JPH03178988A - Physiologically active substance tan-883, preparation and use thereof - Google Patents

Abstract

NEW MATERIAL:A physiologically active substance TAN-883-B having the following physicochemical properties or a salt thereof. The shape: white powder; molecular formula: C20H40N2O6, etc. USE:An angiogenesis-promoting drug. Useful for recovering of wounds accompanied with large surgical operations such as burns, wounds, fractures and organic transplantations, for improving sewed opening disorders and recovery delayed diseases and for the diseases requiring the rapid hyperplasia of angiogenesis, such as ischemic heart diseases, e.g. cardiac infarct and cerebral hemorrhage accompanying arterial sclerosis, and digestive tract ulcer, etc. PREPARATION:A microorganism belonging to the Lysobacer genus, such as Lysobacer gummosus B62-21 strain (FERM BP-2568) is cultured preferably with aerial stirring at 17-28 deg.C at a pH of 5.5-7.0 for 24-144 hours.

Description

[Detailed Description of the Invention] Industrial Application Fields: Repair of burns, wounds, fractures, wounds associated with major surgical operations such as organ transplants, improvement of suturing failure, delayed repair, myocardial infarction accompanied by arteriosclerosis, The present invention relates to a physiologically active substance TAN-883 that is effective for ischemic diseases such as stroke, and diseases such as gastrointestinal ulcers that require an immediate increase in angiogenesis, a method for producing the same, and uses thereof.

Angiogenesis is a physiological phenomenon of vascular (capillary) formation by vascular endothelial cells, and is known to be involved in various pathological conditions seen in many medical fields. , these pathological conditions are collectively referred to as Geo.
Polkman, J.
isease) J [Science (S
Science), 235.442 (1987)].

For example, growth of solid tumors.
rheumatoid arthritis (tumor), rheumatoid arthritis
arthritis), diabetic retinopathy (D 1a
betic retinopathies), psoriasis (P.
5 oriasis), angiogenesis is known to be abnormally accelerated.

On the other hand, angiogenesis is not normally observed in healthy adults, except during normal physiological conditions such as placentation, embryonic development, and ovulation associated with the female sexual cycle. However, the repair process of tissue damage such as wounds, burns, and fractures
g), Chronic inflammation
n), immune reaction
), angiogenesis based on host defense is observed. Acidic and basic fibroblast growth factors are factors that promote angiogenesis.
Under normal conditions, these are bound to sulfated mucopolysaccharides such as heparin and stored in an inactive state in the basement membrane, and are released when necessary, such as during tissue damage. [Procedures of the National Academy of Sciences]
roc, Na11. Acad, Sci, USA
), 842292 (1987)].

In the surgical field, postoperative suture failure and aplastic fractures (non-regenerative fractures)
Healing I'racture); Delayed wound healing
al ing) is often a problem. In addition, angiogenesis plays an important role in the repair and regeneration process of thrombus formation sites and arteriosclerosis sites in the vascular network, and if angiogenesis is inadequate, these will eventually lead to myocardial infarction, cerebral infarction (stroke), etc. leading to serious illness.

Therefore, factors that induce or promote angiogenesis are considered to be effective therapeutic agents for wound healing and ischemic diseases as described above. It may also be used to prevent and treat gastrointestinal ulcers.

Many angiogenic factors have been reported so far, but
Among these, biologically derived substances include protein growth factors, prostaglandins derived from body fluids at injured sites, and lipids derived from adipose tissue [Science
e), 235.442(] 987)']0 In recent years, proteinaceous growth factors with angiogenic activity, such as acidic,
Basic fibroblast growth factor (acidic and
basic FGF), angiogenin (A n
giogenin), transfecting growth factors α, β (TGFα, β), epidermal growth factor (E
GF), tumor necrosis factor (TNF'), etc., their genes were successively cloned, and their amino acid sequences were determined from their base sequences [
Science, 235.

442 (1987)].

On the other hand, low-molecular angiogenic factors include lipids such as prostaglandin E, E2, etc.
Journal of Natl, Cancer I
nst,).

69 475 (1982)], omentum
um)-derived factor [Livid(1,1pid), 22
, 229 (+987)], Walker
) 256 Rat Tumor-Derived Factors
Chem, ), 259605 (1981)], limited degradation product of hyaluronic acid [Science,
228, +324 (+985)], adenosine 1 rculati
on Research), 59, l 63 (1
986)], Nicodinamide [Scien
ce), 236, 843 (1987)], plasma-derived glycyl-I7-histidyl-L=-lysine Cu''''' [
Methods
in Enzymology), 147, 31
4 (1987)], but nothing has been used as an angiogenesis-promoting agent yet.

