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This is to certify that the
5 thesis entitled
Studies in the Isolation of an

Anti-tumor Growth Factor From
Boletus'gggilg

‘ ,' presented by
Alfred Ernest Ritchie

has been accepted towards fulfillment
of the requirements for

__E._ degree hwy

‘-__.
\\\J
a

Major grofessor

Date Aug. 29. 195l__

‘ \ 0-169

 

 

- -
l

STUDIES IN THE ISOLATION OF AN ANTI-TUMOR
GROWTH FACTOR FROM BOLETUS EDULIS

BY

Alfred Ernest Ritchie

A THESIS

Submitted to theSchool of. Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree or

MASTER OF SCIENCE
Department of Chemistry

1951

’7! IFMISTRY DFPT.

Tbla‘0\ E
Reefi

PREFACE

The work to be described was undertaken as a cooper-
ative project of the departments of Chemistry and Horti-
culture at Michigan State College. It was initiated as a
result of the observation of Dr. E. H. Lucas that a tumor
growthrinhibitor could be extracted from the mushroom
Boletus edulis.

The primary aim of the study was to determine, if
possible, a practical means of concentrating or isolating
this principle.

This thesis will deal with the evolution of a pro-
cedure for the separating from crude material extracts
the factor or factors responsible for the inhibitory
activity.

The author wishes to eXpress his sincere thanks to
Dr. E. H. Lucas for the origin of the problem and the
financial assistance given the author throughout the
study and for his friendly counsel and encouragement.

The author also wishes to express his sincere thanks to
Dr. R. U. Byerrum for his intimate association and helpful
suggestions that stimulated the author in his work.

TABLE OF CONTENTS
CHAPTER

I. CHEMICAL STUDIES OF BOLETUS EDULIS . e e . . .

HISTORICAL BACKGROUND. . e . e . e e e e . . .

Tumor growthvinhibitors from sources other

thanBoletusedulis...........

Tumor growth-inhibitor from.Boletus edulis

METHODS AND MATERIALS . . . 0
Method of Biological Assay.
Experimental details. . .
Interpretation of assays.
Source of Materials . . . .
Methods of Concentration. .
Extraction. . . . . . . .
Preoipitating agents. . .
Acetic acid . . . . . .
Ammonium.su1fate . . . .
Ethanol . . . . . . . .
Acetone . . . . . . . .
Dialysis. . . . . . . . .
Lyophilization. . . . . .
RESULTS. . . . . . . . . . . .
Stability Tests . . . . . .
Stability toward acid . .

Stability toward alkali .

Page

OOOQQQQQQGQQGO‘tbfifi-NH

H
O

TABLE OF CONTENTS CONT.

Page

Thermal stability e e e e e e e e e e e e e e 10

Preparation of Samples. . . . . . . . . . . .
50 Per cent ethanol precipitation . . . . .
Acetic acid precipitation . . . . . . . . .
50 Per cent ammonium sulfate precipitation.
50 Per cent ethanol precipitation . . . . .

Dialysis and absorption techniques of
detoxification. e e e e e e e e e e e e e

50 Per cent ethanol followed by acetone
precipitation e e e e e e e e e e e e e 0

Stability and fractionation (acetone)
tOOhDIQUBSe e 0'. e e e e e e e e e e e e

A01d stability. e e e e e e e e e e e e e
Alkali stability. 0 e e e e e e e e e e 0

Stability at 80 - 90 per cent acetone
concentrations. 0 e e e e e e e e e e 0

Acid hydrolysis e e e e e e e e e e e e e

Re-extraction and modified ethanol
precipitation technique . . . . . . . . .

50 Per cent ethanol followed by dialysis
and acetone fractionation . . . . . . . .

50 Per cent ethanol, dial sis, and acetone)
fractionation (modified e e e e e e e e 0

Direct acetone precipitation (rapid method)

Direct acetone precipitation and
re-precipitation. e e e e e e e e e e e e

Detoxification technique (Pancreatin
d1808t10fl). e e e e e e e e e e e e e e e

Acetone fractionation (2 step method) . . .

Detoxification technique (Trypsin digestion).

11
12
13
13
15

16

2O

22
23
25

27
28

30

33

56
57

39

c“;

4?

TABEE OF CONTENTS CONT.
Page

55 Per cent acetone precipitation
(IiflglOBtOPMB‘bhOd)eeeeeeeeeee 48

Acetone fractionation (variable time
method).................. 48

SUMMARY.....................61
CONCLUSIONS 0 O O 0 O 0 O 0 O O 0 0 O 0 O O O O O 52
BIBLIOGRAPEO O O O O O O O O O O O O O O O O 0 O 54

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

LIST OF

ILLUSTRATIONS

Page

CHAPTER I
CHEMICAL STUDIES OF BOLETUS EDULIS

HISTORICAL BACKGROUND

The origin of studies in the isolation of tumor-
growth inhibiting substances from natural sources is

unknown.

Tumor-growth inhibitors from sources othgr_than Boletus
edulis.

In 1924 Matsushita (7) reported that treatment with a
plant enzyme or enzyme-like substance dispersed in oil
resulted in necrosis and gradual resorption of tumors and
was said to have clinically cured thirty-five per cent of
3,417 patients and others improved. Shortly after, Torrey
and Kahn (ll) injected directly into rat tumors preparations
of proteolytic enzymes from Bacterium.histolyticus with
destruction of the growth in from 50 per cent to 75 per cent
of the animals. However, Gye (2) found this preparation
inactive against other mouse tumors. The chemical nature of
these preparations was never reported since the work was
not confirmed by the later investigation of Gye.

For several years beginning in 1959 Lewisohn (5),
Laszlo (3), and Leuchtenberger (4) studied extracts of

polished rice, pearled barley, and Lactobacillus casei and

 

reported that anti-tumor factors could be demonstrated from

these sources and could be quantitatively compared with
-inositol standards. Although the natural extracts were
impure they were more active unit for unit of weight than
inositol itself. Further study, following this observation,
suggested that the "folic acid concentrates" of these prep-
arations might contain the active principle. This was found
to be the case, but not all of the activity could be ascribed
to these fractions of "folic acid". Other materials studied
by these workers included a yeast and S; Lactis 3;, Sugiua
(10) reported that his work with the preparations of Lewisohn,
Laszlo, and Leuchtenberger could not confirm their findings.
In 1951 Reilly and Stock (8) reported that studies
carried out on culture media in which strains of Aspergillus

fumigatus were grown showed that a substance was present-

 

which exhibited tumor-growth inhibitory preperties. This
activity was measured against the mouse sarcoma 180 and was
found to inhibit the growth of the tumor. The active agent
of Aspergillus fumigatus strain 943 was isolated by complete
saturation of a filtrate of the culture medium.with ammonium
sulfate, suspension of the resulting precipitate in water,
and dialysis.. The activity was non-dialyzable and was lyo-
philized. The anti-tumor substance seemed to be protein

in nature and was not one of the four bactericidal agents
that this fungus is known to produce. Further purification
of the active principle did not result in any appreciable
decrease in its toxicity for mice, and thus it has not
proven to be a promising therapeutic agent.

