CN112961203B - Flavonol glycoside derivative and its use and preparation method - Google Patents

Flavonol glycoside derivative and its use and preparation method Download PDF

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CN112961203B
CN112961203B CN202110537839.9A CN202110537839A CN112961203B CN 112961203 B CN112961203 B CN 112961203B CN 202110537839 A CN202110537839 A CN 202110537839A CN 112961203 B CN112961203 B CN 112961203B
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chrysosplenoside
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liver injury
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任刚
蒋伟
蔡紫嫣
李文艳
龚德梅
吴昊芬
陈思
孙宁喆
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Jiangxi University of Traditional Chinese Medicine
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    • C—CHEMISTRY; METALLURGY
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    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
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    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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Abstract

The invention belongs to the field of medicines, and particularly relates to a Tibetan medicine longpedunculus2 high methoxylated flavonol glycoside derivatives in waist and new application thereof in preparing medicaments for treating cholestasis type liver injury diseases, and simultaneously discloses a preparation method thereof, wherein the derivatives have the following chemical structures:
Figure 53707DEST_PATH_IMAGE001
. The compound is proved to have active substances for resisting intrahepatic cholestasis type liver injury, and provides a basis for research and development of medicaments for treating intrahepatic cholestasis type liver injury related diseases.

Description

Flavonol glycoside derivative and its use and preparation method
Technical Field
The invention belongs to the field of medicines, and particularly relates to 2 high-methoxylated flavonol glycoside derivatives in tibetan medicine long-peduncle Jinyao, a new application of the derivatives in preparation of a medicine for treating cholestasis type liver injury diseases, and a preparation method of the derivatives.
Background
Intrahepatic Cholestasis (IC), a clinical syndrome of liver disease caused by the accumulation of bile acids in the liver due to structural and functional disorders of hepatocytes or capillary bile ducts. Long-lasting IC will progress to severe consequences such as primary biliary cirrhosis, primary cirrhotic cholangitis, liver cancer, liver failure, etc. Modern medicine considers that any factor causing damage to hepatocytes and cholangiocytes can cause IC, and common causes include viral and bacterial infections, drug damage, autoimmune diseases, alcoholism, and the like. Epidemiological investigation results show that the incidence of IC is increasing year by year in China. Therefore, the search for high-efficiency IC treatment medicines has important significance for guaranteeing the health of people.
Modern medicine mainly takes comprehensive treatment as a main treatment for treating IC which lacks specificity and has high curative effect and small side effect. The clinical commonly used medicines mainly comprise ursodeoxycholic acid, glucocorticoid, S-adenosylmethionine and the like. Ursodeoxycholic acid can obviously improve cholestasis, but has high price and slow curative effect, and partial patients have poor response to the cholestasis. Glucocorticoids (such as dexamethasone and the like) are considered as one of traditional medicines for treating acute IC, but rebound phenomenon is easy to generate after the administration, and serious adverse reaction is generated after the long-term administration of the glucocorticoids, so that the glucocorticoids cannot be used for a long time for treating severe IC. Generally, the treatment of IC in western medicine is mainly to improve symptoms and control related complications, no specific medicine is available in clinic, few medicines can be selected, and the long treatment process, the expensive medical cost and adverse reactions accompanied by the treatment process cause serious physical and psychological burden to patients. Therefore, the development of new drugs for the treatment of IC is of great interest.
Disclosure of Invention
Technical problem to be solved
In view of the defects and shortcomings of the prior art, the invention provides the high methoxylated flavonol glycoside derivative, and simultaneously discloses the application and the preparation method thereof, the derivative has the activity of resisting IC type liver injury, and can provide a new medicine research and development direction for the research and development of medicines for treating diseases related to IC type liver injury.
(II) technical scheme
In order to achieve the purpose, the invention adopts the main technical scheme that:
in a first aspect, the present invention provides a highly methoxylated flavonol glycoside derivative having the chemical structure shown below:
Figure 110891DEST_PATH_IMAGE001
in a second aspect, the present invention relates to the use of a highly methoxylated flavonol glycoside derivative having a chemical structure represented by the following formula (I) or formula (II) for the preparation of a medicament for the treatment of cholestatic liver injury diseases:
Figure 525692DEST_PATH_IMAGE002
formula (I) formula (II)
Preferably, the medicament is prepared by mixing the compound with the structure shown in the formula (I) or the formula (II) and pharmaceutic adjuvants to prepare a pharmaceutically acceptable preparation.