Problems to be Solved by the Invention As mentioned above, many reports have been made regarding factors that induce and promote angiogenesis. However, many problems are expected when using protein-based factors clinically.
Furthermore, it is thought that few of the known low-molecular-weight factors have low activity or are highly practical in terms of side effects and specificity of action.

Means for Solving the Problems Based on this background, we aimed to find a novel angiogenic factor with strong activity and high practicality.1. After isolating and exploring a large number of microorganisms, we discovered that certain microorganisms produce novel angiogenesis-promoting factors. Also,
It was discovered that the microorganism is Resobacter gamozas, and that by culturing the microorganism in an appropriate medium, an active substance that strongly induces angiogenesis can be accumulated in the medium. As a result of purifying and isolating this active substance from the culture solution and examining its physicochemical properties, we found that two of the four isolated active substances, components B and D, are new compounds, and the remaining two are new compounds. Components A and C are known compounds catacandin B and A, respectively.
B, A + Journal of Antibiotics
cs) 38.1642(+985), and based on these findings, further research was conducted and the present invention was completed.

The present invention (+) Physiologically active substance TAN-883-B.

(2) Physiologically active substance TA, N-883-D.

(3) Belongs to Rhizobacter genus, TAN-883A, -B
, -C or -D is cultured in a medium, and the compound is produced, accumulated, and collected in the culture TA, N-883
-A, -B, -C or -D or a method for producing a salt thereof, (4) Physiologically active substance TAN-883-A, -B.

The present invention provides an angiogenesis-promoting agent comprising C or itne D or a salt thereof.

The physiologically active substance TAN-883 (hereinafter simply referred to as TA) of the present invention
The microorganism that produces TAN-883 (sometimes abbreviated as II-883) may be any microorganism that belongs to the genus Lysobacter and has the ability to produce TAN-883. Examples include Lysobacter gummosus B62, which was isolated from soil collected in Shiga Prefecture.
-21 strain (hereinafter sometimes abbreviated as rB61-21 strain) is specifically mentioned as a microorganism that can be used. B
The mycological properties of strain 62-21 are described below.

(1) Morphological characteristics When observed after culturing on a broth agar slant at 24° C. for 5 days, the cells are rod-shaped with a diameter of 0.3 to 0.6 μm and a length of 1.0 to 3.0 μm. Durham staining is negative, and flagella are not observed, but motility due to briding is observed. Does not form spores. Does not exhibit anti-acid properties.

(2) Growth status on various media The cells were cultured at 24°C and observed for 1 to 14 days.

(a) Broth agar plate culture: Colonies are translucent, swirly yellow, round, the surface is convex, and the periphery is gold-rimmed to wavy. No diffusible dye is produced.

(b) Juicy agar slant culture showing good spread-like growth;
Opaque, pale yellow in color.

(c) Broth liquid culture: Grows in a cloudy shape and forms a closed ring. Bacterial sediment can also be seen at the bottom.

(d) Meat juice geladin puncture culture - Grows well on the surface, and growth is also observed in the middle and deep parts. No oxidative activity.

(e) No reducing ability, peptonization, or coagulation of lid mass, milk, or mass was observed.

(f) Skito milk acetate agar: shows extremely strong proteolytic activity and grows as a sticky gel-like mass on agar-1-.

(3) Physiological properties (a) Reduction of nitrate.

(b) Denitrification reaction (c) MR (methyl red) test (d) VP (Voges Proscalaer) test: (
e) Production of indole (f) Production of hydrogen sulfide (TSr agar).

TST agar meat extract 4 parts, peptone +59, lactose 10g
, white sugar 10g, glucose 1g, thorium chloride 5g, sodium thiosulfate 0.089, sulfite 0)
, 4 g, ferrous sulfate 0.29, phenol red 0
．． 029, agar 15g, distilled water 10. What should I do? pH
7.4° (g) Starch hydrolysis: Very weak.

(h) Utilization of citric acid Cosel medium Simmons medium, -E (1) Utilization of nitrogen sources Potassium nitrate: Ammonium sulfate + Sodium glutamate: - (Formation of D pigment (King A, B and Mannitol yeast extract agar media) : Formation of diffusible dye (J not observed.

