"Physiologically-active novel substance ""Aldostatin"" and production method thereof"

A physiologically-active novel substance "aldostatin" having an estimated molecular formula of C.sub.20 H.sub.20 N.sub.2 O.sub.8 is produced by culturing an aldostatin-producing microorganism of the Pseudeurotium, for example, the Pseudeurotium zonatum M4109 strain. Aldostatin inhibits the aldose reductase activity and as a consequence, avoids abnormal accumulation of sorbitol, galactitol, etc. It is therefore effective for the treatment of chronic complications such as cataract, retinopathy and neuropathy caused by diabetes renalis.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a physiologically-active novel substance 
"aldostatin", which is an aldose reductase inhibitor, and its salts as 
well as a production method thereof. Aldostatin, which the present 
invention provides, is useful for the treatment of certain chronic 
complications caused by diabetes renalis, such as diabetogenous cataract, 
retinopathy and neuropathy. 
2. Description of the Prior Art 
The reduction of blood sugar level has been the subject of a majority of 
attempts which have heretofore been made to obtain antidiabetic drugs. 
Under the circumstances, almost no means is however known for the 
prevention or reduction of chronic diabetogenous complications such as 
diabetogenous cataract, retinopathy and neuropathy. According to Sakamoto 
et al. ["Pharmacia" 19, 43, (1983)], the metabolism in the polyol system 
is activated by slight sthenia of an aldose reductase in a state of high 
blood sugar level such as diabetic, resulting in further promotion of the 
abnormal accumulation of sorbitol, galactitol and fructose. These 
sorbitol, galactitol and fructose are relatively stable in cells. Once 
they are formed there, their transit to the outside of the cells is 
scarcely observed. This disturbance to the balanced production and 
excretion leads to the intracellular accumulation of such sugar alcohols. 
This is in turn considered to cause accumulation of water in cells so that 
the cells can no longer maintain their normal function, resulting in a 
histionic problem. It has hence been considered that the abnormal 
intracellular accumulation of sorbitol, galactitol and fructose can be 
avoided and the cellular function can be maintained normal if the 
activities of the aldose reductase are inhibited. 
Accordingly, there has been a long-standing demand for the provision of a 
novel aldose reductase inhibitor useful for the prevention or treatment of 
chronic diabetogenous complications. 
SUMMARY OF THE INVENTION 
With a view toward obtaining a novel aldose reductase inhibitor, the 
present inventors isolated a number of microorganisms from soil and 
investigated the aldose reductase inhibitory activities of substances 
produced by the microorganisms. As a result, it has been found that 
certain microorganisms of the Pseudeurotium produce a novel aldose 
reductase inhibitor. 
The inhibitor has also been confirmed to be a novel aldose reductase 
inhibitor from various physiochemical and biological properties thereof 
and has now been named "aldostatin". 
Accordingly, an object of this invention is to provide a 
physiologically-active substance "aldostatin" having the following 
properties: 
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(a) distinction of acidic, neutral or basic: 
acidic substance 
(b) elemental analysis: 
C = 57.20 .+-. 2.0 
H = 4.70 .+-. 1.0 
N = 6.97 .+-. 1.5 
(c) measured molecular weight: 
(M + H).sup.+ 417 (by the FAB.sub.mass method) 
(d) estimated molecular formula: 
C.sub.20 H.sub.20 N.sub.2 O.sub.8 
(e) UV absorption spectrum: 
(1) aqueous neutral solution 
.lambda..sub.max 285 .+-. 2 nm (E.sup.1% .sub.1cm 135 .+-. 20) 
(FIG. 1) 
(2) aqueous acidic solution: 
.lambda..sub.max 285 .+-. 2 nm (E.sup.1% .sub.1cm 135 .+-. 20) 
(FIG. 2) 
(3) aqueous alkaline solution: 
.lambda..sub.max 230 .+-. 4 nm sh, 257 .+-. 4 nm sh, 
316 .+-. 2 nm (E.sup.1% .sub.1cm 195 .+-. 20) 
(FIG. 3) 
(f) IR absorption spectrum (KBr; cm.sup.-1): 
3300, 3030, 2920, 1720, 1650, 1495, 1410, 
1370, 1335, 1220. (FIG. 4) 
(g) .sup.13 C--NMR spectrum (d.sub.6 -DMSO, 100 MHz, internal 
standard DMSO = 39.5 ppm; .delta. ppm): 
171.7(s), 160.1(d), 152.1(s), 131.7(d), 
128.2(d), 127.1(s), 125.1(s), 125.1(s), 115.3(d), 
52.1(d), 35.9(t). (FIG. 5). 
