KR100316790B1 - A composition having an effect of preventing liver fibrosis by containing a extract of Rhodiola sachalinensis A. Bor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition capable of inhibiting liver fibrosis while protecting the liver by containing a lyophilized water extract of algae Rhodiola sachalinensis A. Bor as an active ingredient.

The freeze-dried high-alkaline red water extract of the present invention shows excellent liver protection, and does not cause liver toxicity, and thus can be used safely.

Description

A composition having an effect of preventing liver fibrosis by containing a extract of Rhodiola sachalinensis A. Bor}

The present invention relates to a pharmaceutical composition capable of inhibiting liver fibrosis, in particular liver fibrosis due to chronic liver disease, by protecting the liver by containing the lyophilized water extract of Rhodiola sachalinensis A. Bor as an active ingredient. will be.

Liver fibrosis or cirrhosis is caused by excessive intake of alcohol, abuse of chemicals, viral hepatitis, cholestasis due to biliary tract abnormalities, and in the early stages there are no pains or subjective symptoms. The treatment is not easy, and the death rate is high, causing social problems.

Liver fibrosis is a loss of balance between the synthesis and degradation of connective tissue, which is caused by excessive accumulation of connective tissue in liver tissue and is accompanied by necrosis or inflammation (Popper, Leber Magen Darm, 8, 65, 1978; Schuppan et al., Z. Gastro., 26, Suppl. 3, 28, 1988). In addition, the expression of stellate cells appearing in all forms of liver injury. In particular, stellate cells are activated upon hepatic fibrosis, which increases the expression of α-smoothmuscle actin (α-SMA) (Taub et al., Z, Hepatol., 1445-1446, 1998).

Therefore, the development of therapeutic agents for liver fibrosis is currently focused on whether drugs can inhibit the activity of astrocytic cells, and conventionally such drugs include penicillamine, 16,16-dimethyl prostaglandin E2, and biphenyl dimethyl dicarboxyl. Acid, colchicine, glucocorticoid, malotilate, gamma interferon, pentoxifylline, pyridine-2,4-dicarboxylic-diethylamide, pyridine-2,4-dicarboxylic-di (2-methoxyethyl) amide and the like are known.

However, since these drugs have a weak effect or severe side effects in clinical application, it can be said that there are currently no effective therapeutic agents for liver cirrhosis (Boker et al., J. Hepatol., 13, s35, 1991; Tamayo et al., ibid , 3, 112, 1983; Jenkins et al., ibid , 1, 489, 1985; Kershenobich et al., ibid , 7, 1104, 1987; Svegliati et al., 18, 209A, 1993; Guzelian et al., Gastro., 86, 897, 1984; Ala-Kokko et al., J. Lab. Clin. Med., 113, 177, 1989).

Therefore, the present inventors have been able to effectively inhibit liver fibrosis and search for a new substance with no hepatotoxicity. As a result, old age heart failure, gastric pain, diabetes, joint pain, pulmonary tuberculosis, anemia, hypotension, postpartum disease, A freeze-dried extract of Rhodiola sachalinensis A. Bor, an extract of water, known to have a pharmacological effect on forgetfulness or aphasia, can protect the liver without causing hepatotoxicity and, moreover, can inhibit liver fibrosis. It was found that the present invention was completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition capable of protecting the liver and at the same time inhibiting liver fibrosis, particularly liver fibrosis caused by chronic liver disease.

In order to achieve the above object, the pharmaceutical composition according to the present invention is characterized in that it contains an lyophilized water extract of Rhodiola sachalinensis A. Bor water extract in an amount effective to inhibit liver fibrosis.

Hereinafter, the present invention will be described in detail.

Rhodiola sachalinensis A. Bor, a perennial herbaceous plant of the genus Crossulaceae, inhabiting the rocky areas of the highland forests and canyons of 1,800 ~ 2,300m above sea level, is an adverse environmental factor (physical, chemical, and biological). It has been reported to increase the organism's resistance (adaptation ability), to regulate the physiological function of the organism to maintain the normal life activity, and to have high antioxidant activity (Jinjiang Chinese Herbal Medicine Research Institute, Changbai Mountain Plant Medicine, Jilin People's Publishing House, Changchun, 1979., Cheng-Ming., Jung-Gun-Bah., 15 (1): 34, 1984., Nishioka et al., Chem Pharm Bull., 34, 10, 4083, 1986). As a pharmacological component of such high-altitude Honggyeongcheon, salidroside (Salidroside) and its glycosides, tannins, flavones compounds, about 20 amino acids and 18 bioactive microelements have been isolated.

