CN108030781A - Purposes of the Isomperatorin in drug induced hepatic injury protection medicine is prepared - Google Patents

Abstract

The present invention relates to separation, a kind of natural furocoumarin constituents (Isomperatorin) prepared and its application in drug induced hepatic injury protection medicine and health products are prepared from Chinese medicine (such as notopterygium root, the root of Dahurain angelica).By inside and outside experimental study, find that Isomperatorin monomeric compound has antiradiation drug hepatic injury pharmacological activity first.There is chemical composition of the present invention good antiradiation drug hepatic injury to act on, and be conducive to carry out the research and development of novel medicine physical property liver injury protection medicine and health products.

Description

Purposes of the Isomperatorin in drug induced hepatic injury protection medicine is prepared

Technical field

The present invention relates to drug field, and in particular to a kind of effective monomer compound is preparing Drug liver in the Chinese medicine root of purple-flowered peucedanum Application in injury protection medicine.

Background technology

Liver is maximum glandula digestive in human body, dominates human metabolism, is " chemical plant " huge in human body.Hepatic injury It is a kind of pathological state that a variety of liver diseases share, is mainly shown as that necrosis of liver cells or apoptosis, degeneration of liver cells, liver are thin Born of the same parents' steatosis, silt courage infringement and inflammatory reaction etc..Long-term hepatic injury is to cause liver fibrosis, or even hepatic sclerosis, liver cancer hair It is raw important to make reason plain.

In recent years increase sharply with clinical application species and patient's voluntarily take medicine or arbitrarily increase probability of drug dose increases Add, drug induced hepatic injury incidence also accordingly increases, and has more than 900 kinds medicines (such as carbon tetrachloride, bromobenzene, fluothane, treating tuberculosis Medicine, antibiotic, non-steroidal anti-inflammatory drugs etc.) it can clearly cause drug induced hepatic injury.Hepatic injury especially drug induced hepatic injury is tight Human health and life have been threatened again, and protection liver health has been a very urgent task of the pendulum in face of us.

Modern medicine has no the specific drug for the treatment of hepatic injury, it is more using rest, adjust diet, replenishing vitamins and right Disease treatment (selection antiviral drugs, immunoregulation medicament, protecting liver, lowering enzymes medicine), severe patient need to be forced to terminate medication, Yi Mianjia Hepatic injury again.Current application Chinese herbal medicine and its preparation for treating liver diseases are fairly common in China, modern pharmacological studies have shown that Many Chinese medicines (including compound, single medicinal material and monomer component) can effectively treat or improve liver damage disease, and Chinese medicine then because Liver protection curative effect affirms that toxic side effect is smaller, and multicomponent, Mutiple Targets work and increasingly enjoy favor.

The perfume (or spice) being widely present in Chinese medicine (such as notopterygium root, the root of Dahurain angelica, the root of purple-flowered peucedanum, frutus cnidii, the bark of ash, psoralea corylifolia, Radix Glehniae, fingered citron) Legumin is a kind of compound for having benzene a pair of horses going side by side α-pyranone structure, is considered as the lactone of o-hydroxy cinnamic acid, is widely present in In the higher plants such as Rutaceae, Umbelliferae, composite family, pulse family, Thymelaeceae, Solanaceae.According to ring substituents and its position not Together, cumarin is often divided into simple cumarin (such as umbelliferone, aesculetin, Osthole, dephnetin), furans tonka-bean Plain (such as Imperatorin, Isomperatorin, psoralen, 8-methoxypsoralen), pyranocoumarin (such as praeroside, Xanthoxyletin, decursin) and other cumarins (such as armillarisin A, bisaesculetin).Research shows, cumarin Class compound has AntiHIV1 RT activity, anticancer, decompression, anti-arrhythmia, anti-osteoporosis, eases pain, relievings asthma and antibacterial etc. is looked unfamiliar in many ways Thing activity (CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2005,30 (6)：410-414；Chinese Journal of New Drugs, 2013,22 (20)： 2392- 2404).Separately there are some researches prove some coumarin kind compounds also have liver-protecting activity, including (herbal pharmacology is with facing for Osthole Bed, 2006,22 (2)：21-22), phellopterin (Arch Pharm Res, 1993,16 (1)：13-17), 8- methoxyl groups Psoralen Fat element (Chinese Journal of Modern Applied Pharmacy, 2012,29 (8)：682-686)；, it was found that Praeruptorin B has anti-hyperlipidemia, height Active (the application for a patent for invention number of blood glucose, nonalcoholic fatty liver and diabetes B：201710046028.2), in Radix Codonopsis root skin 5 kinds of furocoumarin compounds, which have, suppresses hepatoma cell proliferation active (Chinese experimental pharmacology of traditional Chinese medical formulae magazine, 2012,18 (6)： 203-205), and Imperatorin and Isomperatorin have inhibitory activity (Chinese medicine to cytochromes P450 enzyme Magazine, 2013,38 (8)：1237-1241).

