AGRIMONIA EUPATORIA EXTRACT PREPARATION METHOD, AND COMPOSITION FOR IMPROVING LIVER FUNCTIONS OR TREATING LIVER DISEASES, CONTAINING EXTRACT

Abstract

The present invention relates to: an Agrimonia eupatoria extract preparation method for promoting the recovery rate and bioavailability of an active ingredient; and a composition for improving liver functions or treating liver diseases, containing the extract. More specifically, the present invention provides an Agrimonia eupatoria extract preparation method capable of extracting Agrimonia eupatoria by using a solvent in which cyclodextrin is dissolved, thereby increasing the recovery rate and bioavailability of an active ingredient, and provides a health functional food for improving liver functions or an agent for treating liver diseases, the food and the agent containing an Agrimonia eupatoria extract and having enhanced efficacy.

Claims

1. A method for preparing an Agrimonia extract comprising step a) of obtaining an extract from Agrimonia using an extraction solvent containing cyclodextrin.

2. The method of claim 1, wherein step a) comprises: step a-1) of preparing an extraction solvent by adding cyclodextrin to an organic solvent; and step a-2) of obtaining the extract by adding Agrimonia to the extraction solvent.

3. The method of claim 1, wherein step a-1) comprises preparing the extraction solvent by heating at 20 to 100° C. for 3 to 8 hours after adding cyclodextrin to the organic solvent.

4. The method of claim 2, wherein the organic solvent in step a-1) is at least one selected from the group consisting of methanol, ethanol, n-propanol, iso-propanol, n-butanol, iso-butanol, 2-butanol, and 3-butanol.

5. The method of claim 2, wherein the cyclodextrin in step a-1) is at least one selected from the group consisting of alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin.

6. The method of claim 2, wherein the cyclodextrin in step a-1) is used in an amount of 0.1 to 40 parts by weight based on the total weight of Agrimonia added in step a-2)

7. The method of claim 2, wherein step a-2) comprises obtaining the extract by extraction at 20 to 100° C. for 1 to 40 hours after adding Agrimonia to the extraction solvent.

8. The method of claim 2, wherein Agrimonia in step a-2) is used in an amount of 0.01 to 50 parts by weight based on the total weight of the extraction solvent.

9. The method of claim 2, wherein the extract in step a-2) contains a sparingly soluble component.

10. The method of claim 2, wherein the extract in step a-2) contains 0.1 to 5% by weight of luteolin-7-O-glucuronide, which is an indicator active ingredient and forms an inclusion complex.

11. The method of claim 1, further comprising, after step a), step b) of filtering the extract.

12. The method of claim 11, further comprising, after step b), step b′) of obtaining an extract from filtration residue using an organic solvent containing cyclodextrin.

13. A food composition for improving liver function containing an extract prepared by the method of claim 1.

14. A pharmaceutical composition for treating liver disease containing an extract prepared by the method of claim 1.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0039] FIG. 1 is a process diagram showing a novel method for preparing an Agrimonia eupatoria extract according to one embodiment of the present invention, which increases the recovery of active ingredients for improving liver function.

[0040] FIG. 2 shows the results of testing Caco-2 cell uptake of luteolin-7-O-glucuronide, which is an indicator active ingredient of an extract obtained using each type of cyclodextrin as an inclusion agent according to Preparation Example 1 of the present invention.

[0041] FIG. 3 shows the results of recovering precipitates and solids depending on each type of cyclodextrin according to Preparation Example 1 of the present invention. (a) depicts photographs showing the results of visually comparing precipitates after standing in 0.5% aqueous solution for 2 hours, and (b) is a graph showing the results of calculating the recovery of solids, dried after centrifuging each supernatant, as a percentage of the weight of a sample added.

[0042] FIG. 4 shows the results of HPLC measurement of the increase in the degree of hydration for fat-soluble substances of the extract according to Preparation Example 1 of the present invention.

[0043] FIG. 5 shows the results of measuring yield under various cyclodextrin concentration and inclusion extraction temperature conditions according to Preparation Example 2 of the present invention.

[0044] FIG. 6 shows the results of testing Caco-2 cell uptake of luteolin-7-O-glucuronide, which is an indicator active ingredient of an inclusion extract, under various cyclodextrin concentration and inclusion extraction temperature conditions according to Preparation Example 2 of the present invention.

