KR20060016551A - Plant extracts for inhibition of alcoholic liver fibrosis - Google Patents

Abstract

The present invention relates to a plant-derived extract for preventing and treating liver fibrosis or cirrhosis by alcohol.

The present invention is a methanol extract of Comus officinalis , Pinus koraiensis and Green tea Camellia Sinensis . By showing the effect of preventing and protecting liver fibrosis or cirrhosis of the present invention, the present invention can be used as a material for a hepatoprotective functional food, a food additive or a therapeutic agent.

Description

Plant Extracts for Alcoholic Liver Fibrosis Prevention and Treatment {Plant Extracts for Inhibition of Alcoholic Liver Fibrosis}

1 is a diagram showing the type I collagen production inhibitory effect of cornus, pine needles, green tea methanol extract on HSC-T6 cells.

Figure 2 is a chart comparing the inhibitory effect of hepatic lactate, pine needles, green tea on the hepatic lipid peroxidation in acute liver cirrhosis-induced rat model.

Figure 3 is a chart comparing the inhibitory effect of collagen production of cornus, pine needles, green tea in acute liver cirrhosis-induced rat model.

Figure 4 is a liver tissue micrograph showing the protective effect of liver, pine needles, green tea in acute liver cirrhosis-induced rat model.

The present invention relates to a plant-derived extract for preventing and treating hepatic fibrosis or cirrhosis by alcohol, and by inhibiting hepatic fibrosis, which is a preliminary stage of cirrhosis of the final stage in the pathological path of liver disease, it is possible to control cirrhosis more effectively. It relates to a plant-derived extract having an activity.

Liver fibrosis is the leading cause of death from liver disease. In Western society, 50% of the cirrhosis of liver cirrhosis is caused by excessive alcohol intake, and 83% of men are over 20 years old in Korea. In addition, in Korea, the drinking population of women is rapidly increasing, so it is urgent to research and develop alcoholic liver fibrosis preventive and therapeutic substances (see Greenwell P. Alcohol.Clin.Exp.Res., 23, 3, 930-). 933, 1999).

The oxidation mechanism of alcohol in liver is divided into two ways: ① alcohol dehydrogenase (ADH) pathway and ② microsomal ethanol oxidizing system (MEOS). Acetaldehyde, the result of each pathway, is highly toxic and is known to be a major source of alcoholic liver disease. This substance is known to cause hepatic fibrosis by inducing the modification of many enzymes and proteins in vivo and stimulating type I collagen synthesis in hepatic stellate cells (HSCs). Recently, the action of hepatic fibrosis-related alcohol has some mechanisms for activating HSCs by inducing c- myb , NF-κB or activating IGF, TGFβ _{1 and} cytokine by oxidative stress. In addition, fat accumulation in the liver after alcohol consumption is a representative case of progression to chronic liver disease. The increase of triglyceride in serum is an indicator of the increase of VLDL in the liver. Metabolism of various fatty metabolites and peroxide lipids in the liver They are also responsible for the generation of aldehydes (Svegliati-Baroni, G. et al. Hepatology, 23, 1189-1199, 1996, Tuma, DJ et al. Hepatology, 23, 872-880, 1996, Casini, A. , Hepatology, 13, 758-765, 1991, Geertss, A. et al., J. Hepatology, 9, 59-68, 1989, Shimizu, I. et al. Hepatology, 29, 149-160, 1999).

On the other hand, liver fibrosis or cirrhosis is accepted as the final pathway in the pathological pathways of various liver diseases. Fibrosis of liver cells is a process in which ECM proteins including collagen type I are synthesized and secreted as hepatic stellate cells are activated. Currently, the main areas of research for the prevention and treatment of liver fibrosis are: ① research on reversing or inhibiting activation of HSCs, ② inhibiting the synthesis of ECM proteins, or ③ promoting the degradation of generated ECM proteins, and ④collagenase. It can be divided into studies to increase the expression of metalloproteinases (MMPS) genes, and to reduce the content of MMPs inhibitors such as tissue inhibitor of metalloproteinase, but to prevent and treat liver fibrosis or cirrhosis of edible biological resources. There are no reports of research and development on phytochemicals (Zhu, J. et al. Gastroenterology, 117, 1198-1204, 1999, Inoue, T. et al. European Journal of Phamacology, 378, 129-135, 1999).