However, so far, the pharmacological action of Isomperatorin confrontation drug induced hepatic injury has not yet to see report.

The content of the invention

The object of the present invention is to provide application of the effective component of chinese medicine in drug induced hepatic injury protection medicine is prepared.

The compound that the present invention is studied is related to separation in Chinese medicine (such as notopterygium root, the root of Dahurain angelica), a kind of furans tonka-bean being prepared Plain constituents, its chemical constitution is as follows, can separate and be prepared from natural drug, can be also prepared by chemical synthesis.

The present invention is by investigating compound to carbon tetrachloride (CCl₄) damage suckling mouse primary hepatocyte survival rate, alanine Aminopherase (ALT), aspartate aminotransferase (AST), the shadow of malonaldehyde (MDA) and superoxide dismutase (SOD) Ring, find that Isomperatorin is acted on antiradiation drug hepatic injury first.

The present invention is by investigating compound to CCl₄ALT, AST content, liver morphology, liver in the mice serum of damage The influence of middle liver index, MDA, SOD and glutathione (GSH), and to MDA, atpase activity and film in liver cell mitochondria The influence of current potential, finds that Isomperatorin is acted on antiradiation drug hepatic injury first.

The present invention has found the antiradiation drug hepatic injury of a kind of monomer component in Chinese medicine (such as notopterygium root, the root of Dahurain angelica) by pharmacological evaluation Pharmacological activity, can be used to prepare medicine and health products with respective action, be conducive to carry out novel medicine physical property liver injury protection The research and development of medicine and health products.

The medicine of the present invention can be any pharmaceutically useful formulation, including tablet, capsule, oral liquid, syrup, particle Agent, pill, powder, paste, sublimed preparation, injection, suppository, creme, spray, pill, patch, sustained release preparation and controlled release system Agent etc..

The form of oral liquid can be water-based or oily solution, suspension, emulsion, syrup or elixir, it can Containing conventional additive, including solvent is such as water, ethanol, suspending agents such as sorbierite, syrup, methylcellulose, bright Glue, hydroxyethyl cellulose etc., emulsifying agent is such as lecithin, anhydro sorbitol monooleate, Arabic gum, and preservative is for example Para hydroxybenzene methyl esters, propylparaben, sorbic acid etc..

Oral solid formulation can contain common excipient, such as adhesive, filler, diluent, lubricant, disintegration Agent, colouring agent, flavor enhancement and wetting agent, can be coated applicable filler to tablet if necessary includes cellulose, mannose Alcohol, lactose and other similar fillers.Suitable disintegrant includes starch, polyvinylpyrrolidone and starch derivatives, example Such as sodium starch glycollate.Suitable lubricant includes magnesium stearate.Suitable wetting agent includes lauryl sodium sulfate.

