KR20030092448A - Composition comprising an extract of Sambucus williamsii HANCE var. coreana NAKAI inhibiting HCV activity - Google Patents

Abstract

PURPOSE: Provided is a composition comprising an extract of Sambucus williamsii HANCE var. coreana NAKAI having HCV inhibiting activity. Therefore, it is useful for medicine and health food supplements for the prevention and treatment of HCV. CONSTITUTION: A composition is characterized by comprising an extract of Sambucus williamsii HANCE var. coreana NAKAI having prevention and treatment effects on HCV. The extract is obtained by using a polar solvent, such as water and butanol or nonpolar solvent, such as ethylacetate and chloroform solvent. The composition contains 0.5-50% of the extract, based on the total weight of the composition.

Description

Composition comprising an extract of graft alley with inhibitory hepatitis activity {Composition comprising an extract of Sambucus williamsii HANCE var. coreana NAKAI inhibiting HCV activity}

The present invention relates to pharmaceutical compositions and dietary supplements effective for hepatitis C disease.

In general, hepatitis is a disease caused by drug, alcohol, or virus that causes abnormal liver function. It is clinically measured on the scale of symptoms such as jaundice, fever, abdominal pain, and increase of amino transferase in serum. If the virus is the cause, it can be diagnosed by immunoassay of antigens and antibodies against the causative virus.

Hepatitis A and B viruses, the main cause of viral hepatitis, were isolated and identified in the 1970s, and development of diagnostic reagents and vaccines in 1989 led to significant progress in the diagnosis and prevention of viral hepatitis. However, despite the development of these diagnostic reagents and vaccines, viral hepatitis patients still developed after transfusion, which has been classified as non-Hepatitis A and Non-Hepatitis B (NANBH) viruses different from the existing hepatitis viruses (Alter. HJ et al; Science, 2, p838, 1975), 10 years after the discovery, the NANBH virus was first isolated from chimpanzee serum by Chiron, USA, and subsequently identified as hepatitis C virus (Choo). QL et al; Science, 244, pp 359-362, 1989).

Hepatitis C virus (abbreviated as "HCV") is a virus that is a major cause of hepatitis known to cause non-A or non-B hepatitis. In addition to causing cirrhosis or the transition to liver cancer (Alter. HJ et al; New Eng. J. Med., 327, pp1899-1905, 1992), the leading cause of viral hepatitis associated with cirrhosis and liver cancer It is known that about 70 to 80% of hepatitis that develops after blood transfusion is caused by HCV.

HCV virus can be recovered by inoculating a chimpanzee with plasma from patients with non-A or non-B hepatitis. Thus obtained HCV has a single linear RNA (single linear RNA) of one positive strand, the RNA has a gene of the positive strand ribonucleic acid form of 9400 bases, and several structural and nonstructural proteins One polypeptide (Polypeptide) consisting of (Hough M. et al; European Patent Publication (A1) 0318216, 1989 Publication: Okamoto H et al; J. Gen. Virol, 60, p1679, 1990). Given this genetic structure, HCV shows significant similarity to flaviviruses or pestiviruses and has been classified as a new genus belonging to Flaviviridae. The HCV-derived polypeptide encoded from the gene is from the amino terminus to Core-envelope (E1) -envelope / non-structure 1 (E2 / NS1) -non-structure 3 (NS3) -non-structure 4 (NS4) -non-structure 5 (NS5) protein (Choo, et al; Proc. Natl. Acad. Sci. USA, 88, pp2451-2455, 1991: Kato, et al; Proc. Natl. Acad. Sci) USA, 87, pp9524-9528, 1990: Simonetti, et al; An. Int. Med., 116, pp97-102, 1992).

It is estimated that about 2% of the world's population is infected, and the number of new HCV infections is estimated at about one million people every year.In particular, in Africa, Japan and Korea, 1.5 to 2.0% of the total population is chronic carriers. It is known to be There is currently no vaccine or effective treatment, and the infection continues to spread. Accordingly, the development of therapeutic agents is one of the factors that increases the mortality rate due to liver cancer. The global market for antiviral and vaccines is $ 5.3 billion in 1998, and by 2008, the market for antiviral drugs is expected to grow to $ 11 billion. Currently, the market for HCV therapeutics is $ 1 billion, with interferon and ribavirin (co-developed by ICN and Shering). It is estimated that the development of HCV therapeutics will be very commercially available as it is expected to grow with the increase of hepatitis C virus infection in the future.

