KR20230117059A - A composition for promoting adiponectin secretion comprising a compound from Colletotrichum gloeosporioides - Google Patents

Abstract

The present invention relates to the prevention, improvement or treatment of hypoadiponectinemia-related diseases comprising as an active ingredient a compound isolated from Colletotrichum gloeosporioides JS0417, an endogenous bacterium that lives symbiotically on a plant cultured in a rice medium. It relates to a composition for improving skin aging, skin elasticity, or skin wrinkles. Specifically, the compound of the present invention promotes differentiation of adipocytes and increases adipose tissue, so it is useful for skin care such as skin aging, skin elasticity or skin wrinkle improvement, and can increase adiponectin secretion and increase insulin sensitivity. It can be usefully used for the prevention, improvement or treatment of hypoadiponectinemia-related diseases such as metabolic diseases, metabolism-related inflammatory diseases, inflammatory diseases, autoimmune diseases, infectious diseases, allergies, kidney diseases, liver diseases, cancer, and the like.

Description

A composition for promoting adiponectin secretion comprising a compound isolated from a symbiotic microorganism of A. adiponectin {A composition for promoting adiponectin secretion comprising a compound from Colletotrichum gloeosporioides}

The present invention relates to the medicinal use of a compound isolated from Colletotrichum gloeosporioides JS0417, an endogenous bacterium symbiotic with Suaeda japonica Makino. A composition for preventing, improving or treating hypoadiponectinemia-related diseases and for preventing or improving skin wrinkles or skin aging, comprising a compound (including 5 new compounds) and a compound having an adiponectin secretion promoting activity among the above compounds as an active ingredient composition is provided.

To develop anti-diabetic and anti-obesity drugs, a phenotypic assay based on adipocyte differentiation and adipogenesis models of human bone marrow mesenchymal stem cells (hBM-MSC) simultaneously measures adiponectin and lipid accumulation. (Biochem Pharmacol 83(5):661-670, Biochem Pharmacol 77(1):125-133).

Adiponectin is an adipocytokine mainly produced in adipocytes and has attracted attention as an integrative regulator that plays an important role in signal transduction between adipose tissue and other metabolic-related tissues (J. Mol. Cell. Biol. 2016 , 8, 93-100; Acta Physiol. 2014, 210, 733-753). Therefore, adiponectin is also used as a diagnostic biomarker in various metabolic diseases, and recombinant adiponectin has shown therapeutic effects in various animal models of human metabolic diseases (Trends Pharmacol. Sci. 2009, 30, 234-239). In addition, adiponectin is known to have anti-atherogenic or anti-inflammatory effects as well as improving insulin resistance by regulating blood glucose levels and being involved in fatty acid decomposition. Therefore, adiponectin has therapeutic potential in various diseases including type 2 diabetes, atherosclerosis and cardiovascular disease (Nat. Med. 2001, 7, 941-946; Arterioscler., Thromb., Vasc. Biol. 2004 , 24, 29-33; Diabetes 2007, 56, 1198-1209).

In this regard, several drugs have been developed, but the mechanism is often not clear. For example, sulfonylurea antidiabetic drugs and peroxisome proliferator activated receptor γ (PPARγ) agonists increase adiponectin biosynthesis and lipid accumulation in the hBM-MSC-based phenotyping system (Biochem Pharmacol 83(5):661- 670, J Biol Chem 280(25):23653-23659). In previous studies, nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen and indomethacin also enhanced adiponectin production during adipogenesis in hBM-MSCs, but ibuprofen and indomethacin were used at high concentrations. It is known to directly bind to PPARγ (J Biol Chem 272(6): 3406-3410), and aspirin has been shown not to bind to PPARγ, so the exact mechanism of the adiponectin secretion stimulating effect of NSAIDs is not clear. In addition, with respect to the function of PPARγ, it has been reported that PPARδ can act as a regulator in PPARγ function by inhibiting the activity of PPARγ when PPARδ is overexpressed or its transcriptional activity is increased (Proc Natl Acad Sci USA 99(5):2613-2618), the function of PPARδ in adiponectin-associated diseases remains controversial.

Thus, a phenotype-based approach is very useful as a successful alternative to target-based approaches for known molecules in drug development (Proc Natl Acad Sci USA 111(13):5048-5053), type 2 diabetes And for complex chronic diseases in which multiple genetic and epigenomic factors act, such as obesity, it has advantages compared to target-based approaches.

The present inventors salted three cyclic peptide-polyketide novel compounds (Compounds 1 to 3) and two new ketiacandin-type polyketides (Compounds 4 to 5) along with six known compounds. It was isolated from the culture of Colletotrichum gloeosporioides JS0417, a plant-related fungus, and compounds 1 to 3 were found to be 3,5,11-trihydroxy-2,6-di through spectroscopic analysis. It was found that methyl dodecanoic acid is a cyclic depsipeptide that is linked to two amino acids through amide and ester bonds to form a 12-membered ring. Since compounds 2 and 6, which are cyclic depsipeptides, exhibit an activity that increases adiponectin secretion in adipocytes, it is confirmed that hypoadiponectinemia-related diseases including various metabolic diseases can be effectively prevented, improved, or treated using these activities, , completed the present invention.

On the other hand, trimethoxy phenyl compounds and indole gamma lactam compounds have been disclosed in Patent Documents 1 and 2, respectively, as compounds exhibiting adiponectin promoting activity, but the adiponectin secretion promoting activity of the 11 compounds isolated in the present invention has not been known. .

Republic of Korea Patent Publication No. 10-2019-0037754 Republic of Korea Patent Publication No. 10-2022-0162059

Accordingly, it is an object of the present invention to provide a composition for stimulating adiponectin secretion containing a compound derived from a symbiotic microorganism of Philippine chinensis as an active ingredient.

Another object of the present invention is to provide a composition for preventing, ameliorating or treating hypoadiponectinemia-related diseases, comprising a compound derived from a symbiotic microorganism of Philippine chinensis as an active ingredient.

Another object of the present invention is to provide a composition for improving skin aging, skin elasticity, or skin wrinkles, comprising a compound derived from a symbiotic microbial plant, as an active ingredient.

In order to solve the above problems, the present invention comprises at least one compound selected from the group consisting of compounds represented by the following formulas 2, 6 and 9, isomers thereof, salts thereof, or fractions containing the compounds as active ingredients. A composition for stimulating adiponectin secretion is provided:

[Formula 2]

,

[Formula 6]

and

[Formula 9]

.

here,

The compound represented by Formula 2 is (3S,6R,9R,10R,12S)-10-hydroxy-12-((2S,7R)-7-hydroxyoctane-2-yl)-3,6-diamond isobutyl-4,9-dimethyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione;

The compound represented by Formula 6 is (3S,6R,9R,10R,12S)-6-benzyl-3-((R) -sec -butyl)-10-hydroxy-12-((2S,7R)- 7-hydroxyoctan-2-yl) -4,9-dimethyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione;

The compound represented by Formula 9 may be 2-(1H-indol-3-yl)ethane-1-ol.

In the present invention, the isomers may include racemates, enantiomers, diastereomers, mixtures of enantiomers, or mixtures of diastereomers.

In the present invention, the compounds represented by Formulas 2, 6 and 9 may be isolated from the endogenous bacterium Colletotrichum gloeosporioides JS0417 ( Colletotrichum gloeosporioides JS0417 ) or a culture thereof coexisting with Chimyeoncho.

In the present invention, the culture of Colletotrichum gloeosporioides JS0417 may be obtained by culturing Colletotrichum gloeosporioides JS0417 in rice medium there is.

In the present invention, the compounds represented by Chemical Formulas 2, 6 and 9 may be separated by performing the following steps a) to c):

a) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium symbiotic with S.

b) obtaining a fraction by performing liquid chromatography on the obtained extract; and

c) isolating at least one compound among the compounds represented by Chemical Formulas 2, 6 and 9 by performing silica gel chromatography on the obtained fraction.

In the present invention, the extraction in step a) may be performed by adding an ethyl acetate solvent to the culture of Colletotrichum gloeosporioides JS0417.

In the present invention, the compound represented by Formula 2 may be separated by performing the following steps a-1) to f-1):

a-1) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium that lives symbiotically with Chimyeoncho;

b-1) Perform liquid chromatography on the obtained extract while gradually changing the volume ratio of normal hexane, acetone and methanol as a mobile phase in a volume ratio of 50:1:0 → 1:1:0.1 → 0:1:1 to obtain a first fraction;

c-1) The obtained first fraction is subjected to silica gel chromatography while gradually changing a mixed solvent of normal hexane, ethyl acetate and methanol as a mobile phase in a volume ratio of 30:1:0 → 1:1:0.1 to obtain a second fraction Obtaining;

d-1) obtaining a third fraction by subjecting the obtained second fraction to C18 chromatography while gradually changing a mixed solvent of water and methanol as a mobile phase in a volume ratio of 4:1 → 0:1;

e-1) The obtained third fraction was subjected to high-performance liquid chromatography using a C18 column while gradually changing a mixed solvent of water and acetonitrile as a mobile phase in a volume ratio of 60:40 → 0:100 to obtain a fourth fraction. obtaining; and

f-1) Silica gel chromatography was performed while gradually changing a mixed solvent of normal hexane and acetone as a mobile phase in a volume ratio of 7: 1 → 6: 1 for the obtained fourth fraction to obtain a compound represented by Formula 2 step.

In the present invention, the compound represented by Formula 6 may be separated by performing the following steps a-2) to e-2):

a-2) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium that lives symbiotically with S. chinensis;

b-2) Liquid chromatography was performed while gradually changing the volume ratio of normal hexane, acetone, and methanol as a mobile phase to the obtained extract in a volume ratio of 50:1:0 → 1:1:0.1 → 0:1:1 to obtain a first fraction;

c-2) The obtained first fraction was subjected to silica gel chromatography while gradually changing a mixed solvent of normal hexane, ethyl acetate and methanol as a mobile phase in a volume ratio of 30:1:0 → 1:0.1 to obtain a second fraction doing;

d-2) Silica gel chromatography while gradually changing a mixed solvent of normal hexane, acetone and methanol as a mobile phase with respect to the obtained second fraction at a volume ratio of 2:1:0.01 → 2:1:0.07 → 0:0:1 to obtain a third fraction; and

e-2) The obtained third fraction is subjected to high-performance liquid chromatography using a mixed solvent of water and acetonitrile as a mobile phase in a volume ratio of 10 to 15:85 to 90 to obtain a compound represented by Formula 6 or 7 step.

In the present invention, the compound represented by Formula 9 may be separated by performing the following steps a-7) to e-7):

a-7) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium symbiotic with S.

b-7) Liquid chromatography was performed on the obtained extract while gradually changing the volume ratio of normal hexane, acetone and methanol as a mobile phase in a volume ratio of 50:1:0 → 1:1:0.1 → 0:1:1 to obtain a first fraction;

c-7) The obtained first fraction was subjected to silica gel chromatography while gradually changing a mixed solvent of normal hexane, ethyl acetate and methanol as a mobile phase in a volume ratio of 30:1:0 → 1:1:0.1 to obtain a second fraction Obtaining;

d-7) obtaining a third fraction by subjecting the obtained second fraction to C18 chromatography while gradually changing a mixed solvent of water and methanol as a mobile phase in a volume ratio of 4:1 → 0:1; and

e-7) The obtained third fraction was subjected to high-performance liquid chromatography using a C18 column while gradually changing a mixed solvent of water and acetonitrile as a mobile phase in a volume ratio of 80:20 → 30:70, represented by Chemical Formula 9 Obtaining a compound that is.

The present invention also provides a pharmaceutical composition for preventing or treating hypoadiponectinemia-related diseases comprising the above-described various types of compositions as active ingredients.

In addition, the present invention provides a health functional food composition for preventing or improving hypoadiponectinemia-related diseases comprising the above-described various types of compositions as active ingredients.

In the present invention, the hypoadiponectinemia-related disease may be any one or more selected from the group consisting of metabolic disease, metabolism-related inflammatory disease, inflammatory disease, autoimmune disease, infectious disease, allergy, kidney disease, liver disease, and cancer. .

In the present invention, the metabolic disease may be any one or more selected from the group consisting of obesity, diabetes, nonalcoholic steatohepatitis (NASH), metabolic syndrome, hypertension, hyperlipidemia, cardiovascular disease and atherosclerosis.

In the present invention, the inflammatory disease may be any one or more selected from the group consisting of degenerative inflammation, exudative inflammation, purulent inflammation, hemorrhagic inflammation and proliferative inflammation.

The present invention also provides a cosmetic composition for improving skin aging, skin elasticity or skin wrinkles comprising the above-described various types of compositions as active ingredients.

The present invention also provides an external skin preparation for preventing or improving skin aging, skin elasticity, or skin wrinkles comprising the above-described various types of compositions as active ingredients.

The present invention also provides a health functional food for improving skin aging, skin elasticity or skin wrinkles comprising the above-described various types of compositions as active ingredients.

Since the composition containing the compound represented by Formula 2, 6 or 9, its isomer, its salt, or a fraction containing the compound as an active ingredient exhibits an effect of promoting adipocyte differentiation, skin wrinkles are improved. , It is not only useful for increasing adipose tissue for the purpose of preventing skin aging, skin beauty, or skin plastic surgery, but also for diseases that can be expected to have therapeutic effects by increasing adiponectin levels, such as inflammatory diseases, cancer, obesity, or diabetes. It can be useful for prevention or treatment.