Brief description of the drawings

The tested monomeric compounds of Fig. 1 are to CCl₄Damage the influence of murine liver tissue pathological change

Normal group (A), model group (B), legalon group (C), Isomperatorin 8mg/kg groups (D), Isomperatorin The mouse liver pathological change (× 200) of 16mg/kg groups (E), Isomperatorin 32mg/kg groups (F)

Normal group hepatic tissue is clear in structure as it can be seen that liver cell aligned orderly, in the same size, and hepatic cell cords is in obvious radiation Shape, portal area is without lesions such as denaturation, necrosis and inflammatory cell infiltrations；The obvious oedema of model group mouse liver cell, balloon sample become, Liver rope is disorganized, and liver cell spotty necrosis, liver parenchyma has more cell infiltration；Legalon group hepatic tissue is visible scorching thin Born of the same parents' infiltration substantially mitigates compared with model group, and liver cell is without significant change；Each dosage group of Isomperatorin has different degrees of inflammation carefully Born of the same parents infiltrate, but substantially mitigate compared with model group.

Embodiment

It can be used for the substantive content for preparing drug induced hepatic injury protection medicine and health products in order to preferably explain the present invention, Further explained below by way of specific experiment embodiment.Following embodiments are illustrative, the present invention are not done Any restriction.

Embodiment 1：Influence of the tested monomeric compound to the suckling mouse primary hepatocyte of carbon tetrachloride-injured

1 material, reagent and instrument

1.1 main materials and reagent

Test compound controls product (Isomperatorin, legalon) are purchased from Chengdu Puffy moral Bioisystech Co., Ltd； L-02 cells (Shaanxi Mai Yuan bio tech ltd)；DMEM/F12 culture mediums (Gibco companies of the U.S.)；Hyclone is (beautiful Gibco companies of state)；CCK-8 cell proliferation detecting kits (Suo Laibao bio tech ltd)；Carbon tetrachloride (CCl₄) (Sigma)；Mycillin is dual anti-(Gibco companies of the U.S.)；PBS (Suo Laibao bio tech ltd)；96 hole cell culture Plate (Thermo companies)；0.22um filter membranes (Corning)；Blood counting chamber (Zhengjiang City Dantu Kodak medical supplies factory)；ALT、 AST, SOD, MDA detection kit (Bioengineering Research Institute is built up in Nanjing).

1.2 key instrument

5%CO₂Incubator (Thermo companies)；Desk centrifuge (Changsha Xiang Yi centrifuges Instrument Ltd.)；Biology Safety cabinet (Purifying Equipment Co., Ltd., Suzhou)；Microplate reader (TECAN)；- 80 DEG C of refrigerators (Thermo companies).

2 methods

2.1 given the test agent solution are prepared

Take each Test compound controls product to be dissolved in DMSO in right amount, take the given the test agent that DMSO dissolves to be arrived with normal saline dilution Required concentration.

2.2 modelings and administration

Plating cells：Cell, which is resuspended, makes its concentration be 1 × 10⁵A/mL, 10000 cells (100 μ L), cell are spread per hole Put 3 repetitions, 37 DEG C of 5%CO₂After cultivating 12h, each experimental drug handles 24h.Final concentration of 10mmol/L is then added per hole CCl₄, put 37 DEG C of 5%CO₂Cultivate 6h.

Experiment packet includes：Blank group (containing only culture medium)；Normal group；Solvent control group (1 ‰ DMSO)；Solvent control group +CCl₄(model group)；Legalon group (60 μm of ol/mL of concentration)+CCl₄(positive controls)；Isomperatorin (80 μ of concentration mol/mL、160μmol/mL、320μmol/mL)+CCl₄。

2.3 cell survival rates measure (CCK-8 methods)

100 μ L/ holes serum free mediums are replaced, add 10 μ L/ holes CCK-8 stostes, 5%CO₂2h is incubated in incubator altogether, Absorbance is measured at 450nm.

2.4ALT, AST, MDA, SOD assay

It is measured by the specific steps of kit specification.

2.5 calculate and count

Cell survival rate (living cells relative populations %)=A (dosing)-A (blank)/A (0 dosing)-A (blank) × 100；

SPSS17.0 softwares are calculated and counted, ANOVA one-way analysis of variance significant differences (P<0.05).

