KR20010025876A - Invention of a new α-Glucosidase inhibitor from a Fungus, Penicillium sp. F70614(KCTC 8918P) - Google Patents

Abstract

PURPOSE: A Penicillium sp. F70614(KCTC 8918P) and an α-glucosidase inhibitor(diketopiperazine derivaives(formula)) are provided, which inhibits α-glucosidase effectively, so can be used for treatment and prevention of diabetes and obesity. CONSTITUTION: A process for the preparation of Penicillium sp. F70614(KCTC 8918P) and α-glucosidase inhibitor comprises the steps of: inoculating Penicillium sp. F70614(KCTC 8918P) on a prepared plate, and culturing for 3days at 28deg.C; inoculating the cultured solution in a fermentor, and culturing for 7days at 28deg.C; centrifuging the final cultured solution to separate cultured supernatant and bacillus body, extracting the bacillus body with acetone and concentrating under decompression to remove acetone, and mixing with the cultured supernatant and extracting with ethylacetate; concentrating the ethylacetate extract, and processing silicagel chromatography, LH-20 column chromatography and high-pressure liquid chromatography to get pure active ingredients; removing solvent using a decompressed dryer, and freeze-drying to get the white diketopiperazine derivaives powder(which can be also prepared by chemical synthesis).

Description

Penicillium sp. (Invention of a new α-Glucosidase inhibitor from a Fungus, Penicillium sp. F70614 (KCTC 8918P)}

The present invention relates to a diketopiperazine derivative represented by the following Chemical Formula 1 having penicillium genus (Penicillium sp.) F70614 (KCTC 8918P) and α-glucosidase inhibitory activity.

Formula 1

Sugar's anmeric hydroxy group is reversibly condensed with sugars to form α, β-glycosides, and glycosidic bonds are used to link the anomer carbon and oxygen. It is called). Hydrolysis of these glycosidic bonds is catalyzed by an enzyme called glycosidase.

Among the various carbohydrate cleavage enzymes, α-glucosidase is an important enzyme involved in the final stage of carbohydrate digestion [Jun. W., Biosci. Biotech. Bioch., 61 (1), 177-178, 1997], which has been used medically for the prevention and treatment of diabetes and obesity [D. J. A. Diabetes, 30, 951-954, 1981. That is, α-glucosidase has a function of cleaving glycosidic bonds between sugars and sugars, sugars and lipids, or sugars and proteins in polysaccharides in the cell, and thus inhibiting the glucosidase enzyme reaction in the small intestine. This delay is effective in treating diabetes and obesity.

On the other hand, the α-glucosidase inhibitor is more specific than the α-amylase in being involved in the suppression of postprandial blood sugar elevation due to the ingestion of starch and saccharides. α-glucosidase inhibitors, unlike sulfonylurease-based compounds reduce insulin secretion, and has the advantage of not causing hyperinsulinemia, which causes atherosclerosis and hypertension in diabetic patients. In addition, diseases caused by lack of glycosidase, such as mannosidosis, lack mannosidase, resulting in the accumulation of sugar chains containing mannose in cells. It will be toxic. Many other diseases have been associated with quantitative changes in glycosidase. For example, high concentrations of glycosidase have been found in serum from cancer patients or secretions between cancer cells. In vitro experiments with β-N-acetyl glucose aminidase and β-glucuronidase found that many cancer cells secrete these enzymes into extracellular fluid. These enzymes not only accelerate the differentiation of cancer cells from the primary site, but also aid in the movement or attack of other secondary sites, and thus the structure of the oligosaccharides on the cell surface. It is also involved in biosynthesis and morphological changes in cancer cells. Thus, glycosidase inhibitors may prevent cancer progression or mutation. It is also involved in the structure and biosynthesis of oligosaccharides on the cell surface and in morphological changes in cancer cells. For example, recently reported nojirimycin, a potent α-glucosidase inhibitor, is known to inhibit HIV infection of T cells [R. A. Gruters, Nature, 330, 74-77, 1987].

