KR100852737B1 - Composition prepared from lythrum salicaia having antioxidant and liver protecting activities - Google Patents

Abstract

An extract of Lythrum salicaria roots and a fraction thereof are provided to show excellent antioxidizing activity and hepatic fibrosis inhibitory effect, thereby being applied as a medicine and a health functional food having antioxidant and liver protecting activities. A pharmaceutical composition for preventing or treating liver cirrhosis comprises 0.001-99.999 wt.% of an extract of Lythrum salicaria roots as an effective ingredient, wherein the extract is obtained by using a solvent such as water, methanol, ethanol, and a mixture thereof. Further, the extract comprises a fraction and a weight ratio of the extract and the fraction is 99.999:0.001 to 50:50.

Description

COMPOSITION PREPARED FROM LYTHRUM SALICAIA HAVING ANTIOXIDANT AND LIVER PROTECTING ACTIVITIES}

FIG. 1 is a graph showing a reduction effect on TNF-α production in rat blood of solvent-specific fractions prepared from methanol extract of hairy roots.

FIG. 2 is a graph showing the effect of alleviating the GST activity in the rat liver of the solvent-specific fractions prepared from the methanol extract of the hairy roots.

Figure 3 is a graph showing the inhibitory effect of t-BHT-induced reactive oxygen (ROS) production in YPEN1 cells of 50% ethanol extract (LSR, 40, 200, 1000 ppm treatment) of hairy roots.

Figure 4 is a graph showing the LPS-induced NO production inhibitory activity in BV2 cells of 50% ethanol extract (LSR, 1, 10, 50, 100, 1000 ppm treatment) of hairy roots.

Figure 5 is a graph showing the effect on the survival rate of animal neurons (SH-SY5Y) of 50% ethanol extract (LSR, 1, 10, 50, 100, 1000 ppm treatment) of hairy roots.

FIG. 6 is a graph showing the effect of 50% ethanol extract (LSR, 0.125, 0.25, 0.5 g / kg administration) of hairy buds roots on the reduction of lipid peroxide (TBARS) production in liver liver induced by carbon tetrachloride.

Figure 7 is a graph showing the effect of maintaining the antioxidant content in liver liver fibrillation induced by carbon tetrachloride of 50% ethanol extract (LSR, 0.125, 0.25, 0.5 g / kg administration) of hairy roots.

8 is a graph showing the effect of detoxifying enzyme (GST) activity in rat liver induced with hepatic fibrosis with carbon tetrachloride of 50% ethanol extract (LSR, 0.125, 0.25, 0.5 g / kg administration) of hairy roots.

Figure 9 is a graph showing the effect of reducing collagen content in rat liver induced fibrosis with carbon tetrachloride of 50% ethanol extract (LSR, 0.125, 0.25, 0.5 g / kg administration) of hairy roots.

FIG. 10 is a micrograph of magnification × 100 showing the effect of reducing 50% ethanol extract (LSR, 0.125, 0.25, 0.5 g / kg) of rat hepatic fibrosis marker (collagen) by immunohistochemical analysis. .

FIG. 11 is a micrograph of magnification × 100 showing the effect of reducing 50% ethanol extract (LSR, 0.125, 0.25, 0.5 g / kg) of rat hepatic fibrosis in hairy roots by histological analysis.

The present invention relates to a composition having an antioxidant and hepatoprotective activity, including a hairy hyacinth extract, and more particularly, to the use of the antioxidant activity and hepatoprotective activity of extracts obtained from the hairy hyacinth root and fractions for each solvent.

Modern people are exposed to various adult diseases due to westernized diet, stress, smoking, and harmful substances, and recently, health problems caused by excessive intake of alcohol are also increasing. In Korea, alcoholic liver disease accounts for 14.5% of all liver diseases, and 39.8% of hepatitis B, which occupies the largest proportion of 68.8%, is also known to show alcoholic liver disease and complications (Lee Chang-hong. ). According to the statistics of the National Statistical Office, the proportion of mortality caused by alcoholic liver disease among the mortality caused by liver disease in Korea is increasing rapidly from year to year (from 2.7% in 1990 to 33.0% in 2004), and preventive measures are necessary.

In general, living organisms use oxygen to generate energy, but some of them are converted to harmful free radicals, which are the main cause of oxidative stress. Oxidative stress can also be caused by factors such as drugs that enter the living body from the environment, smoking, ultraviolet irradiation, etc. Alcohol is one such drug. The reason the free radicals that are the main material of the oxidative stress, as due to the form of not paired in the electron shell unstable electron, that is a radical of molecular oxygen, superoxide anion ^{_{(superoxide anion, O 2 - ·}} ), Examples are hydroxyl radicals (OH ·), singlet oxygen ( ¹ O ₂ ), and others (Evans, CR et. al ., Free radicals and oxidative stress: environment, drugs and food additives. Portland Press. 1-31. 1995). These oxidative stresses, including free radicals, cause mutations due to oxidation of biofilm-forming fatty acids, oxidation and cross-linking of biopolymers such as DNA and proteins, and are thus directly linked to the onset and aging of many diseases including cancer (Frei B). Natural antioxidants in human health and disease.Academic Press. 25-55. 1994).

Oxidative stress is usually eliminated to some extent by the inherent antioxidant mechanisms, but in the case of exceeding the body's ability to respond, it is necessary to supply antioxidants to compensate for this. In other words, it is possible to remove radicals introduced into the living body or to prevent the depletion of biological antioxidants, and antioxidants that affect the activity of antioxidant enzymes may prevent such oxidative stress.