Preferably, the cholestatic liver injury disease is any one or more of primary biliary cirrhosis, primary cirrhotic cholangitis, viral hepatitis, alcoholic and pharmaceutical liver injury, and cholestasis during pregnancy, as described above.
Preferably, the compound with the structure shown in the formula (I) or the formula (II) is extracted and separated from the whole plant of the longstem chrysosporium.
In a third aspect, the present invention also relates to a method for preparing a highly methoxylated flavonol glycoside derivative, wherein the highly methoxylated flavonol glycoside derivative is a compound having a structure represented by the formula (I) or the formula (II), and the preparation method comprises the following steps:
s1, carrying out leakage extraction on the whole plant of the longstem chrysosplenium by using ethanol with the volume concentration of 60-80% (preferably 70%) at room temperature, and carrying out reduced pressure concentration to obtain an extract;
s2, adding a dispersing agent into the extract obtained in the step S1 for dispersing, wherein the dispersing agent is 0-10% of ethanol, and the adding proportion is that the extract: dispersing agent 2-3:1-4, wherein the weight of the extract is kilogram, and the weight of the dispersing agent is liter, so as to obtain dispersing liquid;
s3, passing the dispersion liquid obtained in the step S2 through a macroporous adsorption resin column, and performing gradient elution by using distilled water, 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol and 95% ethanol in sequence, wherein each gradient elution is performed for 3-6 (preferably 5) column volumes, so that 10 fractions frs.H0-H9 are obtained;
s4, adding hot methanol into the 40% ethanol elution fraction fr.H5 obtained in the step S3, kneading and dissolving, and performing suction filtration to obtain a methanol insoluble part fr.H5-1 and a methanol soluble part fr.H5-2;
s5, subjecting fr.H5-1 to silica gel column chromatography, and eluting with a dichloromethane-ethanol solvent system 16:1 to obtain 7 fractions fr.H5-1S 1-H5-1S 7; the fr.H5-1S3 fraction is separated by preparative chromatography to obtain a compound with a structure shown in a formula (II);
S6, further loading the methanol soluble part fr.H5-2 obtained in the step S4 on a Sephadex LH-20 column, and eluting by 90% methanol to obtain 8 fractions fr.H5-2L 1-H5-2L 8; wherein, the compound with the structure shown in the formula (I) is obtained by separating H5-2L4 fraction through preparative chromatography.
Preferably, in step S3, the macroporous adsorbent resin column is an HP-20 macroporous adsorbent resin column or a D101 macroporous adsorbent resin column.
Preferably, in steps S5-S6, the preparative chromatography is reversed-phase ODS HPLC preparative chromatography.
The compound having the structure represented by the above formula (I) is named chrysosplenoside I, and the compound having the structure represented by the formula (II) is named chrysosplenoside a, and these names are described below.
(III) advantageous effects
The invention has the beneficial effects that:
the invention separates and identifies the main chemical components in the long-pedunculus chrysosplenium whole grass, screens out the active substance with the function of resisting intrahepatic cholestasis type liver injury, and provides a foundation for the research and development of the medicine for treating intrahepatic cholestasis type liver injury related diseases.
The isolates of the whole herb of longstemma longirosthornii were evaluated by acute toxicity classification method:
acute toxicity of chrysosplenoside I and chrysosplenoside A to mice given a single oral dose. On this basis, the activity of two test monomers (chrysosplenoside I and chrysosplenoside A) against IC-type liver injury was evaluated using an alpha-naphthalene isothiocyanate (ANIT) induced Intrahepatic Cholestasis (IC) -type liver injury model in mice. The results show that: both the chrysosplenoside I and chrysosplenoside A have LD50 (half lethal dose) of 4-5g/kg, and belong to low-toxicity compounds, but the chrysosplenoside I has certain hepatotoxicity when taken in large dose, and the toxicity of the chrysosplenoside I is lower than that of chrysosplenoside A; secondly, the two tested monomers can obviously reduce the serum biochemical index level of the acute IC type liver injury model mouse caused by ANIT under the conditions of low, medium and high (30 mg/kg, 60 mg/kg and 120 mg/kg) dose of gastric perfusion administration, inhibit the in-vivo oxidative stress and lipid peroxidation of the mouse and obviously improve the liver injury state of the model mouse.