Figure 1 shows the chemical structures of compounds (Compounds 1 to 14) isolated from the endogenous bacterium Colletotrichum gloeosporioides JS0417 ( Colletotrichum gloeosporioides JS0417) symbiotic with Chimyeoncho cultured in a rice medium.
2 to 7 are ¹ H NMR (500 MHz, CD ₃ OD) spectrum, ¹³ C NMR (125 MHz, CD ₃ OD) spectrum, COZY spectrum (500 MHz, CD ₃ OD), HSQC spectrum ( 500 MHz, CD ₃ OD), HMBC spectrum (500 MHz, CD ₃ OD) and HR-ESI-MS spectrum.
8 to 13 are ¹ H NMR (500 MHz, DMSO- d ₆ ) spectrum, ¹³ C NMR (125 MHz, DMSO- d ₆ ) spectrum, COZY spectrum (500 MHz, DMSO- d ₆ ), HSQC spectrum (500 MHz, DMSO- d ₆ ), HMBC spectrum (500 MHz, DMSO- d ₆ ) and HR-ESI-MS spectrum are shown.
14 to 19 show ¹ H NMR (500 MHz, DMSO- d ₆ ) spectrum, ¹³ C NMR (125 MHz, DMSO- d ₆ ) spectrum, COZY spectrum (500 MHz, DMSO- d ₆ ), HSQC spectrum (500 MHz, DMSO- d ₆ ), HMBC spectrum (500 MHz, DMSO- d ₆ ) and HR-ESI-MS spectrum are shown.
Figure 20 shows the MTPA ester Δ δ _S - δ _R of compounds 1 and 2.
21 shows the observed ¹ H- ¹ H COSY and major HMBC correlations of compounds 1-3.
22 to 28 are ¹ H NMR (500 MHz, CD ₃ OD) spectrum, ¹³ C NMR (125 MHz, CD ₃ OD) spectrum, COZY spectrum (500 MHz, CD ₃ OD), HSQC spectrum ( 500 MHz, CD ₃ OD), HMBC spectrum (500 MHz, CD ₃ OD), ROESY spectrum (500 MHz, CD ₃ OD) and HR-ESI-MS spectrum.
29 to 35 are ¹ H NMR (500 MHz, CD ₃ OD) spectrum, ¹³ C NMR (125 MHz, CD ₃ OD) spectrum, COZY spectrum (500 MHz, CD ₃ OD), HSQC spectrum ( 500 MHz, CD ₃ OD), HMBC spectrum (500 MHz, CD ₃ OD), ROESY spectrum (500 MHz, CD ₃ OD) and HR-ESI-MS spectrum.
Figure 36 shows the observed ^1H - ^1H COSY, major HMBC and ROESY correlations for compound 4.
37 to 42 show ¹ H NMR (500 MHz, DMSO- d ₆ ) spectrum, ¹³ C NMR (125 MHz, DMSO- d ₆ ) spectrum, COZY spectrum (500 MHz, DMSO- d ₆ ), HSQC spectrum (500 MHz, DMSO- d ₆ ), HMBC spectrum (500 MHz, DMSO- d ₆ ) and HR-ESI-MS spectrum are shown.
43 to 48 show ¹ H NMR (500 MHz, DMSO- d ₆ ) spectrum, ¹³ C NMR (125 MHz, DMSO -d ₆ ) spectrum, COZY spectrum (500 MHz, DMSO- d ₆ ), HSQC spectrum (500 MHz, DMSO- d ₆ ), HMBC spectrum (500 MHz, DMSO- d ₆ ) and HR-ESI-MS spectrum are shown.
Figure 49 confirms the relative stereochemistry of the long chain moiety of Compound 7, A in Figure 49 shows JBCA of the long chain moiety of Compound 7, and B in Figure 49 shows the main long chain moiety of Compound 7. It shows the NOESY correlation.
50 and 51 show the ¹ H NMR (500 MHz, CD ₃ OD) spectrum and ¹³ C NMR (125 MHz, CD ₃ OD) spectrum of compound 8, respectively.
52 and 53 show the ¹ H NMR (500 MHz, CD ₃ OD) spectrum and ¹³ C NMR (125 MHz, CD ₃ OD) spectrum of Compound 9, respectively.
54 shows the ¹ H NMR (500 MHz, CD ₃ OD) spectrum of Compound 10.
55 shows the ¹ H NMR (500 MHz, CD ₃ OD) spectrum of Compound 11.
Figure 56 confirms the secretion promoting activity of adiponectin by treatment with the compounds isolated in the present invention. Figure 56A shows the result of evaluating the adiponectin level by ELISA in the adipogenesis model of hBM-MSC treated with each compound. 56B shows the visualization of fat accumulation by Compounds 2 and 6 by Oil Red-O staining after induction of adipogenesis, and C of FIG. 56 analyzes the effect of promoting adiponectin secretion according to the concentration of Compounds 2 and 6.
Figure 57 confirms the anti-inflammatory and adiponectin secretion-promoting activities by treatment of the compounds isolated in the present invention. Figure 57 A shows the anti-inflammatory activity by evaluating the level of prostaglandin E2 by ELISA after treating HaCaT with compounds 1 to 14. 57B analyzes the inhibitory effect on prostaglandin E2 production according to the concentration of Compound 9, and C in FIG. 57 promotes adiponectin secretion according to the concentration in the adipogenesis model of hBM-MSC treated with Compound 9. The effect was analyzed, and FIG. 57D is a visualization of fat accumulation by Compound 9 by Oil Red O staining after induction of adipocyte differentiation.

As described above, adiponectin has therapeutic potential in various diseases including type 2 diabetes, atherosclerosis, and cardiovascular disease, and many drugs related to it have been developed, but the mechanism is often not clear. Discovery of new substances for promoting secretion is still required. Therefore, the present inventors isolated 5 new compounds and 9 known compounds isolated from the culture of Colletotrichum gloeosporioides JS0417, an endophyte symbiotic with the plant, and these compounds It exhibits adiponectin secretion promotion activity, adipocyte differentiation promotion, and anti-inflammatory activity, so it is useful for improving skin wrinkles, preventing skin aging, increasing adipose tissue for the purpose of skin beauty or skin plastic surgery, and a therapeutic effect can be expected by increasing the level of adiponectin. A solution to the above problem was sought by confirming that it could be applied to the prevention or treatment of diseases such as inflammatory diseases, cancer, obesity or diabetes.

Accordingly, a first aspect of the present invention relates to compounds represented by Formulas 1 to 5 below or isomers thereof:

[Formula 1]

,

[Formula 2]

,

[Formula 3]

[Formula 4]

[Formula 5]

.

In the present invention, the compound represented by Formula 1 is (3S,6R,10R,12S)-6-benzyl-10-hydroxy-12-((2S,7R)-7-hydroxyoctan-2-yl )-3-isobutyl-4-methyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione,

The compound represented by Formula 2 is (3S,6R,9R,10R,12S)-10-hydroxy-12-((2S,7R)-7-hydroxyoctane-2-yl)-3,6-diamond isobutyl-4,9-dimethyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione;

The compound represented by Formula 3 is (3S,6R,9R,10R,12R)-6-benzyl-10-hydroxy-12-((R)-6-hydroxyheptyl)-3-isobutyl-4, 9-dimethyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione;

The compound represented by Formula 4 is (2S,3S,4R,5R,6R)-2-(2,4-dihydroxy-6-(hydroxymethyl)phenyl)-3,5-dihydroxy-6 -(hydroxymethyl)tetrahydro-2H-pyran-4-yl (2E,6E)-4,8-dimethyldeca-2,6-dienoate;

The compound represented by Formula 5 is (2S,3S,4R,5R,6R)-2-(2,4-dihydroxy-6-methylphenyl)-3,5-dihydroxy-6-(hydroxymethyl ) tetrahydro-2H-pyran-4-yl (2E,6E)-4,8-dimethyldeca-2,6-dienoate.

In the present invention, the isomers may include racemates, enantiomers, diastereomers, mixtures of enantiomers or mixtures of diastereomers.

According to a preferred embodiment of the present invention, the compound may be isolated from Colletotrichum gloeosporioides JS0417, an endogenous bacterium symbiotic with S.

At this time, the culture may be a culture medium in which Colletotrichum gloeosporioides JS0417 is cultured or filtered by a physical method, but is not limited thereto.

A total of 11 compounds, including the above 5 compounds according to the present invention, were isolated from the extract of Colletotrichum gloeosporioides JS0417 culture, and the present invention provides a method for separating these compounds include

Therefore, the second aspect of the present invention is represented by the following formulas 1 to 11 from Colletotrichum gloeosporioides JS0417, an endogenous bacterium symbiotic with Chimyeoncho, comprising the steps of a) to c) below It relates to a method for isolating a compound to be.

a) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium symbiotic with S.

b) obtaining a fraction by performing liquid chromatography on the obtained extract;

c) isolating at least one compound among the compounds represented by Formulas 1 to 11 by performing silica gel chromatography on the obtained fraction;

[Formula 1]

,

[Formula 2]

,

[Formula 3]

[Formula 4]

[Formula 5]

,

[Formula 6]

[Formula 7]

,

[Formula 8]

,

[Formula 9]

,

[Formula 10]

,

[Formula 11]

,

In the present invention, the compound represented by Formula 1 is (3S,6R,10R,12S)-6-benzyl-10-hydroxy-12-((2S,7R)-7-hydroxyoctan-2-yl )-3-isobutyl-4-methyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione,

The compound represented by Formula 2 is (3S,6R,9R,10R,12S)-10-hydroxy-12-((2S,7R)-7-hydroxyoctane-2-yl)-3,6-diamond isobutyl-4,9-dimethyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione;

The compound represented by Formula 3 is (3S,6R,9R,10R,12R)-6-benzyl-10-hydroxy-12-((R)-6-hydroxyheptyl)-3-isobutyl-4, 9-dimethyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione;

The compound represented by Formula 4 is (2S,3S,4R,5R,6R)-2-(2,4-dihydroxy-6-(hydroxymethyl)phenyl)-3,5-dihydroxy-6 -(hydroxymethyl)tetrahydro-2H-pyran-4-yl (2E,6E)-4,8-dimethyldeca-2,6-dienoate;

The compound represented by Formula 5 is (2S,3S,4R,5R,6R)-2-(2,4-dihydroxy-6-methylphenyl)-3,5-dihydroxy-6-(hydroxymethyl ) tetrahydro-2H-pyran-4-yl (2E,6E)-4,8-dimethyldeca-2,6-dienoate,

The compound represented by Formula 6 is (3S,6R,9R,10R,12S)-6-benzyl-3-((R) -sec -butyl)-10-hydroxy-12-((2S,7R)- 7-hydroxyoctan-2-yl) -4,9-dimethyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione;

The compound represented by Formula 7 is (3S,6R,9R,10R,12S)-6-benzyl-10-hydroxy-12-((2S,7R)-7-hydroxyoctan-2-yl)-3 -isobutyl-4,9-dimethyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione;

The compound represented by Formula 8 is (E) -5- (hydroxymethyl) -6- (3-hydroxyprop-1-en-1-yl) -4-methoxy-3-methyl-2H- pyran-2-one,

The compound represented by Formula 9 is 2-(1H-indol-3-yl)ethane-1-ol,

The compound represented by Formula 10 is indole-3-acetate methyl ester,

The compound represented by Chemical Formula 11 may be 3-methoxy indole.

Colletotrichum gloeosporioides JS0417 used in the present invention may be purchased commercially or directly collected or cultivated in nature.

In the present invention, the term "culture" refers to a medium containing by-products produced by culturing Colletotrichum gloeosporioides JS0417 in a medium to ingest nutrients and metabolize, wherein the It includes cultures of all formulations that can be formed using the medium, such as dilutions or concentrates of the medium, dried products obtained by drying the medium, adjusted or purified products of the medium, or mixtures thereof.

In the present invention, the culture of Colletotrichum gloeosporioides JS0417 in step a) is any culture capable of culturing Colletotrichum gloeosporioides JS0417. A medium may also be used, but it may be preferably obtained by culturing in a rice medium.

At this time, the rice medium may be prepared by adding distilled water to rice and sterilizing it under high pressure, but is not limited thereto.

In the present invention, the extraction in step a) may be performed by adding an extraction solvent to the culture of Colletotrichum gloeosporioides JS0417, preferably from the obtained extract. of compounds can be isolated.

The term "extract" as used in the present invention refers to a liquid component obtained by immersing a desired substance in various solvents and then extracting at room temperature, low temperature or elevated temperature for a certain period of time, and removing the solvent from the liquid component. It means the result, such as the obtained solid content. In addition, in addition to the above results, it can be comprehensively interpreted as including all dilutions of the results, concentrates thereof, adjusted products, and purified products thereof.

In the present invention, the method for preparing the extract of Colletotrichum gloeosporioides JS0417 culture is conventional in the art such as immersion extraction, stationary extraction, ultrasonic extraction, filtration, hot water extraction and reflux extraction. extraction method can be used. The extraction solvent may be used without limitation as long as it is a conventional extraction solvent in the art. Non-limiting examples of the extraction solvent include water; C1 to C4 lower alcohols such as methanol, ethanol, propyl alcohol and butyl alcohol; polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; or a mixture thereof, preferably ethyl acetate.

In addition, the extraction solvent may be extracted by adding 1 to 20 times the weight of Colletotrichum gloeosporioides JS0417 or its culture.

In addition, the extraction time may be 0.5 to 48 hours, more preferably 1 to 36 hours, but is not limited thereto, and the number of extractions may be repeated 1 to 10 times.

If necessary, it may be prepared into a powder through additional steps such as filtration, concentration, and freeze-drying according to a method known in the art. The concentration may be concentration under reduced pressure, and the concentration under reduced pressure may be performed using a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

The term "fraction" used in the present invention refers to a product obtained by performing fractionation to separate a specific component or a specific component group from a mixture containing various components.

In the present invention, a fractionation method for obtaining the fraction is not particularly limited, and may be performed according to a method commonly used in the art. A non-limiting example of the fractionation method is a method of obtaining fractions from the extract by treating an extract obtained by extracting Colletotrichum gloeosporioides JS0417 or a culture thereof with a predetermined solvent. can

In the present invention, the type of solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvent include water, alcohol, acetone, acetonitrile, hexane, ethyl acetate, chloroform, dichloromethane, hexane, and the like. . These may be used alone or in combination of two or more. When alcohol is used among the fractionation solvents, C1 to C4 lower alcohols may be specifically used.

