KR102025322B1 - Compounds having anti-inflammatory activity isolated from butterbur extract and isolation method thereof - Google Patents

Abstract

The present invention relates to a fraction separated from a Petasites japonicas extract having anti-inflammatory activity. By providing a novel compound separated from the Petasites japonicas extract, it is possible to have excellent anti-inflammatory activity, and at the same time, by providing a compound having anti-inflammatory activity derived from natural products, it is possible to have an effect of providing a pharmaceutical composition safe for the human body.

Description

Anti-inflammatory Active Compounds Isolated from Butterbur Extracts and Methods for Separation thereof {COMPOUNDS HAVING ANTI-INFLAMMATORY ACTIVITY ISOLATED FROM BUTTERBUR EXTRACT AND ISOLATION METHOD THEREOF}

The present invention relates to novel compounds isolated from coltsfoot extract, and more particularly to fractions and methods of separation from coltsfoot extract with anti-inflammatory activity.

Butterbur (common name: Butterbur or scientific name: Petasites japonicus) is a perennial shrub found in Europe, as well as parts of Asia and North America. Extracts isolated from the roots and leaves of coltsfoot have been used as therapeutics for over 2,000 years. Today, butterbur extract has been reported to be used primarily for muscle relaxation, to treat conditions such as abdominal pain in the stomach, to spasms of smooth muscle, to reduce pain and to be used for headaches (Eaton J., Townsend Lett, 2000, 202, 104-106). Butterbur has also been reported to have therapeutic effects on asthma and allergic diseases (Thomet OA, Simon HU, Int Arch Allergy Immunol, 2002, 129 (2), 108-12).

Butterbur is known for its diverse biological activities, including anti-allergic, anti-inflammatory and antioxidant effects. In addition, the camemphenol isolated from the stem of the coltsfoot, anti-apoptotic factor (B-cell lymphoma 2), proapoptotic factor (Bax-like BH3 protein and apoptosis) in mouse hippocampal cells oxidative stress by regulating -inducing factor and mitogen-activated protein kinases (p38, extracellular signal-regulated kinase) and JNK (c-Jun N-terminal kinase) Reduced neuronal damage to oxidative stress

Both the whole coltsfoot extract and the two extracts (petatewalide B and bakkenolide B) stimulated lipopolysaccharide-stimulated BV2 microglia cells through signaling upregulation of AMP-activated protein kinase / nucleus factor erythroid related factor 2 Park, SY; Choi, MH; Li, M .; Li, K .; Park, G .; Choi, YW AMPK / Nrf2 signaling is involved in the anti-neuroinflammatory action of Petatewalide B from Petasites japonicus against lipopolysaccharides in microglia.Immunopharmacol.Immunotoxicol. 2018, 40, 232-241.). Buds of butterbur are also known to inhibit A1-42 as well as bovine serum albumin and lactalbumin.

Inflammation is an immune response of the human body in response to a wound or disease, and oxidative stress such as ultraviolet rays, free radicals, and free radicals activates inflammatory factors and causes various diseases and aging of the skin. Vascular activating polypeptides, kinin, plasmin or complement, act as vasodilation, contraction and chemotaxis, as well as lymphokahs such as interleukin-6 (IL-6). Phosphorus and arachidonic acid are responsible for the inflammatory response. Arachidonic acid is metabolized to prostaglandin or leukotriene, an inflammatory mediator, through two pathways, Cyclooxygenase or Lipooxygenase, to mediate various inflammatory reactions.

Synthetic anti-inflammatory drugs developed so far can be divided into steroids (hydrocortisone, prednisolone, betamethasone) and nonsteroids (aspirin, indomethacin and ibuprofen), and most of them have side effects such as gastrointestinal, kidney and heart disease (Dagne JM , et al. cardiovascular side-effects: from light to shadow.Currpharm Des. 12: 917-975 (2006) and Makins R, Ballinger A. Gastrointestinal side effects of drugs.Expert Opin Drug Saf. 2: 421-429 (2003 )) There is a need to develop safe and effective anti-inflammatory drugs derived from natural products.

The present invention provides a novel compound having excellent anti-inflammatory activity by providing a novel substance isolated from the butterbur extract, and a method for separating the same.

In order to achieve the problem to be solved, the novel compound of one embodiment of the present invention can be represented by the following formula (1).

[Formula 1]

The compound may be extracted from butterbur.

The compound may be an anti-inflammatory activity, the compound may inhibit the expression of iNOS, the compound may inhibit the expression of cyclooxygenase-2 (COX-2).

