CN1879727B - Novel anti-hepatitis B virus inhibitor and its application - Google Patents

Abstract

The invention relates to an anti-hepatitis B virus (HBV) inhibitor containing coffee extract and application thereof. Pharmacological tests show that this type of inhibitor can effectively inhibit the replication of hepatitis B virus DNA, the synthesis of hepatitis B virus s antigen and e antigen, and has the advantages of high efficiency and low toxicity. Therefore, it can be used as a treatment or auxiliary treatment for hepatitis B, and has a low cost , safety and convenience features.

Description

Novel anti-hepatitis B virus inhibitor and its application

technical field

The invention relates to the field of medicines, in particular to an anti-hepatitis B virus (HBV) inhibitor containing coffee extract and application thereof. The anti-hepatitis B virus inhibitor inhibits HBV DNA replication, inhibits the synthesis and secretion of HBeAg and HbsAg, and can be used as a drug for treating and assisting hepatitis B.

Background technique

The coffee ingredients include chlorogenic acid (A), caffeic acid (B) and quinine (nickel) acid (C), etc., and its chemical structure is as follows. Although statistical studies have reported that drinking coffee for a long time can reduce the incidence of hepatocellular carcinoma, reduce the incidence of chronic hepatitis (including chronic viral hepatitis), and coffee ingredients have anti-HIV, HSV and other active reports, but about coffee and its ingredients It has a direct anti-HBV activity, and there is no research report at home and abroad. The research results of the present invention prove that coffee, coffee crude extract and a class of natural small molecule compounds contained in coffee have obvious anti-hepatitis B virus activity, inhibit HBV DNA replication, and inhibit the synthesis and secretion of HBeAg and HbsAg. Our findings confirm that daily drinking of coffee may prevent HBV-induced HBV hepatitis.

Contents of the invention

The object of the present invention is to provide an anti-hepatitis B virus inhibitor comprising coffee extract.

Another object of the present invention is to provide the application of the above-mentioned anti-hepatitis B virus inhibitor in the preparation of medicaments for treating hepatitis B.

The invention provides an anti-hepatitis B virus inhibitor, which includes coffee extract and auxiliary agent, wherein, based on 100 parts by weight of the anti-hepatitis B virus inhibitor, it contains 10-90 parts by weight of coffee extract, and the balance is adjuvant.

In the anti-hepatitis B virus inhibitor of the present invention, wherein the coffee extract is the extract of ordinary coffee, decaffeinated coffee or decaffeinated coffee; the adjuvant is vitamin C, hydroxypropyl vitamin, pregelatinized starch , micronized silica gel, sucrose, alcohol, preservatives, and/or magnesium stearate.

According to the technical solution of the present invention, the coffee extract is prepared by the following method,

1) Coffee is filtered or boiled in hot water at 80-100°C for 5-10 minutes to obtain a coffee solution, wherein the weight ratio of coffee to water is 1:15-1:5, and the coffee solution is freeze-dried to obtain a powder Shaped coffee extract, wherein, preferably, the weight ratio of coffee to water is 1:10; or

2) Weigh the coffee and place it in a round bottom flask, add 95% edible ethanol, the ratio of ethanol volume to coffee mass is 1:5ml/g, heat the mixture to 90°C for reflux for 1.5 hours, filter, and put the filtrate on a rotary evaporator , Concentrate at 60°C to form an extract, and spray dry to obtain a powdered crude coffee extract; or

3) Weigh the coffee and place it in a round bottom flask, add 40-60% edible ethanol, the ratio of ethanol volume to coffee mass is 1:5ml/g, heat the mixture to 90°C for reflux for 1.5 hours, filter, and the filtrate is rotovaped Concentrate on an instrument at 60°C to form an extract, and spray dry to obtain a powdered coffee crude extract.

The present invention also provides the application of the above-mentioned anti-hepatitis B virus inhibitor in the preparation of medicaments for treating hepatitis B.

