KR20190120967A - Composition for anti-inflammatory treatment comprising sesquiterpene-based metabolites - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition containing sesquiterpene-based metabolites. More specifically, the present invention relates to the anti-inflammatory composition containing sesquiterpene-based metabolites which can be separated from an ethyl acetate fraction of a Nardostachys chinensis methanol extract. A composition of the present invention exhibits an excellent anti-inflammatory effect, thereby being able to be usefully used as a pharmaceutical composition, a cosmetic composition, and a food composition.

Description

Composition for anti-inflammatory treatment comprising sesquiterpene-based metabolites {Composition for anti-inflammatory treatment comprising sesquiterpene-based metabolites}

The present invention relates to an anti-inflammatory composition containing a sesquiterpene-based metabolite, and more particularly, to an anti-inflammatory pharmaceutical composition containing a sesquiterpene-based metabolite that can be separated from the ethyl acetate fraction of the methanol extract of saccharin. , Cosmetics compositions and food compositions.

Inflammatory disease refers to the pathological condition of an abscess formed by the invasion of bacteria. Representative inflammatory diseases include encephalitis, osteoarthritis, rheumatoid arthritis, gout, ankylosing spondylitis, tendinitis, tendonitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, asthma, atopy, Crohn's disease, ulcerative colitis. Inflammation is a manifestation of normal and protective in vivo defense mechanisms that are localized to tissue damage caused by physical trauma, harmful chemicals, microbial infections or irritants in metabolites in vivo.

Various biochemical phenomena are involved in the development of inflammation in the living body. Macrophage is a multi-functional cell that plays an important role in the inflammatory response by generating various cytokines and NOx by chemical stimulation. In particular, inducible nitric oxide synthase (iNOS) expressed by cytokine stimulation such as interferons-gamma (IFN-γ) in macrophages produces large amounts of nitric oxide (NO) for a long time. This oxidative stress is known to promote NF-κB activity, a transcription factor of the inflammatory response inhibited by IκB. Activated NF-κB is known to promote the expression of genes that induce inflammatory responses, such as iNOS, COX-2, and various cytokines such as IL-1β or TNF-α. Inflammatory responses are known to be inhibited (Baeuerle et al., Annu. Rev. Immunol., 12: 141-179, 1994).

NO, one of the inflammatory substances, is produced in endothelial cells or macrophages in a normal state and exhibits various physiological activities in addition to cell killing and bactericidal action. In particular, it plays a role in maintaining homeostasis of blood pressure in the relaxation of vascular endothelial cells It is known to do. Stimulation of lipopolysaccharide (LPS), pro-inflammatory factors, and irradiation, in particular, induces the expression of iNOS, an inducible nitric oxide synthase, continuously producing large amounts of NO for 4-6 hours. NO causes such inflammatory diseases.

The most common inflammatory disease prevention or treatment for treating such inflammatory diseases is classified into steroidal and nonsteroidal inflammatory disease prevention or treatment, and most of these synthetic inflammatory disease prevention or treatment has various side effects in addition to the main action. In many cases, excellent effects and development of drugs for preventing or treating inflammatory diseases with fewer side effects are urgently needed. In particular, in terms of efficacy and side effects, natural product formulations, which have a long history of clinical experience and excellent evaluation in terms of safety, are considered to be good candidates for the development of preventive and therapeutic agents for inflammatory diseases.

Nardostachys jatamansi has traditionally been used for stomach pain, gastrointestinal spasms, chest glands, neuropathy, vomiting, headaches, and the like, and has been used as tonics, irritants and anticonvulsants in several Asian countries (Bagchi, A., Et al., Planta Med., 57, 9697 (1991)).

The roots of the nectar are known to contain various sesquiterpenes, lignans, and neolignans such as Jatamansic acid and Jatamansone (Chatterji, A, et al., National Institute of Science Communication, vol. 5, pp 99-100 (1997); Arora, RB., Indian Council of Medical Research, vol. 51 (1965)).

The inventors of the present invention, while studying a novel substance with excellent anti-inflammatory activity from a metabolite derived from natural products, isolates and purifies sesquiterpene-based metabolites having excellent anti-inflammatory activity from sweet persimmon and confirms the anti-inflammatory effect thereof. Completed.

An object of the present invention to provide a pharmaceutical composition for the treatment or prevention of inflammatory diseases comprising sesquiterpene-based metabolites.

In addition, the present invention is to provide a food composition and cosmetic composition comprising the metabolite.

Another object of the present invention is to provide a novel sesquiterpene-based metabolite having an anti-inflammatory effect isolated from the extract of Persimmon.

The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, which is a novel sesquiterpene family of metabolites.

[Formula I]

Wherein R is an α-OH, β-OH or α-methoxy-α-trifluoromethylphenylacetate group.