In the present invention, the silica gel chromatography in step c) may be to separate compounds through a solvent concentration gradient method.

In the present invention, the compound represented by Formula 2 can be separated by performing the following steps a-1) to f-1):

a-1) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium that coexists symbiotically with Chimyeoncho;

b-1) Perform liquid chromatography on the obtained extract while gradually changing the volume ratio of normal hexane, acetone and methanol as a mobile phase in a volume ratio of 50:1:0 → 1:1:0.1 → 0:1:1 to obtain a first fraction;

c-1) The obtained first fraction is subjected to silica gel chromatography while gradually changing a mixed solvent of normal hexane, ethyl acetate and methanol as a mobile phase in a volume ratio of 30:1:0 → 1:1:0.1 to obtain a second fraction Obtaining;

d-1) obtaining a third fraction by subjecting the obtained second fraction to C18 chromatography while gradually changing a mixed solvent of water and methanol as a mobile phase in a volume ratio of 4:1 → 0:1;

e-1) The obtained third fraction was subjected to high-performance liquid chromatography using a C18 column while gradually changing a mixed solvent of water and acetonitrile as a mobile phase in a volume ratio of 60:40 → 0:100 to obtain a fourth fraction. obtaining; and

f-1) Silica gel chromatography was performed while gradually changing a mixed solvent of normal hexane and acetone as a mobile phase in a volume ratio of 7: 1 → 6: 1 for the obtained fourth fraction to obtain a compound represented by Formula 2 step.

In this case, the first fraction may be obtained as 6 fractions (A to F), and preferably, fraction D may be used in the next step, but is not limited thereto.

In this case, the second fraction may be obtained as 6 fractions (D1 to D6), and preferably, fraction D3 may be used in the next step, but is not limited thereto.

In this case, the third fraction may be obtained as five fractions (D31 to D35), and preferably, fraction D34 may be used in the next step, but is not limited thereto.

In this case, the fourth fraction may be obtained in five fractions (D341 to D345), and preferably, fraction D343 may be used in the next step, but is not limited thereto.

In the present invention, the compound represented by Formula 6 or 7 can be separated by performing the following steps a-2) to e-2):

a-2) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium that lives symbiotically with S. chinensis;

b-2) Liquid chromatography was performed while gradually changing the volume ratio of normal hexane, acetone, and methanol as a mobile phase to the obtained extract in a volume ratio of 50:1:0 → 1:1:0.1 → 0:1:1 to obtain a first fraction;

c-2) The obtained first fraction is subjected to silica gel chromatography while gradually changing a mixed solvent of normal hexane, ethyl acetate and methanol as a mobile phase in a volume ratio of 30:1:0 → 1:1:0.1 to obtain a second fraction Obtaining;

d-2) Silica gel chromatography while gradually changing a mixed solvent of normal hexane, acetone and methanol as a mobile phase with respect to the obtained second fraction at a volume ratio of 2:1:0.01 → 2:1:0.07 → 0:0:1 to obtain a third fraction; and

e-2) The obtained third fraction is subjected to high-performance liquid chromatography using a mixed solvent of water and acetonitrile as a mobile phase in a volume ratio of 10 to 15:85 to 90 to obtain a compound represented by Formula 6 or 7 step.

In this case, the first fraction may be obtained as 6 fractions (A to F), and preferably, fraction D may be used in the next step, but is not limited thereto.

In this case, the second fraction may be obtained as 6 fractions (D1 to D6), and preferably, fraction D4 may be used in the next step, but is not limited thereto.

In this case, the third fraction may be obtained as five fractions (D41 to D45), and preferably, fraction D44 may be used in the next step, but is not limited thereto.

In the present invention, the compound represented by Formula 3 can be separated by performing the following steps a-3) to e-3):

a-3) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium that lives symbiotically with Chimyeoncho;

b-3) Liquid chromatography was performed while gradually changing the volume ratio of normal hexane, acetone, and methanol as a mobile phase to the obtained extract in a volume ratio of 50:1:0 → 1:1:0.1 → 0:1:1 to obtain a first fraction;

c-3) The obtained first fraction is subjected to silica gel chromatography while gradually changing a mixed solvent of normal hexane, ethyl acetate and methanol as a mobile phase in a volume ratio of 30:1:0 → 1:1:0.1 to obtain a second fraction Obtaining;

d-3) obtaining a third fraction by subjecting the obtained second fraction to silica gel chromatography while gradually changing a mixed solvent of chloroform and acetone as a mobile phase in a volume ratio of 10:1 → 2:1; and

e-3) The obtained third fraction was subjected to high-performance liquid chromatography using a C18 column while gradually changing a mixed solvent of water and methanol as a mobile phase in a volume ratio of 20:80 → 0:100 to obtain obtaining a compound.

In this case, the first fraction may be obtained as 6 fractions (A to F), and preferably, fraction D may be used in the next step, but is not limited thereto.

In this case, the second fraction may be obtained as 6 fractions (D1 to D6), and preferably, fraction D5 may be used in the next step, but is not limited thereto.

In this case, the third fraction may be obtained in five fractions (D51 to D55), and preferably, fraction D53 may be used in the next step, but is not limited thereto.

In the present invention, the compound represented by Formula 4 or 5 can be separated by performing the following steps a-4) to e-4):

a-4) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium symbiotic with S.

b-4) Liquid chromatography was performed on the obtained extract while gradually changing the volume ratio of normal hexane, acetone and methanol as a mobile phase in a volume ratio of 50:1:0 → 1:1:0.1 → 0:1:1 to obtain a first fraction;

c-4) The obtained first fraction was subjected to silica gel chromatography while gradually changing a mixed solvent of hexane, chloroform and acetone as a mobile phase in a volume ratio of 5:1:0.1 → 0.3:1:1 to obtain a second fraction obtaining;

d-4) The obtained second fraction was subjected to silica gel chromatography while gradually changing a mixed solvent of normal hexane, chloroform, and ethyl acetate as a mobile phase in a volume ratio of 1:1:0.5 → 1:1:5 to obtain a third obtaining fractions; and

e-4) The obtained third fraction was subjected to C18 column high performance chromatography while gradually changing the mixed solvent of water and acetonitrile as a mobile phase in a volume ratio of 90:10 → 0:100 to obtain obtaining a compound.

In this case, the first fraction may be obtained as 6 fractions (A to F), and preferably, fraction E may be used in the next step, but is not limited thereto.

In this case, the second fraction may be obtained as 8 fractions (E1 to E8), and preferably, fraction E7 may be used in the next step, but is not limited thereto.

In this case, the third fraction may be obtained as 6 fractions (E71 to E76), and preferably, fraction E73 may be used in the next step, but is not limited thereto.

In the present invention, the compound represented by Formula 1 can be separated by performing the following steps a-5) to e-5):

a-5) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium that lives symbiotically with Chimyeoncho;

b-5) Liquid chromatography was performed while gradually changing the volume ratio of normal hexane, acetone, and methanol as a mobile phase to the obtained extract in a volume ratio of 50:1:0 → 1:1:0.1 → 0:1:1 to obtain a first fraction;

c-5) The obtained first fraction was subjected to silica gel chromatography while gradually changing a mixed solvent of hexane, chloroform and acetone as a mobile phase in a volume ratio of 5:1:0.1 → 0.3:1:1 to obtain a second fraction obtaining;

d-5) The obtained second fraction was subjected to silica gel chromatography while gradually changing a mixed solvent of normal hexane, chloroform, and ethyl acetate in a volume ratio of 1:1:0.5 → 1:1:5 as a mobile phase to obtain a third obtaining fractions; and

e-5) The obtained third fraction was subjected to C18 column high performance chromatography while gradually changing the mixed solvent of water and acetonitrile as a mobile phase in a volume ratio of 95:5 → 0:100 to obtain the compound represented by Formula 1 step of obtaining.

In this case, the first fraction may be obtained as 6 fractions (A to F), and preferably, fraction E may be used in the next step, but is not limited thereto.

In this case, the second fraction may be obtained as 8 fractions (E1 to E8), and preferably, fraction E7 may be used in the next step, but is not limited thereto.

In this case, the third fraction may be obtained as 6 fractions (E71 to E76), and preferably, fraction E74 may be used in the next step, but is not limited thereto.

In the present invention, the compound represented by Formula 8 can be separated by performing the following steps a-6) to e-6):

a-6) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium that lives symbiotically with S. chinensis;

b-6) Liquid chromatography was performed on the obtained extract while gradually changing the volume ratio of normal hexane, acetone and methanol as a mobile phase in a volume ratio of 50:1:0 → 1:1:0.1 → 0:1:1 to obtain a first fraction;

c-6) The obtained first fraction was subjected to silica gel chromatography while gradually changing a mixed solvent of hexane, chloroform and acetone as a mobile phase in a volume ratio of 5:1:0.1 → 0.3:1:1 to obtain a second fraction obtaining;

d-6) The obtained second fraction was subjected to silica gel chromatography while gradually changing a mixed solvent of normal hexane, chloroform, and ethyl acetate in a volume ratio of 1:1:0.5 → 1:1:5 as a mobile phase to obtain a third obtaining fractions; and

e-6) The obtained third fraction was subjected to C18 column high performance chromatography while gradually changing the mixed solvent of water and acetonitrile as a mobile phase in a volume ratio of 90:10 → 0:100 to obtain a compound represented by Formula 8 step of obtaining.

In this case, the first fraction may be obtained as 6 fractions (A to F), and preferably, fraction E may be used in the next step, but is not limited thereto.

In this case, the second fraction may be obtained as 8 fractions (E1 to E8), and preferably, fraction E7 may be used in the next step, but is not limited thereto.

In this case, the third fraction may be obtained as 6 fractions (E71 to E76), and preferably, fraction E73 may be used in the next step, but is not limited thereto.

In the present invention, the compound represented by Formula 9 can be separated by performing the following steps a-7) to e-7):

a-7) extracting the culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium that lives symbiotically with S. chinensis;

b-7) Liquid chromatography was performed on the obtained extract while gradually changing the volume ratio of normal hexane, acetone and methanol as a mobile phase in a volume ratio of 50:1:0 → 1:1:0.1 → 0:1:1 to obtain a first fraction;

c-7) The obtained first fraction was subjected to silica gel chromatography while gradually changing a mixed solvent of normal hexane, ethyl acetate and methanol as a mobile phase in a volume ratio of 30:1:0 → 1:1:0.1 to obtain a second fraction Obtaining;

d-7) obtaining a third fraction by subjecting the obtained second fraction to C18 chromatography while gradually changing a mixed solvent of water and methanol as a mobile phase in a volume ratio of 4:1 → 0:1; and

e-7) The obtained third fraction was subjected to high-performance liquid chromatography using a C18 column while gradually changing a mixed solvent of water and acetonitrile as a mobile phase in a volume ratio of 80:20 → 30:70, represented by Chemical Formula 9 Obtaining a compound that is.

In this case, the first fraction may be obtained as 6 fractions (A to F), and preferably, fraction D may be used in the next step, but is not limited thereto.

In this case, the second fraction may be obtained as 6 fractions (D1 to D6), and preferably, fraction D3 may be used in the next step, but is not limited thereto.

In this case, the third fraction may be obtained as five fractions (D31 to D35), and preferably, fraction D32 may be used in the next step, but is not limited thereto.

In the present invention, the compound represented by Formula 10 can be separated by performing the following steps a-8) to e-8):

a-8) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium that lives symbiotically with S. chinensis;

b-8) Liquid chromatography was performed on the obtained extract while gradually changing the volume ratio of normal hexane, acetone and methanol as a mobile phase in a volume ratio of 50:1:0 → 1:1:0.1 → 0:1:1 to obtain a first fraction;

c-8) The obtained first fraction was subjected to silica gel chromatography while gradually changing a mixed solvent of hexane, chloroform and acetone as a mobile phase in a volume ratio of 5:1:0.1 → 0.3:1:1 to obtain a second fraction obtaining;

d-8) The obtained second fraction was subjected to silica gel chromatography while gradually changing a mixed solvent of normal hexane, chloroform, and ethyl acetate in a volume ratio of 5:1:0.1 → 0.3:0.3:1 as a mobile phase to obtain a third obtaining fractions;

e-8) The obtained third fraction was subjected to C18 column high performance chromatography while gradually changing the mixed solvent of water and acetonitrile as a mobile phase in a volume ratio of 70:30 → 20:80 to obtain a compound represented by Formula 10. step of obtaining.

In this case, the first fraction may be obtained as 6 fractions (A to F), and preferably, fraction E may be used in the next step, but is not limited thereto.

In this case, the second fraction may be obtained as 8 fractions (E1 to E8), and preferably, fraction E2 may be used in the next step, but is not limited thereto.

In this case, the third fraction may be obtained as 9 fractions (E21 to E29), and preferably, fraction E24 may be used in the next step, but is not limited thereto.

In the present invention, the compound represented by Formula 11 can be separated by performing the following steps a-9) to e-9):

a-9) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium that lives symbiotically with S. chinensis;

b-9) Liquid chromatography was performed on the obtained extract while gradually changing the volume ratio of normal hexane, acetone, and methanol as a mobile phase in a volume ratio of 50:1:0 → 1:1:0.1 → 0:1:1 to obtain a first fraction;

c-9) The obtained first fraction was subjected to silica gel chromatography while gradually changing a mixed solvent of hexane, chloroform and acetone as a mobile phase in a volume ratio of 5:1:0.1 → 0.3:1:1 to obtain a second fraction obtaining;

d-9) The obtained second fraction was subjected to silica gel chromatography while gradually changing a mixed solvent of normal hexane, chloroform, and ethyl acetate in a volume ratio of 1:1:0.5 → 1:1:5 as a mobile phase to obtain a third obtaining fractions;

e-9) The obtained third fraction was subjected to C18 column high performance chromatography while gradually changing the mixed solvent of water and acetonitrile as a mobile phase in a volume ratio of 70:30 → 0:100 to obtain a compound represented by Formula 11. step of obtaining.

