KR19990071183A - Honggyeongcheon Extract with Efficacy and Prevention of Circulatory Diseases - Google Patents

Abstract

The present invention relates to Rhodiola sp. Extract useful for the prevention and treatment of circulatory diseases and a method of preparing the same. 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.

Description

Honggyeongcheon Extract with Efficacy and Prevention of Circulatory Diseases

The present invention relates to a Rhodiola sp. Extract having a prophylactic and therapeutic effect of a circulatory disease and a method of preparing the same.

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 (蕭 培根; 中國 本草 圖鑑 (第 1 卷), 驪 江 出版社, Seoul, p314, 1994). It is used. Hongkyeongcheon is classified as pizza plant gate, Gyeongcheondae, and Hongkyeongcheon by category, and it is reported that there are 96 species worldwide. Especially, there are 70 species in China and 34 species and 2 varieties in Xinjiang. have.

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

Recently, free radical lipid peroxidation has been found to be a major cause of adult disease 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 monovalent reduction of oxygen, which is essential for life, is a 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 Raton, 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 cellular senescence by causing or accumulating lipofuscin (Gryglewski, RJ et al., Prostaglandins , 12 , 685, 1976; Tappel, AL, Fed. 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 capable of effectively inhibiting superoxide radicals or lipid peroxides in a manner related to such enzymes can be usefully used for preventing and treating aging, carcinogenesis, adult diseases and various vascular diseases.

On the other hand, it is reported that the secretory function of the vascular endothelial system is also affected by oxygen-derived free radicals. This is because the superoxide anion destroys endothelial-derived relaxing factor (EDRF) (Gryglewski, RJ 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 the PDGF causes vascular smooth muscle. 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 the injury site of endothelial cells, devour a large amount of fat and become foam cells, thereby causing fat accumulation to cause atherosclerosis. This proliferation and hypertrophy of vascular smooth muscle cells is one of the important characteristics of hypertensive patients (Owens, GK, Circ. Res ., 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 to separate them from natural products. As a result, the roots of Rhodiola sp. Were extracted with water or methanol, and sequentially obtained by distillation 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.

An object of the present invention is 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 rosiridin of formula 1 isolated and purified in Honggyeongcheon,

Figure 2 shows the infrared absorption spectrum of the Rossiridine,

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

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

FIG. 5 shows the mass spectrometry spectra of rosiridol of formula 2 isolated and purified from Honggyeongcheon 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,

FIG. 8 shows the mass spectrometry spectrum of cinnamyl alcohol of Chemical Formula 3 isolated and purified from Honggyeongcheon extract.

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

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

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

Figure 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 (I) (rosiridine), a compound of formula (2) (Rosiridol) 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 as an active ingredient a compound of formula 1 (rosyridine) and a derivative thereof.

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, all plants commonly used as Hongkyeongcheon may be used as the Hongkyeongcheon, including Changbai Mountain 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 is added to the remaining residue, and 50% methanol is added to the remaining residue, followed by extraction with an ultrasonic extractor and the like to obtain 50% methanol precipitation fraction and 50% methanol aqueous fraction.

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.

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 of Rhodiola sachalinensis.

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 to perform the above compounds (rosyridine, rosiridol, Cinnamic alcohol) is separated and purified.

The honggyeongcheon extract and compounds purified 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 compound of formula 2 and the compound of formula 3 are angiotensin II And platelet-derived growth factor (PDGF)³Although it does not inhibit the influx of H-thymidine, the compound of formula 1³Influx of H-thymidine was dose dependently inhibited (11AndFigure 12Reference).

In addition, the present invention to investigate the inhibitory effect of platelet aggregation of Rhodiola sachalinensis extract,

The degree of aggregation of platelets was measured by Born'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 fraction was fractionated, the activity was higher in the polar fraction of water than in 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 was measured by the method of prido beach and in vivo methods (Fridovich, I. et al., J. Biol. Chem ., 243 , 5753, 1968). In this case, the antioxidant activity in vivo was investigated by inducing lipid peroxidation of animals using carbon tetrachloride and then obtaining a microsome fraction. In addition, the present invention also investigated catalase activity by investigating the rate of H ₂ O ₂ consumption 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 vivo, SOD activity and catalase activity significantly increased in the case of administration of Methanol extract of Rhodiola sachaline (200 mg / kg administration group).

