KR100265385B1 - Extract of rhodiola sp. for prevending and treating circulating diseases - Google Patents

Abstract

PURPOSE: Rhodiola sp. extract, a process for preparing the extract by extracting Rhodiola sp. and a pharmaceutical composition containing the extract are provided. The composition can be widely used for prevention and treatment of circulatory diseases. CONSTITUTION: Rhodiola sp. extract having an effect on preventing and treating on circulatory diseases containing thrombosis and arteriosclerosis as well as senility prevention is obtained by extracting Rhodiola sp with lower alcohol, water, dichloromethane, butanol or the like. A pharmaceutical composition contains rosiridin of formula 1 and derivatives thereof of the Rhodiola sp. extract as an effective component.

Description

Honggyeongcheon Extract with Efficacy and Prevention of Circulatory Diseases

The present invention relates to an extract of Rhodiola sp. Having a prophylactic and therapeutic effect of a circulatory disease and a preparation method thereof.

More specifically, the present invention is to inhibit the formation of thrombus, the growth of vascular smooth muscle cells using the extract obtained by sequentially dividing the roots of honggyeongcheon with methanol, dichloromethane, butanol and water, and the compound isolated and purified therefrom as an active ingredient The present invention relates to a pharmaceutical composition having effects such as inhibition, antioxidant, lipid peroxidation inhibition, and physical strength enhancement, and may be widely used for preventing and treating circulatory system diseases including thrombosis and atherosclerosis as well as preventing aging.

Rhodiola sp. Is a perennial herbaceous plant with fleshy rhizome and is used as a medicinal site with a significant root and stem. Hongkyungcheon is classified as pizza plant gate, Gyeongcheondae, and Hongkyungcheon by category, and there are 96 species worldwide. Especially, there are 70 species in China, and 34 species and 2 varieties in Xinjiang. have.

In addition, Hongkyeongcheon is a unique plant that has a low temperature, dryness, oxygen deficiency, strong ultraviolet light and strong adaptability even under bad conditions of 2,000 ~ 5,000m above sea level, where the temperature difference between day and night is severe. Therefore, after the discovery of ginseng and thorny ginseng, it is known as one of the best health medicinal plants to restore freshness and to overcome disease and poison and have a long name.

Lipid peroxidation caused by free radicals has recently been found to be a major cause of aging and aging (McCord, JM, Surgery, 94, 412, 1983., Halliwell et al., Am. J. Medicine, 91, 14, 1991 ., Harman. D., J. Gerontol., 11, 298, 1956.).

The first product of the monovalent reduction of oxygen, which is essential for life, is the superoxide anion, which interacts with hydrogen peroxide to generate hydroxyl radicals. Free radicals, such as superoxide, react with lipids in a chain reaction to produce peroxides, which can lead to aging, cancer and other diseases (Miquel, J. et al., "Handbook of free radicals"). and antioxidants in biomedicine ", CRC Press Inc. Boca Ration, p177, 1989).

In addition, hydroxyl radicals and lipid peroxides are known to inhibit the production of prostacyclin, and in particular, lipid peroxides denature various enzymes or lipoproteins, destroy cell membranes, and prevent constituent disorders. It has been reported to induce cell senescence by causing or accumulating lipofuscin (Gryglewski, RJ et al., Prostaglandins, 12, 685, 1976; Tappel, AL, Feb. Proc., 32, 1870, 1973; Roubal, WT et al., Arch. Biochem. Biophys., 113, 5, 1966). In addition, It is known that by promoting the generation of thromboxane A ₂ (thromboxane A ₂₎ in platelets causing a number of reaction conditions such as to cause the vascular occlusion.

However, most organisms have antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase to remove toxins caused by these free radicals. (Weisberger, RA et al., J. Biol. Chem., 248, 3582, 1973; Pryor, WA et al., "Free radicals in Biology" Vol. I, Academic Press, Orando, Chapter 6 , 1976). Therefore, drugs that can effectively inhibit superoxide radicals or lipid peroxides in a manner related to such enzymes can be usefully used to prevent and treat aging, carcinogenesis, adult diseases and various vascular diseases.

On the other hand, the secretory function of the vascular endothelial system is also reported to be affected by oxygen-derived free radicals. This is because the superoxide anion destroys endothelial-derived relaxing factor (EDRF) (Gryglewski, R. J. et al., Nature, 320, 454, 1986).

In addition, cardiovascular disease is mainly caused by the proliferation of vascular smooth muscle cells. Specifically, when endothelial cell permeability is enhanced by vascular endothelial injury, platelet aggregation occurs at the site of injury, and platelet-derived growth factor (PDGF) is released from the aggregated platelets and vascular smooth muscle is released by PDGF. Oiling from the media to the lining of the cell occurs, which promotes the proliferation of vascular smooth muscle cells. On the other hand, macrophages derived from monocytes present in the blood invade from injury sites of endothelial cells, devour a large amount of fat, and become arterial cells, causing fat accumulation to cause atherosclerosis. Proliferation and hypertrophy of vascular smooth muscle cells is one of the important characteristics of hypertensive patients (Owens, G. K., Circ. Rec., 56, 525, 1985). In addition, platelet aggregation is an essential step leading to blood coagulation and thrombus formation, and is easily affected by increased physiological activity of platelets or changes in lipid metabolism in plasma.