On the other hand, the reason why alcohol becomes oxidative stress is because it decomposes itself into harmful radicals. That is, the alcoholysis superoxide anion by (microsomal ethanol oxidation system) MEOS in metabolic processes, α- hydroxyethyl radical, a perylene buffer ion ^{(perferryl ion, Fe 3 + O} 2 - ·), a ferrous oxygen -2- Active oxygen species such as ferrous dioxygen ferric complex (Fe ^{2 +} O ^{2 -} Fe ^{3 +} ) are produced to reduce glutathione, vitamin A, vitamin C, vitamin E, carotenoids and protein-thiol And increase the levels of the peroxides malondialdehyde (MDA, malondialdehyde), non-heme iron, CYPIIE1, carbonyl protein. Therefore, if the oxidative stress caused by chronic alcohol intake is not properly eliminated, it may cause a hangover in the short term and liver disease in the long term.

Hairy Buddha (Lee Woo-cheol, Korea Botanical Garden, 1996) is another name for Joombu Buddha (Three persons including Jeong Tae-hyun, Joseon Botanical Garden, 1949), True Buddha (Park Man-gyu, Korean Plant Perspective, 1949), and Tumulus (Kim Hyunsam). Phosphorus, Botanical Primitive Color Book, Encyclopedia of Science, 1988). In the case of Guhwangboncho (救 荒 本草, Ming Dynasty, China), the outposts of hairy buddha and buddha were also called Cheongulchae. The name of the hair bud is called Purple Loosestrife, Spiked Loosestrife, and the scientific name is Lythrum. salicaria L. (Sp. Pl . 446, 1753), but L. argyi , L. intermedium , L. propinquum , L. quadrifolium , L. salicaria subsp . intermedium , L. salicaria var . glabricaule , L. salicaria var. glabrum , L. salicaria var . intermedium , L. salicaria var . mairei , L. salicaria var . tomentosum , L. salicaria var . vulgare , L. salicaria var . vulgare subvar . It is known to use various tinnitus such as glabricaule , L. tomentosum , and Salicaria spicata Lam. (Fl. Fr. 3: 103, 1778) or Salicaria vulgris Moench (Method. 664, 1794).

In the herbal medicine medicinal dictionaries (Jung-sup Lee, Younglimsa, 1990), the outposts and roots of hairy buds have pharmacological effects, and are particularly effective in blue blood, hemostasis, and blood transfusions, dysentery, ulcers, and bacterial dysentery. It contains salicarin and tannins, and the flowers contain vitexin, orientin, malvin, cyanidin-3-monogalactoside, gluttony, elazic acid (ellagic acid), chlorogenic acid and the like are described.

In the research reports on the efficacy of hairy buds in professional journals, domestic research reports were not confirmed, and the results of antioxidant experiments on DPPH and linoleic acid of extracts prepared from hairy buds leaves in foreign journals and anti-animal activity in experimental animals Inflammatory and anti-pain effects [Tunalier et al ., J. Ethnopharmacology (in press), 2006] and reported effects on hyperglycemic mice (Lamela et. al ., J. Ethnopharmacology 15 (2): 153-160, 1986) and effects on normal blood glucose rats (Lamela et al. al ., J. Ethnopharmacology 14 (1): 83-91, 1985), antimicrobial effects (Becker et al ., Fitoterapia 7 (6): 580-584, 2005), antimicrobial compounds (Rauha et al ., International J. Food Microbiol. 56 (1): 3-12, 2000) and antibacterial activity (Nurten et. al ., Pharmaceutical Biol. 44 (2): 91-94, 2006).

On the other hand, if you search for a patent application made of hair-blowing flowers, oral composition (application No. 1019960010675, registered) effective for dental caries and periodontal disease, a method for preparing oral microcapsules, and an oral cavity containing microcapsules prepared by the method Composition (application number 1020020013758, registered), method for producing plant extract powder and composition for oral cavity containing plant extract powder (application number 1019990043040, registered) produced by the method and method for producing confectionery containing floral raw materials ( Application No. 1020057019041, published), and is referred to as a cell excipient (anti-application 1020057013882, published) in the antioxidant composition and the external composition in relation to the functionality based on the similar species of Buddha flower.

Thus, when looking at domestic and international academic papers and domestic patent applications, it is confirmed that there are no known papers or patents dealing with the antioxidant activity and the hepatic protective activity of hairy root extract.

Accordingly, the present inventors made the root of the hair bud flower selected during the search for antioxidant activity in the native plant resources of Korea in Toxicity test by free radical scavenging ability and cell viability in vitro and in rats treated with carbon tetrachloride in vivo Antioxidant activity and hepatoprotective efficacy were assayed. As a result, it was confirmed that the extract and the solvent-specific fractions of hairy roots of the roots have antioxidant and hepatoprotective activity, which led to the completion of the present invention.

It is an object of the present invention to provide a composition of plant origin with antioxidant and hepatoprotective activity.

In order to achieve the above object, the present invention provides a composition having antioxidant and hepatoprotective activity comprising an extract of the root of Lythrum salicaria as an active ingredient.

Here, the extract includes a fraction, and the blending ratio of the extract and the fraction is preferably 99.999: 0.001 to 50:50 by weight. The fraction is an organic solvent fraction, in particular the hexane, ether, ethyl acetate and butanol fractions.

In addition, the extract is preferably 0.001 to 99.999% by weight based on the total composition.

The extract may be obtained by extracting a lower alcohol such as methanol or ethanol or a mixed solvent of water and lower alcohol.

Hereinafter, the present invention will be described in more detail.

The extract of the hair bud root according to the present invention is about 2 hours to 24 hours at a temperature of refrigeration temperature to 100 ℃, preferably 70 to 90 ℃ using a solvent of about 10 to 20 times the volume of the dried hair bud root Extraction may be repeated two to five times by a method such as reflux extraction, shaking extraction, or ultrasonic extraction, and the extract may be obtained by concentrating under reduced pressure. For 50% ethanol extract, the yield is about 15%.

