TW201438725A - Fungal composition with biological activities and a preparation method thereof - Google Patents

Abstract

The present invention relates to biologically active of bisabolane-type sesquiterpenoids, xanthones and phenolic compound. The present invention also provides a method for preparing that compound from Aspergillus sydowii. The subject compositions have a variety of pharmaceutical applications.

Description

Biologically active fungal composition and preparation method thereof

The present invention relates to a biologically active composition for fungi and a process for the preparation thereof, which method, in particular, for the isolation and purification of a metabolite of Aspergillus sydowii for the preparation of a compound.

Aspergillus sydowii , a fungus belonging to the genus Mycobacterium, which grows on soil, manure, wine cellar, food, the inner wall of telescope cylinders and the substrates of various materials; This fungus is a pathogen of coral pathogenicity. In 2010, Cui Chuanming reported on the secondary metabolites and biological activities of five marine fungi in the Graduate School of the Chinese Academy of Sciences (Ocean Institute) and found that its metabolites have antioxidant activity and inhibit the activity of human hepatoma cell lines. Du Fengyu, in the 2011 Chinese Academy of Sciences, conducted a study on the secondary metabolites and biological activities of two marine fungi, and found secondary metabolites with inhibitory activity against Staphylococcus aureus.

There are about 250 million people with diabetes in the world, accounting for 3.5% of the world's total population. Therefore, the development of effective diabetes drugs has become the goal of research and development. The research team has been actively searching for compounds with such physiological activity from fungi over the years; for example, the invention of citron extracts for treating diabetes, or the synthesis of chalcone-type compounds by chemical modification for the invention of diabetes.

For the sake of his position, the inventor, in view of the lack of prior art, is Considering the idea of improving the invention, the "bioactive fungal composition and its preparation method" of the present invention can be invented.

The primary object of the present invention is to provide a use of a composition for the preparation of an agent for increasing glucose utilization, the composition comprising an extract of Aspergillus sydowii .

Another object of the present invention is to provide a compound selected from formula (II) or formula (III), which is provided as an effective amount of a principal component and comprising a pharmaceutically acceptable carrier; for the preparation of a medicament for increasing glucose utilization, II), formula (III), and formula (IV) are structures as shown, Wherein R ₇ is a double bond oxy group a group, R ₁₁ may be selected from one of a hydroxyl group, a C _{1 to 3} alkoxy group; R ₈ may be selected from a hydrogen group, a hydroxyl group, a C _{1 to 3} alkoxy group, and a C _{1 to 5} alkyl group. .

In a specific embodiment, increasing glucose utilization is associated with diabetes and metabolic diseases; or administering with insulin to enhance the effect of insulin; or inhibiting fat accumulation in cells.

A further object of the present invention is to provide a compound of formula (I) which is purified from Aspergillus sydowii . Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ may be selected from the group consisting of a hydrogen group, a hydroxyl group, a C _{1 to 3} alkoxy group, a C _{1 to 5} alkyl group, and a C _{1 to 5} containing a single double One of the alkenyl groups of the bond; Formula (I) may be a methylene group with a double bond (-CH ₂ ) when R ₃ and R _{4 are} combined; or one of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ may be Key oxygen The group R ₁₁ may be selected from one of a hydrogen group, a hydroxyl group and a methoxy group.

The above described objects, features, and advantages of the invention will be apparent from the description and appended claims

First: Flow chart of separation of fungal extracts

Figure 2: Evaluation of glucose utilization of its compounds

Figure 3: Oil droplet accumulation test of compound 4

Figure 4: Quantitative observation of intracellular oil droplets of Compound 4 by fat cell oil droplets.

Preparation of compounds from crude extracts

Aspergillus sydowii was purchased from the Biological Resource Conservation Center (BCRC), and was incubated at 25 ° C and 150 rpm, and was incubated in potato nutrient solution (PDB). The total fermentation broth was collected, and the mycelium and culture solution were separated by filter paper. After filtration, the extract was separated as shown in the first figure, and the culture was extracted with ethyl acetate (EtOAc) to give a crude extract.

The crude extract was concentrated under reduced pressure, and a fraction of F1-F4 was obtained by column chromatography on Sephadex. The F1 layer was separated by gel column chromatography to obtain a layer of F1-1~F1-6. The F1-2 partitioning layer was purified by various column chromatography methods and high performance liquid chromatography (HPLC) to obtain compounds 1 (18.9 mg), 3 (2.1 mg) and 7 (9.4). Mg). Compound 4 (236.2 mg) was purified by chromatography on silica gel column eluting with methylene chloride: ethyl acetate (6:1).

