JPH0283351A - Novel alpha-glucosidase-inhibiting substance benanomicin c and production thereof - Google Patents

Abstract

NEW MATERIAL:Benanomicin C expressed by formula. USE:An alpha-glucosidase inhibitor useful as an antiobese agent. PREPARATION:The compound of formula can be separated form the cultured product of a benanomicin C-producing strain belonging to actinomycete (e.g., MH193-16F4 strain).

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to Benanomicin C, a novel α-glucosidase inhibitor, and a method for producing the same.

(Prior Art) Compounds having similar structural features to the α-glucosidase inhibitor benanomycin C according to the present invention include benanomycin A and B (Japanese Patent Application No. 62-277.692)
Dexylosylbenanomycin B (Patent application 1986-327.
163), 5F-2446 [Takeda et al.: rJ,
AntibioticsJ 41. 417-4
24. 1988, KS-619-1[14ats
uda et al.: rJ, AntibioticsJ 40
．． 1104-1114 (1987)], G-2N and G-2A [Gerber et al.: rCanad, J.

Chem, J 62.2818-2821 (1984)
Ko, etc. are known. However, benanomycin C has different physicochemical and biological properties from these substances and is clearly distinguished from them. Benanomycin A and B and dexylosylbenanomycin B discovered by the present inventors exhibit effective antifungal activity, but benanomycin C of the present invention exhibits only weak antifungal activity.

(Problem to be Solved by the Invention) Various enzyme inhibitors produced by microorganisms have been known, but not many α-glucosidase inhibitors useful as anti-obesity agents have been found. There is a constant demand in the field for new α-glucosidase inhibitors.

An object of the present invention is to provide benanomycin C, a novel α-glucosidase inhibitor, and a method for producing the same.

(Means for Solving the Problems) The first gist of the present invention resides in a novel α-glucosidase inhibitor benanomycin C represented by the above formula (1) and a salt thereof.

1. Listing the physical and chemical properties of benanomycin C:
It is as follows.

(1) Color and shape: Reddish brown powder (2) Molecular formula: C2, H2, No. 2 (3) Mass spectrum (FD-MS): m/z 579 (M”)
(4) Melting point: >220°C (5) Specific optical rotation: [α♂2=unmeasurable (c O, 0
5, DMSO) (6) Ultraviolet and visible absorption spectra λ. ax njEe) [MeO) II: 205 (625), 230 (sh
, 490), 284(380).

300 (sh, 300), 480 (150) [0,
IN HCI-MeOH]: 208 (536), 2
35(487), 287(391).

300 (sh, 350), 476 (162) [0,
IN NaOH-MeOl(]: 214(1252)
, 245 (sh, 500).

290 (sh, 265), 320 (sh, 218)
, 503(182)(7) Infrared absorption spectrum (KBr m-1): 3220.2920. , 171
5.1605.1480.1445゜1390, 135
0.12g0.1250.1200.1160°114
0, 1065.1030. 990. 960. 91
5°860, 840. 770 (8) Jl-NMltschect) L/ (400Ml
(z, DMSO-dG, 40°C) δ (ppm): 1
．． 36(3H,d), 2.34(3t(,s),
3.93 (6H, s).

4.44 (IH, dq), 4.54 (LH, d),
5.06 (LH, d).

6.83 (II (, br s), 6.89 (LH, d)
, 7.26 (ltl, d).

8.48(LH,d), 8.63(], )(,br
), 13.25 (IH, s).

14.67 (IH, br) (9) “C-NMR spectrum # (100MHz, DM
SO-ds, 40'C:) δ (ppm): 187.9
s, 185.2 s, 174. Os, 167.
1 s.

165.3s, 164.5s, 155.3s
, 153.6 s.

151.8 s, 144.1 s, 139.6 s
, 137.2 s.

133.6s, 130.8s, 127.7s
, 123.7 d.

121.7s, 115.3s, 115.3s
, 110.9 s.