Therefore, the inventors of the present invention to secure safety in the development of new food materials required for the development of functional foods, food medicines, and medicines to be used as a liver fibrosis inhibitor in the liver as described above, and to reduce the risk of the clinical Over 200 food and medicinal plants, which have been used for a long time, were searched for inhibitors of liver fibrosis. During the search, the present invention was achieved by finding high hepatic fibrosis inhibitory activity in methanol extracts of cornus, pine needles and pine needles, which have been drinking in Korea for a long time, and confirmed in vitro and in vivo .

It is an object of the present invention to provide a natural plant extract having an inhibitory activity against collagen type I production of hepatic stellate cells, which is a cause of liver fibrosis, from established food and medicinal biological resources.

It is still another object of the present invention to provide a functional food or pharmaceutical composition for preventing or treating alcoholic liver fibrosis or cirrhosis containing the L-FABP enhancer of the present invention.

In order to achieve the above object, the present invention is a collagen type I of hepatic stellate cells containing an extract of cornus ( Comus officinalis ), pine needles ( Pinus koraiensis ), or green tea ( Camellia Sinensis ) as an active ingredient Provide production inhibitors.

In the present invention, the extract may be extracted with water, a hydrophilic organic solvent or a mixed solvent thereof, but is preferably methanol extract of cornus oil, pine needles, or green tea. Here, methanol may be used diluted with water at 50% to 100%, but preferably extracted with 100% methanol to elute the active ingredient.

In the present invention, the extract may be further fractionated with various organic solvents such as nucleic acid, chloroform, ethyl acetate after extracting cornus oil, pine needles, or green tea with methanol, may be further purified by various chromatography after organic solvent fractionation. have.

In the present invention, the collagen type I production inhibitor of hepatic stellate cells is used for the prevention and treatment of alcoholic liver fibrosis or cirrhosis. The mechanism of action of type I collagen, which is closely associated with hepatic fibrosis, and the histological changes of hepatic stellate cells by these are well known in the art (Svegliati-Baroni, G. et al. Hepatology, 23: 1189). -1199 (1996); Shimizu, I. et al. Hepatology, 29: 149-160 (1999); Zhu, J. et al. Gastroenterology, 117: 1198-1204 (1999)), the plant extract of the present invention By inhibiting collagen type I production of astrocytes, it may be useful as an agent for preventing and treating alcoholic liver fibrosis or cirrhosis. In addition, the present invention is a natural material separated from edible plants, cornus oil, pine needles, green tea has no side effects compared to the conventional organic synthetic pharmaceuticals, safety is very high.

In order to achieve another object of the present invention, the present invention provides a functional food for the prevention and treatment of alcoholic liver fibrosis or cirrhosis, containing the collagen type I production inhibitor of hepatic stellate cells according to the present invention as an active ingredient.

In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for the prevention and treatment of alcoholic liver fibrosis or cirrhosis, containing the collagen type I production inhibitor of hepatic stellate cells according to the present invention as an active ingredient. .

Hereinafter, the present invention configured as described above, the liver fibrosis inhibitory substance isolated from green tea, cornus oil, pine needles and its in vitro and in vivo liver fibrosis inhibitory activity will be described in more detail.

The present invention relates to the increase of type I collagen caused by acetaldehyde produced by intrahepatic oxidation after alcohol intake using HSC-T6 cell line, which is a hepatic stellate cell in rat, and liver in vivo using DMN-treated rat, a cirrhosis model. After confirming the inhibitory activity of fibrosis, the present invention was completed by obtaining methanol extracts of Comus officinalis , Pine needles ( Pinus koraiensis ), and green tea ( Camellia Sinensis ).

The present inventors found that methanol extracts of green tea, cornus and pine needles have relatively high hepatic fibrosis inhibitory activity among extracts extracted from 200 food and medicinal plants. Thus, hepatic fibrosis inhibitory activity of the extracts was confirmed using a liver fibrosis rat model treated with DMN.

In the present invention, the cornus oil, pine needles, or green tea extracts include any of all extracts, fractions and purified products obtained in each step of extraction, fractionation and purification, dilution or concentrate thereof, or dried products thereof.

In the present invention, as the hydrophilic organic solvent that can be used as the extraction solvent, for example, lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone ; C2-C5 polyhydric alcohols, such as 1, 3- butylene glycol, a propylene glycol, and glycerin, etc. are mentioned, The mixed solution of these hydrophilic organic solvents, and water can be used.

