Antithrombotic composition comprising <i>Angelica gigas </i>Nakai extract

Abstract

Provided are an antithrombotic composition (platelet aggregation inhibitor) comprising an Angelica gigas Nakai extract and a pharmaceutical composition for prevention and/or treatment of vascular disease, which comprises an Angelica gigas Nakai extract. The compositions are characterized in that they do not cause a gastrointestinal disorder.

Claims

1. A method of antithrombotic treatment comprising administering a pharmaceutically effective amount of an Angelica gigas Nakai extract to a patient in need of inhibition of platelet aggregation, wherein the Angelica gigas Nakai extract is an Angelica gigas Nakai ethanol extract obtained by extracting Angelica gigas Nakai with 90 to 100% (v/v) ethanol solution at 40 to 80° C.

2. The method of antithrombotic treatment according to claim 1, wherein the Angelica gigas Nakai extract is an Angelica gigas Nakai ethanol extract obtained by extracting Angelica gigas Nakai with 90 to 100% (v/v) ethanol solution at 40 to 60° C.

3. The method of antithrombotic treatment according to claim 2, wherein the ethanol solution has a concentration of 96 to 100% (v/v).

4. The method of antithrombotic treatment according to claim 1, which does not cause a gastrointestinal disorder.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1a and 1b are graphs showing the antithrombotic effect of the Angelica gigas Nakai extract in human blood.

(2) FIG. 2 is a photograph showing the appearance of the gastric mucosa when the Angelica gigas Nakai extract was administered in a mouse model in which gastrointestinal disorders are induced by aspirin.

(3) FIG. 3 is a graph quantitatively showing the degree of the damage to the gastric mucosa identified in FIG. 2 (Ulcer index; UI).

(4) FIG. 4 is a photograph showing the appearance of the gastric mucosa after administration of the Angelica gigas Nakai extract in a mouse model in which gastrointestinal disorders were induced by Celebrex.

(5) FIG. 5 is a graph quantitatively showing the degree of the damage to the gastric mucosa identified in FIG. 4 (Ulcer index; UI).

MODE FOR INVENTION

(6) Hereinafter, the present disclosure will be described in detail by way of examples, but these are for illustrative purposes only and are not intended to limit the scope of the present disclosure. It will be obvious to those skilled in the art that the examples described below may be modified without departing from the scope of the essential gist of the invention.

Example 1: Preparation of Angelica gigas Nakai Extract

(7) 1.1. Preparation of Angelica gigas Nakai Ethanol Extract

(8) Angelica gigas Nakai roots were washed with clean water and dried sufficiently. The dried Angelica gigas Nakai roots were crushed, 5 volume times (500 ml) of ethanol (98% (v/v) ethanol (alcohol)) was added to 100 g of the obtained powder, extracted at 40 to 60° C. for 4 hours or more, and then filtered through a 1 um (micrometer) filter, and the filtrate was heated and concentrated until it became 10% of the original weight. Crystalline cellulose was gradually added to the obtained concentrate, continuously concentrated, dried completely, and then powdered to prepare an Angelica gigas Nakai ethanol extract powder (hereinafter, referred to as AGE232).

(9) 1.2. Preparation of Angelica gigas Nakai Ethanol High-Temperature Extract

(10) Angelica gigas Nakai roots were washed with clean water and dried sufficiently. The dried Angelica gigas Nakai roots were crushed, 5 volume times (500 ml) of ethanol (98% (v/v) ethanol (alcohol)) was added to 100 g of the obtained powder, extracted at about 90° C. for 4 hours or more, and then filtered through a 1 um (micrometer) filter, and the filtrate was heated and concentrated until it became 10% of the original weight. Crystalline cellulose was gradually added to the obtained concentrate, continuously concentrated, dried completely, and then powdered to prepare a high-temperature Angelica gigas Nakai ethanol extract powder.

(11) 1.3. Preparation of Angelica gigas Nakai Ethanol (Low-Concentration) Extract

(12) Angelica gigas Nakai roots were washed with clean water and dried sufficiently. The dried Angelica gigas Nakai roots were crushed, 5 volume times (500 ml) of 30% (v/v) ethanol was added to 100 g of the obtained powder, extracted at 40 to 60° C. for 4 hours or more, and then filtered through a 1 um (micrometer) filter, and the filtrate was heated and concentrated until it became 10% of the original weight. Crystalline cellulose was gradually added to the obtained concentrate, continuously concentrated, dried completely, and then powdered to prepare an ethanol (low-concentration) Angelica gigas Nakai extract.