In addition, the pharmaceutical composition for preventing or treating inflammatory diseases according to another embodiment of the present invention may include the above anti-inflammatory active compound as an active ingredient.

The inflammatory diseases are brain diseases caused by nerve cell inflammation, atopic dermatitis, encephilitis, inflammatory enteritis, chronic obstructive pulmonary disease, pulmonary pulmonary shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthritis, arthritis, inflammatory Chronic Inflammation, Colitis, Inflammatory Bowel Disease, Type 1 Diabetes, Rheumatoid Arthritis, Reactive Arthritis, Osteoarthritis, Psoriasis, Scleroderma, Osteoporosis, Atherosclerosis, Myocarditis, Endocarditis caused by osteolysis, chronic viral or bacterial infection , Pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, Borreliosis, anorexia-Borrelia, tuberculosis, Sarcoidosis, lupus, discus lupus Lupus, lupus nephritis, systemic lupus erythematosus lupus, macular degeneration, uveitis, irritable bowel syndrome, Crossie's disease, Shogran's syndrome, fibromyalgia, St. fatigue syndrome, may be chronic fatigue immune dysfunction syndrome, muscular encephalomyelitis, amyotrophic lateral sclerosis, and any disease of multiple sclerosis.

In addition, the separation method of the anti-inflammatory active compound from the coltsfoot extract according to another aspect of the present invention comprises the steps of (a) extracting the coltsfoot reflux in water (H ₂ O) and then lyophilized to obtain a powder; (b) obtaining 15 fractions sequentially of the lyophilized powder through ion-exchange chromatography; (c) separating the ninth fractions of the fractions to obtain ten small fractions sequentially through size exclusion chromatography; And (d) performing a reversed phase high performance liquid chromatography (HPCL) on the sixth small fraction of the small fraction to obtain a compound represented by Chemical Formula 1 below.

[Formula 1]

In step (b), the ion exchange chromatography may be Diaion HP-20 column chromatography.

In step (c), the molecular exclusion chromatography may be Sephadex column chromatography.

In the step (d), the reverse phase high performance liquid chromatography (HPCL) may be a chromatography of the YMC Pack ODS-A column high performance liquid chromatography (HPCL).

According to one embodiment of the present invention, there is an effect of providing a novel compound separated from the butterbur extract.

In addition, the present invention has the effect of having excellent anti-inflammatory activity by providing a novel compound.

In addition, the present invention has the effect of providing a composition for the prevention or treatment of inflammatory diseases comprising a compound having excellent anti-inflammatory activity.

In addition, the present invention has the effect of providing a pharmaceutical composition that is safe for the human body by providing a compound having anti-inflammatory activity derived from natural products.

1 illustrates a heteronuclear multiple bond correlation (HMBC) spectrum of Compound 1 isolated from coltsfoot extract according to one embodiment of the present invention.
FIG. 2 illustrates an electronic circular dichroism (ECD) spectrum of Compound 1 isolated from coltsfoot extract according to one embodiment of the present invention.
FIG. 3 shows a heteronuclear multiple bond correlation (HMBC) spectrum of Compound 2 isolated from coltsfoot extract according to one embodiment of the present invention.
FIG. 4 shows the NOESY (nuclear overhauser spectroscopy) spectrum of Compound 1 isolated from coltsfoot extract according to one embodiment of the present invention.
FIG. 5 shows the NOESY (nuclear overhauser spectroscopy) spectrum of Compound 2 isolated from coltsfoot extract according to one embodiment of the present invention.
6 illustrates an electronic circular dichroism (ECD) spectrum of Compound 2 isolated from coltsfoot extract according to one embodiment of the present invention.
FIG. 7 shows an IR spectrum (A of FIG. 7) and a vibrational circular dichroism (VCD) spectrum (B of FIG. 7) of Compound 2 isolated from coltsfoot extract according to one embodiment of the present invention.
Figure 8 shows the NO production inhibition result (A of Figure 8) and iNOS protein expression inhibition result (B of Figure 8) of Compound 1 isolated from the coltsfoot extract according to an embodiment of the present invention.
9 shows the results of inhibition of PGE ₂ (FIG. 9A) and the inhibition of COX-2 expression (FIG. 9B) of Compound 1 isolated from the coltsfoot extract according to one embodiment of the present invention.
Figure 10 shows the results of cell viability evaluation according to Compound 1 (A of Figure 10) and Compound 2 (B of Figure 10) isolated from the extract of Butterbur according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings and the contents described in the accompanying drawings, but the present invention is not limited or limited to the embodiments.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In this specification, the singular also includes the plural unless specifically stated otherwise in the phrase.