Here, the extraction solvent may be a lower alcohol such as methanol, ethanol or a mixed solvent having a mixing ratio of 99.999: 0.001 to 0.001: 99.999 of water and lower alcohol.

Hair bud extract according to the present invention was confirmed to be safe in a single dose test of rats commissioned by GLP research institute (Korea Institute of Chemical Research Safety Evaluation Institute). The use of furnaces and the non-toxicity of the kidneys and the gastrointestinal circulatory system are mentioned, and some safety to humans is recognized.

In addition, the hepatoprotective activity of the composition of the present invention includes a protective activity against liver diseases such as fatty liver, hepatitis, cirrhosis and the like derived from alcohol.

The composition comprising the extract of the hairy root of the root according to the present invention as an active ingredient may be administered orally or orally by dividing the amount of 0.1 to 2 g / kg 1 to 3 times per day for adults.

In addition, the composition using the extract of the hairy root of the root according to the present invention as an active ingredient may be formulated into conventional injections, powders, granules, capsules, tablets, liquids and the like or external skin preparations such as creams, ointments, lotions.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only illustrative of the present invention, and the scope of the present invention is not limited to these.

Example  One: Hair bud  Methanol Extracts of Roots and Their Solvents Fraction  Produce

Hairy roots were harvested from seed growth at the Suwon Rural Development Administration's Medicinal Crop Experimental Packaging in 2005, freeze-dried and dried to make powder extracts.

Methanol corresponding to 10 times the weight of the sample was added and extracted twice at 74 ° C. for 2 hours. The extract was concentrated under reduced pressure, and the remaining methanol and water were freeze-dried and removed in It was used for in vitro antioxidant experiment. The yield of methanol extract obtained through this process was 9.54%.

A part of the obtained methanol extract was dissolved in distilled water, and then sequentially partitioned into hexane, ether, ethyl acetate and butanol, concentrated under reduced pressure and freeze-dried to obtain fractions for each solvent, and used in activity experiments and animal experiments in chemical reaction systems. Particularly, hexane fraction-1 is a part which is completely dissolved in hexane during the fractionation process, and hexane fraction-2 is only a part that is present between the water layer and the hexane layer when fractionated with hexane.

Example  2: for cell experiments and animal activity assays Hair bud  Preparation of Root Extract

In the same hairy roots as Example 1 in 50% ethanol extract was prepared for performing in vitro cell and animal experiments.

50% ethanol corresponding to 10 times the volume of the sample weight was added thereto, and extracted twice at 85 ° C. for 2 hours. The extract was concentrated under reduced pressure to remove the solvent and freeze dried to remove moisture. The yield was about 15.07%.

Experimental Example  1: Methanol Extract and Solvent Specific Fraction In a chemical reaction system  Antioxidant activity assay

1. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity

According to Blois' method (Antioxidant determinations by the use of a stable free radical.Nature 181: 1199-1200, 1958), 2.97 ml of an ethanol 1.5 × 10 ^-4 M DPPH reaction solution was mixed with 0.03 ml of various concentration samples. After 3 minutes the absorbance at 517 nm was measured. The scavenging ability of the extract to DPPH was expressed as RC ₅₀ , the concentration required to reduce the absorbance of the control using only solvent by 50%, or as the scavenging activity (%) of the absorbance of the control at a certain concentration. Α-tocopherol was used as a control.

2. Inhibitory Activity against Low Density Lipoprotein (LDL) Oxidation

Miller et al . Method of In in vitro antioxidant effects of estrogens with a hindered 3-OH function on the copper-induced oxidation of low density lipoprotein. Steroids 61: 305-308, 1996), human LDL formulated to contain 50-100 μg of protein, 0.02 ml of a constant concentration, 0.115 ml of 10 mM phosphate-buffered saline (PBS). And 0.04 ml of 0.25 mM CuSO ₄ at 37 ° C. for 3 hours. 1 ml of 20% TCA was added to the reaction solution to stop the reaction. Then, 1 ml of 0.67% TBA dissolved in 0.05 N NaOH was added thereto, followed by heating and cooling at 95 ° C. for 15 minutes, followed by centrifugation at 3,000 rpm for 15 minutes. The amount of malondialdehyde (MDA) contained in the separated supernatant was measured at 540 nm and the results were expressed as% inhibition to the control.

3. Total Phenolic Content Analysis

To compare the amount of phenolic compounds that affect antioxidant activity, Kim et al. al . (Effect of solvents and some extraction conditions on antioxidant activity in cinnamon extracts. Korean J. Food Sci. Technol. 25: 204-209, 1993). 0.1 ml of a certain amount of hairy lotus extract and 2 ml of 2% Na ₂ CO ₃ were mixed, and after 2 minutes, 0.1 ml of 50% Folin-Ciocalteau reagent was added. After leaving at room temperature for 30 minutes, the total phenol content was calculated by substituting the calibration curve (r = 0.9973) using tannic acid as a standard with absorbance measured at 750 nm.

4. Analysis result

The results of the antioxidant activity in the chemical reaction system of the methanol extract of the hairy roots and the solvent-specific fractions separated therefrom are shown in Table 1 below.

Antioxidant Activity and Total Phenolic Content in the Chemical Reaction System of Methanol Extracts and Fractions of Hairy Root Flower Analysis item sample DPPH scavenging activity (SC _{50 ,} μg / ml) LDL oxidation inhibition (IC _{50 ,} μg / ml) Total Phenolic Content ^* (%) Methanol extract 7.50 - 20.5 ± 0.30 Hexane Fraction-1 29.62 6.86 11.85 ± 0.07 Hexane Fraction-2 7.60 4.65 24.52 ± 0.78 Ether fractions 6.21 4.15 48.17 ± 0.18 Ethyl acetate fraction 4.90 4.53 46.50 ± 0.08 Butanol fraction 5.22 2.65 44.92 ± 0.28 Water fraction 5.38 3.55 22.94 ± 0.89

* Tannic acid equivalent

① In the case of methanol extract, the scavenging ability (SC ₅₀ ) for DPPH radical is 7.50 μg / mL, which is 13.52 μg / mL of α-tocopherol (Lee et al . , 13 (3): 171-176, 2002).