(h) solubility: 
soluble: water, methanol, and 
dimethylsulfoxide. 
slightly soluble: 
acetone, ethyl acetate, 
and chloroform. 
insoluble: benzene, hexane and 
petroleum ether 
(i) color reaction: 
positive: KMnO.sub.4 and FeCl.sub.3. 
negative: ninhydrin, Molisch and Fehling. 
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Another object of this invention is to provide a method for the production 
of a physiologically-active novel substance "aldostatin", which comprises 
culturing an "aldostatin"-producing microorganism of the Pseudeurotium in 
a culture medium and then collecting "aldostatin" from the culture broth. 
Aldostatin of this invention inhibits the aldose reductase activity and as 
a consequence, avoids abnormal accumulation of sorbitol, galactitol, etc. 
It is therefore effective for the treatment of chronic complications such 
as cataract, retinopathy and neuropathy caused by diabetes renalis.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENT 
As a practical example of the microorganism useful for the production of 
the physiologically-active substance "aldostatin" of this invention, may 
be mentioned the M4109 strain of the Pseudeurotium which the present 
inventors isolated from the soil of a farm land in Kannami, Tagata-gun, 
Shizuoka, Japan. Its mycological characteristics are as follows: 
(A) Growth in Various Culture Media 
(1) Czapek's Agar 
At 26.degree. C., the growth is rather slow. It grows to a diameter of 
16-17 mm in 7 days of culture and to 28-29 mm in 14 days of culture. The 
lawn is thin, flat and velvet-like. Only white hyphae grow. No ascocarp is 
formed. Peripheries are smooth. Neither exudate nor diffusible pigment 
occurs. The reverse is white - yellowish white (1A2). No growth at 
37.degree. C. 
(2) Malt Ex. Agar: 
At 26.degree. C., the growth is rather slow. It grows to a diameter of 
21-22 mm in 7 days of culture and to 30-31 mm in 14 days of culture. The 
lawn is thin and flat but is slightly swollen at a central part. White 
hyphae grow at the beginning but as the culture proceeds, a number of 
ascocarps are formed underneath white aerial mycelia so that the color 
changes to light grey (1C1)-medium grey (1E1). Peripheries are smooth. 
Neither exudate nor diffusible pigment occurs. The reverse is yellowish 
white (4A1)-pale yellow (4A2) at an area bearing hyphae and olive grey 
(1E2) at another area with ascocarps formed therein. No growth at 
37.degree. C. 
(3) Potato Dextrose Agar 
At 26.degree. C., the growth is rather slow. It grows to a diameter of 
19-20 mm in 7 days of culture and to 30-31 mm in 14 days of culture. The 
lawn is thin and flat but is slightly swollen at a central part. White 
hyphae grow at the beginning but as the culture proceeds, a number of 
ascocarps are formed underneath white aerial mycelia so that the color 
changes to medium grey (1E1). Peripheries are smooth. A colorless exudate 
is slightly released but no diffusible pigment occurs. The reverse is 
olive grey (1F2). No growth at 37.degree. C. Color names and signs follow 
the method described by A. Kormerup and J. H. Wanscher in "Methuen 
Handbook of Color" 3rd ed., Eyre Methuen, London, 1978. 
(B) Various Physiological Properties 
Growable pH: 3.2-9.6 
Optimum pH for growth: 3.6-6.4 
Growable temperature: 19.degree.-30.degree. C. 
Optimum temperature for growth: 22.degree.-26.degree. C. 
(C) Microscopic Morphological Characteristics 
Formation of closed ascocarps. The closed ascocarps are blackish brown, 
spherical, 80-180 .mu.m in diameter, and in the form of naked asci. The 
ascocarp walls are of somewhat skin-like nature, are dark brown. They are 
composed of a single layer and do not contain any special stitch 
mechanism. Constituent cells of the ascocarp walls have irregular 
polygonal shapes, the diameters of which range from 5 .mu.m to 15 .mu.m. 