107.3 d, 106.3 d, 71.1 d
, 63.7 d.

62.5q, 56.2q, 47.7d, 1
8.7q.

16.9 q (10) Solubility: Slightly soluble in methanol, chloroform, ethyl acetate, and acetone, soluble in dimethyl sulfoxide, dimethyl formamide, and alkaline water, and insoluble in water.

(11) Distinction between basic, acidic, and neutral: Salts of the acidic substance benanomycin C include metal salts, especially alkali metal salts such as sodium chloride salts, alkaline earth metal salts such as calcium salts, and There are salts with amines as described below.

2. α-Glucosidase inhibitory activity of benanomycin C α of benanomycin C measured by Test Example 1 below
- 50% inhibitory concentration (IC,.) for glucosidase
was 60 μg/mfl.

Test Example 1 The inhibitory activity against α-glucosidase (yeast, type ■, manufactured by Sigma, USA) using p-nitrophenyl-α-D-glucopyranoside as a substrate was determined by the following measurement method. 4μQ of 100mM phosphate buffer (pH 6,8) containing 0.1mg/mu of α-glucosidase,
95μQ of the same buffer solution of 0.7mM p-21-lophenyl-α-D-glucopyranoside and benanomycin C
After mixing 1 μQ of dimethyl sulfoxide solution and reacting at 37°C for 15 minutes, add 1 mQ of 1M sodium carbonate aqueous solution.
was added to stop the reaction. After that, 405n of the reaction solution
The inhibition rate was determined by measuring the absorbance at m. Also, 5
The degree of 0% inhibition a was calculated from the inhibition rate measured by changing the concentration of benanomycin C.

3. Antifungal activity of benanomycin C The minimum inhibitory concentration of benanomycin C according to the present invention against various molds is shown in Table 1.

The gist of the present invention in Table 1, Table 2 is a method for producing benanomycin C, which involves culturing benanomycin C-producing bacteria belonging to actinomycetes and collecting benanomycin C from the culture.

An example of a benanomycin C-producing bacterium is the Actinobacterium Agu 93-16F4 strain described below.

(B) Mycological properties of the production bacterium Ayu 93-16F4 strain This production bacterium was isolated from the soil on the premises of the Institute at the Institute of Microbial Chemistry in March 1980.
ycete) and assigned the strain number MH193-16F4. The mycological properties of this strain are as follows.

[11WLUL Agu 93-16F4 strain forms aerial hyphae from branched basal hyphae under a microscope. No division of basal hyphae was observed.6 The attachment of aerial hyphae was to the extent seen in l5P-medium 2 and l5P-medium 3, and sporulation was normally observed in l5P-medium 3.
Observed from the body on the 18th day after culturing at 7°C. No whorls, spiral formation, or sporangium formation is observed in the aerial hyphae, and short sporophores are formed almost perpendicularly to the aerial hyphae, with usually 3-
Chains of 7 or rarely 2 spores are seen. Note that the spore chain may have a loop shape. Individual spores are cylindrical (0
,8X1.0-1.2μm) to spherical (0,8-12μm
), and the spore surface is smooth.

[2] Regarding the description of growth status colors in various media [The standards shown in the box are for containers and
Corporation of America's Color Harmony Manual
ion of America's Co1or Harm
onyManual) was used.

(1) Sucrose/nitrate agar medium (27°C culture)
The growth is colorless, no aerial mycelia are attached, and no soluble pigments are observed. In the vitamin B group enriched medium, the growth is colorless to pink gray [5ec, Dusty Peach], no aerial mycelia are attached, and the soluble pigment is only slightly red.

(2) Glucose-asparagine agar medium (cultured at 27°C) The growth is colorless, no aerial mycelium is attached, and no soluble pigment is observed. In the vitamin B group-enriched medium, 1 growth is colorless to pale pink [4 gc, Nude Tan 1, aerial mycelia do not attach, and 1 degradable pigments are reddish.