The layer F1-5 was partitioned into a solvent system by dichloromethane and methanol, and then purified by HPLC to obtain compound 6 (5.2 mg) and 11 (70.0 mg).

The layer F1-6 was separated into a mixed solvent system of dichloromethane and methanol (6:1), and the compound 2 (3.1 mg) and 5 (34.9 mg) were obtained by a gel column chromatography. Compound 8 (20.4 mg) was purified from the partitioned layer F2 using dichloromethane and methanol (14:1) as solvent system and further purified by HPLC.

The layer F4 was separated and purified by ruthenium column chromatography of dichloromethane and methanol solvent system, and 9 (8.0 mg) and 10 (4.1 mg) were obtained by increasing polarity. Some of the above compounds are obtained by HPLC conditions of a mixed solution of acetonitrile and water in an octadecylsilyl (ODS hypersil) Thermo column.

The obtained 11 compounds were determined by physical property data and ¹ H-NMR and ¹³ C-NMR nuclear magnetic resonance spectroscopy, respectively, belonging to bisabolane-type sesquiter penoids and xanthones. And phenolic and the like are divided into four types, namely, formula (I), formula (II), formula (III) or formula (IV). Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be selected from the group consisting of a hydrogen group, a hydroxyl group, a C _{1 to 3} alkoxy group, a C _{1 to 5} alkyl group, and C _{1 to 5;} One of the alkenyl groups containing a single double bond; if R ₃ and R _{4 are} combined, they may be a methylene group having a double bond (-CH ₂ ); or R ₁ , R ₂ , R ₃ , R ₄ , One of R ₅ may be a double bond oxy group The group, R _{11 ,} may be selected from one of a hydrogen group, a hydroxyl group, and a methoxy group.

And R ₇ is a double bond oxygen a group, R ₁₁ may be selected from one of a hydroxyl group and a C _{1 to 3} alkoxy group; R ₈ may be selected from a hydrogen group, a hydroxyl group, a C _{1 to 3} alkoxy group, and a C _{1 to 5} alkyl group. .

The 11 compounds isolated and purified from Aspergillus sydowii have the following structural formula: Compound 10 compound 11

The chemical name of the above compounds, compound 1 were Christopher 7-ol ((7 S) - (+ ) - 7- O -methylsydonol); 7, 12-dihydroxy compound is 2-dimethyl-hexanediol Hydroxybenzoic acid ((7 S , 11 S )-(+)-12-hydroxysydonic acid); Compound 3 is 7-deoxy-7,14-didehydrosudyl alcohol (7-deoxy-7,14- Didehydrosydonol); Compound 4 is Stilol (( S )-(+)-sydonol); Compound 5 is 7-Hydroxydimethylhexyloxybenzoic acid (( S )-(+)-sydonic acid); 6 is 7-dehydroxywaltol B (anhydrowaraterpol B); compound 7 is 1-hydroxytrimethylheptenylmethylphenol (( E )-5-(hydroxymethyl)-2-(6'-methylhept- 2'-en-2'-yl)phenol); compound 8 is xanthone methoxylate (AGI-B4); compound 9 is sydowinin B; compound 10 is sadobe A ( 8-hydroxy-6-hydroxymethyl-9-oxo-9H-xanthene-1-carboxylic acid methyl ester, sydowinin A); Compound 11 is dihydroxymethylphenyl ether (3,3'-Dihydroxy-5,5'-dimethyldiphenyl Ether, diorcinol).

Type 2 diabetes is known to be closely related to obesity. Such diabetic patients usually need to use oral anti-diabetic drugs to improve insulin resistance, or use drugs to promote insulin release, increase blood insulin concentration to stabilize blood sugar. The currently commercially available drugs for this type of patient are best known as the insulin sensitizer Thiazolidinedione (TZD). Recent studies have pointed out that because of the anti-diabetic and anti-inflammatory activity of thiazole-type drugs, this type of diabetes drug is evaluated with a focus on similar functions. In addition, thiazole-type drugs have problems such as lipid accumulation and obesity, which causes long-term use of patients to pay attention to a considerable number of contraindications in the diet, to avoid weight gain, massive fat accumulation, edema, anemia and stagnation heart failure.