(13) 1.4. Preparation of Angelica gigas Nakai Hot Water Extract

(14) 5,000 ml of distilled water was added to 2 kg of Angelica gigas Nakai, extracted 4 times for each 8 hours in a water bath (about 90° C.), filtered and the solvent was evaporated with a concentration device to obtain a herbal extract. The obtained extract was freeze-dried for 12 hours in a freeze dryer, and then powdered to prepare a dried powdered Angelica gigas Nakai hot water extract powder.

(15) 1.5. Preparation of Angelica gigas Nakai Ethanol R/T (Room Temperature) Extract

(16) Angelica gigas Nakai roots were washed with clean water and dried sufficiently. The dried Angelica gigas Nakai roots were crushed, 5 volume times (500 ml) of ethanol (98% (v/v) ethanol (alcohol)) was added to 100 g of the obtained powder, extracted at room temperature (about 25° C.) for 4 hours or more, and then filtered through a 1 um (micrometer) filter, and the filtrate was heated and concentrated until it became 10% of the original weight. Crystalline cellulose was gradually added to the obtained concentrate, continuously concentrated, dried completely, and then powdered to prepare a high-temperature Angelica gigas Nakai ethanol R/T extract powder.

Example 2: Antithrombotic Effect Test

(17) 2.1. Ex-Vivo Test

(18) To prepare platelets, human platelet-rich plasma (PRP) was obtained from the Red Cross Blood Center (Seoul, Korea) (PRP concentration: 1×10.sup.9 platelets/ml).

(19) Platelet analysis was performed as follows:

(20) An aggregometer (Instrument: Whole blood/optical Lumi-Aggregometer Model 700 (CHRONO-LOG)) manufactured by Chrono-Log Co. was used as an analytical instrument. The light transmission value of PPP (Platelet Poor Plasma; Red Cross Blood Center (Seoul, Korea)) was used as the baseline value, and the maximum value interfering with light transmission due to PRP (Platelet Rich Plasma) aggregation was converted into a percentage (%). Collagen (12.5 μg/ml) was used as an aggregation agent.

(21) The inhibitory effect of each extract on platelet aggregation was measured using a dual channel aggregometer. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were used. 360 μl of the prepared PRP was put into a cuvette, and 40 μl of 0.25% (w/v) PEG200 (vehicle), aspirin (10 ug/ml), or each extract (80 ug/ml) was added thereto. For comparison, instead of the above extract, aspirin (10 ug/ml), which is well known as an antithrombotic agent, or decursin (80 ug/ml; single compound) or decursinol (80 ug/ml; single compound), which is an active ingredient of the Angelica gigas Nakai extract, were used to perform the same experiment.

(22) The mixture was cultured with stirring at 1000 rpm and 37° C. in an aggregometer for 2 minutes. After culturing, 12.5 μg/ml of collagen was added to induce platelet aggregation, and after 6 minutes, changes in light transmission were confirmed, and the degree of platelet aggregation was measured. The obtained platelet aggregation (%) is shown in FIGS. 1a and 1b (con: non-administration group). As shown in FIGS. 1a and 1b, the Angelica gigas Nakai extract, in particular, the extract using ethanol as an extraction solvent showed a lower platelet aggregation rate compared to the vehicle-administered group and the control group (non-administered group). In particular, it can be confirmed that the Angelica gigas Nakai extract (AGE232) had remarkably low platelet aggregation, as compared with the extracts of other extraction conditions (type of extraction solvent, concentration of extraction solvent, and/or extraction temperature), and not only it showed a platelet aggregation equal to that of aspirin (ASA) used as an antithrombotic agent, but also showed remarkably low platelet aggregation than decursin and decursinol which are the single compounds. These effects show that Angelica gigas Nakai extract, especially ethanol extract (e.g., AGE232) not only has an excellent antithrombotic effect, but also has an antithrombotic effect equivalent to that of aspirin (ASA) used as a conventional antithrombotic agent, and has a remarkably higher antithrombotic effect than decursin and decursinol which are the single compounds.

(23) 2.2. In-Vivo Test

(24) Preparation of Experimental Animals

(25) As for the experimental animals, 5-week-old female ICR mice provided by YoungBio were stabilized in an animal laboratory at a temperature of 24±1° C., a humidity of 55±5% in a 12-hour day-night cycle for 1 week and used in the experiments.

