Anti-angiogenesis composition containing sepsis acid A isolated from Prunella vulgaris as an active ingredient

Abstract

The present invention relates to an anti-angiogenesis composition comprising sepsis A isolated from Prunella vulgaris as an active ingredient. The composition of the present invention has an excellent angiogenesis inhibitory effect as confirmed by cell invasion reduction effect, tube formation inhibitory effect, angiogenesis reduction in an in vivo angiogenesis model, and hemoglobin reduction effect.

Description

Technical Field

[0001] The present invention relates to an anti-angiogenesis composition, which comprises sepsis acid A isolated from Prunella vulgaris and having excellent angiogenesis inhibitory effect as an effective ingredient and has excellent angiogenesis inhibitory effect. Background Art

[0002] Angiogenesis is the process of generating new blood vessels from existing blood vessels, which is an essential process for maintaining the homeostasis of an individual as part of normal physiological functions. This angiogenesis is accomplished through a series of consecutive steps including the following steps: the breakdown of the vascular basement membrane by proteases, the migration and proliferation of vascular endothelial cells that form the vascular wall, and the differentiation of vascular endothelial cells to form tubes to reorganize blood vessels to generate new capillaries.

[0003] Since blood vessels have the function of supplying oxygen and nutrients to cells, all cells in the body exist within about 100 μm of the range of oxygen diffusion from blood vessels. When a cell exceeds this range, it is exposed to a hypoxic environment and secretes a substance called hypoxia inducible factor 1α (HIF1α) to promote angiogenesis.

[0004] In addition to its physiological role, angiogenesis is a very important phenomenon in the course of various diseases such as tumor growth, pulmonary hypertension, diabetic retinopathy, age-related macular degeneration, etc. Due to the importance of angiogenesis in various disease processes, many studies have been conducted on inhibiting the angiogenesis process.

[0005] Prunella vulgaris is obtained by drying the whole herb of Prunella vulgaris L., a plant of the Labiatae family. Prunella vulgaris, also known as honey grass, evening grass, Naidong, and Yanmian, grows in the mountains and roadsides of various places. When the flowers bloom, cut the whole herb and dry it in a cool place. It tastes bitter and is cold in nature.

[0006] Prunella vulgaris acts on the liver meridian, and has antipyretic, detoxifying, and eye-improving effects. In addition, it is well known that it has antihypertensive, diuretic, and antibacterial effects, and is also effective for edema, hypertension, tuberculosis, and epidemic hepatitis.

[0007] Regarding the pharmacological effects of Prunella Vulgaris, Korean Patent Publication No. 10-2005-0119488 discloses a pharmaceutical composition for preventing and treating degenerative arthritis, the pharmaceutical composition comprising a tallow extract mixture containing glucosamine and tallow extract, shark cartilage extract, Prunella Vulgaris extract and St. John's wort extract. In addition, Korean Patent Publication No. 10-2008-0056462 discloses a composition for preventing, treating and inhibiting cancer diseases, the composition comprising Prunella Vulgaris extract as an active ingredient. In addition, Korean Patent Publication No. 10-2012-0058756 discloses a pharmaceutical composition for preventing and treating diabetic atherosclerosis.

[0008]

Prior art documents

[0009] [Patent Documents]

[0010] (Patent Document 1) Korean Patent Publication No. 10-2005-0119488

[0011] (Patent Document 2) Korean Patent Publication No. 10-2008-0056462

[0012] (Patent Document 3) Korean Patent Publication No. 10-2012-0058756 Summary of the invention

[0013] An object of the present invention is to provide an anti-angiogenesis composition comprising, as an active ingredient, sepsis acid A which is isolated from Prunella vulgaris and has an excellent angiogenesis inhibitory effect.

[0014] To achieve the above object, the present invention provides an anti-angiogenesis composition comprising pygenic acid A isolated from Prunella vulgaris as an effective ingredient.

[0015] Preferably, in the composition, the sepsis acid A is included in a concentration of 20 to 40 μM.