The present invention isolates the new strain Penicillium sp. F70614 (KCTC 8918P) from soil fungi, diketopiperazine represented by the formula (1) obtained from the new strain or synthesized by chemical methods The present invention has been completed by knowing that the derivative has α-glucosidase inhibitory activity.

1 is a micrograph of Penicillium sp. F70614 (KCTC 8918P).

Figure 2 is a graph showing cell growth and α-glucosidase inhibition rate according to the penicillium sp. (Penicillium sp.) F70614 (KCTC 8918P) strain culture period.

3 is a FAB mass spectrometry spectrum of a diketopiperazine derivative represented by Chemical Formula 1. FIG.

FIG. 4 is a 300 MHz hydrogen nuclear magnetic resonance spectrum of a diketopiperazine derivative represented by Chemical Formula 1 in a CD ₃ OD solution. FIG.

FIG. 5 is a 300 MHz carbon nuclear magnetic resonance spectrum of a diketopiperazine derivative represented by Chemical Formula 1 in a CD ₃ OD solution. FIG.

6 is a graph showing the activity of pig small intestine α-glucosidase according to the dose of diketopiperazine derivatives represented by the formula (1).

The present invention is characterized by Penicillium sp. F70614 (KCTC 8918P), which produces α-glucosidase inhibitors.

In addition, the present invention includes a diketopiperazine derivative represented by the following formula (1) having an α-glucosidase inhibitory activity, and a preparation method thereof.

Formula 1

In addition, the present invention includes an α-glucosidase inhibitor containing a diketopiperazine derivative represented by the formula (1).

Referring to the present invention in more detail as follows.

1. Identification of Mycobacteria

The results observed after inoculation of fungal strains isolated from soils in Korea were inoculated in potato-dextrose agar, Czapek's agar and malt extract agar.

Growth was very good and colony diameter reached 30-40 mm at 7 ° C. for 7 days. The surface color was yellowish green and the backside color was dark green.

According to the results of observation using optical and electron microscopy in potato-dextrose agar medium for morphological observation [see FIG. 1], the conidiophore started from the basal hyphae, and penicillia was the only monoverticillate). Phialides range in size from 2 to 3 × 7.5 Μm, the shape of conidia changed from globose to subglobose, and the diameter was 2.0-2.5 μm.

In view of the above characteristics, it was found that the producing strain belongs to the genus Penicillium sp. Thus, in the present invention, the new strain was named Penicillium sp. F70614, and was deposited on December 16, 1998 at the Genetic Resource Center in the Institute of Biotechnology and was given an accession number of KCTC 8918P.

2. Cultivation of Mycobacteria

Strain culture in the present invention is cultured in a medium containing a nutrient source that can be used by ordinary microorganisms. For example, glucose is used as the carbon source, and yeast extract and polypeptone are used as the nitrogen source. And inorganic salts, such as magnesium sulfate and dipotassium phosphate, are added and cultured. As a culture method, shaking culture or aeration stirring culture in aerobic conditions is preferable. The culture temperature was slightly different depending on the conditions, but it was appropriate to incubate at 25 ℃ ~ 30 ℃, it was incubated at 28 ℃. In addition, in the case of incubation period or shaking culture, the production of the new compound represented by Chemical Formula 1 reached the highest by culturing for 5 to 7 days [see FIG. 2].

3. Isolation and Purification of New Compounds

Penicillium sp. F70614 (KCTC 8918P) strains were cultivated under the culture conditions as described above to obtain a novel diketopiperazine derivative represented by Chemical Formula 1. Since the diketopiperazine derivative represented by Chemical Formula 1 is present in the bacterial culture as well as the strain culture solution, the culture solution is centrifuged to separate the culture supernatant and the bacteria and then efficiently extracted and purified by the method described below. That is, since the diketopiperazine derivative represented by Chemical Formula 1 is a fat-soluble substance, the culture solution is extracted by adding an organic solvent such as ethyl acetate or chloroform. On the other hand, the cell part is agitated by adding an organic solvent such as acetone, and extracting the active ingredient from the cell, the solvent such as acetone is evaporated and extracted again with ethyl acetate, chloroform and then combined with the culture solution. After solvents such as ethyl acetate and chloroform are evaporated to dryness from the mixture containing the obtained active ingredient, silica gel column chromatography is performed using a solvent of chloroform / methanol = 100/1 to 10/1 (V / V). Then, the obtained active site was subjected to Sephadex LH-20 column chromatography again with chloroform / methanol = 1/1 (V / V). The pure effective compound can then be obtained by HPLC. By repeating the separation and purification method as described above alone or in combination, it is possible to obtain a pure purified product more easily.