② As a result of analyzing total phenolic content which has a high correlation with antioxidant activity, total phenolic content of methanol extract of hairy root was 20.5%.

③ All fractions except hexane fraction-1 among the solvent fractions of hairy roots showed excellent scavenging ability against DPPH radicals, followed by ethyl acetate fraction, butanol fraction, water fraction, ether fraction and hexane fraction-2. Was excellent (SC ₅₀ , 4.90-7.60 μg / ml).

④ The inhibitory activity (IC ₅₀ ) on the oxidation of LDL of fractions was also 2.65∼6.86 ㎍ / ml, and all solvent fractions of hairy roots showed very effective activity.

⑤ In addition, the total phenolic content (%) for 100 g of fractions was relatively high in the ethylene fraction, ethyl acetate fraction and butanol fraction as 48.2%, 46.5% and 44.9%, respectively, hexane fraction-1 and hexane fraction- 2 and water fractions showed a relatively low content of 11.9 to 24.52%. Therefore, the antioxidant activity is expected to be excellent in the ether fraction, ethyl acetate fraction and butanol fraction.

Experimental Example  2: By solvent Fraction In rats  Hepatoprotective Activity Assay

1. Animal experiment plan

Sprague Dawley male rats weighing about 200 g were used as experimental animals, and the fractions and positive control (Silymarin, Silymarin) doses were 100-300 mg / kg body weight / rat / day (1 time / day, total). Toxicity was induced by intraperitoneal injection of 1 ml of CCl ₄ per kg body weight in 2 ml of corn oil. For the feed, a commercial solid feed (Sam taco) was used, and the experimental group was configured as shown in Table 2, and rats were randomly assigned to 8 animals in each experimental group. Hexane fraction-1 and hexane fraction-2 were pulverized and mixed to use in the experiment.

Normal Negative Control (NC) Positive control group Solvent fraction administration group Oral administration carrier carrier Silymarin + carrier Hexane Fraction (Hfr.) + Carrier Ethyl Fraction (Efr.) + Carrier Ethyl acetate fraction (EAfr.) + Carrier Butanol fraction (Bfr.) + Carrier Water Fraction (Wfr.) + Carrier CCl ₄ injections No treatment process process process process process process process

The experimental animals were adapted to the laboratory by preliminary breeding for one week, and the experiment was conducted. The laboratory maintained a constant temperature at 22 ± 2 ° C. Twenty four hours before the sacrifice of animals for activity analysis, feeding was stopped and only water was supplied. After anesthesia with an ether, the stomach was opened, blood was collected from the abdominal vein, perfused with 0.9% saline, and the liver was extracted.

2. Serum Separation and Preparation of Enzyme Solution from Soy Sauce

The collected blood was left in the refrigerator for at least 30 minutes, centrifuged at 3,000 rpm for 10 minutes, and serum was separated and used for analysis of TNF-α (tumor necrosis factor-α). Extracted soy sauce for the analysis of glutathione-S-transferase (GST) activity was homogenized in a teflon homogenizer with 0.1 M sodium phosphate buffer (pH 7.4) in volume four times its weight, and then 20 to 10,000 g. The supernatant (cytosol fraction) obtained by ultracentrifuging the supernatant obtained by centrifugation for 10 minutes at 105,000 g was separated.

3. Analysis of TNF-α Content in Serum

To see the inhibitory activity of hairy roots on TNF-α production, one of inflammation-related cytokines, the kit (Endogen ER3TNFA5) was analyzed.

4. Analysis of GST Activity in Soy Sauce

The extracted liver cytosol was mixed with 2800 μl of potassium phosphate buffer (pH 6.5) and 75 μl of 0.04 M reduced glutathione, preincubated at 25 ° C. for 5 minutes, and then 0.12 M 2,4-dinitrochlorobenzene (DNCB) 25 The reaction was terminated by adding μl to 25 ° C. for 2 minutes and terminating the reaction by adding 500 μl of 20% TCA, sequentially centrifuged at 1,500 × g for 10 minutes, and the supernatant was measured for absorbance at 340 nm. The results were calculated according to the formula of enzyme activity (conjugated DNCB nM / mg protein) = (absorbance / 9.6) × (mg protein in 1 / enzyme solution / 2 minutes).

5. Analysis result

(1) Reduction Effect of Solvent Fractions Prepared from Methanol Extract of Hairy Root Flower on TNF-α Production in Rat Blood

FIG. 1 is a graph showing a reduction effect on TNF-α production in rat blood of solvent-specific fractions prepared from methanol extract of hairy roots. Where Normal is the carbon tetrachloride-free normal group, NC is the carbon tetrachloride-treated control group, Sil is the carbon tetrachloride and silymarin (commercial soy sauce) control group, Hfr. Etel fraction administration group, EAfr. Is the ethyl acetate fraction administration group of carbon tetrachloride and hairy roots, Bfr. Is the butanol fraction administration group of carbon tetrachloride and hairy roots, and Wfr.

