阅读说明：本技术 木豆素在制备预防和/或治疗药物性肝损伤的药物中的应用 (Application of cajanin in preparation of medicine for preventing and/or treating drug-induced liver injury ) 是由 闫明珠 常琪 靳苏维 王智 夏天吉 刘永广 于 2021-08-30 设计创作，主要内容包括：本发明属于医药技术领域,具体涉及木豆素在防治药物性肝损伤中的新用途。本发明通过动物实验首先证明并公开了木豆素能够有效地治疗对乙酰氨基酚所致的药物性肝损伤。本发明进一步公开了木豆素用于制备预防和/或治疗药物性肝损伤药物的用途。本发明为研制以木豆素为活性成分的保肝药物提供了科学依据,具有重大的应用价值。(The invention belongs to the technical field of medicines, and particularly relates to a new application of cajanin in preventing and treating drug-induced liver injury. Animal experiments prove and disclose that the cajanin can effectively treat drug-induced liver injury caused by acetaminophen. The invention further discloses application of the cajanin in preparing a medicine for preventing and/or treating drug-induced liver injury. The invention provides scientific basis for developing the liver-protecting medicine taking the cajanin as the active ingredient and has great application value.)

1. Use of cajanin and its composition in preparing medicine for preventing and/or treating drug-induced liver injury is provided.

2. Use according to claim 1, characterized in that: the chemical name of cajaningstilbene (cajaningstilbene acid) is 2-carboxyl-3-hydroxy-4-isopentenyl-5-methoxystilbene, 2-carboxyl-3-hydroxy-4-phenyl-5-methoxystilbene).

3. Use according to claim 1, characterized in that: the cajanin can be extracted and separated from Cajanus cajan (L.) Millsp or other plants containing cajanin; it can also be synthesized by chemical synthesis.

4. The pharmaceutical composition for preventing/treating drug-induced liver injury according to claim 1, wherein: the medicine is composed of the therapeutically effective cajanin and a pharmaceutically acceptable carrier, and can be capsules (soft capsules), granules (dry suspensions), tablets (dispersible tablets, effervescent tablets, chewable tablets, orally disintegrating tablets), solutions (syrup), pills (concentrated pills, dripping pills, micro-pills), or injections and the like which are processed by the cajanin.

5. The activity of the compound cajanin and the composition thereof in preventing and treating the drug-induced liver injury of the claims 1 to 4 and the application thereof in preparing the drugs for preventing and treating the drug-induced liver injury.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of cajaningiline acid (CSA) (chemical name: 2-carboxyl-3-hydroxyl-4-isopentenyl-5-methoxyl stilbene, 2-carboxyl-3-hydroxyl-4-prenyl-5-methoxyl stilbene) in preparation of a medicine for preventing and/or treating drug-induced liver injury.

Background

Drug-induced liver injury (DILI) refers to liver injury induced by various prescriptive or non-prescription chemical drugs, biological agents, traditional Chinese medicines, natural medicines, health products, dietary supplements, metabolites thereof, and adjuvants. The acute DIL1 reported in China at present accounts for about 20% of the hospitalization rate of acute liver injury, and the incidence rate is gradually increased. DILI is one of the most common and serious adverse drug reactions, and severe patients can cause acute liver failure and even death. Thus, DILI has become a non-negligible public health problem. Acetaminophen (APAP) is the most widely used and most used antipyretic analgesic over-the-counter medicine in the world. However, improper APAP administration can cause severe liver damage and is the most common cause of acute liver failure in many countries. In China, the incidence of liver injury caused by APAP is on the trend of rising year by year. In addition, APAP-induced acute liver injury is also a common drug-induced liver injury model and is commonly used for evaluating the liver protection potential of drugs.

Cajaninstiline acid (shown in figure 1) is a stilbene compound separated and extracted from folium Cajani. The research shows that the cajanin has the pharmacological activities of resisting depression, improving learning and memory, resisting inflammation, easing pain, resisting tumor, resisting herpes virus and reducing blood sugar. The applicant prepares the cajanin from cajanus cajan leaves by using a modern advanced extraction technology at the earlier stage, researches the protection effect of the cajanin on drug-induced liver injury caused by APAP, and finds that the cajanin can obviously reduce the levels of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase in serum of a mouse with the APAP liver injury and reduce the area of liver necrosis, which indicates that the cajanin has good protection and treatment effects on the drug-induced liver injury caused by the APAP.

Disclosure of Invention

The invention aims to provide a new medicinal application of a stilbene compound cajanin in cajanus cajan leaves. The above purpose is realized by the following technical scheme:

the new application of the cajanin medicine provided by the invention is the application of the cajanin in preparing a product with the following functions:

1) preventing and/or treating drug-induced liver injury;

2) inhibiting the elevation of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase levels in blood serum caused by drug-induced liver injury;

3) inhibit hepatocyte necrosis caused by drug-induced liver injury.

In the application, the drug-induced liver injury can be acute liver injury caused by drugs.

