H),2.96(m,2H),3.87(s,3H),7.04(s,1H),7.47-7.59(m,10H),7.61(d,J＝6.69Hz,1H),7.65(s,1H),7.74(d,J＝6.69Hz,1H),7.96(s,1H),8.01(s,1H)
13 C-NMR(75MHz,DMSO-d6):
11.23,23.16,44.20,56.23,94.52,103.31,104.95,119.15,124.35,126.21,126.30,126.49(2C),128.79,129.04(4C),129.13(4C),139.27(2C),141.95,145.62,152.76,152.94,154.41,155.77,164.01,182.44
experimental example
1. Main reagent
Compounds 1 to 15 prepared in examples 1 to 5;
compound glycyrrhizin tablet (Le Pu pharmaceutical company, national drug standard H20073723);
ALT, AST, TC, TG kit (Beckmann coulter Co., ltd., U.S.A.);
MDA kit (Nanjing institute of biological engineering) adopts thiobarbituric acid colorimetric method (TBA) and detects according to the requirement of the kit.
2. Pharmaceutical formulation
Test drug solution: preparing the compounds 1-15 into a tested drug solution with the drug concentration of 50mg/mL by adopting an aqueous solution of sodium carboxymethylcellulose (CMC-Na) with the mass percentage of 0.5%;
tectorigenin solution: preparing tectorigenin into tectorigenin solution with concentration of 50mg/mL by adopting CMC-Na water solution with mass percent of 0.5%;
compound glycyrrhizin solution: the compound glycyrrhizin is prepared into a compound glycyrrhizin solution with the concentration of 6mg/mL by adopting a CMC-Na aqueous solution with the mass percentage of 0.5 percent.
3. Experimental animals and raising
190 SPF-class SD rats with 8 weeks of age, weight of 180-220 g, animal laboratory temperature of 22-25 ℃ and relative humidity of 55-70%.
4. Grouping, modeling and administration of animals
After the SD rats are adaptively fed for 7 days, the SD rats are randomly divided into 19 groups according to the body weight, wherein 10 groups are respectively a blank control group, an alcohol model group, an experimental group (comprising the compounds 1-15 prepared in the embodiments 1-5 of the invention), an tectorigenin control group and a compound glycyrrhizin positive control group. Wherein 10 blank groups are administered with physiological saline in the morning, the dosage of 8mL/kg is given in the first 2 weeks, and the dosage of 12mL/kg is given in the last 4 weeks; the remaining 18 groups (180) were model mice given 50% alcohol daily at noon, 8mL/kg for the first 2 weeks and 12mL/kg for the second 4 weeks.
After molding for 2 weeks (i.e. 4 weeks later), the treatment drug was administered daily in afternoon at a dose of 10mL/kg, wherein the blank control group and the alcohol model group were administered with physiological saline, and the test group (compound groups 1-15), tectorigenin control group and compound glycyrrhizin positive control group were respectively administered with the test drug solution (compound groups 1-15), tectorigenin solution and compound glycyrrhizin solution, and the specific administration modes were as shown in table 1:
table 1 experimental treatment modes of each group
Note that: the gastric lavage time interval between morning and afternoon is about 6 hours.
5. Index detection and method
5.1 observations after dosing
Observation is carried out before daily administration, and mental state, exercise cleaning, hair color, stool and other conditions are carried out.
5.2 Biochemical index
After the last gastric lavage, the patients are fasted and not forbidden for 12 hours, after weighing, isoflurane anesthesia is performed, abdominal aorta blood is taken, whole blood is separated, and a full-automatic biochemical analyzer (AU 480) is used for detecting serum glutamic-oxaloacetic transaminase (AST), alanine Aminotransferase (ALT), triglyceride (TG) and Total Cholesterol (TC); malondialdehyde (MDA) was measured by 10% liver tissue homogenization.
5.3 organ coefficients
After blood sampling is completed, the liver is rapidly dissected and separated, the liver is rinsed by cold normal saline, and the liver coefficient is calculated by weighing after the filter paper is wiped dry.
6. Experimental results
6.1 test results of biochemical indicators and organ coefficients of each group are shown in Table 2.