The present invention finds that the freeze-dried extract of the extract obtained by extracting high-altitude red gyeongcheoncheon having the above efficacy does not cause hepatotoxicity, and can inhibit liver fibrosis (liver cirrhosis) induced by carbon tetrachloride and simultaneously protect the liver. It is to provide a pharmaceutical composition having this as an active ingredient.

The freeze-dried product of the high-acid honggyeongcheon water extract used in the present invention, after drying the root portion of the high-acid honggyeongcheon sufficiently in the shade, finely crushed or pulverized with a cutter, and using water to heat the pulverized product or crushed water at a temperature of 60-80 ° C. Obtained by freeze-drying the extract extracted for 2-4 hours at.

On the other hand, the freeze-dried product of the high-acid honggyeongcheon water extract obtained by the above-mentioned method contains salidroside in an amount of 7.5 to 8.5% by weight.

The composition according to the present invention contains the lyophilized water extract of the high-alkaline red ginseng extract in an amount effective for inhibiting liver fibrosis and liver protection by chronic liver disease, and the effective amount is the route of administration, the formulation, the purpose of use, the degree of disease of the patient. Can be determined within a wide range. In general, however, the lyophilisate can be formulated to be administered at a concentration of 1-2000 mg / kg / day, preferably 10-400 mg / kg / day.

The preparation for administering the lyophilisate of the high-alkaline red ginseng extract in the above range may have a conventional form, and may be used, for example, in the form of a pill, a drink, a medicine or a dietary supplement. They can be administered for the prevention and treatment of cirrhosis of the liver by liver fibrosis via oral or various parenteral routes of administration, which are suitable for the dosage form and are well known to those skilled in the art and can be easily selected by those skilled in the art. Excipients, carriers, diluents and the like.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited to these examples.

Example 1 Lyophilized Water Extract of Alpine Honggyeongcheon Water Extract

A four-year-old successful cultivation in Helong City, Jilin Province, China, the roots of alpine Rhodiola sachalinensis A. Bor, verified by Professor Kim Moon-il of the Medicinal Plants Department of Pharmacy, Yanbian University, China, were dried and sectioned under natural conditions. Then, 300 g of the mixture was warmed and extracted with 1,800 mL of distilled water at 80 ° C. for 3 hours, and filtered through a 150 mm filter paper to separate the filtrate (1). 1,500 ml of distilled water was added to the rest, followed by warm extraction at 80 ° C. for 3 hours as described above, followed by filtration with 150 mm of filter paper to obtain a new filtrate (2). After the filtrates (1) and (2) were mixed, they were left in a freezer at 4 ° C. for 24 hours, and then the precipitate was removed and the supernatant was collected and lyophilized at -50 ° C. using FD-5N eyela (Japan). The freeze dried 18g of the alpine red ginseng water extract was obtained.

Example 2 Determination of Salidroside in Lyophilized Products

The amount of salidroside contained in the freeze-dried water extract of algae Honggyeongcheon water extract was measured according to the method of Seong-gu et al. (11, 4, P47, 1994). That is, the freeze-dried extract of the high-altitude Honggyeongcheon water extract was refluxed with 70% ethanol in a water bath at 80 ° C., and the extract was concentrated under reduced pressure four times for 30 minutes each time to obtain an ethanol extract extract. The same amount of distilled water was added to the ethanol extract extract, the mixture was stirred, left at 4 ° C. for 2 hours, filtered through a 0.45 μm filter, and then reverse-phase chromatography was performed. The conditions for reverse phase chromatography are as follows:

product; LC-6A High Performance Liquid Chromatography

Column: Shim-Pack CLC-ODS (6.0 mm × 15 cm)

Mobile phase: methanol-water (0.3: 0.7)

Detector: UV Detector 225 nm

Flow rate: 1ml / min

Sample injection volume: 5 μl

As a result, it was confirmed that salidroside was contained in an amount of 7.5 to 8.5% by weight in the freeze-dried product of the high-alkaline red water extract.

Example 3 Liver Protective Effect Test

Exposure to experimental animals to carbon tetrachloride, ethanol, methotrexate, dimethylnitrosamine or deficiency of vitamin B ₁₂ results in experimental animals (called "liver fibrotic (liver cirrhosis) animals"), mainly treated with carbon tetrachloride. In the present invention, rats treated with carbon tetrachloride were used to examine liver toxicity. Rats treated with carbon tetrachloride were treated with lyophilized water extracts of alpine rhodiola saline extract, and the amounts of ALT (Alanine transaminase), AST (Aspartate transaminase) and albumin in serum were measured.

In other words, male rats (Korea Experimental Animal Center; Chungbuk Negative) weighing 190-220 g were adapted to the laboratory environment for one week, and enough feed and water were supplied. The number of each group and experimental animals used in the experiment is as follows.