The novel compound of one embodiment of the present invention can be represented by the following formula (1).

[Formula 1]

The compound may be extracted from butterbur. More specifically, the butterbur may be extracted from a freeze-dried powder by reflux extraction in water (H ₂ O).

The compound may have anti-inflammatory activity, and more particularly, may inhibit the expression of iNOS, and may also inhibit the expression of cyclooxygenase-2 (COX-2).

In addition, the pharmaceutical composition for preventing or treating inflammatory diseases according to another embodiment of the present invention may include the above anti-inflammatory active compound as an active ingredient.

The inflammatory diseases are brain diseases caused by nerve cell inflammation, atopic dermatitis, encephilitis, inflammatory enteritis, chronic obstructive pulmonary disease, pulmonary pulmonary shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthritis, arthritis, inflammatory Chronic Inflammation, Colitis, Inflammatory Bowel Disease, Type 1 Diabetes, Rheumatoid Arthritis, Reactive Arthritis, Osteoarthritis, Psoriasis, Scleroderma, Osteoporosis, Atherosclerosis, Myocarditis, Endocarditis caused by osteolysis, chronic viral or bacterial infection , Pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, Borreliosis, anorexia-Borrelia, tuberculosis, Sarcoidosis, lupus, discus lupus Lupus, lupus nephritis, systemic lupus erythematosus lupus, macular degeneration, uveitis, irritable bowel syndrome, Crossie's disease, Shogran's syndrome, fibromyalgia, St. fatigue syndrome, may be chronic fatigue immune dysfunction syndrome, muscular encephalomyelitis, amyotrophic lateral sclerosis, and any disease of multiple sclerosis.

In addition, the separation method of the anti-inflammatory active compound from the coltsfoot extract according to another aspect of the present invention comprises the steps of (a) extracting the coltsfoot reflux in water (H ₂ O) and then lyophilized to obtain a powder; (b) obtaining 15 fractions sequentially of the lyophilized powder through ion-exchange chromatography; (c) separating the ninth fractions of the fractions to obtain ten small fractions sequentially through size exclusion chromatography; And (d) performing a reversed phase high performance liquid chromatography (HPCL) on the sixth small fraction of the small fraction to obtain a compound represented by Chemical Formula 1 below.

[Formula 1]

The step (a) is to extract the butterbur to reflux in water (H2O) and then lyophilized to prepare a powder (powder), can be dissolved in the powder lower alcohol. The lower alcohol may be methanol (MeOH) or ethanol (EtOH), preferably ethanol (EtOH).

In the step (b), the ion exchange chromatography uses a column filled with an ion exchange resin, and may be a column filled with a nonionic adsorptive resin, and preferably, Diaion HP-20 column chromatography Can be.

In step (c), the molecular exclusion chromatography may be Sephadex column chromatography.

In the step (d), the reverse phase high performance liquid chromatography (HPCL) may be a chromatography of the YMC Pack ODS-A column high performance liquid chromatography (HPCL).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited by these examples.

Reagents and Instruments

Diaion HP-20 and Sephadex LH-20 (Amersham Pharmacia Biotech) were used for column chromatography. HPLC was performed using a Gilson HPLC system using a YMC Pack ODS-A column (250 × 20.0 mm id, 5.0 μm , YMC) and a Luna 10 μm C18 (2) 100A column (250 × 21.2 mm id, 10.0 μm , Phenomenex). Was performed. TLC analysis was performed using Silica gel 60 glass plates (Merck) and Silica gel 60 RP 18 F _254s glass plates (Merck), and then immersed in 20% (v / v) H ₂ SO ₄ reagent (Aldrich) and then heated to 110 ° C. It developed for a minute. All organic solvents used in the experiment were used after distillation. NMR experiments were performed with a JEOL 500 MHz NMR spectrometer, and chemical shifts were measured based on residual NMR solvents (acetone- d _6, CD ₃ OD) or TMS.

Sample Preparation

The butterbur ( Petitesites japonicus ) used in the present invention was lyophilized after extraction with reflux of H ₂ O, and 100 g of the resulting powder was used.

Preparation Cell culture

RAW 264.7 macrophage cell lines were obtained from the Korea Cell Line Bank (Seoul, South Korea), DMEM (in 10% FBS, penicillin-streptomycin (100 units / mL) at 37 ° C with 5% CO ₂ ) Was cultured using as a medium.

