CN104491496B - Application of gastrodin/gastrodia elata powder in the preparation of anti-hepatic fibrosis medicine - Google Patents

Abstract

The invention belongs to the field of medicine, and relates to the application of gastrodin in the preparation of anti-hepatic fibrosis drugs, through the inhibitory effect on the activity of type I collagen α1 promoter, the influence on the ALT and AST content in rat serum, and the effect on rat liver The effects of histopathological structure, the effect on the content of hydroxyproline in rat liver tissue, the effect on the oxidative stress response in rat liver tissue, and the effect on the degree of liver fibrosis in rats have proved that gastrodin has a good The anti-hepatic fibrosis activity is expected to be developed as a new anti-hepatic fibrosis drug.

Description

Application of gastrodin/gastrodia elata powder in the preparation of anti-hepatic fibrosis medicine

Technical field:

The invention belongs to the field of medicine and relates to the application of gastrodin/gastrodia elata powder in the preparation of anti-hepatic fibrosis drugs.

Background technique:

Liver fibrosis is a pathological process of excessive deposition of extracellular matrix (especially type I collagen α1) in the liver, and it is a repair response of the body to acute and chronic liver injury. Chronic liver disease caused by various etiologies and liver injury caused by certain toxic substances can cause inflammation and necrosis of liver cells, and the pathological features of secondary fibrosis. Hepatic fibrosis (hepatic fibrosis, HF) is an important link in the development of liver cirrhosis and even primary hepatocellular carcinoma. In my country, the main disease leading to liver fibrosis is hepatitis B virus. The traditional view is that once the human liver fibrosis occurs, it is impossible to reverse it. However, the famous liver disease expert Rogking proposed after in-depth research on liver fibrosis that typical human liver fibrosis can be reversed. It is of great significance to effectively control the malignant development of liver diseases from hepatitis to cirrhosis and liver cancer. Over the years, scholars at home and abroad have conducted a lot of research on the pathogenesis, regulatory factors, diagnosis and treatment of liver fibrosis. Although there are a lot of drugs used in experimental research, there is still no western medicine that can be used in clinical practice with high efficiency and no obvious side effects except for the cause of liver fibrosis. Clinical practice shows that the treatment of liver fibrosis with traditional Chinese medicine has shown great potential in improving clinical symptoms and so on. At present, the anti-hepatic fibrosis traditional Chinese medicines in clinical use in my country include: Fuzheng Huayu Capsules, Fufang Biejiaruangan Tablets, Dahuang Zhechong Pills, Xiao Bupleurum Preparations, Qianggan Capsules, etc. The medicines are all traditional Chinese medicine compound preparations, which are composed of multi-flavored traditional Chinese medicines, and all the compound preparations do not contain Gastrodia elata.

Gastrodia elata is a plant of the genus Gastrodia elata in the family Orchidaceae. Its dried tubers are also known as Gastrodia elata Blume. It is a commonly used and relatively expensive traditional Chinese medicine. Gastrodin is one of the effective monomer components of Gastrodia elata, and it is mainly used clinically to treat cardiovascular and cerebrovascular diseases and nervous system diseases. Gastrodin CAS No. 62499-27-8, molecular formula C ₁₃ H ₁₈ O ₇ , molecular weight 286.3, molecular structural formula:

In recent years, through the research on the system biological activity of gastrodin/gastrodia powder, our laboratory found that gastrodin/gastrodia powder, as a single drug, has anti-hepatic fibrosis activity, and then carried out follow-up in-depth research work. The application of gastrodin/gastrodia elata powder in anti-hepatic fibrosis has not been reported so far at home and abroad.

Invention content:

The invention provides the application of gastrodin/gastrodia elata powder in the preparation of anti-hepatic fibrosis medicine.

The invention also provides the application of the composition composed of gastrodin/gastrodia powder as an active ingredient and a pharmaceutically acceptable carrier in the preparation of anti-hepatic fibrosis medicine.