3 experimental results

Compound is to CCl₄Cause the influence of suckling mouse the primary hepatocyte survival rate, ALT, AST, MDA, SOD of damage

Legalon (positive control) can play hepatocytoprotection, the study find that it is remarkably improved CCl₄Damage Hepatocyte viability, while significantly reduce ALT, AST, MDA content, and dramatically increase SOD contents, illustrate cell pathology mould Type is reliable.The same with legalon, each dosage group of Isomperatorin is remarkably improved CCl₄The hepatocyte viability of damage, And it can significantly reduce ALT, MDA content；160 μ g/mL of Isomperatorin, 320 μ g/mL dosage groups can significantly reduce AST and contain Amount, and dramatically increase SOD contents.The result shows that the compound has antiradiation drug hepatic injury activity (table 1).

1 test-compound of table is to CCl₄Cause the influence of suckling mouse the primary hepatocyte survival rate, ALT, AST, MDA, SOD of damage (n=3,)

Compared with model group,^#P<0.05,^##P<0.01,^###P<0.001。

Embodiment 2：Influence of the tested monomeric compound to carbon tetrachloride-injured mouse liver and serum indices

1 material, reagent and instrument

1.1 animal

5~6 week old male cleaning grade Kunming mouses, 18~22g of weight, purchased from the limited public affairs of the long-living biotechnology in Liaoning Department, the certification of fitness number：SCXK (the Liao Dynasty) 2015-0001.Before experiment, the equal adaptability of animal used is raised 3 days.Raised in adaptability During supporting, the free feeding of animal, free water.Keep quite in receptacle, indoor temperature maintains 23-25 DEG C, humidity 55% ~75%, fluorescent lamp lighting, keeps in daily 24h 12h illuminations 12h dark.

1.2 main materials and reagent

Test compound controls product (Isomperatorin, legalon) are purchased from the limited public affairs of Chengdu Puffy moral biotechnology Department；ALT、AST、 MDA、SOD、GSH、Na⁺-K⁺- ATP enzyme, Ca²⁺-Mg²⁺- atpase assay kit is purchased from Nanjing and builds up life Thing Graduate School of Engineering；BCA determination of protein concentration kit is purchased from green skies biotechnology research institute；CCl₄Analyze pure.

1.3 key instrument

MILLI-Q ultrapure water systems (Millipore companies of the U.S.)；Electronic analytical balance, PB-10pH meters (Germany Sartorius)；Glass homogenizer (NingBo XinZhi Biology Science Co., Ltd)；UV-1800PC types UV, visible light is divided Photometer (Shanghai Ao Yi Instrument Ltd.)；(eastern KingMax neoformation science and technology has Infinite M200Pro multi-function microplate readers Limit company)；Plus384 light absorbs microplate reader (Molecular Devices companies of the U.S.)；HR/16M is at a high speed Refrigerated centrifuge (Hunan He Xi instrument and equipment Co., Ltd).

2 experimental methods

Male Kunming strain mice, is randomly divided into 6 groups, every group 10, i.e. normal group (physiological saline), model group (0.1% CCl₄Peanut oil), legalon (positive controls, 16mg/kg), Isomperatorin low middle high dose group (8mg/kg, 16mg/ kg、32mg/kg)；Each group mouse is with the dosage gastric infusion of 10mL/kg, continuous 7d, 1 time a day, after last dose 1h Start modeling.By CCl₄It is dissolved in peanut oil, is made into 0.1% CCl₄Peanut oil solution, abdominal cavity is carried out by the dosage of 10mL/kg Injection, establishes acute liver model.Normal group intraperitoneal injection equivalent peanut oil.

Fasting for solids but not liquids after poisoning, plucks eyeball after 16h and takes blood, whole blood room temperature to separate serum after putting 2h, -80 DEG C of preservations are standby With；Take after blood that cervical dislocation puts to death mouse immediately, take liver rapidly, eliminating surface with the physiological saline rinsing of 4 DEG C of precoolings floats Blood, filter paper wipe it is dry after weigh, calculate liver index.3 mouse are chosen from every group and take same area liver, cutting tissue block is placed in 24h is fixed in 10% formalin, for check pathological section；Remaining liver is placed in -80 DEG C of refrigerators and preserves, and is taken when to be detected Go out, weigh its 0.2g and be put in glass homogenizer and shred, add the physiological saline of 2mL precoolings that 10% tissue is made in ice-water bath Homogenate, 3000rpm centrifugation 10min, supernatant are put 4 DEG C and are saved backup.