The glucose utilization test was carried out using the mouse pre-adipocyte cell line 3T3-L1, and the extracellular glucose concentration was analyzed. Eleven compounds isolated and purified from Aspergillus sydowii were evaluated to see if the glucose concentration after compound addition was observed. change. As shown in the second figure, the extracellular glucose concentration of the control group was 427.4±1.1 mg/dL when no test compound was added. When the concentration of 0.32 μM insulin was added, the glucose concentration was decreased to 404.9±11.5 mg/dL. When the concentration of each test compound was 30 μg/mL and 0.32 μM insulin, the extracellular glucose concentration was significantly different from that of the control group, especially when the compound 4 and insulin were combined to reduce the glucose concentration. Significantly, a decrease of about 24% or more compared with the control group showed that the compound had a better glucose utilization rate and the use of insulin in combination enhanced the effect of insulin. Compound 8 was combined with 0.32 μM insulin and observed under a microscope. It was found that fat cells had apoptosis, and it is speculated that this compound may have a cytotoxic or anti-cancer effect.

While observing the increase in glucose utilization rate, it was evaluated whether glucose could be converted into fat and stored in cells. As shown in the third figure, the accumulation of oil droplets in compound 4 was significantly less than that in the control group and the insulin group, compared with the insulin group. The oil droplet content was found to decrease by approximately 48%. As shown in the fourth figure, Compound 4 not only effectively increases the glucose utilization effect but also inhibits fat accumulation in cells.

Eleven compounds were obtained from Aspergillus sydowii , and the expression of anti-inflammatory activity was evaluated by measuring the release of superoxide radical anion. As shown in Table 1, data except for compounds 3 and 10 have the activity of inhibiting the release of more than half of superoxide radical anions, and compounds 4 , 7 , and 8 are compared with the positive control group sorafenib. It is preferred to inhibit superoxide radical anion release activity, wherein compound 4 has a better inhibitory effect, and its IC ₅₀ value is 5.23 ± 0.55 μM. On the performance of elastase release, as shown in Table 2, the performance of compound 8 is better, and its IC ₅₀ value is 6.60 ± 0.16 μM. This result shows that compound 4 has potential for development not only in antidiabetic drugs, but also in anti-diabetic drugs. A superior candidate drug is presented on the inflammatory effect.

Note: [a] The percentage of inhibition (%) is 10 μg/ml, and the data results are expressed as mean ± SEM (n = 3-4). [b] When the percentage of inhibition exceeds 50%, it is expressed as a semi-inhibitory concentration (IC ₅₀ ). [c] The chemical name of sorafenib is 4-{4-[(3-{[4-chloro-3-(trifluoromethyl)phenyl]amino}carbonyl)amino]phenoxy}-N- 4-(4-(3-Chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)-N2-methylpyridine-2-carboxamide (Sorafenib) was used as a positive control group. [d] ND means no measurement.

Note: [b] When the percentage of inhibition exceeds 50%, it is expressed as the semi-inhibitory concentration (IC ₅₀ ). [c] Sorafenib was a positive control group. [d] ND means no measurement.

Assess glucose utilization

Mouse pre-adipocyte cell line 3T3-L1, purchased from the Biological Resource Conservation Center (BCRC), with 450 mg/dL glucose (D-glucose), 0.32 μM insulin (insulin), 0.5 mM 3-isobutyl-1- Methylxanthine (3-isobutyl-1-methylxanthine, IBMX) and 1 μM dexamethasone were induced to differentiate into mature adipocytes for 48 hours and then replaced with cell culture medium. The culture medium contains glucose 100 mg/dL and cultured in DMEM. Base, 10% fetal bovine serum (Fetal Bovin Serum, FBS), 100 U/mL penicillin (penicillin), and 100 μg/mL streptomycin.

The cells were divided into the following groups: (i) 450 mg/dL glucose was added to the culture medium as a control group; (ii) 450 mg/dL glucose and 0.32 μM insulin were added to the insulin group; (iii) in the culture medium. 450 mg/dL glucose, 0.32 μM insulin, and 30 μg/mL of the test compound were added. After 24 hours, the glucose concentration in the cell culture medium was measured with a blood analyzer (Roche Cobas Integra 400 Chemistry Analyzer, Roche Diagnostics, Taipei, Taiwan) to determine whether the test compound promoted increased glucose utilization.

Fat cell oil drop staining test

The mouse pre-adipocyte cell line 3T3-L1 of the above test was subjected to oil droplet staining by oil red dye. The fat cells were fixed with 10% formaldehyde and stained with oil red staining, and the oil in the cells was stained red. After lysing the cells with 100% isopropanol, the absorbance was measured at 490 nm using a Thermo Multiskan Ascent microplate reader. The higher the absorbance value, the more fat cell droplet formation.