Tumor-growth inhibitor £322 Boletus edulis

In 1950 Lucas (6) prepared water extracts of dry, crude
material from.§; edulis which consistently inhibited the
growth of the mouse sarcoma 180. Although the inhibition
was not complete and the dose levels were toxic, these obser-
vations warranted further investigation. Treatment of the
extract with 0.01 per cent formaldehyde destroyed the
activity of the samples. Likewise, refluxing for two hours
at 100°C. resulted in loss of the activity. 'The activity
however was not destroyed by treating the dry, crude material
with.absolute ethanol prior to the water extraction. These
observations led to a preliminary belief that the substance
responsible for the activity might be proteinaceous or closely

associated with the protein extracted.from.the crude material.

METHODS AND MATERIALS
Method of Biological Assay

Experimental details.

An in vivo test (1, 8, 9) was employed by the Sloan-
Kettering Institute for Cancer Research in New'York to
ascertain the tumor-growth inhibiting activity of the
samples prepared.

Preliminary to the inhibition test, single dose toxicity
limits were determined so that the inhibition test could be
carried out at a maximum.tolerated dosage. As will be seen
from.the results reported here, the single-dose toxicity
test could not be perfectly correlated with the in vivo
inhibition test results.

The inhibition test consisted of treating groups of
five mice (20 g.1:2 White Swiss), 24 hours previously sub-
cutaneously implanted with the Crocker Mouse sarcoma 180,
with intraperitoneal injections of the various fractions
prepared from §:_edulis. Injections were usually 1 cc. or
less administered twice daily for seven days. Mice of
similar age and weight Were implanted with the tumor but
received equivalent amounts of physiological saline to
serve as controls. At the end of seven days the tumors
of both the treated and control animals were measured in

two diameters by means of calipers. The inhibition of the

5

tumors in the treated animals was based upon the develOpment
of the tumors in relation to the untreated controls. ‘leight
changes of the treated animals and controls were determined
and reported.

Interpretation 93: m.

The single dose toxicity test results were reported as
numbers within the limits of 0.5 and 5.0. Five-tenths
represented extreme toxicity accompanying a single injection
01:10.5 cc. of the sample, and 5.0 represented non-toxicity
at dosages of 5.0 cc. Numbers between these two figures
represented gradations of the toxicity within these arbi-
trary limits.

For the inhibition test an arbitrary system‘cf grading
was also adopted. It was as follows:

flanked inhibition (+) - The average diameters of the
tumors in the treated animals were one-quarter or less than
that of the control animal tumors.

Good inhibition.(t*) - ‘Average tumor diameters in the
treated animals were one-fourth.to one-half of those of the
controls.

Slight inhibition (1?) -- Average tumor diameters in the
treated animals were one-half to three-fourths of those of
the controls.

Negative (-) -- Average tumor diameters in the treated
animals were three-fourths of or equal in diameter of those
of the controls.

mew

Boletus M grows naturally in wooded areas of
Germany from which the material was obtained. After the
mushrooms were gathered, they were thoroughly air dried
and packed for shipment.

. Samples for this work were taken from three different
shipments from Germany all of which were shown to contain
the active principle.

Methods 9; COncentration "

htraction.

In all instances reported here the dry material after
grinding to 60 mesh in a Wiley mill was extracted with dis-
tilled water. The extractions were made either in a ceramic
morter with sand or in a Waring Blender without the use of
sand. The ratio of weights of sample to extrath was be-
tween the limits of 1:10 and 1:30 with the mjority of ex-
tractions carried out at 1:20. Hereafter the solution
obtained from the extraction or the fungus with water .111

be referred to as the extractive.

Precipitatigg agents.
As discussed previously (page 5), the possibility that

the active principle might have been proteinaceous led to

the use of common protein precipitants as trial methods for
the concentration studies. All precipitations were carried
out in the cold at 7.0013. in standard beakers or Erlenmeyer

 

‘ Condensation,gconoentration, condensation and concentrate
are used synonymously in this paper.

flasks.

Acetic acid: the use of acetic acid was limited to
lowering the pH of the extractive to ca. pH 4.0 and collect-
ing the resulting precipitated materials.

Armenian sulfate: fifty per cent saturation with ammon-
ium sulfate was used as a method of precipitation followed
by dialysis to remove the excess salt from the collected
precipitate. Il'he method used consisted of adding an equal
volume of a saturated solution of amonium sulfate to the
extractive of the §_._ M material.

The saturated solution of amonium sulfate was clarified
by the addition of ammonium hydroxide (cone.) to a pH of 9.0,
heating for two hours on a steam bath, allowing to stand for
48 hours and decanting to remove the hydrous oxides of the
heavy metals. The pH of the decanted solution was readjusted
with C.P. sulfuric acid to ca. 5.2 (to the pH of a satur-
ated solution of G.P. ammonia sulfate). After the pH
adjustment crystalline 0.P. ammonium sulfate was added to
insure complete saturation.

Ethanol: ethanol was used in a concentration up to 50
per cent by volume by the addition of the calculated amount
of '70 or 95 per cent ethanol to the extractive.

Acetone: acetone was used over a wide range of concen-
trations from 30 per cent by volume to 90 per cent by volume
as. an attempt to fractionate the precipitated materials.

Dialysis: the apparatus used for dialysis was of the
rotating tank type driven by a fractional horsepower motor

geared down by either reduction drives or pulleys to a
speed at ca. 50 rpm. The dialyzing bag was imersed in the
rotating tank excentrically to facilitate agitation with
consequent shorter equilibration time. The ratio of the
solution in the bag to the dialysate varied within wide
limits depending upon the nature of the material in the bag
and the purpose for which the dialysis was made. The lower
limit of ratio was ca. 1:1 and the higher limit greater
than 1:45. Usually the dialysis was carried out at room
temperature with a few runs made in the cold at 7.0°0.
'The dialysate was distilled water in all cases.
Lyophilizaticn: the apparatus used in 1yophilization
of some of the samples utilized a standard Cenco vacuum
pump with a pistol type mercury diffusion pump in series
to develop a vacuum of ca. 0.001 mm Hg pressure. The
freezing unit consisted of a steel tank of ca. five liters
capacity into which ethanol and solid carbon dioxide were
placed. The temperature of this system was ca. 42.0%.
The plan of this equipment as designed and constructed by
Diang is shown in Figure 1.

b Figure 1*

 

 

C: x d

 

 

 

 

 

A

Figure l. Freezingodehydration tank A. Side section view B. Top section view

a. freaing chamber. b. center pipe. c. freezing cone, d. 250 ml centrifuge bortle. e. adapter, f. rubber ring

 

 

 

 

b
.-
C

d
bUclamp F
csetdngscrew
dwccdecblod:

Cid-'00

 

 

 

4L

 

 

re

l
to mechanical pump ’0
.—
P P
B
l—J
l

k.

