CN109432082B - Pharmaceutical composition for preventing and treating chemical liver injury - Google Patents

Pharmaceutical composition for preventing and treating chemical liver injury Download PDF

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CN109432082B
CN109432082B CN201811504184.XA CN201811504184A CN109432082B CN 109432082 B CN109432082 B CN 109432082B CN 201811504184 A CN201811504184 A CN 201811504184A CN 109432082 B CN109432082 B CN 109432082B
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林朝展
祝晨蔯
顿珠
泽仁达瓦
康萨索朗其美
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Guangzhou University of Chinese Medicine
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Abstract

The invention relates to a pharmaceutical composition for preventing and treating chemical liver injury, which consists of active ingredients and medically acceptable auxiliary materials, and is characterized in that the active ingredients consist of the following chemical ingredients in percentage by weight: 70-80% of medicanin and 20-30% of luteoloside; wherein the chemical structure of the medicanin is shown as the following formula (I), and the chemical structure of the luteolin is shown as the following formula (II). The pharmaceutical composition provided by the invention can obviously reduce transaminase of a rat with a chemical liver injury model, increase the contents of superoxide dismutase and glutathione in the serum of the rat, inhibit the reduction of inflammatory factors in the liver, and has a remarkable effect of preventing and treating chemical liver injury.