In this case, the first fraction may be obtained as 6 fractions (A to F), and preferably, fraction E may be used in the next step, but is not limited thereto.

In this case, the second fraction may be obtained as 8 fractions (E1 to E8), and preferably, fraction E6 may be used in the next step, but is not limited thereto.

In this case, the third fraction may be obtained as 6 fractions (E61 to E66), and preferably, fraction E64 may be used in the next step, but is not limited thereto.

Since the compound according to the present invention exhibits an activity of increasing adiponectin production and secretion in cells capable of producing adiponectin, including adipocytes, it can be provided as a composition for stimulating adiponectin secretion.

Accordingly, a third aspect of the present invention is adiponectin comprising, as an active ingredient, one or more compounds selected from the group consisting of compounds represented by Formulas 2, 6 and 9, isomers thereof, salts thereof, or fractions containing the compounds. It relates to a composition for promoting secretion:

[Formula 2]

,

[Formula 6]

and

[Formula 9]

,

here,

The compound represented by Formula 2 is (3S,6R,9R,10R,12S)-10-hydroxy-12-((2S,7R)-7-hydroxyoctane-2-yl)-3,6-diamond isobutyl-4,9-dimethyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione;

The compound represented by Formula 6 is (3S,6R,9R,10R,12S)-6-benzyl-3-((R) -sec -butyl)-10-hydroxy-12-((2S,7R)- 7-hydroxyoctan-2-yl) -4,9-dimethyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione;

The compound represented by Formula 9 is 2-(1H-indol-3-yl)ethane-1-ol.

In this regard, the present invention provides a pharmaceutical composition for preventing or treating hypoadiponectinemia-related diseases, comprising the above-described composition for promoting secretion of adiponectin as an active ingredient.

In this regard, the present invention further provides a health functional food composition for preventing or alleviating hypoadiponectinemia-related diseases, comprising the above-described composition for promoting secretion of adiponectin as an active ingredient.

In the present invention, the hypoadiponectinemia-related disease may be any one or more selected from the group consisting of metabolic disease, metabolism-related inflammatory disease, inflammatory disease, autoimmune disease, infectious disease, allergy, kidney disease, liver disease, and cancer. , but is not limited thereto.

In the present invention, the metabolic disease may be selected from the group consisting of obesity, diabetes, nonalcoholic steatohepatitis (NASH), metabolic syndrome, hypertension, hyperlipidemia, cardiovascular disease and atherosclerosis, but is limited thereto It is not.

In the present invention, the inflammatory disease may be any one or more selected from the group consisting of degenerative inflammation, exudative inflammation, purulent inflammation, hemorrhagic inflammation and proliferative inflammation.

In the present invention, the metabolism-related inflammatory disease may include inflammation occurring in chronic inflammation or metabolic disease that causes the onset of metabolic disease. For example, the metabolism-related inflammatory disease may be any one or more selected from the group consisting of chronic inflammation, obese inflammation, diabetic inflammation, and vascular inflammation, but is not limited thereto.

Adiponectin attenuates the inflammatory response to multiple stimuli by regulating signaling pathways in various cell types. The anti-inflammatory properties of adiponectin are a major factor in its beneficial effects on cardiovascular and metabolic disorders, including atherosclerosis and insulin resistance (Ouchi N, Walsh K. Adiponectin as an anti-inflammatory factor. Clin Chim Acta . 2007;380 (1-2):24-30). Therefore, a compound that promotes adiponectin secretion in vivo can help treat not only metabolic diseases but also metabolic inflammatory diseases.

In the present invention, the method for isolating any one compound selected from the group consisting of compounds represented by Formulas 2, 6 and 9 and the method for obtaining a fraction containing the compound are the same as described above. omit

As used herein, the term "prevention" refers to any action that suppresses or delays the onset of a disease or condition. In the present invention, it means delaying the onset of hypoadiponectinemia-related diseases or suppressing the onset of them.

The term "improvement" used in the present invention refers to all activities that improve or beneficially change a disease or condition, and in the present invention, it means improving symptoms of hypoadiponectinemia-related diseases.

As used herein, the term "treatment" refers to any action that delays, stops, or reverses the progression of a disease or condition, and in the present invention, reduces, alleviates, eliminates, or reverses symptoms of hypoadiponectinemia-related diseases. means that

As used herein, the term "salt thereof" includes "pharmaceutically acceptable salts", "food acceptable salts" or "cosmetically acceptable salts" of the above-mentioned compound, and is relatively inexpensive to patients. It refers to any organic or inorganic addition salt of the compounds of this invention in which the beneficial effects of the compounds of this invention are not diminished by the side effects attributable to these salts at concentrations which have a toxic and harmless effect. For these salts, inorganic and organic acids can be used as free acids, hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, perchloric acid, and phosphoric acid can be used as inorganic acids, and citric acid, acetic acid, lactic acid, maleic acid, and fumarin as organic acids. Acids, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesul For example, phonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid or malonic acid may be used. In addition, these salts include alkali metal salts (sodium salt, potassium salt, etc.) and alkaline earth metal salts (calcium salt, magnesium salt, etc.) and the like. For example, acid addition salts include acetate, aspartate, benzate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, Gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, malic Eight, malonate, mesylate, methyl sulfate, naphthylate, 2-naphsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharide Lates, stearates, succinates, tartrates, tosylates, trifluoroacetates, aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, Potassium, sodium, tromethamine, zinc salts and the like may be included, among which hydrochloride or trifluoroacetate may be used.

The "pharmaceutically acceptable salts" are generally safe, non-toxic, and are not unsuitable from biological or other aspects, so that they can be used in the preparation of pharmaceutical compositions.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The pharmaceutical composition including a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration may include tablets, patients, powders, granules, capsules, troches, etc., and such solid preparations may contain at least one excipient such as starch, calcium carbonate, It may be prepared by mixing sucrose or lactose or gelatin. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used. Liquid formulations for oral administration may include suspensions, internal solutions, emulsions, or syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives are used. can be included

Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspension solutions, emulsions, freeze-dried formulations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerol, gelatin, and the like may be used.

The pharmaceutical composition of the present invention may be administered through any general route capable of reaching a target tissue or cell in a subject or sample. The administration may include systemic or local administration, and may include intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. Not limited. The dosage of the pharmaceutical composition may vary depending on the patient's age, weight, sex, dosage form, health condition and disease severity.

The term “patient” refers to any single individual in need of treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included are any subjects participating in clinical research trials who do not show any clinical signs of disease or subjects participating in epidemiological studies or subjects used as controls.

In the present invention, the term "functional health food" includes both the meanings of "functional food" and "health food".

In the present invention, the term "functional food" is the same term as food for special health use (FoSHU), and in addition to nutritional supply, medicine and medical effects processed to efficiently display bioregulatory functions Means high food.

In the present invention, the term "health food" refers to a food having an active health maintenance or promotion effect compared to general food, and health supplement food refers to a food for health supplement purposes. In some cases, the terms functional food, health food, and health supplement food are used interchangeably. The food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, and pills in order to obtain useful effects for preventing or improving hypoadiponectinemia-related diseases.

As a specific example of such a functional food, by using the compound of the present invention, its isomer, or its food-acceptable salt, a processed food with improved shelf life while modifying the characteristics of agricultural products, livestock products, or aquatic products can be prepared.

The health functional food composition of the present invention may also be prepared in the form of nutritional supplements, food additives and feeds, and is intended for consumption by humans or animals, including livestock.

Food compositions of this type can be prepared in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruits, bottled products, jams, marmalades, etc.), fish, meat and their processed foods (e.g. ham, sausages) Corned beef, etc.), breads and noodles (e.g. udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine , Vegetable protein, retort food, frozen food, various seasonings (eg, soybean paste, soy sauce, sauce, etc.), etc., can be prepared by adding the compound of the present invention, its isomer, or its food-acceptable salt.

In addition, nutritional supplements are not limited thereto, but may be prepared by adding the compound of the present invention, an isomer thereof, or a food chemically acceptable salt thereof to capsules, tablets, pills, etc.

In addition, health functional foods are not limited thereto, but, for example, the compounds of the present invention, their isomers, or their food-acceptable salts are prepared in the form of tea, juice and drinks and liquefied so that they can be consumed (healthy drinks) , can be ingested by granulating, encapsulating and powdering. In addition, in order to use the compound of the present invention, its isomer or its food chemically acceptable salt in the form of a food additive, it can be prepared and used in the form of a powder or concentrate. In addition, the compound of the present invention, its isomer or its food chemically acceptable salt can be prepared in the form of a composition by mixing it with a known active ingredient known to be effective in preventing or improving hypoadiponectinemia-related diseases.

When the food composition of the present invention is used as a health beverage composition, the health beverage composition may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

The compound of the present invention, its isomer or its food chemically acceptable salt may be contained as an active ingredient in a health functional food composition for preventing or improving hypoadiponectinemia-related diseases, and the amount is effective to obtain the above preventive or improving effect. The amount, for example, is preferably 0.01 to 100% by weight based on the total weight of the total composition, but is not particularly limited thereto. The health functional food composition of the present invention can be prepared by mixing the compound of the present invention, its isomer or its food chemically acceptable salt together with other active ingredients known to be effective in preventing or improving hypoadiponectinemia-related diseases. there is.

In addition to the above, the health functional food of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH regulators, stabilizers, and preservatives. , glycerin, alcohol or carbonation agent, and the like. In addition, the health food of the present invention may contain fruit flesh for producing natural fruit juice, fruit juice beverage, or vegetable beverage. These components may be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The term "food acceptable salt" used in the present invention may be included in the kind of "pharmaceutically acceptable salt", is generally safe, non-toxic, and is not unsuitable in terms of biological or other aspects, so it is not suitable for food It means that it can be used in the preparation of the composition.

Since the compound according to the present invention exhibits an effect of promoting differentiation of adipocytes, it can be provided as a composition for improving skin wrinkles, preventing skin aging, increasing adipose tissue for the purpose of skin beautification or skin plastic surgery.

Accordingly, a fourth aspect of the present invention relates to a cosmetic composition for improving skin aging, skin elasticity, or skin wrinkles, comprising the above-described composition for promoting adiponectin in various forms as an active ingredient.

In this regard, the present invention also provides an external skin preparation for improving skin aging, skin elasticity, or skin wrinkles, comprising the above-described composition for promoting adiponectin secretion in various forms as an active ingredient.

In this regard, the present invention also provides a health functional food composition for improving skin aging, skin elasticity, or skin wrinkles, comprising the above-described composition for promoting adiponectin secretion in various forms as an active ingredient.

In the present invention, "improving skin aging" may mean any action that prevents or inhibits skin aging. Skin aging refers to both tangible and intangible changes that appear in the skin with aging, such as thinning of the epidermis, number of cells or blood vessels in the dermis, DNA damage repair ability, cell replacement cycle, wound healing, skin barrier function , decrease in epidermal moisture retention, sweat secretion, sebum secretion, vitamin D production, physical damage defense, chemical substance elimination ability, immune response, sensory function, and thermoregulation. The skin aging includes endogenous aging over time and extrinsic aging caused by an external environment. The extrinsic factor refers to various external factors, such as ultraviolet rays (light), and the endogenous factor is also referred to as a chronological factor and refers to a factor mainly caused by the passage of time. That is, the skin aging is specifically, not only early aging symptoms induced by external stimuli such as ultraviolet rays, pollution, cigarette smoke, chemicals, etc., but also natural aging that occurs as the proliferation of skin cells decreases with age. It is a concept that includes wrinkles, loss of elasticity, skin sagging, and dryness. The aging may be skin photoaging by exposure to ultraviolet rays (including UVA, UVB, and UVC).

In the present invention, the term "improvement of skin elasticity" may refer to any action that suppresses or inhibits a decrease in skin elasticity or promotes the expression of skin elasticity-related factors.

In the present invention, the term “improvement of skin wrinkles” may mean inhibiting or inhibiting the formation of wrinkles on the skin, or alleviating wrinkles that have already been created.

In the present invention, the term "improvement" may mean any activity that at least reduces a parameter related to alleviation or treatment of a condition, for example, the severity of a symptom.

In the present invention, the method for obtaining any one compound selected from the group consisting of the compounds represented by Chemical Formulas 2, 6 and 9 or a fraction containing the same is the same as described above, so the description thereof is omitted.

The term "cosmetically acceptable salt" used in the present invention may be included in the type of "pharmaceutically acceptable salt", is generally safe, non-toxic, and is not unsuitable in terms of biological and other aspects, so it is used in cosmetics. It means that it can be used in the preparation of the composition.

The cosmetic composition is cosmetic water (skin lotion), skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, moisture cream, eye cream, hand cream, foundation, essence, nutrient essence It can be prepared in a formulation including a mask, eye essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cream, body cleanser, suspension, gel, powder, paste, mask pack or sheet or aerosol composition. there is. Compositions of such formulations may be prepared according to conventional methods in the art.

Ingredients included in the cosmetic composition may include ingredients commonly used in cosmetic compositions in addition to the composition as active ingredients, for example, conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments and fragrances, and A carrier may be included.

The external skin preparation may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof. The external skin preparation is a component commonly used in external skin preparations such as cosmetics or pharmaceuticals, for example, water-based components, oil-based components, powder components, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, and fragrances. , colorants, various skin nutrients, or combinations thereof and may be suitably blended as needed. The external skin preparations include metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridin, and carlin. Hot water extracts of fruits, various herbal medicines, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts, etc. drugs, vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose , saccharides such as trehalose, etc. can also be blended appropriately.