 

 

 

 

 

 

 

 

 

 

 

 

f”:'721_____i:a I
Civ :3

Figure 3. Assembly of apparatus A. Freezing-dehydration tank B. mcruxry condensation pump

 

 

 

 

 

C

 

 

 

 

 

 

 

 

C. mechanical pump D. coil trap E. McLeod gaub-e F. dry air inlet G. we Way stopcock

* Courtesy of Stephen Ljang (M.S.C. thesis in preparation)

RESULTS

Stability Tests

Previous experience and the reported findings of
other investigations had suggested that the anti-tumor
factor or factors from natural sources were of an unstable
nature. In order to obtain definite information concom-
ing the risk of losing the activity in the course of the
isolation processes, samples were prepared and tested in
order to ascertain stability limits of the material under

various conditions.

Stabilin toward acid. ‘
An extraction was made of the crude material and the

extractive made acid to a pH of 2.0 by the addition of
concentrated hydrochloric acid. After a short period of
time (ca. 2 hours), the pH was adjusted to 5.1 with 10 per
cent sodium hydroxide added dropwise. The upper limit of
pH 5.1 was taken since the pH of the extractive was deter-
mined as 5.1 with a pH meter. The tumor inhibition test
result of this sample was negative.

Another extractive was made acid to a pH less than
1.0 with concentrated hydrochloric acid and allowed to stand
in the cold (7°C.) for four days. The solution was then
dialyzed until the pH had risen above 4.0 and no test for

10

carbohydrates (Molisch) was observed. The tumor inhibition
test result was negative. From these results, it was assumed
that the active principle was acid labile and all subsequent
preparations were made without lowering the pH of the

extractive.

Stability toward alkali.
An extractive was made alkaline to a pH of 8.9 for about

two hours with 10 per cent sodium hydroxide, then adjusted
to 5.1 with concentrated hydrochloric acid. The inhibition
test result was negative and further preparations were made
without raising the pH of the extracting medium.

Thermal stability.

The stability toward heat was determined by Lucas as
described previously (page 3). As a result of the loss of
anti-tumor activity by refluxing for two hours, all samples
prepared in this work were kept from obtaining a temperature
greater than 35°C. in any step in their preparation. Loss
of activity by low temperatures (7.000.) was never observed,
except through the action of extreme concentration of acid

in the cold.

Stabilig toward dialysis.
It was observed that the active principle was non-

dialysable, but no specific experiment to determine its
stability during dialysis was carried out. Activity was
demonstrated after three days of dialysis at room temperature.

Preparation pf samples

The sequence followed here is in the same order as the
samples were prepared in the development of a generalized
procedure for concentrating or isolating the active principle
from Boletus 293153.

The description of the procedures followed will be
divided into appropriate parts with separate headings to
facilitate referencing and marative examination of the
work. These headings will include the method of extraction
and filtration, dialysis, precipitation, special treatments,
collection of fractions, solubilization and results of the
tuner inhibition tests. A discussion of the motives for
modification of the procedures will also be presented under
the description of the methods of sample preparation.

A descriptive title will be given to a specific type of
preparation or to groups of samples similarly prepared.

Illustrations were prepared for representative pro-
cedures throughout this study to summarise the treatments
followed in the preparation of the samples. These will
show the method of preparation and the results of the tumor
inhibition test for the sample described.

g2 29}; 9333 ethanol precipitation Lng M 9gp precipi-
m (R-B 1 and 8-8 2).

. Extraction and filtration: a 34 g. sample was tritur-
ated in a porcelain morter with 340 ml. of distilled water
for 20 minutes and then filtered on a Buchner funnel using

12

No. 2 Ihatman paper. Recovery was 260 ml. Filtration was
extremely slow*due to the small particle size of the sus-
pended material. The recovered extractive was divided
equally for treatment separately as follows:

A. 30 per cent ethanol precipitation (R-B 1) Fig. 2

Precipitation: a 150 ml. portion of the ex-
tractive was placed in an Erlenmeyer f1ask.which.was immersed
in a beaker of crushed ice in the cold (7.0°0.) for ca.

20 minutes. One hundred.ml. of 70 per cent ethanol was
then added and the mdxture allowed to stand overnight in the
cold.

Collection of precipitate: the precipitate was
centrifuged, but the supernatant was not decanted. The
bottles were placed on the lycphilizer, after freezing, for
10 hours to remove the ethanol and water. The sample was
removed and allowed to stand overnight in a vacuum.desic-
eator over P205. The sample was shipped in the dry state.
‘leight ca. 0.6 g.

Solubilization: solubility was determined in
water, physiological saline, and phosphate buffer of pH 7.5.
The material was readily soluble in the phosphate buffer,
but slightly less soluble in water or saline.

 

 

Result:.
Single weight change
dose Dose Result in .
toxicity’ ‘ cc. Treated Controls~ Remarks
3.0 0.5-0.2 - -3.0 ' 1 died

-1e5

Figure 2

RAB l and 348 2

Crude Haterial

1. Ground to 60 mesh
2. Extracted with H20 (1:10) in.morter with sand
3. Filtered with suction (No. 2 Whatman paper)

 

Solid residue discarded Extractive (filtrate)

Divided equally

R-i3 1 ., R—L 2

H

1. Eade 30% ethanol at 7°C . pH lowered to h.3 with Glacial
2. Stood overnight at 7°C acetic acid
3. Centrifuged 2. Stood overnight at 7°C
h. Lyophilized 10 hours to 3. Centrifuged

dryness h. Lyophilized 10 hours to dryness
5. Stood overnight in descicator 5. Stood overnight in descicatcr

over P205 over P205
Inhibition test results (3-8 1) Inhibition test results (R98 2)
Toxicity - (3.0) Toxicity - (3.0)

Inhibition - (-) Inhibition — (-)

B. Acetic acid precipitation (R-B 2) Fig. 2

Precipitation: a 130 ml. portion of the extrac-
tive was cooled in the cold (7.0%.) and then treated with
glacial acetic acid to a pH of 4.5 as determined by a pH
meter. The solution was allowed to stand overnight in the
cold.