Therefore, the present inventors continued to study new substances useful for circulatory diseases from natural products. As a result, the roots of Rhodiola sp. Were extracted with water or methanol, and sequentially obtained with dichloromethane, butanol and water. The present invention was completed by confirming that the extract and the compound isolated and purified therefrom were effective in inhibiting thrombus formation, inhibiting proliferation of vascular smooth muscle cells, antioxidant, inhibiting lipid peroxidation and enhancing physical strength.

It is an object of the present invention to provide a pharmaceutical composition useful for the prevention and treatment of aging and circulatory diseases, which comprises the extract of Rhodiola sachalinen and the compound isolated and purified therefrom.

Figure 1 shows the mass spectrometry spectrum of rosiridine (rosiridin) of formula 1 isolated and purified in Honggyeongcheon,

2 shows the infrared absorption spectrum of the Rossiridine,

Figure 3 shows the hydrogen nuclear magnetic resonance spectrum of the Rossiridine,

Figure 4 shows the carbon nuclear magnetic resonance spectrum of the Rossiridine,

Figure 5 shows the mass spectrometry spectrum of rosiridol of formula (2) isolated and purified from the Hong Kyungcheon extract,

Figure 6 shows the hydrogen nuclear magnetic resonance spectrum of the Rossiridol,

Figure 7 shows the carbon nuclear magnetic resonance spectrum of the Rossiridol,

8 shows the mass spectrometry spectra of cinnamic alcohol of Formula 3 isolated and purified from Honggyeongcheon extract,

9 shows a hydrogen nuclear magnetic resonance spectrum of the cinnamil alcohol,

Figure 10 shows the carbon nuclear magnetic resonance spectrum of the cinnamil alcohol,

Figure 11 shows the inhibitory effect of 3H-thymidine influx by the treatment of angiotensin II and platelet-derived growth factor (PDGF) by the siriridine,

12 shows dose-dependent inhibition of ³ H-thymidine influx by the PDGF by the rosyridine.

In order to achieve the above object, the present invention provides a honggyeongcheon extract that can be used for the prevention and treatment of circulatory diseases extracted Rhodiola sp. With lower alcohol or water.

In addition, the present invention also provides a honggyeongcheon extract fractionated back to dichloromethane, butanol or water.

The honggyeongcheon extract includes a compound of formula (1) (rosiridine), a compound of formula (2) (roshiridol) and a compound of formula (3) (cinnamil alcohol) as an active ingredient.

In addition, the present invention is obtained by extracting the honggyeongcheon with lower alcohol to obtain an alcohol extract or residue, and suspended the honggyeongcheon alcohol extract again in distilled water to obtain a variety of solvent fractions by repeatedly fractionating with an organic solvent, the residue is extracted again with methanol to precipitate fractions and water-soluble fractions It provides a method for preparing the Honggyeongcheon extract to obtain.

In this case, methanol or ethanol is used as the lower alcohol, dichloromethane or butanol is sequentially used as the organic solvent, and the residue is extracted using methanol.

Rhodiola sachalinensis extract of the present invention has the effect of preventing and treating atherosclerosis due to the inhibition of thrombus formation and vascular smooth muscle cell proliferation inhibitory activity.

In addition, the honggyeongcheon extract of the present invention has an anti-aging and fatigue recovery effect due to the antioxidant activity.

The present invention also provides a pharmaceutical composition for treating a circulatory disease, comprising the compound of formula 1 (rosyridine) and derivatives thereof as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a honggyeongcheon extract useful for circulatory diseases and a method for producing the same.

First of all, the present invention cuts Rhodiola sp. And extracts with lower alcohol or water to obtain Rhodiola sp. At this time, it is preferable to use methanol or ethanol as the lower alcohol. In addition, as the honggyeongcheon, all plants commonly used as honggyeongcheon can be used, including Changbai Mountain Rhogyeongcheon (Rhodiola sachalinensis) and Seojang Honggyeongcheon.

In another aspect, the present invention provides a honggyeongcheon extract obtained by fractionating the honggyeongcheon alcohol extract again with an organic solvent or water. At this time, it is preferable to use dichloromethane, butanol and the like as the organic solvent.

Specifically, in the present invention, the methanol extract is suspended in distilled water and repeatedly fractionated with dichloromethane to obtain a dichloromethane fraction of honggyeongcheon, which is then fractionated with butanol to obtain a butanol fraction and a water fraction. In addition, 50% methanol precipitated and 50% methanol water-soluble fractions are obtained by extracting the red gyeongcheoncheon used above with methanol, adding 50% methanol to the remaining residue, extracting with an ultrasonic extractor, and filtering.