Currently, there are no treatments for the effective treatment of chronic infection of HCV, and the administration of α-interferon as a treatment for HCV has been carried out. However, recently, the treatment has recently been performed using the interferon resistance sequence. It appears that there is no effect on HCV which has, and the development of a new therapeutic agent is required. The new form of HCV therapy will be most effective with inhibitors of proteins such as proteases or RNA polymerases for the treatment of RNA viruses such as the AIDS virus.

On the other hand, the graft alley used in the present invention (Sambucus williamsii HANCE var.coreana NAKAI) is also called elder tree, elder tree, is a deciduous tree. Stimulation of the graft alley in the stomach of the mouse 20g / ㎏ sedative effect, strength is less than morphine, but better than sulpyrin (sulpyrin), after administration the rat is stable. It is effective in treating gout, improves blood circulation and stops pain. It is known to be effective in treating musculoskeletal pain, low back pain, hand and foot pain, fracture, arthritis, neuralgia, edema, gout, nephritis, nervous breakdown, postpartum anemia, long-term pain from bruises and wound bleeding caused by rheumatism. Doha Chinese Herbal Dictionary, Younglim History, pp.941-942, 1998).

Although grafting is known to be beneficial to the liver through folk remedies, there has been no report on the therapeutic effect of grafting on hepatitis C in the literature and research reports.

Therefore, the present inventors completed the present invention by finding out that the polar and non-polar solvent soluble extracts of the grafts show strong inhibitory activity against proteolytic enzymes of HCV, while trying to obtain a therapeutic agent for HCV from Korean native medicinal plants. It was.

It is an object of the present invention to provide a pharmaceutical composition and dietary supplement showing a prophylactic and therapeutic effect of hepatitis C.

Figure 1a is a diagram showing the overall protein pattern by Coomassie blue staining after expression of the replication enzyme of hepatitis C virus (HCV), 1b is Western blotting of the replication enzyme of hepatitis C virus (HCV) (Western blotting) shows the results,

2 is a diagram showing the relative activity effect on the total activity of 36 extracts (50 μM) obtained from 14 plants and the enzyme activity (2.5 × 10 ⁴ CPM set at 100% basis) measured in the absence of the extract. ,

Figure 3 is a diagram showing the relative activity effect of the extract of Graft Allyl ethyl acetate at a concentration of 500μM,

Figure 4 is a diagram showing the relative activity effect of each concentration of Graft Allyl ethyl acetate extract,

Figure 5 is a diagram showing the cytotoxic effect of each concentration of the grafted ethyl acetate extract.

In accordance with the above object, the present invention provides a polar and non-polar solvent extract of Sambucus williamsii HANCE var. Coreana NAKAI effective for the prevention and treatment of hepatitis C.

In addition, the present invention provides a composition for the prevention and treatment of hepatitis C, including the extract of the graft. The grafted extract refers to extracts sequentially extracted using a polar solvent such as water, methanol, butanol, and the like, and a non-polar solvent such as ethyl acetate, chloroform, and the like.

Lower alcohol extracts of Sambucus williamsii HANCE var.coreana NAKAI were sequentially extracted with ethyl acetate, butanol, and water, and the purified HCV transcriptase, activity titer and inhibitory effect were measured. .

Hereinafter, specifically describing the separation process of the graft alley extract,

A first step of extracting the grafts with a polar solvent such as methanol, extracting with hot water, and then concentrating under reduced pressure to obtain X;

A second step of dissolving the X in a nonpolar solvent such as ethyl acetate and suspending it, followed by filtration to separate the ethyl acetate soluble extract layer;

A third step of sequentially fractionating and filtering using a polar solvent such as butanol, water, and the like, to obtain a butanol soluble extract and a water soluble extract;

And a fourth step of concentrating and drying the ethyl acetate soluble extract, butanol soluble extract, and water soluble extract under reduced pressure.