Since the health functional food composition for improving skin aging, skin elasticity, or skin wrinkles is the same as the health functional food composition according to the third aspect, the description thereof is omitted.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

[Preparation example]

HR-ESI-MS spectra were obtained with a UHR ESI Q-TOF mass spectrometer (Bruker, Billerica, MA, USA) and a Q-TOF micromass spectrometer (Waters, Milford, MA, USA). NMR spectra were recorded in DMSO- d ₆ , CD ₃ OD, and chemical shifts showed the corresponding signals ( δ H 3.30/ δ C 49.00 for CD ₃ OD; δ H 2.50/ δ C 39.50 for DMSO- d ₆ ) ( Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA) and measured with a Varian VNS 500 spectrometer (Varian, Palo Alto, CA, USA) at the Center for Analytical and Life Science Instruments, Korea University. All HPLC analyzes were performed using a Waters HPLC instrument equipped with a reversed-phase C18 column (Agilent Technologies ZORBAX, 250 Х 21.2 mm; Phenomenex Luna, 250 Х 10 mm). Open column chromatography was performed using silica gel 60 (70-230 mesh, Merck, Germany) and LiChroprep RP-18 gel (40-63 μm, Merck, Germany).

Separation of compounds derived from cultures of commensal endophytes of S.

1-1. Preparation of a culture of commensal endophytes of S.

Endogenous bacteria that live symbiotically on S. Colletotrichum gloeosporioides JS0417 ( Colletotrichum gloeosporioides JS0417) was collected from a wetland in Suncheon, Korea (34° 83'79'' N, 127° 44'95'' E) in September 2011 ( Suaeda japonica ). Makino). Rhizome tissues were cut into small pieces (0.5 Х 0.5 cm), sterilized with 2% sodium hypochlorite for 1 minute and 70% ethanol for 1 minute, and then washed with sterile distilled water. Fungal strains were grown on malt extract agar (MEA, Difco) supplemented with 50 ppm kanamycin, 50 ppm chloramphenicol and 50 ppm Rose Bengal at 22 °C for about 7 days and then cultured on plant tissue. The actively growing edge of the agar block of the newly formed colony was cut and then transferred to fresh PDA (Potato dextrose agar, Difco) for pure culture. Thereafter, the culture medium was stored as a 20% glycerol stock solution in a liquid nitrogen tank of the Wildlife Genetic Resources Bank at the National Institute of Biological Resources (Incheon, Korea) and used.

1-2. Extraction and Separation

Colletotrichum gloeosporioides JS0417, an endogenous bacterium commensal to S. chinensis, was prepared in a rice medium (prepared by autoclaving 80 g of rice in a 500 mL Erlenmeyer flask with 120 mL of distilled water) at room temperature. cultured for 3 weeks. To this culture, 200 mL of ethyl acetate was added to each flask, and sonicated and extracted three times for 1 hour. The ethyl acetate extract was evaporated in vacuo to give 106.4 g of an oily material.

The extract was separated by silica gel vacuum liquid chromatography (VLC) using a n-hexane-acetone-MeOH gradient (v/v/v, 50:1:0 → 1:1:0.1 → 0:1:1) solvent. Six fractions (A to F) were generated. Fraction D was separated by silica gel column chromatography using a gradient system of n-hexane-EtOAc-MeOH (v/v/v, 30:1:0 → 1:1:0.1) into six small fractions (D1 to D6). ) was obtained. Sub-fraction D3 was separated by C-18 column chromatography using a H ₂ O-MeOH gradient (v/v, 4:1 → 0:1) solvent system to obtain 5 sub-fractions (D31 to D35). Sub-fraction D34 was separated by HPLC with a C-18 column using a H ₂ O-acetonitrile gradient (v/v, 60:40 → 0:100) solvent to obtain 5 sub-fractions (D341 to D345). Small fraction D343 was purified by silica column chromatography using an n-hexane-acetone gradient (v/v, 7:1→6:1) system to obtain compound 2 (4.4 mg).

Small fraction D4 was separated by silica gel column chromatography using n-hexane-acetone-MeOH (v/v/v, 2:1:0.01 → 2:1:0.07 →0:0:1) gradient system to obtain five Small fractions (D41 to D45) were obtained. Small fraction D44 was purified by HPLC with a C-18 column using H ₂ O-acetonitrile isocratic (v/v, 13:87) solvent to obtain compound 6 (75.2 mg) and compound 7 (26.7 mg). .

Sub-fraction D5 was subjected to silica column chromatography using a gradient system of CHCl ₃ -acetone (v/v, 10:1 → 2:1) to yield 5 sub-fractions (D51 to D55). Compound 3 (1.0 mg) was purified by HPLC using a C-18 column using a H ₂ O-MeOH gradient (v/v, 20:80 → 0:100) solvent.

Fraction E was separated into 8 small fractions (E1 to E8) by silica gel vacuum liquid chromatography using a hexane-CHCl ₃ -acetone gradient (v/v/v, 5:1:0.1 → 0.3:1:1) solvent. got The small fraction E7 was separated by silica gel column chromatography using n-hexane-CHCl ₃ -EtOAc (v/v/v, 1:1:0.5 → 1:1:5) solvent to form six small fractions (E71 to E76). ) was obtained. Small fraction E73 was purified by HPLC with a C-18 column using a H ₂ O-acetonitrile gradient (v/v, 90:10 → 0:100) solvent to give compound 4 (8.3 mg) and compound 5 (2.0 mg). ) was obtained.

The small fraction E74 was purified by HPLC with a C-18 column using a H ₂ O-acetonitrile gradient (v/v, 95:5 → 0:100) solvent to obtain Compound 1 (3.0 mg).

Sub-fraction E7 was separated by silica gel column chromatography using a solvent of hexane-CHCl ₃ -EtOAc (v/v, 1:1:0.5 → 1:1:5) to obtain 6 sub-fractions (E71 to E76). The small fraction E73 was purified by HPLC with a C-18 column using a H ₂ O-acetonitrile gradient (v/v, 90:10→0:100) solvent to obtain Compound 8 (2.0 mg).

Small fraction D32 was purified by HPLC with a C-18 column using a H ₂ O-acetonitrile gradient (v/v, 80:20→30:70) solvent to obtain compound 9 (10.0 mg).

Small fraction E2 was separated by silica gel column chromatography using a solvent of hexane-CHCl ₃ -EtOAc (v/v/v, 5:1:0.1 → 0.3:0.3:1) to obtain 9 small fractions (E21 to E29). got it The small fraction E24 was purified by HPLC with a C-18 column using a H ₂ O-acetonitrile gradient (v/v, 70:30 → 20:80) solvent to obtain compound 10 (1.8 mg).

Small fraction E6 was separated by silica gel column chromatography using a solvent of hexane-CHCl ₃ -EtOAc (v/v/v, 1:1:0.1 → 1:1:3) to obtain six small fractions (E61 to E66). got it The small fraction E64 was purified by HPLC with a C-18 column using a H ₂ O-acetonitrile gradient (v/v, 70:30 → 0:100) solvent to obtain compound 11 (2.5 mg).

Structure determination of isolated compounds 1 to 3

The chemical structures of compounds 1 to 5 among the 13 isolated compounds were determined by analyzing 1H-NMR and 13C-NMR and analyzing 2D NMR. In addition, the relative and absolute arrangements of compounds 1 to 3 were confirmed by a combination of spectroscopic analysis, chemical reaction and chromatography methods.

Table 1 shows ^{the 1} H-NMR and ¹³ C-NMR results for compounds 1 to 3, and in FIGS. 2 to 7, ¹ H NMR (500 MHz, CD ₃ OD) spectrum, ¹³ C NMR ( 125 MHz, CD ₃ OD) spectrum, COZY spectrum (500 MHz, CD ₃ OD), HSQC spectrum (500 MHz, CD ₃ OD), HMBC spectrum (500 MHz, CD ₃ OD) and HR-ESI-MS spectrum. was

8 to 13, ¹ H NMR (500 MHz, DMSO- d ₆ ) spectrum, ¹³ C NMR (125 MHz, DMSO- d ₆ ) spectrum, COZY spectrum (500 MHz, DMSO- d ₆ ), HSQC spectrum (500 MHz, DMSO- d ₆ ), HMBC spectrum (500 MHz, DMSO- d ₆ ) and HR-ESI-MS spectrum are shown.

14 to 19 show ¹ H NMR (500 MHz, DMSO- d ₆ ) spectrum, ¹³ C NMR (125 MHz, DMSO- d ₆ ) spectrum, COZY spectrum (500 MHz, DMSO- d ₆ ), HSQC spectrum (500 MHz, DMSO- d ₆ ), HMBC spectrum (500 MHz, DMSO- d ₆ ) and HR-ESI-MS spectrum are shown.

No. One ^a 2 ^a 3 ^b δ _C δ _H , multi
( J in Hz) δ _C δ _H , multi
( J in Hz) δ _C δ _H , multi
( J in Hz) One 173.4 172.8 173.3 2a
2b 47.7 2.62, dd(3.5, 11.0)
2.08, t (11.0) 46.2 2.56 m 46.1 2.59, dt (3.5, 7.0) 3 67.2 4.03, ddd (3.5, 7.5, 11.0) 67.5 3.90 m 67.3 3.92, t(8.5) 3-OH 4.47, d (5.5) 4.52, d (5.5) 4a
4b 37.8 1.86, ddd(2.3, 7.5, 16.0)
1.76, dd(3.5, 16.0) 32.2 1.54 m
1.39, dd(3.5, 10.5) 34.5 1.54 m
1.35 m 5 78.4 4.73, dt, (10, 2.3) 76.4 4.64, dt (7.5, 10) 73.0 4.75, d (12.0) 6 35.6 2.00 m 33.7 1.89 m 31.6 1.55 m 7a
7b 33.4 1.64 m
1.09 m 31.8 1.53 m
1.02 m 29.1 1.23 m
1.20 m 8a
8b 27.7 1.37 m
1.28 m 26.2 1.24 m 25.3 1.27 m
1.18 m 9a
9b 27.1 1.37 m 25.7 1.24 m 25.3 1.27 m
1.18 m 10a 40.1 1.47, dd(8.0, 16.0) 39.2 1.34 m 38.9 1.28 m 10b 1.38 m 1.25 m 1.24 m 11 68.5 3.70, dq (6.0, 11.5) 65.8 3.54, quint (5.5) 65.7 3.53, t(5.0) 11-OH 4.28, d (5.0) 4.29, d (4.5) 12 23.5 1.14, d (6.0) 23.7 1.02, d (6.0) 23.7 1.01, d (6.0) 13 16.2 0.82, d (6.5) 7.5 0.85, d (6.5) 7.4 0.83, d (6.5) 14 15.7 0.71, d (6.5) N -Me-l-Leucine N -Me-l-Leucine N -Me-l-Leucine One' 171.3 169.9 169.8 2' 55.1 5.32, dd (5.5, 10.5) 53.1 5.28, dd(5.5, 10) 53.3 5.21, dd(5.0, 11.5) 3'a
3'b 36.3 1.60, ddd(4.5, 10.0, 14.5)
1.42, ddd(5.5, 10.5, 14.5) 35.0 1.61, dq (5.0, 14)
1.47 m 34.5 1.53 m
1.37 m 4' 25.5 1.10 m 24.3 1.39, dd(3.5, 10.5) 23.9 1.04 m 5' 23.5 0.84, d (6.5) 23.0 0.89, d (7.0) 23.1 0.77, d (6.5) 6' 22.1 0.84, d (6.5) 21.6 0.85, d (6.5) 21.2 0.74, d (6.5) N -Me 30.6 2.75, s 29.9 2.88, s 29.8 2.66, s d-Phenylalanine d-Leucine d-Phenylalanine One'' 174.5 172.7 172.6 2'' 51.7 5.27, dd(6.0, 10.0) 45.8 4.92, dqui (7.0, 10) 49.2 5.12, dd(4.0, 16.0) 3''a
3''b 37.4 3.22, dd(10.0, 13.0)
2.94, dd(6.0, 13.0) 38.8 1.70 m
1.45 m 36.1 3.08, dd(4.5, 8.0)
2.85, dd(6.5, 12.5) 4'' 138.5 24.6 1.46, dd(6.0, 14) 137.8 5'' 130.3 7.26 m 22.5 0.85, d (6.5) 129.1 7.22 m 6'' 129.5 7.26 m 22.4 0.87, d (6.5) 128.1 7.24 m 7'' 127.8 7.21 m 126.3 7.18, d (6.5) 8'' 129.5 7.26 m 128.1 7.24 m 9'' 130.3 7.26 m 129.1 7.22 m N -H 8.35, d (9.5) 8.61, d (9.5)

^a at CD ₃ OD, ¹ H at 500 MHz, ¹³ C at 125 MHz

^b DMSO- d ₆ for ¹ H at 500 MHz and ¹³ C for 125 MHz

In addition, the relative and absolute arrangements of compounds 1 to 3 were confirmed by a combination of spectroscopic analysis, chemical reaction and chromatography methods.

ckner, H. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2011, 879, 3148-3161.).The absolute arrangement of the amino acids of compounds 1 to 3 was confirmed by the advanced Marfey's method (Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K. Anal. Chem.1997 , 69 , 5146-5151;Bhushan, R. ; Br

ckner, H. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci . 2011 , 879 , 3148-3161.).

Compound 1 (1.0 mg) was hydrolyzed with 500 μL of 6N HCl at 120 °C. After 1 hour, the reactants were immersed in ice water for 3 minutes to cool. Then, the reaction material was suspended in 250 μL of H ₂ O three times to completely remove residual HCl. After dividing the hydrolyzate into two parts in a 5 mL vial, add 60 µL of 1N NaHCO ₃ and 60 µL of 3.8 mg/mL l-FDLA (1-fluoro-2,4-dinitrophenyl-5- l-leucine amide) or d-FDLA (1-fluoro-2,4-dinitrophenyl-5-d-leucine amide) was added to each hydrolyzate. The reaction mixture was incubated in an 85 °C water bath for 3 minutes, then the reaction was quenched for neutralization by adding 20 μL of 2N HCl. In addition, 500 μL of acetonitrile was added to the solution to dissolve the mixture, which was filtered (0.25 μm PETE) and analyzed. A 10 μL aliquot of each reaction mixture was run in a gradient solvent system (30–50% CH ₃ CN with 0.1% trifluoroacetic acid for 40 min, C18 reversed-phase column 150 Х 4.6 mm, temperature 40 °C, flow rate 1 mL/min). rate, UV detection at 340 nm). The same method was used for compound 2. The elution sequence of the FDAA derivatives from HPLC analysis established that Phe has the D-configuration, while the other residues, N-Me-Leu and Leu, have the L-configuration.