The invention also provides a medicament for preventing and/or treating drug-induced liver injury, which contains the cajanin. The pharmaceutical composition is prepared by mixing an effective amount of the cajanin with a pharmaceutically acceptable carrier, excipient or diluent.

The invention discovers that the cajanin has good treatment effect on liver injury caused by APAP for the first time. The invention discloses that an active ingredient of the cajanus cajan in cajanus cajan leaves has the function of preventing and treating the drug-induced liver injury and has good treatment effect on APAP-induced acute liver injury, and the invention provides scientific basis for developing the liver-protecting drug based on the cajan component and has great application value.

Drawings

FIG. 1 chemical Structure of cajanin

FIG. 2 Experimental example 1H & E staining chart of liver tissue section of each group of mice

FIG. 3 Experimental example 2H & E staining chart of liver tissue section of each group of mice

Detailed Description

The present invention is further described with reference to the following specific embodiments, and it will be understood by those skilled in the art that changes in the details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but the changes and substitutions are within the scope of the present invention.

Experimental example 1 therapeutic Effect of intragastric administration of cajanin on drug induced hepatic injury due to APAP

1. Experimental Material

Experimental animals: c57BL/6N male mouse, weight 20-22g, 24, purchased from Beijing Wittingle laboratory animal technology Co., Ltd, production license: SCXK (Kyoto) 2016-. The conventional feed for mice was provided by Witongliwa laboratory animal technology, Inc. Mice were housed in SPF-level environment (laboratory animal house of institute for medicinal plants of the chinese academy of medical sciences [ SYXK (kyo) 2018-: the water and food are freely drunk and eaten in 6 groups/cage at the temperature of 23-25 ℃ and the humidity of 45-65% in the dark circadian rhythm of 12 hours. The experiment was approved by the animal administration and animal welfare committee of the institute for medicinal plants research, academy of medical sciences (review batch number: SLXD-20191219001).

Drugs and reagents: cajanin: prepared by the laboratory, and the HPLC purity is up to 98 percent. The medicine is dissolved and prepared by normal saline containing 5 percent of Solutol HS-15 before administration. APAP (cat # HY66005) was purchased from MedChemexpress and was prepared with 55 deg.C physiological saline prior to administration.

2. Experimental methods

Grouping and administration: mice were divided into a blank control group, an APAP liver injury model group, a cajanin low dose treatment group (50mg/kg) and a high dose treatment group (75mg/kg) and acclimatized in the animal room for 7 days. Mice were fasted for 16 hours before the official experiment. The blank control group is injected with normal saline, APAP solution is injected into each group of APAP liver injury according to the dosage of 300mg/kg, and the mice of the cajanin treatment group are gavaged for 30 minutes after the APAP administration to be administered with cajanin. The administration volumes were all 0.2mL/10 g. Mice were anesthetized 24 hours after APAP molding, blood was taken from the abdominal aorta, and euthanasia was performed after liver was harvested.

Detecting serum biochemical indexes: whole blood was centrifuged at 1500 Xg for 15 minutes at 4 ℃ and the supernatant was collected for subsequent testing. Serum contents of Alanine transaminase (ALT) and Aspartate Aminotransferase (AST) were measured using a full-automatic biochemical analyzer (Beckman AU480) and Zhongsheng North control kit (cat # 020003, 020013).

And (3) pathological detection: mouse livers were fixed with formalin for 24 hours, and then subjected to conventional paraffin embedding, sectioning, Hematoxylin-eosin staining (H & E staining).

And (3) data analysis: necrotic area in H & E staining results was calculated using Image J software. All data were statistically analyzed using SPSS19.0 software and finally expressed as mean ± SEM. The data are first tested for normality by the Shapiro-Wilk method, if normal distribution is satisfied, then tested for homogeneity of variance. If the data simultaneously meet normal distribution and the data of each group have uniform variance, adopting one-factor variance analysis and selecting an LSD method to compare the means between the groups two by two. If the data does not meet normal distribution or the variance of each group of data is not uniform, adopting nonparametric test.

3. Results of the experiment

(1) Effect of cajanin on Biochemical indicators of mouse serum

The results (see table 1) show that the ALT and AST levels in the serum of mice after the intraperitoneal injection of APAP are obviously increased (P is less than 0.001) compared with the blank control group, which indicates that the model of the drug-induced liver injury model is successfully modeled. The administration of low dose and high dose of cajanin by gavage can obviously reduce the level of ALT (P is less than 0.01, P is less than 0.001) and AST (P is less than 0.01) in the blood serum of mice, and the cajanin has the function of treating APAP drug-induced liver injury.

TABLE 1 Effect of cajanin on ALT and AST in serum of APAP liver-injured mice (mean + -SEM, n ═ 5-6)

Compared with the blank control group, the composition of the composition,^***p is less than 0.001; in comparison with the set of models,^##P＜0.01，^###p is less than 0.001; i.p.: intraperitoneal injection, i.g.: and (5) performing intragastric administration.