Cornus, pine needles, or green tea extract of the raw material of the present invention may be formulated in the form of granules, tablets, capsules or drinks by conventional methods known in the art. Moreover, in order to make storage and handling easy, you may add the carrier used for normal formulation, such as dextrin and cyclodextrin, and other arbitrary preparations. In addition, auxiliary raw materials or additives conventionally added to foods or drugs are not particularly limited, for example, glucose, fructose, sucrose, maltose, sorbitol, stevioside, loxoside, corn syrup, lactose, citric acid, tartaric acid, malic acid , Succinic acid, lactic acid, L-ascorbic acid, d1-α-tocopherol, sodium elisolvinate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, gum arabic , Casein, gelatin, pectin, agar, vitamin Bs, nicotinic acid amide, calcium panthenate, amino acids, calcium salts, pigments, flavors, preservatives and the like.

The plant extract of the present invention can be added to foods as it is to produce foods for the prevention and treatment of alcoholic liver fibrosis or cirrhosis. Since the food and drink of the present invention thus obtained can be consumed on a daily basis, a high liver function improvement effect can be expected and is very useful.

The addition amount of the plant extract in such foods cannot be regulated uniformly depending on the type of foods to be used, but may be added within a range that does not impair the original taste of foods. Preferably, it is the range of 0.1-20 mass%. In the case of food in the form of granules, tablets or capsules, it is usually 0.1 to 100% by mass, preferably added in the range of 5 to 100% by mass.

In addition, the bellflower extract, which is an active ingredient of the liver L-FABP enhancer of the present invention, is appropriately administered so that the daily intake amount of adult is 1 ~ 3000mg. In addition, the dosage can be appropriately increased or decreased depending on age, symptoms, and the like.

Next will be described in more detail with reference to the embodiment of the present invention configured as described above. These examples are only for illustrating the present invention in detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1: Cornus ( Comus officinalis ), Pine needles ( Pinus koraiensis ), green tea ( Camellia sinensis ) Type I collagen inhibitory activity in methanol extract

In order to determine the type I collagen inhibitory activity of cornus, pine needles and green tea selected from 200 kinds of food and medicinal biomass, the following method was performed. Blasting the cornus fruit part, pine and green tea leaves at 100 ° C. for 5 minutes and standing at room temperature with 5 times 100% methanol for 24 hours were repeated three times, and the extract was concentrated under reduced pressure. (CM). In the concentrate, 1 L of nucleic acid, chloroform, and ethyl acetate were exchanged three times for 36 hours in each solvent, followed by fractionation with a funnel (MH, MC, ME), and the remaining dissolved in methanol (MM). The solvent fractions of were filtered through a filter cloth, and the filtrate was concentrated under reduced pressure and dried to obtain an extract.

HSC-T6 cells (provided by Mount Sinai Medical Center, NY, Dr. Friedman), a liver hepatic stellate cell line, were seeded in 24 wells at 1.5 x 10 ⁴ cells / well and then passed for 40 hours at 37 ° C. Acetaldehyde, β-aminopropionitrile fumarate (100 μg / ml) and L-ascorbic acid (50 μg / ml) were added and incubated for 24 hours. The culture was diluted with carbonate / bicarbonate buffer (pH 9.6) and measured as follows by ELISA method. 100 μl of 0.01% glutaraldehyde was injected into the immuno-well plate and reacted at 37 ° C. for 1 hour, followed by washing the plate with distilled water and removing water. After diluting the diluted culture solution and reacting for 2 hours, the plate was washed again and reacted with blocking solution (0.5% BSA) at 37 ° C. for 1 hour. After the reaction, the collagen type I polyclonal antibody (Abcam, Cambridge, UK) was diluted to 1: 4,000 (0.5% BSA), and 100 µl was dispensed and reacted at 37 ° C for 1 hour. Anti-rabbit IgG conjugated peroxidase (Sigma, St. Louis, USA) was diluted 1: 0.5 in 0.5% BSA and reacted at 37 ° C for 1 hour. In each step, the plate was washed and drained three times. Substrate (TMB 10 mg / ml DMSO, 3% H ₂ O ₂ , 50 mM sodium acetate buffer, pH 5.1) was added at 100 μl per well for 15 minutes, and 50 μl of 1 M sulfuric acid was added for reaction. After complete stop, absorbance was measured at 450 nm using a microplate reader (Model 550, BIO-RAD laboratories, USA). As a result of the above method, the inhibitory activity in cornus (28.0%), pine needles (23.8%) and green tea (26.8%) was confirmed when each extract was treated at a concentration of 0.1 mg / ml (not shown). ). In addition, the inhibitory activity was examined when the concentration was diluted to 50 ug / ml and 100 ug / ml. 1 is a diagram showing the type I collagen production inhibitory effect of cornus, pine needles, green tea methanol extract on HSC-T6 cells. Here, the production inhibitory activity (%) of Type I collagen was converted according to the following equation.