Asci are scattered at random in closed ascocarps, have egg-like, pear-like 
or oval shapes, contain 8 spores, and are 8-11.times.7-9 .mu.m. Ascospores 
are spherical and are each formed of a single cell. Their walls are thick 
and are dark brown. Diameters: 3-4 .mu.m. Neither germ pores nor slits are 
contained. They look like Sporothrix in the conidial stage. Conidia are 
formed in the form of the sympodiosporae type at small tooth-like tips of 
conidium-forming cells. The conidia are colorless, are each formed of a 
single cell, are 5-7.5.times.2.5-3.5 .mu.m, have either oval shapes or 
inverted egg-like shapes, and are slightly thinner at basal portions 
thereof. Walls are smooth. Conidium-forming cells occur vertically from 
aerial hyphae, are 8-40.times.3-5 .mu.m, are not branched, are slightly 
swollen at central parts, and are tapered toward their free ends. The 
conidiumforming portions lengthen new growing points one after another. 
The mycological characteristics of the above microorganism have been 
described above. Since it grows through the ascigenous stage (complete 
stage), the above microorganism is considered to belong to the genus of 
Ascomycetes. Owing to such characteristic properties that closed ascocarps 
are formed, ascocarps are irregularly scattered therein, and ascospores 
are formed of single cells and contain no germ pores or slits, the above 
microorganism is classified under the ordo of Eurotiales. There are about 
50 genera under the ordo of Eurotiales. The above microorganism has 
however been judged to fall under the genus of Pseudeurotium for such 
characteristic properties that its closed ascocarps are dark brown, are 
formed of polygonal cells and have no stitch mechanism, and ascospores are 
small, brown and smooth, and conidial stages are Sporothrix like. Three 
species have been recognized by now under the genus of Pseudeurotium, 
namely, Pseudeurotium ovalis, Pseudeurotium punctatum and Pseudeurotium 
zonatum. Although ascospores of Pseudeurotium ovalis and Pseudeurotium 
punctatum have oval - egg-like shapes, the present microorganism M4109 
contains spherical ascospores characteristic to Pseudeurotium zonatum. As 
a result of detailed observation, the present strain M4109 was identified 
as a Pseudeurotium zonatum and was hence named "Pseudeurotium zonatum 
M4109". This microorganism has been deposited under FRI Deposition FERM 
P-8614, Fermentation Research Institute, Agency of Industrial Science and 
Technology, Ministry of International Trade and Industry, the Japanese 
Government. 
Upon culture of such a microorganism of the Pseudeurotium which produces 
the physiologically-active substance "aldostatin", a material which the 
microorganism can metabolize, such as glucose, starch, sucrose, dextrin, 
molasses, glycerol, oil or fat, an organic acid or the like may be used as 
a carbon source for a culture medium. As a nitrogen source, an organic or 
inorganic nitrogen-containing compound such as soybean meal, cotton seed 
flour, CSL, meat extract, peptone, yeast extract, germ, urea, ammonium 
sulfate, ammonium nitrate, ammonium phosphate, ammonium chloride or the 
like may be employed. Besides, inorganic salts such as NaCl, KCl, 
CaCO.sub.3, MgSO.sub.4, KH.sub.2 PO.sub.4, Na.sub.2 HPO.sub.4, FeSO.sub.4, 
MnCl.sub.2, CoCl.sub.2, ZnSO.sub.4 and CaSO.sub.4 may also be used 
suitably either singly or in combination. Vitamins such as vitamin B.sub.1 
and biotin, a deforming agent such as silicone oil and/or a surfactant 
such as a polyalkylene glycol ether may also be added to culture media as 
needed. In addition, it is also feasible to add one or more organic and/or 
inorganic substances which promote the growth of the microorganism and 
stimulate the production of aldostatin. 
For the culture of the above microorganism, any one of conventional culture 
methods for microorganisms may be employed. Although both solid culture 
and liquid culture are usable, aerated stirring culture is preferred. A 
suitable culture temperature can be chosen within a temperature range in 
which the microorganism is allowed to grow and to produce aldostatin. The 
culture temperature may however be 19.degree.-30.degree. C., especially, 
22.degree.-26.degree. C. The culture may be terminated by judging the time 
at which aldostatin has reached its maximum potency. Although the actual 
number of days required for the culture varies to a certain extent 
depending on conditions, 2-7 days, especially, 3-5 days are preferred. 