HPLC analysis of FDLA derivatives of compounds 1-3 using the improved Mapei method. One 2 3 Amino acids Elution order tRL ₍ min) t _RD (min) Amino acids Elution order tRL ₍ min) t _RD (min) Amino acids Elution order tRL ₍ min) t _RD (min) Phe(d) D→L 35.2 25.3 Leu(d) D→L 37.9 24.3 Phe(d) D→L 35.2 25.3 N -Me-Leu (l) L→D 29.6 35.1 N -Me-Leu(l) L→D 29.5 35.0 N -Me-Leu (l) L→D 29.5 35.0

Thus, the absolute configuration of the α carbons of amino acids could be established as indicated.

In addition, the absolute configuration of the two oxymethine carbons C-3 and C-11 was determined using a modified Mosher's method. Compound 1 (1.0 mg) was added to (S)-(+)-MTPA chloride (10 μL) and DMAP (4-dimethylaminopyridine; 1 grain) dissolved in pyridine (550 μL) and the solution was left at room temperature did After drying the mixture for 15 minutes to obtain (R)-MTPA ester (1a), 1H-NMR was performed by adding 550 μL of CD ³ OD. A similar treatment of 1 (1.0 mg) with (R)-(+)-MTPA-Cl yielded the (S)-MTPA ester (1b). Compound 2 was also subjected to Mosher reaction to obtain (R)-MTPA ester (2a) and (S)-MTPA ester (2b) in the same manner as in Compound 1.

(S)-MTPA ester 1a : pale yellow solid; ¹ H-NMR (500 MHz, CD ₃ OD) (Figure S9) δ 7.255 (m, H-5′′/ H-6′′/ H-8′′/ H-9′′), 7.209 (m, H-7′′), 5.344 (dd, J = 10.5, 5.5 Hz, H-3/H-2′), 5.255 (m, H-2′′), 5.087 (septet, J = 6.0 Hz, H-2′) 11), 4.751 (ddd, J = 10.0, 7.0, 2.5 Hz, H-5), 3.241 (m, H-3a′′), 2.956 (m, H-3b′′), 2.826 (dd, J = 11.5 , 3.5 Hz, H-2a), 2.733 (s, N -Me), 2.146 (t, J = 6.0 Hz, H-2b/H-4), 1.654 (m, H-6), 1.599 (ddd, J = 15.0, 11.0, 4.5, H-3a′), 1.525 (m, H-7a), 1.513 (m, H-10a), 1.439 (dd, J = 9.0, 6.0 Hz, H-3b′), 1.436 ( m, H-10b), 1.309 (d, J = 6.0 Hz, H-12), 1.279 (m, H-9), 1.071 (m, H-7b/H-4′), 0.827 (d, J = 6.5 Hz, H-5′/H-6′), 0.728 (d, J = 6.5 Hz, H-14), 7.365–7.577 (5H, m, phenyl group), 3.540 (s, OCH ₃ ).

(R)-MTPA ester 1b : pale yellow solid; ¹ H-NMR (500 MHz, CD ₃ OD) (Figure S10) δ 7.257 (m, H-5′′/ H-6′′/ H-8′′/ H-9′′), 7.205 (m, H-7′′), 5.325 (dd, J = 10.5, 5.5 Hz, H-2′), 5.280 (m, H-3), 5.255 (m, H-2′′), 5.039 (sextet, J = 6.5 Hz, H-11), 4.694 (dt, J = 10.0, 3.0 Hz, H-5), 3.241 (m, H-3a′′), 2.956 (m, H-3b′′), 2.948 (dd, J = 11.5, 3.5 Hz, H-2a), 2.722 (s, N -Me), 2.213 (t, J = 11.5 Hz, H-2b), 2.084 (t, J = 3.0 Hz, H-4), 1.620 (m, H-7), 1.581 (ddd, J = 14.5, 10.5, 4.0, H-3a′), 1.534 (m, H-10a), 1.514 (m, H-8), 1.502 (m, H-8) 10b), 1.414 (ddd, J = 12.0, 9.5, 6.0 Hz, H-3b′), 1.329 (m, H-6), 1.323 (m, H-9), 1.221 (d, J = 6.5 Hz, H -12), 1.071 (m, H-7b/H-4′), 0.818 (d, J = 6.5 Hz, H-5′/H-6′), 0.604 (d, J = 7.0 Hz, H-14 ), 7.380-7.566 (5H, m, phenyl group), 3.539 (s, OCH ₃ ).

(S)-MTPA ester 2a : pale yellow solid; ¹ H-NMR (500 MHz, DMSO- d ₆ ) (Figure S17) δ 8.638 (d, J = 8.5 Hz, NH), 5.402 (t, J = 5.0 Hz, H-3), 5.327 (dd, J = 10.0, 5.5 Hz, H-2′), 5.063 (dd, J = 12.0, 6.5 Hz, H-11), 4.921 (dd, J = 17.0, 4.0 Hz, H-2′′), 4.699 (m, H -5), 2.916 (s, N -Me), 2.808 (dd, J = 6.5, 4.5 Hz, H-2), 1.906 (m, H-4), 1.699 (m, H-7a/3a′′) , 1.618 (m, H-3a′), 1.567 (m, H-10a/ H-3b′′), 1.542 (m, H-6), 1.522 (m, H-4′′), 1.498 (m, H-3b′), 1.452 (m, H-10b), 1.419 (m, H-8/ H-9), 1.399 (m, H-4′), 1.281 (d, J = 6.5 Hz, H-12 ), 1.068 (m, H-7b), 0.896 (d, J = 7.0 Hz, H-5′), 0.890 (d, J = 6.5 Hz, H-6′′), 0.867 (d, J = 7.0 Hz) , H-6′), 0.851 (d, J = 7.0 Hz, H-5′′), 0.708 (d, J = 6.5 Hz, H-13), 0.684 (d, J = 7.0 Hz, H-14) , 7.206-7.663 (5H, m, phenyl group), 3.484 (s, OCH ₃ ).

(R)-MTPA ester 2b : pale yellow solid; ¹ H-NMR (500 MHz, DMSO- d ₆ ) (Figure S18) δ 8.615 (d, J = 9.5 Hz, NH), 5.356 (t, J = 5.5 Hz, H-3), 5.306 (dd, J = 11.0, 5.5 Hz, H-2′), 5.009 (dd, J = 11.5, 6.5 Hz, H-11), 4.921 (m, H-2′′), 4.652 (m, H-5), 2.908 (s , H-2/ N-Me), 1.870 (m, H-4), 1.690 (m, H-3a′′), 1.625 (m, H-7a/ H-3a′), 1.613 (m, H-3a′′) 10), 1.565 (m, H-3b′′), 1.535 (m, H-3b′), 1.514 (m, H-4′′), 1.456 (m, H-9), 1.384 (m, H-9) 8/ H-4′), 1.345 (m, H-6), 1.168 (d, J = 6.0 Hz, H-12), 1.032 (m, H-7b), 0.929 (d, J = 7.0 Hz, H -13), 0.883 (d, J = 6.5 Hz, H-5′), 0.878 (d, J = 6.5 Hz, H-6′′), 0.854 (d, J = 7.0 Hz, H-6′), 0.832 (d, J = 6.5 Hz, H-5′′), 0.606 (d, J = 6.5 Hz, H-14), 7.283–7.659 (5H, m, phenyl group), 3.482 (s, OCH ₃ ).

Both C-3 and C-11 had an R configuration based on the ΔδH values (ΔδH = δS - δR) of bis-S-(1a-2a) and bis-R-MTPA esters (1b-2b) (FIG. 20 ). Thus, the chiral centers C-5 and C-6 of the long chain moiety were both found to have the S configuration based on established relative stereochemistry.

Compound 1 was isolated as a white needle, and its molecular formula was determined from 1H and 13C NMR spectroscopic data (Table 1) and HRESIMS with 8 degrees of unsaturation (m/z, 541.32483 [M + Na]+, calculated [calcd. ], 541.32481) as C ₂₉ H ₄₆ N ₂ O ₆ . The ¹ H NMR spectrum showed two α-amino proton signals (δH 5.32 and 5.27) representing the peptide. This was supported by ¹³ C NMR resonances for three amide/ester carbonyl carbon signals (δC 174.5, 173.4 and 171.3) and two α-amino carbon signals (δC 55.1 and 51.7). Aromatic signals (δH 7.21-7.26; δC 138.5, 130.3, 129.5 and 127.8) were downshifted methylene (δH 3.22 and 2.94; δC 37.4), second methylene (δH 1.60 and 1.42; δC 36.3), methine (δH 1.10; δC 25.5) and two double methyls (δH 0.84; δC 23.5 and 22.1) suggested that this peptide contains phenylalanine and leucine moieties. The remaining signals were all sp3 methyl, methylene and methine for long lipophilic chains. C-1/C-2/C-3/C-4/C-5/C-6(C-13)/C-7/C-8/C-9/C-10/C-11/C Correlation spectroscopy (COSY) of isolated CH2-CH(O)-CH2-C(O)-CH(CH3)-CH2-CH2-CH2-CH2-CH(O)-CH3 corresponding to -12 was obtained using The long chain and the two amino acids were linked by analysis of the heteronuclear multiple bond correlation HMBC correlation (FIG. 21). The leucine moiety was found to be methylated at the N position based on the HMBC correlation from N-methyl protons (δH 2.75) to C-2'. The α-amino proton H-2' (δH 5.32) of N-Me-Leu showed an HMBC correlation with the amide carbonyl carbon C-1" (δC 174.5) of Phe, allowing linkage of N-Me-Leu to Phe. H-2" (δH 5.27), the α-amino proton of Phe and the long-chain moiety with the same amide carbonyl carbon C-1 (173.4), the HMBC correlation of methylene H2-2 (δH 0.82) has a long It was verified that the chain moiety was linked to the Phe moiety through an amide bond. In addition, the HMBC correlation of oxymethine H-5 (δH 4.73) with the carbonyl carbon C-1' (171.3) of the N-Me-Leu moiety was observed to form a 12-membered lactone ring system. . The structure of compound 1 was very similar to that of choletopeptide B. Compound 1 differed from choletopeptide B in that the secondary methyl was missing at C-2 of the long polyketide chain. The molecular weight obtained by HRMS was 14 amu less than choletopeptide B, which corresponds to the difference of CH2 units in the molecular formula. The lack of a methyl group at C-2 was further confirmed by HMBC correlations from the methylene proton (H2-2) to C-1, C-3 and C-4 in the lipid chain. Therefore, instead of 3,5,11-trioxygenated-2,6-dimethyl-dodecanoic acid (3,5,11-trioxygenated-6-methyl-dodecanoic acid), 3,5,11-trioxygenated -6-methyl-dodecanoic acid (3,5,11-trioxygenated-2,6-dimethyl-dodecanoic acid) was combined to form a cyclic depsipeptide.

The molecular formula of Compound 2 was determined to be C ₂₇ H ₅₁ N ₂ O ₆ by HRESIMS and HSQC data observed at m/z 521.35614 for [M + Na]+ (calcd, 521.35611). The spectral profile of compound 2 was similar to that of compound 1 except that the signal for the phenylalanine moiety disappeared. Signals for additional leucine moieties appeared instead: one α-amino proton at δH 4.92 (dq, J = 7.0, 10.0 Hz, H-2"), δH 1.70 (m, H-3"a) and 1.45 (m , methylene at H-3"b), and two secondary methyl groups at δH 0.87 (d, J = 6.5 Hz, H-6") and 0.85 (d, J = 6.5 Hz, H-5"). In addition, An additional secondary methyl group appeared at δH 0.85, unlike compound 1. The HMBC correlations of methyl protons with amide carbonyl carbon C-1 and oxymethine C-3 at δH 0.85 were 3,5,11-trioxygenated- The presence of 2,6-dimethyl-dodecanoic acid (3,5,11-trioxygenated-2,6-dimethyl-dodecanoic acid) was suggested by the methyl proton (δH 2.88) of N-Met-Ieu and the presence of leucine moiety. HMBC correlation of the C-1 of the long chain part with the α-proton (H-2") of the leucine moiety, together with the HMBC correlation of C-1", the leucine moiety replaced the phenylalanine of compound 1 in this molecule. showed As a novel compound, compound 2 was named choletopeptide F.

The spectral data of compound 3 also showed signals for a cyclic lipodesipeptide as shown for compounds 1 and 2, sharing the same molecular formula C ₂₉ H ₄₆ N ₂ O ₆ as compound 1 based on (+)HRESIMS data. did Interpretation of 1H-1H COZY revealed the absence of substituents at C-6 in this molecule, which correlated with HMBC correlations of C-4, C-5 and C-7 with H2-6 at δH 1.55 (2H, m). confirmed by Thus, the planar structure of compound 3 was established as shown in FIG. 21 .

Compound 1 is (3S,6R,10R,12S)-6-benzyl-10-hydroxy-12-((2S,7R)-7-hydroxyoctan-2-yl)-3-isobutyl-4- Methyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione, compound 2 is (3S,6R,9R,10R,12S)-10-hydroxy-12-((2S ,7R)-7-hydroxyoctan-2-yl)-3,6-diisobutyl-4,9-dimethyl-1-oxa-4,7-diazacyclododecane-2,5,8- Trione, compound 3 is (3S,6R,9R,10R,12R)-6-benzyl-10-hydroxy-12-((R)-6-hydroxyheptyl)-3-isobutyl-4,9- It was identified as dimethyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione. These compounds were identified as new peptide-based substances, in order, compound 1 is colletopeptide E, compound 2 is colletopeptide F, compound 3 is colletopeptide G named as

Structure determination of compounds 4 and 5

Among the 13 compounds separated, the chemical structures of compounds 4 and 5 were determined by analyzing ¹ H-NMR and ¹³ C-NMR and analyzing 2D NMR.