[0045] FIG. 7 shows the results under various cyclodextrin concentration and inclusion extraction temperature conditions according to Preparation Example 2 of the present invention. (a) depicts photographs showing the results of visually comparing precipitates after standing in a 0.5% aqueous solution for 2 hours, and (b) is a graph showing the results of calculating the recovery of solids as a percentage of the weight of a sample added, after drying of the solids obtained by centrifugation of each supernatant.

[0046] FIG. 8 shows the results of performing the measurement of AST and ALT, which are liver injury indicators, for an Agrimonia eupatoria extract according to Experimental Example 1 of the present invention.

[0047] FIG. 9 depicts photographs showing the results of analyzing the liver injury protective effect of an Agrimonia eupatoria extract according to Experimental Example 1 of the present invention, and shows the results of performing histopathological examination of the white rat liver.

MODE FOR INVENTION

[0048] Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. However, the following examples are described for the purpose of illustrating the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

Preparation Example 1. Selection of Cyclodextrin Type

[0049] In order to prepare an Agrimonia eupatoria inclusion extract using each type of cyclodextrin, Agrimonia eupatoria leaves purchased through Jiyu Boncho Co., Ltd. were used.

[0050] Specifically, foreign substances were removed from the leaves of Agrimonia eupatoria, and each of dextrin as a control and alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin as treatment groups was added to 50% fermented alcohol in an amount of 0.13% relative to the liquid amount (2% relative to raw material), thus preparing solutions. A 15-fold weight of each prepared solution was added to 50 g of the Agrimonia eupatoria sample, and each mixture was stirred and extracted at 300 rpm at 80° C. for 5 hours, and then filtered through filter paper to obtain each primary extract. A 14-fold weight of each fresh dextrin or cyclodextrin solution was added to the filtration residue, and each mixture was stirred and extracted at 300 rpm at 80° C. for 5 hours, and filtered through filter paper, and the filtrate was combined with the primary extract, concentrated under reduced pressure at 60° C., and then freeze-dried, thus preparing final extracts.

[0051] 1-1. Measurement of Yield and Content

[0052] The extraction yield was determined by calculating the weight of the freeze-dried extract as a percentage of the weight of the sample used for preparing the extract.

[0053] To measure the content of the active ingredient luteolin-7-O-glucuronide in each of the prepared Agrimonia eupatoria extract powder (control group) and inclusion extract powder (treatment group), high-performance liquid chromatography (Infinity 1260, Agilent, USA) with X Select HSS C18 Column (Waters, 4.6*250 mm, 5 μm particle size) was used, and the detection spectrum was measured at 350 nm UV. Distilled water containing 0.1% phosphoric acid was used for mobile phase A, and 100% ACN was used for mobile phase B. The conditions of the mobile phases with time were as follows. Mobile phase A was kept at 90% at 0 to 20 min, 90% to 88% at 20 to 35 min, 88% at 35 to 60 min, 88% to 70% at 60 to 70 min, 70% to 20% at 70 to 72 min, 20% at 72 to 75 min, 20% to 90% at 75 to 76 min, and 90% at 76 to 90 min. The low rate was 1.6 ml/min, and the sample amount was 5 μl. For standard solution preparation, an active ingredient and 20 mg of a precursor standard were placed in a 20-ml flask, and dissolved in DMSO to make a standard solution, and diluted at each concentration. The standard solution was analyzed, and then a calibration curve was prepared to measure the content of each active ingredient. To prepare a sample solution, 500 mg of the sample was placed in a 50-ml flask, dissolved in 70% MeOH, shaken with ultrasonic waves, and filtered through a 0.45 μm filter, followed by measuring the content of the active ingredient luteolin-7-O-glucuronide. The results are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Control α-CD β-CD γ-CD Actual yield 22.7 23.2 22.0 24.6 (%) Active 6.5 6.7 6.1 7.1 ingredient content (mg/g) Yield (yield X 147.4 155.2 134.0 174.9 content)

[0054] As shown in Table 1 above, it was conformed that, when extraction with the solvent containing gamma-cyclodextrin was performed, the yield and content tended to increase compared to those of the control, and in particular, the yield increased by 18% compared to that of the control.

[0055] 1-2. Bioavailability Measurement

[0056] Cell uptake of the Agrimonia eupatoria inclusion extract was measured using Caco-2 cells.

[0057] First, Caco-2 cells were seeded into Trans-wells and cultured using MEM medium containing 20% FBS, and then culture was performed while replacing the MEM medium until a membrane was formed. The Agrimonia eupatoria extract dissolved in medium was added to insert wells, and after 30 minutes, the medium of the basolateral well was collected. Uptake evaluation was performed by measuring the value transferred from the insert well to the basolateral well through HPLC analysis of each collected medium. The results are shown in FIG. 2.