In order to collect the physiologically-active substance "aldostatin" from 
a culture broth obtained in the above-described manner, it is possible to 
follow suitably any one of separation methods which are routinely used 
upon collection of intermediates, produced by general microorganisms, from 
their culture broths. For example, cells are removed from a culture broth 
by either filtration or centrifugation since aldostatin shows properties 
of water-soluble acidic substances and is primarily contained in the 
filtrate of the culture broth. The thus-obtained filtrate of the culture 
broth is adsorbed on a suitable carrier, followed by selective desorption 
of the effective substance with a suitable solvent. As a carrier of a 
chromatography employed for the desorption, a material making use of 
differences in adsorptiveness such as activated carbon, silica gel, 
alumina, cellulose powder or a synthetic adsorptive resin, a material 
utilizing differences in functional groups such as an anion exchange resin 
or anion exchange cellulose, or a material relying upon differences in 
molecular weight such as a molecular sieve carrier can be advantageously 
used. In order to elute the target compound "aldostatin" from such a 
carrier, water-containing organic solvents, namely, water-containing 
acetone, water-containing methanol and water-containing acetonitrile, 
acids, alkalis and buffers, aqueous solutions containing inorganic or 
organic salts, etc. may be used in a suitable combination, although their 
combination varies depending on the kind and properties of the carrier. 
The inhibitor of this invention obtained in a crude form by the 
above-mentioned chromatography can be subjected further to a preparative 
high-performance liquid chromatography so as to purify same. In detail, 
the pH of the filtrate is adjusted to about 4. It is then caused to pass 
through a column packed with a synthetic adsorptive resin as a carrier, 
for example, "DIAION HP-20" (trade name; product of Mitsubishi Chemical 
Industries, Ltd.), "Amberlite XAD-II" (trade name; product of Rohm and 
Haas Company) or the like, whereby the inhibitor of this invention 
contained in the filtrate is adsorbed. The thus-adsorbed inhibitor of this 
invention is then eluted with a water-containing alcohol or 
water-containing acetone. Fractions containing the thus-eluted inhibitor 
are concentrated and are then adsorbed on alumina or silica gel. It can 
thereafter be eluted with a solution of aqueous ammonia in acetonitrile or 
with a water-containing alcohol solution. The thus-fractionated eluate 
fractions are processed into powder through steps such as concentration 
and lyophilization. 
If the purity of the thus-obtained powder is low, high-performance liquid 
chromatography can be advantageously used for further purification. As 
useful carrier, may be mentioned "Lichroprep RP-18 Gel" (trade name; 
product of MERCK & CO. INC.) or "YMC Gel ODS 30/60" (trade name; product 
of Yamamura Chemical Laboratories, Inc.) by way of example. As a mobile 
phase, it is possible to use a mixture of methanol or acetonitrile and an 
aqueous solution of a salt or the like. Aldostatin obtained in the above 
manner usually contains one or more salts employed for its purification. 
For desalting same, it is advantageous to use chromatography which employs 
a synthetic adsorbent. Namely, an aqueous aldostatin solution containing 
the salts is adjusted to about pH 4.0 with dilute hydrochloric acid, 
followed by its chromatography on "DIAION HP-20". After washing the 
carrier with water, active fractions are eluted with a water-containing 
alcohol. Upon concentration and lyophilization of the eluate, aldostatin 
is obtained in its free form. 
The thus-obtained aldostatin of this invention has the following physical 
and chemical properties. From these properties, it is considered to be a 
compound represented by the following estimated structural formula: 
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##STR1## 
Physiochemical properties 
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(1) external appearance: white powder. 
(2) distinction of acidic, neutral or basic: 
acidic substance 
(3) melting point: 144-146.degree. C. 
(4) elemental analysis: 
C = 57.20 .+-. 2.0 
H = 4.70 .+-. 1.0 
N = 6.97 .+-. 1.5 
(5) measured molecular weight: 
(M + H).sup.+ 417 (by the FAB.sub.mass method) 
(6) estimated molecular formula: 
C.sub.20 H.sub.20 N.sub.2 O.sub.8 
(7) specific rotation [.alpha.].sub.D.sup.26 
+ 59.0.degree. .+-. 10.degree. (C = 0.50, H.sub.2 O) 
(8) UV absorption spectrum: 
(1) aqueous neutral solution: 
.lambda..sub.max 285 .+-. 2 nm (E.sub.1 cm.sup.1% 135 .+-. 20) 
(FIG. 1) 
(2) aqueous acidic solution: 
.lambda..sub.max 285 .+-. 2 nm (E.sub.1 cm.sup.1% 135 .+-. 20) 
(FIG. 2) 
(3) aqueous alkaline solution: 
.lambda..sub.max 230 .+-. 4 nm sh, 257 .+-. 4 nm sh, 
316 .+-. 2 nm (E.sub.1 cm.sup.1% 195 .+-. 20) 
(FIG. 3) 
(9) IR absorption spectrum (KBr; cm.sup.-1): 
3300, 3030, 2920, 1720, 1650, 1495, 1410, 
1370, 1335, 1220. (FIG. 4) 
(10) .sup.13 CNMR spectrum (d.sub.6 -DMSO, 100 MHz, 
internal standard DMSO = 39.5 ppm; .delta. ppm): 
171.7(s), 160.1(d), 152.1(s), 131.7(d), 
128.2(d), 127.1(s), 125.1(s), 115.3(d), 
52.1(d), 35.9(t). (FIG. 5). 