Superoxide radical release assay

Containing 0.5 mg / ml ferricytochrome c (ferricytochrome c), 1 mM calcium chloride (CaCl ₂₎ and magnesium chloride (MgCl ₂₎ of neutrophil suspension ^{(1 × 10 6 cells / ml} ), Preheat for 2 minutes at 37 ° C with stirring. After adding the test compound for 5 minutes, 0.1 μM N-formyl-methionyl-leucyl-phenylalanine (fMLP)/1 μg/ml cells were added. Relaxation (cytochalasin B, CB) or 50 nM phorbol 12-myristate 13-acetate (PMA) was reacted for 10 minutes on an ultraviolet/visible spectrophotometer ( Hitachi, U3010) continuously monitors changes in absorbance at a wavelength of 550 nm. Evaluate the superoxide anion radical (O ₂ ^·- ) content released by neutrophils, and the extinction coefficient is 21.1/mM/cm, which can pass 100 U/ml superoxide dismutase (SOD). ) Suppressing the reduction of ferricytochromec, the calculation is known. The calculation formula is as follows: △A550=extinction coefficient×1×△C

△C=△A550 (21.1/mM/cm×1 cm)

△C=△A550×47.4 nmol/ml

Where ΔA550 is the measured absorbance minus the absorbance of the SOD inhibition group, and ΔC is the amount of O ₂ ^·- . As a result, a superoxide generation, expressed in nmol, was produced every 10 minutes at A550 or 1 x 10 ⁶ cells. Sorafenib was used as a positive control group.

Elastase enzyme assay

Human leukocyte elastase (PME elastase, HLE.EC3.4.21.37) is the enzyme target, MeO-Suc-Ala-Pro-Val-p-nitroanilide is the substrate required for the elastase, according to the enzyme The principle of the reaction, the specific absorption value is determined by a high efficiency of the microplate reader, and the anti-inflammatory effect is evaluated.

A neutrophil suspension (1×10 ⁶ cells/ml) containing MeO-Suc-Ala-Pro-Val-p-nitroanilide was preheated for 2 minutes at 37 ° C with stirring, and the compound was added for 5 minutes. Thereafter, the reaction was further carried out by fMLP (0.1 μM)/cytochalasin B (CB, 0.5 μg/mL) for 10 minutes, and the change in absorbance was measured by an ultraviolet/visible spectrophotometer (Hitachi, U3010) at a wavelength of 405 nm. Sorafenib was used as a positive control group.

The above excipients are referred to as "pharmaceutically acceptable carriers or Excipients, "bioavailable carriers or excipients", including solvents, dispersing agents, coatings, antibacterial or antifungal agents, preserving or delaying the absorption of any suitable compound for the preparation of a dosage form. . Usually such carriers or excipients do not themselves have the activity of treating diseases, and the derivatives disclosed in the present technology, together with pharmaceutically acceptable carriers or excipients, are prepared for administration to animals or humans. Causes adverse reactions, allergies or other inappropriate reactions. Thus, the derivatives disclosed in the present technology, in combination with pharmaceutically acceptable carriers or excipients, are suitable for clinical and human use. "Effective dose" means a dose sufficient to ameliorate or prevent a medical condition or a biological state. An effective dose also means that the dose of the administered compound is sufficient for the diagnosis. Unless otherwise stated in the specification, "active compound" and "pharmaceutically active compound" are used interchangeably herein to refer to a substance having a pharmaceutical, pharmacological or therapeutic effect.

The dosage form of the compound can be administered intravenously, orally, by inhalation or by local or sublingual administration such as nasal, rectal, vaginal, etc., to achieve a therapeutic effect. For patients with different conditions, about 0.1 mg to 100 mg of active ingredient is administered daily.

The carrier will vary with each dosage form, and the sterile injectable compositions may be solution or suspended in a non-toxic intravenous diluent or solvent such as 1,3-butanediol. A carrier acceptable therebetween may be Mannitol or water. In addition, fixed oils or synthetic single or diglyceride suspension media are common solvents. Fatty acids, such as oleic acid, olive oil or castor oil, and their glyceride derivatives, especially in the form of polyoxyethylation, are useful as injections and are natural pharmaceutically acceptable oils. These oil solutions or suspensions may contain long chain alcohol diluents or dispersants, carboxymethyl cellulose or similar dispersing agents. Other commonly used surfactants such as Tween, Spans or other similar emulsifiers or used in general pharmaceutical manufacturing A bioavailable enhancer can be used in solid, liquid or other bioavailable formulations.