NMR analysis for structural analysis was carried out using Varian UNITY 300, the solvent was measured using CD ₃ OD. The molecular weight was determined by electrospray ionization mass spectroscopy (ESIMS), and the UV spectrum (Shimazu UV-260) was measured by methanol as a solvent.

The active substance produced by Penicillium sp. F70614 (KCTC 8918P) strain was white powder and showed the maximum absorption peak at UV λ _max (MeOH) 208 nm. On the HR-mode, the calculated value was 182.1055 and found 182.1056. As a result, the molecular formula was determined as C ₉ H ₁₄ N ₂ O ₂ , and the structure was confirmed by ¹ H, ¹³ C, and heteronuclear multiple bond coherence (HMBC). .

The physicochemical characteristics of the other active substances are as follows:

1) Property: White powder

2) Molecular Weight: 182

3) Molecular Formula: C ₉ H ₁₄ N ₂ O ₂

4) Mass spectrometry (M + H): 182 [See FIG. 3]

5) UV absorption spectrum (UV λ _max (MeOH)): 208, 350 nm

6) Infrared absorption spectrum (KBr) cm ^-1 : 3447, 2928, 1678, 1637, 1458, 1337

7) nuclear magnetic resonance (NMR) absorption spectrum: a CD ₃ OD as solvent and TMS (Si (CH _{3) 4),} hydrogen nuclear magnetic resonance ⁽¹ H-NMR) spectra and carbon nuclear magnetic resonance ⁽¹³ to a standard C-NMR) spectra were examined and shown in FIGS. 4 and 5, respectively.

8) Solubility

Soluble: Acetone, Chloroform, Ethyl Acetate, Methanol

Insoluble: water

9) Thin layer chromatography: R _f value 0.39

Adsorbent: Merck Silica Gel 60F 254

Developing Solvent: CHCl ₃ / MeOH = 10/1 (V / V)

10) Chemical Structural Formula

4. Chemical Synthesis of Novel Compounds

The diketopiperazine derivative represented by Chemical Formula 1 may be obtained from Penicillium sp. F70614 (KCTC 8918P) strain culture, and may also be prepared by chemical synthesis. That is, the diketopiperazine derivative represented by Chemical Formula 1 has a structure in which dehydroalanine and leucine are bound. First, cyclization of t-boc-serine and leucine methyl ester is performed, followed by tos-chloride. It synthesize | combines by removing OH group of serine residue using

5. Assay of α-glucosidase inhibitory activity on novel compounds

Α-glucosidase inhibitory activity of the diketopiperazine derivative represented by Formula 1 was 30 μl of the sample, 250 μl of buffer (0.1 M phosphate buffer pH 7.0) and p-nitrophenyl-α-D-glucose as a substrate. 250 μl of ranoside (5 mM, pH 7.0) was added, followed by 10 μl of enzyme solution (α-glucosidase (4.75 unit / ml, Sigma). 2 ml of sodium hydroxide solution (pH 10.4) was added to stop the reaction, and the absorbance was measured at 410 nm, and the amount of glucose liberated by enzymatic action was measured when enzyme activity was measured using maltose, a natural substrate. The enzyme was measured at 500 nm using the GLzyme kit, and then the results were substituted into the following equation to calculate the enzyme activity inhibition rate.