(26) The change in behavioral capacity was evaluated for the prepared thrombus-induced mouse model using Rota-rod, and the antithrombotic effect of the Angelica gigas Nakai extract was evaluated. The thrombus-induced mouse model was prepared as follows. A 6-week-old ICR mouse (about 20 to 25 g) stabilized as an experimental mouse was prepared, and subjected to Rota-rod training, and the reference value was created three times, and the rotation speed was set to 24 rpm, and objects withstanding without falling at a rotation speed of 300 or more were selected. The selected mice were divided into treatment groups, and after administration of each drug (see Table 1), after 90 minutes, collagen (13.2 μg) and epinephrine (114 μg) were mixed and injected into the tail vein to induce thrombus. After 15 minutes, Rota-rod testing was repeated 3 times (rotation speed: 24 rpm), and the difference in athletic performance was analyzed by the following Equation:
Protection rate (%)=[(Total number of individuals−Number of killed or paralyzed individuals)/Total number of individuals]×100

(27) (Number of total individuals: the number of individuals that did not fall at a rotation speed of 300 or more at a rotation speed of 24 rpm in Rota-rod testing;

(28) Number of killed or paralyzed individuals: The number of individuals who fell at a rotation speed of 24 rpm or less in Rota-rod testing at a rotation speed of 150 or less)

(29) The obtained protection rate is shown in Table 1 below:

(30) TABLE-US-00001 TABLE 1 Number of killed or paralyzed individuals / Number of total Protection Group Dose individuals (%) Control PEG 15/18 16.7 25% (w/v) ASA (Aspirin) 100 mg/kg  11/20 45 AGE232 10 mg/kg 10/19 47.3 Decursin 10 mg/kg 11/18 38.9 Decursinol 10 mg/kg 12/19 36.8 Hot water 10 mg/kg 15/19 21.1 extract Ethanol high- 10 mg/kg 12/18 33.4 temperature extract 30% ethanol 10 mg/kg 13/20 35 extract Ethanol R/T 10 mg/kg 11/18 38.9 extract

(31) As shown in Table 1, Angelica gigas Nakai extract showed excellent protection rate compared to the control group. In particular, the Angelica gigas Nakai extract ethanol extract (AGE232) not only showed remarkably superior protection rate compared to the extract under other extraction conditions, but also exhibited protection rates equal to or higher than those of aspirin used as a conventional antithrombotic agent, and decursin and decursinol which are single compounds.

Example 3. Gastrointestinal Disorder Inhibition Testing (In-Vivo)

(32) Experimental Animals

(33) Male ICR mice, 5 per cage, room temperature (22±0.5° C.), bred under the conditions of a 12-hour day-night cycle.

(34) Water and food were fed without limitation, the animals were adapted to the experiment at least 2 hours before experiment, and to reduce the variation, proceeded in a light phase (10:00-17:00),

(35) The test drug was orally administered for each treatment group once daily for 5 days using syringes as follows (ASA (Aspirin) and Celebrex were used to induce gastrointestinal disorders):

(36) Treatment of Experimental Animal

(37) ASA 300 or Cel 300: Aspirin™ (ASA, Bayer AG) 300 mg/kg or Celebrex™ (Cel, Pfizer) 300 mg/kg, administered for 5 consecutive days;

(38) Con: control;

(39) AGE232 1000: Angelica gigas Nakai extract AGE232 1000 mg/kg, administered for 5 consecutive days;

(40) ASA+AGE232 or Cel+AGE232:

(41) ASA+AGE232 or Cel+AGE232: combined administration of Angelica gigas Nakai extract AGE232 1000 mg/kg and Aspirin™ (ASA) 300 mg/kg or Celeblex™ (Cel) 300 mg/kg, administered for 5 consecutive days;

(42) Decursin 300: decursin 300 mg/kg, administered for 5 consecutive days;

(43) ASA+Decursin or Cel+Decursin: combined administration of dercursin 300 mg/kg and Aspirin™ (ASA) 300 mg/kg or Celebrex™ (Cel) 300 mg/kg, administrated for 5 consecutive days;

(44) Decursinol 300: decursinol 300 mg/kg, administered for 5 consecutive days;

(45) ASA+Decursinol or Cel+Decursinol: combined administration of dercursinol 300 mg/kg and Aspirin™ (ASA) 300 mg/kg or Celebrex™ (Cel) 300 mg/kg, administrated for 5 consecutive days.

(46) Dissection and Observation of Gastric Mucosa

(47) Immediately after dislocation of the cervical spine, the stomach was taken out, opened along the greater curvature, and developed on a cork plate. The inner surface was rinsed with iced cold saline, blood and the like were wiped off, the inner wall of the stomach was observed using a high-resolution microscope, etc. The results are shown in FIG. 2 (aspirin-administered gastrointestinal disorder-inducing group) and FIG. 4 (Celebrex-administered gastrointestinal disorder-inducing group).