Hepatitis C preventive and therapeutic composition of the present invention, the extract comprises 0.5 to 50% by weight based on the total weight of the composition.

The composition comprising the grafted graft extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The composition comprising the extract of the present invention may further comprise a suitable carrier, excipient and diluent according to conventional methods.

Carriers, excipients and diluents that may be included in the composition comprising the extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Compositions comprising extracts according to the invention are formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be used.

The amount of the extract of the present invention may vary depending on the age, sex, and weight of the patient, but may be administered once to several times in an amount of 0.1 to 100 mg / kg. In addition, the dosage of the extract can be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The composition containing the extract of the present invention can be used in various ways, such as drugs, foods and drinks for the prevention and treatment of hepatitis C. Examples of foods to which the present graft allotment extract can be added include various foods, beverages, gums, teas, vitamin complexes, and health supplements.

The graft alley extract of the present invention is a drug that can be used with confidence even when taken for a long time for the purpose of prevention because there is little toxicity and side effects.

The extract of the present invention may be added to food or beverages for the purpose of preventing hepatitis C. At this time, the amount of the extract in the food or beverage is generally added to the health food composition of the present invention 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 10 g, preferably based on 100 ml Can be added in a ratio of 0.3 to 1 g.

The health beverage composition of the present invention is not particularly limited in the liquid component except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention is described in more detail based on the following examples, although the present invention is not limited thereto.

Example 1. Plant Extract Preparation and Library Construction

After extracting 21 samples (100g) tested by Korea Institute of Herbal Medicine (100g), methanol extracting step was completed, extracting useful materials sequentially with ethyl acetate, butanol, and water, and completed the extraction library consisting of 63 extracts. It was.

The samples used for the primary extraction were selected from the seaweed, wild berry leaves, juniper tree, wild berry tree, wild oyster mushroom, sesame tree, kkujippong, sesame leaf, black sea buckwheat, and chopped, 100 g of each sample, and 0.2 liter of 20% methanol. The extract obtained by heating at reflux twice at 50 ° C. for 2 hours was concentrated under reduced pressure with a vacuum concentrator (Eyela, N-1000, Japan) to obtain a dried extract, which was suspended in 50 ml of water and filtered. Filtered by Whatman, USA).

The extract obtained above was fractionated twice by adding 0.1 L of ethyl acetate, and the ethyl acetate soluble extract layer and the water soluble extract layer were separated. The obtained water-soluble extract layer was further fractionated by sequentially adding butanol and water, and as a result, ethyl acetate soluble extract, butanol soluble extract and water soluble extract were obtained.

The second extracted sample list was performed by the same method as in the first extraction with ginger tree, grafting tree, cattle tree, Mokcheoncho, Cheongmirae vine, wild mulberry leaf, sewage, soybeans, Jules, Bokyeong, and Schisandra chinensis per each sample. The ethyl acetate soluble extract, butanol soluble extract and water soluble extract eluted in three solvents were obtained.

Thus, 63 kinds of extracts obtained in the first and second concentrated under reduced pressure and dried to build an extract library.

Reference example. Expression Purification of HCV Recombinase Recombinant Protein

For expression of the NS5B protein, a hepatitis C virus (HCV) transcriptase, these genes were cloned into the pTrcHisB vector, transformed into E. coli BL21, and recombinant proteins fused with His (histidine) × 6. Expression.