Table 3 shows the ¹ H-NMR and ¹³ C-NMR results for compounds 4 and 5.

22 to 28, ¹ H NMR (500 MHz, CD ₃ OD) spectrum, ¹³ C NMR (125 MHz, CD ₃ OD) spectrum, COZY spectrum (500 MHz, CD ₃ OD), HSQC spectrum ( 500 MHz, CD ₃ OD), HMBC spectrum (500 MHz, CD ₃ OD), ROESY spectrum (500 MHz, CD ₃ OD) and HR-ESI-MS spectrum.

29 to 35, ¹ H NMR (500 MHz, CD ₃ OD) spectrum, ¹³ C NMR (125 MHz, CD ₃ OD) spectrum, COZY spectrum (500 MHz, CD ₃ OD), HSQC spectrum ( 500 MHz, CD ₃ OD), HMBC spectrum (500 MHz, CD ₃ OD), ROESY spectrum (500 MHz, CD ₃ OD) and HR-ESI-MS spectrum.

No. 4 5 δ _C δ _H , multi ( J in Hz) δ _C δ _H , multi ( J in Hz) One 78.6 4.79, d (9.5) 79.1 4.69, d (9.5) 2 71.8 3.94, t(9.5) 71.9 4.04, t(9.5) 3 80.9 5.08, t(9.0) 80.9 5.07, t(9.5) 4 69.6 3.68, t(9.5) 69.7 3.65, t(9.5) 5 82.3 3.50, ddd(9.5, 4.5, 2.0) 82.2 3.46, ddd(10, 5.0, 2.5) 6a
6b 62.0 3.85, d d (12, 2.0)
3.77, d d (12, 4.5) 62.2 3.84, d d (12, 2.5)
3.76, dd(12, 5.0) 7 114.5 115.1 8 158.7 158.5 9 104.3 6.26, d (2.5) 102.6 6.14, d (2.5) 10 159.3 158.9 11 109.3 6.40, d (2.5) 110.5 6.15, d (2.5) 12 143.1 140.9 13 63.6 4.63, d (12)
4.54, d (12) 21.0 2.29, s One' 168.4 168.5 2' 120.8 5.88, d d (16, 1.5) 120.9 5.87, d d (16, 1.5) 3' 155.8 6.97, dd(16, 7.0) 155.7 6.97, dd(16, 7.0) 4' 37.9 2.38, sept (6.5) 37.9 2.38, sept (6.5) 4′-CH ₃ 19.0 1.05, d (7.5) 19.0 1.05, d (7.5) 5'a
5'b 40.2 2.13, quint (6.5)
2.05, quint (6.5) 40.2 2.13, quint (6.5)
2.06, quint (6.5) 6' 126.9 5.31, d d (16, 6.5) 126.9 5.34, d d (16, 6.5) 7' 139.9 5.29, d d (16, 6.5) 139.9 5.29, d d (16, 6.5) 8' 40.0 1.96, sept (6.5) 40.0 1.97, sept (6.5) 8'-CH ₃ 21.0 0.95, d (6.5) 21.0 0.95, d (7.0) 9' 30.9 1.30 m 30.9 1.28 m 10' 12.3 0.84, t(6.5) 12.3 0.85, t(6.5)

The molecular formula of compound 4 was determined to be C ₂₅ H ₃₆ O ₉ by ¹ H and ¹³ C NMR spectroscopic data (Table 3), which was (+)HRESIMS (m/z, 503.22518 [M + Na]+, calcd., 503.22515 ) was further confirmed by The 1H NMR spectrum of compound 4 showed characteristic signals for resorcinol glycoside acylate: two meta-bonded aromatic protons at δH 6.40 and 6.26 for the resorcinol moiety, to the sugar moiety. 5 oxymethine protons at δH 5.08, 4.79, 3.94, 3.68 and 3.50, 4 olefin protons at δH 6.97, 5.88, 5.31 and 5.29, 2 secondary methyl groups at δH 1.05 and 0.95, and a terminal triplet at δH 0.84. (triplet) Aliphatic methylene and methine signals at δH 2.38, 2.13, 2.05, 1.96 and 1.30 for methyl and long-chain acyl moieties. 13 C NMR and HSQC spectra showed 25 carbon signals, including 1 ester, 6 aromatics, 4 olefins, 7 methane, 3 methylene and 3 methyl carbons (Table 3). The long-chain acyl moiety, CH/CH/CH(CH3)/CH2/CH/CH/CH(CH3) corresponding to C-2' to C-10' is 1H-1H with a hexose moiety It was obtained by analysis of COZY (FIG. 36). The long-chain acyl moiety, hexose and resorcinol moiety were linked together by the HMBC correlation. Hexose was identified as glucose based on its proton adjacent coupling constant ( ³ J _H,H ) and ¹³ C shift.

Oxymethylene protons isolated at δH 4.54 and 4.63 (d, J = 12 Hz, respectively) show strong HMBC correlations with aromatic carbons C-7, C-11 and C-12 of the resorcinol ring, indicating that the oxidized orci It suggests that it has an orcinol backbone (FIG. 36). In addition, HMBC correlations of C-7, C-8 and C-12 of the aromatic ring with the anomeric proton indicated that the glucose moiety was attached to C-7 of the resorcinol ring via a C-glycosidic linkage. At δH 5.08 assigned by 1H-1H COSY and HMBC, the H-3 of glucose showed a strong HMBC correlation with the carbonyl carbon of the long-chain acyl residue, suggesting that the attachment of the acyl moiety at glucose C-3 to an ester linkage. showed up The long chain moiety from C-1' to C-10' is dimethyl decadienoic acid, and the position of the olefin group was confirmed by HMBC data and 1H-1H COSY. HMBC correlations of H-2' and C-1' ester carbons at δH 5.88 (d, J = 16 Hz), and secondary methyl protons with olefinic carbons C-3' at δH 1.05 (J = 7.5 Hz) It was shown that there is one double bond between -2' and C-3' and a methyl group is bonded to C-4'. Additional HMBC correlations of the terminal triplet methyl proton with the methyl containing methine carbon and the olefinic carbon C-7' at δC 139.9 with the remaining secondary methyl protons at δH 0.95 indicate that the double bonds at C-6' and C-7' present, suggesting the presence of a methyl substituent at C-8'. Glucose was in the relative β direction due to the anomeric proton's adjacent coupling constant (JH-1,H-2 = 9.5 Hz).

The molecular formula of compound 5 was determined to be C ₂₅ H ₃₆ O ₈ by 1H and 13C NMR spectroscopic data (Table 3) together with HRESIMS (m/z, 487.23026 [M + Na]+ , calcd., 487.23024). The structure of compound 5 was similar to that of compound 4 based on the NMR spectrum (Table 3). Compound 5 differed only in the presence of a singlet methyl signal at δH 2.29 instead of the oxygenated methylene group of compound 4. Therefore, compound 5 had a methyl group in the resorcinol moiety instead of a hydroxymethyl group, which was determined by HMBC correlations between the methyl proton at δH 2.29 and C-7, C-11 and C-12 of the resorcinol moiety. has been supported

Compound 4 is (2S,3S,4R,5R,6R)-2-(2,4-dihydroxy-6-(hydroxymethyl)phenyl)-3,5-dihydroxy-6-(hydroxy Methyl) tetrahydro-2H-pyran-4-yl (2E,6E)-4,8-dimethyldeca-2,6-dienoate, compound 5 is (2S,3S,4R,5R,6R)- 2-(2,4-dihydroxy-6-methylphenyl)-3,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl (2E,6E)-4,8 -Identified as dimethyldeca-2,6-dienoate. These compounds were identified as novel papulacandin backbone polyketides, and compound 4 was named colletotricandin A and compound 5 was named colletotricandin B.

Structure determination of compounds 6 and 7

Among the 13 compounds separated, the chemical structures of compounds 6 and 7 were determined by analyzing ¹ H-NMR and ¹³ C-NMR and analyzing 2D NMR.

37 to 42 show ¹ H NMR (500 MHz, DMSO- d ₆ ) spectrum, ¹³ C NMR (125 MHz, DMSO- d ₆ ) spectrum, COZY spectrum (500 MHz, DMSO- d ₆ ), HSQC spectrum (500 MHz, DMSO- d ₆ ), HMBC spectrum (500 MHz, DMSO- d ₆ ) and HR-ESI-MS spectrum are shown.

43 to 48 show ¹ H NMR (500 MHz, DMSO- d ₆ ) spectrum, ¹³ C NMR (125 MHz, DMSO- d ₆ ) spectrum, COZY spectrum (500 MHz, DMSO- d ₆ ), HSQC spectrum (500 MHz, DMSO- d ₆ ), HMBC spectrum (500 MHz, DMSO- d ₆ ) and HR-ESI-MS spectrum are shown.

In addition, the stereochemistry of compounds 6 and 7 was established by J-based array analysis (JBCA), ROESY and modified Mosher's method. The association constants ( ³ J _HH , ³ J _HC and ² J _HC ) were determined for JBCA, and the ROESY correlation was analyzed to establish the relative stereochemistry for compound 7 (FIG. 49). The small 3JH-2,H-3, small 3J2-CH3,H-3, and large 2JC-3,H-2 values suggest that the methyl group at C-2 and the hydroxyl group at C-3 are on the same side of the molecule. (A in FIG. 49). This was also supported by the strong ROESY correlation between the hydroxyl protons of C-3 and the methyl protons of C-2 (FIG. 49B). In the case of H-5 and H-6 of compound 7, JBCA was not meaningful because the value of the adjacent coupling constant (3JH-5, H-6 = 10 Hz) was large. The only important point was whether the two moieties attached to C-5 and C-6 were in close proximity to each other, which was determined by the ROESY spectrum. The strong ROESY correlation between H2-4 and H2-7 suggested that they were in opposite directions of the molecule (FIG. 49). Additionally, strong NOESY correlations for H-3/H-6 and H-5/14-CH3 also supported the relative stereochemistry of the long chain moieties as indicated.

Compound 6 is (3S,6R,9R,10R,12S)-6-benzyl-3-((R) -sec -butyl)-10-hydroxy-12-((2S,7R)-7-hydroxy Octan-2-yl) -4,9-dimethyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione, the compound 7 is (3S, 6R, 9R, 10R, 12S)-6-benzyl-10-hydroxy-12-((2S,7R)-7-hydroxyoctan-2-yl)-3-isobutyl-4,9-dimethyl-1-oxa-4; It has been identified as 7-diazacyclododecane-2,5,8-trione.

Isolation confirmation of compounds 8 to 11

The chemical structures of compounds 8 to 13 among the 13 isolated compounds were determined by analyzing ¹ H-NMR and ¹³ C-NMR and analyzing 2D NMR. As a result of analysis, compound 8 is (E) -5- (hydroxymethyl) -6- (3-hydroxyprop-1-en-1-yl) -4-methoxy-3-methyl-2H-pyran- 2-one (also known as 'cladobotrin IV'), compound 9 is 2-(1H-indol-3-yl)ethane-1-ol (also known as 'triptopol'), and compound 10 is indole-3 -acetate methyl ester, compound 11 was identified as 3-methoxy indole.

¹ H-NMR and ¹³ C-NMR results for Compounds 8 to 11 are shown below.

Compound 8: Amorphous; ¹ H NMR (500 MHz, CD ₃ OD) δ 4.18 (3H, s, 4-OCH ₃ ), 2.64 (2H, t, J = 7.5 Hz, H-7), 1.71 (2H, sxt, J = 7.5 Hz , H-8), 0.99 (3H, t, J = 7.5 Hz, H-8), 4.54 (2H, s, H-10), 4.41, (s, H-11); ¹³ C NMR (125 MHz, CD ₃ OD) δ 167.5 (C-2), 110.2 (C-3), 171.7 (C-4), 63.1 (4-OCH ₃ ), 115.3 (C-5), 165.9 ( C-6), 33.5 (C-7), 22.2 (C-8), 13.9 (C-9), 55.3 (C-10), 55.2 (C-11).

Compound 9: Amorphous; ¹ H NMR (500 MHz, CD ₃ OD) δ 7.52 (1H, d, J = 7.5 Hz, H-4), 7.31 (1H, d, J = 7.5 Hz, H-7), 7.06 (1H, dt, J = 7.5, 1.0 Hz, H-6), 7.05 (1H, s, H-2), 6.98 (1H, dt, J = 7.5, 1.0 Hz, H-5), 3.80 (2H, t, J = 7.5 Hz, H-9), 2.96 (2H, dt, J = 7.5, 1.0 Hz, H-8); ¹³ C NMR (125 MHz, CD ₃ OD) δ 138.1 (C-7a), 128.9 (C-3a), 123.5 (C-2), 122.2 (C-5), 119.5 (C-6), 119.2 (C -4), 112.7 (C-3), 112.1 (C-7), 63.7 (C-9), 29.8 (C-8).

Compound 10: Amorphous; ¹ H NMR (500 MHz, CD ₃ OD) δ 7.50 (1H, d, J = 8.0 Hz, H-4), 7.33 (1H, d, J = 8.0 Hz, H-7), 7.14 (1H, s, H-2), 7.09 (1H, dt, J = 7.5, 1.0 Hz, H-6), 7.00 (1H, dt, J = 7.5, 1.0 Hz, H-5), 3.75 (2H, s, H-8 ), 3.66 (3H, s, 9-OCH ₃ ).