King A medium: 10 g glycerin, 20 g peptone, 1.4 g magnesium chloride, ammonium sulfate IO9, 15 g agar, and adjust to IQ with distilled water.

pH 7.2° King B medium 10 g glycerin, peptone 202, potassium hydrogen phosphate 1.5 g, magnesium sulfate 1.
5g, agar 15g, and distilled water to determine IQ.

pi-r 7, 2° (k) urease (1) oxodase: + (m) catalase: (n) Growth range pH: pH 5, grows at 0 to 8.3, but the optimum pH is 5
．． 2-6.7 Temperature: Grows at 14-34°C, but optimal temperature is 16-34°C
25℃ (o) Attitude towards oxygen: Aerobic (p) O-F (oxidative fermentative)
Guess l-[Hiko Leifson (LTugh-Lei)
q) Production of acid and gas from sugar and utilization of sugar Acid Gas Utilization (Peptone water) (Davis medium) L-Arabinose D-Xylose D-Glucose D-Mannose D -Fructose D-Galactose Maltose Sucrose Lactose Trehalose D-Sorvit D-Mannitinoscilla)/Glycerin Starch (r) GC (guanine/cytosine) content of DNA = 66
9% Literature “FEMS Microbiology Letters (P.
EMS Microb, Letters), 25-
According to the HPI, C method described in 125 (1984).

(s) Ability to form microcysts: (1) Degradability of carboxymethylcellulose 2+ (liquefies) (U) Degradability of colloidal chitin: + (v) Degradation of Tween 80: 10 (w) Assimilation of L-arginine The B62-21 strain, which has mycological properties equal to or higher than that of B.
ofDeterminative F3acteri
ology) 8th edition, Bersey's Manicocal of
Systematic Bacteriology (Bergy's
Manual of Systematic B
Acteriology, 1st Edition and International Journal of Systematics Batteriology, 1st Edition.
nalof Systematic Bacteria
30, pp. 225-420 (1980),
32, pp. 146-149 (1982) and subsequent validation lists.
B61-
Strain 21 is a whirlpool-yellow, Durum-negative bacillus that does not have flagella and exhibits motility that involves briding.It is aerobic, has a high GC content in its DNA, and has no ability to form microcysts, so it belongs to the Rhizobacter genus. It is reasonable to assume that

Therefore, strain B61-21 was compared with known species of the genus Rhizobacter. The only known species of the genus Rhizobacter are four species and one subspecies, which are described in International Journal of Systematic Bacteriology, Vol. 28, pp. 367-393 (1978). Therefore, these five species (46 strains are described in the above literature) and 862-
When we compared the properties of 21 stocks, we found the following: 61-2
Characteristics of the strain: i) cell morphology does not become filamentous; 2) it does not produce soluble pigment; 3)
Lysobacter gamosus (Lysobacter gamosus) is a strain of Lysobacter gamosus (Lysobacter
acLer gummosus). Rhizobacter gamosus strain B61-21 was introduced in 1988 (+9
88) This microorganism was deposited with the Institute of Fermentation Research Foundation (IFO) on December 12th under the accession number IPOI4804, and this microorganism was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on August 28th, 1999 (+989). 1" RI, 1-1-3, Tsukuba Kuranori, Ibaraki Prefecture) under deposit number FERM BP-2568 under the Budapest Treaty.

Rhizobacter used in the method of the present invention is generally susceptible to changes in its properties, and can be easily mutated by artificial mutagenesis means using, for example, ultraviolet rays, X-rays, chemicals (e.g., nitrosoguanidine, edylmethanesulfonic acid), etc. However, any mutant strain capable of producing the compound targeted by the present invention can be used in the method of the present invention.

When culturing the bacteria producing TAII-883, carbon sources such as glucose, maltose, lactose, blackstrap molasses, oils and fats (e.g., soybean oil, olive oil, etc.), organic acids (e.g., citric acid, succinic acid, etc.) are recommended. , gluconic acid, etc.) that can be assimilated by bacteria are used as appropriate. Examples of nitrogen sources include soybean flour, cottonseed flour, corn steep liquor, dried yeast, yeast extract, meat extract, peptone, urea, ammonium sulfate, ammonium nitrate, ammonium chloride,
Organic nitrogen compounds and inorganic nitrogen compounds such as ammonium phosphate can be used. In addition, as inorganic salts, for example, inorganic salts normally required for culturing bacteria such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, potassium phosphate, and disodium phosphate are used alone or in appropriate combinations. Ru. In addition, sulfur compounds that can be assimilated by bacteria that produce the target compound, such as sulfates (e.g., ammonium sulfate, etc.), diosulfates (e.g., ammonium thiosulfate, etc.), and sulfites (e.g., ammonium sulfite)
Inorganic sulfur compounds such as amino acids (e.g. cystine,
When organic sulfur compounds such as cysteine, L-thiazolidine-4-carboxylic acid), hypotaurine, and peptides (e.g., geltadeone), or mixtures thereof, are added to the medium, the production amount of the target product may be increased.