Description

Pharmaceutical composition for preventing and treating chemical liver injury
Technical Field
The invention relates to the field of traditional Chinese medicines, in particular to a medicine containing flavonoid glycoside compounds.
Background
Chemical liver injury is liver injury caused by chemical hepatotoxic substances. These chemicals include alcohol, environmentally toxic chemicals and certain drugs. The liver, which is an important detoxification organ of the human body, has dual blood supplies of hepatic artery and hepatic vein.
Chemical substances can enter the liver through the portal vein of the gastrointestinal tract or systemic circulation for transformation, and thus the liver is vulnerable to toxic substances in the chemical substances. In the production process of both nature and human industry, some substances with toxicity to liver exist, which are called as "hepatotropic poisons", the poisons are generally susceptible in people, the incubation period is short, the pathological process is directly related to the infection dosage, and the hepatonecrosis, fatty deformation, liver cirrhosis and liver cancer of the liver can be caused to different degrees.
At present, the drugs for treating chemical liver injury are mainly western medicines, such as: 1) 10% glucose, vitamin C and the like are instilled into the vein, and 12 units of regular insulin and 1 gram of potassium chloride can be added into 1000ml of 10% glucose; 2) dexamethasone is taken at a dose of 20-40 mg/day, the curative effect and the reaction are observed after administration, the dosage is adjusted in time, special attention needs to be paid to protecting gastric mucosa and preventing upper gastrointestinal hemorrhage; 3) oral administration of lactulose, neomycin, metronidazole or ampicillin to inhibit intestinal bacterial growth and reduce bacterial decomposition of proteins and thus ammonia production; 4) administration of fresh plasma or human albumin to correct hypoproteinemia; 5) intravenous drip of branched chain amino acids to reverse the plasma branched chain/aromatic amino acid ratio; 6) the levodopa is used for eliminating and replacing pseudoneurotransmitter in the brain of a hepatic coma patient, and the patient is enabled to be conscious.
Compared with western medicines, the traditional Chinese medicine has the advantage of small toxic and side effects. The patent application with the publication number of CN 101502627A discloses a traditional Chinese medicine health product preparation for preventing and treating alcoholic liver diseases, which is prepared from flos puerariae lobatae, semen hoveniae, radix puerariae, fructus amomi, fructus mume, fructus jujubae and liquorice, and can effectively prevent the GSH exhaustion and MDA increase of the liver caused by ethanol and relieve the steatosis of liver cells. However, the traditional Chinese medicine health product preparation has more medicinal ingredients and complicated chemical components, and the quality of the product is difficult to control.
Tagetes erecta is a dry inflorescence of Pyrethrum tatsienense (Bur. et Franch.) Ling of West yellow-flower of Begonia of Compositae, is mainly produced in Qinghai, Sichuan, Yunnan and Tibet places in China, is a Tibetan common medicament, has the effects of activating blood circulation, dissipating blood stasis, dispelling wind, eliminating dampness, diminishing inflammation and relieving pain, is clinically used for treating headache, head injury, traumatic injury, damp-heat, pyocutaneous disease, wound yellow water, impetigo, hepatitis and the like, and is prepared by separating 18 flavonoids such as alfalfa, luteolin-7-O- β -D-glucoside and the like from Tagetes erecta through a chemical component-target-disease network analysis, and predicting that the 18 flavonoids have pharmacological activity of protecting liver (Xiyuyu Yiyu, Zhuyu Ying, Yingying, Sakung and the like, and a plurality of compounds are selected from a research technology of researching on a total flavonoids in the period of Chastew.7 th years, namely, and a synergistic compound of Tokutshi.8. a traditional Chinese medicinal material with synergistic effect of a synergistic technology of research on a target-MS-TOF, and a total flavonoids of No. 5.
Disclosure of Invention
The invention aims to solve the technical problem of providing the pharmaceutical composition for preventing and treating the chemical liver injury, and the pharmaceutical composition has obvious effect of preventing and treating the chemical liver injury.
The technical scheme for solving the problems is as follows:
the pharmaceutical composition for preventing and treating chemical liver injury comprises active ingredients and medically acceptable auxiliary materials, and is characterized in that the active ingredients comprise the following chemical ingredients in percentage by weight: 70-80% of medicanin and 20-30% of luteoloside; wherein the chemical structure of the medicanin is shown as the following formula (I), and the chemical structure of the luteolin is shown as the following formula (II):
Figure BDA0001899007370000021
the optimal weight ratio of the chemical components of the pharmaceutical composition is 75 percent of lucernetin and 25 percent of luteolin.
The dosage form of the pharmaceutical composition can be tablets, granules, capsules, pills or dripping pills.
The pharmaceutical composition has the double target activities of anti-inflammation and inhibition of oxidative stress, on one hand, the pharmaceutical composition obviously inhibits the over-expression anti-inflammation effect of inflammatory factors such as TNF- α, IL-1 β and the like, on the other hand, the pharmaceutical composition also has the function of regulating the in vivo oxidative stress level, regulates the liver metabolism, detoxifies and promotes the liver cell regeneration, keeps the liver function stable and prevents the occurrence and development of liver diseases through the regulated expression of phase II detoxification enzyme and antioxidant enzyme genes.
Experiments prove that the traditional Chinese medicine composition has the technical effects of the tricin and the luteolin.
Pharmacodynamics of pharmaceutical composition on rat oxidative stress liver injury and mechanism research thereof
60 male SD rats (200-220 g) with SPF level are randomly divided into 6 groups, namely ① normal group, ② model control group, ③ positive drug control group, namely biphenyldicarboxylate group (50mg/kg), ④ drug A group, ⑤ drug B group, ⑥ drug C group, ⑦ control group 1 and ⑧ control group 2, wherein the drug A group is capsules of the following example 1, the drug B group is tablets of the following example 2, the drug C group is soft capsules of the following example 3, the drugs of the control group 1 are prepared by the following method, 75g of lucernin is prepared into capsules according to the method of the example 1, and the drugs of the control group 2 are prepared by the following method, 25g of luteolin is prepared into capsules according to the method of the example 1.