It is worth mentioning that the effect of the two tested monomers on resisting the IC liver injury of the mice caused by ANIT under the condition of medium dose (60 mg/kg) is better than that of ursodeoxycholic acid (UDCA, 100 mg/kg) which is a first-line medicament for clinically treating the IC liver injury diseases on the whole, which indicates that the chrysosplenoside I and chrysosplenoside A have the potential of being further developed into medicaments for treating the IC liver injury, particularly chrysosplenoside I with lower toxicity.
Drawings
FIG. 1 is a structural formula of chrysosplenoside I;
FIG. 2 is a structural formula of chrysosplenoside A;
FIG. 3 is a HPLC fingerprint of an extract of chrysosplenoside, wherein 1 and 2 are fingerprints of chrysosplenoside I and chrysosplenoside A, respectively; :
FIG. 4 shows a hydrogen NMR spectrum of chrysosplenoside I (1H NMR)。
FIG. 5 is a nuclear magnetic resonance carbon spectrum of chrysosplenoside I (C:)13C NMR)。
FIG. 6 is the primary HMBC (H → C) correlation of chrysosplenoside I.
Detailed Description
Example 1 separation of major components in Long-stemmed Golike waist
The embodiment is a method for separating main components from long-peduncle golden waist, which comprises the following steps:
(1) taking 6.0 kg of dried longstem chrysosplenium whole plant, appropriately crushing, placing in a percolation barrel, adding 70% ethanol for percolation extraction, combining extracting solutions, concentrating under reduced pressure and recovering a solvent to obtain 2.5 kg of extract. Dissolving a small amount of the extract with 70% methanol, filtering with 0.45 μ M filter membrane, and injecting 10 μ L of the obtained filtrate into High Performance Liquid Chromatograph (HPLC) to obtain HPLC fingerprint of 70% ethanol extract of radix seu caulis Henryi (figure 3). As shown in fig. 1, 2, and 3, the main components thereof are compound 1 and compound 2.
(2) 2.4 kg of extract is taken, heated and kneaded by 500 mL of 10% ethanol, and then 500 mL of distilled water is added for dispersion.
(3) Passing the dispersion through HP-20 macroporous adsorbent resin column, gradient eluting with distilled water, 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, and 95% ethanol, and eluting 5 column volumes per gradient to obtain 10 fractions frs.H0-H9.
(4) By HPLC analysis, target compounds 1 and 2 were mainly concentrated at 40% ethanol elution site fr.h 5. Adding fr.H5 (92.8 g) into hot methanol, kneading, and vacuum filtering to obtain methanol insoluble part fr.H5-1 and methanol soluble part fr.H5-2.
(5) Subjecting fr.H5-1 (32.4 g) to silica gel column chromatography, and eluting with dichloromethane-ethanol solvent system 16:1 to obtain 7 fractions fr.H5-1S 1-H5-1S 7. Wherein fr.H5-1S3 fraction was subjected to reversed-phase ODS HPLC preparative chromatography (5 μm, i.d.20 mm. times.250 mm, YMC Co., Ltd., mobile phase 30% acetonitrile) to repeatedly prepare Compound 2(12.5 g,t R 19 min)。
(6) and (3) taking fr.H5-2 (38.4 g) and further loading the fr.H5-2 (38.4 g) on a Sephadex LH-20 column, and eluting by 90% methanol to obtain 8 fractions of frs.H5-2L 1-H5-2L 8. Wherein the H5-2L4 fraction was repeatedly subjected to reversed-phase ODS HPLC preparative chromatography (5 μm, i.d.20 mm. times.250 mm, YMC Co., Ltd., mobile phase 30% acetonitrile) to obtain Compound 1(14.8 g, t R 14 min)。
Example 2 this example is a structural characterization of compound 1 and compound 2 above, including two parts:
(1) structural characterization of Compound 1