(3) Glycerin-asparagine agar medium (I5P-medium 5.27°C culture) Growth is colorless, no aerial mycelia are attached, and no soluble pigment is observed. In the vitamin B group-enriched medium, the grayish red-purple [8g,
Rose Mauve]~Nibu red purple [8e, Rose
A slight white aerial mycelium grows on the growth of the red-purple [8pc, Cranberry].
] produces soluble pigments.

(4) Starch/Inorganic Salt Agar Medium (ISP-Medium 4.2
(cultivated at 7°C) The growth is colorless, no aerial mycelia are attached, and no soluble pigments are observed. In vitamin B group enriched media, the soluble pigments have a slight reddish tinge.

(5) Tyrosine agar medium (ISP-medium 7, 27°C culture) Growth is colorless to light yellowish brown [3ic, Lt Ambcr
No aerial mycelia were attached, and no soluble pigment was observed. In the vitamin B group-enriched medium, the growth is colorless to grayish reddish-purple, no aerial mycelia are attached, and soluble pigments are slightly reddish.

(6) Nutrient agar medium (27°C culture) Growth is pale yellow [2gc, Bamboo], no aerial mycelia are attached, and no soluble pigment is observed. Similar properties were observed in the case of the vitamin B group-enriched medium.

(7) Yeast/malt agar medium (l5P-medium 2,27
℃ culture) Light yellow [2ec, B15cuit] to dark red [7pi
, Dk Wine-7Tpg, Dk Red] to grayish red [7pgt Wine], grayish-white aerial mycelium grows from the body on the 14th day after culturing.

The soluble pigment of Tomato Red is seen only around the growth in the early stage of culture, but gradually diffuses. Both the developmental pigment and the soluble pigment gradually change color with HCI, but no change is observed with NaOH. Similar properties are observed in the vitamin B group-enriched medium.

(8) Oatmeal agar medium (ISP-medium 3.27℃
Culture) Light pink [4gc, Nude Tan1 to gray red [
6e.

Cedar ~ dark red [7pe, Cherry Wi
On the development of Mauve Gray, the body became lighter and grayer on the 10th day after culture [3c].
b, 5and] aerial mycelia are attached. The soluble pigment is only slightly reddish. Both growth color and soluble pigment are H
The color gradually changes with CI, but no change is observed with NaOH. A vitamin B group-enriched medium also showed similar properties.

(9) Glycerin/nitrate agar medium (cultured at 27°C) Growth is colorless, no aerial mycelia are attached, and no soluble pigment is observed. Similar properties are observed in the vitamin B group-enriched medium.

(10) Starch agar medium (27℃ culture) Growth is colorless;
No aerial mycelia are attached, and no soluble pigments are observed. In the vitamin B group-enriched medium, 1 growth was colorless and no aerial mycelia were attached.
The soluble pigment has a slight reddish tinge.

(11) Malate lime agar medium (27°C culture) Growth is colorless, aerial mycelia do not adhere, and soluble pigment is slightly reddish. In the vitamin B group-enriched medium, the growth is colorless, no aerial mycelia are attached, and no soluble pigments are observed.

(12) Cellulose (synthetic solution with filter paper added, cultured at 27°C)
Growth is not recognized.

(13) Gelatin puncture culture Growth was poor in both 15% simple gelatin medium (20'C culture) and glucose peptone gelatin medium (27°C culture), growth was colorless, aerial mycelia did not attach, and soluble No dyes are recognized.

(14) Skimmed milk (cultured at 37°C) Growth is extremely poor, colorless, no aerial mycelium attached, and no soluble pigments are observed.

[3] 1 ri roasted 1 (1) Growth temperature range Starch yeast agar (1.0% soluble starch, 0.2% yeast extract, 3.0% string agar, pH 7.0
), 20°C, 24°C, 27°C, 30°C, 37°C,
As a result of testing at various temperatures of 50℃, growth occurred at all temperatures except 50℃, but the optimal growth temperature was 27℃ to 37℃.
It seems to be around ℃.