(11) .sup.1 HNMR spectrum (d.sub.6 -DMSO, 400 MHz, 
internal standard TMS; .delta. ppm): 
8.02(1H,S), 7.98(1H,d), 7.05(1H,s), 
6.97(1H,d), 6.80(1H,d), 4.50(1H,q), 
2.96(2H,m). (FIG. 6) 
(12) thin layer chromatography (TLC): 
spot film, "Silica Gel f" (trade name; 
product of Tokyo Kasei Kogyo Co., Ltd.) 
Solvent system Rf value 
BuOH:AcOH:H.sub.2 O (4:1:1) 
0.63 
CHCl.sub.3 :MeOH:H.sub.2 O (5:5:1) 
0.31 
EtOAc:MeOH:H.sub.2 O (5:5:1) 
0.27 
CHCl.sub. 3 :MeOH:AcOH:H.sub.2 O (10:5:1:1) 
0.36 
PrOH:H.sub.2 O (4:1) 0.25 
(13) high-performance liquid chromatography (HPLC): 
carrier: "Hitachi Gel #3056" (trade name; 
product of Hitachi Ltd.) 
mobile phase: 
8% methanol with 1% ammonium ace- 
tate, 0.8 ml/min, Rt = 4.2 (min). 
(14) solubility: 
soluble: water, methanol, and dimethylsulfoxide. 
slightly soluble: 
acetone, ethyl acetate, and chloroform. 
insoluble: benzene, hexane and petroleum ether 
(15) color reaction: 
positive: KMnO.sub.4 and FeCl.sub.3. 
negative: ninhydrin, Molisch and Fehling. 
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Biological Properties 
(1) Aldose Reductase Inhibitory Activity 
The preparation of an aldose reductase and the quantitative analysis of its 
enzymatic activity were effected in accordance with the method described 
by Hayman et al. in "Journal of Biological Chemistry" 240, 877 (1965). It 
was a partially purified aldose reductase derived from bovine lenses that 
was employed as an enzyme. Placed in a quartz cell in a spectrophotometer 
photometer was 2.4 ml of a 5.times.10.sup.- 5M NADPH solution dissolved in 
2/15M phosphate buffer (pH 6.0), 0.4 ml of 15 milli-units/ml solution of 
the enzyme and 0.1 ml of an aqueous aldostatin of one of various 
concentration. The contents were pre-incubated at 37.degree. C. for 4 
minutes. The reaction started by adding 0.1 ml of 0.015M 
D,L-glyceraldehyde into the quartz cell. The reduction of the absorbance 
at 340 nm was traced at 37.degree. C. for 4 minutes. At that time, the 
value (B) of tan .theta. was determined. Similarly, the reduction of the 
absorbance at 340 nm without addition of the inhibitor "aldostatin" was 
tracked. At that time, the value (A) of tan .theta. was determined. 
Percent inhibition (%) was calculated as follows. 
##EQU1## 
The amount of inhibitor for 50% inhibition was expressed as IC.sub.50. 
The aldose reductase inhibitory activity of aldostatin determined in the 
above-described manner is as follows: 
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Substrate (substrate concentration) 
IC.sub.50 (.mu.g/ml) 
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glyceraldehyde (5.0 .times. 10.sup.-4 M) 
0.5 
glucose (5.0 .times. 10.sup.-2 M) 
1.1 
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(2) Acute Toxicity 
Aldostatin showed no toxic effect when it was administered i.v. into mice 
at a dose of 200 mg/kg. 
Aldostatin of this invention may also be converted into its metal salts and 
ammonium salt a needed. Exemplary metal salts include its sodium salt, 
potassium salt, lithium salt, calcium salt, etc. 