Compound 1 is a yellowish brown amorphous powder, readily soluble in methanol. HR-ESI-MS gives the peak of the excimer ionm/ z: 553.1545([M+H]+，C25H29O14Theoretical calculation value of 553.1552), and the molecular formula is determined to be C25H28O14。
Nuclear Magnetic Resonance (NMR) hydrogen spectrum of compound 1: (1H) [ as shown in FIG. 2 ] and carbon spectrum (C [)13C) [ FIG. 3 ] the data are very similar to a known pentamethoxyflavonol glycoside chrysosplenoside H. With chrysosplenoside H1Compared with the H NMR spectrum, the compound 1 has 1 more phenolic hydroxyl proton signal in the low field regionδ H9.00 (1H, brs) signal with one less methoxy group in the high fieldδ H3.93 (3H, s); with chrysosplenoside H13Compound 1 has a signal of one methoxy group less than that of C NMR spectrumδ C56.9. This suggests that compound 1 is a demethylated derivative of chrysosplenoside H. Analysis of the HSQC, HMBC NMR spectrum of compound 1, with a global assignment of all its hydrogen and carbon signals, supports this hypothesis. In addition, in the HMBC NMR spectrum of Compound 1, the terminal glucose proton signalδ H 4.77 (1H, d, J = 7.8 Hz, H-1 '') with aromatic carbon signalsδ C149.0 (C-2 ') has a long-range correlation demonstrating that the glucosyl group is attached at the 2' position of the B-ring; the type of oxidative substitution of rings a and B and the attachment position of the four methoxy groups are determined by the HMBC correlation shown in fig. 4. Thus, Compound 1 was finally identified as 5,2 ', 5 ' -trihydroxy-3, 6,7,4 ' -tetramethoxyflavonol-2- O-βD-glucoside, designated here as chrysosplenoside I. The following table shows the NMR data (DMSO-) for Compound 1d 6Making a music score)
Table 1:
Figure 264978DEST_PATH_IMAGE004
through the above identification procedure, the structural formula of compound 1 was determined as follows:
Figure 234071DEST_PATH_IMAGE005
。
(2) structural characterization of Compound 2
Compound 2 is a yellowish brown amorphous powder, insoluble in methanolAnd can be dissolved in DMSO. HR-ESI-MS gives the peak of the excimer ionm/z：523.1434 ([M+H]+, C24H27O13Theoretical calculation value of 523.1452), and the molecular formula is determined to be C24H26O13. The nmr data are as follows:1H NMR (600 MHz, DMSO-d 6) δ H: 12.69 (1 H, s, HO-5), 8.96 (1 H, s, HO-5′), 6.98 (1 H, s, H-3′), 6.83 (1 H, s, H-6′), 6.58 (1 H, d, J = 2.2 Hz, H-8), 6.35 (1 H, d, J = 2.2 Hz, H-6), 3.81 (6 H, s, MeO-7,4'), 3.70 (3 H, s, MeO-3), 4.74 (1 H, d, J = 7.8 Hz, H-1’’), 3.07-3.11 (1 H, m, H-2’’), 3.17-3.21 (1 H, m, H-3’’), 3.02-3.06 (1 H, m, H-4’’), 3.38-3.42 (1 H, m, H-5’’), 3.42 (1 H, m, Ha-6’’), 3.73 (1 H, m, Hb-6’’); 13C NMR (150 MHz，DMSO-d 6) δ C: 156.4 (C-2), 139.3 (C-3), 178.4 (C-4), 161.1 (C-5), 97.6 (C-6), 164.9 (C-7), 92.4 (C-8), 157.0 (C-9), 105.9 (C-10), 111.2 (C-1′), 148.9 (C-2′), 101.8 (C-3′), 150.3 (C-4′), 140.9 (C-5′), 115.9 (C-6′), 102.1 (C-1’’), 73.1 (C-2’’), 76.7 (C-3’’), 70.1 (C-4’’), 77.3 (C-5’’), 60.9 (C-6’’)，60.0 (MeO-3), 56.1 (MeO-7), 56.6 (MeO-4′)。
by comparison, compound 2 was identified as the following structural formula and was named chrysosplenoside a.
Figure 451425DEST_PATH_IMAGE006
。
Example 3
This example relates to the acute toxicity evaluation of Chrysosplenoside a and Chrysosplenoside I by the following methods: according to the technical guide principle of chemical Drug acute toxicity test and the guide principle of acute toxicity grading method issued by China Food and Drug Administration (CFDA), 2g/kg is used as the initial Administration dose of chrysosplenoside I and chrysosplenoside A, and the mice are subjected to one-time oral gavage for acute toxicity test.