[0016] Preferably, in the composition, the septic acid A is separated by the following method; the method comprises: a step of extracting Prunella vulgaris with alcohol to obtain a Prunella vulgaris extract; a step of filtering the Prunella vulgaris extract to obtain a filtrate; a step of concentrating the filtrate under reduced pressure to obtain a Prunella vulgaris extract concentrate; a step of adding distilled water in a volume 20 to 25 times the weight of the concentrate to the Prunella vulgaris extract concentrate and shaking to obtain a suspension; a step of separating a dichloromethane (CH2Cl2) fraction from the suspension; a step of gradient eluting the dichloromethane fraction to obtain a septic acid A fraction; and a step of purifying the septic acid A fraction and drying under reduced pressure to obtain septic acid A.

[0017] Preferably, in the composition, the alcohol is used in an amount of 6 to 8 times the weight of Prunella vulgaris.

[0018] Preferably, in the composition, the extraction is repeatedly performed 2 to 4 times.

[0019] Preferably, in the composition, the distilled water is used in an amount of 20 to 25 times the weight of the Prunella Vulgaris extract concentrate.

[0020] Effects of the Invention

[0021] The sepsis acid A isolated from Prunella vulgaris of the present invention has an excellent angiogenesis inhibitory effect as confirmed by cell invasion reducing effect, tube formation inhibitory effect, angiogenesis factor inhibitory effect, angiogenesis reduction in an in vivo angiogenesis model, and hemoglobin reduction effect.

[0022] Therefore, since the anti-angiogenesis composition of the present invention comprising sepsis acid A isolated from Prunella vulgaris as an active ingredient has an excellent angiogenesis inhibitory effect, it can be effectively used for preventing, treating and ameliorating diseases associated with angiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the structural formula of sepsis acid A (CAS No. 52213-27-1, 3-epicorosolic acid).

[0024] Figure 2 This is to confirm the results of the cell invasion-reducing effect of septic acid A on cell invasion induced by fibronectin (FN).

[0025] Figure 3 These are the results of confirming the tube formation inhibitory effect of septic acid A.

[0026] Figure 4 The results are a comparison of the tube formation inhibitory effects of septic acid A and the angiogenesis inhibitor SU5416.

[0027] Figure 5 This is a result of confirming the angiogenesis factor inhibitory effect of septic acid A.

[0028] Figure 6 The results are a comparison of the angiogenesis factor inhibitory effects of septic acid A and SU5416.

[0029] Figure 7 Results to confirm the morphology and color of Matrigel in the in vivo Matrigel plug assay.

[0030] Figure 8 To confirm the results of the in vivo Matrigel plug assay for vessel density within Matrigel.

[0031] Fig. 9 Results to confirm hemoglobin concentration in the in vivo Matrigel plug assay. DETAILED DESCRIPTION

[0032] The present invention relates to an anti-angiogenesis composition, which comprises sepsis acid A separated from Prunella vulgaris as an effective ingredient.

[0033] In the composition of the present invention, septic acid A is a substance also known as CAS No.52213-27-1, 3-epicorosolic acid, which is preferably separated by the following method; the method comprises: a step of extracting Prunella vulgaris with alcohol to obtain a Prunella vulgaris extract; a step of filtering the Prunella vulgaris extract to obtain a filtrate; a step of concentrating the filtrate under reduced pressure to obtain a Prunella vulgaris extract concentrate; a step of adding distilled water in a volume of 20 to 25 times the weight of the concentrate to the Prunella vulgaris extract concentrate and shaking to obtain a suspension; a step of separating a dichloromethane (CH2Cl2) fraction from the suspension; a step of gradient eluting the dichloromethane fraction to obtain a septic acid A fraction; and a step of purifying the septic acid A fraction and drying it under reduced pressure to obtain septic acid A.

[0034] Hereinafter, the present invention will be described in detail.

[0035] The herb is preferably cut and used. The herb is extracted by adding 6 to 8 volumes of alcohol, which is equal to the weight of the herb. As the alcohol, an alcohol having 1 to 5 carbon atoms is preferably used, and more preferably, one or more of methanol, ethanol and isopropanol can be used.

[0036] As the extraction method, conventional extraction methods such as cold soaking extraction, hot water extraction, ultrasonic extraction, reflux extraction, cooling extraction, etc. can be used, and extraction is more preferably performed by reflux cooling extraction. The extraction is performed for 2 to 4 hours, more preferably for 3 hours. Moreover, the extraction can be repeated once or twice or more, preferably 2 to 4 times, more preferably 3 times.

[0037] After extraction, the mixture was filtered to obtain a filtrate, and the filtrate was concentrated under reduced pressure to obtain a dealcoholized Prunella vulgaris extract concentrate.