In addition, the present invention was measured lipid peroxidation inhibitory activity of Rhodiola oleracea extract by the method of Nose using linoleic acid as a substrate and 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 reduced in the case of administration of methanol extract of Rhodiola sachaline (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.

Toxicity experiments were carried out on the Hong Kyungcheon of the present invention and the compounds 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, 50% lethal dose (LD ₅₀ ) 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, forming agent It may be prepared and used in the form of a powder, granules, tablets, capsules or injections by mixing with diluents and the like. 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. 30-300 mg is administered, but it is preferable to administer about 50-200 mg. Therefore, the unit dosage form of the present invention is prepared to have a content of 20-100 mg of the active substance of the present invention in consideration of the above-mentioned effective amount range. Preferably 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

1 kg 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 methanol extract of Hongkyeongcheon (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 give 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 50% methanol soluble fraction (RJ-6).

Example 2 Isolation and Purification of Biologically Active Substances in 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 performed, 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) afforded a compound of formula 1 (rosyridine, rosiridin), a compound of formula 2 (rosiridol, rosiridol), and a compound of formula 3 (cinnamyl alcohol, cinnamyl alcohol).

Example 3 Physicochemical Properties of Compounds Isolated and Refined from Rhodiola sachalinensis

(3-1) A compound of formula 1: rosiridin

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-glucopyranoside)

(1) Property of the substance: white powder

(2) Molecular weight: 332

(3) Molecular formula: C ₁₆ H ₂₈ O ₇

(4) Mass Spec. (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 nm): 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 detromethanol (CD ₃ OD) as a solvent are shown in FIG . 3 .

δ5.57 (1H, t, J = 6.6 Hz, H-2), 5.10 (1H, t, J = 7.0 Hz, H-6), 4.34 (2H, m, H-1), 4.29 (1H, d , J = 7.5 Hz, H-1 '), 3.97 (1H, d, J = 6.9 Hz, H-4), 3.87 (1H, dd, J = 1.5, 11.9 Hz, Ha-6'), 3.66 (1H , dd, J = 5.2, 11.9 Hz, Hb-6 '), 3.34 (1H, m, H-3'), 3.28 (1H, m, H-5 '), 3.26 (1H, m, H-4' ), 3.18 (1H, m, H-2 '), 2.24 (2H, t, J = 6.9 Hz, H-5), 1.69 (3H, s, H-9), 1.66 (3H, s, H-8 ), 1.62 (3H, s, H-10)

(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 .

δ 142.9 (C-7), 134.1 (C-3), 122.9 (C-2), 121.6 (C-6), 102.8 (C-1 '), 78.1 (C-3'), 78.0 (C- 4, C-4 '), 75.0 (C-2'), 71.6 (C-5 '), 66.1 (C-1), 62.7 (C-6'), 34.8 (C-5), 26.0 (C- 9), 18.0 (C-10), 12.0 (C-8)

(7) chemical structure

(3-2) A compound of formula (II): rosiridol

3,7-dimethyl-2,6-octadiene-1,4-diol (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.%):

Mass spectrometry measured by the EIMS method is as shown in FIG . 5 .

170 (M ⁺ , 0.19), 135 (10.2), 101 (42.4), 83 (78.3), 55 (100)

(5) hydrogen nuclear magnetic resonance spectrum (NMR)

Hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectra measured using dutromethanol (CD ₃ OD) as solvents are shown in FIG . 6 .

δ 5.35 (1H, t, J = 6.3 Hz, H-2), 5.11 (1H, t, J = 7.0 Hz, H-6), 4.12 (2H, t, J = 6.3 Hz, H-1), 3.93 (1H, dd, J = 6.6, 7.2 Hz, H-4), 2.23 (1H, dd, J = 6.6, 7.2 Hz, H-5), 1.68 (3H, s, H-9), 1.64 (3H , s, H-8), 1.62 (3H, s, H-10)

(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 .

δ 140.6 (C-7), 134.0 (C-3), 126.2 (C-2), 121.7 (C-6), 78.1 (C-4), 59.2 (C-1), 34.8 (C-5), 26.0 (C-9), 18.0 (C-10), 11.8 (C-8)

(7) chemical structure

(3-3) A compound of the formula (3): cinnamic alcohol

3-phenyl-2-propen-1-ol (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 .