In the present invention, the COL1A1 promoter of type I collagen α1 gene is used as the drug screening target, and a cell screening model for anti-hepatic fibrosis drugs is constructed, and the gastrodin/gastrodia elata powder is screened out to have a significant inhibitory effect on the COL1A1 promoter. In order to further confirm the anti-hepatic fibrosis effect of gastrodin/gastrodia elata powder, the present invention prepares the SD rat model of common bile duct ligation, and affects rat liver function, liver tissue pathological structure, liver tissue fibrosis degree, collagen fiber content and oxidation The stress degree was detected, and the results showed that gastrodin/gastrodia elata powder could inhibit the generation of liver fibrosis.

When the present invention is used for anti-hepatic fibrosis treatment, oral administration of Gastrodia elata powder and oral or non-oral administration of gastrodin are all safe and effective. Oral administration can be made into any conventional dosage form, such as tablets, capsules, powders, granules, etc.; non-oral administration can be made into injections, etc.

The dosage of gastrodin in the present invention can be adjusted and changed according to factors such as the mode of administration, the severity of the disease, and the age of the patient. Generally, the adult oral dosage is selected from 1-100 mg/day; the adult injection dosage is selected from 1-50 mg/day.

When the present invention is used to prepare the anti-hepatic fibrosis drug, its adjuvant and preparation method can be in any pharmaceutically acceptable form.

Description of drawings:

Figure 1 - Inhibitory effect of gastrodin on type Ⅰ collagen α1 promoter activity

Wherein: *p<0.05 compared with the control group (dosing concentration is zero).

Figure 2 - Effect of gastrodin on the pathological structure of rat liver tissue

Among them: A is the HE staining result of liver tissue sections of rats in the sham operation group; B is the HE staining results of liver tissue sections of rats in the model group; C is the HE staining results of liver tissue sections of rats in the gastrodin-administered group; D is the HE staining results of all animals Statistical graph of tissue bile duct hyperplasia; E is statistical graph of tissue necrosis in all animals *p<0.05, **p<0.01 compared with BDL group; ##p<0.01 compared with sham operation group.

Figure 3 - Effect of gastrodin on the degree of liver fibrosis in rats

Among them: A is the result of Sirius red staining of liver tissue sections of rats in the sham operation group; B is the result of Sirius red staining of liver tissue sections of rats in the model group; C is the result of Sirius red staining of liver tissue sections of rats in the gastrodin group; D is Statistical map of the Sirius red staining area of all animal tissues

*p<0.05 vs. BDL group; ##p<0.01 vs. sham group.

Figure 4 - Effect of gastrodin on hydroxyproline content in rat liver tissue

Where: *p<0.05 compared with BDL group; ##p<0.01 compared with sham group.

Figure 5 - Effect of gastrodin on oxidative stress response in rat liver tissue

Where: *p<0.05 compared with BDL group; ##p<0.01 compared with sham group.

Figure 6 - Inhibitory effect of Gastrodia elata powder on the activity of type Ⅰ collagen α1 promoter

Wherein: *p<0.05, **p<0.01 compared with the control group (dosing concentration is zero).

Figure 7-Effect of gastrodia elata powder on the pathological structure of rat liver tissue

Among them: A is the HE staining result of the liver tissue section of rats in the sham operation group; B is the HE staining result of the liver tissue section of the model group; C is the HE staining result of the liver tissue section of the rat in the gastrodia elata powder group; D is the HE staining result of all animals Statistical graph of tissue necrosis

**p<0.01 vs. BDL group; ##p<0.01 vs. sham group.

Figure 8 - Effect of Gastrodia elata powder on the degree of liver fibrosis in rats

Among them: A is the result of Sirius red staining of liver tissue sections of rats in the sham operation group; B is the result of Sirius red staining of liver tissue sections of rats in the model group; C is the result of Sirius red staining of liver tissue sections of rats in the Gastrodia elata powder group; D is Statistical map of the Sirius red staining area of all animal tissues

## p<0.01 vs. sham group.

Figure 9 - Effect of gastrodia elata powder on hydroxyproline content in rat liver tissue

Where: ##p<0.01 vs. sham group.

detailed description:

The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but the content is an explanation of the present invention rather than a limitation.