In the above formula (I) of the present invention, R may be β-OH, and is referred to as compound 1 or kanshone J.

In the above formula (I) of the present invention, R may be an α-methoxy-α-trifluoromethylphenylacetate group (MTPA), and (S) MTPA is referred to as Compound 1a, and (R) MTPA is referred to as Compound 1b.

In the above formula (I) of the present invention, R may be α-OH, and is referred to as compound 2 or kanshone K.

The present invention also provides the following sesquiterpene family metabolites or pharmaceutically acceptable salts thereof:

Compound 3: desoxo-narchinol A

Compound 4: narchinol B

Compound 5: Bullatantriol

Compound 6: 1β, 4β, 7α-trihydroxyendesman (1β, 4β, 7α-trihydroxyeudesmane)

Compound 7: teclalatriol

Compound 8: 4β-hydroxy-8βmethoxy-10-methylene-2,9-dioxatricyclo [4.3.1.03,7] decane (4β-hydroxy-8β-methoxy-10-methylene-2,9- dioxatricyclo [4.3.1.03,7] decane)

Compound 9: Yatamanin A

The present invention relates to compounds 1 to 9, specifically Kanshone J, Kanshone K, Desoxo-narchinol A, Nachinol B, Bulatantriol (bullatantriol), 1β, 4β, 7α-trihydroxyendesman, 1β, 4β, 7α-trihydroxyeudesmane, teclalatriol, 4β-hydroxy-8β-methoxy-10-methylene-2,9 Dioxatricyclo [4.3.1.03,7] decane (4β-hydroxy-8β-methoxy-10-methylene-2,9-dioxatricyclo [4.3.1.03,7] decane), yatamanin A And it provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising at least one sesquiterpene-based metabolite selected from the group consisting of pharmaceutically acceptable salts thereof as an active ingredient.

Here, Kanshon J may comprise the MTPA ester of Kanshon J.

Preferably, the composition may include desoxo-narchinol A, nachinol B, or a pharmaceutically acceptable salt thereof as an active ingredient.

In this case, the sesquiterpene-based metabolites may be separated from the ethyl acetate fraction of the extract of Methanol.

The compositions of the invention may also show anti-inflammatory effects by inhibiting the production of NO (Nitric oxide), or PGE ₂ (prostaglandin E _2).

In addition, the composition of the present invention can inhibit the expression of iNOS (inducible NO synthase) or COX-2 (Cyclooxygenase-2) protein.

In addition, the composition of the present invention can inhibit the expression of IL-1β (Interleukin 1β), IL-6 (Interleukin 6) or TNF-α (Tumor Necrosis Factor α).

At this time, the pharmaceutical composition is a powder, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, external preparations, external preparations, external preparations, ointments, creams, gels, warnings It may be formulated in the form of a dressing, a patch, a spray or an injection.

The present invention also provides a cosmetic composition comprising the sesquiterpene-based metabolite as an active ingredient.

The cosmetic composition can be formulated in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations or sprays. .

The present invention also provides a food composition containing the sesquiterpene-based metabolite as an active ingredient.

At this time, the food composition may be formulated in the form of tablets, granules, pills, capsules, solutions or powders.

Inflammatory diseases of the present invention may also be encephalitis, osteoarthritis, rheumatoid arthritis, gout, ankylosing spondylitis, tendonitis, tendonitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, asthma, atopy, Crohn's disease or ulcerative colitis , Preferably encephalitis.

The composition comprising a sesquiterpene-based metabolite of the present invention as an active ingredient is excellent in inhibiting the production of NO or PGE ₂ , iNOS or COX-2 protein expression, and IL-1β, IL-6 or TNF-α expression. Has an inflammatory effect.

In particular, it exhibits an excellent anti-inflammatory effect in microglia, and has an excellent effect on the improvement, treatment and prevention of encephalitis.

Therefore, by using the anti-inflammatory composition comprising the sesquiterpene-based metabolite of the present invention as an active ingredient, it is possible to develop eco-friendly and human-friendly medicines, cosmetics and foods.

FIG. 1 shows a method for separating sesquiterpene-based metabolites from ethyl acetate fractions of methanol extract of Gamsong-hyang.
2 is NMR data of novel compounds 1 and 2. FIG.
FIG. 3 confirms the steric structure of Compound 1 by calculating the difference between Compounds 1a and 1b generated by NOESY spectral data analysis and Mosher's method.
Figure 4 shows the inhibitory effect of NO in microglia cells with IC ₅₀ values for 9 sesquiterpene family metabolites.
Figure 5 shows the effect of inhibiting NO production in primary microglia for compounds 3 (a) and 4 (b).
Figure 6 shows the effect of inhibiting the production of inflammatory cytokine PGE ₂ in microglia (a and b) and primary microglia (c and d) for the compounds 3 and 4.
Figure 7 shows the effect of inhibiting the expression of iNOS and COX-2 protein of compounds 3 and 4.
FIG. 8 shows the inhibitory effect of the production of inflammatory cytokines IL-1β (a and d), IL-6 (b and e) and TNF-α (c and f) in microglial cells and primary microglial cells. It is shown.
Figure 9 shows the inhibitory effect of the production of inflammatory cytokines IL-1β (a and d), IL-6 (b and e) and TNF-α (c and f) in microglia and primary microglia for compound 4 It is shown.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are provided only for the purpose of illustration to help the understanding of the present invention, the scope of the present invention is not limited by the following examples.