134 (M ⁺ , 55.1), 105 (42.1), 92 (100)

(5) hydrogen nuclear magnetic resonance spectrum (NMR)

Edu Trojan dimethyl sulfoxide (DMSO-d ₆₎ hydrogen nuclear magnetic resonance ⁽¹ H-NMR) spectrum was measured by the solvent is the same as shown in Fig.

7.46 (2H, d, J = 7.5 Hz, H-2, H-6), 7.36 (2H, d, J = 7.5 Hz, H-3, H-5), 7.25 (1H, m, H-4) , 6.59 (1H, d, J = 15.9 Hz, H-7), 6.41 (1H, dt, J = 15.9, 4.8 Hz, H-8), 4.15 (2H, m, H-9)

(6) carbon nuclear magnetic resonance spectrum (NMR)

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

136.86 (C-1), 130.77 (C-8), 128.58X2 (C-3, C-5), 128.37 (C-7), 127.17 (C-4), 126.09X2 (C-2, C-6 ), 61.45 (C-9)

(7) chemical structure

Example 4 Measurement of Vascular Smooth Muscle Cell Proliferation Inhibitory Activity 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 fat tissue, 0.2% collagenase (collagenase) Endothelial cells were removed by treatment in a thermostat at 37 ℃ for 20 minutes. The inner and outer membranes were then crushed and crushed again, followed by shaking in an orbital shaker for 4 hours with complete cell culture (10% FBS, DMEM medium containing antibiotics and glutamine) containing collagenase. Reacted. Next, the DMEM solution was centrifuged to wash the collagenase, and the precipitate was mixed in a culture dish in which an appropriate amount of the complete DMEM solution was dispensed, and then 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 medium, and the same amount of trypan blue was added to the hemocytometer, which was measured under a microscope. 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 sacrum on rat arotic vascular smooth muscle cells (RVSMs) of rat aorta, which were left unsustained for 24 hours in serum-free medium. And pretreated with a compound of formula 3 (cinnamyl alcohol) at a concentration of 10 μg / ml for 1 hour and then angiotensin Ⅱ 10^-5M and PDGF 50 ng / ml were treated for 20 hours, then 1 μCi / ml of [methyl-³H] Thymidine was labeled for 4 hours. Cells were labeled with cold phosphate buffer (PBS; 1 mmol / L CaCl₂, 1 mmol / L MgCl₂, 10% trichloroacetic acid, ethanol / ether (2: 1 vol / vol)), insoluble in acid³H-thymidine was extracted by adding 250 µl of 0.5 M NaOH per petri dish, and 5 ml of scintillation cocktail was added to 0.1 ml of the extract and measured by a β-ray meter. Among the compounds isolated from Hong Kyungcheon, the compound of Formula 2 and the compound of Formula 3 are angiotensin II And by platelet-derived growth factor (PDGF).³Although it did not inhibit the influx of H-thymidine, the compound of formula 1 was shown to significantly inhibit the influx of thymidine (11Reference).

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 μg / ml concentration of the results of processing by the Procedure ³ naphthyridin 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 vol / vol) 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. The platelet count of PRP was adjusted to a concentration of 3 × 10 ⁸ cells / ml.

(5-2) Measurement of platelet aggregation inhibitory ability

Platelet aggregation inhibitory activity was measured according to the 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.

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 by ADP, and when the solvent fractionation was performed, the active substance was present in the polar fraction of water rather than the nonpolar solvent fraction. there was.