Inhibitory activity of Type I collagen production (%)

                  = [1-(As-Ab / Ac-Ab)] × 100

Ac: absorbance of the culture medium without acetaldehyde treatment

Ab: Absorbance of non-sampled cultures after acetaldehyde treatment

As: absorbance of sample treated culture solution after acetaldehyde treatment

Example 2 Assay of Hepatic Fibrosis Prevention Effect in DMN-treated Rats

In order to induce cirrhosis, 8 male wister rats (Sam taco) weighing 240 ± 7 g in each group were treated with 10 mg / kg body weight (0.15 mol / l pyrogen free sterile NaCl) Dilution) was administered intraperitoneally once a day for 3 consecutive days per week. The experiment lasted for 4 weeks, and the sample was divided into two groups, 1.5 mg / l and 3 mg / l, for free drinking with drinking water. The control group received the same amount of NaCl. After sacrifice, liver was removed, homogenized with 10 times the volume of phosphate buffer (0.5 M), homogenized with Teflon glass homogenizer (Glas-Col), centrifuged at 10,000 rpm, and supernatant was taken to obtain intrahepatic lipid peroxide. The effect of the three extracts was confirmed by measurement and hydroxyproline assay, which is an indicator of the decrease in collagen in liver (Spector et al. Cells, a laboratory manual, Vol. 1, 34.1 ~ 55.1, CSHL Press, 1998). .

Lipid peroxide measurement was performed by mixing 3.5 ml of 10% trichloroacetic acid with 0.5 ml of homogenized tissue, followed by centrifugation at 3,000 rpm for 10 minutes. 2 ml of the supernatant was taken and mixed again with 2 ml of 0.6% thiobarbituric acid reagent (prepared with 0.25 N HCl), reacted in a 100 ° C. water bath for 15 minutes, and then absorbed at 532 nm (FIG. 2). Figure 2 is a chart comparing the inhibitory effect of hepatic lactate, pine needles, green tea on the hepatic lipid peroxidation in acute liver cirrhosis-induced rat model. DC: Acute liver cirrhosis model (DMN-treated), AL: Green tea low concentration (0.15%) group, AH: Green tea high concentration (0.3%) group, BL: Cornus low concentration (0.15%) group, BH: Cornus high concentration (0.3%) group , CL: pine needle low concentration (0.15%) administration group, CH: pine needle high concentration (0.3%) administration group. Where MDA is Under the influence of toxic acetaldehyde that occurs when alcohol is ingested, lipid peroxidation occurs due to a decrease in the protection mechanism against free radicals in cells, which is an indicator of one of the preliminary stages of liver cirrhosis. MDA, the product of this lipid peroxidation, was expressed according to the following equation.

MDA (%) = [1-A _b / A _c ] x 100

A _b: 156 mM-1 cm-1 (extinction coefficient for MDA-reactive product) x

     Absorbance of sample treatment group or liver cirrhosis model

Ac: 156 mM-1 cm-1 (extinction coefficient for MDA-reactive product) x

     Absorbance of the control group

In FIG. 2, the MDA was significantly decreased when the sample was administered at a higher concentration than the MDA of the cirrhosis model group. In particular, the MDA in the green tea concentration group (AH) was reduced to 50% of the cirrhosis model group.