[0038] The Prunella Vulgaris extract concentrate is added with distilled water in a volume of 20 to 25 times the weight of the concentrate, and then shaken to obtain a suspension.

[0039] An equal amount of dichloromethane (CH2Cl2) was added to the suspension to perform solvent fractionation to separate the dichloromethane fraction.

[0040] The dichloromethane fraction was subjected to gradient elution in a column made of silica gel suspended in a mixed solution of ethyl acetate and alcohol as a mobile phase, and the fraction containing septic acid A was separated and then concentrated to obtain a septic acid A fraction. The separated septic acid A fraction was purified by preparative high performance liquid chromatography (prep HPLC) and dried under reduced pressure at 40° C. for 6 hours to obtain pure septic acid A.

[0041] The structural formula of sepsis acid A is shown in Figure 1 .

[0042] Septin A has an excellent angiogenesis inhibitory effect, as confirmed by its cell invasion reducing effect in Transwell migration assay, tube formation inhibitory effect in tube formation assay, angiogenesis factor inhibitory effect, angiogenesis reducing effect and hemoglobin reducing effect in an in vivo angiogenesis model.

[0043] Therefore, the anti-angiogenesis composition of the present invention comprising sepsis acid A isolated from Prunella vulgaris as an active ingredient has an excellent angiogenesis inhibitory effect and can be effectively used for preventing, treating and ameliorating diseases associated with angiogenesis.

[0044] The composition preferably comprises sepsis A at a concentration of 20 to 40 μM, more preferably comprises sepsis A at a concentration of 30 to 40 μM, and particularly preferably comprises sepsis A at a concentration of 40 μM.

[0045] The present invention will be described in more detail below by way of examples. These examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

[0046] <Example 1>

[0047] Isolation of Septic A

[0048] Prunella vulgaris was purchased in a cut state. 2 L of methanol was added to 300 g of the purchased Prunella vulgaris, and the mixture was reflux-cooled with a heating mantle and extracted three times over 3 hours.

[0049] The Prunella Vulgaris extract obtained under reduced pressure was filtered through a Buchner funnel with filter paper, and the obtained Prunella Vulgaris filtrate was concentrated under reduced pressure to obtain 22.4 g of a concentrated extract from which methanol had been removed.

[0050] 500 mL of distilled water was added to 22.4 g of the obtained concentrated extract, and the mixture was shaken and suspended. Then, an equal amount of dichloromethane (CH 2 Cl 2 ) was added, and 8.4 g of a dichloromethane (CH 2 Cl 2 ) fraction was separated by solvent fractionation.

[0051] 250 g of silica gel (230 to 400 mesh) was suspended in a mixture of ethyl acetate (EtOAC): methanol (MeOH) = 1:0 (mobile phase) to prepare a 9:1 size column. 8.4 g of dichloromethane (CH2Cl2) fraction was dissolved in the initial mobile phase and loaded on the column, and then gradient elution was performed using the mobile phase solvent [EtOAC: MeOH = 10: 0: 0: 10]. The fraction containing septic acid A was confirmed by TLC (RP solvent MeOH: H2O = 90: 10, Rf 0.3) in the eluted fraction, and the fractions were combined and concentrated to obtain a purified septic acid A fraction. The septic acid A fraction was purified by preparative high performance liquid chromatography (prep HPLC) and dried under reduced pressure at 40°C for 6 hours to obtain 60 mg of pure septic acid A. The yield of septic acid A obtained from the herbal extract was 0.027%.

[0052] [Experimental example]

[0053] Prepare cells

[0054] SVEC4-10 cells were used in the experiment and were purchased from ATCC.

[0055] <Experimental Example 1>

[0056] Cell invasion reduction effect

[0057] The cell invasion-reducing effect of septic acid A on cell invasion induced by fibronectin (FN) was confirmed by Transwell migration assay as follows.

[0058] Transwells were coated with 5 μg / mL fibronectin and left overnight at 4° C. The fibronectin-coated Transwells were blocked with 1% BSA for 60 minutes and then washed with PBS.

[0059] 2×10 4 SVEC4-10 cells per cell / well were pretreated with 10 μM, 20 μM and 40 μM septic acid A for 30 minutes, respectively, and then divided in the upper chamber of the washed Transwell and cultured at 37°C, 5% humidity and CO2 for 16 hours.