For the anti-hepatitis B virus inhibitor of the present invention, its administration form includes tablet (comprising effervescent tablet), capsule (comprising soft capsule), electuary, oral liquid, pill (comprising dropping pill) etc. dosage form.

The study of the present invention proves that coffee and coffee extract can effectively inhibit the replication of hepatitis B virus DNA and the synthesis of hepatitis B virus s antigen and e antigen. Coffee is a commonly used beverage and food without obvious toxic and side effects, and its extract can be used to make therapeutic Or adjuvant drugs for the treatment of hepatitis B.

Detailed ways

Example

Embodiment 1 extracts coffee crude extract

Regular coffee (Maxwell Coffee, 100% pure coffee, Maxwell Coffee Company, USA) and decaf coffee (Melaleuca Classic Decaf Coffee, medium roast, 100% pure Melaleuca coffee, containing no more than 0.4% caffeine , Germany Melitta Group) was purchased from Metro Supermarket. The extraction method of the crude extract is as follows: (1) Weigh 40g of coffee, add about 400ml of water, boil fresh coffee with a coffee pot (18k gold-coated metal filter for coffee pot), freeze-dry, and obtain powdered coffee crude extract Or, (2) Weigh 100g of coffee and place it in a 1L round bottom flask, add about 500ml of 95% edible ethanol, heat to 90°C for reflux for 1.5 hours, filter, and carry out the filtrate on a rotary evaporator at 60°C Concentrate into an extract, spray dry to obtain a powdered crude coffee extract; or, (3) Weigh 100g of coffee into a 1L round bottom flask, add about 500ml of 40-60% edible ethanol, heat to 90°C and reflux for 1.5 hours, filtered, and the filtrate was concentrated on a rotary evaporator at 60°C to form an extract, and spray-dried to obtain a powdered coffee crude extract. Weighing, compared with the quality of the original coffee, to get the recovery rate. The contents of chlorogenic acid, caffeic acid and quinine (ninic) acid in the crude coffee extract were compared with respective standard substances to make a standard curve, and the HPLC-ESI-MS/MS method was used for content determination.

Example 2-3 extract coffee crude extract

The crude coffee extract was extracted in the same manner as in Example 1, except that the weight ratio of coffee to water was 1:15 and 1:5.

Example 4

Choose the following dosage of coffee or decaffeinated coffee extract, and use the medicinal and edible plant honeysuckle extract with auxiliary materials, and mix in the following ratio:

Coffee or Decaffeinated Coffee Extract 400g

Honeysuckle Extract 400g

Hydroxypropyl Vitamin 20g

Micropowder silica gel 60g

120g pregelatinized starch

Magnesium Stearate Appropriate amount

             

Made into 1000 capsules

Mix decaffeinated coffee extract, honeysuckle extract and additives evenly, use 10% pregelatinized starch slurry as binder, wet granulate, dry, add appropriate amount of magnesium stearate to mix, and press into tablets.

Example 5

Choose the following amounts of coffee or decaffeinated coffee extract, vitamin C and excipients, and mix in the following proportions:

Coffee or Decaffeinated Coffee Extract 400g

Vitamin C 400g

Hydroxypropyl Vitamin 20g

Micropowder silica gel 48g

Alcohol Appropriate amount

Magnesium Stearate Appropriate amount

               

Made into 900 capsules

Mix coffee with hydroxypropyl cellulose and micropowder silica gel, use appropriate amount of 70% ethanol as wetting agent, wet granulate, sieve through 40 mesh to form granules, dry, add magnesium stearate, mix, and pack into hard capsules.

Example 6

Choose the following amounts of coffee or decaffeinated coffee extract, vitamin C and additives and mix in the following proportions:

Coffee or Decaffeinated Coffee Extract 800g

Vitamin C 400g

Sucrose 40 grams

Preservatives Appropriate amount

Appropriate amount of distilled water

             

Made 20000ml

Take an appropriate amount of distilled water, add coffee, stir while adding to dissolve, and filter. In addition, heat and dissolve sucrose and preservatives with distilled water, slowly add the above solution under stirring, add distilled water to the full amount, mix, refrigerate, filter, seal in pots, and sterilize to obtain oral liquid.