(2) Liquefaction of gelatin (15% simple gelatin medium, 20%
℃ culture; glucose peptone gelatin medium, 27℃
Culture) Both the simple gelatin medium and the glucose peptone gelatin medium were observed for 3 months after culture, but no liquefaction was observed.

(3) Hydrolysis of starch (starch/inorganic salt agar medium and starch agar medium, both cultured at 27°C) No ice-melting properties were observed in either the starch/inorganic salt agar medium or the starch agar medium.

(4) Coagulation and peptonization of skimmed milk (skimmed milk, 37℃
Culture) Growth was poor, and neither coagulation nor peptonization was observed during observation for 3 months after culture.

(5) Production of melanin-like pigments (tryptone, yeast,
Broth, l5P-culture 1; peptone yeast iron agar, l5P-medium 6; tyrosine agar, l5P-medium 7;
Both cultures were negative at 27°C).

(6) Utilization of carbon sources (Pridham-Gotlieb agar medium, l5P-medium 9.27°C culture) Utilization of glucose, L-arabinose, D-xylose, D-fructose, sucrose, rhamnose, raffinose, D-mannitol and does not utilize inositol.

(7) Dissolution of malic acid lime (malic acid lime agar, 27℃
Culture) is negative.

(8) Nitrate reduction reaction (peptone water containing 0.1% potassium nitrate, 15P-medium 8, 27°C culture) is positive.

(9) Decomposition of cellulose (synthetic salt added to filter paper, cultured at 27°C) No growth.

To summarize the above characteristics, strain MH193-l6P4 forms a spore chain consisting of 3-7 (rarely 2) spores almost perpendicular to the main axis of aerial hyphae, and uses whorling, spiral formation, and spore formation. No carcinogenesis was detected. In addition, no division of the basal hyphae is observed. The spore surface is smooth. l5P-medium 2
．． In two types of medium, 15P-medium 3, gray-white aerial mycelia are formed on the gray-red to dark-red growth. In addition, slant medium (yeast extract 0.2%, soluble starch 1.0
%, string agar 3.0%, pH 7.0), a slight pink aerial mycelium may grow. Also, l5
P-Medium 2 produces a dark red soluble pigment. On various other media, the growth is colorless, no aerial mycelia are attached, and almost no soluble pigments are observed. However, vitamin B in the medium
There is a medium in which growth is improved by adding a group of seeds, and the growth becomes red. Both growth color and soluble pigment are HC
The color gradually changes from red with I, and no change is observed with NaOH. Production of melanin-like pigment, proteolytic ability, and starch ice-melting ability were all negative, and nitrate reduction reaction was positive. In addition, growth may be good on a vitamin B group-enriched medium, and it is thought that the plant has a vitamin requirement.

By the way, the Kawaki 93-16F4 strain contains meso-diaminopimelic acid in the whole cell as a cell component, and glucose, ribose, and madulose as sugar components.

Lechevalier et al.
, rInternational Journal o
f Systematic Bacteriology
20, p. 435, 1970), the main constituents of the cell wall are type IIIB. The type of phospholipid is PIV type (contains phosphatidyl/ethanolamine and unknown glucosamine-containing phospholipid, but does not contain phosphatidyl/glycerol), and the composition of menaquinone is MK-9 (H,) as the main component. (
H, ), MK-9 (H4), MK-9 (H2), MK
-9 (H□.), the GC content of DNA was 71.5%. As a result of gas chromatography analysis of bacterial cells, iso-branched fatty acids (i-16:0), anti-iso-branched fatty acids (a-17:O), and 10-methyl fatty acids (10M
e-17:O).