Compound 11: amorphous; ¹ H NMR (500 MHz, CD ₃ OD) δ7.54 (1H, d, J = 8.0 Hz, H-4), 7.34 (1H, d, J = 8.0 Hz, H-7), 7.18 (1H, s , H-2), 7.10 (1H, dt, J = 7.5, 1.0 Hz, H-6), 7.01 (1H, dt, J = 7.5, 1.0 Hz, H-5), 3.65 (2H, s, H-5). 8).

50 and 51 show ^{the 1} H NMR (500 MHz, CD ₃ OD) spectrum and ¹³ C NMR (125 MHz, CD ₃ OD) spectrum of compound 8, respectively, and FIGS. 52 and 53 show ^{the 1} H NMR of compound 9, respectively. (500 MHz, CD ₃ OD) spectrum and ¹³ C NMR (125 MHz, CD ₃ OD) spectrum, and FIG. 54 shows ^{a 1} H NMR (500 MHz, CD ₃ OD) spectrum of Compound 10, FIG. 55 ¹ H NMR (500 MHz, CD ₃ OD) spectrum of compound 11 is shown in .

Analysis of the adiponectin secretion promoting effect of the isolated compound

In order to analyze the effect of increasing the adiponectin level of the isolated compound, ELISA for the adiponectin level was performed.

Specifically, human mesenchymal stem cells (hBM-MSC; Lonza, Inc., Walkersville, MD, USA) cells were treated with penicillin-streptomycin, Glutamax ^™ (Invitrogen, Carlsbad, CA, USA) and 10% fetal bovine serum (FBS). It was cultured in low glucose (1 g/L) containing DMEM (Dulbecco Modified Eagle's Medium). To promote adipogenesis, medium was replaced with DMEM containing high glucose (4.5 g/L) supplemented with 10% FBS, Glutamax ^™ , 10 μg/mL insulin, 1 μM dexamethasone and 0.5 mM IBMX (IDX conditions); The separated compounds were treated with 30 μM each and cultured for 5 days. Then, the cell supernatant was harvested and the concentration of adiponectin was determined using the Quantikine ^™ immunoassay kit (R&D Systems, Minneapolis, Minn., USA) according to the manufacturer's instructions. Lipid droplet formation during adipogenesis was evaluated in hBM-MSCs using Oil red O staining.

As a result, as shown in FIG. 56A, among the 14 compounds, compounds 2, 5, and 6 significantly reduced adiponectin secretion during adipogenesis in hBM-MSC by 116%, 78%, and 123%, compared to the IDX control group. promoted. In addition, as can be seen in B of FIG. 56, compounds 2 and 6 increased the formation of lipid droplets in the adipocyte differentiation model when compared to the control group.

The concentration-dependent effects of compounds 2 and 6 on adiponectin secretion were analyzed by setting the maximum adiponectin secretion promoting activity of antidiabetic glibenclamide to 100% effect. As shown in C of FIG. 56 , the EC ₅₀ values of compounds 2 and 6 for promoting secretion of adiponectin were 29.4 μM and 22.7 μM, respectively.

Analysis of anti-inflammatory activity of compounds and analysis of adiponectin secretion promoting effect

ELISA was performed on the concentration of prostaglandin E2 (PEG2) in order to analyze the anti-inflammatory activity associated with adiponectin for the isolated compound. PGE2 is an essential lipid mediator that regulates acute pro-inflammatory processes in response to various endogenous or exogenous toxic stimuli.

e, France)을 사용하여, 자외선 B를 20 mJ/cm² 세기로 조사한 뒤 2시간 후의 상등액을 수확하여 제조사의 지시 (Cayman Chemical, Ann Arbor)에 따라 프로스타글란딘 E2 농도를 결정하였다. Specifically, HaCaT cells were cultured in high glucose (4.5 g/L) DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 °C, 5% CO _{2 .} conditions were cultivated. Cells in 48-well plates were pretreated with aspirin (300 μM) and rinsed 3 times with DPBS. Subsequently, the separated compounds 2, 4-6, and 8-11 were replaced with media treated with 10 μM concentrations, respectively, and UV irradiation systems (Bio-Sun, Vilber Lourmat, Marne-la-Vall

e, France), UV B was irradiated at an intensity of 20 mJ/cm ² , and the supernatant was harvested after 2 hours to determine the concentration of prostaglandin E2 according to the manufacturer's instructions (Cayman Chemical, Ann Arbor).

As a result, as shown in FIG. 57A , compound 9 significantly inhibited PGE2 production in HaCaT keratinocytes and exhibited anti-inflammatory activity. The PEG2 inhibitory activity of compound 9 was shown in a concentration-dependent manner along with the control group, and as shown in FIG. 57B, the half maximal inhibitory concentration (IC ₅₀ ) for the PGE2 production inhibitory effect was 6.55 μM, compared to aspirin (21.27 μM). and resveratrol (18.31 μM).

PGE2 inhibits adiponectin production during adipogenesis in hBM-MSCs, suggesting that compound 9 may have adiponectin secretion promoting activity similar to that of aspirin. From the observation that compound 9 exhibited concentration-dependent anti-inflammatory activity, in order to re-observe the adiponectin secretion promoting effect of compound 9, the effect of increasing adiponectin level of compound 9 at a concentration higher than the concentration treated in Example 6 was analyzed, As a result, as shown in FIG. 57C , a concentration-dependent secretion-stimulating effect of adiponectin was observed. As a result of observing the phenotype of Compound 9, which increased adiponectin production, through a microscope, as shown in FIG. 57D, Compound 9 increased lipid accumulation at a concentration of 300 μM, which indicates improvement in insulin sensitivity. Accordingly, Compound 9 exhibited a stronger effect than aspirin because it had both adiponectin secretion promoting and anti-inflammatory activities.

Claims (18)

A composition for stimulating adiponectin secretion comprising at least one compound selected from the group consisting of compounds represented by Formulas 2, 6, and 9, an isomer thereof, a salt thereof, or a fraction containing the compound as an active ingredient:
[Formula 2]
,
[Formula 6]
and
[Formula 9]
,
here,
The compound represented by Formula 2 is (3S,6R,9R,10R,12S)-10-hydroxy-12-((2S,7R)-7-hydroxyoctane-2-yl)-3,6-diamond isobutyl-4,9-dimethyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione;
The compound represented by Formula 6 is (3S,6R,9R,10R,12S)-6-benzyl-3-((R) -sec -butyl)-10-hydroxy-12-((2S,7R)- 7-hydroxyoctan-2-yl) -4,9-dimethyl-1-oxa-4,7-diazacyclododecane-2,5,8-trione;
The compound represented by Formula 9 is 2-(1H-indol-3-yl)ethane-1-ol. The composition for stimulating adiponectin secretion according to claim 1, wherein the isomer comprises a racemate, an enantiomer, a diastereomer, a mixture of enantiomers, or a mixture of diastereomers. The adiponectin according to claim 1, wherein the compounds represented by Formulas 2, 6 and 9 are isolated from Colletotrichum gloeosporioides JS0417, a symbiotic endogenous bacterium that lives on S. chinensis or a culture thereof. A composition for promoting secretion. According to claim 3, wherein the culture of Colletotrichum gloeosporioides JS0417 ( Colletotrichum gloeosporioides JS0417) is obtained by culturing Colletotrichum gloeosporioides JS0417 in rice medium A composition for stimulating phosphorus and adiponectin secretion. The composition for stimulating adiponectin secretion according to claim 1, wherein the compounds represented by Formulas 2, 6 and 9 are separated by performing the following steps a) to c):
a) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium symbiotic with S.
b) obtaining a fraction by performing liquid chromatography on the obtained extract; and
c) isolating at least one compound among the compounds represented by Chemical Formulas 2, 6 and 9 by performing silica gel chromatography on the obtained fraction. The composition for stimulating adiponectin secretion according to claim 5, wherein the extraction in step a) is performed by adding an ethyl acetate solvent to the culture of Colletotrichum gloeosporioides JS0417. The composition for stimulating adiponectin secretion according to claim 1, wherein the compound represented by Formula 2 is isolated by performing the following steps a-1) to f-1):
a-1) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium that lives symbiotically with Chimyeoncho;
b-1) Perform liquid chromatography on the obtained extract while gradually changing the volume ratio of normal hexane, acetone and methanol as a mobile phase in a volume ratio of 50:1:0 → 1:1:0.1 → 0:1:1 to obtain a first fraction;
c-1) The obtained first fraction is subjected to silica gel chromatography while gradually changing a mixed solvent of normal hexane, ethyl acetate and methanol as a mobile phase in a volume ratio of 30:1:0 → 1:1:0.1 to obtain a second fraction Obtaining;
d-1) obtaining a third fraction by subjecting the obtained second fraction to C18 chromatography while gradually changing a mixed solvent of water and methanol as a mobile phase in a volume ratio of 4:1 → 0:1;
e-1) The obtained third fraction was subjected to high-performance liquid chromatography using a C18 column while gradually changing a mixed solvent of water and acetonitrile as a mobile phase in a volume ratio of 60:40 → 0:100 to obtain a fourth fraction. obtaining; and
f-1) Silica gel chromatography was performed while gradually changing a mixed solvent of normal hexane and acetone as a mobile phase in a volume ratio of 7: 1 → 6: 1 for the obtained fourth fraction to obtain a compound represented by Formula 2 step. The composition for stimulating adiponectin secretion according to claim 1, wherein the compound represented by Formula 6 is separated by performing the following steps a-2) to e-2):
a-2) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium that lives symbiotically with S. chinensis;
b-2) Liquid chromatography was performed while gradually changing the volume ratio of normal hexane, acetone, and methanol as a mobile phase to the obtained extract in a volume ratio of 50:1:0 → 1:1:0.1 → 0:1:1 to obtain a first fraction;
c-2) The obtained first fraction is subjected to silica gel chromatography while gradually changing a mixed solvent of normal hexane, ethyl acetate and methanol as a mobile phase in a volume ratio of 30:1:0 → 1:1:0.1 to obtain a second fraction Obtaining;
d-2) Silica gel chromatography while gradually changing a mixed solvent of normal hexane, acetone and methanol as a mobile phase with respect to the obtained second fraction at a volume ratio of 2:1:0.01 → 2:1:0.07 → 0:0:1 to obtain a third fraction; and
e-2) The obtained third fraction is subjected to high-performance liquid chromatography using a mixed solvent of water and acetonitrile as a mobile phase in a volume ratio of 10 to 15:85 to 90 to obtain a compound represented by Formula 6 or 7 step. The composition for promoting adiponectin according to claim 1, wherein the compound represented by Formula 9 is isolated by performing the following steps a-7) to e-7):
a-7) extracting a culture of Colletotrichum gloeosporioides JS0417, an endogenous bacterium symbiotic with S.
b-7) Liquid chromatography was performed on the obtained extract while gradually changing the volume ratio of normal hexane, acetone and methanol as a mobile phase in a volume ratio of 50:1:0 → 1:1:0.1 → 0:1:1 to obtain a first fraction;
c-7) The obtained first fraction was subjected to silica gel chromatography while gradually changing a mixed solvent of normal hexane, ethyl acetate and methanol as a mobile phase in a volume ratio of 30:1:0 → 1:1:0.1 to obtain a second fraction Obtaining;
d-7) obtaining a third fraction by subjecting the obtained second fraction to C18 chromatography while gradually changing a mixed solvent of water and methanol as a mobile phase in a volume ratio of 4:1 → 0:1; and
e-7) The obtained third fraction was subjected to high-performance liquid chromatography using a C18 column while gradually changing a mixed solvent of water and acetonitrile as a mobile phase in a volume ratio of 80:20 → 30:70, represented by Chemical Formula 9 Obtaining a compound that is. A pharmaceutical composition for preventing or treating hypoadiponectinemia-related diseases, comprising the composition of any one of claims 1 to 9 as an active ingredient. The method of claim 10, wherein the hypoadiponectinemia-related disease is at least one selected from the group consisting of metabolic disease, metabolism-related inflammatory disease, inflammatory disease, autoimmune disease, infectious disease, allergy, kidney disease, liver disease, and cancer Phosphorus, a pharmaceutical composition for preventing or treating diseases related to hypoadiponectinemia. The method of claim 11, wherein the metabolic disease is at least one selected from the group consisting of obesity, diabetes, nonalcoholic steatohepatitis (NASH), metabolic syndrome, hypertension, hyperlipidemia, cardiovascular disease and atherosclerosis, wherein the Inflammatory disease is any one or more selected from the group consisting of degenerative inflammation, exudative inflammation, purulent inflammation, hemorrhagic inflammation and proliferative inflammation, a pharmaceutical composition for preventing or treating hypoadiponectinemia-related diseases. A health functional food composition for preventing or improving hypoadiponectinemia-related diseases, comprising the composition of any one of claims 1 to 9 as an active ingredient. The method of claim 13, wherein the hypoadiponectinemia-related disease is at least one selected from the group consisting of metabolic disease, metabolism-related inflammatory disease, inflammatory disease, autoimmune disease, infectious disease, allergy, kidney disease, liver disease, and cancer A health functional food composition for preventing or improving phosphorus, hypoadiponectinemia-related diseases. The method of claim 14, wherein the metabolic disease is at least one selected from the group consisting of obesity, diabetes, nonalcoholic steatohepatitis (NASH), metabolic syndrome, hypertension, hyperlipidemia, cardiovascular disease, and atherosclerosis, wherein the Inflammatory disease is any one or more selected from the group consisting of degenerative inflammation, exudative inflammation, purulent inflammation, hemorrhagic inflammation and proliferative inflammation, hypoadiponectinemia-related disease prevention or improvement for health functional food composition. A cosmetic composition for improving skin aging, skin elasticity or skin wrinkles comprising the composition of any one of claims 1 to 9 as an active ingredient. An external skin preparation for improving skin aging, skin elasticity, or skin wrinkles, comprising the composition of any one of claims 1 to 9 as an active ingredient. A health functional food composition for improving skin aging, skin elasticity or skin wrinkles, comprising the composition of any one of claims 1 to 9 as an active ingredient.