Collection of the precipitate: the precipitate
was centrifuged and with the supernatant was lycphilized
for ten hours followed by standing overnight in a vacuum
desiccator over P205. The sample was shipped in the dry
state. Weight ca. 0.5 g.

Sclubilisaticn: solubility was determined in
water, saline, and phosphate buffer of pH 7.3. The material
was readily soluble in the buffer, but less soluble in

water or saline.

 

 

Result:
Single Weight change
dose Dose Result in . Remarks
toxicity ' cc. ~ ‘ Treatedfiontrcls
5.0 0.8-0.3 . .400

.1e5

 

53 Le; M ammonium sulfate precipitation (R-B 5).

Extraction and filtration: an 18 3. sample was ex-
tracted with 160 m1. of distilled water for 20 minutes in
a morter and filtered on a Buchner funnel with No. 2 What-
.man paper. Recovery was ca. 120 ml.

Precipitation: the extractive was cooled in the cold

14

rocm.and then.an.equal volume of saturated ammonium.sulfate
was added and the mdxture allowed to stand overnight in the
cold. The precipitate was filtered off with.suotion and
placed in a cellophane dialysing bag. The sample was dial-
yzed against 5 liters of distilled water with one change
after ca. 2 hours. Thermaterial was then lyophilised for
ten hours and dried over P205 overnight. The sample was
shipped in the dry state. 'Weight ca. 0.4 g.
Solubilization: solubility was determined in water,
saline, and a phosphate buffer of pHIV.5. The material was
readily soluble in the buffer but less soluble in water
and saline.

 

 

Result:
Single ‘Weight change
dose Dose Result in . Remarks
toxicity cc. Treated/gontrcls
3.0 00"002 . p '6 .U

.105

 

Discussion: although.the results of these three methods
‘were all negative, ethanol as a.precipitating agent was
selected for further study at higher concentrations due to
its ease of removal from.the precipitated material. Also the
extreme difficulty of filtration through.paper prompted its
replacement with.unbleached muslin to increase the speed of
filtration. The overall time required for the preparation
of the samples was possibly a detriment to the demonstration
of anti-tumor activity.

93 £11; 9335 ethanol precipitation (R-B 4 and R-B 5)

Extraction and filtration: a sample of ca. 28 g. was
extracted with distilled water at a ratio of 1:10 in a
flaring Blendor. lt was found difficult to carry out the
extraction at this low ratio and water was added to raise
the ratio to 1:15. After ca. 3 minutes of blending the
material was transferred to a clean square of unbleached
muslin and was squeezed by hand (maxim pressure) to
attempt clarification. Considerable solids were extruded
in this process. Recovery ca. 370 ml.

Precipitation: the extractive was cooled to 7°C. and
made 50 per cent by volume with redistilled ethanol and
allowed to stand overnight in the cold.

Collection of fractions: the precipitate was centri-
fuged off and dried on the lyophilizer. The precipitate
was sent for testing as sample 3-3 4.

The supernatant was concentrated from a volume of ca.
650 ml. to 10 ml. under a vacuum.aspirator at room.temper-
ature (ca. 22°C.) or at 35°C. on a water bath. The conden-
sation required ca. 18 hours. The 10 ml. condensate was
diluted to 40 ml. for shipment as R-B 5.

 

 

Results:
Single ‘ieight change
Sample dose Dose Result in . Remarks
‘ toxicity cc. Treated Controls
R-B 4* - 0.5 - -5.5
dil. 1:10 -1.5
R-B 4* - 0.2 - ‘ -2.5

dlle 1:10 -1.5

16

Results: cont.

 

 

Single weight change
Sample dose Dose Result in . Remarks
- toxicity cc. Treatedfiontrols
11-3 5*” . 1.0-0.5 + -5.0 1 died
dil. 1:10 , 1'- :3

 

4* Suspended in 100 cc. saline
H Diluted 1:2 with distilled water

Discussion: the results indicated that the activity
was apparently not precipitated with the "crude protein”
at this concentration of ethanol, however the level of ~
dosage of the precipitated sample (R-B 4) was lower than
that of the supernatant (R-B 5) which exhibited activity
and might have accounted for the inability to demonstrate
inhibition with the precipitate.

The excessive solids extruded during clarification of
the extractive resulted in a large amount of precipitate
upon the addition of ethanol which was but slightly soluble
and my have contributed to the results obtained with R-B 4.

Since the activity was apparently not destroyed by
the ethanol during this process and the length of time
required for a preparation comparatively short, further
studies using ethanol were attempted. The toxicity of

R-B 5 was extremely high even at the dilution (1:20) used
in the inhibition test and suggested the study of methods
of decreasing this toxicity.

Dialysis and absorption techniQue g; detoxification (R-B 6 ,
R-B 7, R-B a, R-B 9)

17

Extraction and filtration: a 40 g. sample was ex-
tracted with distilled water (1:15) in a Haring Blender
for 5 minutes and filtered with suction followed by addition-
a1 filtration through a muslin bag pressed by hand.

Precipitation: an equal volume of 95 per cent ethanol
was added to the extractive and the mixture allowed to stand
overnight in the cold. The precipitate was filtered off
on a Buchner funnel and was discarded.

The filtrate was condensed in a split-resin flask
under reduced pressure with an aspirator to a thick syrup
after 44 hours of evaporation. The condensation was carried
out at room temperature. The condensate was washed into
a graduate cylinder up to a volume of ’75 ml. It was noted
that a lipid-like film developed on the walls of the flask
and an attempt was made to scrape it off and combine it
with the condensate.

Separation and special treatment: the condensate was
divided into three equal portions of 25 ml. each and treated
as follows:

3. 11-5 6 -- 25 m1. of the condensate was packaged
in a sterile bottle for testing without further treatment.

B. R-B 7 -- a 25 m1. portion was trout-cc with 0.5 g.
of Ruohar m and shaken for ca. 10 minutes and filtered
twice with suction. About 7.0 ml. of the original treated
solution was lost due to a water pressure drop at the as-
pirator which allowed tap water to back into the filtering
flask. The remaining sample was diluted to 55 ml. and

18

filtration continued. The filtrate was bottled for testing.
The sample weight represented'by this fraction was ca. 9.5 g.
whereas the samples R-B 6, R-B 8, and R98 9 represented ca.
15 g. of crude material.