A drug containing aldostatin of this invention may be formulated into 
dosable preparations such as injections, nasal drops, suppositories and 
the like in addition to its orally administrable preparations and eye 
drops. The drug may be prepared by a method known per se in the art while 
incorporating aldostatin or a salt thereof suitably in an amount of 100 
.mu.g-500 mg per administration. 
EXAMPLE 
This invention will next be described by the following Example. It should 
however be borne in mind that the present invention is not limited by the 
following Example. 
EXAMPLE 1 
(i) The strain of Pseudeurotium zonatum M4109 (FRI Deposition FERM P-8614), 
which had been allowed to grow by slant culture on nutrient agar, was 
inoculated in a 500 ml Erlenmeyer flask containing 100 ml of a sterilized 
culture medium of 2% glucose, 2% starch, 2% soybean flour, 0.25% NaCl, 
0.5% yeast extract and 0.35% CaCO.sub.3 (pH 6.5). The contents were 
subjected to shaking culture at 26.degree. C. for 3 days. The resultant 
culture broth was used as a seed culture. 
(ii) Next, twenty liters of a culture medium containing 2% glucose, 1% 
peptone, 1% CSL, 0.2% KH.sub.2 PO.sub.4 and 0.1% MgSO.sub.4.7 H.sub.2 O 
(pH 6.5) were poured 100 ml by 100 ml in 500 ml Erlenmeyer flasks. After 
sterilizing the contents at 120.degree. C. for 20 minutes, each flask was 
inoculated with 4 ml of the seed culture, followed by shaking culture a 
26.degree. C. for 4 days. 
(iii) The culture broth (20 l) obtained in the above procedure (ii) was 
adjusted to pH 4, followed by filtration to obtain 17 l of a culture broth 
filtrate. A majority of aldostatin was contained in the filtrate. The 
culture broth filtrate was subjected.to chromatography on "DIAION HP-20" 
(1.6 l). After the column was washed with 8 l of water, active fractions 
were eluted with 50% methanol water. The active eluates (4 l) were 
concentrated to 1 l under reduced pressure. After adjustment with aqueous 
ammonia to pH 6.5, the resultant solution was charged in a column of 500 
ml of "DIAION HP-20". Upon elution with water, a majority of the activity 
was contained in both the eluate (1 l) and the water-eluted fractions (1.3 
l). 
(iv) Under reduced pressure, 2.3 l of these active fractions was 
concentrated to about 20 ml, followed by an addition of 200 ml of 
methanol. The resulting precipitate was removed by centrifugation so as to 
obtain a centrifugal supernatant. The supernatant was charged in an 
alumina column (200 ml) which had in advance been filled with methanol. 
After washing the column with 1 l of methanol and 1 l of acetonitrile, the 
column was developed with a 2:1 mixed solvent of acetonitrile and 1N 
aqueous ammonia. The eluate was fractionated 18 g by 18 g so that the 
active substance of this invention was eluted in Fraction Nos. 61-190. 
These fractions were collected and then concentrated under reduced 
pressure. The residue was charged in a Sephadex G-15 column (1650 ml), 
followed by development with water. The eluate was fractionated 15 g by 15 
g. The active substance was eluted in Fraction Nos. 65-80. These fractions 
were collected and then concentrated under reduced pressure, followed by 
lyophilization to obtain 350 mg of white powder. 
(v) The white powder obtained in the above procedure (iv) was subjected to 
preparative high-performance liquid chromatography on a column of 
"Lichroprep RP-18" (trade name; product of MERCK & CO., INC.) as a 
carrier, the particle sizes of which ranged from 25 .mu.m to 40 .mu.m. The 
column was then eluted and fractionated with a 3% aqueous solution of 
methanol, which contained 0.5 of ammonium acetate. The resultant fractions 
were each analyzed by liquid chromatography. Fractions showing the single 
peak characteristic to aldostatin were collected. The thus-collected 
active fraction was adjusted to pH 4.0 with 0.5N hydrochloric acid. It 
was thereafter caused to pass through a column packed with 50 ml of 
"DIAION HP-20". After washing the column with 250 ml of water, the column 
was eluted with 150 ml of a 50% aqueous solution of methanol. The eluate 
was concentrated, followed by lyophilization to obtain 75 mg of aldostatin 
as white powder in its free form. 
Having now fully described the invention, it will be apparent to one of 
ordinary skill in the art that many changes and modifications can be made 
thereto without departing from the spirit or scope of the invention as set 
forth herein.