Group 1 (n = 5)

Freeze-dried product of Group 2 (n = 4) alpine red ginseng extract (Example 1)

(50 mg / kg / day, p.o.)

Lyophilized product of group 3 (n = 4) alpine red ginseng extract (Example 1)

(100 mg / kg / day, p.o.)

Lyophilized product of Group 4 (n = 4) alpine red ginseng extract (Example 1)

(200 mg / kg / day, p.o.)

Group 5 (n = 8) Induced Liver Fibrosis by Carbon Tetrachloride

After induction of liver fibrosis by group 6 (n = 8) carbon tetrachloride, the freeze-dried product of the high-alkaline red ginseng extract of the present invention (Example 1) dosage group (50 mg / kg / day, p.o.)

After induction of liver fibrosis by group 7 (n = 8) carbon tetrachloride, the freeze-dried product of the high-alkaline red ginseng extract of the present invention (Example 1) dosage group (100 mg / kg / day, p.o.)

After induction of liver fibrosis by group 8 (n = 8) carbon tetrachloride, the freeze-dried product of the high-alkaline red ginseng extract of the present invention (Example 1) dosage group (200 mg / kg / day, p.o.)

Groups 1 and 5 administered the corresponding amount of distilled water, and the other group orally administered each drug using sonde. Each drug was administered on the 4th day, and after 2 hours, 1 to 4 groups of corn oil (made by Dongyang Flow Co., Ltd.) were mixed, and 5 to 8 groups of carbon tetrachloride were mixed in the same amount of corn oil to 1.0 ml / kg. Twice a week orally (10 am Monday, 10 am Thursday). Finally, after 72 hours of oral administration of carbon tetrachloride, rats were anesthetized, cardiac punctured, blood was collected, left at room temperature for at least 1 hour, centrifuged to obtain serum, and stored at minus 20 ° C.

The amount of ALT, AST, ALP and albumin in the serum was measured by a spotcare analyzer II (Spotchem-SP4410, Japan) using the kit of AKRAY FACTORY (Japan), and the results are shown in Table 1. .

Experimental group ALT (IU / L) AST (IU / L) ALP (IU / L) Albumin (mg / dl) One 36 ± 7 92 ± 9 314 ± 65 5.29 ± 0.43 2 34 ± 5 83 ± 15 285 ± 69 5.38 ± 0.33 3 38 ± 5 82 ± 17 287 ± 55 5.49 ± 0.51 4 33 ± 4 80 ± 8 276 ± 87 5.75 ± 0.33 5 851 ± 374 ^a 1019 ± 307 ^a 2238 ± 971 ^a 3.9 ± 0.60 ^a 6 532 ± 342 ^b 615 ± 411 ^b 1643 ± 699 ^b 4.90 ± 0.23 ^b 7 287 ± 113 ^b 379 ± 100 ^b 1300 ± 932 ^b 4.73 ± 0.40 ^b 8 327 ± 157 ^b 416 ± 148 ^b 1289 ± 778 ^b 4.69 ± 0.40 ^b (reliable on interval a = p <0.01, b = p <0.05)

From Table 1, in the 6-8 groups treated with 50 mg, 100 mg, and 200 mg of the lyophilisate of the present invention, the ALT values decreased by 37%, 66%, and 62%, respectively, compared to the 5 groups, and the AST value was 40%. , 63%, 59% decreased, ALP values decreased 26%, 21%, 20%, albumin amount was increased by 26%, 21%, 20%, respectively. In other words, it can be seen that the lyophilized water extract of the alpine red ginseng stream extract of the present invention is excellent in protecting liver in carbon tetrachloride-induced liver cirrhosis animals.

Example 4 Histological Examination of Hepatic Fibrosis Inhibitory Effect

Exposure of the experimental animals to carbon tetrachloride results in fibrosis of the liver after 8-12 weeks. When liver becomes fibrin, total collagen content increases.

Therefore, the amount of collagen in liver tissue can be estimated by quantifying hydroxyproline, which accounts for 10% of the collagen constituting the connective tissue of the liver (Schuppan, J. Hepatol., 13, s17, 1991). To determine the effect of liver fibrosis inhibition by quantifying the amount of.

Hepatic hydroxyproline was quantified according to the method of Jamal et al. (Jamall et al., Anal. Biochem., 112, 70, 1981). That is, 0.2 g of each liver tissue isolated in Example 3 was homogenized with 6 ml of 6 N hydrochloric acid, hydrolyzed at 110 ° C. and filtered. 50 μl of the filtrate was then evaporated to dryness at 80 ° C. under reduced pressure, dissolved in 1.2 ml of 50% isopropanol solution and oxidized to 200 μl of Chloramine-T (Sigma). Hydroxyproline was quantified by color development with 0.1 ml of Erlich reaction solution and absorbance at 558 nm. The results are shown in Table 2.