3 Indexs measures

3.1 liver index

Liver is taken respectively, is weighed, and liver index is calculated by liver index=[liver weight g/ weight g] × 100%.

3.2 Biochemical Indices In Serums measure

Serum alt, the measure of AST are measured using improvement reitman-frankel method by the specific steps of kit specification.

3.3 hepatic tissue Biochemical Indexes

100 μ L of liver tissue homogenate's liquid are taken, are operated according to kit specification, MDA, SOD and the GSH measured in hepatic tissue contains Amount.

3.4 pathological study

Hepatic tissue is fixed graded ethanol after 24h is rinsed with water in 10% formalin fixer and is eluted, and dimethylbenzene is transparent, The thin slice of 4 μ m-thicks, HE dyeing observations are cut into after progress paraffin embedding processing.

3.5 liver cell mitochondria indexs of correlation

3.5.1 liver cell mitochondria separates

The mouse liver for being stored in -80 DEG C of refrigerators is taken out, clip each group liver organization, is filled with the physiological saline of precooling respectively Divide and clean residual blood, prune away connective tissue, with filter paper suck dry moisture, weigh (about 0.1g)；Hepatic tissue is transferred to the glass of precooling The glass homogenizer mouth of pipe, hepatic tissue is shredded to without obvious tissue block, add the mitochondria separation of 1mL precoolings with the scissors of precooling Liquid is (0.21M mannitol, 0.07M sucrose, 10mM Tris base, 1mM EDTA, 0.5mM EGTA, pH to 7.4), in frozen water It is homogenized 10 times in bath；Tissue homogenate is transferred in 1.5mL centrifuge tubes respectively, in 4 DEG C, 1000 × g centrifugation 10min, discard Precipitation, supernatant are transferred in another centrifuge tube, 4 DEG C, and 10000 × g centrifugation 10min, gained precipitation is liver cell line grain Body.

3.5.2 liver cell mitochondria film potential measures

Extract liver cell mitochondria, ledger line mitochondrial membrane potential measure medium (0.25M sucrose, 5mM MgCl2,10mM KCl, 5mM KH2P04,10mM Hepes, 10mM succinates, pH 7.4) suspension is mixed into, with BCA determination of protein concentration reagents Box measures mitochondrial protein concentration；100 μ L mitochondrial suspensions are taken, 2.9mL measure media is added, adds 5 μ L rhodamines 123 mix, and after 37 DEG C of insulation 30min, fluorescence intensity is surveyed under excitation wavelength 484nm, launch wavelength 534nm.

3.5.3 liver cell mitochondria MDA contents, atpase activity measure

Liver cell mitochondria is extracted, adds physiological saline to be mixed into suspension, with BCA determination of protein concentration kit measurement lines Mitochondrial protein concentration；100 μ L mitochondrial suspensions are taken respectively, are operated according to kit specification, are measured in liver cell mitochondria MDA, Na⁺-K⁺- ATP enzyme and Ca²⁺-Mg²⁺The activity of-ATP enzyme.

4. statistical procedures each group of data is represented using mean ± standard deviation (x ± s), using 11.0 statistical softwares of SPSS Analyzing and processing data, comparison among groups, P are carried out using one-way analysis of variance<0.05 thinks there is significant difference.

5 experimental results

5.1 tested monomeric compounds are to CCl₄Damage the influence of mouse liver index

Compared with normal group, the liver weight and liver index of model group mouse significantly raise, and prompt liver damage；With model group phase Than the liver weight and liver index of legalon group (positive control) mouse significantly reduce, and illustrate that legalon can effectively be alleviated CCl₄Caused hepatic injury；The same with legalon, the liver index of each dosage group of Isomperatorin is significantly reduced (with model Group is compared), show that Isomperatorin has antiradiation drug hepatic injury activity (table 1).