The known actinomycetes that have spore chains and exhibit cell wall type nlB are Actinomadu (Actinomadu).
ra), Microbispora
, Microtetraspora
There are three genera a). MH93-l6P4 strain and the above 3
Characteristics of the genus are shown in Table 2. The * mark for menaquinone indicates that it is included as a main component.

References 1) Experimental Methods for Identification of Actinomycetes 9 edited by the Japan Actinomycete Research Society.

1985 2) J, Po5chner et al.: rDNA-DNA
Reassociation and Chemotax
onomic 5tudies on Actinom
adura.

Microbispora, Microtetras
pora rMicropolyspora and
Nocardiopsis.

Systematic and Applied Mi
crobiologyJ volume 6.

pp. 246-270, 1985 3) A., Fischer et al.: rMolecular
-genetic and Chemotaxonomi
c 5tudies on Actinomad
uraand Nocardiopsis, Journ
al of General Microbiology
J Vol. 129, pp. 3433-3446, 1983 4) Thiemann et al.: rA New Genu
s of the Actinomycetales:
Microtetrasporagen, nov.

Journal of General Microb
iologyJ volume 50, pages 295-303, 196
8th year 5) Nonomura et al.: Distribution of actinomycetes in soil (11th report) ActinoIIIadura Lacheval
ier et al., Several new species of the genus, "Journal of Fermentation Engineering" 4
Vol. 9, pp. 904-912, 1971 As shown in Table 2, there is no genus to which the 1M old 93-16F4 strain easily falls. These genera have undergone very subtle changes and are described in Bergey's Manual.
annual of DeterminativeBac
We are looking forward to seeing how it will be published in the new edition of TERIOLOGY. However, many species have been described in the genus Actinomajula, and there is a wide range of properties, but among them, Actinomajula spasix
adura 5padix) and there are considerable similarities in morphology, fatty acid composition and menaquinone composition of the bacterial cells (references 1), 3) and 5) above). from now on.

We plan to first conduct a comparative experiment between the MH193-16F4 strain and Actinomadura spazix. Furthermore, M
An application was made for depositing the H193-16F4 strain to the Institute of Microbial Technology, Agency of Industrial Science and Technology, and it was deposited on August 21, 1986 as Microbiological Research Institute No. 9529.

(0) Cultivation method of strain MH193-16F4 By inoculating benanomycin C-producing bacteria belonging to actinomycetes into a medium containing nutrients that can be used by ordinary microorganisms and growing them under aerobic conditions, mainly culture solution Benanomycin C is produced and accumulated in the culture medium, and the target product is further separated from the culture, especially the culture solution. As the nutrient source in the medium used, any known nutrient source used as a nutrient source for actinomycetes can be used. For example, commercially available nitrogen sources such as soybean flour, nissan meat, peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour, dried yeast, yeast extract, NZ amine, casein, sodium nitrate, ammonium sulfate, ammonium nitrate, etc. , and carbon sources such as glycerin, sucrose, starch, glucose, galactose, maltose, dextrin, lactose, molasses, soybean oil, fats, amino acids, and salt, phosphates, calcium carbonate, magnesium sulfate, cobalt chloride, chloride. Inorganic salts such as manganese can be used. Other trace amounts of metal salts, anti-foaming agents, and other substances as necessary.
Mineral oil etc. can be added. Any of these materials may be used as long as they are useful for the production of benanomycin C, and all known culture materials for actinomycetes can be used.

Liquid culture is preferred for mass production of benanomycin C;
The culture temperature can be applied within a range that allows the producing bacteria to grow and produce benanomycin C, and is usually 20-40°C, preferably 25-40°C.
The temperature is 37°C. Cultivation can be carried out by appropriately selecting the conditions described above depending on the properties of the benanomycin C-producing bacteria.