The honggyeongcheon extract obtained above comprises a compound of formula (1) (rosyridine, rosiridin), a compound of formula (2) (rosiridol, rosiridol) and a compound of formula (3) (cinnamyl alcohol, cinnamyl alcohol) as an active ingredient.

[Formula 1]

[Formula 2]

[Formula 3]

The present invention also provides a method for preparing the compound of formula 1 (rosiridine), the compound of formula (2) (rosiridol) and the compound of formula (3) (cinnamil alcohol) from the extract.

Specifically, the dichloromethane fraction of honggyeongcheon was concentrated and re-divided with hexane and methanol to obtain a methanol fraction of honggyeongcheon, and then subjected to silica gel column chromatography and preparative high-speed liquid chromatography and the like (Rosiridine, Rossiridol, Cinnamic alcohol) is separated and purified.

The honggyeongcheon extract and purified compounds therefrom have anti-thrombotic formation, anti-proliferative activity of vascular smooth muscle cells, antioxidant, lipid peroxidation, physical strength enhancing activity and the like.

Specifically, in order to investigate the vascular smooth muscle cell proliferation inhibitory activity of Rhodiola sachaline extract, ³ H-thymidine influx was measured in primary cultured cells of rat aortic smooth muscle. In particular, at this time, the dose-dependent effects of the compounds purified in Honggyeongcheon were investigated together.

As a result, the compounds of Formula 2 and Formula 3 do not inhibit the influx of ³ H-thymidine by Angiotensin II and platelet-derived growth factor (PDGF), but the compounds of Formula 1 are significantly The influx of ³ H-thymidine was inhibited dose dependently (see FIGS. 11 and 12).

In addition, the present invention, to investigate the platelet aggregation inhibitory effect of Rhodiola sachalinensis extract, the degree of aggregation of platelets was measured according to the Bourne's turbidity measurement method using a platelet aggregation tester (Born, GVR et al., J. Physiol., 168, 178, 1963).

As a result, as shown in Table 1, the methanol extract of Rhodiola sachaline significantly reduced the aggregation of human platelets stimulated with adenosine diphosphate, and when the solvent was fractionated, the activity was higher in the water fraction of higher polarity than the nonpolar solvent fraction. It can be seen that the material exists.

Therefore, the extract of red gyeongcheoncheon of the present invention and the compound purified therefrom can be usefully used to prevent and treat diseases of the circulatory system including thrombosis and arteriosclerosis.

In addition, the present invention, the superoxide dismutase (SOD) activity in order to investigate the antioxidant activity of the Rhodiola extract, was measured by the method of prido beach and in vivo methods (Fridovich, I. et al., J. Biol. Chem., 243, 5753, 1968). At this time, the antioxidant activity in vivo was investigated by inducing the lipid and oxidation of the animal using carbon tetrachloride and then obtaining a microsome fraction. In addition, the present invention also examined the catalase activity by examining the rate of consumption of H ₂ O ₂ in vivo.

As a result, the SOD activity in each solvent fraction of Hong Kyung-cheon was 12-18 times higher than that of Rooibos tea. In addition, in vivo, SOD activity and catalase activity significantly increased when the methanol extract of Rhodiola sachaline (200 mg / kg administration group) was administered.

In the present invention, lipid peroxidation inhibitory activity of Rhodiola sachalinensis extract was measured by the method of Nose using linoleic acid as a substrate and the in vivo method using rats (Nose, M. et al., Nippon Shokuhin Kogyo Gakkaishi). , 29, 507, 1982). In addition, the content of lipid peroxide in vivo was measured by a method such as Masugi (Masugi, F. et al., Vitamin, 51, 21, 1977).

As a result, the inhibition of peroxide generation in each solvent fraction of Hong Kyung-cheon was found to be approximately 40-57% evenly in each solvent fraction. Also, in vivo, the production of lipid peroxide was significantly decreased when the methanol extract of Rhodiola sachaline was administered (200 mg / kg administration group).

In order to investigate the fatigue recovery effect of Rhodiola sachalinensis extract, the effects of the mouse's exercise performance and physical strength enhancement were used by slightly modifying Richard's swimming test method (Richard, HB et al., Am. J. Physiol , 168, 178, 1963).

As a result, swimming time was significantly increased even in the 50 mg / kg administration group.

Therefore, the honggyeongcheon extract of the present invention and the compound purified therefrom can be usefully used to prevent aging and to prevent aging and to recover from fatigue as well as to prevent and treat diseases of the circulatory system.

In the present invention, toxicological experiments were carried out on the Rhodiola sachaline and the compound purified therefrom. Specifically, the composition was dissolved in water and administered to mice (5 mice per group), and then observed for 14 days to determine mortality. At this time, when the composition was administered by the concentration step, the 50% lethal dose (LD50) was found to be 5 g / kg or more.

In order to use the composition of the present invention prepared by the above process as a therapeutic agent, it may be prepared by a known method in the pharmaceutical field, and by itself or a pharmaceutically acceptable carrier, excipient (forming agent) It may be mixed with a diluent and the like to be prepared and used in the form of a powder, granule, tablet, capsule or injection. In addition, they may be administered orally or parenterally.

