WO2007128191A1 - Anti-hepatitis b virus inhibitorand use thereof - Google Patents

Abstract

An anti-hepatitis B virus inhibitor containing coffee extract for treating hepatitis B.

Description

 BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is directed to the field of pharmaceuticals, and in particular, the present invention contemplates an anti-hepatitis B virus (HBV) inhibitor containing coffee extract and its use. The anti-hepatitis B virus inhibitor inhibits HBV DNA replication, inhibits the synthesis and secretion of HBeAg and HbsAg, and is useful as a medicament for the treatment and adjuvant treatment of hepatitis B. Background Art Coffee components include chlorogenic acid (A), caffeic acid (B), and quinine (C), etc., and their chemical structural formulas are as follows. Although there are statistical studies that long-term drinking coffee 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 activity reports, but about coffee and its ingredients It has direct anti-HBV activity, and no research reports have been reported at home and abroad. The results of the present invention demonstrate that coffee, coffee extracts and a class of natural small molecule compounds contained in coffee have significant anti-HBV activity, inhibit HBV DNA replication, and inhibit the synthesis and secretion of HBeAg and HbsAg. Our results confirm that daily coffee consumption may have hepatitis B virus hepatitis caused by hepatitis B virus.

发明内容 本发明的目的是提供一种包含咖啡提取物的抗乙肝病毒抑制剂。 本发明的再一目的是提供上述抗乙肝病毒抑制剂在制备用于治疗乙肝 的药物中的应用。 本发明提供了一种抗乙肝病毒抑制剂， 其包括咖啡提取物和辅剂， 其 中， 基于 100重量份的所述抗乙肝病毒抑制剂， 含有 10 - 90重量份的咖啡 提取物， 余量为辅剂。 在本发明的抗乙肝病毒抑制剂中， 其中所述咖啡提取物为普通咖啡、 低咖啡因咖啡或脱咖啡因咖啡的提取物； 所述辅剂为维生素 C、 羟丙维生 素、 预胶化淀粉、 微粉硅胶、 蔗糖、 乙醇、 防腐剂、 和 /或硬脂酸镁。 根据本发明的技术方案， 所述咖啡提取物通过以下方法制备，

SUMMARY OF THE INVENTION It is an object of the present invention to provide an anti-hepatitis B virus inhibitor comprising a coffee extract. A further object of the present invention is to provide the use of the above-mentioned anti-hepatitis B virus inhibitor for the preparation of a medicament for the treatment of hepatitis B. The present invention provides an anti-hepatitis B virus inhibitor comprising a coffee extract and an adjuvant, wherein 10 to 90 parts by weight of the coffee extract is contained based on 100 parts by weight of the anti-hepatitis B virus inhibitor, and the balance is Adjuvant. In the anti-hepatitis B virus inhibitor of the present invention, wherein the coffee extract is an extract of ordinary coffee, low caffeine coffee or decaffeinated coffee; the auxiliary agent is vitamin C, hydroxypropyl vitamin, pregelatinized starch , micronized silica gel, sucrose, ethanol, preservatives, and / or magnesium stearate. According to the technical solution of the present invention, the coffee extract is prepared by the following method,

1) The coffee is filtered in hot water at 80-100 °C or boiled in hot water 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. a powdered coffee extract, wherein preferably the weight ratio of coffee to water is 1:10; or

2) Weigh the coffee into a round bottom flask, add 95% edible ethanol, the ratio of ethanol volume to coffee mass is 1: 5 ml/g, 'The mixture is heated to 90 ° C for 1.5 hours, filtered, and the filtrate is rotated. Concentrated into an extract on an evaporator at 60 ° C, and spray-dried to obtain a crude powdered coffee extract; or