The composition for oral administration is in any orally acceptable dosage form, and the form thereof includes a capsule, a tablet, a tablet, an emulsifier, a liquid suspension, a dispersing agent, and a solvent. Oral dosage forms are generally used as carriers, and in the case of tablets, lactose, corn starch, and a lubricant such as magnesium stearate are basic additives. The diluent used in the capsules includes lactose and dried corn starch. The liquid suspension or emulsifier dosage form is prepared by suspending or dissolving the active substance in an oily interface combined with an emulsifier or a suspending agent, and adding a moderate sweetener, flavor or pigment as needed.

Nasal gasifying sprays or inhalant compositions can be prepared according to known formulation techniques. For example, the composition is dissolved in physiological saline, benzyl alcohol or other suitable preservative, or an absorbent is added to enhance bioavailability. Compositions of the compounds disclosed in the present technology may also be formulated as a suppository for rectal or vaginal administration.

The compounds disclosed in the present technology may also be administered "intravenous administration", including subcutaneous, intraperitoneal, intravenous, intramuscular, or intra-articular, intracranial, intra-articular, intraspinal injection, aortic injection, intrathoracic injection, diseased parts. Intra-injection, or other suitable drug delivery techniques.

Example: Example 1 Preparation of crude extract

Aspergillus sydowii purchased from the Breast Cancer Resource Center (BCRC) was cultured in potato dextrose agar (PDB) at a rotational speed of 150 rpm at 25 ° C for 10 days. After that, a total of 12 liters of fermentation broth was collected. The mycelium was separated from the culture solution by a filter paper, and the culture solution was filtered, and extracted with ethyl acetate (EtOAc) three times to obtain a crude extract of 0.8 g.

The detailed composition of the potato nutrient solution (PDB), 24 grams of potato nutrient solution is dissolved in 1 liter of water. ^{Difco TM Potato Dextrose Broth (PDB)} , purchased from BD Biosciences (Becton, Dickinson and Company).

Example 2 Separation of column chromatography

0.8 g of the crude extract was loaded on Sephadex as a chromatographic material, methanol was used as a column for the extract, and separated by column chromatography to obtain four partition layers of F1-F4.

The F1 partition layer was placed on a silica gel column, and dichloromethane and methanol (29:1) were used as the extracting liquid, and separated by a gel column chromatography to obtain six divided layers of F1-1 to F1-6.

The F1-2 was divided into layers, and various divisional layers were obtained through various column chromatography methods.

Compound 1 (18.9 mg), 3 (2.1 mg) and 7 (9.4 mg) were obtained by HPLC purification.

Compound 4 (236.2 mg) was purified by chromatography on silica gel column eluting with methylene chloride: ethyl acetate (6:1).

The layer F1-5 was partitioned into a solvent system by dichloromethane and methanol, and then purified by HPLC to obtain compound 6 (5.2 mg) and 11 (70.0 mg).

The partition layer F1-6 was separated by a mixed solvent system of dichloromethane and methanol (6:1), and separated by a silica gel column chromatography method, and confirmed by a thin layer chromatography (TLC) method to obtain a compound 2 (3.1 mg) and 5 (34.9). Mg). Compound 8 (20.4 mg) was separated from the layer F2 by a silica gel column chromatography of dichloromethane and methanol (14:1) as solvent system. This compound was further purified by HPLC.

The fractionation layer F4 was separated and purified by ruthenium column chromatography, and extracted from increasing polarity by dichloromethane and methanol solvent system, and confirmed by thin layer chromatography (TLC) method to obtain 9 (8.0 mg) and 10 (4.1 mg). ).

Example 3 HPLC separation

Compounds 1, 3, 4, 6, 7, 8, and 11 were obtained according to the conditions of HPLC, and the reference of the fingerprint was established by the HPLC method. The sample concentration was prepared at 50 mg/ml, and 100 μL was sampled, using an ODS column having an inner diameter (I.D.) of 10 mm, a length (L) of 250 mm, and a particle size of 5 μm, and a detection wavelength of 215 nm.

The HPLC conditions for the separation of compounds 1, 3, and 7 were octadecylsilyl (ODS Hypersil) Thermo column (250 × 10 mm, 5 μm, 2.5 mL / min), and the mobile phase composition was acetonitrile. 80:20 (v/v) solution of water. HPLC conditions for separation of compound 4, octadecyl decane (ODS Hypersil) Thermo column (250 x 10 mm, 5 μm, 2.5 mL/min), mobile phase composition of acetonitrile and water 60:40 (v/v) . HPLC conditions for separation of compounds 6, 11 , octadecyl decane (ODS Hypersil) Thermo column (250 × 10 mm, 5 μm, 2.5 mL / min), mobile phase composition of acetonitrile and water 35:65 (v /v). The HPLC conditions for the isolation of compound 8, the octadecyl decane (ODS Hypersil) Thermo column (250 x 10 mm, 5 μm, 2.5 mL/min), and the mobile phase composition was 50:50 (v/v) of acetonitrile and water.