The dosage of the active ingredient according to the present invention is appropriately selected according to the absorption of the active ingredient in the body, the rate of water activation and excretion, the age, sex and condition of the patient, the severity of the disease to be treated, etc. About 30-300mg is administered, but it is preferable to administer about 50-200mg. Therefore, the unit dosage form of the present invention is prepared in the amount of 20-100 mg of the active substance of the present invention in consideration of the above-mentioned effective amount range. Preferably it is formulated to contain 10-50 mg. The unit dosage form thus formulated may be administered several times at regular intervals or by using specialized dosing methods according to the judgment of the expert who monitors or observes the administration of the drug as needed and the needs of the individual.

Hereinafter, the present invention will be described in detail by examples.

The following examples illustrate the invention, but the scope of the invention is not limited by the examples.

Example 1

Preparation of Honggyeongcheon Extract

1kg of Hongkyeongcheon root was chopped and placed in an Erlenmeyer flask, methanol was added until the sample was immersed, extracted at room temperature for 2 days, filtered, and concentrated under reduced pressure to obtain Hongkyeongcheon methanol extract (RJ-1). In addition, the methanol extract of Honggyeongcheon was suspended in 20-fold amount of distilled water, and the same amount of dichloromethane was added thereto to repeat the distillation three times to obtain a dichloromethane fraction (RJ-2), which was then fractionated with butanol to obtain a butanol fraction (RJ- 3) and a water fraction (RJ-4) were obtained. In addition, the Hong Kyungcheon was extracted with methanol, 50% methanol was added to the remaining residue, extracted with an ultrasonic extractor, filtered and left to obtain a precipitate fraction (RJ-5) and a methanol soluble fraction (RJ-6) of 50%.

Example 2

Isolation and Purification of Biologically Active Substances from Rhodiola sachalinensis

The dichloromethane fraction of Honggyeongcheon obtained in the above example was concentrated and re-divided into hexane and 90% methanol to obtain 90% methanol fraction of Honggyeongcheon. Then, a mixed solvent of dichloromethane and methanol (30: 1 → 1: 1) was used as a developing solvent. Silica gel column chromatography was carried out, and 20 fractions were obtained from the fractions, and one fraction was collected by preparative high performance liquid chromatography (equipment: Waters Model, 600 pump, 996 photodiode array detector, 600 controller; column: Prep Nova-pak HR). C18, solvent: 25% methanol) to obtain a compound of formula 1 (rosiridine, rosiridin), a compound of formula 2 (rosiridol, rosiridol), a compound of formula 3 (cinnamyl alcohol, cinnamyl alcohol).

Example 3

Physicochemical Properties of Compounds Isolated and Refined from Hong Kyung-cheon

(3-1) Compound of Formula 1: Rossiridine

3,7-dimethyl-2,6-octadiene-1,4-diol-1-o-β-D-glucopyranoside

(3,7-dimethyl-2,6-octadien-1,4-diol-1-o-β-D-gulucopyranoside)

(1) Property of the substance: white powder

(2) Molecular weight: 332

(3) Molecular formula: C ₁₆ H ₂₈ O ₇

(4) Mass spectrometry (m / z, rel. Int.%):

Mass spectrometry measured by the EIMS method is shown in FIG. 1.

315 (1.3), 263 (10.3), 69 (100)

(5) UV absorption spectrum (UVmax mn): 208

The ultraviolet absorption spectrum measured in methanol is as shown in FIG. 2.

(6) hydrogen nuclear magnetic resonance spectrum (NMR)

Hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectra measured using dutromethanol (CD ₃ OD) as a solvent are shown in FIG. 3.

(7) carbon nuclear magnetic resonance spectrum (NMR)

Carbon nuclear magnetic resonance ( ¹³ C-NMR) spectra measured using detromethanol (CD ₃ OD) as a solvent are shown in FIG. 4.

(7) chemical structure

(3-2) Compound of Formula 2: Rossiridol

3,7-dimethyl-2,6-octadiene-1,4diol (3,7-dimethyl-2,6-octadien-1,4-diol)

(1) Property of the substance: white powder

(2) Molecular Weight: 170

(3) Molecular formula: C ₁₀ H ₁₈ O ₂

(4) Mass spectrometry (m / z, rel. Int.%):

The mass spectrometry measured by the EIMS method is shown in FIG. 5.

(5) hydrogen nuclear magnetic resonance spectrum (NMR)

Carbon nuclear magnetic resonance ( ¹ H-NMR) spectra measured using detromethanol (CD ₃ OD) as a solvent are shown in FIG. 6.

(6) carbon nuclear magnetic resonance spectrum (NMR)

Carbon nuclear magnetic resonance ( ¹³ C-NMR) spectra measured using detromethanol (CD ₃ OD) as a solvent are shown in FIG. 7.