Example 1 Extraction and Component Separation

7.0 × 44.8 cm, Acetone/H₂O = 0/10 → 2/8 → 4/6 → 6/4 → 8/2 → 10/0)를 실시하여 15개의 분획물(F1 내지 F15)로 나누었다. 이 중 분획물 F9 (2.36 g)에 대하여 세파덱스(Sephadex) 컬럼 크로마토그래피(

4.5 × 56.2 cm, Ethanol/H₂O = 1/1)를 실시하여 10개의 소분획물(F9-1 내지 F9-10)로 나누었다. 소분획물 F9-6 (0.23 g)을 YMC Pack ODS-A 컬럼을 이용한 HPLC를 실시하여 화합물 1(4.2 mg)을 분리하였다. The coltsfoot was extracted with reflux in water (H ₂ O) and then lyophilized to obtain a powder. Column chromatography packed with Diaion HP-20 for 100 g of powder (

7.0 × 44.8 cm, Acetone / H ₂ O = 0/10 → 2/8 → 4/6 → 6/4 → 8/2 → 10/0), and divided into 15 fractions (F1 to F15). Sephadex column chromatography on fraction F9 (2.36 g)

4.5 × 56.2 cm, Ethanol / H ₂ O = 1/1), and divided into 10 small fractions (F9-1 to F9-10). Small fraction F9-6 (0.23 g) was subjected to HPLC using a YMC Pack ODS-A column to separate compound 1 (4.2 mg).

4.5 Х 56.2 cm, Ethanol/H₂O = 1/1)를 실시하여 12개의 소분획물(F8-1~F8-12)로 나누었다. 소분획물 F8-9 (0.21 g)를 Luna 10μm C18(2) 100A 컬럼을 이용한 HPLC를 실시하여 화합물 2(61.6 mg)을 분리하였다.On the other hand, Sepadex column chromatography on fraction F8 (3.22 g)

4.5 Х 56.2 cm, Ethanol / H ₂ O = 1/1) was divided into 12 small fractions (F8-1 ~ F8-12). Small fraction F8-9 (0.21 g) was subjected to HPLC using a Luna 10 μm C18 (2) 100A column to separate compound 2 (61.6 mg).

Compound 1 and compound 2 were obtained in the form of dark brownish powder.

Measurement Example 1 IR Spectrum Analysis

For the compounds 1 and 2 according to Example 1, IR spectra were analyzed using an Agilent Cary 630 FTIR spectrometer (Agilent Technologies, Santa Clara, Calif., USA).

Measurement Example 2 UV Spectral Spectroscopic Analysis

Ultraviolet spectral spectroscopic analysis was performed on Compound 1 and Compound 2 according to Example 1 using an Optizen pop instrument (Mecasys, Daejeon, Korea).

Measurement Example 3 Optical Activity Measurement

Using the JASCO P-2000 polarimeter for the compound 1 and compound 2 according to Example 1, the emission spectrum D line (λ = 589.3 nm) of sodium at 23 ° C. in a 10 cm (1 dm) microcell. Optical rotation ([α] _D ²³ ) was measured and the solvent was measured in methanol (MeOH), 1.0 g / 100 ml concentration ( c ).

Measurement Example 4 Structure Analysis of Compound 1

Mass spectrometry

The high resolution mass spectrometry (HRESIMS) was used to define the molecular formula of Compound 1 of the Example and the HRESIMS spectra was measured using a Q-TOF micro mass spectrometer (Waters, Milford, MA, USA).

Mass spectrometry (negative mode, m / z = 325.0714 [M ⁻ H] ⁻ ; calculated for C ₁₈ H ₁₃ O ₆ , 325.0712), the molecular formula of Compound 1 may be defined as C ₁₈ H ₁₄ O ₆ .

NMR analysis

NMR analysis of the compound 1 obtained in the Example (JEOL 500 MHz) The following chemical shift values were obtained.

Dark brownish powder; ¹ H-NMR (500 MHz, acetone- d ₆ ) δ 3.62 / 3.86 (2H, d, J = 23.0 Hz, H-7), 4.53 (1H, s, H-7 ′), 4.89 / 4.97 (2H, d, J = 17.0, H-9), 6.45 (1H, dd, J = 8.0, 2.0 Hz, H-6 '), 6.59 (1H, d, J = 2.0 Hz, H-2'), 6.60 (1H , s, H-3), 6.63 (1H, d, J = 8.0 Hz, H-5 '), 6.72 (1H, s, H-6); ¹³ C-NMR (125 MHz, acetone- d ₆ ) δ 29.0 (C-7), 42.3 (C-7 '), 72.4 (C-9), 115.7 (C-6), 116.1 (C-5') , 116.2 (C-2 '), 116.9 (C-3), 120.3 (C-6'), 123.0 (C-1 '), 128.2 (C-8'), 129.8 (C-1), 136.9 (C -2), 144.6 (C-4 '), 145.1 (C-4), 145.3 (C-5), 145.9 (C-3'), 160.9 (C-8), 173.9 (C-9 ')

Through mass spectrometry and NMR analysis, Compound 1, two quaternary carbons ( δ _C 160.9, 128.2) are connected to each other by a double bond, it can be confirmed that the lignan-type structure exists, Heteronuclear multiple bond correlation (HMBC) correlation of Compound 1 can be seen to exist as an aryltetralin lactone type lignan having a double bond between C-8 and C-8 ′ (see FIG. 1). .