C. R-B 8 - a 25 m1. aliquot was dialyzed against
5 successive portions of 150 ml. of distilled water. The
volume of this solution increased to ca. 52.5 ml. and was
bottled for testing.

D. R-B 9 - the dialysate from.R-B 8 was condensed
under vacuum to a volume less than '70 ml. and was bottled

 

 

for testing.
Results: ......
_F' Single leight change
Sample dose Dose Result in . Remarks
- - toxicity- cc.‘ ‘ . ‘ -Treated-Controle
R98 6 5.0 T Dil.to
, 80 OOe
R-B 7 5.0 0.6-0.5 17 -5.0 2 died
0
RpB 8 5.0 0.4-0.2 ? 5 died
3-3 s (retest) 1.0-0.5 t“ -e.5 1 died
* 0
3-3 9 sec 0e4 " '2e5
0

 

Discussion: the inconclusive result reported for R-B 6
was probably due to its high toxicity. Treatment with
Ruchar xxx apparently lowered the toxicity but not within
practical limits for therapeutic use; however it did not
result in loss of the activity of the sample. This lower

19

toxicity of the IIch sample might have resulted from the
loss of a portion of the sample during filtration. Dialysis
resulted in no significant decrease in toxicity. The nega-
tive report of R-B 9 indicated that the active principle
was non-dialyzable under the conditions of preparation. The
discarding of the 50 per cent ethanol precipitate with the
demonstration of the activity in the supernatant was taken
as confirmation of the results obtained with R-B 4 and R-B 5.
Pressing the extractive through muslin seemed to be a rapid
means of crude clarification of the extractive only if further
treatment provided for discarding the initial precipitate of
”crude protein".
1 The dialysis apparently removed the major portion of
soluble carbohydrate since it was carried out until a
Molisch test was negative, which tentatively suggested that
the active principle was in the non-carbohydrate fraction.
Consideration of the lowering of the surface tension
and di-electrio constant of the extractive by the addition
of ethanol with resultant precipitation of “crude protein“
suggested a study ofthe effects of acetoneas a preoipi-e
tating agent since it likewise lowered the surface tension
and di-electric constant of aqueous solutions. Since the
hydroxyl and carbonyl functional groups in compounds confer
different characteristics to them, the properties of materials
precipitated by these two classes of compounds should be
somewhat different.

20

52 P33 gent ethanol followed 13; acetone precipitation (R-B 10)

Extraction and filtration: a 10 g. sample was extracted
with 200 ml. of distilled water in a Haring Blender for 5
minutes and filtered with suction. Recovery ca. 1’70 ml.

Initial precipitation: to the filtrate from above
was added an equal volume of 95 per cent ethanol in the cold
and allowed to stand ca. 6 hours. The precipitate was
filtered off on a Buchner funnel with No. 2 whatnnn paper
and discarded. The filtrate was collected and treated as
follows:

Condensation: the filtrate was condensed under re-
duoed pressure in a split-resin flask at room temperature
to a volume of ca. 18 m1.

Dialysis: the condensate was washed into a dialysing
bag and dialysed against successive changes of distilled
water until the dialysate gave a negative Holisch test.

Collection of fractions (R-B 10 and R-B 11): the
residue in the dialysing membrane was collected in a beaker
and cooled to 7°C. in equal volum of cold acetone was
then added and the mixture allowed to stand 1} hours in the
cold. The precipitate was dark brown in color and was very
smll in weight and fine in texture. The precipitate was
dissolved from the surface of the filter paper with distilled
water (ca. 50 ml.) and filtered again to remove the inso1uble
granules and paper. This fraction was sent for testing as
R-B 10.

The acetone supernatant from R-B 10 was condensed under

21

reduced pressure to a volume of ca. 10 m1. and transferred
with washings toga sterile bottle for testing as R-B 11.
Final volume 25 m1.

 

 

Results:
. Single ‘Weight change
Sample dose Dose Result in . Remarks
' toxicity* cc. ‘ Treated Controls
R-B 10 5.0 0.8-0.4 is" 4.0 2 died
D11. to 80 Elle .5 o
with dis. 320 ’
R“B 11 3.0 Oe5 2 3 diOd

Dile to 80 Ille .
with dis. 320

 

Discussion: although the weight of the precipitate of
R-B 10 was small, the results indicated that the use of
acetone as a precipitating agent should be further studied
since it seemed not to destroy the activity.

in interesting observation was noted in the results of
the inhibition test of sample R-B 10. The apparent non-
toxicity of the single dose treatment actually resulted in
a cumulative toxicity during the course of the test killing
two mice which suggested that an anaphylactic shock might
have arisen from the repeated injections of the sample.

That the dialysate fraction was extremely toxic and the
test results inconclusive could not be interpreted as indica-
ting the ability of 50 per cent acetone to effect complete
separation of the active principle from the supernatant of
the 50 per cent ethanol treatment.

22

Stability g; fractionation (acetone) techniques (R-B l2,
ReB 13, R-B 17, R-B 13, R-B 19, R-B 20, Res 21, R-B 22,
8-3 25, 3-3 24, R-B 25).

Extraction and filtration: a sample of ca. 45 g. was
extracted with water (1:20) in a Waring Blendor for 5 minutes.
After blending, the mixture was squeezed through a muslin
bag to speed up the clarification. Considerable extrusion
of the crude solids was noted. The extractive was measured
at ca. 8'70 ml.

Precipitation: after cooling the extractive to 7°c.
an equal volume of 95 per cent ethanol was added and the
mixture allowed to stand overnight in the cold. The pre-
cipitate was filtered off with suction and discarded. The
filtrate was transferred to a split-resin flask for con-
densation.

Condensation: the filtrate was evaporated under re-
duced pressure for 6 hours in the flask. This process was
not well adapted to such a large volume of solution and an
alternate method attempted. The contents of the flask were
transferred to a porcelain-lined photographic pan 14" x 18"
x 2‘? and placed on a water bath at 55°C. under an exhaust.
hood for 17 hours until the volume was reduced to ca. so ml.