[0058] As shown in FIG. 2, it was confirmed that all the experimental groups showed higher Caco-2 cell uptake than the control group, and in particular, the uptake of the group extracted using gamma-cyclodextrin as an inclusion agent was about 18%, which was about 3 times higher than that of the experimental group extracted using alpha- or beta-cyclodextrin as an inclusion agent.

[0059] 1-3. Water Solubility Measurement

[0060] 1 g of the Agrimonia eupatoria inclusion extract prepared using each type of cyclodextrin was dissolved in 50 mL of purified water and centrifuged for 20 minutes at 4,000 rpm at room temperature. A supernatant of the centrifuged solution was collected and dried using a freeze dryer. The weight of the dried sample was measured, and the recovery rate was determined by calculating the weight of the sample, obtained by drying the supernatant, as a percentage of the weight of the sample added. The results are shown in FIG. 3.

[0061] As shown in FIG. 3, it was confirmed that the amount of the precipitate in the group extracted using gamma-cyclodextrin as an inclusion agent was smaller than that in the control group, and the recovery rate of solids in the group was significantly higher than that in the control group.

[0062] 1-4. HPLC Measurement

[0063] To measure the water solubility of the fat-soluble substances of the prepared Agrimonia eupatoria extract powder and inclusion extract powder, analysis was performed in the same manner as in Preparation Example 1-1 using high-performance liquid chromatography (Infinity 1260, Agilent, USA). The results are shown in FIG. 4.

[0064] As shown in FIG. 4, it was confirmed that the water solubility of the fat-soluble component of the group extracted using gamma-cyclodextrin as an inclusion agent increased compared to that of the control group.

[0065] 1-5. Analysis of Content of Indicator Active Ingredient Forming Inclusion Complex

[0066] Many literatures and patents have already demonstrated that cyclodextrin inclusion complexes maintain their water solubility even at low pH compared to non-inclusion complexes. Thus, in order to measure the content of the indicator active ingredient luteolin-7-O-glucuronide forming a inclusion complex in each of the prepared Agrimonia eupatoria extract powder (control group) and the inclusion extract powder (treatment group), 100 g of each inclusion extract powder was dissolved in 1 L of purified water, and then adjusted to pH 2.7 by addition of 2N HCl, and centrifuged at 4,000 rpm at room temperature for 20 minutes, and the precipitate and the supernatant were separately collected. The supernatant was concentrated and freeze-dried, and the precipitate was diluted with purified water and freeze-dried. Each dried sample was weighed and the yield was calculated. In the yield calculation method, the yield was determined by calculating the weight of each dried sample, obtained from each of the supernatant and the precipitate, as a percentage of the weight of the initially added sample.

[0067] The content of the indicator active ingredient luteolin-7-O-glucuronide in the dry powder obtained from each of the obtained supernatant and precipitate was analyzed in the same manner as in Preparation Example 1-1 using high-performance liquid chromatography (Infinity 1260, Agilent, USA). The results are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Control α-CD β-CD γ-CD Precipitate Total yield (g) 71 59 62 32 (non- of precipitate inclusion Luteolin-7-0- 7.7 7.9 6.5 6.5 complex) glucuronide content (mg/g) in precipitate Yield of luteolin-7- 545.6 466.9 404.4 206.6 O-glucuronide in precipitate Supernatant Supernatant 29 41 38 68 (inclusion yield (g) complex) Luteolin-7-0- 3.6 5.3 4.9 8.3 glucuronide content in supernatant (mg/g) Yield of luteolin-7- 104.4 217.3 186.2 564.4 O-glucuronide in supernatant Inclusion Inclusion rate 16.1 31.8 31.5 73.2 rate (supernatant calculation luteolin-7-0- glucuronide yield/ total yield * 100)

[0068] As shown in Table 2 above, it was confirmed that the inclusion rate of the extract obtained using gamma-cyclodextrin as an inclusion agent was more than 5 times higher than that of the control, and the content of the indicator active ingredient (luteolin-7-O-glucuronide) forming an inclusion complex was also more than doubled.

[0069] Through the above experiments, it was confirmed that the use of the gamma-cyclodextrin solution could effectively extract the active ingredient of Agrimonia eupatoria. Thus, gamma-cyclodextrin was used in subsequent experiments.