Intrahepatic collagen was measured by hydrolysis of homogenized tissues in 6 N hydrochloric acid at 110 ° C. for 16 hours, then evaporated again in 100 ° C. water bath to remove acid, and the residue was diluted in distilled water to a certain volume. . 1.2 ml of 50% isopropanol was added and mixed, followed by mixing with chloramine-T solution and allowed to stand at room temperature for 10 minutes. Finally, p- dimethylamino-benzaldehyde (60% perchloric acid) was added and reacted at 60 ° C. for 30 minutes, and the absorbance was measured at 558 nm. The absorbance was determined according to the measured hydroxyproline standard, and the unit was expressed in μg / g wet liver. As a result, collagen decreased by 11% and 42% in green tea, 6% and 27% in cornus oil, 26% and 24% in pine needles at 1.5 mg / l and 3.0 mg / l compared to liver cirrhosis group. The inhibitory activity of was confirmed. In particular, the green tea methanol extract showed a similar value to the normal group at a concentration of 3.0 mg / ml (Fig. 3). Figure 3 is a chart comparing the inhibitory effect of collagen production of cornus, pine needles, green tea in acute liver cirrhosis-induced rat model.

The isolated livers of the sacrificed rats were fixed in 10% formaldehyde, dehydrated with alcohol according to concentration, embedded with paraffin, and then paraffin-fixed tissue was cut into 7 μm thick sections with Microme HM 340E, and then the Mayer's hematoxylin and eosin staining was performed to detect changes in liver tissue. The cornus and pine needles showed liver damage compared to the normal group, but liver damage was insignificant compared to the liver cirrhosis model, and green tea showed the same morphology as the normal group when 0.3% was administered. (Figure 4). Figure 4 is a liver tissue micrograph showing the protective effect of liver, pine needles, green tea in acute liver cirrhosis-induced rat model. A: normal rat, A-1: acute liver cirrhosis-induced model, B: pine needle low concentration (0.15%) fed rats, B-1: pine needle high concentration (0.3%) fed rats, C: cornus low concentration (0.15%) fed rats, C-1: pine needle high concentration (0.3%) fed rats, D: green tea low concentration (0.15%) fed rats, D-1: green tea high concentration (0.3%) fed rats.

Example 3: Formulation of Plant Extracts (Tablets)

Tablets of the following formulations were prepared by conventional tablet presses.

20 parts by mass of powdered cornus oil, pine needles and green tea extract of Example 1

Dextrin 72 parts by mass

80 parts by mass per minute

8 parts by mass of glycerin fatty acid ester

Mixing and tableting of raw materials were easy, and refined refinement | purification was obtained.

Example 4: Formulation of Plant Extracts (Capsules)

By the usual method, the capsule which has the following compositions was manufactured. In addition, No. 1 hard gelatin capsule was used for the capsule.

Composition in 1 Capsule (200 mg)

5 mg of cornus, pine needle, green tea extract of Example 1

Cornstarch 60.0mg

Lactose 100.0mg

Calcium Lactate 10.0mg

Hydroxypropyl cellulose (HPC-L) 10.0 mg

Example 5: Formulation of Plant Extracts (Granules)

By a conventional method, instant tea granules of the following formulation were prepared by a fluid bed granulator.

20 parts by mass of powdered cornus oil, pine needles and green tea extract of Example 1

40 parts by mass of oligosaccharide

50 parts by mass of citric acid

50 parts by mass of sugar

Dextrin 810 parts by mass

Mixing of the raw materials and granulation with a fluidized bed granulator were easy, and a fine grain of instant tea was obtained.

As described above, methanol extract obtained from cornus oil, pine needles and green tea according to the present invention is excellent in the prevention and treatment of liver fibrosis or cirrhosis, so that it can be used in functional foods or food additives or drugs for preventing alcoholic liver fibrosis as an active ingredient. It will be effective.

Claims (5)

Inhibitor of collagen type I production of hepatic stellate cells containing the extract of Comus officinalis , Pinus koraiensis , or Green tea ( Camellia Sinensis ) as an active ingredient. 2. The inhibitor of collagen type I production of hepatic stellate cells according to claim 1, wherein the extract is methanol extract of cornus, pine needle or green tea. The inhibitor of collagen type I production of hepatic stellate cells according to claim 1, which is used for the prevention and treatment of alcoholic liver fibrosis or cirrhosis. A functional food for preventing and treating alcoholic liver fibrosis or liver cirrhosis, comprising the inhibitor of collagen type I production of hepatic stellate cells according to any one of claims 1 to 3. A pharmaceutical composition for the prevention and treatment of alcoholic liver fibrosis or cirrhosis, comprising the inhibitor of collagen type I production of hepatic stellate cells according to any one of claims 1 to 3.

Images