ECD (electronic circular dichroism) spectrum

ECD spectra (using a J-2200 circular dichroism spectrophotometer, JASCO, Tokyo, Japan) were calculated by Spartan'14 (Wavefunction, Inc., Irvine, CA; 2014).

The absolute configuration of C-7 'for Compound 1 is calculated using the calculated spectra of the (7'R) and (7'S) models using the time-dependent density functional theory (TDDFT) method. It can be confirmed by comparing the ECD spectrum (FIG. 2) by the experiment of compound 1.

Conformational searches are performed in the MMFF (Molecular mechanics forcefield ) in Spartan'14 software, and the geometric optimization of the shape selected by Gaussian 09 software (Revision E.01; Gaussian, Inc., Wallingford, CT; 2009) B3LYP / 6- Perform at 31 + G (d, p) level. Theoretical ECD spectra were measured at the CAM-B3LYP / SVP level with a conductor like polarizable continuum solvent model (CPCM) in acetonitrile. The measured ECD spectrum of Compound 1 was found to have a positive Coton effect (CE) of 239 nm (ㅿ ε +2.1) and a negative Coton effect (CE) of 214 nm (ㅿ ε- 10.4) and (ㅿ ε-5.3) at 254 nm (see Figure 2).

The calculated ECD spectra of the (7'R) model correspond to the experimental results shown in Figure 2, and the absolute configuration of compound 1 is (7'R).

Based on the above data, the chemical structure of New Compound 1 is ( R ) -9- (3,4-dihydroxyphenyl) -6,7-dihydroxy-4,9-dihydronaphtho [2,3- c ] furan-1 It may be defined as (3 H ) -one and may be represented by the formula (1).

[Formula 1]

Measurement Example 5 Structure Analysis of Compound 2

Mass spectrometry

The molecular formula of Compound 2 of this Example was defined via high resolution mass spectrometry (HRESIMS) and the HRESIMS spectra was measured using a Q-TOF micro mass spectrometer (Waters, Milford, MA, USA).

Mass spectrometry (negative mode, m / z = 343.0810 [M ⁻ H] ⁻ calcd for C ₁₈ H ₁₅ O ₇ , 343.0818), the molecular formula of Compound 1 may be defined as C ₁₈ H ₁₆ O ₇ .

NMR analysis

NMR analysis of the compound 2 obtained in Example 1 (JEOL 500 MHz) The following chemical shift values were obtained.

Dark brownish powder; ¹ H-NMR (500 MHz, CD ₃ OD) δ 2.67 / 2.83 (2H, d, J = 14.5 Hz, H-7), 3.24 (1H, d, J = 3.0 Hz, H-8 ′), 3.93 / 4.17 (2H, d, J = 10.0, H-9), 4.18 (1H, d, J = 3.0 Hz, H-7 '), 6.52 (1H, dd, J = 8.0, 2.0 Hz, H-6') , 6.55 (1H, s, H-3), 6.58 (1H, d, J = 2.0 Hz, H-2 '), 6.64 (1H, s, H-6), 6.68 (1H, d, J = 8.0 Hz , H-5 '); ¹³ C-NMR (125 MHz, CD ₃ OD) δ 39.6 (C-7), 47.4 (C-7 '), 56.2 (C-8'), 78.0 (C-8), 80.1 (C-9), 116.0 (C-2 '), 116.2 (C-5'), 117.0 (C-6), 117.1 (C-3), 120.2 (C-6 '), 127.0 (C-1), 130.1 (C-2 ), 135.5 (C-1 '), 144.8 (C-4'), 145.5 (C-4), 145.7 (C-5), 146.3 (C-3 '), 181.0 (C-9')

Through mass spectrometry and NMR analysis, Compound 2 showed NMR data similar to that of Compound 1, and from the ¹³ C-NMR and molecular weights of Compounds 1 and 2, carbon C-8 and C-8 ′ of compound 1 double-bonded It can be seen that ( δ _C 160.9 and 128.2) were substituted with oxygen-bonded quaternary carbon ( δ _C 78.0) and methine carbon ( δ _C 56.2).