3) Weigh the coffee into a round bottom flask, add 40-60% edible ethanol, the ratio of the volume of ethanol to the mass of the coffee is 1: 5 ml / g, and the mixture is heated to 90 ° C for 1.5 hours, filtered, The filtrate was concentrated to an extract at 60 ° C on a rotary evaporator, and spray-dried to obtain a crude powdery coffee extract. The present invention also provides the use of the above anti-hepatitis B virus inhibitor for the preparation of a medicament for treating hepatitis B. For the anti-hepatitis B virus inhibitor of the present invention, the dosage forms thereof include tablets (including effervescent tablets:), capsules (including soft tablets, sputum), granules, oral liquids, pills (including dropping pills), and the like. Any of the oral dosage forms. The research of the invention proves that the coffee and coffee extract can effectively inhibit the DNA replication of hepatitis B virus, the synthesis of hepatitis B virus s antigen and e antigen, and coffee is a commonly used beverage food without obvious toxic and side effects, and the extract can be used for treatment. Or a drug that aids in the treatment of hepatitis B. EXAMPLES Example 1 Extraction of coffee crude extracts Common coffee p (Misswell coffee, 100% pure coffee, American Maxwell coffee company) and low caffeine coffee (Mellot classic caffeine coffee, medium roasted , 100% pure Merlot coffee, containing no more than 0.4% of caffeine, German Melitta Group) 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 it into fresh coffee with a coffee pot (a special 18k gold metal filter for coffee pot 4), freeze-dry, and obtain powdered coffee. Or (2) Weigh 100g of coffee into a 1L round bottom flask, add about 500ml of 95% edible ethanol, heat to 90 ° C for 1.5 hours, filter, and filtrate on a rotary evaporator at 60 ° C down Concentrate into an extract and spray-dry to obtain a crude powdered coffee extract; or, (3) Weigh 100g of coffee into a 1L round bottom flask, add 40-60% edible ethanol to about 500ml, and heat to 90 °C. After refluxing for 1.5 hours, it was filtered, and the filtrate was concentrated to an extract at 60 ° C on a rotary evaporator, and spray-dried to obtain a crude powdery coffee extract. Weighing, compared to the original coffee quality, the recovery rate. The content of chlorogenic acid, caffeic acid and quinine (ni) acid in the crude coffee extract was determined by using the respective standard products as a standard curve by HPLC-ESI-MS/MS method. Example 2 to 3 Extraction of crude coffee extract A 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 The following amounts of coffee or decaffeinated coffee extract were used, and the edible and edible plant honeysuckle extract was mixed with an auxiliary material and mixed in the following ratios:

^P coffee or coffee off by non-coffee extract 400 g

Honeysuckle Extract 400g

Hydroxypropylated vitamin 20g

Micronized silica gel 60g

Pregelatinized starch 120g

Magnesium stearate

Made of 1000 capsules The decaffeinated coffee extract, the honeysuckle extract and the additive are uniformly mixed, and 10% pregelatinized starch slurry is used as a binder, wet granulated, dried, mixed with an appropriate amount of magnesium stearate, and compressed into tablets. Example 5 The following amounts of coffee or decaffeinated coffee extract, vitamin C and excipients were used and mixed in the following proportions:

Coffee or Decaffeinated Coffee Extract 400g

Vitamin C 400g

Hydroxypropylated vitamin 20g

Micronized silica gel 48g

Ethanol

Magnesium stearate

Make 900 capsules, mix coffee with hydroxypropyl cellulose and micro-silica gel, use appropriate amount of 70% ethanol 4, humectant, wet granulation, sieve into granules after 40 mesh, dry, add magnesium stearate, mix, load Hard plastic. Example 6 The following amounts of coffee or decaffeinated coffee extract, vitamin C and additives were used and mixed in the following ratios:

Coffee or Decaffeinated Coffee Extract 800g

Vitamin C 400g Sucrose 40 g

Preservative

Distilled water

Prepare 20,000 ml of steamed water, add coffee, stir while stirring, and filter. Further, sucrose and a preservative are dissolved by heating in distilled water, and the above solution is slowly added under stirring, distilled water is added to the whole amount, mixed, refrigerated, filtered, potted, and sterilized to obtain an oral solution. Example 7 The same procedure as in Example 4 was carried out 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 The same procedure as in Example 4 was carried out 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

 First, the purpose of the experiment

 Sample compounds were screened for anti-hepatitis B virus (HBV) activity. The test includes: In the test at the virus-cell level, the cytotoxicity of the sample compound, the secretion of the hepatitis B virus surface and the core antigen, and the effect of the viral nucleic acid (DNA) replication level are examined.

Second, the experimental principle Hepatitis B virus (HBV) transgenic human hepatoma cell HepG2.2.15 cell line can secrete hepatitis B virus particles (containing antigen and DNA) in culture supernatant during culture.

 Under the intervention of antiviral drugs, the levels of HBsAg, HBeAg and viral DNA secreted by the cells into the culture supernatant were detected, and the reference was weak. The content of the drug control group can observe the antiviral activity of the sample drug and simultaneously detect the cytotoxic effect of the sample drug. The MTT method is used to detect the 50% cytotoxic death of the sample drug, and the concentration is 5050. The ELISA method is used to detect the drug to inhibit the secretion of HBsAg and HBeAg, and the real-time PCR method is used to detect 50% of the virus replication of the sample drug. The concentration value is IC50.