Animal groups and dosing regimens were as follows: randomly selecting 40 mice, each half of the mice is male and female, and marking the mice with 3% picric acid solution; the experiment is divided into 4 groups, each group is 5 male and female, and the groups are respectively a blank control group, a solvent control group, a chrysosplenoside I administration group and a chrysosplenoside A administration group; ultrasonically dissolving the tested medicine by using 0.5 percent CMC-Na solution, wherein the administration dose is 2g/kg, and carrying out single intragastric administration; the solvent control group is perfused with a 0.5% CMC-Na solution with the same volume as the perfusate, the blank control group is perfused with an aqueous solution with the same volume as the perfusate, the perfusion volume is 40mL/kg, and the single perfusion is carried out; fasting is carried out for 12 h before administration, and water is not forbidden.
The results are as follows:
(1) effect of Chrysosplenoside I and Chrysosplenoside A on the health status of mice
After single intragastric administration, compared with mice in a blank control group and a solvent control group, the mice in the chrysosplenoside I administration group have the phenomena of eye closure, tachypnea, accelerated heartbeat, weakened activity and the like, after 5 min, the symptoms are obviously relieved, after 10 min, the symptoms basically disappear, and the activity behaviors of the mice are normal; in 2 min after administration of the chrysosplenoside A administration group mice, symptoms such as slow action, accelerated heartbeat, weakened external stimulation and the like appear, and the symptoms of female mice are more obvious; after 5 min of administration, the heartbeat of the mice in the chrysosplenoside A administration group is gradually gentle, and the response time to external stimulation is shortened; after 10 min, the symptoms basically disappear, and the mouse gradually moves normally and climbs in a mouse cage. After 2 h after the gavage, the mice of each group recovered to eat, and the diet condition was not greatly different. In the observation period, the animals have normal behavior, food intake, drinking and respiration, have no obvious difference compared with a blank control group, and have no toxic reaction and death phenomenon. Compared with a blank control group, the observation indexes of the mice in the solvent group have no significant difference. Indicating that chrysosplenoside I and chrysosplenoside A belong to the same low toxicity compounds.
(2) Effect of Chrysosplenoside I and Chrysosplenoside A on mouse body Mass
Statistics were made on the body mass of each group of mice, and the results are shown in table 2. The body mass of each group of mice before gastric lavage has no significant differenceAll mice did not die within 14 days after single intragastric administration, and the growth trend was substantially consistent with that of blank and vehicle controls and gradually increased. The body mass of the Chrysosplenoside I and Chrysosplenoside A administration group within administration 14 d was not significantly different from that of the blank and vehicle control group ((ii) ((iii)P >0.05), indicating that both chrysosplenoside I and chrysosplenoside A had no significant effect on the growth of mice when administered intragastrically at a dose of 2g/kg (Table 2).
Table 2: effect of Chrysosplenoside I and Chrysosplenoside A on mouse body Mass
Figure 720733DEST_PATH_IMAGE008
(3) Effect of Chrysosplenoside I and Chrysosplenoside A on mouse organ index
14 d after administration, taking blood, removing neck, killing the mouse, dissecting, observing pathological changes of main organs of the mouse such as liver, heart, spleen, lung, kidney and the like with naked eyes, weighing the wet weight of each organ, and calculating the index of each organ. The organ index calculation formula is as follows: organ index = organ mass/animal mass × 100%. The texture and color of each organ of mice in the Chrysosplenoside I and Chrysosplenoside A administration groups are not obviously different from those of mice in the blank and solvent control groups, and have no obvious toxic reaction and no obvious difference in organ coefficient of each tissue (A) P >0.05). The data are shown in Table 3.
Table 3: influence of Chrysosplenoside I and Chrysosplenoside A on mouse organ index
Figure 630920DEST_PATH_IMAGE010
(4) Effect of Chrysosplenoside I and Chrysosplenoside A on mouse liver and Kidney function indices
Compared with the vehicle control group, the serum ALT, AST, ALP, TP, ALB and BUN levels of male mice in the chrysosplenoside I administration group have no significant change and have no statistical significance (P >0.05); female mouse serum ALT, TP, ALB, BUN waterNo significant change in average: (P >0.05); there was no significant change in ALP, TP, ALB, BUN levels in the chysosplenoside A-administered group mice: (P >0.05), mice in the male administration group had significant changes in AST levels (P <0.01). Compared with the mice of the blank control group, the water average of the mice of the same sex of the vehicle control group ALT, ALP, TP, ALB and BUN has no significant change ((P >0.05). In addition, due to individual differences, serum AST of individual mice in the group administered female chrysosplenoside I and chrysosplenoside A mice was not statistically significant, although there was a clear difference between the groupsP >0.05). The data are shown in Table 4.