As shown in Figure 1, the result of analyzing the content of the inflammatory cytokine TNF-α in the rat serum, TNF- which is higher than the normal group (Normal, 54 pg / ㎖) in the negative control group (NC, 83 pg / ㎖) α content was decreased 78.5% compared to the negative control group by the administration of the ether fraction (Efr., 18 pg / ㎖), which is the most effective, 44% reduction of the silymarin administration group (Sil. 47 pg / ㎖), a positive control It is superior to the effect. Also slightly weaker than the ether fraction, the ethyl acetate fraction (EAfr., 64 pg / ml), the hexane fraction (Hfr., 74.2 pg / ml) and the butanol fraction (Bfr., 75.3 pg / ml) were 23.1% and 9.5, respectively. % And 10.8% showed a relatively decreasing effect.

(2) Effect of Solvent Fractions Prepared from Methanol Extract of Hairy Root Flower Root on GST Activity Mitigation in Rat Liver

FIG. 2 is a graph showing the effect of alleviating the GST activity in the rat liver of the solvent-specific fractions prepared from the methanol extract of the hairy roots. Here, Normal, NC, PC (= Sil), Hfr., Efr., EAfr., Bfr., And Wfr. Are as defined above, respectively.

As shown in Figure 2, the GST activity showed increased activity in the negative control group (NC) than the normal group (Normal) by injection of carbon tetrachloride is the majority of the experimental groups administered the solvent-specific fractions of the hairy roots (Hfr., EAfr., Bfr., Wfr.) Was found to be alleviated, and the degree of relaxation was found to be as effective as the silymarin-administered group (PC). Etel fraction administration group (Efr.) Also confirmed a better effect than the negative control group.

Experimental Example  3: Buddha  50% of ethanol extract In a chemical reaction system  Antioxidant Analysis

In According to the results of preliminary experiments in vitro (omitted from the experimental results), 85 ° C. and 50% ethanol extracts were selected as the optimal extraction conditions of hairy roots, and the activities in the chemical reaction system were analyzed as follows.

1. Analysis of ROS (reactive oxygen species) scavenging activity in DCFDA (2 ', 7'-dichlorofluorescin diacetate) system

Samples prepared at three concentrations and 10 μl of control material (Trolox, Aldrich, 238813-1G) were dispensed into 96 wells each, and the liver tissues of rats treated with LPS (lipopolysaccharide) were homogenized and then 100-fold with 50 mM PBS. After diluting and dispensing 190 μl, 50 μl of 0.125 mM DCFDA was dispensed and mixed for 5 minutes, and then excitation at 485 nm and emission at 530 nm were measured for 5 minutes using a fluorophotometer (Genios). 50 mM PBS was used as a blank, and the results were obtained by averaging the percentage of change per second of Ex _{485 nm} / Em _{530 nm} per concentration for each of the three samples for a blank of Ex _{485 nm} / Em _{530 nm} per second. After calculating the equation between the percentage of change per second of Ex _{485 nm} / Em _{530 nm} , the concentration was reduced by 50%.

2. Peroxynitrite (ONOO ^-) scavenging analysis

After preparing three samples, 10 μl of the sample was dispensed into a 96 well black plate, 180 μl of the DHR mixture containing Rhodamine buffer, 5 mM DTPA, and DHR 123 at a constant ratio, and 10 μl of 200 μM peroxynitrie. After each _aliquot , Ex _{485 nm} / Em _{535 nm} was measured with a fluorophotometer. The results were calculated in the same way as DCFDA.

3. Experimental Results

The experimental results are shown in Table 3 below.

Lythrum hair root (LSR) with 50% of active oxygen in the reaction system of the ethanol extract of DCFDA (ROS) and peroxynitrite (ONOO ^-) prepared in the scavenging Extract & Control ROS scavenging activity (SC ₅₀ , μg / ml) ONOO ^- scavenging capacity (SC ₅₀ , μg / ml) Hairy Buddha Extract 2.42 ± 0.03 -23.3 ± 5.4 Trolox 3.43 ± 0.02 - Penicillamine - -43.1 ± 5.6

(1) It was confirmed that 50% ethanol extract of the hairy root of Root-bloom was very effective in the antioxidant activity of 2.42 ㎍ / ml in DCFDA reaction system, showing better activity than 3.43 ㎍ / ml of Trolox, a positive control.

(2) In addition, the scavenging activity of peroxynitrie was 50% ethanol extract of hairy root, -23.3 ㎍ / ml, which was somewhat higher than -43.1 ㎍ / ml of the positive control penicillamine. It showed peroxynitrite scavenging ability.

Experimental Example  4: Hair bud  Root 50% Ethanol Extract Cells In the reaction system  Active black

1. Inhibitory Effect of Reactive Oxygen Generation in YPEN1 Cells

YPEN1 cells, endothelial cells isolated from bovine prostate, were seeded in 96 wells at 1.5 × 10 ⁻⁴ cells / well, incubated overnight, discarded and washed with PBS, and 150 μl of serum-free medium was added. Buddha root 50% ethanol extract was dispensed into each well and incubated for 1 hour. Thereafter, t-BHT (t-buthyl hydroperoxide), which is an inducer of active oxygen generation, was dispensed to a final concentration of 10 μM, mixed, incubated for 1 hour, medium removed, washed with PBS, and 180 μl of SFMedia was added, followed by fluorescent staining. 50 μl of the reagent was dispensed to measure Ex _{485 nm} / Em _{535 nm} on a fluorophotometer. The results were calculated in the same way as in DCFDA.

2. Inhibitory Effect of Nitric Oxide (NO) Production in BV2 Cells

BV2 cells were adjusted to 1.0 × 10 ⁶ cells / ml with DMEM medium, then inoculated into 60 mm dishes and treated with extracts (0.8, 4.0, and 20.0 μg / ml) and cytokine mixture and incubated for 16 hours. It was. The amount of NO (Nitric oxide) produced was performed according to Liang's method using Griess reagent. Measured in the form of (NO ^{2 −} ) present in the cell culture, 100 μl of the cell culture supernatant and Griess reagent [0.1% N- (1-naphthyl) ethylenediamine. 2HCl, 1% sulfanylamide in 5% conc. After 100 μl of H ₃ PO ₄ in H ₂ O was mixed and reacted in 96 wells for 10 minutes, the absorbance was measured at 540 nm. The amount of NO produced was confirmed by comparing sodium nitrite (NaNO ₂ ) with a standard product.