Preparation Example 2. Determination of Cyclodextrin Concentration and Inclusion Extraction Temperature

[0070] The following experiment was performed to select the concentration of gamma-cyclodextrin and the inclusion extraction temperature. The amounts of gamma-cyclodextrin used were 0%, 2%, and 4% of the weight of the raw material (Agrimonia eupatoria leaves), and the inclusion extraction temperatures were 60° C., 70° C., and 80° C.

[0071] Specifically, foreign substances were removed from the leaves of Agrimonia eupatoria, and a solution was prepared by adding dextrin and/or gamma-cyclodextrin (see Table 3 below) to 50% fermented alcohol. 50 g of the Agrimonia eupatoria sample was added to each of three bottles, and a 15-fold weight of the prepared solution was added to the sample, and the mixture was stirred and extracted for 5 hours at 300 rpm at each of 60° C., 70° C. and 80° C., and then filtered through filter paper to obtain a primary extract. A 14-fold weight of the same fresh solution was added to the filtration residue, and the mixture was stirred and extracted at 300 rpm for 5 hours at the same temperature as the first extraction temperature (each of 60° C., 70° C., and 80° C.) in each bottle, and filtered through filter paper, and then the filtrate was combined with the primary extract, concentrated under reduced pressure at 60° C., and then freeze-dried, thus preparing final extracts.

[0072] 2-1. Measurement of Yield and Content

[0073] The extraction yield was determined by calculating the weight of the freeze-dried sample as a percentage of the weight of the sample used to prepare the extract.

[0074] To measure the content of the active ingredient luteolin-7-O-glucuronide in each of the prepared Agrimonia eupatoria extract powder (control) and inclusion powder (treatment group), analysis was performed in the same manner as in Preparation Example 1-1 using high-performance liquid chromatography (Infinity 1260, Agilent, USA). The results are shown in Table 3 below and FIG. 5.

TABLE-US-00003 TABLE 3 γ-CD 0% γ-CD 2% γ-CD 4% Solution Dextrin* 0.26% 0.13% — Gamma-cyclodextrin* — 0.13% 0.26% Yield 60° C.** 134.1 188.8 168.5 (yield X 70° C.** 141.2 177.0 160.9 content) 80° C.** 147.7 174.8 165.2 *Amount of dextrin and/or gamma cyclodextrin relative to liquid amount **Inclusion extraction temperature

[0075] As shown in Table 3 above and FIG. 5, it was confirmed that the highest yield was obtained when extraction was performed at 60° C. and at a gamma-cyclodextrin concentration of 2%.

[0076] 2-2. Measurement of Bioavailability

[0077] Cell uptake of the Agrimonia eupatoria inclusion extract was measured in the same manner as in Preparation Example 1-2 using Caco-2 cells. The results are shown in FIG. 6.

[0078] As shown in FIG. 6, it was confirmed that the bioavailability was the highest when extraction was performed at 60° C. and at a gamma-cyclodextrin concentration of 2%.

[0079] 2-3. Water Solubility Measurement

[0080] The recovery rate was calculated in the same manner as in Preparation Example 1-3 using 2.5 g of each of the Agrimonia eupatoria inclusion extracts obtained under various inclusion conditions. The results are shown in FIG. 7.

[0081] As shown in FIG. 7, it was confirmed that the solids recovery rate was the highest when extraction was performed at 60° C. and at a gamma-cyclodextrin concentration of 2%.

[0082] Through the above experiments, the gamma-cyclodextrin concentration was set to 2% of the raw material, the inclusion extraction temperature was set to 60° C., and subsequent experiments were conducted.

Example 1. Preparation of Agrimonia eupatoria Inclusion Extract

[0083] In order to prepare an optimized Agrimonia eupatoria inclusion extract, Agrimonia eupatoria leaves purchased through Jiyu Boncho Co., Ltd. were used.

[0084] Specifically, foreign substances were removed from the leaves of Agrimonia eupatoria, and gamma-cyclodextrin was added to 50% fermented alcohol in an amount of 0.13% relative to the liquid amount (2% relative to raw material), thus preparing a gamma-cyclodextrin solution. A 15-fold weight of the prepared gamma-cyclodextrin solution was added to 50 g of the Agrimonia eupatoria sample, and the mixture was stirred and extracted at 300 rpm at 60° C. for 5 hours, and then filtered through filter paper to obtain a primary extract. A 14-fold weight of a fresh gamma-cyclodextrin solution was added to the filtration residue, and the mixture was stirred and extracted at 300 rpm at 60° C. for 5 hours, and filtered through filter paper, and the filtrate was combined with the primary extract, concentrated under reduced pressure at 60° C., and then freeze-dried, thus preparing a final extract. The extraction yield was 25.93%, which was determined by calculating the weight of the freeze-dried sample as a percentage of the weight of the sample used to prepare the extract.