Further, heavy metals such as ferrous sulfate and copper sulfate, vitamins such as hitamine R, and pyotin are added as necessary. Furthermore, silicone oil, polyalkylene glycol ether agents, and surfactants may be added to the medium. Organic or inorganic substances that aid the growth of the bacteria and promote the chemical production of the target compound may be added as appropriate.

The culture method 7 may be carried out in the same manner as in general antibiotic production methods, and solid culture or liquid culture may be used. In the case of liquid culture, any method such as agitation culture, shaking culture, or aeration culture may be performed when the culture is left standing, but aeration agitation culture is particularly preferred. The culture temperature is preferably in the range of about 15°C to 32°C, more preferably about 17°C to 28°C.
℃, the pII of the medium is in the range of about 5 to 8.3, more preferably in the range of about 55 to 70, and the culture is carried out for about 8 to 168 hours, preferably about 24 to 144 hours.

The produced physiologically active substance TAN-883 can be separated into a supernatant liquid and bacterial cells by centrifugation or filtration of the culture, and purified from the supernatant liquid and bacterial cells, respectively. In some cases, it may be more advantageous to purify the product directly from an extract obtained by adding an organic solvent such as methanol, acetone, ethyl acetate, or citanol directly to the product.

Since TAN-883 is a fat-soluble acidic substance, separation and purification means commonly used to collect such microbial metabolites are appropriately used. For example, methods that utilize the difference in solubility with impurities, methods that use various carriers such as nonionic high-porous resins, activated PIE carbon, silica gel, alumina, and dextran gel, and L:J madography may be used alone or in combination. used.

To specifically explain the method for collecting the physiologically active substance TAN883 produced in the culture, first, the pH of the culture solution is made acidic, and extraction is performed with an organic solvent that is immiscible with water, such as ethyl acetate. Examples of organic solvents used to extract TAN883 from the culture solution include fatty acid esters such as ethyl acetate and isobutyl acetate, isobutanol, n
Examples include alcohols such as -butanol, halogenated hydrocarbons such as chloroporm and methylene chloride, and ketones such as methyl isobutyl ketone.

After filtering the extract containing TAN-883 using Hyfloth-Parcel or the like, the organic solvent layer is separated. Next, weakly alkaline water such as dilute sodium bicarbonate solution is added to this, and the substance is stirred into the aqueous layer. Concentrate the aqueous layer (7
After removing the remaining organic solvent, Diaion II
Pass through a column of adsorbent resin such as P-20,
A crude substance is obtained by developing and fractionating with 30% to 70% methanol/water, and combining the two fractions containing the active substance, concentrating and drying.

This crude material is dissolved in 40% methanol/water and developed for column chromatography on MCI gel CI (1)-201) (tL, 60% to 80% methanol/water. Both fractions eluted first) When concentrated, TAN-
8 A mixture containing 83-A, -B and 10 was obtained,
A mixture containing TAN883-D is obtained from both fractions that are eluted later.

Both fractions containing TAN-883-A, -B, and -C were subjected to preparative high performance liquid chromatography (color YMCSH-343
0DS), mobile phase: 60% methanol 0,05M p
H7.5 phosphate buffer, flow rate 10ffc/min),
Both components containing only component A were concentrated, the pH was made acidic and extracted with ethyl acetate, washed with water and concentrated, resulting in TAN-8 8
A purified powder of 3-A is obtained. By collecting both components containing only the C component and treating them in the same way as the A component, TAN-8
A purified powder of 83-C is obtained.

Both fractions containing mainly component B were collected, treated in the same manner as A and C, and then subjected to preparative high performance liquid chromatography again under the same conditions as above, and both fractions containing only component B were collected and treated in the same manner as A and C. A purified powder of TAN-[83-B is obtained.