The position of these carbons was confirmed by the COSY spectrum of the relationship between H-7 '( δ _H 4.18) and H-8' ( δ _H 3.24), and the HMBC spectrum showed a correlation with aryltetrarin lactone type lignans. It was additionally confirmed (see FIG. 3). Considering the biogenic relationship with Compound 1, the absolute configuration at C-7 'of Compound 2 is proposed as (R) -configuration. The coupling constant of 3.0 Hz between H-7 ′ and H-8 ′ represents the relative arrangement of the cis -geometry of C-7 ′ and C-8 ′, and the NOESY (nuclear Overhauser spectroscopy) By further confirming the relationship between H-7 'and H-8' (see Figs. 4 and 5).

ECD (electronic circular dichroism) spectrum

ECD spectra (using a J-2200 circular dichroism spectrophotometer, JASCO, Tokyo, Japan) were calculated by Spartan'14 (Wavefunction, Inc., Irvine, CA; 2014).

Absolute configuration of C-8 for Compound 2 was determined by the measurement of Compound 2 and the calculated spectra of the (8R) and (8S) models using the time-dependent density functional theory (TDDFT) method. This can be confirmed by comparing the ECD spectra.

Conformational searches are performed in the molecular mechanics forcefield (MMFF) in Spartan'14 software, and the geometric optimization of the shape selected by Gaussian 09 software is performed at the B3LYP / 6-31 + G (d, p) level. Theoretical ECD spectra were measured at the CAM-B3LYP / SVP level with a conductor like polarizable continuum solvent model (CPCM) in acetonitrile. The measured ECD spectrum of Compound 2 shows that the positive Cotonton effect (CE) is (ㅿ ε +4.0) at 203 nm, (ㅿ ε +1.9) at 229 nm, and the negative nose pain effect ( CE) has (ㅿ ε -5.4) at 210 nm and (ㅿ ε -4.4) at 222 nm.

The calculated ECD spectrum of the (8S) model corresponds to the experimental results shown in FIG. 6.

VCD (vibrational circular dichroism) spectrum

Theoretical VCD spectra are calculated on a DFT [B3LYP / 6-31 + G (d, p)] basis set level in Gaussian 09 software (Revision E.01; Gaussian, Inc., Wallingford, CT; 2009). .

IR according to the measured value for compound 2, VCD spectrum (using a ChiralIR-2X ™ FT-VCD spectrometer, BioTools, Jupiter, FL, USA) (see FIG. 7) and theoretical spectrum according to the measured value The arrangement is proposed as (8S).

Based on the above data, the chemical formula of the new compound 2 is (9 R , 3a S , 9a R ) -9- (3,4-dihydroxyphenyl) -6,7,3a-trihydroxy-4,9,3a, 9a-tetrahydronaphtho [2,3- c ] furan-1 ( 3H ) -one and may be represented by the following Chemical Formula 2.

 [Formula 2]

In summary, if the above is summarized,

Compound 1 can be summarized as follows.

Compound 1: dark brownish powder; [a] _D ²³ : −11.5 ° ( c 0.1, MeOH); mp: 240 ° C .; UV (MeOH) λ _max (log ε ) 204 nm (3.85), 262 nm (3.57); CD (CH ₃ CN) λ _max (ㅿ ε ) 214 nm (-10.4), 239 nm (2.1), 254 nm (5.3); IR (ATR) ν _max 3333, 2915, 2847, 1718, 1524, 1240 cm ⁻¹ ; ¹ H and ¹³ C NMR data, see Table 1; Negative mode (HRESIMS) m / z = 325.0714 [M−H] ⁻ (calcd for C ₁₈ H ₁₃ O ₆ , 325.0712).

Compound 2 can be summarized as follows.

Compound 2: dark brownish powder; [α] _D ²³ : -20.6 ° ( c 0.1, MeOH); mp: 160 ° C .; UV (MeOH) λ _max (log ε ) 206 nm (4.34), 287 nm (3.48); CD (CH ₃ CN) λ _max (ㅿ ε ) 203 nm (4.0), 210 nm (-5.4), 222 nm (-4.4), and 229 nm (1.9); VCD ( c 0.5 M, DMSO- d ₆ ) (FIG. 3); IR (ATR) ν _max 3305, 1768, 1606, 1514 cm ⁻¹ ; ¹ H and ¹³ C NMR data, see Table 1; HRESIMS (negative mode) m / z = 343.0810 [M-H] ^- (calcd for C ₁₈ H ₁₅ O ₇ , 343.0818).