Example 4 Data of purified compounds

Hydrogen nuclear magnetic resonance spectroscopy ( ¹ H-NMR) was determined by deuterated chloroform (CDCl ₃ ) 400 MHz, 600 MHz, carbon nuclear magnetic resonance spectroscopy ( ¹³ C-NMR) deuterated chloroform 100 MHz, 150 MHz, heteronuclear single quantum Relationship (heteronuclear singular quantum correlation, HSQC), Heternuclear Multiple Bonding Correlation (HMBC).

Compound 1 7-methylsuditol ((7 S )-(+)-7- O- Methylsydonol): colorless gum; +1.87 ( c 1.69, CH ₂ Cl ₂ ); infrared spectrum (IR, Neat) v _max 3308, 2952, 2869, 1627, 1575, 1459, 1265, 1160, 1042 cm ^-1 ; UV spectrum (UV, CH ₃ OH) λ _max (log ε ) 207 (4.30), 223 (3.99), 279 (3.64) nm; ¹ H-NMR (CDCl ₃ , 600 MHz) and ¹³ C-NMR (CDCl ₃ , 150 MHz) data, respectively Table 3; positive ion mode mass spectrometry (MS-ESI ⁺ ) m/z 289 [M+Na] ⁺ ; HRMS (ESI ⁺ ) m/z calcd for C ₁₆ H ₂₆ O ₃ Na[M+Na] ⁺ 289.1779, Found 289.1780

Compound 2 7,12-dihydroxydimethylhexane hydroxybenzoic acid ((7 S , 11 S )-(+)-12-Hydroxysydonic acid): colorless oil; +3.9 ( c 0.39, CH ₃ OH); IR (Neat) v _max 3385, 2928, 1704, 1575, 1392, 1228, 1030 cm ^-1 ; UV(CH ₃ OH) λ _max (log ε ) 210 (4.59) , 241 (4.15), 294 (3.70) nm; ¹ H-NMR (CD ₃ OD, 400 MHz) and ¹³ C-NMR (CD ₃ OD, 100 MHz) data are shown in Table 3, respectively.

(MS-ESI ⁺ ) m/z 305 [M+Na] ⁺ ; HRMS (ESI ⁺ ) m/z calcd for C ₁₅ H ₂₂ O ₅ Na[M+Na] ⁺ 305.1362,found 305.1365

Compound 3 7-Deoxy-7,14-didehydrosydonol: colorless oil; IR(Neat) v _max 3335, 2953, 2927, 1608, 1423, 1289 , 1027 cm ^-1 ; UV(CH ₃ OH) λ _max (log ε ) 209 (4.75), 238 (4.22), 283 (3.95), 333 (3.58) nm; ¹ H-NMR (CDCl ₃ , 600 MHz) Data with ¹³ C-NMR (CDCl ₃ , 150 MHz) are listed in Table 3; MS (ESI ⁺ ) m/z 257 [M+Na] ⁺ ; HRMS (ESI+) m/z calcd for C ₁₅ H ₂₂ O ₂ Na[M+Na] ⁺ 257.1516, found 257.1517

The structural formula is determined according to the relevant literature information; the reference of compound 4 is Kudo, S. et al., Biosci. Biotech. Bioch. 2009, 73 , 203; the reference of compound 5 is Hamasaki, T. et al., Agr. Biol. Chem. 1978, 42 , 37 and Li, D. et al., Mar. Drugs 2012, 10 , 234; reference to compound 6 is Herne, P. et al., Liebigs Ann. Chem. 1993, 1993 , 565; reference to compound 7 is Sumarah, MW et al., Phytochemistry 2011, 72 , 1833; reference to compound 8 is Kim, HS et al., J. Antibiot. 2002, 55 , 669; The literature is Nakanishi, S. et al., J. Antibiot. 1993, 46 , 1775; reference to compound 10 is Hamasaki, T. et al., Agr. Biol. Chem. 1975, 39 , 2341; The literature is Itabashi, T. et al., Chem. Pharm. Bull. 1993, 41 , 2040.