Example 1

Preparation of Persimmon Ethyl Acetate Fraction

5 kg of persimmon perfume was added to 24 L of methanol, and ultrasonic extraction was performed for 2 hours, thereby obtaining 663 g of a persimmon extract.

The obtained methanol extract was dissolved in 8 L of water and 1 L of methanol, and partitioned in the order of hexane (18 L), chloroform (18 L), ethyl acetate (18 L) and butanol (18 L) to obtain 21.8 g of ethyl acetate fraction.

Example 2

Isolation of Nine Sesquiterpene-Based Metabolites from Persimmon Ethyl Acetate Fraction

As in the method described in Fig. 1, nine sesquiterpenes were separated from the sweetened ethyl acetate fraction obtained in Example 1.

Stage 1

The ethyl acetate fraction obtained in Example 1 was subjected to silica gel column chromatography using hexane ethyl acetate mixed solution (hexane: ethyl acetate = 3.5: 1-0: 1, v / v) as 13 extraction fractions. Got.

2 steps

The hexane ethyl acetate mixed solution (hexane: ethyl acetate = 3.5: 1-0: 1, v / v) of the third fraction [NJ (E) -S3] of the 13 fractions of step 1 was subjected to silica gel column chromatography. Nine fractions were obtained as extraction solvents.

3 steps

The 4th and 5th fractions [NJ (E) -S (45)] of the 13 fractions of the above step 1 were purified using C18 column chromatography (water: methanol = 9: 1-0: 1, v / 15 fractions were obtained using v) as the extraction solvent.

4 steps

The third fraction [NJ (E) -S (45) -3] of the fifteen fractions of step 3 was subjected to silica gel column chromatography using dichloromethane acetone mixture (dichloromethane: acetone = 5: 1-0: 6 fractions were obtained using 1, v / v) as the extraction solvent.

5 steps

Sixth fraction [NJ (E) -S (45) -6] of the fifteen fractions of the third step was prepared using hexane ethyl acetate mixed solution (hexane: ethyl acetate = 1: 1-0: 1, using silica gel column chromatography). 7 fractions were obtained using v / v) as the extraction solvent.

6 steps

The 7th fraction [NJ (E) -S (45) -7] of the 15 fractions of step 3 was subjected to silica gel column chromatography using hexane ethyl acetate mixture (hexane: ethyl acetate = 3: 1-0: 1, 15 fractions were obtained using v / v) as the extraction solvent.

7 steps

The ninth fraction [NJ (E) -S (45) -9] of the fifteen fractions in step 3 was subjected to C18 column chromatography using a mixture of water methanol (water: methanol = 6: 4-4: 6, v / Four fractions were obtained using v) as the extraction solvent.

Isolation of Compound 1

The second fraction of the four fractions of step 7 [NJ (E) -S (45) -9-2] was subjected to silica gel column chromatography using chloroform ethyl acetate mixed solution (chloroform: ethyl acetate = 1.5: 1-0: 8 fractions were obtained using 1, v / v) as the extraction solvent. The fourth fraction [NJ (E) -S (45) -92-4] was extracted using hexane acetone (hexane: acetone = 7: 3-1: 1, v / v) using silica gel column chromatography. Compound 1 (6.6 mg) was isolated as a solvent.

Isolation of Compound 2

The fifth fraction [NJ (E) -S (45) -7-5] of the fifteen fractions in step 6 was replaced with water methanol solvent gradient conditions [water (0.1% formic acid): methanol = 65: 35-40: 60, v / v] was subjected to reverse phase HPLC for 30 minutes to separate compound 2 (5.2 mg).

Isolation of Compound 3

 The 3, 4 and 5th fractions [NJ (E) -S3-A] of the 9 fractions of step 2 were subjected to C18 column chromatography using a water methanol mixture (water: methanol = 8: 2-0: 1, v / v). Nine fractions were obtained as the extraction solvent, and the fifth fraction [NJ (E) -S3A-5] was subjected to the water methanol solvent gradient condition [water (0.1% formic acid): methanol = 45:55-35: 65, v / v] was subjected to reverse phase HPLC for 18 minutes to separate compound 3 (12 mg).