Position Compound 1 (in acetone- d ₆ ) Compound 2 (in CD ₃ OD) δ _C δ _H Multi ( J in Hz) δ _C δ _H Multi ( J in Hz) One 129.8 - 127.0 - 2 136.9 - 130.1 - 3 116.9 6.60 s 117.1 6.55 s 4 145.1 - 145.5 - 5 145.3 - 145.7 - 6 115.7 6.72 s 117.0 6.64 s 7 29.0 3.86 d (23.0) 39.6 2.83 d (14.5) 3.62 overlapped 2.67 d (14.5) 8 160.9 - 78.0 - 9 72.4 4.97 d (17.0) 80.1 4.17 d (10.0) 4.89 d (17.5) 3.93 d (9.5) One' 123.0 - 135.5 - 2' 116.2 6.59 d (2.0) 116.0 6.58 d (2.0) 3 ' 145.9 - 146.3 - 4' 144.6 - 144.8 - 5 ' 116.1 6.63 d (8.0) 116.2 6.68 d (8.0) 6 ' 120.3 6.45 dd (8.0, 2.0) 120.2 6.52 dd (8.0, 2.0) 7 ' 42.3 4.53 s 47.2 4.18 d (3.0) 8' 128.2 - 56.2 3.24 d (3.0) 9 ' 173.9 - 181.0 -

Experimental Example 1. NO Formation of Compound 1 and mRNA Expression Inhibition of iNOS Protein

RAW 264.7 macrophage lines were dispensed into 24 well plates at a cell density of 1 × 10 ⁵ cells / well. Cells were pretreated for 1 hour at various concentrations (6.25 μM, 12.5 μM, 25 μM, 50 μM) of the extract (Compound 1), and 24 hours with and without LPS (Lipopolysaccharide) (1 μg / ml) Activated during

To determine the amount of nitrite produced in the culture supernatant of RAW 264.7 cells activated by LPS, 100 μl of the culture supernatant was collected, and then mixed with grease reagent for 10 minutes. Absorbance was measured at 540 nm by a microplate reader to investigate the effect of inhibiting nitrogen monoxide (NO) production. A positive control was L-N6- (1-Iminoethyl) lysine dihydrochloride (L-NIL) (Sigma Aldrich, St. Louis, MO, USA), known as an inducible nitric oxide synthase (iNOS) inhibitor (NIL in Figure 7A). This was used.

8A (x-axis represents the concentration (μM) of Compound 1 and L-NIL added to the LPS-treated sample (cell culture medium), y-axis represents the concentration of nitrogen monoxide (μM) remaining in each sample) Referring to the present invention, Compound 1 was treated by concentration (6.25 μM, 12.5 μM, 25 μM, 50 μM), and as a result, it can be seen that Compound 1 inhibits NO production in a concentration-dependent manner.

FIG. 8B (x-axis represents the concentration (μM) of Compound 1 added to the LPS-treated sample (cell culture medium), and y-axis represents the relative quantitative ratio of iNOS mRNA expression to β-actin). For reference, mRNA expression of iNOS was investigated by RT-PCR to investigate the correlation between the inhibition of nitrite production by iNOS and iNOS expression. The mRNA of iNOS was markedly increased by LPS, and the expression of iNOS protein was inhibited at concentrations of 25 μM and 50 μM.

Experimental Example 2. PGE of Compound 1 ₂  Formation and Inhibition of mRNA Expression of COX-2 Proteins

10⁵ cells/well의 세포밀도로 분주하였다. 추출물(화합물 1)의 다양한 농도(6.25μM, 12.5μM, 25μM, 50μM)로 1시간 동안 세포를 전처리 하고, LPS(1 μg/ml)가 존재하는 경우와 존재하지 않는 경우로 24 시간 동안 활성화하였다.RAW 264.7 macrophage lines into 24 well plates 1

The cell density was 10 ⁵ cells / well. Cells were pretreated for 1 hour with various concentrations (6.25 μM, 12.5 μM, 25 μM, 50 μM) of the extract (Compound 1) and activated for 24 hours with and without LPS (1 μg / ml). .

To measure the production of PGE ₂ in RAW 264.7 cells activated by LPS, it was investigated using an ELISA (Enzyme Linked Immunosorbent Assay, R & D Systems, MN, USA) kit. As a positive control, N- [2- (Cyclohexyloxy) -4-nitrophenyl] methanesulfonamide (NS-398) (Sigma Aldrich, St. Louis, Mo., USA), known as a selective COX-2 (cyclooxygenase) inhibitor, was used.