Example 7

It was formulated in the same manner as in Example 4, except that 200 g of coffee or decaffeinated coffee extract and 600 g of honeysuckle extract were used based on 1000 g of the total mixture.

Example 8

It was formulated in the same manner as in Example 4, except that 600 g of coffee or decaffeinated coffee extract and 200 g of honeysuckle extract were used based on 1000 g of the total mixture.

Experimental Example

1. Purpose of the experiment

Anti-hepatitis B virus (HBV) activity screening of sample compounds. The test includes: in the virus-cell level test, the cytotoxicity of the sample compound, the effect on the secretion of the surface and core antigens of hepatitis B virus, and the replication level of viral nucleic acid (DNA) are detected.

2. Experimental principle

Hepatitis B virus (HBV) transgenic human liver cancer cell line HepG2.2.15 can secrete hepatitis B virus particles (containing antigen and DNA) in the culture supernatant during culture.

Under the intervention of antiviral drugs, the content of HBsAg, HBeAg and viral DNA secreted by cells into the culture supernatant can be detected, and the antiviral activity of the sample drug can be observed with reference to the content of the control group without drug addition. Toxic effects. Using the MTT method to detect the numerical concentration of 50% cytotoxic death caused by the sample drug is CC50; use the ELISA method to detect the sample drug to inhibit the secretion of HBsAg and HBeAg, and use the fluorescence quantitative PCR method to detect the sample drug to inhibit 50% of the amount of viral DNA replication Concentration values are IC50.

3. Experimental samples

When ready for use, the drug concentration of the sample required for the experiment was prepared, and each sample drug was tested with 7 dilution concentrations, and antiviral drugs such as lamivudine were set as positive control drugs for the experiment.

4. Experimental method

1. Collection of culture supernatant

HepG2.2.15 cells were inoculated in a 96-well plate, the sample drug was added the next day, the culture medium and the sample drug of the same concentration were replaced regularly, and the culture supernatant was collected on the eighth day for testing. Add MTT to the cells in the 96-well plate, add MTT lysate to react overnight after 4 hours, and measure OD ₅₇₀ on a microplate reader the next day. Calculate the toxic effect of the sample drug on HepG2.2.15 cells according to the OD value, affect the condition of cell growth, and cause the required concentration (CC ₅₀ ) of half the amount of cell death.

2. Collection of processed cells

HepG2.2.15 cells were inoculated in culture flasks, and the sample drug was added the next day. After regular replacement of the culture medium and the sample drug at the same concentration, the cells were collected on the eighth day for testing.

3. Detection of HBsAg and HBeAg content in culture supernatant (ELISA method):

HBsAg and HBeAg detection kits (Huamei Bioengineering Company, Luoyang, Henan) were used for detection. Add samples to the coated strip plate, and add an equal amount of enzyme-labeled conjugate, react at 37°C for 1 hour, wash the plate, and repeat 5 times. Chromogenic solutions A and B were added, and the reaction was terminated after 15 minutes. OD _450/630 was measured, and the half inhibitory rate (IC ₅₀ ) of the sample to HBV antigen was calculated according to the OD value.

4. Detection of HBV-DNA content in samples by fluorescent quantitative PCR method:

(1) Detection of HBV DNA in extracellular culture supernatant:

Take an appropriate amount of culture supernatant and add it to an equal volume of virus extract, mix well and boil, then centrifuge at room temperature at 10,000rpm for 5 minutes, take an appropriate amount of supernatant for PCR amplification, and set 5 HBV-DNA standard samples at the same time, Make a standard curve. And according to the viral DNA replication value that detects gained, calculate the inhibitory rate to HBV-DNA replication when each concentration of each sample drug, then carry out sample drug half inhibitory rate and calculate and obtain it (IC ₅₀ ), can not carry out IC ₅₀ value For the calculated samples, give the IC _X representation and give the corresponding concentration values.