1 tested monomeric compound of table is to CCl₄Damage mouse liver index influence (N=10)

Compared with model group,^#P<0.05,^##P<0.01,^###P<0.001。

5.2 tested monomeric compounds are to CCl₄Damage the influence of mice serum ALT, AST

Compared with normal group, ALT, AST in model group mice serum are significantly raised, modeling success；Compared with model group, ALT, AST in legalon group mice serum are significantly reduced, and show that legalon can effectively alleviate CCl₄Caused hepatic injury； Compared with model group, ALT, AST content significantly reduce in Isomperatorin 8mg/kg dosage group mice serums, show different Europe It is active (table 2) that the root of purple-flowered peucedanum is known as antiradiation drug hepatic injury.

2 tested monomeric compound of table is to CCl₄Damage mice serum ALT, AST influence (N=10)

Compared with model group,^#P<0.05。

5.3 tested monomeric compounds are to CCl₄Damage the influence of mouse liver MDA, SOD and GSH

Compared with normal group, the MDA contents in model group mouse liver significantly raise, and the content of SOD and GSH significantly drop It is low, modeling success；Compared with model group, the MDA contents in legalon group mouse liver significantly reduce, and SOD and GSH's contains Amount significantly rise, shows that legalon can effectively alleviate CCl₄Caused hepatic injury；Compared with model group, Isomperatorin 8mg/ Kg dosage groups can significantly reduce MDA contents, and increased SOD content；And its 16mg/kg dosage group can significantly raise GSH contents, Also illustrate that Isomperatorin has antiradiation drug hepatic injury activity (table 3).

3 tested monomeric compound of table is to CCl₄Damage mouse liver MDA, SOD and GSH content influence (N=10)

Compared with model group,^#P<0.05,^##P<0.01。

5.4 tested monomeric compounds are to CCl₄Damage the influence of mouse liver mitochondria MDA, ATP enzyme and film potential

Compared with normal group, the MDA contents in model group mouse liver mitochondria significantly raise, Na⁺-K⁺- ATP enzyme and Ca^{2 +}-Mg²⁺The activity of-ATP enzyme significantly reduces, and liver cell mitochondria fluorescence intensity significantly increases, and causes mitochondrial membrane potential to collapse, Show modeling success；Compared with model group, the MDA contents in legalon group mouse liver significantly reduce, Na⁺-K⁺- ATP enzyme And Ca²⁺-Mg²⁺The activity of-ATP enzyme significantly rise, mitochondria fluorescence intensity are remarkably decreased, so as to alleviate the journey of film potential collapse Degree, shows that legalon can effectively alleviate CCl₄Caused hepatic injury.Compared with model group, Isomperatorin 8mg/kg dosage groups MDA contents can be significantly reduced, and significantly raise Na⁺-K⁺- ATP enzyme and Ca²⁺-Mg²⁺The activity of-ATP enzymes；Its 16mg/kg at the same time Dosage group can significantly reduce fluorescence intensity, alleviate the collapse degree of mitochondrial membrane potential, as a result illustrate that Isomperatorin can be protected Mitochondrial function and to play antiradiation drug hepatic injury active (table 4).

4 tested monomeric compound of table is to CCl₄Damage mouse hepatic mitochondria MDA, ATP enzyme and film potential influence (N= 10)

Compared with model group,^#P<0.05,^##P<0.01。

Claims (5)

1. application of the Isomperatorin in drug induced hepatic injury protection medicine and health products are prepared, its structural formula are as follows：

A kind of 2. medicine of drug induced hepatic injury protection, it is characterised in that：Including Isomperatorin.

A kind of 3. medicine of drug induced hepatic injury protection, it is characterised in that：Including Isomperatorin and other with Drug liver The medicine of injury protection effect.

4. medicine as claimed in claim 2 or claim 3, its formulation is oral formulations.

5. medicine as claimed in claim 4, it is characterised in that its formulation is tablet, granule, capsule, oral liquid, suspension Deng.