Table 2 results of biochemical measurements and organ coefficients (n=10, mean±standard deviation)
Group of AST ALT TG TC MDA Liver coefficient
Blank control group 96.5±13.49 47.3±11.75 0.5±0.13 2.0±0.4 1.8±0.21 3.7±0.17
Alcohol model group 198.2±40.2 94.4±19.43 1.3±0.27 4.1±0.9 3.2±0.59 4.5±0.21
Compound 1 102.5±20.51 *** 53.2±10.87 *** 0.7±0.14 *** 2.1±0.46 *** 1.9±0.33 *** 3.8±0.21 ***
Compound 2 126.6±24.02 *** 61.4±12.75 *** 0.8±0.15 *** 2.6±0.58 *** 2.0±0.43 *** 4.0±0.26 ***
Compound 3 134.3±28.1 *** 62.0±13.83 *** 0.8±0.16 *** 2.8±0.64 ** 2.1±0.41 *** 4.0±0.27 ***
Compound 4 147.4±29.69 *** 63.6±14.23 *** 0.8±0.17 *** 2.8±0.54 *** 2.1±0.49 *** 3.9±0.32 ***
Compound 5 151.2±32.22 ** 72.3±14.23 ** 1.0±0.19 * 3.3±0.57 * 2.6±0.56 * 4.2±0.34 *
Compound 6 153.9±30.82 ** 73.2±14.74 * 0.9±0.2 ** 3.1±0.65 ** 2.4±0.5 ** 4.1±0.22 **
Compound 7 157.9±31.8 ** 71.1±15.79 ** 1.0±0.18 * 3.1±0.84 * 2.4±0.45 ** 4.1±0.3 **
Compound 8 141.7±30.14 *** 66.7±14.05 ** 0.9±0.21 ** 2.9±0.6 ** 2.3±0.45 ** 4.0±0.21 ***
Compound 9 149.5±31.6 ** 71.3±14.64 ** 0.9±0.2 ** 3.1±0.53 ** 2.4±0.43 ** 4.1±0.25 **
Compound 10 148.5±28.91 ** 69.9±14.81 ** 0.9±0.19 ** 3.0±0.73 ** 2.5±0.46 ** 4.2±0.26 *
Compound 11 171.0±36.67 * 72.8±15.31 ** 0.8±0.19 ** 3.2±0.65 ** 2.5±0.58 ** 4.0±0.27 **
Compound 12 122.5±24.29 *** 51.6±12.18 *** 0.6±0.13 *** 2.3±0.45 *** 1.9±0.38 *** 3.8±0.22 ***
Compound 13 127.2±31.06 *** 51.5±12.55 *** 0.6±0.16 *** 2.3±0.46 *** 1.8±0.36 *** 3.8±0.25 ***
Compound 14 176.5±36.26 * 77.7±17.88 * 0.9±0.21 * 3.5±0.69 * 2.7±0.48 * 4.2±0.19 *
Compound 15 167.8±32.73 ** 72.0±11.31 ** 0.8±0.23 ** 3.4±0.7 * 2.5±0.56 ** 4.1±0.2 **
Tectorigenin 181.0±41.83 * 78.8±14.93 * 0.9±0.2 * 3.5±0.71 * 2.7±0.51 * 4.1±0.2 *
Positive control group 131.7±32.73 *** 55.8±11.73 *** 0.6±0.16 *** 2.4±0.35 *** 1.9±0.38 *** 3.8±0.33 ***
Note that: * Represents P <0.05 compared to model control group (t-test);
* P <0.01 compared to model control group (t-test);
* P <0.001 (t-test) compared to model control group.
As shown in table 2, each biochemical index observation of the alcohol model group rats: serum glutamic-oxaloacetic transaminase (AST), alanine transaminase (ALT), triglyceride (TG), total Cholesterol (TC), malondialdehyde (MDA) are all significantly increased compared to the blank, and liver index is also significantly increased. The serum glutamic-oxaloacetic transaminase (AST), alanine transaminase (ALT), triglyceride (TG), total Cholesterol (TC), malondialdehyde (MDA) and liver index of the rats in the group 1-15 are obviously different from those in the alcohol model group, so that the improvement effect is improved to different degrees, and the improvement effect is superior to that of tectorigenin (each index of tectorigenin Huang Suzu is P <0.05 compared with that of the model control group); especially, compared with the alcohol model group, the rats of the groups 1, 2, 12 and 13 have better improvement effect compared with P <0.01 or P < 0.001. In addition, the improvement degree of each index of the compounds 12 and 13 is better than that of the compound glycyrrhizin positive control group. The above proves that the 6, 7-position cyclo-isoflavone compound provided by the invention has good activity of treating alcoholic liver injury and has good liver protection effect.
6.2 observations after dosing
6.2.1 observations of results in rats in alcohol model group
Rats in the alcohol model group showed different levels of listlessness; the ingestion and water intake are reduced, and the stool is loose; the haircolor of the rat is not glossy, a little standing condition occurs, and the rat is generally represented by scratching the mouth with the front paws and drunk: the light person walks unstably, acts hard, the heavy person overturns, the reflection disappears, and the individual rats have shortness of breath.
6.2.2 observations of the results from rats of the experimental group
Compared with the alcohol model group, the compound feed has the advantages that the listlessness degree is relieved, the ingestion water intake is reduced compared with the model group, and loose stool is improved; the frequency of the front paw scratching mouth is greatly reduced compared with the model group under the condition of partial hair color and non-gloss, and the drunk person almost has no overturning condition.
The 6, 7-position cyclic isoflavone compound provided by the invention has obvious anti-alcohol effect through observing and comparing the behaviors of rats in an alcohol model group and an experimental group.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims (4)

1. An application of isoflavone compounds and pharmaceutically acceptable salts thereof in preparing medicines for preventing or treating alcoholic liver injury or relieving or protecting liver, wherein the isoflavone compounds have the following structures:
2. the use according to claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier.
3. The use according to claim 1, wherein the medicament is formulated into clinically acceptable tablets, capsules, oral liquid, powder, drop pills, granules or injections by adding conventional excipients according to conventional processes.
4. The use according to claim 1, wherein the alcoholic liver injury comprises: alcoholic fatty liver, alcoholic liver fibrosis, alcoholic hepatitis, and alcoholic cirrhosis.
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