TABLE 4 influence of Chrysosplenoside I and Chrysosplenoside A on mouse serum marker levels
Figure 87309DEST_PATH_IMAGE012
Example 4
This example investigates the protective effects of Chrysosplenoside I and Chrysosplenoside a on ANIT-induced mouse IC-type liver injury according to the following protocol:
90 male Kunming mice were randomly divided into the following 9 groups: a control group, a model group, a positive drug group (ursodeoxycholic acid UDCA, first-line drug for clinical treatment of IC-type liver injury), a chrysosplenoside I low dose group (30 mg/kg), a chrysosplenoside I medium dose group (60mg/kg), a chrysosplenoside I high dose group (120 mg/kg), a chrysosplenoside A low dose group (30 mg/kg), a chrysosplenoside A medium dose group (60mg/kg), and a chrysosplenoside A high dose group (120 mg/kg).
Dosing regimen: chrysosplenoside I, Chrysosplenoside A and UDCA were suspended in a 0.5% CMC-Na solution. Each group was perfused continuously for 7 days, 1 time per day, with a perfusion volume of 15 mL/kg, a blank group and a model group perfused with 0.5% CMC-Na solution of the same volume, and the rest groups were perfused with corresponding solutions. 1 h after the 5 th day of intragastric administration, except the blank group of the olive oil with the same volume of intragastric administration, the other groups of the olive oil solution with 60mg/kg of ANIT are intragastric administered for molding. And (3) after 48 h, taking blood from the abdominal aorta of the mouse, standing the whole blood for 1 h, centrifuging at 3500 r/min for 10 min at 4 ℃, sucking the supernatant into an EP (EP) tube, storing in a refrigerator at 4 ℃, and detecting. Dissecting mouse, rapidly separating mouse liver on ice bench, selecting liver tissue of the same part, and storing in refrigerator at-80 deg.C for preparing liver tissue homogenate.
The kit is used for detecting biochemical index components of serum and tissue homogenate, and the result is as follows:
(1) influence of test compound on serum biochemical indexes of mice with acute IC (acute liver injury) caused by ANIT (ananas disease)
Compared with a control group, the levels of serum ALT, AST, ALP, TBA, DBIL and TBIL of the model group mice are obviously increased (P<0.01), indicating that the molding is successful; the UDCA group and the test compound-containing groups showed a reduction in serum ALT, AST, ALP, TBA, DBIL, TBIL levels in mice compared with the model group (P<0.01 orP<0.05) and has obvious dose dependence, which shows that both chrysosplenoside I and chrysosplenoside A have obvious protective effect on mice with acute IC liver injury caused by ANIT. It is worth mentioning that the medium (60 mg/kg) and high (120 mg/kg) dose groups of the two test compounds have overall better improvement effect on serum biochemical indicators than the positive drug UDCA group (100 mg/kg). The specific data are shown in tables 5 and 6.
Table 5: effect of test Compounds on serum ALT, AST and ALP levels in mice with ANIT-induced acute IC-type liver injury
Figure 394442DEST_PATH_IMAGE014
TABLE 6 Effect of test Compounds on serum TBA, DBIL and TBIL levels in mice with ANIT-induced acute IC-type liver injury
Figure 987097DEST_PATH_IMAGE016
As shown in tables 5-6, compared with the model group, the two tested compounds can obviously reduce the serum biochemical index level of the acute IC type liver injury model mouse caused by ANIT under the conditions of low, medium and high (30 mg/kg, 60 mg/kg and 120 mg/kg) dose gastric lavage administration, inhibit the oxidative stress and lipid peroxidation in vivo and obviously improve the liver injury state of the model mouse. Compared with the UDCA (ursodeoxycholic acid) group, the tested two compounds have better effect on resisting IC liver injury of mice caused by ANIT under the condition of medium dose (60 mg/kg).