The PCR primers used in the test are:

P1: 5'ATCCTGCTGCTATGCCTCATCTT3'

P2: 5'ACAGTGGGGAAAGCCCTACGAA3',

The PCR probes used in the test are:

5'TGGCTAGTTTACTAGTGCCATTTTG3'

(2) Detection of DNA in HBV core particles in cells:

Solution A: 1ml 10mM Tris (pH 7.5)/1mM EDTA/50mM NaCl/8% sucrose/0.25% Nonidet (Nonidet detergent) P-40, lysate

Solution B: DNase I (50g/ml) and RNase A (20g/ml), extract

Solution C: 26% polyethylene glycol/1.4M NaCl/25mM EDTA, precipitation solution

Solution D: 10mM Tris(pH 7.5)/6mM MgCl2, suspension

The method adopted is: cells treated with drugs for 8 days, digested with trypsin, and DNA in the core particles extracted from the cells: the digested cells were lysed with solution A, centrifuged at 4°C for 3min, the supernatant was taken, adjusted to a concentration of 6mM MgCl2, added Solution B, 37°C for 30min. Add 330 μl solution C to precipitate core particles, centrifuge for 4 minutes at 4°C for 30 minutes. The precipitate was resuspended in 100 μl solution D, and DNase I was added to digest at 37°C for 15 minutes. The obtained samples were subjected to real-time fluorescent quantitative PCR, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) in the total DNA of HepG2.2.15 cells was used as the internal standard. Detection of changes in DNA levels in HBV core particles.

The test primer is: P3: 5'-GGTATCGTGGAAGGACTCATG AC-3'

P4: 5'-ATGCCAGTGAGCTTCCCGTTCAGC-3'

5. Experimental results

Table 1, the coffee extract prepared by Example 1 and the coffee component chlorogenic acid, the inhibitory effect of caffeic acid and quinine (nickel) acid on HepG2.2.15 cell HBV

^a CC ₅₀ is the effect of the sample drug on the growth of HepG2.2.15 cells, 50% lethal concentration.

^b _IC50 is the concentration at which the sample inhibits DNA copying by 50%. SI is the selection coefficient of sample biological activity.

Table 2. Contents of chlorogenic acid, caffeic acid and quinine (ninic) acid in coffee

40g of coffee is heated and filtered through an 18k gold-coated metal filter coffee pot to obtain about 400ml of fresh drinking coffee, which is then freeze-dried and concentrated into a crude coffee extract. Ordinary coffee is recovered to obtain 10g of coffee crude extract; decaffeinated coffee is recovered to obtain 8.6g of coffee crude extract. Detect the content of the corresponding active compound in the crude extract, and then convert it to the content in 100ml of fresh drinking coffee.

Claims (3)

1. coffee-extract is used for the treatment of purposes in the medicine of hepatitis B in preparation, and described coffee-extract prepares by the following method:

1) coffee filters or hot water boiled 5～10 minutes at 80～100 ℃ hot water, obtains coffee solution, and wherein, the weight ratio of coffee and water is 1: 15～1: 5, and lyophilizing coffee solution, acquisition powdered coffee extract; Or

2) taking by weighing coffee places in the round-bottomed flask, add 95% edible ethanol, the ratio of ethanol volume and coffee quality is 1: 5ml/g, mixture heated to 90 ℃ refluxed 1.5 hours, filter, filtrate is carried out spray drying and is got the powdered coffee crude extract on the Rotary Evaporators, is condensed into extractum under 60 ℃; Or

3) taking by weighing coffee places in the round-bottomed flask, add the 40-60% edible ethanol, the ratio of ethanol volume and coffee quality is 1: 5ml/g, mixture heated to 90 ℃ refluxed 1.5 hours, filter, filtrate on Rotary Evaporators 60 ℃ be condensed into extractum, carry out spray drying and get the powdered coffee crude extract.

2. purposes according to claim 1 is characterized in that, described coffee is normal coffee, low-caffeine coffee or decaf.

3. purposes according to claim 1 is characterized in that, in method 1) in, the weight ratio of coffee and water is 1: 10.