Each group of rats is raised in stainless steel metal cages with 5 rats per cage, room temperature is adjusted to 26 +/-2 ℃, humidity is controlled to 40-70%, and free diet and water intake are realized. From the first day of the experiment, rats in each group were gavaged at 1ml/100g for 3 days (rats in the normal group were gavaged with the same volume of distilled water). Starting the model building on the 4 th day, and injecting 20% CCl into the abdominal cavity of rats except the normal group4Soybean oil solution 0.1ml/100g (0.2 ml/100g for the first time), 2 times/week, and 6 weeks. During the molding period, the rats in each group are subjected to gastric lavage administration according to the experimental method, the administration is continuously carried out for 6 weeks, after 24 hours of last administration (12 hours of fasting without water), blood is taken from abdominal aorta, the rats are dissected, and thymus, spleen and liver are separated. Wherein, the administration dosage of the drug A group, the drug B group, the drug C group, the control group 1 and the control group 2 is 40mg per kilogram of the rat (body weight) based on the weight of the corresponding chemical components. The experimental data obtained were statistically processed using SPSS17.0 software and expressed in x. + -. s.
(1) Influence on rat organ coefficients
The organ coefficient of rats is an index reflecting the relationship between the body weight of rats and the weight of liver, spleen and thymus in each group. The magnitude of the organ coefficient can indirectly reflect whether the lung tissue is diseased or not and the degree of the disease. The liver coefficient of the model group rat is obviously higher than that of the normal group, and the thymus coefficient is obviously lower than that of the normal control group, which indicates that the liver of the model group is enlarged and the thymus is atrophied. Compared with the model group, the Arcisaprine has the effects of inhibiting liver enlargement and thymus gland atrophy in the high, medium and low dose groups of total flavonoids, and the effects are not shown in the bifendate group. Alfalfa essence had no significant effect on spleen coefficients, and the results are shown in table 1.
TABLE 1 Effect of Chinese medicinal composition on organ coefficients of rats of each group: (
Figure BDA0001899007370000031
N＝8)
Figure BDA0001899007370000032
Note: in comparison with the normal group,##P<0.01，#P<0.05; compared with model group**P<0.01，*P<0.05; compared with control group 1⊿⊿P<0.01，⊿P<0.05; compared with control group 2★★P<0.01，★P<0.05。
(2) Influence on the content of AST, ALT, MDA, SOD and GSH in serum
AST and ALT are 'gold indexes' for evaluating liver injury, when the liver is injured, the content of AST and ALT in the liver is abnormally increased, and the content of AST and ALT is measured to reflect the degree of the liver injury. The experiment initially reflects the degree of liver injury by measuring the AST and ALT content in serum. The experimental result shows that the content of AST and ALT in the serum of the rat in the model group is obviously higher than that of the rat in the normal control group, which indicates that liver injury is formed and the model is successfully replicated. The A, B, C groups of the medicines can obviously reduce the abnormal increase of AST and ALT content caused by model building, and the A, B, C groups of the medicines show a certain dose-dependent relationship (P is less than 0.05), which indicates that the traditional Chinese medicine composition can reduce the liver injury degree of model rats.
MDA is an index of lipid peroxidation of organisms, and SOD and GSH indexes reflect the oxidation resistance of the organisms and are the most common indexes for detecting the oxidation and oxidation resistance imbalance of the organisms. The experimental result shows that compared with the normal group of rats, the MDA content in the serum of the model group is obviously higher than that of the normal group of control group; the contents of SOD and GSH are obviously lower than those of a normal control group, which shows that the oxidation resistance of the rat after model building is weakened, the lipid peroxidation degree is enhanced, and each administration group can obviously improve the oxidation resistance of the rat after model building and inhibit lipid peroxidation. The specific results are shown in tables 2 and 3.
TABLE 2 influence of the Chinese medicinal composition on the serum AST and ALT content of rats of each group: (
Figure BDA0001899007370000041
N＝10)
Figure BDA0001899007370000042
Note: in comparison with the normal group,##P<0.01，#P<0.05; compared with model group**P<0.01，*P<0.05; compared with control group 1⊿⊿P<0.01，⊿P<0.05; compared with control group 2★★P<0.01，★P<0.05。
TABLE 3 influence of the Chinese medicinal composition on the contents of MDA, SOD and GSH in the serum of each group of rats: (
Figure BDA0001899007370000043
N＝10)
Figure BDA0001899007370000044
Figure BDA0001899007370000051
Note: in comparison with the normal group,##P<0.01，#P<0.05; compared with model group**P<0.01，*P<0.05; compared with control group 1⊿⊿P<0.01，⊿P<0.05; compared with control group 2★★P<0.01，★P<0.05。
(3) For the content of inflammatory factors TNF- α and IL-1 β in liver homogenate
The test judges whether inflammation exists by measuring the contents of inflammatory factors TNF- α and IL-1 β in liver tissues, and the result shows that compared with a normal group, the contents of TNF- α and IL-1 β in the liver tissues of a model group are obviously increased (P <0.01) and the degree of TNF- α is more obvious than that of the normal group, and after the treatment of the traditional Chinese medicine composition, the contents of TNF- α and IL-1 β in the liver tissues of each administration group are obviously reduced (P <0.05) compared with the liver tissues of the model group and are closer to the liver tissues of the normal group, so that the traditional Chinese medicine composition is supposed to have better early intervention from the formation of the chronic hepatitis, and the specific result is shown in Table 4.
TABLE 4 TNF- α and IL-1 β levels in rat liver tissue
Figure BDA0001899007370000052
n＝10)
Figure BDA0001899007370000053
Note: in comparison with the normal group,##P<0.01，#P<0.05; compared with model group**P<0.01，*P<0.05; compared with control group 1⊿⊿P<0.01，⊿P<0.05; compared with control group 2★★P<0.01，★P<0.05。
(4) HE staining
The pathological section microscopic observation of rat liver shows that the liver cords of the rats in the normal group are arranged regularly, the liver tissue structure is complete, and inflammatory infiltration in the area of the junction is occasionally seen; the rat liver cord of the model group is irregularly arranged, liver cells are loosely arranged, cells near a central vein are dissolved and necrotic, partial inflammatory infiltration and large-scale liver cell necrosis appear near a junction area, the most obvious liver cell lipid change is represented by the fact that vacuoles with different sizes, round shapes and tension appear in cell cytoplasm, and cell nucleuses are pushed to one side. The A, B, C groups and the biphenyldicarboxylate group can both obviously reduce the liver injury degree of rats, which indicates that the traditional Chinese medicine composition can treat CCl4Resulting chronic liver in ratThe injury has a certain protective effect, wherein the protective effect of the medicine A group is more obvious. Meanwhile, the results of the four experiments show that the pharmaceutical composition for preventing and treating chemical liver injury has a synergistic effect. The representative pathological patterns and the pathological degree score criteria of liver tissues are shown in FIG. 1 (A-H).