"Example 1" Gastrodin Inhibits Type I Collagen α1 Promoter Activity Test

1.1 Construction of monoclonal cell line LX2-COL stably expressing type Ⅰ collagen α1 promoter COL1A1P

Genomic DNA was extracted from LX2 cells (Mount Sinai School of Medicine) using a genomic DNA extraction kit. The first about 2400 bp of the COL1A1 sequence (sequence number: NC_000017.11) of the human whole genome information of the NCBI database human type I collagen α1 gene COL1A1 was used as the COL1A1 promoter sequence. Primer Premier 5.0 was used to design primers for the COL1A1 promoter. The upstream and downstream primer sequences were 5'AAGAGCTCGTGGGAAAGCCTGGATGG3' (including the SacⅠ restriction site), 5'AAAGATCTTTTGGGACTTACTGTCTTCGT 3' (including the BglⅡ restriction site). Using the genomic DNA of LX2 cells as a template, PCR amplification was carried out to obtain the target fragment type Ⅰ collagen α1 gene COL1A1 promoter, and the PCR product was initially identified by agarose gel electrophoresis.

The COL1A1 promoter fragment of type I collagen α1 gene was ligated with the pGL4.17[luc2/Neo] vector after SacⅠ and BglⅡ double enzyme digestion, and the ligation reaction solution was transformed into Escherichia coli DH5α competent medium, and cultured on LB solid plate at 37°C upside down 16-20h, pick ampicillin-resistant single clones, and extract plasmid DNA. Positive clones were preliminarily identified by colony PCR, enzyme digestion and other techniques, and a recombinant plasmid was obtained, named pGL4.17-COL1A1P

LX2 cells were cultured in DMEM (Gibco) medium containing 10% fetal bovine serum at 37°C and 5% CO2. Spread 5×10 ⁴ cells per well on a 48-well plate and culture for 24 hours. When the LX2 cells were cultured in a 6-well plate and reached close to 90%-95% confluency, cell transfection was performed. After 24 hours, a concentration gradient of G418 (AMERCO) was added, and finally G418 with a concentration of 100 μg·mL ^-1 was selected to continue culturing the LX2 cells transfected with the pGL4.17-COL1A1P plasmid, and the medium was changed in time. After the cells in the 6-well plate were confluent, spread the single cells to the 96-well plate. After the single cells grow into clones (about 1-2 weeks), add D-luciferin (Thermo Fisher) to a final concentration of 60 μg·mL ^-1 , and use an in vivo bioluminescent imaging system (IVIS 200, Xenogen) to take pictures, and select the most signal. Strong monoclonal cell line LX2-COL, and expanded culture.

1.2 Spread the monoclonal cell LX2-COL stably expressing the type I collagen α1 promoter COL1A1P constructed above on a 96-well white plate, with 2×10 ⁴ cells per well. After the confluence of the cells is about 90%, culture them without serum for 24 hours, add TGF-β1 (2ng/ml) to induce, and add gastrodin (commercially available product) in a gradient concentration at the same time, the concentrations are 1 μg/ml, 3 μg/ml, and 30 μg respectively /ml, and three replicate wells were set up for each experiment. Follow the operating steps of the Bright-Glo ^TM Luciferase Assay System manual, discard the medium after 24 hours, and add 50 μl/well of any medium. Add 50 μl/well of luciferase substrate, and detect after 2 min. The results showed that as the gastrodin concentration increased, the fluorescence intensity weakened, and when the gastrodin concentration was 3 μg/ml and 30 μg/ml, the fluorescence intensity decreased significantly (Figure 1). It shows that gastrodin has obvious inhibitory effect on COL1A1 promoter activity when the concentration is 3μg/ml and 30μg/ml.