Both fractions containing components 1) that were eluted late in MCI Gel CI-I P-20P column chromatography were purified by preparative high performance liquid chromatography [1. Purification of TAN-883-D was carried out in the same manner as for the A component, by collecting both fractions containing only the D component and treating them in the same manner as the A component described above. A powder is obtained which can be crystallized from methanol as pale yellow crystals.

TAN-883-B thus isolated and -
1) The physicochemical properties of the educt are as follows.

(1) 'T'AN-883-B l) Shape: White powder 2) Melting point: 244°C (decomposed) 3) Specific optical rotation: [α]do+I 61' (c = 0.3
4 minutes MF) 4) Molecular weight: 512 (ETMS) 5) Elemental analysis value (%) Actual value c, 64.45; H, 7,80; N, 5.13 Calculated value・(C2゜l-14, N, O, -1,5T (as 20) C64,,54F ■ 8.03 N 5 1
96) Molecular formula: C3JrtoNpOe 7) Ultraviolet absorption spectrum (Figure 1) 8) Infrared absorption spectrum (KRr tablet method): As shown in Figure 2.

9) 'H nuclear magnetic resonance spectrum (300MHz,
As shown in Figure 3.

1.0) I'C nuclear magnetic resonance spectrum (75 MHz, chemical soft in deuterated pyridine, δppm) 12.9 (
q), ! 8.9(q), 26.6(t), 29.1
(t).

32.7(t), 37.8(t), 37.9(t), 4
0.8(t) 42.3(d), 43.2(t), 44.
5(d), 4.6.3(d)4, 7.4(d), 48.
6(d), 54.3(d), 59.1(d) 60.2(
d), 70.3(d),”/ 1.7(d), 74.0
(d) 101.8(s), 123.3(d), 1 24
．． 9(d) 140.1(d), I 50.1(d)
, I 66.9 (s) 174.6 (s), I 77
．． 7 (s), I 94.4 (s) 11) Soluble Soluble / dimethyl sulfide, dimethyl formamide, pyridino, methanol Poorly soluble: water, n-hexane 12) Thin layer chromatography (carrier, silica gel glass spray) 60 P vs 4.0.25mm, manufactured by Merck & Co., West Germany) Ryoaki Kashu Dan Echloroform-methanol-0.32 Ammonia water (6:4: Chloroform-methanol-acetic acid 0.20 (8
・21) 13) High performance liquid chromatography: Carrier 2L 1chrospherl OOYIP-18
(e) 5 μm (4X I 25 mm) Solvent system: 65% MeOH-0.02 M phosphate buffer (p
i-(7,2) Flow rate: 1. OmQ 1 minute retention time (tR) + 5.8 minutes 14) Color reaction positive, ferric chloride, 17.2,4-nonitrophenylhydrazine, Ehrlich reagent negative:
Ninhydrin reagent 15) Acidic, neutral, basic: Acidic (2) TAN-883-D: 1) Shape: pale yellow crystal 2) Melting point: 196°C (decomposition) 3)) J:, Optical rotation: [α ]3+724°(c=0.3
6DMF) 4) Molecular weight 494 5) Elemental analysis value (%): Actual value: C, 68, 19; H, 7, 57, N, 5.3 Calculated value (C79H,, N2O5・I (as 20) C67
,95・H7,86・N 5゜46) Molecular formula: C2゜T
-13e N 2057) Ultraviolet absorption spectrum (4th
Figure) 370 (sh, 370) 8) Infrared absorption spectrum (KBr tablet method) As shown in Figure 5.

9) 'H nuclear magnetic resonance spectrum (300 MI (in z-heavylene) as shown in FIG. 6.

1o) 13c nuclear magnetic resonance spectrum (75MHz, chemical shift in deuterated pyridine, δppm): 12.9
(q), 19.2 (q), 21.7 (t), 26.8
(t) 27.0 (t), 39.3 (t), 40.7 (t
), 41.3(t).

48(t), 45.5(d), 48.1(d), 54.
5(d) 56.5(d), 56.5(d), 62.2(
d), 75.4(d).

102.1(s), 123.1(d), 126.5(d
).

126.6(d), +29.4. (d), 134
．． ．． 6(d).

139.7(d), 143.2(d), 147.5
(d).

166.4 (s), + 74.0 (s), 177
．． 0(s).