 Third, the actual sample

 At the time of use, the concentration of the sample required for the experiment is determined, and each sample is tested for 7 dilutions, and an antiviral drug such as lamivudine is used as a positive control for the experiment.

 Fourth, the actual inspection method

 1. Cultivate the collection of supernatants

The HepG2.2.15 cells were seeded in a 96-well plate, the sample drug was added the next day, the culture solution and the same concentration of the drug were periodically replaced, and the culture supernatant was collected on the eighth day for testing. MTT was added to the cells in the 96-well plate, and after 4 hours, the MTT solution was added to react overnight, and the next day, OD ₅₇₀ was measured on a microplate reader. The calculated sample OD value toxic effects of drugs on HepG2.2.15 cells, cell growth conditions, resulting in half the required amount of cell death in a concentration (CC _50).

 2, handle the collection of fine ^

 HepG2.2.15 cells were inoculated into the culture flask, and the sample drug was added the next day. The culture medium and the same concentration of the sample drug were periodically replaced, and the cells were collected on the eighth day for testing.

3. Detection of HBsAg and HBeAg content in culture supernatant (ELISA method): The kits for detection of HBsAg and HBeAg (Huamei Bioengineering Co., Luoyang, Henan) were used for testing. The sample was added to the coated strip, and an equal amount of the enzyme-labeled conjugate was added, and the plate was washed for 1 hour, and the plate was washed 5 times. The color developing solutions A and B were added, and after 15 minutes, the reaction was terminated, and OD _{45Q/63 was} measured. And calculate the half-inhibition rate (IC ₅₀ ) of the sample against the HBV antigen based on the OD value.

 4. Fluorescence quantitative PCR method for detecting HBV-DNA content in samples:

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

Add appropriate amount of culture supernatant to an equal volume of virus extract, mix and boil, then centrifuge at room temperature for 1000 minutes at 1000 rpm, take appropriate amount of supernatant for PCR amplification, and set up 5 HBV-DNA standard samples. Make a standard curve. According to the detected viral DNA replication value, the inhibition rate of HBV-DNA replication at each concentration of each sample drug was calculated, and then the half-inhibition rate of the sample drug was calculated to obtain (IC _5Q ), and IC ₅ could not be performed _{( The} value of the sample is calculated, given to IC _X and given the corresponding concentration value.

 The PCR primers used for the test are:

 P 1: 5 'ATCCTGCTGCTATGCCTCATCTT3 '

 P2: 5 'AC AGTGGGGAAAGCCCTACGAA3 ' ,

 The PCR probe used for the test is:

 5 'TGGCTAGTTTACTAGTGCCATTTTG3 '

 (2) Intracellular HBV core particles DNA detection:

 Solution A: lml 10 mM Tris (pH 7.5) / l mM EDTA / 50 mM NaCl / 8% sucrose / 0, 25% Nonidet (Nona detergent) P-40, lysate

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

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

Solution D: 10 mM Tris (pH 1.5) 16 mM MgC12, suspension The method used was as follows: the cells were treated with the drug for 8 days, trypsinized, and the DNA in the core particles was extracted from the cells: the digested cells were lysed with solution A, 4. C was centrifuged for 3 min, the supernatant was taken, adjusted to a concentration of 6 mM MgC12, and solution B, 37 was added. C 30min. The core particles were precipitated by adding 330 μL of solution C, and after centrifugation at 4 ° C for 30 min, it was centrifuged for 4 min. The pellet was resuspended in 100 μL of solution D, and digested with DNase I at 37 ° C for 15 min. The obtained sample was subjected to real-time fluorescent quantitative PCR with GAPDH (glycerol casein-3-phosphate dehydrogenase) in the total DNA of HepG2.2.15 cells. Target, detection of changes in DNA levels in HBV core particles.

The test primers were: P3: 5'-GGTATCGTGGAAGGACTCATGAC-3'

 P4: 5 '-ATGCCAGTGAGCTTCCCGTTCAGC-3 ' V. Actual results

 Table 1. Example 1 Preparation of coffee extract and coffee ingredients chlorogenic acid, caffeic acid and quinine (ni) acid inhibited HBV in HepG2.2.15 cells

Extracellular (in culture supernatant) intracellular compound CC50 ⁰ DNA replication HBsAg HBeAg DNA replication

IC ₅₀ (SI) ^A IC50 (SI) IC50 (SI) IC ₅₀ (SI) Ordinary coffee rough 82.4 ±23.2 62.5 ±6.5

 447 ± 110 128 soil 45 (3.5) 141 ± 38 (3.2) ( g / ml) (5.4) (7.2)