Example 5 Composition for preparing a tablet

The following components are separately weighed according to the amount, mixed and filled in a tableting machine to prepare a tablet

In summary, the present invention uses the mouse pre-adipocyte cell line 3T3-L1 to evaluate 11 compounds isolated and purified from Aspergillus sydowii , and whether it can promote the increase of glucose utilization rate as a model for developing drugs. The isolated crude extract or purified compound can effectively increase the glucose utilization effect and inhibit the accumulation of fat in the cells. The elastase enzyme assay and the superoxide radical release assay found that the compound isolated and purified from the fungal extract also inhibited inflammation. It is shown that the crude extract or compound of the present invention can be used in industrial products, and is indeed progressive and novel.

Therefore, even though the present invention has been described in detail by the above-described embodiments, it can be modified by those skilled in the art, and is not intended to be protected as claimed.

Other embodiments

A use of a composition for the preparation of an agent for increasing glucose utilization, the composition comprising an extract of Aspergillus sydowii .

2. Use of a composition for the preparation of an agent for increasing glucose utilization, the composition comprising a compound selected from the group consisting of formula (II) or formula (III);

Wherein R ₇ is a double bondoxy group a group, R ₁₁ may be selected from one of a hydroxyl group, a C _{1 to 3} alkoxy group; R ₈ may be selected from a hydrogen group, a hydroxyl group, a C _{1 to 3} alkoxy group, a C _{1 to 5} alkyl group. .

3. The use of the composition according to items 1 and 2 of the present embodiment relates to diabetes and metabolic diseases; or in combination with insulin, which can enhance the effect of insulin; or inhibit the accumulation of fat in cells; or anti-inflammatory.

4. A composition for increasing glucose utilization selected from a compound of formula (I); Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be selected from the group consisting of a hydrogen group, a hydroxyl group, a C _{1 to 3} alkoxy group, a C _{1 to 5} alkyl group, and a C _{1 to 5} containing a single double. One of the alkenyl groups of the bond; if R ₃ and R _{4 are} combined, it may be a methylene group having a double bond (-CH ₂ ); or one of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ Can be double-bonded with oxygen The group, R _{11 ,} may be selected from one of a hydrogen group, a hydroxyl group, and a methoxy group.

5. The composition according to item 4 of the present embodiment, for use in the treatment of diabetes and metabolic diseases; or in combination with insulin, which enhances the effect of insulin; or inhibits fat accumulation in cells; or anti-inflammatory.

6. A compound, as shown in formula (I), purified from Aspergillus sydowii extract; Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be selected from the group consisting of a hydrogen group, a hydroxyl group, a C _{1 to 3} alkoxy group, a C _{1 to 5} alkyl group, and a C _{1 to 5} containing a single double. One of the alkenyl groups of the bond; if R ₃ and R _{4 are} combined, it may be a methylene group having a double bond (-CH ₂ ); or one of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ Double bond oxygen The group R ₁₁ may be selected from one of a hydrogen group, a hydroxyl group and a methoxy group.

7. Use of a composition for the preparation of an agent for increasing glucose utilization, the composition comprising an HPLC chromatogram of an ethyl acetate extract of Aspergillus sydowii .

8. Use of a composition for the preparation of an agent for increasing glucose utilization, the composition comprising a salt selected from the group consisting of stenol (( S )-(+)-sydonol), 7-hydroxydimethylhexyl Hydroxybenzoic acid (( S )-(+)-sydonic acid), 7-dehydroxywaltol B (anhydrowaraterpol B), 1-hydroxytrimethylheptenylmethylphenol (( E )-5-( Hydroxymethyl)-2-(6'-methylhept-2'-en-2'-yl)phenol), xanthone methoxy ester (AGI-B4); compound 9 is sydowinin B, A compound of Sydowinin A, diorcinol or a mixture thereof.

9. A method of increasing the utilization of glucose composition, selected from 7-ol Christopher ((7 S) - (+ ) - 7- O -methylsydonol), 7,12- dihydroxy dimethyl adipate Alcoholic acid ((7 S , 11 S )-(+)-12-hydroxysydonic acid) 7-methylsuditol ((7 S )-(+)-7- O -methylsydonol); compound 2 is 7 , 12-dihydroxydimethylhexane hydroxybenzoic acid ((7 S , 11 S )-(+)-12-hydroxysydonic acid); compound 3 is 7-deoxy-7,14-didehydrogens A compound of 7-deoxy-7,14-didehydrosydonol, 7-deoxy-7,14-didehydrosydonol or a mixture thereof.

A compound, Christopher 7-ol ((7 S) - (+ ) - 7- O -methylsydonol), based Sabouraud purified from Aspergillus (Aspergillus sydowii) extract.

11. A compound, 7,12-dihydroxydimethylhexane hydroxybenzoic acid ((7 S , 11 S )-(+)-12-hydroxysydonic acid), purified from Aspergillus sydowii Things.