196.6 (s) 11) Solubility: Soluble Nidimethyl sulfide, dimethylformamide, pyridine, methanol Poorly soluble: water, n-hexane 12) Thin layer chromatography (carrier, silica gel glass plate 60 F 264.0. (25 mm, manufactured by Merck & Co., West Germany) Developing solvent R: Chloroform-methanol-0.42 Aqueous ammonia (6:4:1) Chloroform-methanol-acetic acid 0.42 (8
:2:1) 13) High performance liquid chromatography; Carrier: Lichrospherl 00RP-18(e
) 5 μm (4x ] 25++un) Solvent system: 65% MeOH-0.02M phosphate buffer (p
H7, 2) Flow rate: 1.0 t (1/min retention time (tR)・7.9 min 14) Positive color reaction: ferric chloride, I3.2.4-dinitrophenylhydrazine, Ehrlich reagent negative:
Ninhydrin reagent 15) Distinction between acidic, neutral, and basic: acidic Based on these physicochemical properties, TAN-883B and -D were judged to be new compounds of the Ikarugamycin family. In addition, TAN-88 isolated at the same time
3-A and 10 were consistent with katacandins B and A from their chemical properties [Journal of Antibiotics, J.
cs), 38, 1642 (1985)].

] Since TAN-883-B, -D, and -A-C are all acidic substances, alkali metal salts (sodium salts, potassium salts, lithium salts, etc.), alkaline earth metal salts (calcium salts with inorganic or organic bases such as ammonium salts, triethylamine salts, ethanolamine salts, etc.) can be formed.

=29- TAN-883 shows no toxicity in rats after oral administration up to 200 H/9, has very low toxicity, and has strong angiogenesis-promoting activity, and is effective against various types of wounds described above. It is extremely useful as a therapeutic agent for curing and preventing ischemic diseases.

The compound may be administered orally or parenterally to a mammal (e.g.
It is administered to rats, rabbits, monkeys, and humans) in the form of tablets, granules, capsules, syrups, powders, injections, or topical creams and ointments. Generally, TAN-883 is administered orally to adult humans at a dose of 5 to 1.000 mg per day, and when administered parenterally,
It is administered in an amount of about 0.2 to 200 mg per day in one to several divided doses. Depending on the dosage form, these pharmaceutical compositions may contain suitable commonly used additives (raw materials) such as excipients, binders, disintegrants, lubricants, colorants, flavoring agents, stabilizers, etc. may be included. In addition, the composition can be used for sustained release using sustained release polymers.
It may also be administered using a tained release technique. Additionally, for example, the composition can be incorporated into polyethylene-vinyl acetate copolymer pellets and the pellets can be advantageously implanted into the tissue to be treated.

The proposed angiogenesis induction promoting activity was measured by the following two methods.

Method 1) Shell-less chicken embryo chorioallantoic membrane (Shell-1ess
Chickchorioallantoic mem
The test using the brain, CAM) was performed according to the method of J. Folkman et al. [R, Crum et al., Science, 230, 1375 (198
5)] by a slightly modified method. That is,
The eggshells of fertilized chicken eggs cultured for 3 days are broken and the resulting chicken embryos are placed in a plastic cup (inner diameter 7.3 am, height 6.0 am).
The cells were hung in a hammock shape using a polyvinylidene chloride film (Ocm, with two windows on the side), and cultured aseptically for an additional 6 days. A medyl cellulose disk containing a test sample prepared by the method described below was gently placed on the chorioallantoic membrane of this chicken embryo, and culture of the chicken embryo was continued aseptically, and after 24 and 481 hours, the area around the disk was Angiogenesis-promoting zones (Spoke wheel-1ike cap) formed radially in the
The presence or absence of illary format 1on) was observed under a microscope (X20, SMZlOlNikon).

The test sample was prepared as follows. That is, a solution of TAN-883 prepared aseptically and a 1% methylcellulose (4000 centivoise) aqueous solution are mixed in equal volumes, and the resulting solution IOμQ is gently dropped onto a sterile Teflon plate. A 4 mm disc was used.

Angiogenesis-promoting activity (%) was calculated as the ratio of the number of samples in which an angiogenesis-promoting effect was observed to the number of samples used in the test. The angiogenesis-promoting activity of each segment of TAN-883 is the first
Shown in the table.

32~ Table 1 *Measurement value after 48 hours Method 2) Rat corneal micropocket method is performed by Gimbron (
[Journal of National Cancer Institute (J.