(48) For the quantitative measurement of stomach injury, the gastric mucosal morbidity rate (Ulcer index (UI)) was measured by the following method:

(49) (I) Viscous bleeding and erosions site <1 mm: gastric mucosal edema, hyperemia, presence of one bleeding;

(50) (II) Erosion site 1˜3 mm: When small mucosal bleeding was accompanied by small gastric mucosal erosion;

(51) (III) Erosion site >3 mm: A bleeding edge was accompanied by severe erosion, erosive injuries or ulcers are present.

(52) Ulcer index (UI): (1*n*(I))+(2*n*(II))+(3*n*(III))/number of animals (n is Means the number of animals corresponding to the standard of I, II, and III)

(53) The Ulcer index (UI) obtained for the results of FIGS. 2 and 4 using the above method was shown in FIG. 3 (aspirin (ASA)-administered gastrointestinal disorder-inducing group) and FIG. 5 (celecoxib (cel)-administered gastrointestinal disorder-inducing group), respectively.

(54) As shown in FIGS. 2 to 5, (1) when comparing the results of ASA 300 or Cel 300 and ASA+AGE232 or Cel+AGE232, the gastrointestinal disorders (gastric bleeding) induced by aspirin or celebrex were significantly reduced when administered together with Angelica gigas Nakai extract, and (2) when aspirin or celebrex was administered in combination with Angelica gigas Nakai extract, the gastrointestinal disorders (gastric bleeding) induced by aspirin or celebrex were significantly reduced as compared with the combined administration of decursin and decursinol which are single compounds.

Example 4. Analysis of Active Ingredients Between Each Extract (HPLC)

(55) <Preparation of Sample Solution>

(56) 1 g of 5 types of samples (Angelica gigas Nakai water extract, Angelica gigas Nakai ethanol high temperature extract, Angelica gigas Nakai 30% ethanol extract, Angelica gigas Nakai ethanol extract (AGE232), or Angelica gigas Nakai R/T extract) were exactly taken and put in a 50 ml volumetric flask, and then dissolved by adding about 30 ml of methanol (100%), filled with methanol to a marked line, filtered, and used as a sample solution. The sample solution was prepared by varying the degree of dilution according to the type of sample and analysis items.

(57) <Preparation of Standard Solution>

(58) 10 mg of decursinol standard product (purity 98% or more), 5 mg of decursinol standard product (purity 98% or more), and 5 mg of decursinol angelate standard product (purity 98% or more) were taken and put in a 25 ml flask, and dissolved by adding 100% methanol, filled with methanol to a marked line, filtered, and used as a sample solution. A standard solution having a concentration of 12.5-25-50-100-200 μg/ml was prepared from this standard solution and used for measuring the calibration curve.

(59) <HPLC Operating Conditions>

(60) Testing was performed with the sample solution and standard solution according to the operating conditions of the liquid chromatography below to calculate the content of decursinol, and decursinol angelate:

(61) Column: Cadenza CW C18, (150*4.6 mm, 3 μm) or equivalent column;

(62) Detector: Ultraviolet spectrophotometer (detection wavelength: 330 nm);

(63) Flow rate: 0.7 ml/min;

(64) Mobile phase: Water (A %), Acetonitrile (B %), 0-5 min (20, B), 5-6 min (20.fwdarw.40, B), 6-22 min (40.fwdarw.55, B), 22-23 min (55.fwdarw.80, B), 23-25 min (80, B), 25-27 min (20, B); Sample injection amount: 10 μl.

(65) <Result>

(66) The ingredient analysis results of each of the obtained extracts are shown in Table 2 below.

(67) TABLE-US-00002 TABLE 2 Analysis results of major ingredients according to the extraction solvent and extraction method of Angelica gigas Nakai Ethanol high- Water tem- Ethanol (hot perature 30% extract Ethanol water) extract ethanol (AGE R/T Extraction Method extract (90° C.) extract 232) extract Ext. vol. before 1,800 650 1210 465 401 concent. (ml) Ext. weight after 42.0 24.2 21.7 30.5 25.1 concent. (g) Total Decursin 19 1640 1101 3243 1570 extracted (mg) compounds Decursinol 10 790 865 1995 811 from angelate 100 g (mg) dried Nodakenin 559 1332 543 2982 792 Angelica (mg) gigas β- 13 85 35 324 255 Nakai Sitosterol (mg) (mg)

(68) As shown in Table 2, it was confirmed that the Angelica gigas Nakai extract had a relatively high content (on weight base) of active ingredients such as decursin, decursinol angelate, nodakenin, and beta-sitosterol, in particular, the content of the active ingredient of Angelica gigas Nakai ethanol extract (AGE232) was remarkably high.