Dialysis: the condensate, which was dark brown in
color and viscous, was washed into a dialysing membrane and
dialyzed for 5 days against 2 liter portions of distilled
water changed at intervals of ca. 6 hours except for 11 to
12 hour intervals overnight. The dialysis was discontinued

23

after the dialyzable portion of the pigment was removed
(no apparent color change in.dialysate). The volume of
the solution in the membrane was measured at 210 ml. which
was an increase in volume of ca. 150 per cent. This solution
was still extremely viscous.
The solution was divided into 4 equal portions of ca.
50 ml. each and treated separately as follows:
A. Acid stability (R-B l2, R-B l7, R-B 21) Fig. 3
.A 50 ml. portion was treated with ca. 5 drops
of concentrated hydrochloric acid until the pH dropped to
2.0. ‘While at this low'pH the solution was shaken with.ca.
2 - 3 per cent Nuohar'xxx and filtered with.cuotion. The
Huohar was added to the solution in a 250 ml. centrifuge
bottle and shaken.by hand for ca. 2 minutes. After filtra-
tion, the pH‘lIl raised to 5.1 with.lO per cent sodium
hydroxide (pH of the solution prior to acid addition) and
the solution condensed under a vacuum.to a volume of 14:ml.
Separation of fractions (3-3 12, RAB 17, 8-3 21)
Fig. 3: the 14 ml. condensate was made 80 per cent by
volume of acetone by the addition.cf'55 ml. cf’acetone and
allowed to stand 15 - 30 minutes at room.temperature. The
precipitate was centrifuged and the supernatant decanted
for further treatment as RAB 17. The precipitate was dried
under a stream.of air, dissolved in 50 m1. of water and
bottled for testing as R-B 12.
The acctone supernatant from.R-B 12 was evaporated

under a stream.of air to remove the acetone for ca. 3 hours

ligure 3
fi—B l2, d-L 17 and 1+: 21

Crude Laterial

1. Ground to 60 mesh _

2. Extracted with H20 (1:20) in Waring filendor
3. Clarified through uncleached muslin

h. fade hQZ by volume with 95% ethanol at 703

5. Stood overnight at 7°C

6. Filtered with suction (Ho. 2 Hhatman paper)

5’ v 6
Extractive
l. Concentrated under reduced pressure
2. Concentrated on 35°C water bath
3. cialyzed 3 days at ca. 2103

I
t . it

Lembrane solution oialySite discarded
1. pH lowered to 2.0 with NC
a. Shaken with Huchar liX
b. ?iltered with suction -

Solid residue discarded

 

 

 

l 1
Iuchar discarded Filtrate .
1. pH raised to 5.1 with HaOH
2. Condensed under vacuum
3. Lade 30» acetone by volume
h. stood 15-30 minutes at ca. 2103
S. Centrifuged
7—3 12 l
{—7 I
Lrecipitate Supernitint
l. tried under air current I
2. Lissolved in H20 fl ‘ .
l. concentrated under air current
.. . . .. a ""4, ‘3’)! ' a --
Inhlzutlon test results (.i—y; 12)- 2. ..:;-de ,5 ace/torque by volmaem
Toxicity _ < 1.0) ,. icood' or ca. 2 hours at 7 t
Inhioition _ (’) b. centrifuged
1—7 17 _2:B 2l
Yrecipitate Supernatant
1. dried under air current 1. Iried under air current
2. Lissolved in HQO 2. Residue dissolved in H2O

Inhibition test results (3-3 1?) Inhibition test results (24% 21)
Toxicity — ()l.O) Toxicity — C)l.O)
Inhibition - (-) ' Inhioition - (-)

24

and it was observed that as the volume lowered white cry-
stalline materials appeared at dry surfaces left by the
receding solution. The condensate, however, remained a
clear straw color. After the condensate was evaporated to
6 ml., 54 m1. of acetone were added giving a solution
90 per cent acetone by volume which.was allowed to stand in
the cold for ca. 2 hours. A precipitate was formed and
was centrifuged, decanted, air dried and dissolved in.40 ml.
of water and shipped as 3-3 17. -

The decanted supernatant from R-B 17 was evaporated
to dryness and the residue dissolved (with difficulty)
in 30 ml. of water and shipped as R-B 21.

 

 

Results:
_fl' Single weight change
Sample dose Dose Result in . Remarks
toxicity co.' Treated/gontrols

R-B 12 1.0 0.8-0.4 - -5.0

dil. 1:5 ‘5'—
R-B 17 1.0 0.8 - g

dile 135 O
R-B 21 1.0 Oe5.0e25 - -3.0 1 d16d

0'

Discussion: the results prompted two major con-
siderations of the procedure. First, it was not known
whether the activity was actually present in the medium
after the prolonged periods of condensation and dialysis,
but previoue experience suggested that its stability under
these processes was very good and that it could be demen-

25

strated just prior to the acid treatment. Secondly, the
high concentrations (80 and 90 per cent) of acetone were not
known to be injurious to the active principle, but concen-
tration of 50 per cent acetone had not been shown to destroy
the active principle (R-B 10).

These results, assuming-the activity present prior to
acid addition and nonpdostruotion of activity by 80 - 90 per
cent acetone, were interpreted as indicating that the active
principle was acid labile.

B. Alkali stability (R-B 15, R-B 18, R-B 22)

A 50 ml. aliquot of the original extractive
after treatment with.50 per cent ethanol, condensation,
and dialysis was treated with a 10 per cent solution of
sodium hydroxide to a pH of 8.9 and then shaken with 2 - 5
per cent Euchar'xxx.and filtered with.suotion. The pH was
lowered to 5.1 with concentrated hydrochloric acid after
filtration, and the solution condensed under vacuum.to 21 ml.

Separation of fractions (R-B l5, RAB 18, R-B 22):
the 21 md. condensate was made 80 per cent acetone and
allowed to stand 15‘ - so minutes then centrifuged. The
supernatant was decanted and treated further as R-B 18.
The precipitate was dried under an air current and dissolved
(with.difficulty) in.4o ml. of water for testing as 8-3 15.

The supernatant was evaporated to a volume of 11.5 ml.
similarly to R-B 17 above, cooled to 7°C. and acetone added
to a concentration of 80 per cent. It was noted that the
cloudiness observed in the medium.at this concentration of

26

acetone during the acid treatment was not present and the
concentration of acetone was increased to 90 per cent.
After standing in the cold for ca. 2 hours, a slight gummy
precipitate, dark brown in color, was centrifuged down.
The supernatant was decanted and treated as R-B 22. The
precipitate was dried under an air current and dissolved in
water for testing as R-B 18.

The supernatant was not further fractionated but was
evaporated to dryness in a stream.of air and the residue
dissolved in water as 3-8 22.

 

 

Results:
Single ‘Weight change
Sample dose Dose Result in . Remarks
toxicity cc. Treated Controls,
R-B 15 0.5 0.8-0.4 - -l.5
dil. 1310 o
8-3 18 I 1.0 0.5 - -c.5
0
3-8 22 1.0 0.5 - g
0

 

_ Discussion: the difference in weight of the
precipitates collected from the acid and alkali treated
samples suggested that either the alkaline medium enhanced
the absorptive ability of the Euchar’xxx toward the material
in solution or that appreciable hydrolysis occurred during
the short interval (ca. 15 hours) that the medium.was
alkaline to a pH of 8.9. That the latter was the case was
suggested by the viscoue nature of the alkaline treated

27

precipitate as compared to the fluffy nature of the acid
precipitate.