National Cancer Institute
e), 52, 413 (1974)] as follows. Sprague Uda Ray (Sp
rague-Dawley) line mature male ll- (1m
-16 weeks old) was anesthetized with Nembutal, and xylocaine eye drops were dropped into the eyeballs for local anesthesia. An approximately 2 mm incision is made with a syringe needle into the cornea, approximately 2 mm inside the corneal limbus.
A slow-release pellet of the specimen was inserted. A pellet containing no specimen was inserted into the cornea of rats in the control group. After 10 days, the cornea was observed under a stereomicroscope, and if angiogenesis was observed after administration of the sample, it was determined that the cornea had angiogenic activity. Sustained-release pellets were created by the following method.

The sample was dissolved in ethanol to a concentration of 2 ng/2 μQ, mixed with 6 μQ of ethylene-vinyl acetate copolymer (Narita Pharmaceutical) solution, air-dried on a glass petri dish, and the resulting sheet was rolled.

An example of the test results for TAN-883-A is shown in Table 2.

0 (control pellet) 0/4 0.1 5/7 0.5 6/6 5 7/7 20 3/3 Next, anti-ulcer activity (effect on water immersion restraint ulcer) was measured.

1- yz wa r,, ra h 1 m 6 animals.
A predetermined amount of TAN883-C was orally administered, and the ulcer index was determined by a conventional method. The results are shown in Table 3.

Table 3 Dose Qtg/kg) Ulcer index (to) Ulcer index 11
-Rate (%) 0 (control) 18.7 3.0 +3.7 2710.0
4.0 79 Examples The present invention will be explained in more detail with reference to Examples below.

Example 1 Curcose 2%, soluble starch 3%, corn steep
Liquor I%, raw soybean flour 1%, peptone 0.5%, thorium chloride 0.3%, precipitated calcium carbonate 0.5% (p
Seed culture medium consisting of I (7,0) 40 ml (200 t
rt (dispense into a 1-volume Erlenmeyer flask, sterilize at 120°C for 20 minutes, and inoculate with strain B 62-21 (culture at -128°C for 40 hours on a rotary shaker. 25% seed culture, 3% dextrin, 1 part raw soybean flour
．． 5%, corn gluten meal 15%, peptone o
2% sodium thiosulfate, 0.1% sodium thiosulfate, 0.5% precipitated calcium carbonate (pT-17.0) to 200 mO. The mixture was transferred to an Erlenmeyer flask and cultured at 24°C for 90 hours on a rotary shaker to obtain a culture of 4.50%.

Example 2 pT of cultured limb (4,5C) obtained by the method of Example 1
-I was adjusted to 3.0, and ethyl acetate (450 was added to
After stirring for 0 minutes, the mixture was filtered using Hyfloth-Purcel (Johns Manpil, USA). The ethyl acetate layer (4,0Q) of the filtrate was extracted twice with a 2% sulfur/lium hydrogen carbonate aqueous solution (1,5&), and the aqueous layers were combined and concentrated to distill off the ethyl acetate. T-IP
-20 (Mitsubishi Kaeisha, Japan) (200m&) column. After washing the column with water (1,012), 30% methanol (800m0., fractions N091-4)
and 70% methanol (1,0c, fraction no.
, 5=9) and fractionated onto 200z12. Fraction Nos. 3 to 8 (1,2C) were concentrated and dried to obtain 1.51 g of crude powder.

The obtained coarse powder was dissolved in 40% methanol (207!12), and MCI gel CI-IP-20P (■50-30
0μ, Mitsubishi Kasei Co., Japan, +ooytra) Column [1] was inserted into the magnetic head. After washing the column with 40% methanol (300+), rinse with 60% methanol (
300 m12, fraction No. I~6), and 8
0% methanol (300 m (1, fraction No. 7
~+2) and fractionated at 50 m (addressed to 50 m). Fraction Nos. 4 to 7 (200 mQ) were collected, concentrated, and dried to yield a powder (678o) containing TAN-883-A, -B, and C. On the other hand, fraction No. 8
~9 (100xf2) was concentrated and dried, TAN-8
A powder (201*g) containing 83-D was obtained.

Powder containing TAN-883-A1-B and -C (67
0x9) was divided into 6 times and subjected to preparative high performance liquid chromatography (column: YMC5H-34301), mobile phase: 60% methanol - 0.05 Mpl - + 7.5 phosphate buffer, flow rate 10 μm/min). The sample was attached and fractionated to 15xQ. Each fraction was analyzed by high-performance liquid chromatography, and both fractions (135mc) containing only component A were collected and concentrated. ) for 3 times, and after washing with water,
Upon concentration, TAN883-A (82 my) was obtained as a white powder.