The pathological condition of the inflammation activity of the liver tissue of the sample is judged according to a scoring scheme of the inflammation activity and the fibrosis degree of the chronic hepatitis proposed by Wangtai, and the result is shown in a table 5. The scoring principle is as follows:
Figure BDA0001899007370000061
scoring formula: p + L +2(PN + BN)
TABLE 5 hepatic injury score scores for various groups of rats
Figure BDA0001899007370000062
(5) Western blot experiment result of related target spots in Nrf2-ARE signal path in liver tissue
Total protein expression of Nrf2 in rat liver tissue: compared with the normal group, the expression level of the Nrf2 protein in the liver tissue of the rat in the model group is obviously increased (P < 0.05); the expression level of Nrf2 protein was also significantly increased in rat liver tissue upon administration compared to the model group (P <0.05), see fig. 3A. Total protein expression of Keap 1: compared with the normal group, the liver tissue of the model group has no significant difference of Keap 1; compared with the model group, the expression level of the Keap1 protein in the liver tissue of the rat in the administration group is remarkably increased (P < 0.05). Total protein expression of Bach 1: compared with a normal group, the expression level of the Bach1 protein in the liver tissue of the model group is obviously increased, (P < 0.05); compared with the model group, the expression level of Bach1 protein in the liver tissue of rats in the administration group is obviously increased (P <0.05), and the figure 2A shows.
The expression conditions of HO-1, NQO1 and GCLC in liver tissues of rats are as follows: compared with the normal group, the expression levels of HO-1, NQO1 and GCLC proteins in the liver tissues of the model group are obviously increased, and the difference has statistical significance (P <0.01 or P < 0.05); compared with the model group, the expression levels of HO-1, NQO1 and GCLC proteins in the liver tissues of the drug group are obviously increased, and the difference has statistical significance (P <0.05), which is shown in figure 2B.
(6) Cytoplasmic and nuclear protein expression results of Nrf2 nuclear transcription factor
Compared with a normal group, the expression of Nrf2 in nucleus after rat molding is obviously increased, and the expression of Keap1 protein is obviously reduced; in cytoplasm, Nrf2 protein expression is obviously reduced, and Keap1 protein expression has no obvious difference. Compared with a model group, the PTTF can obviously increase the expression of Nrf2 protein in a nucleus and obviously reduce the expression of Keap1 protein; while the expression of Nrf2 protein was significantly reduced in the cytoplasm, there was no significant difference in the expression of Keap1 protein, see fig. 3.
(7) RT-PCR experiments
Expression of Nrf2, Keap1, Bach1, HO-1, NQO1, GCLC mRNA in rat liver tissue: compared with the normal group, the expression levels of the mRNA of Nrf2, Bach1, HO-1, NQO1 and GCLC in the liver tissues of the model group are obviously increased, and the difference has statistical significance (P <0.01), while the expression of the mRNA of Keap1 has no obvious difference; compared with the model group, the expression levels of Nrf2, Keap1, Bach1, HO-1, NQO1 and GCLC mRNA in the liver tissues of the drug group are obviously increased, and the difference has statistical significance (P <0.01), which is shown in figure 4.
Drawings
FIG. 1 is a micrograph of HE staining of rat liver tissue in each group (. times.100).
FIG. 2 shows the Western blot experiment results of the Chinese medicinal composition on relevant targets in the Nrf2-ARE signal path in liver tissues.
FIG. 3 is a graph showing the effect of a Chinese medicinal composition on the cytoplasmic and nuclear protein expression of Nrf2 nuclear transcription factors.
FIG. 4 is a graph showing the effects of the Chinese medicinal composition on the expression of Nrf2, Keap1, Bach1, HO-1, NQO1 and GCLC mRNA in rat liver tissues.
Detailed Description
Example 1
1. Prescription of effective components: 75g of medicaginine and 25g of luteolin.
2. The preparation method comprises the following steps:
taking tricin and luteolin, adding excipient HPMC and silica gel micropowder according to equivalent progressive method respectively according to a ratio of 1: 7.5: 2.5, sieving with No. 6 sieve, granulating, and drying at 60 deg.C for 3 hr. Get 1#And (3) filling the empty enteric-coated capsules with the dry granules respectively, wherein each 1 capsule is 0.2g, coating a layer of acacia mucilage at the mouth of each capsule, sealing the capsules, wiping the capsules with dry gauze, and filling the capsules into a sealed brown bottle to obtain the enteric-coated capsules. The preparation is administered orally twice a day, 2 granules each time.
Example 2
1. Prescription of effective components: 80g of lucernetin and 20g of luteolin.
2. The preparation method comprises the following steps:
taking the medicatin and the luteolin, adding 60g of starch, 20g of dextrin, 15g of cane sugar and a proper amount of 65% ethanol, uniformly mixing, granulating, drying to obtain granules, sieving, granulating, adding 1.0g of superfine silica gel powder, uniformly mixing, and pressing into common tablets. The preparation is administered orally twice a day, 2 tablets each time.
Example 3
1. Prescription of effective components: 70g of lucernetin and 30g of luteolin.
2. The preparation method comprises the following steps:
taking the medicanin and the luteolin, adding 40g of soybean oil, mixing, adding 10.0g of beeswax and 0.6g of tween-80, and pressing into 300 soft capsules (0.2 g/capsule) by a rotary capsule-making machine. The preparation is administered orally twice a day, 4 granules each time.
Example 4
1. Prescription of effective components: 80g of lucernetin and 20g of luteolin.
2. The preparation method comprises the following steps:
taking tricin and luteolin, adding PVP 50g, CMC-Na 40g, L-HPC 15g, CMS-Na 10g and appropriate amount of 65% ethanol to prepare soft material, sieving with a screen, drying at 60 deg.C to obtain granule, sieving, grading, and packaging with packaging equipment to obtain granule, each bag is 5 g. The preparation is administered twice a day, 1 bag each time, with warm boiled water.
Example 5
1. Prescription of effective components: 75g of medicaginine and 25g of luteolin.
2. The preparation method comprises the following steps:
taking the medicatin and the luteolin, adding 50g of honey, 40g of rice paste, 40g of flour paste and a proper amount of 65% ethanol to prepare soft materials, and then pressing into pills with the weight of 0.1g per pill. The preparation is administered orally twice a day, 6 pills each time.
Example 6
1. Prescription of effective components: 70g of lucernetin and 30g of luteolin.
2. The preparation method comprises the following steps:
taking the medicatin and the luteolin, adding 150g of gelatin and 3g of polyethylene glycol, and preparing into dripping pills of 0.05g per pill. The preparation is administered orally twice a day, 20 pills each time.
Example 7
1. Prescription of effective components: 80g of lucernetin and 20g of luteolin.
2. The preparation method comprises the following steps:
taking the medicatin and the luteolin, adding 40g of gelatin, 45g of Arabic gum and 8g of sodium carboxymethylcellulose, and preparing into microcapsules of 0.06 g/granule. The preparation is administered orally at a dose of 20 granules twice a day.