3. Confirmation of viability of SH-ST5Y cells

2 × 10 ⁴ cells per well were added to 96 wells, and only one medium was used as a blank for absorbance measurement. A sample solution dissolved in a solution dissolved in water was made 10 times the desired concentration, diluted with PBS, and 20 μl of each solution was added to each of the six wells. After incubating the sample plate for 48 hours at 37 ° C and 5% CO ₂ incubator, 50 µl of 2 mg / ml MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) solution / Well was added and incubated again for 4 hours. At the end of the culture, centrifuge at 3,000 rpm for 5 minutes, remove the medium, add 150 μl / well of DMSO (dimethylsulfoxide) to dissolve and homogenize the formazan crystals, and then use IZ ₅₄₀ with ELIZA reader (TECHAN, USA). The value was measured. The cell survival rate of the experimental group compared to the control group was OD ₅₄₀ The values were obtained and expressed as a percentage of the mean OD ₅₄₀ value of the control.

4. Experimental Results

(1) Figure 3 is a graph showing the inhibitory effect of t-BHT-induced reactive oxygen (ROS) production in YPEN1 cells of 50% ethanol extract (LSR, 40, 200, 1000 ppm treatment) of hairy roots.

As shown in Fig. 3, the 50% ethanol extract of the hairy irises was normal compared to the negative control group (111.4) treated only with t-BHT at 40, 200 and 1,000 ppm concentrations for endothelial cells (YPEN1 cells) isolated from bovine prostate. Levels (54.5, 54.5 & 62.8) have been shown to reduce the production of free radicals. This showed 43.6-51.1% less active oxygen production than the negative control group, and the effect in the chemical reaction system was found to be very effective in animal cells.

(2) FIG. 4 is a graph showing LPS-induced NO production inhibitory activity in BV2 cells of 50% ethanol extract (LSR, 1, 10, 50, 100, 1000 ppm treatment) of hairy roots.

As shown in Figure 4, it was confirmed that the 50% ethanol extract of the hairy roots of the roots generally reduced in concentration depending on the NO production in BV2 cells, the most 23% reduction of nitrogen oxide was confirmed at 1,000 ppm concentration.

(3) MUT analysis was performed to determine whether 50% ethanol extract of hairy roots exerts the effect on animal cells according to toxicity to cells. FIG. 5 shows 50% ethanol extract of hairy roots (LSR, 1). , 10, 50, 100, 1000 ppm treatment) is a graph showing the effect on the survival rate of animal neurons (SH-SY5Y).

As shown in FIG. 5, the hair hyacinth extract did not significantly reduce the survival of SH-ST5T cells. Rather, when the concentration was 1,000 ppm, it was confirmed that the survival of the cells was increased compared to the control group.

Experimental Example  5: Buddha  50% of ethanol extract Liver fibrosis  Inhibitory Activity Assay

1. Animal experiment plan

Experiments were conducted to determine whether 50% ethanol extract of hairy roots had inhibitory activity against fibrosis-induced rat liver.

As experimental animals, Sprague Dawley male rats weighing about 100 g were randomly assigned to 8 animals in each experimental group. The experimental group was configured as shown in Table 4 below, and the 50% ethanol extract and the positive control doses of the hairy roots were 0.125, 0.25 and 0.5 g / kg body weight / rat / day (once / day, five times / week, 5 weeks of administration). Toxicity induction was intraperitoneally injected with 1 ml / kg body weight / rat of 40% CCl ₄ mixed with 1 ml of corn oil. Commercial solid feed (Sam taco) was used, and breeding conditions and serum separation were performed under the same conditions as in Experimental Example 2.

Normal Negative Control Positive control Hair Buddha Root Extract Administration Group Substance carrier carrier Carrier + Silymarin (0.125, 0.25, 0.5 g / kg) Carrier + Extract (0.125, 0.25, 0.5 g / kg) Carbon tetrachloride injection No treatment process process process

2. Preparation of homogenate from soy sauce

In order to analyze the contents of thiobarbituric acid reactive substance (TBARS) and glutathione, homogenates were obtained from the liver by homogenizing in a teflon homogenizer with 0.1 M sodium phosphate buffer (pH 7.4) of 4 times the volume of the extracted soy. .

3. Analysis of thiobarbituric acid reactive substance (TBARS) in liver

2 ml of soy homogenate was added to a 25 ml centrifuge tube, and 3 ml of 10% TCA aqueous solution and 2.5 ml of hexane containing 0.8% BHT were added sequentially, mixed for 30 seconds, and then mixed at 600 × g (300 rpm). Centrifuged for minutes, discarded the hexane layer of the upper layer, and the aqueous layer was allowed to settle 10 ml with 5% TCA. 2.5 ml of this solution was taken into a test tube with a lid, 1.5 ml of 0.8% TBA was added thereto, and then reacted in boiling water for 15 minutes according to a conventional method, followed by quenching and absorbance at 521.5 nm. The results were calculated according to the calibration curve formulated by MDA, the standard.

4. Analysis of Antioxidant Content in Soy Sauce

The total glutathione content was deproteinized by adding an equal amount of 4% sulfosalicylic acid to 1 ml of homogenate prepared from soy sauce, and 39.636 mg of disulfide reagent (5,5'-dithiobis (2-nitro benzoic acid)) was added 0.1M phosphoric acid to the obtained supernatant. The blue compound obtained by adding 2.7 ml of sodium buffer (pH 8.0) dissolved in 1 L was measured at 421 nm, and the result was calculated by substituting absorbance into the calibration curve using glutathione standard.