Isolation of Compound 4

The fifth fraction [NJ (E) -S (45) -7-5] of the fifteen fractions in step 6 was replaced with water methanol solvent gradient conditions [water (0.1% formic acid): methanol = 65: 35-40: 60, v / v] was subjected to reverse phase HPLC for 30 minutes to separate compound 4 (8.2 mg).

Isolation of Compound 5

The third fraction [NJ (E) -S (45) -9-3] of the four fractions of step 7 was subjected to silica gel column chromatography using chloroform ethyl acetate mixed solution (chloroform: ethyl acetate = 2: 1-0: 8 fractions were obtained using 1, v / v) as the extraction solvent. The seventh fraction [NJ (E) -S (45) -93-7] was extracted using C18 column chromatography using a mixture of water methanol (water: methanol = 65:35, v / v) as an extraction solvent. 5 (2.8 mg) was isolated.

Isolation of Compound 6

Fourth and fifth fractions [NJ (E) -S6- (45)] of the seven fractions of step 5 were subjected to hexane acetone mixture (hexane: acetone = 5: 2, v / v) using silica gel column chromatography. Compound 6 (25 mg) was isolated as an extraction solvent.

Isolation of Compound 7

The third fraction [NJ (E) -S (45) -9-3] of the four fractions of step 7 was subjected to silica gel column chromatography using chloroform ethyl acetate mixed solution (chloroform: ethyl acetate = 2: 1-0: 8 fractions were obtained using 1, v / v) as the extraction solvent. The third fraction [NJ (E) -S (45) -93-3] was purified using prep silica gel TLC and extracted with hexane acetone (hexane: acetone = 1.8, v / v) as a solvent. mg) was isolated.

Isolation of Compound 8

The fifth fraction [NJ (E) -S (45) -5] of the fifteen fractions of step 3 was subjected to silica gel column chromatography using hexane ethyl acetate mixed solution (hexane: ethyl acetate = 2: 1-0: 1, v / v) was used as an extraction solvent to separate compound 8 (11.2 mg).

Isolation of Compound 9

The first fraction [NJ (E) -S (45) -3-1] of the six fractions of step 4 was subjected to water methanol solvent gradient conditions [water (0.1% formic acid): methanol = 70: 30-50: 50, v / v] was subjected to reverse phase HPLC for 30 minutes to separate compound 9 (6.9 mg).

Example 3

Confirmation of structure of isolated sesquiterpene metabolites

In the process of identifying compounds 1 and 2, they were identified by NMR data and Mass data that were not previously reported, and their structures were identified using Moser's method, and the results are shown in FIGS. 2 and 3. Compounds 1 and 2 were named kanshone J and kanshone K.

Compounds 1 to 9 were identified by the ¹ H-NMR, ¹³ C-NMR and Mass data are shown in Table 1 below.

compound
number denomination Chemical formula Measures One Kanshon J
(kanshone J)

brownish oil;

-98.3 (c 0.3, MeOH); HRESIMS m / z 273.1463 [M + Na] ⁺ , calcd. for C ₁₅ H ₂₂ O ₃ Na, 273.1467. 1a MTPA S ester

¹ H NMR (400 MHz, MeOH- d ₄ ) data, δ 7.14 (H-7), 6.82 (H-1), 6.16 (H-8), 5.59 (H-2), 3.01 (H-4) , 2.68 (H-6), 1.86 (H-3), 1.78 (H-3), 1.10 (H-12), 1.04 (H-14), 1.01 (H-15), 0.75 (H-13). 1b MTPA R ester

¹ H NMR (400 MHz, MeOH- d ₄ ) data, δ 7.17 (H-7), 6.86 (H-1), 6.19 (H-8), 5.61 (H-2), 2.84 (H-4) , 2.70 (H-6), 1.81 (H-3), 1.67 (H-3), 1.12 (H-12), 1.05 (H-14), 1.00 (H-13), 0.95 (H-15). 2 Kanshone K

yellow oil;

-76.2 (c 0.3, MeOH); HRESIMS m / z 273.1457 [M + Na] ⁺ , calcd. for C ₁₅ H ₂₂ O ₃ Na, 273.1467. 3 Desoxo-narchinol A

¹ H NMR (400 MHz, chloroform- d ) δ7.07 (H-7), 7.05 (H-1), 6.16 (H-8), 4.07 (H-6), 2.33 (H-4), 2.28 ( H-2), 1.56 (H-3), 0.97 (H-11), 0.96 (H-12); ¹³ C NMR (100 MHz, chloroform- d ) δ 187.5 (C-9), 146.8 (C-8), 140.8 (C-7), 138.5 (C-10), 130.5 (C-1), 68.4 ( C-6), 42.5 (C-5), 30.9 (C-4), 26.4 (C-2), 25.6 (C-3), 19.8 (C-11), 15.0 (C-12); HRESIMS m / z 193.1224 [M + H] ⁺ , calcd for C ₁₂ H ₁₇ O ₂ , 193.1229. 4 Narchinol B