When the fraction containing only the C component (1365i0.) is collected and treated in the same manner as the A component, TAN-883c (+
9exy) was obtained as a pale yellow powder. (2) A fraction (270m) containing the three components was collected and treated in the same manner as the component B to obtain a powder (53mg) mainly containing component B. This powder was re-separated under the same conditions as above. Both fractions (6) containing only component B were subjected to chromatography.
By collecting 071 and processing in the same way as the Δ component,
TAN-883B (zffg) was obtained as a white powder.

Powder containing TAN-883-D obtained previously (201s
g) was divided into two batches and subjected to preparative high-performance liquid lithography (mobile phase: 65% methanol 0.05M pi-1).
7,5 phosphate buffer and other conditions (same as Mamoeki), and fracnoyone (225M &
) was collected and processed in the same manner as for component A to obtain TAN-88.
3-1) (13 mg) was obtained as a pale yellow powder. Furthermore, when this was crystallized from methanol, pale yellow crystals were obtained.

Effects of the Invention TAN-883, which has an angiogenesis-promoting effect, is useful for various wound healing, ischemic diseases, and prevention and treatment of gastrointestinal ulcers.

[Brief explanation of drawings]

The first factor is i"AN-883-II's ultraviolet absorption spectrum, Figure 2 is its extra-ray absorption spectrum), Figure 3 is its'!- (nuclear magnetic resonance spectrum, Figure 4 is TAN-
883-1), FIG. 5 shows its infrared absorption spectrum, and FIG. 6 shows its '+1 nuclear magnetic resonance spectrum.

Claims (1)

[Claims] (1) Physiologically active substance TAN having the following physicochemical properties
-883-B or a salt thereof. 1) Shape: White powder 2) Molecular formula: C_2_9H_4_0N_2O_83) Ultraviolet absorption spectrum: λ^M^e^O^H_m_a_x: 233,318 nm
λ^N^/^5^0^H^C^l^-^M^e^O^H
_m_a_x:218,320nmλ^N^/^5^0
^H^C^l^-^M^e^O^H_m_a_x:28
3,315nm4)^1^3C nuclear magnetic resonance spectrum (
75MHz, chemical shift in heavy pyridine, δpp
m): 12.9(q), 18.9(q), 26.6(t
), 29.1(t), 32.7(t), 37.8(t)
, 37.9(t), 40.8(t), 42.3(d),
43.2(t), 44.5(d), 46.3(d), 4
7.4(d), 48.6(d), 54.3(d), 59
．． 1(d), 60.2(d), 70.3(d), 71.
7(d), 74.0(d), 101.8(s), 123
．． 3(d), 124.9(d), 140.1(d), 1
50.1(d), 166.9(s), 174.6(s)
, 177.7(s), 194.4(s) (2) A physiologically active substance TAN-883-D or a salt thereof having the following physicochemical properties. 1) Shape: Pale yellow crystal 2) Molecular formula: C_2_9H_3_8N_2O_53) Ultraviolet absorption spectrum: λ^M^e^O^H_M_a_x: 259,340 nm
λ^N^/^5^0^H^C^l^-^M^e^O^H
_m_a_x: 265, 355, 370 (sh) nmλ
^N^/^5^0^H^C^l^-^M^e^O^H_
m_a_x: 259,338nm4)^1^3C nuclear magnetic resonance spectrum (75MHz, chemical shift in heavy pyridine, δppm): 12.9(q), 19.2(q
), 21.7(t), 26.8(t), 27.0(t)
, 39.3(t), 40.7(t), 41.3(t),
42.7(d), 45.5(d), 48.1(d), 5
4.5(d), 56.5(d), 56.5(d), 62
．． 2(d), 75.4(d), 102.1(s), 12
3.1(d), 126.5(d), 126.6(d),
129.4(d), 134.6(d), 139.7(d
), 143.2(d), 147.5(d), 166.4
(s), 174.0(s), 177.0(s), 196
．． 6(s) (3) Belongs to the genus Rhizobacter, TAN-883-A)-
B) culturing a microorganism capable of producing -C or -D in a medium, producing and accumulating the compound in the culture;
Physiologically active substance TAN-883 characterized by being collected
A method for producing -A, -B, -C or -D or a salt thereof. (4) An angiogenesis promoting agent containing a physiologically active substance TAN-883-A, -B, -C or -D or a salt thereof.