Experimental group Hydroxyproline (μg / g) One 275 ± 34 2 285 ± 45 3 274 ± 56 4 255 ± 64 5 574 ± 110 ^a 6 382 ± 67 ^b 7 357 ± 46 ^b 8 342 ± 35 ^b (reliable on interval a = p <0.01, b = p <0.05)

From Table 2, the amount of hydroxyproline in the liver fibrosis-induced animals was 33.4%, 37.9 dose-dependently compared to group 5 in groups 6 to 8 by the amounts of 50 mg, 100 mg, and 200 mg of lyophilisates of the present invention. It can be seen that liver fibrosis is inhibited since%, 40.4% is reduced. On the other hand, in groups 2 to 4, a slight increase or decrease in the amount of hydroxyproline before or after administration of the lyophilized product of the present invention was observed, compared to the first group, and thus it was found that it did not affect much.

Example 5 Masson trichrome staining of liver tissue

In Example 3, carbon tetrachloride-induced hepatic fibrotic tissue (groups 5 to 8) was deparaffinized, washed with water and distilled water, and the sections fixed in formalin were transferred to a Bouin solution and buried at 56 to 60 ° C for 1 hour. Transfer to 50-70% ethyl alcohol to remove picroic acid and wash with tap water for 10 minutes in Weigert iron heatoxylin solution, and in Biebrich scarlet-acid fuchsin solution. Rinse with distilled water for 10 minutes, then 15 minutes in phosphomolybdic-phosphotungstic acid solution, 2-5 minutes in aniline blue solution, 3 in 1% acetic acid solution After dyeing for 5 minutes, it was dehydrated, transparent, and sealed. Next, the number of inflammatory cells, necrosis, and degree of fibrosis were observed under an optical microscope. That is, the degree of fibrosis, necrosis and inflammation was evaluated according to the following evaluation criteria, and the results are shown in Table 3.

<Evaluation Criteria>

+: Slight ++: Medium +++: Severe

Experimental group Dose (mg / kg) Inflammation Necrosis Fibrosis 5 groups 0 +++ ( n = 6) ++ ( n = 2) +++ ( n = 4) ++ ( n = 2) + ( n = 2) +++ ( n = 5) ++ ( n = 2) + ( n = 1) 6 groups 50 +++ ( n = 1) ++ ( n = 3) + ( n = 1) ++ ( n = 2) + ( n = 6) ++ ( n = 3) + ( n = 5) Group 7 100 ++ ( n = 3) + ( n = 5) ++ (n = 1) + ( n = 7) ++ ( n = 2) + ( n = 6) 8th group 200 ++ ( n = 2) + ( n = 6) ++ ( n = 1) + ( n = 7) ++ ( n = 1) + ( n = 7) *n: Number of animals

From Table 3, it can be seen that liver fibrosis, necrosis, and inflammation are significantly improved in dose-dependently administered groups of 5 to 8 liver fibroblasts administered with the lyophilisate of the present invention.

Example 6 Acute Toxicity Test

The mice were treated with the lyophilized water extract of the alpine honggyeongcheon extract of Example 1 at the dosage of Table 4 through the oral administration route to the experimental group 5 consisting of 10 animals per group, and observed for 7 days to determine the maximum allowable amount. The results are shown in Table 4.

Count Dose (mg / kg) Lethal Maximum dose (mg / kg) Mouse 10 1000 0/10 - 10 2000 0/10 - 10 3000 0/10 - 10 6000 0/10 More than 6000 Control 10 0 0/10

From Table 4, it can be seen that the maximum allowable dose at the time of oral administration of the lyophilized water extract of the high-alkaline red ginseng extract of the present invention is 6,000 mg / kg or more, which indicates that the biological stability is very high. Therefore, the pharmaceutical composition having a liver fibrosis inhibitory effect and a hepatoprotective effect containing an effective amount of the lyophilized water extract of the algae red ginseng extract of the present invention can be safely administered to a living body.

As described above, the lyophilized water extract of alpine Rhodiola sachalinensis A. Bor can protect the liver and simultaneously inhibit liver fibrosis without causing toxicity in the liver. The composition can be provided.

Claims (3)

Hepatic fibrosis inhibiting composition comprising a freeze-dried product of Rhodiola sachalinensis A. Bor water extract in an amount effective to inhibit liver fibrosis. The composition of claim 1, wherein the lyophilisate contains salidroside in an amount of 7.5 to 8.5% by weight. The method of claim 1, wherein the water extract is dried in the root portion of the alpine Rhodiola sachalinensis A. Bor, cut, and then extracted with water for 2 to 4 hours at a temperature of 60 ~ 90 ℃ Composition.