Following the assumptions made in the interpretation
of the results of R-B 12, R-B 17, and R-B 21, the results of
these tests were interpreted as indicating the alkali lability
of the active principle.

0. Stability at 80 and 90 per cent acetone concen-
trations (R-B 19, R-B 20, R-B 23)

The pH of a 50 ml. portion of the extractive
after 50 per cent ethanol treatment, condensation and dialysis
was determined as 5.1 with a pH meter.

The sample was treated with.Nuchar*xxx.as described
under 3-8 12 (acid stability) above. The filtrate was con-
centrated to 15 ml. and.made 80 per cent acetone and allowed
to stand in the cold for ca. 2 hours. The precipitate was
considerably heavier than observed in the acid or alkali
treatments and was centrifuged for collection. The super-
natant was decanted for further treatment as BpB 20. After
drying the precipitate in an air current, it was taken up
in 40 m1. of water for testing as R-B 19.

The supernatant of 3-8 19 was evaporated to a volume
of 9 ml. and.made 90 per cent acetone by the addition of
81 ml. of acetone and.allowed to stand overnight in the cold.
The supernatant was decanted from the precipitate and treated
as R-B 23.

The precipitate was taken up.in.45 ml. of water for
testing as RPB 20.

28

The supernatant from.R-B 20 was evaporated to remove
the acetone but was not carried to dryness. The final
volume was less than 45 ml. and was bottled for testing

 

 

as 3-3 23s
Results:

Single 'Weight change
Sample dose Dose Result in g. Remarks

toxicity co. . Treated/Controls
3-13 19 1.0 0.5-0.1 t’ -:5.0 1 died
3-3 20 1.0 0.5 - -1.0

0
3-8 23 1.0 1.0 - -1 0
an. 1.35 "'6':-

 

Discussion: the results seemed to confirm the
stability of the active principle through.extended periods
of time under the conditions described for condensation
and dialysis at room.temperature. Also its stability at 80
per cent acetone concentrations was indicated and suggested
further study of acatone as a direct precipitating agent.

The negative result at the 90 per cent acetone level
could not be interpreted as indicating instability but
rather that the active principle was precipitated from.the
medium.at the lower concentration. The result of RPB 19
was taken as confirmation of the activity of fraction RPB 10.

D..Acid hydrolysis (R93 24, R-B 25)

From.the original sample after 50 per cent

ethanol treatment, condensation, and dialysis the fourth

29

aliquot of 50 ml. was treated with concentrated hydrochloric
acid to a pH less than 1.0 and allowed to stand in the cold
for 4 days. The solution was dialysed to a pH ca. 4.0 until
the dialysate gave no test for carbohydrates (Molisch).

After dialysis, the solution was concentrated and then made

'70 per cent acetone and allowed to stand in the cold over-
night. The precipitate was centrifuged and the supernatant
decanted and treated as R-B 25. The precipitate was dissolved
in 45 m1. of water and bottled for testing as R-B 24.

The supernatant was evaporated to 7.5 ml. and made 80
per cent acetone without any sign of formation of a pre-
cipitate (cloudiness). The concentration of acetone was
increased to 90 per cent and the mixture allowed to stand
overnight in the cold. No precipitate was formed and the
solution was evaporated to remove the acetone and the
residue (ca. 6 ml.) was diluted with water to 45 m1. and
bottled for testing as R-B 25.

 

 

Results:
Single Weight change
Sample dose Dose Result in 3. Remarks
toxicity cc. A Treated/Controls

R-B 24 1.0 0.5-0.25 - 9_
O

R’B 25 1.0 0.5 - 9.
0

 

Discussion: the results clearly confirmed the
acid} lability of the active principle.

30

Re-extrac_t:_igp_ _an_g._ modified ethanol precipitation W
(R-B 26, 11-13 27, R-B 28, and R-B 29).

Extraction.and centrifugation: a 20 g. sample was
water extracted (1:21) in a'waring Blender for 5 minutes
and was centrifuged. The precipitate was separated from
the supernatant and was treated as R-B 27. The supernatant
was treated as follows:

Precipitation: the supernatant was made 50 per cent
by volume with 95 per cent ethanol and allowed to stand
overnight in the cold. The precipitate was filtered off
with suction and the filtrate condensed.under an air current
to 15 ml. The condensate was washed.into a dialyzing meme
brane and dialysed for several days at room.temperature
against 2 liter portions of water with.frequent changes
until a cloudiness was observed in the dialysate. It was also
noted that a precipitate had formed in the bag.

Collection of fractions (R-B 28, RAB 29): the solution
in the dialyzing bag was centrifuged and the supernatant
decanted and treated as R-B 29. The precipitate was dried
in an air current and taken up in physiological saline for
it appeared to be globulin-like in.nature. This fraction
was numbered 3-8 26 for testing.

The supernatant.frcm R-B 26 was made 60 per cent ace-
tone and allowed to stand in the cold. The precipitate
formed was centrifuged, dried in air and taken.up in water
for testing as R98 29.

Re-extraotion (R-B 27 and R-B 28): the solid residue

31

after separation from the extractive by centrifugation was
re-extracted with.water (1:20) in a flaring Blender for 5
minutes to ascertain if the method of extraction was quan-
titative with a single extraction.

The further treatment of this additional extractive
was similar to that as described under R-B 26 and R-B 29.
It consisted of filtration, treatment of the extractive
with 50 per cent ethanol, condensation and dialysis for the
same periods of time. It was observed that the cloudinets
of the dialysate develOped and a precipitate formed in the
dialyzing bag similarly to fractions R-B 26 and R-B 29.

Collection of fractions: the precipitate in the bag
was centrifuged, dried and taken up in water for testing
as R-B 27. The supernatant of R-B 27 was not further treated
with acetone to 60 per cent to precipitate the active prin-
ciple since the concentration of the active principle in
the second extraction was unknown and the quantitative
separation with.acetone had not been confirmed by previous
treatments. This supernatant was diluted to 45 ml. with
water and bottled for testing as R-B 28.

 

 

Results:
Single weight change
Sample dose Dose Result in . Remarks
toxicity cc. Treated/gentrols
R-B 26 5.0 0.6-0.5 1;" -2.0
orig. dile O
1:100
R-B 26 (retest) 0.6 - -3=5
orig. dil. 1.5

1:1000

52

Results: cont.