¹ H NMR (400 MHz, methanol- d ₄ ) δ7.14 (H-7), 6.86 (H-1), 6.14 (H-8), 4.21 (H-2), 4.08 (H-6), 2.62 (H-4), 1.74 (H-3), 1.66 (H-3), 0.96 (H-12), 0.91 (H-11); ¹³ C NMR (100 MHz, methanol- d ₄ ) δ 189.6 (C-9), 149.9 (C-7), 142.7 (C-10), 137.7 (C-1), 130.5 (C-8), 68.6 (C-6), 64.1 (C-2), 43.9 (C-5), 36.1 (C-3), 26.7 (C-4), 18.3 (C-11), 15.0 (C-12); HRESIMS m / z 231.0991 [M + Na] ⁺ , calcd for C ₁₂ H ₁₆ O ₃ Na, 231.0997. 5 Bullatantriol

¹ H NMR (400 MHz, pyridine- d ₅ ) δ 3.75 (H-9), 2.85 (H-4), 2.45 (H-8), 2.41 (H-3 and 10), 1.99 (H-2) , 1.96 (H-8), 1.91 (H-7), 1.66 (H-3), 1.64 (H-1 and 14), 1.62 (H-7), 1.60 (H-10), 1.59 (H-15) ), 1.55 (H-2), 1.45 (H-12 and 13), 1.13 (H-5); ¹³ C NMR (100 MHz, pyridine- d ₅ ) δ 77.9 (C-9), 71.3 (C-6), 70.6 (C-11), 59.8 (C-5), 52.3 (C-10), 48.2 (C-1), 42.4 (C-7), 40.2 (C-2), 33.2 (C-3 and 14), 32.6 (C-15), 32.4 (C-4), 31.0 (C-12), 30.7 (C-13), 29.4 (C-8); HRESIMS m / z 279.1935 [M + Na] ⁺ , calcd for C ₁₅ H ₂₈ O ₃ Na, 279.1936. 6 1ββαα-trihydroxyeudesmane

¹ H NMR (400 MHz, pyridine- d ₅ ) δ3.72 (H-1), 2.58 (H-2), 2.29 (H-9), 2.12 (H-6), 2.01 (H-9), 2.00 (H-6), 1.97 (H-3), 1.93 (H-2), 1.80 (H-5 and 11), 1.79 (H-8), 1.68 (H-3), 1.65 (H-14), 1.37 (H-15), 1.13 (H-12 and 13); ¹³ C NMR (100 MHz, pyridine- d ₅ ) δ79.6 (C-1), 73.1 (C-7), 70.8 (C-4), 45.8 (C-5), 41.1 (C-3), 40.13 (C-11), 40.10 (C-10), 35.7 (C-9), 30.7 (C-6), 30.5 (C-15), 29.7 (C-8), 28.4 (C-2), 17.6 ( C-13), 17.5 (C-12), 12.6 (C-14); HRESIMS m / z 279.1931 [M + Na] ⁺ , calcd for C ₁₅ H ₂₈ O ₃ Na, 279.1936. 7 Teclalatriol

¹ H NMR (400 MHz, pyridine- d ₅ ) δ4.46 (H-6), 2.29 (H-2), 2.26 (H-1), 2.25 (H-5), 2.23 (H-9), 2.05 (H-11), 2.04 (H-3), 1.82 (H-2 and 3), 1.81 (H-9), 1.79 (H-8), 1.52 (H-8), 1.49 (H-15), 1.44 (H-14), 1.33 (H-7), 1.22 (H-12), 1.11 (H-13); ¹³ C NMR (100 MHz, pyridine- d ₅ ) δ 80.9 (C-4), 74.4 (C-10), 71.5 (C-6), 56.2 (C-5), 52.6 (C-7), 49.1 (C-9), 46.5 (C-1), 42.0 (C-3), 30.6 (C-11), 24.2 (C-2), 23.6 (C-15), 22.6 (C-14), 22.0 ( C-13), 21.5 (C-8), 21.4 (C-12); HRESIMS m / z 279.1936 [M + Na] ⁺ , calcd for C ₁₅ H ₂₈ O ₃ Na, 279.1936. 8 4βhydroxy-8βmethoxy-10-methylene-2,9-dioxatricyclo [4.3.1.03,7] decane (4ββdioxatricyclo [4.3.1.03,7] decane)