Inhibitory Activity of Human Platelet Aggregation by ADP Stimulation of Rhodiola sachalinensis Extract sample density Cohesion Rate (%) Inhibition Rate (%) Methanol fraction 1 mg / ml 2.5 97.1 Dichloromethane fraction 1 mg / ml 69.6 30.4 Water fraction 1 mg / ml 2.0 98.0

Example 6 Determination of the Effect of Honggyeongcheon Extract on 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 xanthine / xanthine oxidase reaction. Specifically, SOD activity was 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 N NaOH ), 100 ml of 50 mM sodium phosphate buffer (pH 7.8, 0.1 mM EDTA) was added, and 50 µl of the diluted fraction of Rhodiola savonsis extract was added to 2 ml of the mixed substrate solution, and stabilized for 2 minutes. 50 μl of ml, 0.1 mM EDTA) was added to the cuvette and mixed, and the absorbance was measured at 550 nm for 2 minutes, whereby the absorbance (OD) on the X-axis and the absorbance (OD) on the Y-axis were constant (linear). Changes in absorbance per minute (ΔAbs / min) in the) section were determined to determine relative SOD activity. In this case, SOD 1 U was defined as an amount that inhibits the reduction of cytochrome c by 50%, and the SOD titer (specific activity = U per mg) was determined in the experiment with an amount that inhibits the absorbance per minute (OD) of the reference group by 50%. .

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 very high and even 12-12 times, especially butanol fraction (RJ-3) was the highest 18 times.

Superoxide Dismutase (SOD) Activity Treated with Solvent Fraction of Rhodiola sachalinensis Extract sample SOD activity (U / g) Rooibos tea 861.5 Methanol Fraction (RJ-1) 14072.6 Dichloromethane Fraction (RJ-2) 872.5 Butanol Fraction (RJ-3) 15964.2 Water Fraction (RJ-4) 10508.6 50% Methanol Precipitation Fraction (RJ-5) 15101.2 50% Methanol Water Soluble Fraction (RJ-6) 13234.5

Example 7 Measurement 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 (sample 5 mg / ml concentration) were placed in a test tube of 1.6 cm × 6 cm for 24 hours at 50 ° C incubator to promote oxidation, followed by chloroform / acetic acid (2: 3, V / V) Dissolve in 35 ml and place this solution in a 250 ml idle conical flask to replace air with nitrogen. 1 ml of saturated aqueous potassium iodide solution was added thereto, mixed vigorously for 1 minute, allowed to stand in the dark for 5 minutes, and then 75 ml of distilled water and 1 ml of starch solution were added, followed by iodine (I₂), POV was measured by reverse titration and the lipid peroxidation inhibitory effect of each sample was measured.

POV (meq / kg) = [(Tv-Bv) × 0.01 × 1000] / W

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

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

W: amount of substrate used in experiment (g)

As a result, 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.

Inhibitory Effect of Rhodiola sachalinensis Extract Fraction sample POV (meq / kg) Inhibition Rate (%) Reference group 1312.5 - Quercetin 223.4 82.9 BHT 131.4 90.0 Methanol Fraction (RJ-1) 562.4 57.2 Dichloromethane Fraction (RJ-2) 672.8 48.8 Butanol Fraction (RJ-3) 714.8 45.6 Water Fraction (RJ-4) 788.4 40.0 50% Methanol Precipitation Fraction (RJ-5) 772.6 41.2 50% Methanol Water Soluble Fraction (RJ-6) 804.2 38.8

*% Inhibition = [1-(sample _POV / reference _POV )] × 100

Example 8 Measurement of Lipid Peroxidation Inhibitory Activity at the Cell Level of Honggyeongcheon Extract

(8-1) Preparation of rat liver microsome fraction

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 in 4-fold buffer solution (50 mM Tris-HCl, pH) 7.4) and homogenized the 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 microsomal fractions. This microsome fraction was quantified according to the Biuret method (Ohnishi, ST et al., Anal Biochem. , 86 , 193, 1978) and stored frozen at -70 ° C.

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

1.6 ml of 50 mM Tris-HCl buffer (pH 7.5) 0.1 ml of the sample solution, 0.1 micrograms of liver microsome fraction (containing 1 mg of protein in 1 ml), 0.1 ml of 0.1 mM ascorbic acid, 0.1 ml of 5 mM FeSO ₄ To this was added sequentially to prepare a reaction solution. 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, 0.5 ml of a mixed solution of 3 M TCA and 2.5 N hydrochloric acid was added, and centrifuged 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, a sample solution was not added. The reaction solution was used as a blank sample, and malondialdehyde (MDA) was quantified using tetramethoxypropane as a standard solution.