(7) chemical structure

(3-3) Compound of Formula 3: Cinnamic alcohol

3-phenyl-2-propen-1-ol

(1) Property of the substance: white powder

(2) Molecular weight: 134

(3) Molecular formula: C ₉ H ₁₀ O

(4) Mass spectrometry (m / z, rel. Int.%):

Mass spectrometry measured by the EIMS method is shown in FIG. 8.

(5) hydrogen nuclear magnetic resonance spectrum (NMR)

Hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectra measured using DUPROdimethylsulfoxide (DMSO-d ₆ ) as a solvent are shown in FIG. 9.

(6) carbon nuclear magnetic resonance spectrum (NMR)

Carbon nuclear magnetic resonance ( ¹³ C-NMR) spectra measured using deutlodimethylsulfoxide (DMSO-d ₆ ) as a solvent are shown in FIG. 10.

(7) chemical structure

Example 4

Determination of Vascular Smooth Muscle Cell Proliferation of Rhodiola sachalinensis Extract

(4-1) Primary vascular cell culture

Inhalation of carbon dioxide in rats, killing and disinfecting the incision site, dissecting the aorta, immediately transfer to sterilized physiologic salt solution (PSS) to remove the adipose tissue, 0.2% collagenase (collagenase) Endothelial cells were removed by treatment in a thermostat at 37 ° C. for 20 minutes. The inner and outer membranes were then crushed and crushed again, followed by shaking with an orbital shaker for 4 hours by adding complete cell culture medium (10% FBS, DMEM medium containing antibiotics and glutamine) containing collagenase. Reacted. Next, the collagenase was washed by centrifugation of the DMEM solution, and the precipitate was mixed in an appropriate dish of an appropriate amount of the complete DMEM solution, and immediately placed in a CO ₂ incubator at 37 ° C., and the cells were cultured. At this time, the number of viable cells was taken in a portion of the culture solution, and the same amount of trypan blue was added to the hemocytometer and measured under a microscope. The cells used in the experiment were cells of 20 passages, which were specific for smooth muscle. Β-actin was identified using antibodies.

(4-2) Measurement of ³ H-thymidine influx

Compounds of Formula 1 (rosiridin) and Compounds of Formula 2 (rosiridol) isolated from Rhodiola sac on rat arotic vascular smooth muscle cells (RVSMs) of rat aorta, which had been suspended for 24 hours in serum-free medium. And a compound of Formula 3 (cinnamyl alcohol) at a concentration of 10 ug / ml for 1 hour, and 10 ^-5 M of angiotensin II and 50 ng / ml of PDGF for 20 hours, followed by 1 μCi / ml [Methyl- ³ H] thymidine was labeled for 4 hours. Cells were labeled and washed with cold phosphate buffer (PBS; 1 mmol / L CaC _l2 , 1 mmol / L MgC _l2 , 10% trichloroacetic acid, ethanol / ether (2: 1 volume / volume)) and insoluble in acid. Phosphorus ³ H-thymidine was extracted by adding 250 μl of 0.5 M NaOH per petri dish, and 5 ml scintillation cocktail was added to 0.1 ml of the extract and measured by β-ray measuring instrument. Compounds of formula (2) and (3) did not inhibit the influx of ³ H-thymidine by angiotensin II and platelet-derived growth factor (PDGF), but compounds of formula (1) It was shown to inhibit the influx of thymidine (see FIG. 11).

In order to examine the dose-dependent effects of ^three compounds of formula I for the inflow of H- thymidine by the following PDGF, the compounds 1, 10, 50 ug / ml Procedure After a treatment with a concentration of ³ naphthyridin this by PDGF It was confirmed that the influx of H-thymidine was dose-dependently (see FIG. 12).

Example 5

Platelet aggregation inhibitory effect

(5-1) Preparation of Platelet Rich Plasma

Blood was drawn from the vein using a tube treated with 3.8% sodium citrate (1: 9 volume / volume) in healthy volunteers who had not received drug for at least 15 days. Platelet rich plasma (PRP) was obtained by centrifuging the collected blood at 120 g for 10 minutes, and platelet poor plasma (PPP) was prepared by PRP and then centrifuged at 1000 g for 25 minutes. Platelet count of PRP was adjusted to 3 × 10 ⁸ cells / ML concentration.

(5-2) Measurement of platelet aggregation inhibitory ability

Platelet aggregation inhibitory activity was measured by a turbidity measurement method such as Bonn using a platelet aggregometer (whole blood lumi-ionized calcium aggregometers, Chrono-Log Corp., USA). 440 μl of the PRP was taken and first incubated at 37 ° C. for 3 minutes, 10 μl of sample was added, and 2 minutes later, 5 μl of platelet aggregation promoter was induced. The aggregation reaction was measured for 5 minutes, and the degree of inhibition was determined by obtaining the maximum aggregation point. While the aggregation was measured, the reaction solution was agitated at 1,000 rpm, and ADP (20 μM, Sigma) was used as a platelet aggregation promoter. The degree of inhibition was calculated using the following equation.