"Example 2" SD rat common bile duct ligation-induced liver fibrosis model preparation

Select 19 SD male rats with a body weight of 180-220g, and randomly divide them into a sham operation group, a model group, and a gastrodin administration (500mg/kg) group, wherein 5 rats were in the sham operation group, 7 rats in the model group, and 7 rats in the gastrodin group. 7 only. After fasting for 12 hours before the animal experiments, the animals were anesthetized with isoflurane for surgery. The common bile duct ligation (BDL) was performed in the model group and the treatment group. Under aseptic operation, a midline incision was made on the upper abdomen, the liver margin was raised, the duodenum was pulled apart, and the common bile duct was separated by 2-3 cm. Two 000-gauge silk sutures were ligated near the duodenum and near the porta hepatis, and the common bile duct was cut from the middle. In the sham operation group, only the midline upper abdominal incision was made and sutured, and the common bile duct was not ligated. After the animals wake up from anesthesia, eat normally, drink water freely, and inject penicillin intramuscularly for 3 days to prevent infection. The experimental temperature is (22±2)°C. On the 2nd day after modeling, intragastric administration was started, and normal saline (sham operation group, model group) and gastrodin 500 mg/kg (gastrodin administration group) were administered, once a day, for 14 days.

"Example 3" Gastrodin is on the inhibitory effect of BDL-induced rat serum ALT/AST level increase

Fasting for 12 hours before sampling, rats were anesthetized with 10% chloral hydrate, and abdominal aortic blood was collected. Centrifuge at 800rpm for 5min, and take serum to detect the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The results showed that gastrodin can significantly inhibit the contents of ALT and AST in rat serum (Table 1), indicating that gastrodin can improve rat liver function.

Table 1. Effect of gastrodin on BDL-induced serum ALT/AST levels in rats

**p<0.01 vs. BDL group; ##p<0.01 vs. sham group.

"Example 4" Inhibitory effect of gastrodin on BDL-induced changes in rat liver pathological structure

Before sampling, the rats were fasted for 12 hours, and the rats were killed, and the liver tissue was taken, and the large lobe of the liver was excised and fixed in 10% formalin. After dehydration, paraffin embedding, slicing, baking slices, etc. to make paraffin sections. Hematoxylin-eosin (HE) staining solution was used for staining to observe the changes of rat liver tissue pathological structure. The results showed that the hepatic cells in the liver tissue of rats in the sham operation group were arranged regularly, the hepatic lobule was intact, and there was no inflammatory cell infiltration and bile duct hyperplasia. ; The liver tissue structure of the rats in the gastrodin administration group was significantly improved, the bile duct hyperplasia was significantly inhibited, and the degree of tissue necrosis was significantly reduced (Figure 2). It shows that gastrodin can significantly improve the pathological structure of liver tissue in BDL rats.

《Example 5》The inhibitory effect of gastrodin on BDL-induced rat liver fibrosis

The paraffin sections were stained with Sirius red to observe the fibrosis of rat liver tissue. The results showed that the degree of fibrosis in rat liver tissue was significantly increased after BDL, and the degree of fibrosis was significantly inhibited after administration of gastrodin (Figure 3). Show that gastrodin can significantly inhibit BDL-induced liver fibrosis in rats.

"Example 6" the impact of gastrodin on hydroxyproline content in rat liver tissue

The rats were fasted for 12 hours before sampling, and the rats were sacrificed. The liver tissue was taken, and 30-100 mg was accurately weighed, and the operation was performed according to the instructions of the hydroxyproline test kit (purchased from Nanjing Jiancheng Bioengineering Institute). The results showed that compared with the rats in the sham operation group, the content of hydroxyproline in the liver tissue of the rats in the BDL group was significantly increased; compared with the BDL group, after the administration of gastrodin, the content of hydroxyproline in the liver tissue of the rats significantly reduced (Figure 4). It shows that gastrodin can significantly reduce the content of hydroxyproline in liver tissue, that is, gastrodin can significantly inhibit the production of collagen fibers in BDL rats.

"Example 7" Inhibitory Effect of Gastrodin on BDL-Induced Rat Liver Tissue Oxidative Stress Response

The rats were fasted for 12 hours before sampling, and the rats were sacrificed, and the liver tissues were taken, cut into small pieces and temporarily stored in liquid nitrogen, and then transferred to -80°C for storage. Accurately weigh the tissue weight, add 9 times the volume of pre-cooled saline according to the ratio of weight (g): volume (ml) = 1:9, grind in an ice bath to prepare a homogenate, centrifuge at 3000-4000rpm for 10-15min , take 10% homogenate supernatant to be tested. Malondialdehyde (MDA), nitric oxide ( NO) and superoxide dismutase (SOD) content changes. The results showed that compared with the rats in the sham operation group, the contents of MDA and NO in the liver tissue of the rats in the BDL group were significantly increased, and the contents of SOD were significantly lower; The contents of MDA and NO were significantly reduced, and the contents of SOD were significantly increased (Figure 5). It shows that gastrodin can significantly inhibit the oxidative stress response of rat liver tissue.