¹ H NMR (400 MHz, chloroform- d ) δ5.01 (H-3), 4.97 (H-1), 4.84 (H-11), 4.77 (H-11), 3.91 (H-7), 3.39 ( 1-OCH3), 3.09 (H-5), 2.28 (H-9), 2.11 (H-6), 1.82 (H-6), 1.38 (H-10); ¹³ C NMR (100 MHz, chloroform- d ) δ 149.3 (C-4), 107.2 (C-11), 97.2 (C-1), 93.8 (C-3), 82.1 (C-8), 79.2 ( C-7), 55.4 (1-OCH3), 43.2 (C-9), 42.9 (C-6), 36.6 (C-5), 19.0 (C-10); HRESIMS m / z 235.0945 [M + Na] ⁺ , calcd for C ₁₁ H ₁₆ O ₄ Na, 235.0946. 9 Yatamanin A

¹ H NMR (400 MHz, methanol- d ₄ ) δ 5.09 (H-3 and 11), 5.05 (H-11), 4.41 (H-3), 3.74 (H-7), 3.33 (H-5) , 2.98 (H-9), 2.16 (H-6), 1.97 (H-6), 1.46 (H-10); ¹³ C NMR (100 MHz, methanol- d ₄ ) δ174.9 (C-1), 144.2 (C-4), 113.6 (C-11), 86.1 (C-8), 81.4 (C-7), 71.0 (C-3), 53.6 (C-9), 41.0 (C-6), 40.7 (C-5), 21.9 (C-10); HRESIMS m / z 221.0789 [M + Na] ⁺ , calcd for C ₁₀ H ₁₄ O ₄ Na, 221.0790.

Example 4

Culture of BV2 Cells, a Mouse-derived Microglial Cell

Mouse-derived microglia BV2 cells (2 × 10 ⁵ cells / well) were treated with 10% heat-inactivated FBS, penicillin G (penicillin G, 100 IU / ml, Gibco, 15240062), and streptomycin (100 μg / ml, Gibco, 15240062) were dispensed in DMEM medium and incubated for 24 hours at 37 ° C. in a 5% carbon dioxide incubator (Sanyo, MCO175).

Example 5

Inhibition of NO Production by Sesquiterpene Metabolites

The method of Kim et al., European Journal of Pharmacology, 721, pp. 267-276 (2013) was used to identify the inhibitory effect of NO, the inflammation mediator in BV2 cells of sesquiterpene family metabolites. The experiment was carried out using.

Nine sesquiterpene-based metabolites were treated with BV2 cells at a concentration that showed no cytotoxicity and incubated for 3 hours, followed by inflammatory reaction with lipopolysaccharide (LPS), and then 5% carbon dioxide for 24 hours. The culture was carried out in an incubator.

The inhibition of NO production after the culture was confirmed, and the results are shown in FIG. 4. Compounds 1 to 9 showed NO production inhibitory effect as shown in Figure 4 within 60 μM concentration, it was confirmed that the compound 3 and 4 shows the best effect.

Example 6

Inhibition of NO Production by Compounds 3 and 4 in Primary Microglial Cells

To confirm the production of inflammatory mediators and inhibition of protein expression of iNOS and COX-2 in BV2 cells and primary microglia for compounds 3 and 4 (Kim et al., European Journal of Pharmacology, 721, pp. 267). -276 (2013)) was conducted using the method.

It was confirmed whether the compounds 3 and 4 show an anti-inflammatory effect in the primary microglia cells. Primary microglia cells were isolated from the cerebral cortex of 1-day-old rats and cultured in a 75cm ² flask for 2 weeks, and only microglia were isolated through a cell filter.

Then, incubated for 3 days in DMEM medium containing 10% heat-inactivated FBS, penicillin G and streptomycin with B27 additive (Thermo, 17504044), and then incubated for 3 days after replacing with DMEM medium without B27 additive. It was used for the experiment.

After pre-treatment of the cultured primary microglial cells 3 and 4 in a concentration of 1.3, 2.5, 5.0 and 10.0 μM for 3 hours, the inflammatory reaction with lipopolysaccharide (LPS) induced 5% for 24 hours The culture was carried out in a carbon dioxide incubator. After incubation, NO production inhibition was confirmed by a grease reaction, and the results are shown in FIG. 5.

As shown in Figures 5A and 5B, it was confirmed that the production of NO is significantly inhibited as the concentration of compounds 3 and 4 increases.

Example 7

PGE of compounds 3 and 4 ₂  Confirmation of generation suppression

In order to confirm the inhibitory effect of PGE2 production according to the content of compounds 3 and 4, the experimental method performed using an enzyme-linked immunosorbent assay (ELISA) kit (R & D System, KGE004B) is as follows.