The selected E. coli transformants were incubated at Luria-Bertani medium (with 100 μg / ml ampicillin) and at 37 ° C., and the absorbance at 600 nm was 0.5 mM IPTG (isopropyl-β-D). -thiogalactopyranoside) was incubated at 25 ° C. in a medium to which 6- 12 hours of protein expression was incubated and the cells were recovered from the culture. Cells obtained by centrifugation were washed once with PBS (phosphate buffered saline), followed by lysis buffer (50 mM Na-phosphate buffer (pH 8.0), 1% Nonidet P-40, 10 mM mercaptoethanol, 10% glycerol, 10 mM The cells were suspended by sonication in suspension, and centrifuged (12000 rpm, 30 minutes, 4 ° C.) to remove precipitated cell residue and supernatant was taken. The supernatant obtained therefrom was adsorbed onto an affinity column, Ni2 + -NTA (nitrilotriacetic acid) column (Qiagen, USA), eluted with 100-500 mM imidazole, and buffer A (50 mM Tris-HCl) pH 8.0), 50 mM sodium chloride, 5 mM magnesium chloride, 1 mM DTT) and bind to a heparine-agarose column, an ion-exchanger column treated with the same buffer, and 0.1 to Eluted with Buffer A with 1.0 M sodium chloride. The obtained fractions were finally purified using an ion exchange column, Sp-Sepharose column (Amersham Pharmacia Biotech) in a similar manner. The first extract (crude extract), the precipitate, the supernatant and the purified protein were electrophoresed (10% SDS-PAGE) and stained with comasie blue (commassie blue) to confirm the expression of the protein. Protein separated on 10% SDS-PAGE gel was transferred to Nitrocellulose membrane (Amersham Pharmacia Biotech) using protein electroblotter (Hoefer) and recognized His residues attached to N-terminus. The antibody (anti-His6 antibody, Santa Cruz biotechnology) was used as the primary antibody and peroxidase-labeled secondary antibody (Roche) was added to react with each other. Western blot analysis was performed using a lotting kit (ECL western blotting kit, Amersham Pharmacia Biotech). The results showed that NS5B was present at high concentration in the purified protein fraction as shown in FIGS. 1A and 1B. This purified fraction was used for medicinal plant extract screening to inhibit the activity of hepatitis C virus transcriptase.

Experimental Example 1. Determination of Hepatitis C Virus Replication Enzyme Activity of Extracts

Conditions for measuring the activity of RNA-dependent RNA transcriptase, a hepatitis C virus transcriptase, can be found in the literature (JW Oh et al .; Journal of Virology, 73 (9), pp7694-7702, 1999).

In the reference example, the active hepatitis C virus transcriptase protein NS5B and homopolymeric RNA were used to evaluate the activity potency of plant-specific toxic plant extracts in Korea. Homozygous RNA was synthesized using polyA RNA (Pharmacia) and oligo (U) 20 primer (Pharmacia), which act as a substrate in a primer dependent manner against NS5B protein of hepatitis C virus. Details of the reaction are as follows.

1 μg of polymorphic RNA poly (A) (Pharmacia) and 2 pmol of NS5B enzyme and 10 μM ATP, CTP, GTP and UTP, respectively, were added to the reaction, and 10 μCi of ³² P-UTP or ³ was used to label the synthesized RNA. H-UTP (3000 Ci / mmol, NEN Life science Products) was added to buffer buffer (50 mM Tris-hydrochloric acid (pH 8.0), 50 mM sodium chloride, 100 mM potassium glutamate, 5 mM magnesium chloride, 1 mM dithiothreitol (DTT), 20 ㎍ / ㎖ actinomycin D (Sigma), 20 units (RNase inhibitor, Promega, 10% glycerol) was added to the reaction at 25 ℃ for 1 hour. Subsequently, the polymer RNA reactant synthesized with 10 μg of calf thymus DNA was precipitated with 5% trichloroacetic acid (TCA) and filtered with a GF / C glass filter (Whatman). It was. Wash the filter with cold 5% TCA solution, wash with 1% TCA solution, wash with 95% ethanol and dry it, and then use the scintillation counter (LC 600IC, Beckman) Was measured.

Experimental Example 2. Examination of HCV replication enzyme inhibitory effect of the second sample extract

Example 1 HCV replication enzymes for the ethyl acetate extracts for three primary samples (acid blue-leaf leaves, juniper, seaweed), three kinds of solvent extracts for 11 secondary samples, a total of 36 extracts Determination of the activity inhibitory activity titer showed that the sample was estimated at 50 μM final concentration and 100 molecular weight than the activity of the enzyme (2.5 × 10 ⁴ CPM was set as 100% standard) measured in the absence of the extract. Concentration)) Three samples showing low activity could be detected (graft Ethyl Acetate extract, Zhulul ethyl acetate extract, Bokryeong water extract) (see Fig. 2).