12. A compound, 7-deoxy-7,14-didehydrosydonol, purified from Aspergillus sydowii extract.

references

Kudo, S. et al., Biosci. Biotech. Bioch. 2009, 73 , 203

Hamasaki, T. et al., Agr. Biol. Chem. 1978, 42 , 37

Li, D. et al., Mar. Drugs 2012, 10 , 234

Henne, P. et al., Liebigs Ann. Chem. 1993, 1993 , 565

Sumarah, MW et al., Phytochemistry 2011, 72 , 1833

Kim, HS et al., J. Antibiot. 2002, 55 , 669

Nakanishi, S. et al., J. Antibiot. 1993, 46 , 1775

Hamasaki, T. et al., Agr. Biol. Chem. 1975, 39 , 2341

Itabashi, T. et al., Chem. Pharm. Bull. 1993, 41 , 2040.

Claims (10)

A use of a composition for the preparation of an agent for improving diabetes, the composition comprising an extract of Aspergillus sydowii . Use of a composition for the preparation of an agent for ameliorating metabolic diseases, the composition comprising an extract of Aspergillus sydowii . Use of a composition for the preparation of a medicament for improving diabetes, the composition comprising a compound selected from the group consisting of formula (II), formula (III) or formula (IV); Wherein R ₇ is a double bond oxy group a group, R ₁₁ may be selected from one of a hydroxyl group, a C _{1 to 3} alkoxy group; R ₈ may be selected from a hydrogen group, a hydroxyl group, a C _{1 to 3} alkoxy group, a C _{1 to 5} alkyl group. . The use of the composition as described in claims 1, 2, and 3 relates to increasing the utilization rate of glucose; or in combination with insulin, the effect of insulin is enhanced; or the accumulation of fat in cells is inhibited; or anti-inflammatory. A composition for improving metabolic diseases, which is selected from the group consisting of compounds of formula (I); Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be selected from the group consisting of a hydrogen group, a hydroxyl group, a C _{1 to 3} alkoxy group, a C _{1 to 5} alkyl group, and a C _{1 to 5} containing a single double. One of the alkenyl groups of the bond; if R ₃ and R _{4 are} combined, it may be a methylene group having a double bond (-CH ₂ ); or one of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ Can be double-bonded with oxygen The group, R _{11 ,} may be selected from one of a hydrogen group, a hydroxyl group, and a methoxy group. The composition as claimed in claim 5 is for improving diabetes; increasing glucose utilization; or administering the drug in combination with insulin to enhance the effect of insulin; or inhibiting fat accumulation in cells; or anti-inflammatory. a compound, as shown in formula (I), purified from Aspergillus sydowii extract; Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be selected from the group consisting of a hydrogen group, a hydroxyl group, a C _{1 to 3} alkoxy group, a C _{1 to 5} alkyl group, and a C _{1 to 5} containing a single double. One of the alkenyl groups of the bond; if R ₃ and R _{4 are} combined, it may be a methylene group having a double bond (-CH ₂ ); or one of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ Double bond oxygen The group R ₁₁ may be selected from one of a hydrogen group, a hydroxyl group and a methoxy group. A use of a composition for the preparation of a medicament for improving diabetes comprising a composition selected from the group consisting of stenol (( S )-(+)-sydonol), 7-hydroxydimethylhexyl hydroxybenzoic acid ( ( S )-(+)-sydonic acid), 7-dehydroxywaltol B, 1-hydroxytrimethylheptenylmethylphenol (( E )-5-(hydroxymethyl)-2 -(6'-methylhept-2'-en-2'-yl)phenol), xanthone methoxylate (AGI-B4); compound 9 is sydowinin B, Sadovin A (sydowinin A), a compound of diorcinol or a mixture thereof. Use of a composition for the preparation of an agent for improving metabolic disorders, comprising a composition selected from the group consisting of stenol (( S )-(+)-sydonol), 7-hydroxydimethylhexyl hydroxybenzene Formic acid (( S )-(+)-sydonic acid), 7-dehydroxywaltol B (anhydrowaraterpol B), 1-hydroxytrimethylheptenylmethylphenol (( E )-5-(hydroxymethyl) -2-(6'-methylhept-2'-en-2'-yl)phenol), xanthone methoxy ester (AGI-B4); compound 9 is sydowinin B, Sado A compound of sydowinin A, diorcinol or a mixture thereof. The composition as described in claims 8 and 9 is used to increase the utilization rate of glucose; or to be combined with insulin to enhance the effect of insulin; or to inhibit the accumulation of fat in cells; or to resist inflammation.