In Equation 1: A is the absorbance before the reaction of the inhibitor; B is the absorbance after the reaction with the inhibitor; C is the absorbance before the reaction without the inhibitor; D is the absorbance after the reaction with no inhibitor.

Accordingly, the present invention includes an α-glucosidase inhibitor containing the diketopiperazine derivative represented by Formula 1 as an active ingredient. In addition to the diketopiperazine derivative represented by Chemical Formula 1, the medicament according to the present invention contains a pharmaceutically acceptable carrier or excipient which can be easily understood by those skilled in the art, and includes tablets, powders, It may be formulated into granules, capsules, suspensions, emulsions or formulations for parenteral or parenteral administration for parenteral administration. The effective dosage of the medicament may vary depending on the age, physical condition, weight, etc. of the patient, and is generally administered within the range of 1 to 10 mg / kg (weight) / day. In addition, within a daily effective dosage range, it can be injected once or several times a day.

The present invention as described above will be described in more detail based on the following examples, but the present invention is not limited by the following examples.

Example 1 Microbial Culture

It was used after adjusting to pH 5.6 in a medium containing 2% glucose, 0.2% yeast extract, 0.5% polypeptone, 0.05% magnesium sulfate, and 0.1% potassium diphosphate as seed and production medium. 100 ml of two 500 ml Erlenmeyer flasks were sterilized for 20 minutes at 121 ° C., and then inoculated with 1 to 2 platinum seeds of penicillium sp. F70614 strain. Shaking culture for 3 days at 28 ℃ was used as species culture of the incubator. 200 ml of the seed culture solution was inoculated into a 5 L fermentor containing 3 L production medium sterilized at 121 ° C. for 30 minutes, and incubated at 28 ° C. for 7 days under aeration at 400 rpm.

Example 2 Determination of Structure of Culture Material

The culture supernatant and the cells were separated by centrifuging 6 l of the culture filtrate obtained in Example 1 at 12,000 × g for 15 minutes, and the cells were again extracted twice with the same amount of 100% acetone and concentrated under reduced pressure to remove acetone. Combined and extracted again three times with the same amount of ethyl acetate. The ethyl acetate extract was concentrated, dissolved in chloroform, and developed on silica gel (70-230 mesh, Merck) chromatography with an organic solvent composed of chloroform / methanol = 100/1 → 10/1 (V / V) to obtain an active fraction. Collected. The active fractions were concentrated and developed on Sephadex LH-20 (Pharmacia) column chromatography using a developing solvent of chloroform / methanol = 1/1 (V / V) to collect the active fractions. The active fractions were concentrated and separated on high pressure liquid chromatography (column: YMC-pack ODS 250 × 100 mm, 5 µm, flow rate 1 ml / min, 254 nm detection, eluent 50% methanol) at a residence time of about 20 minutes. The active substance was purely separated, the solvent was removed with a reduced pressure drier, and lyophilized to obtain 5 mg of white powder.

NMR analysis for structural analysis was carried out using Varian UNITY 300, the solvent was measured using CD ₃ OD. Molecular weight was determined by fast atom bombardment (FAB) and electrospray ionization mass spectroscopy (ESIMS), and UV spectrum (Shimazu UV-260) was measured by methanol as a solvent.

As a result, the physicochemical characteristics of the active material produced by Penicillium sp. (Penicillium sp.) F70614 (KCTC 8918P) strain is shown in Table 1. That is, the active material showed a maximum absorption peak at a UV λ _max (MeOH) 208 nm as a white powder, the molecular weight was found to be 182 by FAB, ESIMS analysis. In addition, as a result of structural analysis by NMR, the molecular formula was determined as C ₉ H ₁₄ N ₂ O ₂ , and the structure was confirmed by heteronuclear multiple bond coherence (HMBC).