After incubating the BV2 cells and the primary microglia for 12 hours at a concentration of 3 X 10 ⁶ cells / ml in a 60 mm dish, the compounds 3 and 4 were treated by concentration (1.3 to 10.0 μM) and pretreated for 3 hours. Then, lipopolysaccharide was treated with 1 and 0.5 μg / ml respectively and induced an inflammatory reaction in a 5% carbon dioxide incubator for 24 hours.

After incubation, the culture solution was collected, 150 μL was placed in a 96 well plate of an ELISA Kit, and then 50 μL of the primary antibody solution was added and reacted at room temperature for 1 hour at 500 ± 50 rpm. After the reaction was stopped, 50 μL of a PGE ₂ conjugated solution was added to the well plate, and reacted at room temperature for 2 hours at 500 ± 50 rpm. After the reaction was stopped, 400 μL of the washing solution was added to the well plate and washed, and this was repeated three times.

Subsequently, 200 μL of the substrate reaction solution was added to react for 30 minutes in the shaded space, and then 100 μL of the reaction stop solution was added to stop the reaction, and the absorbance was measured by measuring the absorbance at 450 nm using a microplate reader. Converted to PGE ₂ concentration, the results are shown in FIG.

As shown in FIG. 6, as the concentration of compounds 3 and 4 increases, the production of PGE ₂ by lipopolysaccharide in BV2 cells (FIGS. 6A and 6B) and primary microglia (FIGS. 6C and 6D) It was confirmed that it is suppressed dependently.

Example 8

Inhibition of iNOS and COX-2 Protein Expression by Compounds 3 and 4

In order to confirm the iNOS and COX-2 protein expression inhibitory effect according to the content of the compounds 3 and 4, the experimental method performed the Western blot analysis is as follows.

First, BV2 cells and primary microglial cells were incubated for 12 hours at a concentration of 3 X 10 ⁶ cells / ml in a 60 mm dish, followed by pretreatment for incubation for 3 hours after treatment of compounds 3 and 4 by concentration, followed by lipopoly The saccharides were treated with 1 and 0.5 μg / ml respectively and induced an inflammatory response in a 5% carbon dioxide incubator for 24 hours.

After incubation, RIPA (89900, Thermo) buffer was added to the BV2 cells and the primary microglia except the culture medium, and centrifuged at 4 ° C. and 14,000 × g for 25 minutes to separate the supernatant. Protein quantification in the isolated supernatant was performed using a BSA protein experiment kit (Pierce Biotechnology).

Specifically, the supernatant was electrophoresed for 2 hours using 7.5% SDS-polyacrylamide gel, and then transferred from polyacrylamide gel using a nitrocellulose membrane (NC membrane). . The transferred nitrocellulose membrane was quenched for 1 hour in a fresh blocking buffer containing 0.1% Tween 20 in Tris-buffered saline containing 5% nonfat milk. After stopping the reaction, the membrane was washed three times every 10 minutes with PBST (PBS, 0.1% Tween 20), and then iNOS (SC-650, Santacruz biotechnology, USA), COX-2 (SC-1745, Santacruz biotechnology) , USA) antibody (Ab) was diluted 1: 1000 and the reaction was performed for 3 hours. After the reaction, the plate was washed three times with PBST once every 10 minutes, and the secondary antibody (Anti-rabbit IgG, AP132, Millipore, USA) was diluted 1: 1000 and reacted for 1 hour.

After the reaction, washed three times with PBST once every 10 minutes, and then mixed ECL (Amersham Pharmacia Biotech) solution 1: 1 well, poured onto a nitrocellulose membrane (NC membrane) to emit light, X-ray in the dark room It developed after photosensitive to a film. In the same manner, the content of Actin (Actin) was measured using an antibody to Actin (SC-1616, Santacruz biotechnology, USA), and the results are shown in FIG. 7.

As shown in Figure 7, as the concentration of compounds 3 and 4 was confirmed that the protein expression of iNOS and COX-2 known as inflammatory mediators in BV2 cells and primary microglia cells is significantly inhibited.

Example 9

Inhibition of cytokine production of compounds 3 and 4

Enzyme-linked immunosorbent assay (ELISA) kits (R & D System, MLB00C, M6000B, MTA00B, RLB00, R6000B, RTA00) were used to confirm the inhibitory effect of the production of inflammatory cytokines according to the contents of compounds 3 and 4. The experimental method performed using the following is as follows.