[0060] The cultured Transwell cells were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet.

[0061] As a negative control group (NC), SVEC4-10 cells coated with 2% BSA and cultured on a Transwell plate at 4°C overnight were used, and as a positive control (PC), SVEC4-10 cells treated with fibronectin without pretreatment with septic acid A were used.

[0062] The degree of cell invasion was confirmed by comparing the percentage with the positive control group, and the results are shown in Figure 2 .

[0063] like Figure 2 As shown in the results, cell invasion induced by fibronectin was reduced by sepsis A, and the higher the concentration of sepsis A, the greater the effect of reducing cell invasion. In particular, at a concentration of 40 μM, the degree of cell invasion was lower than that of the negative control group.

[0064] <Experimental Example 2>

[0065] Tube formation inhibition effect

[0066] The tube formation inhibitory effect of septic acid A was confirmed by tube formation assay as follows.

[0067] After adding 50 μL of Matrigel to each well of the 96-well plate, the plates were left to stand at 37° C. for 30 minutes to allow polymerization to proceed.

[0068] 4×10 4 SVEC4-10 cells were split at cells / well and pretreated with 10 μM, 20 μM and 40 μM septic acid A for 30 minutes.

[0069] SVEC4-10 cells pretreated with septic acid A were seeded on Matrigel and cultured at 37°C, 5% humidity, and CO2 for 3 hours.

[0070] As a negative control group (NC), SVEC4-10 cells not treated with fetal bovine serum (FBS) were used, and as a positive control group (PC), SVEC4-10 cells not pretreated with septic acid A but treated with FBS were used.

[0071] The degree of tube formation was confirmed as a percentage compared with the positive control group, and the results are shown in Figure 3 .

[0072] like Figure 3 As shown in the results, when septic acid A was treated, the degree of tube formation was inhibited, and the higher the concentration of septic acid A, the greater the tube formation inhibition effect.

[0073] <Experimental Example 3>

[0074] Tube formation inhibition effect

[0075] The tube formation inhibitory effects of sepsis A and the angiogenesis inhibitor SU5416 (Semaxanib) were compared using a tube formation assay as follows.

[0076] After adding 50 μL of Matrigel to each well of the 96-well plate, the plates were left to stand at 37° C. for 30 minutes to allow polymerization to proceed.

[0077] 4×10 4 Cells / well were divided into SVEC4-10 cells, and pretreated with 40 μM septic acid A and 4 μM SU5416 for 30 minutes respectively.

[0078] The pretreated SVEC4-10 cells were seeded on the matrigel and cultured at 37°C, 5% humidity, and CO2 for 3 hours.

[0079] As a control group (Control), SVEC4-10 cells treated with Matrigel alone were used.

[0080] The degree of tube formation was confirmed as a percentage compared with the control group, and the results are shown in Figure 4 .

[0081] like Figure 4 As shown in the results, the tube formation degree was inhibited when septic acid A and SU5416, an inhibitor of angiogenesis, were treated. Moreover, the tube formation degree was further inhibited when septic acid A was treated compared to the case of SU5416 treatment.

[0082] SU5416 used as a positive drug is a well-known angiogenesis inhibitor. The concentration of SU5416 is based on a previous research paper. (Ref: Penduliflaworosin, a Diterpenoid from Crotoncrassifolius, Exerts Anti-Angiogenic Effect via VEGF Receptor-2 Signaling Pathway)

[0083] <Experimental Example 4>

[0084] Angiogenic factor inhibitory effect

[0085] The angiogenic factor inhibitory effect of septic acid A was confirmed by western blotting analysis as follows.

[0086] SVEC4-10 cells were treated with 10 μM, 20 μM, and 40 μM sepsis A for 24 hours, respectively, and then lysed using RIPA lysis buffer.

[0087] Equal amounts of proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel. The separated proteins were transferred to a nitrocellulose membrane, blocked with 5% skim milk in TBST (10mmol / L Tris pH 8.0, 150mmol / L NaCl, 0.1% Tween2) for 1 hour, and then reacted with primary antibodies against angiogenic factors FAK, p-FAK, Src, p-Src, Akt, p-Akt, ERK, and p-ERK at 4°C overnight. β-actin was used as a standard substance.

[0088] After washing with TBST, the nitrocellulose membrane was incubated with HRP-conjugated secondary antibody for 2 hours at room temperature.