Oxidized glutathione (GSSG) was mixed with 1 ml of the centrifuged supernatant and 20 µl of 1 M 2-vinyl pyridine, and left at room temperature for 60 minutes. The content of reduced glutathione (GSH) was calculated by subtracting the content of oxidized glutathione from the total glutathione content, and the results are expressed as their ratio (GSH / GSSG).

5. GST activity analysis of liver

It carried out by the same method as specified in Experimental Example 2.

6. Analysis of Collagen Content in Soy Sauce

About 100 mg of liver tissue was added with 2 ml of 6N HCl, hydrolyzed at 110 ° C. for 24 hours, evaporated to dryness in a 50 ° C. water bath, and the remaining residue was dissolved in 10 ml of distilled water and filtered through a 0.45 μm Millipore filter. 1 ml of the mixture was mixed with 1 ml of freshly prepared 1.4% chloramine-T solution, left at room temperature for 20 minutes, mixed with 1 ml of 19% perchloric acid, and reacted at room temperature for 5 minutes, and finally in the newly prepared methyl cellosolve. 1 ml of 20% p-dimethylaminobenzaldehyde (or ethylene glycol monomethyl ether) was added. The reaction solution was reacted for 20 minutes in a 60 ° C. water bath, cooled, and the absorbance measured at 560 nm. The results were obtained after a calibration curve was prepared using a standard product (hydroxyproline) and expressed in μg / tissue 100 mg.

7. Analysis of collagen reduction effect of liver by immunohistochemistry

(1) Extraction between Rats

After removing the lobe of the same position in all the experimental groups, soaked in 10% buffered formalin solution (0.1M PBS, pH 7.0) and left for 24 to 48 hours, the sample was washed with PBS and graded alcohol 50% → 75% → 90% → 95% → 100%), and then immersed in paraffin to obtain a block, which was then sliced into 5 μm using a microtome.

(2) Confirmation of collagen reduction effect in liver of rat by immunohistochemical analysis

Using the peroxidase anti-peroxidase (PAP) method, paraffin was removed with xylene from slides with 5 μm sections attached to the above paraffins for hybridization, followed by a series of staged ethanol solutions (100% → 90%). → 75% → 50% → 0%) and then rinsed with distilled water. To destroy endogenous peroxidase activity, the mixture was treated with methanol containing 0.3% hydrogen peroxide for 10 minutes at 4 ° C. and treated with 5% bovine serum albumin (BSA) in PBS for 30 minutes. Sections were monoclonal anti-collagen, type III, Sigma C7805, clone FH, a mouse-derived antibody diluted in phosphate buffered saline (PBS) containing 1% bovine serum albumin. -7A, dilution 1: 2,000) and incubated at room temperature for 1 hour and washed with PBS. Incubated with diluted goat-derived secondary anti-mouse IgG (Fab specific) antibody (Sigma M6898, 1: 1,000) at room temperature for 30 minutes, washed with PBS, and then mouse-derived. Incubated with PAP complex (Sigma P2416 1: 500) at room temperature for 1 hour. This was washed with PBS, reacted with 0.05% 3,3'-diaminobenzidine hydrochloric acid solution (DAB, Sigma D4418) substrate chromogen at room temperature for 5 minutes, washed with water and counterstained with hematoxylin. staining). It was then washed with water and then mounted using a mounting medium and sealed with a cover slip. The degree of collagen production was observed on a light microscope with dried slide specimens.

8. Analysis of Collagen Reduction Effect of Rat Liver by Histological Analysis

As described in Experiment 5 of Example 7, paraffin blocks were prepared between the rats, and then paraffin was removed from the 5 μm sections attached to the slides by xylene treatment, and a series of stepwise ethanol solutions (100% → 90% → 75%). → 50% → 0%). After primary dyeing with 0.1% sirius red, washed and counter staining with fast green, a series of staged ethanol solutions (50% → 75% → 90% → 95%) 100%) and dehydrated with xylene and then mounted and sealed with cover slip. The degree of collagen production was observed on a light microscope with dried slide specimens.

9 . Experiment result

(1) Effects on Reduction of Lipid Peroxide Production in Rat Liver

FIG. 6 is a graph showing the effect of 50% ethanol extract (LSR, 0.125, 0.25, 0.5 g / kg administration) of hairy buds roots on the reduction of lipid peroxide (TBARS) production in liver liver induced by carbon tetrachloride. Here, Normal is a carbon tetrachloride-free normal group, NC is a carbon tetrachloride-treated control group, Sil-0.125, Sil-0.25, Sil-0.5 is carbon tetrachloride and silymarin 0.125, 0.25, 0.5 g / kg administration control, LSR-0.125, LSR- 0.25, LSR-0.5 refers to the administration of carbon tetrachloride and hairy root 50% ethanol extract 0.125, 0.25, 0.5 g / kg respectively.

As shown in Figure 6, 50% ethanol extract of the hairy iris flower root was administered 0.125 g / kg, 0.25 g / kg and 0.5 g / kg in rat liver fibrosis induced with carbon tetrachloride, the hairy iris flower extract was Peroxide contents of 7.3, 8.1 and 5.8 μg / g tissues were shown. Compared with the negative control group (9.1 μg / g tissue, 100%), hepatic lipid peroxide production was reduced by 11 to 36.3%, and the peroxide content when the positive control silymarin was administered 9.1, 6.6 And 0-27.5%, which is a reducing effect of 6.6 μg / g tissue.

(2) Antioxidant Maintenance Effect in Liver of Rat

Figure 7 is a graph showing the effect of maintaining the antioxidant content in liver liver fibrillation induced by carbon tetrachloride of 50% ethanol extract (LSR, 0.125, 0.25, 0.5 g / kg administration) of hairy roots.