(c) When collecting benanomycin C from a culture of benanomycin C obtained according to the present invention, conventional separation methods utilizing its properties such as solvent extraction method, ion exchange resin method, adsorption or distribution column chromatography method, Extraction and purification can be carried out using a gel filtration method, a dialysis method, a precipitation method, etc. alone or in appropriate combinations. For example, benanomycin C is extracted from cultured bacterial cells with acetone-water or methanol-water. Benanomycin C accumulated in the culture solution is adsorbed to a synthetic adsorbent such as Diaion HP-20 (manufactured by Mitsubishi Kasei Corporation). Furthermore, if extraction is performed with an organic solvent that does not mix with water, such as butanol or ethyl acetate, benanomycin C will be extracted into the organic solvent layer.

To further purify benanomycin C, silica gel (
Chromatography using an adsorbent such as Wako Gel C-300 (manufactured by Wako Pure Chemical Industries, Ltd.), alumina, or Sephadex LH-20 (manufactured by Pharmacia) may be performed.

Benanomycin C produced in culture in this way
can be isolated in free form, i.e. as benanomycin C itself, and the solution containing benanomycin C or its concentrate can be treated with a base, i.e. an alkali metal compound such as sodium hydroxide, canium hydroxide, calcium hydroxide, During each step of the operation, for example, extraction,
When treated during the separation or purification steps, benanomycin C is converted to its corresponding salt form and separated. Separately, the salts of benanomycin C produced in this way can be converted into free form and benanomycin C by a conventional method.
It can change itself. Furthermore, benanomycin C obtained in free form may be converted into the corresponding salt thereof using the above-mentioned base in a conventional manner. Therefore, benanomycin C as well as its salts as described above are included within the scope of the present invention.

Examples of the production of benanomycin C of the present invention are shown below, but since the properties of benanomycin C have been clarified by the present invention, various methods for producing benanomycin C can be devised based on those properties. . Therefore, the present invention is not limited to the examples, and may of course be modified by the examples.

It covers all methods for producing, concentrating, extracting, and purifying benanomycin C by applying known means based on the properties of benanomycin C revealed by the present invention.

Akuru 93-16, which belongs to actinomycetes, was cultured on agar slant medium.
F4 strain (Feikoken Bacteria No. 9529) with 1.0% starch
, a liquid medium containing 3.0% soybean flour, 0.01% KM7, and 0.01% Adekanol (80 mQ in a 500 mQ Sakaro flask).

The cells were inoculated at a pH of 7.0 before sterilization, and cultured with shaking (135 rpm) at 28°C for 3 days to obtain a type 1 culture. Three of each of these seed cultures were inoculated into the same medium as above and cultured with shaking under the same conditions for 3 days to obtain a second type culture. 1 liter of this seed culture
It was inoculated into a 100 L culture tank containing 50 L of the above-mentioned medium that had been sterilized at 20°C for 15 minutes, and then kept at 28°C for 2 days with an aeration rate of 5.
A third type culture was obtained by aeration stirring culture at 0 L/min and 200 rp+a. Glycerin 2.0%, Nisanmeat 1.5%, K, HPO40, previously sterilized at 125°C for 30 minutes
,025%.

KH, PO40, 1125%, CoC1, 6H, OO
,0005%, KM 720.03%, Adekanol 0
．． 12 L of the above type 3 culture was inoculated into a 570 L culture tank containing 300 L of production medium consisting of 0.01% (pH 6.2 before sterilization), and maintained at 28°C for 7 days with an aeration rate of 1 for the initial 24 hours of culture.
50L/min, 300L/min after 24 hours, 300rpm
The cells were cultured with aeration and agitation. After the culture was completed, diatomaceous earth was added as a filter aid and the mixture was filtered to obtain 25 OL of filtrate (pH 6.0).