(8-4) Effect of Lipid Peroxidation Inhibitory Activity

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

Lipid Peroxidation Inhibitory Activity of Rhodiola sachalinensis Extract (Non-enzymatic Fe ²⁺ / ascorbic Acid System) sample MDA (nmol / mg protein) Inhibition Rate (%) Reference group 3.73 - BHT 0.41 89.1 Methanol Fraction (RJ-1) 2.95 46.6 Dichloromethane Fraction (RJ-2) 4.33 20.9 Butanol Fraction (RJ-3) 2.62 55.4 Water Fraction (RJ-4) 3.61 32.7 50% Methanol Precipitation Fraction (RJ-5) 1.60 70.1 50% Methanol Water Soluble Fraction (RJ-6) 3.02 47.3

*% Inhibition = [1-(sample _POV / reference _POV )] × 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. 0.5% 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 normal 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-cold 0.9% saline solution, and the liver microsomal 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 microsome 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, and the liver microsome fraction diluted in the cytochrome c and xanthine mixed solution was added thereto, and the absorbance was measured at 550 nm by adding xanthine oxidase. As a result, CCl ₄ administration showed that liver SOD activity was reduced by 35% compared to the normal control group. Methanol extract of Rhodiola sachalinensis was increased by 29% compared to CCl ₄ administration group, but there was no significance, and 42% was increased in 200 mg / kg administration 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 μl of 30% hydrogen peroxide was dissolved in 5.0 ml of 0.05 M potassium phosphate buffer (pH 7.0) 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 0.05 M potassium phosphate buffer (pH 7.0), 330 μl of substrate and 100 μl of a dilute solution of homogenate as enzyme source were placed in a cuvette and mixed for 40 seconds at 240 nm for 10 seconds.

Active U / ml = 22.94 × amount of change in absorbance / min × dilution factor

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

As a result, CCl ₄ administration decreased hepatic catalase activity by 44% compared to the normal control group, and the methanol extract of Rhodiola savona 50 mg / kg increased 27% compared with CCl ₄ administration group, but was not significant, 200 mg / kg There was a significant increase of 50% in the administration group (P <0.05).

(9-3) Determination of lipid peroxide content

The content of lipid peroxides was measured by Masugi et al . (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, followed by addition of 0.1 M hydrochloric acid and 0.375% thiobarbituric acid solution. The mixture was then cooled in boiling water for 45 minutes, cooled, and mixed with butanol. The mixture was centrifuged at 1,000 g, the upper butanol layer was taken, and the absorbance was measured at 532 nm. The amount of MDA was increased using tetramethoxypropane as a standard solution. Was quantified.

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

Example 10 Investigation of Exercise Performance and Physical Strength Enhancement Effect of Honggyeongcheon 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 50 mg / kg administration group and 200 mg / kg administration group, and CMC administration group to mice After oral administration once daily for 7 days, swimming time was measured on the 8th day. The test method was a slight modification of Richard's swimming test method, and male ICR mice weighing 21-27 g were used as test animals (Richard, HB et al ., Am. J. Physiol., 168 , 178, 1963). The pool was 25 × 40 × 13 cm in width, length, and 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 surfactant (trio, aekyung) at a concentration of 0.2% on the morning of the test day and immediately transfer to a normal pool with only tap water for another 1 minute. Swimming was removed to remove fat from the hair of the mouse. After about 3 hours of physical recovery and hair drying period, the mice were weighed and the test substance was administered. Test substance was homogeneously suspended in 0.5% CMC-Na aqueous solution and orally administered to mice at a volume of 0.1 ml per 10 g, and in a pool containing surfactant (trio) at a concentration of 0.01% 1 hour after administration of the test substance. The swimming time of the mice 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 that was not administered the Rhodiola extract (p <0.05). 136.6 ± 11.6 minutes, significantly increased than the control group (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 separated 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 red rose 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 the honggyeongcheon with lower alcohol to obtain an alcohol extract or residue, and suspended the honggyeongcheon alcohol extract again in distilled water and fractionated repeatedly with an organic solvent to obtain a variety of solvent fractions, the residue is extracted again with methanol to obtain a precipitate fraction and water-soluble fraction of claim 1 Preparation method of the Honggyeongcheon extract. 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 antioxidant activity. A pharmaceutical composition for treating a circulatory disease, comprising as an active ingredient a compound of formula 1 (rosyridine) and a derivative thereof.