[0089] After incubation, each protein was detected using enhanced chemiluminescence (ECL) reagent.

[0090] As a negative control group (NC), SVEC4-10 cells not treated with fetal bovine serum (FBS) were used, and as a positive control group (PC), SVEC4-10 cells not pretreated with septic acid A but treated with FBS were used.

[0091] The results are shown in Figure 5 .

[0092] like Figure 5 As shown in the results, when septic acid A was treated, the expression levels of proteins p-FAK, p-Src, p-Akt, and p-ERK involved in angiogenesis were inhibited, and the higher the concentration of septic acid A, the greater the inhibitory effect.

[0093] <Experimental Example 5>

[0094] Angiogenic factor inhibitory effect

[0095] The angiogenic factor inhibitory effect of septic acid A was compared with that of the angiogenic factor inhibitor SU5416.

[0096] SVEC4-10 cells were treated with 40 μM sepsis A and 4 μM SU5416 for 24 hours, respectively, and FAK, p-FAK, ERK, and p-ERK were used as angiogenesis factors. As a control group, SVEC4-10 cells treated with only Matrigel were used. The experiment was performed in the same manner as in Experimental Example 4. The results are shown in Figure 6 .

[0097] like Figure 6 As shown in the results, the expression levels of p-FAK and p-ERK were further reduced when septic acid A was treated compared with the case of treatment with the angiogenesis inhibitor SU5416.

[0098] SU5416 used as a positive drug is a well-known angiogenesis inhibitor. The concentration of SU5416 is based on a previous research paper. (Ref: Penduliflaworosin, a Diterpenoid from Crotoncrassifolius, Exerts Anti-Angiogenic Effect via VEGF Receptor-2 Signaling Pathway)

[0099] <Experimental Example 6>

[0100] In vivo Matrigel plug assay

[0101] In an in vivo angiogenesis model, the effects of septic acid A on angiogenesis and hemoglobin content were confirmed as follows.

[0102] The mice used were purchased from Coretech and were 6 weeks old. 20 μM and 40 μM septic acid A were used as experimental samples, and 4 μM SU5416 was used for comparison.

[0103] At 4°C, each sample was mixed with a mixture of 400 ng / mL VEGF (vascular endothelial growth factor) and 20 U heparin in a matrix gel mixture, and then placed on ice to remove bubbles. Anesthetized mice were removed from the ribs, and 200 μL of the mixture of the matrix gel mixture and the experimental sample was subcutaneously inserted into each site.

[0104] As a negative control group (NC), SVEC4-10 cells not treated with fetal bovine serum (FBS) were used, and as a positive control group (PC), SVEC4-10 cells not pretreated with septic acid A but treated with FBS were used.

[0105] After one week, the mice were dissected and the morphology and color of the matrix gel were confirmed. The results are shown in Figure 7 , confirm the density of blood vessels in the matrix gel, and the results are shown in Figure 8 .

[0106] from Figure 7 and Figure 8 The results confirmed that when VEGF was treated, many blood vessels were formed in the matrigel. When septic acid A and SUS5416 were treated, angiogenesis was reduced, and when 40 μM septic acid A was treated, the angiogenesis reduction effect was better than that of SUS5416.

[0107] In addition, a portion of the matrix gel was removed and placed in a tube containing 500 μL of DW (+0.2% heparin), and placed in a refrigerator overnight, and then centrifuged at 15,000 rpm for 20 minutes. 100 μL of the supernatant after centrifugation was mixed with 100 μL of Drabkin kit solution, and the hemoglobin concentration was measured at 540 nm. The results are shown in Fig. 9 .

[0108] like Fig. 9 As shown in the results, it was confirmed that in the in vivo angiogenesis model, the VEGF-treated group showed a higher hemoglobin content, but when treated with sepsis acid A, the hemoglobin content was significantly reduced, and when treated with 40μM sepsis acid, the hemoglobin content was lower than that of SUS5416.

[0109] SU5416 used as a positive drug is a well-known angiogenesis inhibitor. The concentration of SU5416 was determined based on a previous research paper (Ref: Penduliflaworosin, a Diterpenoid from Crotoncrassifolius, Exerts Anti-Angiogenic Effect via VEGF Receptor-2 Signaling Pathway).

Claims

1. Application of sepsis acid A in the preparation of anti-angiogenic drugs.

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