Example 3 Chemical Synthesis of Diketopiperazine Derivatives of Formula 1

205 mg of t-Boc-serine (205 mg, 1 mmol) was added to methylene chloride (10 ml), leucine methyl ester (181 mg, 1 mmol), triethylamine (140 μl, 1 mmol) and N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride (192 mg, 1 mmol) was added. The reaction solution was reacted under ice bath for one day. Methylene chloride (10 mL) was added and washed with distilled water (40 mL), then the organic layer was washed with 1N citric acid (40 mL), washed with 1N sodium bicarbonate (40 mL) and dried to give a liquid (240 mg). Got. Formic acid (20 mL) was added and then maintained at room temperature for 2 hours 30 minutes, sec-butanol (20 mL) and toluene (10 mL) were added and refluxed for 2 hours 30 minutes. Ether (20 mL) was added, washed with methanol and dried to obtain L-ser-L-leu as a yellow liquid cycle. The above synthesized cycle was added to the L-ser-L-leu at 0 ℃ p-toluenesulfonyl chloride / pyridine solution for 2 hours, maintained at 0 ℃ for 12 hours and dried. The resultant was washed with a mixed solution of ether and petroleum ether (1: 1) to obtain L-leu-ser-Tos as a cycle. L-leu-ser-Tos was added to and dissolved in dimethylformamide (DMF), and diethylformamide (10.45 ml) was added thereto, maintained at room temperature for 5 hours, and then dried to represent the above formula (1). Diketopiperazine derivatives were synthesized.

Example 4: Measurement of α-glucosidase inhibitory activity

The degree of inhibitory activity of the diketopiperazine derivatives represented by the formula (1) on α-glucosidase was investigated. Enzyme inhibition rate according to the diketopiperazine derivative dosage was calculated as shown in Equation 1 shown in Figure 6, the IC ₅₀ value was 35 ㎍ / ㎖ (yeast enzyme), 50 ㎍ / ㎖ (pork small intestine).

In addition, a comparison of the activity of the diketopiperazine derivative represented by Formula 1 with respect to other glucosidase is shown in Table 2 below.

Example 6 Acute Toxicity Study of α-Glucosidase Inhibitors

The experimental animals used 6-week-old female ICR mice with SPF (Specific Pathogens Free) maintained by the Biotechnology Institute. The experimental animals were maintained in an animal room with a temperature of 22 ± 1 ° C., a humidity of 55 ± 5% and illumination of 12L / 12D. Mice were purified prior to use in experiments. The sample was prepared at a dose of 30 mg / kg. The administration method was first divided into three mice per group and then orally administered once by 0.1 ml based on the 10 g-weight of the mouse. Mouse weight change and death were observed for 10 days after drug administration. On the day of dosing, 1 hour, 4 hours, 8 hours, and 12 hours after the administration, the change of general symptoms and the presence of dead animals were observed at least once in the morning and afternoon every day from the next day to the 10th day of administration. In addition, the change in body weight was measured at intervals of 3 days from the day of administration to observe the weight change of the animal by the sample. As a result of the acute oral toxicity test, no mortality was observed for 10 days after oral administration of this sample at a dose of 100 mg / kg. At necropsy, there were no gross lesions, and normal body weight gain was observed without any weight loss for 10 days from the day after administration. In conclusion, no toxicity was observed in this sample upon oral administration (see Tables 3 and 4).

As described above, the diketopiperazine derivative represented by Chemical Formula 1 synthesized by Penicillium genus Penicillium sp. F70614 (KCTC 8918P) or synthesized by a chemical synthesis method according to the present invention is α- Because of its excellent inhibitory activity against glucosidase, it is useful as an effective drug for the treatment and prevention of diabetes and obesity.

Claims (4)

Penicillium sp. F70614, characterized by producing α-glucosidase inhibitors (KCTC 8918P). Diketopiperazine derivatives, characterized in that represented by the formula (1). Formula 1 Α-glucosidase inhibitor, characterized in that it contains a diketopiperazine derivative represented by the following formula (1). Formula 1 After performing the cyclization reaction of t-boc-serine and leucine methyl ester, diketopipera represented by the following Chemical Formula 1 is characterized by removing the serine residue (OH group) using p-toluenesulfonyl chloride. Method for preparing gin derivatives. Formula 1