 Caffeine coffee

 9.5 ±8.5 41.7 ±20.3 46.1 ±21.89 Crude extract 403 ± 47 136 ± 112(3)

 (42.4) (9.7) (8.7) ( g/ml)

 Lamivudine 0.29 ± 0.04 264.3 ± 35.2 234.4 soil 28.5 0.5 + 0.1

 >1000

 (μΜ) (>3448) (>3.8) (>4.3) (>2000) Adefovir 1.1 ±0.3 3.6 ±0.5 213.2+18.3 1.0 ±0.7

 182+21

 (μΜ) (165) (50.1) (0-9) (182)

 1.2 ±0.4 65.9 ±1.8 1.3 ±0.01 Chlorogenic acid (μΜ) >1000 >1000

 (>833) (>15.2) (>769) Quinine (ni) acid 2.5 + 0.5 293.6 ± 126.4 1.6 ±0.04

 >1000 >1000

 (μΜ) (>400) (3.4) (345)

 116.4 ±38.6 0.7 + 0.2 caffeic acid (μΜ) 500 ± 200 109 + 56 (4.6)

(4.3) (714) is the effect of sample drug on the growth of HepG2.2.15 cells, 50% lethal concentration. ^b IC ₅₀ is the concentration at which the inhibition of DNA copies by the sample reaches 50%. SI is the sample bioactivity selection factor. Table 2. Contents of chlorogenic acid, caffeic acid and quinine in coffee

 Concentration in crude extracts in fresh coffee

 Crude extract source compound concentration

 (mg/g)

 (mg/100 ml) chlorogenic acid 43.3 ± 0.7 108.3

 Ordinary coffee quinine (ni) acid 41.6 ± 1.0 104.0

 Caffeic acid 1.2 ± 0.03 3.0

 Chlorogenic acid 63.4 ± 0.4 158.5

 4氐Caffeine coffee quinine (ni) acid 28.7 ± 0.2 71.8

 Caffeic acid 1.5 ± 0.00 3.2

40g of coffee was heated and filtered through a 18k gold-coated metal filter coffee pot to obtain about 400ml of fresh drinking coffee, which was lyophilized and concentrated into a crude coffee extract. Ordinary coffee was recovered with 10 g of crude coffee extract; low caffeine coffee was recovered with 8.6 g of crude coffee extract. The content of the corresponding active compound in the crude extract is measured and then converted to the content in 100 ml of fresh drinking coffee.

Claims

Claim An anti-hepatitis B virus inhibitor characterized by comprising a coffee extract and an adjuvant. The anti-hepatitis B virus inhibitor according to claim 1, wherein the anti-hepatitis B virus inhibitor contains 10 to 90 parts by weight of the coffee extract, and the balance is an adjuvant. The anti-hepatitis B virus inhibitor according to claim 1 or 2, wherein the coffee extract is an extract of ordinary coffee, low caffeine coffee or decaffeinated coffee. The anti-hepatitis B virus inhibitor according to claim 1, wherein the adjuvant comprises vitamin (, hydroxypropyl vitamin, pregelatinized starch, micronized silica gel, sucrose, ethanol, preservative, and/or hard). Magnesium citrate. The anti-hepatitis B virus inhibitor according to claim 1, wherein the coffee extract is prepared by the following method, 1) The coffee is filtered in hot water at 80 ~ 100 °C or boiled in hot water 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. Powdered coffee extract; or 2) Weigh the coffee into a round bottom flask, add 95% edible ethanol, the ratio of ethanol volume to coffee mass is 1: 5 ml/g, the mixture is heated to 90 ° C for 1.5 hours, filtered, and the filtrate is rotary evaporated. Concentrated into an extract at 60 ° C, spray-dried to obtain a crude powdered coffee extract; or 3) Weigh the coffee into a round bottom, add 40-60% edible ethanol, the ratio of ethanol volume to coffee mass is 1: 5 ml/g, and the mixture is heated to 90 ° C for 1.5 hours, filtered, and the filtrate is filtered. The extract was concentrated to an extract at 60 ° C on a rotary evaporator, and spray-dried to obtain a crude powdered coffee extract. The anti-hepatitis B virus inhibitor according to claim 5, wherein in the method 1), the weight ratio of coffee to water is 1:10. The use of the anti-hepatitis B virus inhibitor of claim 1 for the preparation of a medicament for the treatment of hepatitis B. The dosage form of the anti-hepatitis B virus inhibitor according to claim 1, wherein the dosage form is a tablet, a capsule, a granule, a broth, or a granule.