(2) Influence on levels of SOD, MDA and GSH-Px in liver homogenate of mice with acute IC (acute liver injury) caused by ANIT (animal and animal information)
Compared with the control group, the contents of SOD and GSH-Px in the liver tissue homogenate of the model group mice are obviously reduced (P <0.01), the MDA content is obviously increased (P <0.01), indicating that the molding is successful; compared with the model group, the content levels of SOD and GSH-Px in liver homogenates of mice in the UDCA group and the test compound groups with different dosages are obviously increased (P <0.01 orP <0.05) and the MDA content level are all obviously reduced (P <0.01 orP <0.05) and there was some dose dependence (table 7). These results suggest that chrysosplenoside I and chrysosplenoside A may improve the liver damage state of mice induced by ANIT by scavenging free radicals in vivo and inhibiting lipid peroxidation.
Table 7: effect of tested compounds on liver tissue homogenate SOD, MDA and GSH-Px of mice with ANIT-induced acute liver injury
Figure 68185DEST_PATH_IMAGE018
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (8)

1. A highly methoxylated flavonol glycoside derivative, characterized in that it has the following chemical structure:
Figure DEST_PATH_IMAGE002
。
2. the application of the high methoxylated flavonol glycoside derivative in preparing the medicament for treating cholestatic liver injury diseases is characterized in that the high methoxylated flavonol glycoside derivative has a chemical structure shown as the following formula (I):
Figure DEST_PATH_IMAGE004
。
3. the use of claim 2, wherein the medicament is a pharmaceutically acceptable formulation prepared by mixing a compound having a structure represented by formula (I) with a pharmaceutically acceptable adjuvant.
4. The use according to claim 2 or 3, wherein the cholestatic liver injury disease is any one or more of primary biliary cirrhosis, primary cirrhotic cholangitis, viral hepatitis, alcoholic and pharmaceutical liver injury, and cholestasis during pregnancy.
5. The use according to claim 2, wherein the compound of formula (I) is isolated from the whole plant of the long-stemmed euonymus japonicus.
6. A preparation method of a highly methoxylated flavonol glycoside derivative is disclosed, wherein the highly methoxylated flavonol glycoside derivative is a compound with a structure shown in a formula (I) or a formula (II),
Figure DEST_PATH_IMAGE006
The preparation method is characterized by comprising the following steps:
s1, carrying out leakage extraction on the whole plant of the longstem chrysosplenium by using ethanol with the volume concentration of 60-80% at room temperature, and carrying out reduced pressure concentration to obtain an extract;
s2, adding a dispersing agent into the extract obtained in the step S1 for dispersing, wherein the dispersing agent is 0-10% of ethanol, and the adding proportion is that the extract: dispersing agent 2-3:1-4, wherein the weight of the extract is kilogram, and the weight of the dispersing agent is liter, so as to obtain dispersing liquid;
s3, putting the dispersion liquid obtained in the step S2 on a macroporous adsorption resin column, and performing gradient elution by using distilled water, 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol and 95% ethanol in sequence, wherein each gradient elution is 3-6 column volumes, so as to obtain 10 fractions fr.H0-H9;
s4, adding hot methanol into the 40% ethanol elution fraction fr.H4 obtained in the step S3, kneading and dissolving, and performing suction filtration to obtain a methanol insoluble part fr.H4-1 and a methanol soluble part fr.H4-2;
s5, subjecting fr.H4-1 to silica gel column chromatography, and eluting with a dichloromethane-ethanol solvent system 16:1 to obtain 7 fractions fr.H4-1S 1-H4-1S 7; the fr.H4-1S3 fraction is separated by preparative chromatography to obtain a compound with a structure shown in a formula (II);
s6, further loading the methanol soluble part fr.H4-2 obtained in the step S4 on a Sephadex LH-20 column, and eluting by 90% methanol to obtain 8 fractions fr.H4-2L 1-H4-2L 8; the fr.H4-2L4 fraction is separated by preparative chromatography to obtain the compound with the structure shown in the formula (I).
7. The method according to claim 6, wherein in step S3, the macroporous adsorbent resin column is HP-20 macroporous adsorbent resin column or D101 macroporous adsorbent resin column.
8. The production method according to claim 6, wherein in the steps S5-S6, the preparative chromatography is reversed-phase ODS HPLC preparative chromatography.
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