[Equation 1]

Inhibition% = [(A-B) / A] × 100

A:% aggregation level of reference group

B: degree of aggregation at the time of drug treatment

As a result, as shown in Table 1, the methanol extract of Rhodiola sachaline significantly reduced human platelet aggregation stimulated with ADP, and when the solvent fractionation was performed, it was found that the active substance was present in the highly polar water fraction rather than the nonpolar solvent fraction. there was.

TABLE 1

Inhibitory Activity of Human Platelet Aggregation by ADP Stimulation of Rhodiola sachalinensis Extract

Example 6

Determination of the Effect of Rhodiola sachalinensis Extract on the Superoxide Dismutase Activity

In the present invention, the superoxide dismutase (SOD) activity of the Rhodiola sachaline extract was measured according to the method of Pridovic (Fridovich, I. et al., J. Biol. Chem., 243, 5753, 1968). ). This method measures the reduction of cytochrome c by the superoxide anion produced by the xanthi ne / xanthine oxidase reaction. Specifically, SOD activity is measured using a phenomenon in which the amount of superoxide anion is reduced by SOD and the rate of change of cytochrome c is decreased. The composition of the reaction solution is cytochrome c 24.8 mg, 5 μM xanthine 10 ml (0.001 NNaOH), Add 100 ml of 50 mM sodium phosphate buffer (pH 7.8, 0.1 mM EDTA), add 50 μl of the distilled distilled water extract to 2 ml of the mixed substrate solution, stabilize for 2 minutes, and then xanthine oxidase (0.2U / ml, 0.1 ㅡ) EDTA) 50μl into the cuvette and mixed, measure the absorbance at 550nm for 2 minutes, the absorbance per minute in the linearly increasing part with time (min) on the X-axis and absorbance (OD) on the Y-axis The change in ΔAbs / min was determined to determine the relative SOD activity. At this time, the amount of inhibiting cytochrome c reduction by 50% was defined as SOD 1U, the SOD titer (specific activity = U per mg) was determined in the experiment by the amount of 50% inhibition of absorbance per minute (OD) of the reference group.

As a result, as shown in Table 2, the SOD activity of each solvent fraction of honggyeongcheon was compared to the rooibos tea commercially available Rooibos tea, all fractions except dichloromethane fraction The SOD activity of was 12-18 times very high and even, especially butanol fraction (RJ-3) was the highest 18 times.

TABLE 2

Superoxide Dismutase (SOD) Activity Treated with Solvent Fraction from Rhodiola sachalinensis Extract

Example 7

Determination of Lipid Peroxidation Inhibitory Activity of Rhodiola sachalinensis Extract

The present invention is a method of Nose using a lipid peroxidation inhibitory activity (Peroxide value, POV) of linoleic acid as a substrate (Nose, M. et al., Nippon shokuhin Kogyo Gakkaishi, 29, 507, 1982). 100 μl of linoleic acid and 5 μl of each sample (5 mg / ml concentration of sample) were placed in a test tube of 1.6 cm × 6 cm for 24 hours in a 50 ° C. thermostat to promote oxidation, followed by chloroform / acetic acid (2: 3, V / V Dissolve in 35 ml and place the solution in a 250 ml orthogonal Erlenmeyer flask to replace air with nitrogen. 1 ml of saturated aqueous potassium iodide solution was added thereto, mixed vigorously for 1 minute, left in the dark for 5 minutes, 75 ml of distilled water and 1 ml of starch solution were added, and iodine (I2) was reversely titrated with 0.01 N sodium thiosulfate solution. The lipid peroxidation inhibitory effect of each sample was measured.

[Equation 2]

Tv: Consumption (0.01) of 0.01 N sodium thiosulfate solution consumed for titration by sample

Bv: Consumption (0.01) of 0.01 N sodium thiosulfate solution consumed for titration of linoleic acid

W: amount of substrate used in the experiment (g)

As a result, as shown in Table 3, the inhibitory effect of peroxide production on linoleic acid substrate of each solvent fraction of Rhodiola saline was approximately 40-57% in each solvent fraction. % Inhibition effect, dichloromethane fraction and butanol fraction also showed a relatively large inhibitory effect.

TABLE 3

Inhibitory Effect of Rhodiola sachalinensis Extract Fraction

* Inhibition rate (%) = [1- (sample _POV / reference group _POV )] x 100

Example 8

Measurement of Lipid Peroxidation Inhibitory Activity at the Cell Level of Rhodiola sachalinensis Extract

(8-1) Preparation of microsomal fraction in white liquor

Liver of 8-week-old rat females weighing 200-220 g was perfused with 0.15 M potassium chloride, and then chopped and pre-cooled with a 4-fold buffer solution (50 mM Tris-HCl, pH 7.4) Into and homogenized tissue. This was centrifuged at 8,000 g for 15 minutes and the filtrate was further centrifuged at 10-000 g for 20 minutes to remove unhomogenized tissue sections and mitochondria. The supernatant was then centrifuged at 100,000 g for 1 hour, the precipitated pellet was suspended in homogenization solution, centrifuged once again at 100,000 g, and the precipitated pellet was aliquoted into microsome fractions. This microsome fraction was quantified according to the Biuret method (Ohnishi, S. T. et al., Anal Biochem., 86, 193, 1978) and stored frozen at -70 ° C.