After incubating BV2 cells and primary microglia cells at a concentration of 3 X 10 ⁶ cells / ml in a 60 mm dish for 12 hours, compounds 3 and 4 were treated by concentrations (1.3 to 10.0 μM) and pretreated for 3 hours. Then, lipopolysaccharide was treated with 1 and 0.5 μg / ml respectively and induced an inflammatory reaction in a 5% carbon dioxide incubator for 24 hours. After incubation, the culture solution was collected, and 50 μL of the reaction solution and 50 μL of the culture solution were added to the 96 well plate of the ELISA Kit, followed by reaction at room temperature for 1 hour. After the reaction, 400 μL of the washing solution was added to the well plate and washed, and this was repeated five times. Thereafter, 100 μL of the cytokine-containing solution was added and reacted at room temperature for 2 hours. After the reaction, 400 μL of washing solution was added and washed, which was repeated five times. Then, 100 μL of the substrate reaction solution was added to react for 30 minutes in the shaded space, and 100 μL of the reaction stop solution was added to stop the reaction, and then the absorbance was measured at 450 nm using a microplate reader. The absorbance thus obtained was converted into concentration, and shown in FIGS. 8 and 9.

As shown in FIG. 8, IL is an inflammatory cytokine increased by lipopolysaccharide in BV2 cells (FIGS. 8A, 8B and 8C) and primary microglia (FIGS. 8D, 8E and 8F) as the concentration of compound 3 increases. It was confirmed that the production of −1β (FIGS. 8A and 8D), IL-6 (FIGS. 8B and 8E) and TNF-α (FIGS. 8C and 8F) were inhibited.

In addition, as shown in FIG. 9, inflammatory cytokines increased by lipopolysaccharide in BV2 cells (FIGS. 9A, 9B and 9C) and primary microglia (FIGS. 9D, 9E and 9F) as the concentration of compound 4 increased. It was confirmed that the production of phosphorus IL-1β (FIGS. 9A and 9D), IL-6 (FIGS. 9B and 9E) and TNF-α (FIGS. 9C and 9F) were inhibited.

Claims (13)

A pharmaceutical composition for preventing or treating an inflammatory disease containing as an active ingredient at least one sesquiterpene-based metabolite selected from the group consisting of a compound of Formulas 1 to 9, and pharmaceutically acceptable salts thereof.
[Formula 1]

(R is a β-OH or α-methoxy-α-trifluoromethylphenylacetate group).
[Formula 2]

(R is α-OH).
[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

According to claim 1, wherein the composition is a pharmaceutical composition for the prevention or treatment of inflammatory diseases, characterized in that it contains a compound of formula 3, a compound of formula 4 or a pharmaceutically acceptable salt thereof as an active ingredient.
The method of claim 1, wherein the inflammatory disease is encephalitis, osteoarthritis, rheumatoid arthritis, gout, ankylosing spondylitis, tendinitis, tendonitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, asthma, atopy, Crohn's disease or ulcerative colitis Pharmaceutical composition, characterized in that.
The pharmaceutical composition of claim 3, wherein the inflammatory disease is encephalitis.
The method of claim 1, wherein the sesquiterpene-based metabolite is a pharmaceutical composition for the prevention or treatment of inflammatory diseases, characterized in that separated from the ethyl acetate fraction of methanol extract.
According to claim 1, wherein the composition is a pharmaceutical composition for the prevention or treatment of inflammatory diseases, characterized in that to inhibit the production of NO (Nitric oxide) or PGE ₂ (prostaglandin E ₂ ).
According to claim 1, wherein the composition is a pharmaceutical composition for the prevention or treatment of inflammatory diseases, characterized in that by inhibiting the expression of inducible NO synthase (iNOS) or COX-2 (Cyclooxygenase-2) protein.
According to claim 1, wherein the composition is for the prevention or treatment of inflammatory diseases, characterized in that the suppression of the expression of IL-1β (Interleukin 1β), IL-6 (Interleukin 6) or TNF-α (Tumor Necrosis Factor α) Pharmaceutical compositions.
A food composition for preventing or ameliorating an inflammatory disease containing as an active ingredient at least one sesquiterpene-based metabolite selected from the group consisting of a compound of Formulas 1 to 9, and pharmaceutically acceptable salts thereof.
[Formula 1]

(R is a β-OH or α-methoxy-α-trifluoromethylphenylacetate group).
[Formula 2]

(R is α-OH).
[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

The food composition of claim 9, wherein the food composition may be formulated in the form of tablets, granules, pills, capsules, liquids or powders.
A cosmetic composition for preventing or ameliorating an inflammatory disease containing as an active ingredient at least one sesquiterpene-based metabolite selected from the group consisting of a compound of Formulas 1 to 9, and pharmaceutically acceptable salts thereof.
[Formula 1]

(R is a β-OH or α-methoxy-α-trifluoromethylphenylacetate group).
[Formula 2]

(R is α-OH).
[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

The formulation of claim 11 wherein the cosmetic composition is in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation or spray Cosmetic composition, characterized in that can be converted.
A compound of formula (I) or a pharmaceutically acceptable salt thereof:
[Formula I]

Wherein R is an α-OH, β-OH, or α-methoxy-α-trifluoromethylphenylacetate group.

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