The active substance in this culture filtrate is Diaion HP-20 (
After washing with water (100 L) and 50% methanol (45 L), absorb into 70% methanol (45 L).
), then eluted with methanol (90 L) to obtain the first fraction (
53L), second fraction (38L) and third fraction (27L)
) was obtained. The first fraction containing benanomycin A was concentrated under reduced pressure to 3 L, and the pH was adjusted to 3.5 using IN hydrochloric acid to obtain a red precipitate. This precipitate was collected by filtration and dried under reduced pressure to obtain a brown coarse powder (152 g) containing mainly benanomycin A. This crude powder (+50 g) was dissolved in dimethylformamide (600+nR) and saturated with water vapor in a desiccator at room temperature for 3 days, resulting in the precipitation of a crystalline precipitate. This precipitate was collected by filtration and dried under reduced pressure to obtain benanomycin A dimethylformamide turbate (29 g). The second fraction was treated in the same manner as the first fraction to obtain benanomycin A/dimethylformamide/turbate (14 g).

Benanomycin A/dimethylformamide/turbate (1 g) obtained from the first fraction was dissolved in dimethyl sulfoxide (5++Q) and added dropwise to methanol (300 mQ) with stirring. After further stirring for 10 minutes, a reddish brown precipitate was deposited. . This precipitate was collected by filtration and dried under reduced pressure at 50°C for 2 days to obtain pure benanomycin A reddish brown powder (935
mg) was obtained.

The third fraction was concentrated under reduced pressure to 1.5 L and adjusted to pl+ 3.5 using IN hydrochloric acid to obtain a red precipitate.

This precipitate was collected by filtration and dried under reduced pressure to obtain benanomycin B.
A brown coarse powder (99 g) containing C and C was obtained. This coarse powder (1 g) was mixed with dimethylformamide (10 mQ).
The solution was dissolved by heating (40'C), applied to a Sephadex LH-20 column packed with dimethylformamide, and developed with dimethylformamide. Fraction Nα64-72 (1 fraction 6+J) containing the active substance was collected and concentrated to dryness under reduced pressure to form benanomycin B, dimethylformamide,
A brown coarse powder (657 mg) containing turbate and benanomycin C dimethylformamide turbate was obtained. This crude powder was dissolved in methanol (100+nR), IN hydrochloric acid (1 mfl) was added, and the mixture was concentrated to dryness under reduced pressure. The obtained brown coarse powder was mixed with dimethyl sulfoxide (
Dissolved in 3m(1) of chloroform (200mQ) under stirring.
) and stirred for further 20 minutes, a reddish brown precipitate was deposited. The precipitate was collected by filtration and dried under reduced pressure to obtain pure benanomycin B hydrochloride (565 mg).

The filtrate was concentrated under reduced pressure to approximately 5 mQ, and then filtered with Sephadex LH-
20 (600 mQ), and chromatography was performed using methanol as a developing solvent. Concentration of the single red fraction to dryness on TLC yielded benanomycin C (38
, 5111 g) was obtained as a reddish brown powder.

The 50% inhibitory concentration for glucosidase is 60μa/m
It was u.

[Brief explanation of the drawing]

Figure 1: Benanomycin C in methanol (20 gg/
resistance, shown by solid line), 0. IN hydrochloric acid-methanol (20
tt g/mQ, indicated by the dashed line) and 0. Ultraviolet and visible absorption spectra in IN sodium hydroxide-methanol (20 gg/a+Q, indicated by the dashed line) are shown. Figure 2: Shows the infrared absorption spectrum of benanomycin C in potassium bromide tablets. FIG. 3: Shows the 400 M) Iz hydrogen nuclear magnetic resonance spectrum of benanomycin C in deuterated dimethyl sulfoxide solution. FIG. 4: Shows the 100M) lz carbon nuclear magnetic resonance spectrum of benanomycin C in deuterated dimethyl sulfoxide solution.

Claims (1)

[Claims] 1. α-glucosidase inhibitor benanomycin C and its salts represented by the following formula ▲ Numerical formula, chemical formula, table, etc. ▼ (I). 2. Benanomycin C, which is characterized by culturing benanomycin C-producing bacteria belonging to Actinomycete and collecting benanomycin C from the culture.
manufacturing method.