Comparative Example 1. Preparation of Agrimonia eupatoria Extract

[0085] In order to prepare an Agrimonia eupatoria extract, Agrimonia eupatoria leaves purchased through Jiyu Boncho Co., Ltd. were used.

[0086] Specifically, foreign substances were removed from the leaves of Agrimonia eupatoria, and dextrin was added to 50% fermented alcohol in an amount of 0.13% relative to the liquid amount (2% relative to raw material), thus preparing a dextrin solution. A 15-fold weight of the prepared dextrin solution was added to 50 g of the Agrimonia eupatoria sample, and the mixture was stirred and extracted at 300 rpm at 80° C. for 5 hours, and then filtered through filter paper to obtain a primary extract. A 14-fold weight of a fresh dextrin solution was added to the filtration residue, and the mixture was stirred and extracted at 300 rpm at 80° C. for 5 hours, and filtered through filter paper, and the filtrate was combined with the primary extract, concentrated under reduced pressure at 60° C., and then freeze-dried, thus preparing a final extract. The extraction yield was 22.68%, which was determined by calculating the weight of the freeze-dried sample as a percentage of the weight of the sample used to prepare the extract.

[0087] Experimental Example 1. Analysis of Liver Injury Protective Effect of Agrimonia eupatoria Extract

[0088] As experimental animals, 6-7 week-old Sprague-Dawley white rats were used and bred at room temperature of 22±2° C. For the experiment, white rats having similar weights were divided into 4 groups: a positive control group not treated with carbon tetrachloride (CCl4); a negative control group treated with CCl4 alone; a group treated with 50 mg/kg of the Agrimonia eupatoria extract powder of Comparative Example 1; and a group treated with 50 mg/kg of the Agrimonia eupatoria inclusion extract powder of Example 1. Experimental diet was provided by free drinking water, and the intake by each group was recorded at a fixed time once a day.

[0089] In order to examine the acute hepatotoxicity preventive effect and antioxidant effect of each of the Agrimonia eupatoria extract powder and the inclusion extract powder, the experimental diet was supplied every week for one week (see Korean Patent Application Publication No. 10-2012-0108797), and CCl4 was administered at the last week. Then, the rats were fasted for 24 hours and then sacrificed. For administration of CCl4, 10% CCl4 was mixed with olive oil at a ratio of 1:1 and administered at a dose of 1 ml/kg. After fasting, blood was collected from the abdominal aorta, and the collected blood was immediately centrifuged at 3,000 rpm for 15 minutes, and serum was separated therefrom and used as an analysis sample. In addition, the liver, kidney, heart, and spleen were extracted immediately after blood collection, rinsed in physiological saline, and weighed after removal of water from the surfaces thereof, and then stored frozen at −70° C.

[0090] For AST (aspartate aminotransferase) and ALT (alanine aminotransferase) measurement in serum, blood collected from the abdominal aorta was immediately centrifuged at 3,000 rpm for 15 minutes, and serum was separated therefrom and used as samples for AST and ALT analysis. The results are shown in Table 4 below and FIG. 8.

TABLE-US-00004 TABLE 4 Comparative CON CCL.sub.4 Example 1 Example 1 AST (IU/L) 105.3 573.4 416.0 321.5 ALT (IU/L) 35.6 547.3 391.8 306.7

[0091] As shown in Table 4 above and FIG. 8, it was confirmed that the AST and ALT levels tended to decrease in the group treated with the Agrimonia eupatoria extract powder (Comparative Example 1) compared to the CCL group, and in particular, were significantly low in the group treated with the Agrimonia eupatoria inclusion extract powder (Example 1).

[0092] Liver histopathological analysis was performed to examine the acute hepatotoxicity preventive effect and antioxidant effect of each of the Agrimonia eupatoria extract powder and inclusion extract powder, and the results are shown in FIG. 9.

[0093] As shown in FIG. 9, as a result of performing histopathological analysis of the experimental animals of each group, rapid changes in liver injury, including cell disruption and bile duct proliferation, could be observed in the liver tissue of the control group (CCl4) treated with carbon tetrachloride (CCl4) alone. On the other hand, some injury appeared in the group treated with the Agrimonia eupatoria extract powder (Comparative Example 1), but was similar to that in the control group, and in particular, the group treated with the Agrimonia eupatoria inclusion extract powder (Example 1) showed almost the same tissue findings as the control group.