As shown in Fig. 7, as a result of administering 0.125 g / kg, 0.25 g / kg and 0.5 g / kg of the extract of hairy roots to the rat model induced liver fibrosis with carbon tetrachloride, GSH / GSSG of 6.1, 7.8 and 12.5, respectively Rain was shown. This is an increase of 277 to 568% compared with 2.2 of the negative control group at 100%, which is much better than the increase effect of the GSH / GSSG ratio of 4.9, 7.5 and 6.5 of the positive control silymarin, 223 to 341%.

(3) Efficacy of Detoxifying Enzyme (GST) Activity in Rat Liver

8 is a graph showing the effect of detoxifying enzyme (GST) activity in rat liver induced with hepatic fibrosis with carbon tetrachloride of 50% ethanol extract (LSR, 0.125, 0.25, 0.5 g / kg administration) of hairy roots.

As shown in FIG. 8, administration of hairy root extract of 0.125 g / kg, 0.25 g / kg and 0.5 g / kg showed GST activity of 5.2, 4.9 and 4.8 μM conjugated DNCB / mg protein / min, respectively. This resulted in 1.9%, 8.5%, and 9.4% relaxation of normal control level when the 5.3 μM conjugated DNCB / mg protein / min of the negative control group was 100%, which is active in the amphibian control silymarin. It is more effective than 1.9, 5.7, and 3.7% of the mitigating effects of 5.2, 5.0, and 5.1 μM conjugated DNCB / mg protein / min.

(4) Relaxation Effect of Collagen Production in Rat Liver

① Confirmation of collagen production reduction effect through quantification of total collagen content

Figure 9 is a graph showing the effect of reducing collagen content in rat liver induced fibrosis with carbon tetrachloride of 50% ethanol extract (LSR, 0.125, 0.25, 0.5 g / kg administration) of hairy roots.

As shown in Figure 9, the extract of the root of the hairy iris flower was administered to liver fibrosis-induced rats in amounts of 0.125 g / kg, 0.25 g / kg and 0.5 g / kg for 5 weeks, and then the collagen of the extracted liver was L-hydroxy. Analysis of the total collagen content after digestion with proline showed L-hydroxyproline content of 9.6, 5.3 and 10.1 μg / 100 mg tissues, respectively. This is a decrease of 46.1%, 70.2% and 43.3% for the 17.8 μg / 100 mg tissues of the negative control group, respectively, and 45.5% and 14.0%, which is the reduction effect of 9.7, 15.3 and 13.4 μg / 100 mg tissues of the positive control silymarin. And 24.7% showed much better collagen production inhibition.

② Confirmation of collagen production reduction effect by immunohistochemistry

FIG. 10 is a micrograph of magnification × 100 showing the effect of reducing 50% ethanol extract (LSR, 0.125, 0.25, 0.5 g / kg) of rat hepatic fibrosis marker (collagen) by immunohistochemical analysis. .

10, when observed under a microscope, 50% ethanol extract of the hairy root of the hairy roots, as in the results of ①, brown collagen and two antibodies, PAP complex and dose-dependently at 0.25 and 0.5 g / kg Reaction with DAB chromogen was reduced compared to the negative control (NC, negative control), which was confirmed to be superior to the silymarin administration.

③ Confirmation of collagen production reduction effect by histology

FIG. 11 is a micrograph of magnification × 100 showing the effect of reducing 50% ethanol extract (LSR, 0.125, 0.25, 0.5 g / kg) of rat hepatic fibrosis in hairy roots by histological analysis.

Collagen production in rats induced liver fibrosis of 50% ethanol extracts of hairy roots by treatment with sirius red, a staining reagent that reacts specifically to all collagen proteins, and its control staining agent The reduction effect was confirmed. As shown in FIG. 11, the hair root extract was significantly reduced in red-brown pigmentation site showing total collagen as compared to the negative control group as the dose was increased, which was much better than that of the positive control silymarin.

The results obtained in the experimental example of the present invention was tested for significance at the level of P <0.05 by Duncan's multiple range test of the SAS program.

Above Examples 1 and 2, and when these results of Experimental Example 1-5, the extract and fraction of the hair roots is in Lythrum In vitro chemical reaction system, cell response system and liver fibrosis animal model, it can be said that it shows the antioxidant and liver fibrosis inhibitory activity which is very effective against the damage of liver by drug including alcohol. Moreover, this effect is superior to the commercially available soy sauce silymarin, and therefore, the hair-blowing root extract of the present invention is highly likely as a plant-derived natural liver protectant that can replace silymarin.

As described above, the extracts and fractions of the hair bud root of the present invention is excellent in antioxidant activity and hepatic fibrosis inhibitory effect, it can be industrialized as a medicine and health functional food material having antioxidant activity and hepatoprotective function.

Claims (5)

A pharmaceutical composition for preventing or treating liver cirrhosis, comprising an extract of the root of Lythrum salicaria as an active ingredient. According to claim 1, wherein the extract is a liver cirrhosis prevention or treatment pharmaceutical composition, characterized in that 0.001 to 99.999% by weight of the total composition. The method of claim 1, wherein the extract comprises a fraction, the ratio of the extract and the fraction of the liver cirrhosis preventive or therapeutic pharmaceutical composition, characterized in that the weight ratio of 99.999: 0.001 to 50:50. 4. The pharmaceutical composition for preventing or treating liver cirrhosis according to claim 3, wherein the fraction is an organic solvent fraction selected from hexane, ether, ethyl acetate and butanol. The pharmaceutical composition for preventing or treating liver cirrhosis according to claim 1, wherein the extract is prepared by extracting any one of lower alcohols selected from methanol and ethanol, or a mixed solvent of water and the lower alcohols.

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