9A (x-axis represents the concentration (μM) of Compound 1 and NS398 added to the LPS-treated samples (cell culture medium), y-axis represents the concentration of PGE ₂ (pg / ml) remaining in each sample) Referring to the present invention, Compound 1 was treated by concentration (6.25 μM, 12.5 μM, 25 μM, 50 μM), and as a result, it can be seen that Compound 1 inhibits the formation of PGE _{2 in} a concentration-dependent manner.

9B (x-axis represents the concentration (μM) of Compound 1 added to the LPS-treated sample (cell culture medium), y-axis represents the relative quantitative ratio of β-actin of the amount of COX-2 mRNA expression) ), The mRNA expression of COX-2 was investigated by RT-PCR to determine the correlation between the inhibition of PGE ₂ production and COX-2 expression by Compound 1. The mRNA of COX-2 was significantly increased by LPS, and the expression of COX-2 protein was inhibited at concentrations of 12.5μM, 25μM and 50μM.

Experimental Example 3. IC ₅₀ evaluation

The compounds 1 and was the amount of NO and PEG ₂ of Compound (2) determine the concentration (IC ₅₀₎ (μM) required to reduce by 50%, together the measurement results and L-NIL and the IC ₅₀ value of NS-398 Table Summarized in 2.

Compound IC ₅₀  (μM) NO PGE ₂ Compound 1 14.71 ± 1.41 16.75 ± 3.24 Compound 2 > 50 > 50 L-NIL 1.62 ± 0.08 - NS-398 - 3.27 ± 0.15

Experimental Example 4. Cell viability

Cytotoxicity was evaluated using MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-dipheyltetrazolium bromide) assay. MMT reagents are available from Molecular Probes Inc. (Eugene, OR, USA). RAW 264.7 macrophages were seeded at a cell density of 1 × 10 ⁵ cells / well for each 50 μL of DMEM medium in a 96 well plate. Cells were pretreated for 1 hour at various concentrations of Compound 1 and Compound 2 (0-100 μM) and then stimulated with LPS (1 μg / ml). After 24 hours, 20 μL of MTT solution (5 mg / ml) was added to each well and incubated for an additional 4 hours. Supernatants were removed from each well, and the formazan remaining on the plate was dissolved in 50 μL of DMSO. Absorbance in each well was measured at 540 mm by a microplate spectrophotometer (SpectraMax; Molecular Devices, Sunnyvale, Calif.) And the results are shown in FIG. 10.

Referring to FIG. 10, in the case of Compound 1, it was confirmed that cell viability was maintained high. Through this, in the case of Compound 1, it was confirmed that NO and PEG2 were inhibited and cell viability was maintained high.

On the other hand, the embodiments of the present invention disclosed in the specification and drawings are merely presented specific examples for clarity and are not intended to limit the scope of the present invention. It is apparent to those skilled in the art that other modifications based on the technical idea of the present invention can be carried out in addition to the embodiments disclosed herein.

Claims (11)

A compound represented by the following formula (1).
[Formula 1]

The method of claim 1,
The compound is extracted from butterbur.
The method of claim 1,
The compound is characterized in that it has an anti-inflammatory activity.
The method of claim 1,
The compound is characterized in that the inhibition of the expression of iNOS.
The method of claim 1,
The compound is characterized in that to inhibit the expression of COX-2 (cyclooxygenase-2).
delete delete (a) extracting coltsfoot under reflux with water (H ₂ O) and freeze drying to obtain a powder;
(b) obtaining 15 fractions sequentially of the lyophilized powder through ion-exchange chromatography;
(c) separating the ninth fractions of the fractions to obtain ten small fractions sequentially through size exclusion chromatography; And
(d) performing a reversed phase high performance liquid chromatography (HPCL) on the sixth small fraction of the small fraction to obtain a compound represented by the following Chemical Formula 1;
A method for obtaining an anti-inflammatory active compound represented by the following Chemical Formula 1 and isolated from coltsfoot.
[Formula 1]

The method of claim 8,
In step (b),
The ion exchange chromatography is a method of obtaining an anti-inflammatory active compound isolated from butterburs, characterized in that Diaion HP-20 column chromatography.
The method of claim 8,
In the step (c),
The molecule exclusion chromatography is a method for obtaining an anti-inflammatory active compound isolated from coltsfoot, characterized in that the Sephadex column chromatography.
The method of claim 8,
In step (d),
The reversed phase high performance liquid chromatography (HPCL) is chromatograph YMC Pack ODS-A column high performance liquid chromatography (HPCL) characterized in that the method for obtaining an anti-inflammatory active compound isolated from butterbur.

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