(8-2) Preparation of Fe / ascorbic acid system reaction solution

To 1.6 ml of 50 mM Tris-HCl buffer solution (pH 7.5), 0.1 ml of the sample solution, 0.1 ml of liver microsome fraction (containing 1 mg of protein in 1 ml), 0.1 ml of 0.1 mM ascorbic acid, 5 mM FeSO ₄ , and 0.1 ml were added sequentially. The reaction solution was prepared. At this time, the concentration of the sample was adjusted to 200 μg in the reaction system (Wong, SF et al., J. Inorg. Biochem., 14, 127, 1981).

(8-3) Assay of lipid peroxidation inhibitory activity

Each reaction solution prepared above was mixed well and reacted at 37 ° C. for 1 hour, and 0.5 ml of a mixed solution of 3M TCA and 2.5N hydrochloric acid was added thereto, followed by centrifugation at 1,000 g for 10 minutes. Next, 1 ml of the supernatant was mixed with 1 ml of 0.67% TBA solution, heated for 30 minutes in boiling water, developed, cooled to room temperature, and absorbed at 532 nm (Esterbauer, H. et al., Methods in enzymology, Academic Press, New). York, 150, 319, 1981). As a control sample, no sample solution was added, and no reaction solution was used as a blank sample. Malondialdehyde (MDA) was quantified using tetrametho xypropane as a standard solution. .

(8-4) Effect of Lipid Peroxidation Inhibitory Activity

The results of assaying the non-enzymatic lipid peroxidation inhibitory activity using each solvent fraction of Hong Kyung-cheon are shown in Table 4. In particular, the methanol fraction of Honggyeongcheon showed an inhibitory effect of 46.6%, and the butanol fraction (RJ-3), which was re-fractionated, showed a high inhibitory effect of 55.4%. The fraction extracted with methanol also showed high inhibitory activity.

TABLE 4

Lipid peroxidation inhibitory activity of honggyeongcheon acetate fraction (non-enzymatic Fe ²⁺ / ascorbic biksan system)

* Inhibition rate (%) = [1- (sample _POV / reference group _POV )] x 100

Example 9

Antioxidant Activity of Rhodiola sachalinensis Extract in Vivo

Five rats were used in 1 group, and two groups were dissolved in 0.5% carboxymethylcellulose (CMC) for 5 days in methanol extract of Honggyeongcheon. Orally, 50 mg / kg and 200 mg / kg were administered orally. % CMC was administered. On the last day of dosing, 25 ml of carbon tetrachloride (CCl ₄ ) fasted for 6 hours in advance and dissolved in corn oil was orally administered at 4 ml / kg. The control group received only CCl ₄ instead of the sample solution, and the settlement group received only corn oil without the CCl ₄ and the sample solution. After 12 hours of administration of CCl ₄ , anesthesia was inhaled with ether, the abdomen was excised, the liver was extracted, washed several times with ice-cooled 0.9% saline solution, and the liver microsome fraction was obtained to obtain the SOD and catalase described above. And as a substrate for measuring lipid peroxidation. At this time, the protein content of the microsomal fraction was measured using the Burette method.

(9-1) Superoxide Dismutase Activity Measurement

SOD activity was measured according to the cytochrome c method of the prido beach. Specifically, 50 μl was used as an enzyme by diluting the liver microsome fraction. The diluted liver microsome fraction was added to a mixture of citchrome c and xanthine, and xanthine oxidase was added to measure absorbance at 550 nm. As a result, CCl ₄ administration showed that liver SOD activity was reduced by 35% compared to the normal control. Methanol extract of Rhodiola sachaline extract was increased by 29% compared with CCl ₄ tug group, but there was no significant difference, but there was a significant increase of 42% in 200 mg / kg group (P <0.05).

(9-2) Catalase Activity Measurement

The catalase activity of microsomal liver by measuring the rate of consumption of H ₂ O ₂ and the H ₂ O ₂ as a substrate was measured (Luck, H. et al., Methods of Enzymatic Analysis, Academic Press, New York, p885, 1971). 30% of 30% hydrogen peroxide (Hydrogen peroxide) was dissolved in 5.0 ml (ph 7.0) of 0.05 M potassium phosphate buffer and used as a substrate. At this time, the enzyme was used by diluting the liver microsome fraction to 100-fold concentration. 570 μl of 0.05M potassium phosphate buffer (pH 7.0), 330 μl of substrate, and 100 μl of a dilute solution of homogenate as an enzyme source were put into a cuvette and mixed for 10 seconds at 240 nm for 40 seconds.