Experimental Example 3. Evaluation of titer on the extract of Graft Allyl Ethyl Acetate

It showed inhibition of HCV transcriptase activity at the final concentration of 25 μM extract in the primary sample in Example 1, inhibited at the second screening (50 μM final concentration) in the selected scavenger ethyl acetate extract, courageous butanol extract and Experimental Example 2 Reconfirmation of inhibitory effect of Glycophyll ethyl acetate extract, Zulgo ethyl acetate extract, and Bokyeong water extract showed 500 g effects, showing that 100% of the total activity of the positive control HCV clone enzyme was 100%. It was confirmed that the effect of HCV synthase was significantly inhibited to less than 10% at a high concentration of 500μM (see Figure 3).

Experimental Example 4. Examination of effect by concentration on ethyl acetate extract

In order to see the inhibitory effect of HCV replication enzyme activity according to the concentration of ethyl acetate extract, the activity was measured according to Experimental Example 1 for each series of final concentrations of 0μM, 50μM, 100μM, 250μM, 500μM. The extract was shown to inhibit at least 50% of HCV transcriptase from in vitro testing when 100-250 μM was added at final concentration. One of a molecular weight within the useful component extract below estimated as 100 and was calculated the molar concentration (concentration required to inhibit 50% activity) typically several hundred to as saenggakdoem there are thousands of materials, IC ₅₀ after further purification the extract is 1μM There seems to be a possibility of finding a substance.

Experimental Example 5. Cytotoxicity Test of Different Grafts of Ethyl Acetate Extract by Concentration

Huh7, a liver cancer cell, was treated with increasing concentrations of ethyl acetate acetate extract, and then the toxicity to the cells was determined by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide. Thiazolyl blue) test method was confirmed that the cytotoxicity is not large.

Huh7 cells were put into 10- ⁵ amendments in 96-well plates, 100 μM and 250 μM of grafted ethyl acetate extract were incubated for 16 hours, and 25 μl of 2 mg / ml MTT solution was added to each well and further incubated for 4 hours. . After centrifugation, the supernatant was removed, 100 μl of DMSO was added, the precipitate layer was dissolved for 30 minutes, shaken, and the absorbance (Optical Density; OD) was measured at a wavelength of 540 nm. Using the resulting absorbance value, the concentration for the OD value corresponding to half of the control OD value is IC ₅₀ (concentration required for 50% activity inhibition) and compared and reported as 100% cytotoxicity.

5, the cytotoxicity of the grafted ethyl acetate extract showing IC ₅₀ was lower than that of the control group, and 100 μM was lower than 250 μM in comparison with the final concentration.

In conclusion, the graft alley extract and the polar and non-polar soluble extracts were found to inhibit the activity of hepatitis C and reaffirmed that it is effective in the prevention and treatment of hepatitis C.

The grafting plant extract according to the present invention and the pharmaceutical preparations containing the polar and non-polar soluble extract thereof is a herbal that has been widely used from conventional, there is no toxicity problem, can be effectively used for the prevention and treatment of hepatitis C disease.

Claims (10)

Soluble extract of polar and nonpolar solvents of Maclura tricuspidata CARR. Showing the prophylactic and therapeutic effect of hepatitis C. The extract of claim 1, wherein the polar solvent is water, butanol and the nonpolar solvent is ethyl acetate. Pharmaceutical composition for the prevention and treatment of hepatitis C, comprising grafting extract as an active ingredient. The composition of claim 3, wherein the grafting extract is extracted with a polar solvent selected from water or butanol. 4. The composition of claim 3, wherein the grafting extract is extracted with a nonpolar solvent selected from ethyl acetate or chloroform. A pharmaceutical composition for the prevention and treatment of hepatitis C, comprising a polar and non-polar solvent soluble extract of the grafting graft of claim 1. A pharmaceutical composition for the prevention and treatment of hepatitis C, comprising water extract, butanol extract or ethyl acetate extract of the grafts of claim 2. 8. A composition according to claim 7, comprising 0.5-50% of said extract relative to the total weight of the composition. A dietary supplement comprising an extract or a polar solvent soluble extract, a nonpolar solvent soluble extract, and a food acceptable additive for foods that exhibit the prophylactic and therapeutic effect of hepatitis C. 10. The health supplement food according to claim 9, which is a health drink.