"Example 8" Gastrodia elata powder inhibits type Ⅰ collagen α1 promoter activity

According to the method described in "Example 1", the inhibitory effect of Gastrodia elata powder (commercially available product, particle size 250-800 mesh) on type I collagen α1 promoter activity was detected, wherein the concentration gradient of Gastrodia elata powder was 1 μg/ml, 30 μg /ml, 100μg/ml. The results showed that with the increase of gastrodia powder concentration, the fluorescence intensity weakened, and when the gastrodia powder concentration was 30 μg/ml and 00 μg/ml, the fluorescence intensity weakened obviously (Figure 6). It showed that Gastrodia elata powder had obvious inhibitory effect on COL1A1 promoter activity when the concentration was 30μg/ml and 00μg/ml.

"Example 9" Gastrodia elata powder on the inhibitory effect of BDL-induced increase in serum ALT/AST levels in rats

According to the methods in "Experimental Example 2" and "Example 3", the influence of Gastrodia elata powder on BDL-induced serum ALT/AST levels in rats was detected. Wherein, the intragastric administration dose of Gastrodia elata powder is 5 g/kg per rat per day. The results showed that Gastrodia elata powder could significantly inhibit the levels of ALS and AST in rat serum (Table 2). It shows that Gastrodia elata powder can improve liver function in rats.

Table 2. Effect of Gastrodia elata powder on BDL-induced serum ALT/AST levels in rats

**p<0.01 vs. BDL group; ##p<0.01 vs. sham group.

"Example 10" Effect of Gastrodia elata powder on BDL-induced rat liver histopathological structure

According to the method described in "Example 4", the influence of Gastrodia elata powder on the pathological structure changes of rat liver induced by BDL was observed. The results showed that in the sham operation group, the liver tissue cells were arranged regularly, the hepatic lobule was intact, and there was no inflammatory cell infiltration and bile duct hyperplasia; the pathological structure of the rat liver tissue changed significantly after BDL, the bile duct hyperplasia was very obvious, and the tissue necrosis increased significantly; The liver tissue structure of the rats in the Gastrodia elata powder administration group was improved, and the degree of tissue necrosis was significantly reduced (Figure 7). It was shown that Gastrodia elata powder significantly improved the necrosis of liver tissue in BDL rats.

《Example 11》Effect of Gastrodia elata powder on BDL-induced rat liver fibrosis

The paraffin sections were stained with Sirius red to observe the fibrosis of rat liver tissue. The results showed that the degree of fibrosis in the liver tissue of rats was significantly increased after BDL, and the degree of fibrosis was improved after the administration of Gastrodia elata powder, but there was no statistical significance (Figure 8). It shows that Gastrodia elata powder has a certain inhibitory effect on BDL-induced liver fibrosis in rats.

"Example 12" Gastrodia elata powder on the impact of hydroxyproline content in rat liver tissue

According to the method described in "Example 6", the influence of Gastrodia elata powder on the content of hydroxyproline in rat liver tissue was detected. The results showed that compared with the rats in the sham operation group, the content of hydroxyproline in the liver tissue of the rats in the BDL group was significantly increased; decreased, but not statistically significant (Figure 9). It shows that Gastrodia elata powder has a certain inhibitory effect on the production of collagen fibers in rat liver.

Claims (3)

1. application of the Gastrodin in anti-hepatic fibrosis medicines are prepared.

The composition that 2. Gastrodin described in claim 1 is active ingredient to be constituted with pharmaceutically acceptable carrier is in system

Application in standby anti-hepatic fibrosis medicines.

3. the application described in claim 1 or 2, it is characterized in that, Gastrodin peroral dosage form can be made and parenteral in clinical application Formulation；Peroral dosage form is tablet, capsule, powder, particle, and non-oral dosage forms is injection liquid.