(2) Effect of cajanin on pathological injury of liver of mouse

H & E staining of the liver tissue sections of the mice shows that liver tissues of the mice in the blank control group have no hepatocyte necrosis, the liver cell boundary plate is complete, inflammatory cell infiltration is not seen in the sink area, and no obvious pathological abnormality exists on the whole (figure 2A). Excessive APAP can lead to blood stasis/hemorrhage of hepatic sinusoids, massive necrosis of the center of hepatic lobules, and moderate amounts of mononuclear and lymphocytic foci infiltration (FIG. 2B; Table 2, P < 0.001). Liver lobule structures of livers of mice in low-dose and high-dose treatment groups of the cajanin exist, and liver cell steatosis is not seen; focal hepatocyte necrosis and a small amount of mononuclear lymphocytic focal infiltration were occasionally seen (fig. 2C and 2D). Compared with the APAP drug-induced liver injury model group, the cajanin can obviously reduce the area of liver necrosis (the P is less than 0.001 in table 2) and reduce the degree of liver necrosis. The results show that the cajanin can treat hepatocyte necrosis caused by APAP.

TABLE 2 Effect of cajanin on the area of liver necrosis in APAP liver-injured mice (mean + -SEM, n ═ 6)

Compared with the blank control group, the ratio of the active ingredients,^***p is less than 0.001; in comparison to the set of models,^###p is less than 0.001; i.p.: intraperitoneal injection, i.g.: and (5) performing intragastric administration.

Experimental example 2 therapeutic Effect of intraperitoneal injection of cajanin on drug-induced liver injury caused by APAP

1. Experimental Material

Experimental animals: the experiment was approved by the animal administration and animal welfare committee of the institute for medicinal plants research, academy of medical sciences (review batch number: SLXD-20200103001). The rest is the same as in experimental example 1.

Drugs and reagents: the same as in experimental example 1.

2. Experimental methods

Grouping and administration: mice were divided into a blank control group, an APAP liver injury model group, a cajanin low dose treatment group (50mg/kg) and a high dose treatment group (75mg/kg) and acclimatized in the animal room for 7 days. Mice were fasted for 16 hours before the official experiment. The blank control group was injected with physiological saline, the APAP liver injury groups were injected with APAP solution at a dose of 300mg/kg, and the mice in the cajanin-treated group were further injected with cajanin 1.5 hours after APAP administration. The above administration modes are all intraperitoneal injection, and the injection volume is 0.2mL/10 g. Mice were anesthetized 24 hours after APAP molding, blood was taken from the abdominal aorta, and euthanasia was performed after liver was harvested.

Detecting serum biochemical indexes: the same as in experimental example 1.

And (3) pathological detection: the same as in experimental example 1.

And (3) data analysis: the same as in experimental example 1.

3. Results of the experiment

(1) Effect of cajanin on Biochemical indicators of mouse serum

The results (see table 3) show that the ALT and AST levels in the serum of mice after the intraperitoneal injection of APAP are obviously increased (P is less than 0.001) compared with the blank control group, which indicates that the model of the drug-induced liver injury model is successfully modeled. The low dose and the high dose of the cajanin injected into the abdominal cavity can obviously reduce the levels of ALT and AST in the serum of a mouse (P is less than 0.001), which indicates that the cajanin has the function of treating APAP drug-induced liver injury.

TABLE 3 Effect of cajanin on ALT and AST in serum of APAP liver-injured mice (mean + -SEM, n ═ 5-6)

Compared with the blank control group, the composition of the composition,^***p is less than 0.001; in comparison with the set of models,i.p.: and (5) carrying out intraperitoneal injection.

(2) Effect of cajanin on pathological injury of liver of mouse

H & E staining of the liver tissue sections of the mice shows that liver tissues of the mice in the blank control group have no hepatocyte necrosis, the liver cell boundary plate is complete, inflammatory cell infiltration is not seen in the sink area, and no obvious pathological abnormality exists on the whole (figure 3A). Excessive APAP causes significant liver sinus congestion/hemorrhage, massive necrosis in the center of the liver lobule, and moderate amounts of mononuclear and lymphocytic foci infiltration (FIG. 3B; Table 4, P < 0.001). After the low-dose and high-dose cajanin is injected into the abdominal cavity, liver lobular structures in the liver of the mouse exist, and liver cell steatosis is not seen; focal hepatocyte necrosis and a small amount of mononuclear lymphocytic focal infiltration were occasionally seen (fig. 3C and 3D). Compared with the APAP drug-induced liver injury model group, the cajanin can obviously reduce the area of liver necrosis (Table 4, P is less than 0.001) and reduce the degree of liver necrosis. The results show that the cajanin can treat hepatocyte necrosis caused by APAP.

TABLE 4 Effect of cajanin on the area of liver necrosis in APAP liver-injured mice (mean + -SEM, n ═ 6)

Compared with the blank control group, the ratio of the active ingredients,^***p is less than 0.001; in comparison to the set of models,^###p is less than 0.001; i.p.: and (5) carrying out intraperitoneal injection.