Claims (3)

1. The pharmaceutical composition for preventing and treating chemical liver injury comprises active ingredients and medically acceptable auxiliary materials, and is characterized in that the active ingredients comprise the following chemical ingredients in percentage by weight: 70-80% of medicanin and 20-30% of luteoloside; wherein the chemical structure of the medicanin is shown as the following formula (I), and the chemical structure of the luteolin is shown as the following formula (II):
Figure FDA0001899007360000011
2. the pharmaceutical composition for preventing and treating chemical liver injury according to claim 1, wherein the effective components comprise the following chemical components in percentage by weight: 75% of medicanin and 25% of luteoloside.
3. The pharmaceutical composition for preventing and treating chemical liver injury according to claim 1 or 2, wherein the pharmaceutical composition is in the form of tablets, granules, capsules, pills or dripping pills.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107397740A (en) * 2017-07-17 2017-11-28 华南理工大学 A kind of antitumor polyphenol compositions of Synergistic and application

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107397740A (en) * 2017-07-17 2017-11-28 华南理工大学 A kind of antitumor polyphenol compositions of Synergistic and application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
基于UPLC-Triple TOF MS/MS和网络药理学技术分析打箭菊总黄酮部位保肝活性成分及其潜在靶点研究;夏玉英等;《中药材》;20180731;第41卷(第7期);参见第1609-1614页 *
木犀草素对对乙酰氨基酚诱导的L02肝细胞损伤的保护作用;贺兰芝;《中国中药杂志》;20161130;第41卷(第22期);参见第4234-4239页 *

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