[Equation 3]

Active U / m1 = 22.94 × change in absorbance / min × dilution factor

Active U / mg = protein = (U / m1) / mg protein / ml

As a result, CCl ₄ administration decreased hepatic catalase activity to 44% compared to the normal control group, while the Methanol extract 50mg / kg group in Hong Kyung-cheon increased 27% compared to CCl ₄ group, but it was not significant, but 200mg / kg group. There was a significant increase of 50% (P <0.05).

(9-3) Measurement of lipid peroxidation content

The content of lipid peroxides was measured by the method such as Masugi (Masugi, F. et al., Vitamin, 51, 21, 1977). To the homogenate of the liver microsome fraction, 10% sodium dodecyl sulfate solution was added and left at room temperature for 30 minutes, and then 0.1M hydrochloric acid and 0.375% thiobarbituric acid solution were added. Next, the mixture was reacted with boiling water for 45 minutes, cooled, mixed with butanol, and then centrifuged at 1,000 g to obtain an upper butanol layer. The absorbance was measured at 532 nm. The amount of MDA was measured using tetramethoxypropane as a standard solution. Quantification

Specifically, the effects of oral administration of Honggyeongcheon extract on the production of MDA, a marker of lipid peroxidation, in the liver microsome fraction were investigated. CCl ₄ administration resulted in a 2.3-fold increase in liver peroxide content compared with the normal control group. The Methanol extract 50mg / kg of Hongkyungcheon was 32.9% lower than the CCl ₄ group, but was not significant, but 54% in the 200mg / kg group. A significant decrease of was shown (P <0.05).

Example 10

Exercise Performance and Physical Strength Enhancement Effect of Rhodiola sachalinensis Extract

In order to evaluate the effect of honggyeongcheon extract on the exercise performance and physical strength enhancement of the mouse, 12 mice were placed in each group, divided into 3 groups, such as 50mg / kg administration group and 200mg / kg administration group, CMC administration group to the mouse and placed in the mouse for 7 days Swimming time was measured on day 8 following oral administration once daily. As a test method, a slight modification of Richard's swimming test method was used, and a male ICR mouse weighing 21-27 g was used as a test animal (Richard. HB et al., Am. J. Physiol., 168, 178, 1963).

The pool was 25 × 40 × 13 cm in width × length × depth and the water temperature was maintained at 25 ° C. To reduce swimming time and reduce individual differences, swim mice for 1 minute in a pool containing 0.2% surfactant (Trio, Aekyung) in the morning of the test day and immediately transfer to a regular pool with only tap water for another 1 minute. Swimming removes fat from the hairs of mice. After about 3 hours of physical recovery and hair drying period, the mice were weighed and the test substance was administered. The test substance was homogeneously suspended in 0.5% CMC-Na aqueous solution and orally administered to the mouse at a volume of 0.1 ml per 10 g, and the mice were placed in a pool containing a surfactant (trio) at a concentration of 0.01% 1 hour after administration of the test substance. The swimming time of the was measured.

As a result, the swimming time in the 50 mg / kg administration group was 135.9 ± 15.5 minutes, which was significantly increased compared to 95.5 ± 6.2 minutes in the control group not administered the Rhodiola extract (p <0.05), and 136.6 ± in the 200 mg / kg group. 11.6 minutes, significantly increased than the control (p <0.01).

As described above, the present invention is not only methanol extract of Rhodiola sp., But also extracts obtained by sequentially dividing with dichloromethane, butanol and water, and the compounds isolated and purified therefrom are antioxidant, lipid peroxidation inhibitory, stamina. Enhancement, inhibition of thrombus formation, and inhibition of vascular smooth muscle cell proliferation, as well as preventing and treating diseases of the circulatory system including anti-aging and thrombosis and atherosclerosis, as well as widely used as a fatigue recovery agent, etc. Also usefully applicable.

Claims (7)

Rhodiola sp. Extract which can be used for the prevention and treatment of circulatory diseases obtained by extracting Rhodiola sp. With lower alcohol or water. According to claim 1, in addition to the above process, red gyeongcheoncheon extract, which is fractionated again with dichloromethane, butanol or water. The honggyeongcheon extract according to claim 1 or 2, which comprises as a component a compound of formula 1 (rosiridine), a compound of formula 2 (rosiridol) and a compound of formula 3 (cinnamil alcohol). Extracting honggyeongcheon with low alcohol to obtain alcohol extract or residue, and suspending honggyeongcheon alcohol extract again in distilled water and dividing it repeatedly with organic solvent to obtain various solvent fractions. Method for preparing the extract of Hong Kyungcheon. According to claim 1, Hongkyeongcheon extract having a prophylactic and therapeutic effect of atherosclerosis due to the inhibition of thrombus formation and vascular smooth muscle cell proliferation inhibitory activity. The extract of Honggyeongcheon according to claim 1 or 5, which further has an anti-aging and fatigue recovery effect due to homeostasis activity. A pharmaceutical composition for treating circulatory disease, comprising the compound of formula 1 (rosyridine) and derivatives thereof as an active ingredient.