 

 

Single Weight change
Sample dose Dose Result in . Remarks
toxicity cc. Treated/gontrols
8.3 27 5.0 0.5-0.25 " -105
0
R-B 28 1.0 0.25 1? -5.0
0
R'B 29 3.0 005.002 $.- 7.0
orig. dil. 0
1:100
3-3 29 (retest) 0.3-0.7 ;* -3.o
orig. dile 1.5
1:1000

 

Discussion: the most significant result reported to
date in this study was that of fraction R-B 29 which.was
diluted 1000 fold and, given at a relatively low'dosage
level, yet produced greater inhibition than at a higher
concentration. This result suggested that the active
factor was not identical with.the toxic portion of the prep-
aration and could possibly be separated from.it; but this
could not be interpreted to mean that the active factor was
inherently non-toxic.

The results of R-B 26 and R-B 27 seemed to be conflicting
in that the two precipitates formed upon prolonged dialysis
gave quite different results; however, the relative concen-
tration of the active principle in the first extractive was
perhaps several times greater than in the second extractive
which might account for these results.‘ Since the amounts of

,V

33

the two precipitates were comparable, it was improbable
that they were in themselves actually the inhibitory factor,
but that some of the activity was occluded in the formation
of the precipitate from.the more concentrated solution of
the first extractive.

No explanation was given for the dilution of samples
R-B 26 and R-B 27 which were apparently non-toxic in .
single dose. This dilution may have accounted for the
negative results obtained with these fractions.

The precipitate formed in the dialyxing.memhrane may
have been the result of insufficient clarification of the
extractive by centrifugation.

§_C_)_ £35; gent ethanol followed by dialysis and acetone f__1:_a_g-
tionation (R-B 30, R-B 31, R-B 32, R-B 33) Fig. 4.

Extraction and filtration: a 10 g. sample was extracted
with.water (1:20) in a flaring Blender for 3 minutes and
filtered on a Buchner funnel with.Re. 2 Whatman paper.

Precipitation, concentration, and dialysis: the fil-
trate from.abeve was made 50 per cent ethanol and allowed
to stand overnight in the cold. The precipitate was filtered
off and discarded. The filtrate was condensed under an air
current to remove the ethanol and then dialyzed for 3i-days
against successive 6 liter portions of distilled water. No
precipitation occurred during this dialysis as observed
with R-B 26 and R-B 27.

Fractionation: the solution in the dialyzing membrane

was fractionated as described below:

Figure h
R—S 30, R—B 31, R-B 32 and 3.3 33
Crude Katerial
l. Extracted with H 0 (1:20) in Waring Blendor
2. Extractive made 0% ethanol at 7°C

3. Stood overnight at 7°C
h. Filtered with suction

r +

Supernatant

 

 

 

Precipitate discarded 1. Condensed under air current
2. Dialyzed 3% days at ca. 21°C
3. Kade hBZ acetone at 7°C
h. Stood overnight at 7°C
5. Centrifuged
+ I 8:3 30
Supernatant Precipitate
l. Eade 55$ acetone at 7°C :' éir diieg . q 0
2. Stood ca. 8 hours at 7°C ' “1880 vs 1n ‘2
3. Centrifuged Inhibition test results (R-B 30)
Toxicity - (5.0)
ng3¥31 1 Inhibition - (—)
Precipitate Supernatant
1, Air dried . l. Iade 70% acetone at 7°C
2. sissolved in qu 2. Stood overnight at 7°C
Inhibition test résuite: 3. Centrifused
Toxicity -(S.0)
Inhibition - (-) R~3_32 3—3 337
Precipitate Supernatant
1. Air dried l. Acetone evaporated in air
2. Dissolved in H20 2. Residue diluted with H20

Inhibition test results (3-8 32) Inhibition test results (2-3 33)
Toxicity - (5.0) Toxicity — (5.0)
Inhibition - (-) ’ Inhibition - (-)

54

Acetone was added to the solution until a cloudiness
developed at a concentration of 48 per cent and the mixture
allowed to stand overnight in the cold. The precipitation
was very slight but was centrifuged and the supernatant
decanted and treated as R-B 31. The precipitate was dried
in air and taken up in water for testing as R-B 30.

To the supernatant was then added sufficient acetone
to develop a cloudiness (55 per cent) and the solution
allowed to stand in the cold for ca. 8 hours. The precip-
itation was slight but was centrifuged and the supernatant
decanted. The precipitate was taken up in water for testing
as R-B 31.

The supernatant from R-B 31 was made 70 per cent ace-
tone (first cloudiness) and allowed to stand overnight in
the cold. The precipitation was again slight and the pre-
cipitate collected by centrifugation, dried in air and
taken up in water for testing as R-B 32.

The supernatant from R-B 31 was evaporated to remove
the acetone, diluted to ca. 40 ml. with distilled water and
bottled and sent for testing as R-B 33.

 

 

Results:
Single 7 weight change V
Sample dose Dose Result in . Remarks
" ' * toxicity cc. Treated Controls

R-B 30 5 00 0 .8 - .230
'0

R-B 31 5.0 0.8-0.4 - O 5* 1 died
5.5

R-B 32 500 0.6.0.4 "' -100

Oe

0|

55

Results: cont.

 

 

Single ‘Weight change
Sample dose Dose Result in 3. Remarks
toxicity cc. Treated/Controls .n
3.3 53 5.0 0.0 - -105
0.5

 

Discussion: since activity had been.demonstrated in
the supernatant after treatment with 50 per cent ethanol
and dialysis for extended periods (R93 29) an attempt was
made to use acetone as a fractionating agent. The criteria
of fractionation was taken as first apparent cloudiness of
the mixture. The results reported here indicated that the
activity was apparently lost before the fractionation was
attempted, however several factors were considered in the
interpretation of these results. First, the weight of crude
sample represented by these was small. Secondly, the
amount of’precipitate collected at each.fracticnating con-
centration of acetone was extremely slight. Also, the
length of time that the material was in contact with high
concentrations of acetone may have resulted in loss of
activity. This total time at concentrations above 48 per
cent acetone was greater than 36 hours.

These results suggested three modifications in the
general procedure: (1) to increase the weight of crude sample
represented by the fractions, (2) direct precipitation with
acetone after the 50 per cent ethanol treatment excluding
the process of dialysis, and (:5) to decrease the length of
time required for concentration and dialysis.

36

§_(_)_ 33;; gent etflol followed by dialysis 2.99. acetone gagg-
tionation (modified - sample weight doubled) (R-B 34, R-B
35, R-B 36).

Extraction and filtration: a 20 g. sample was ex-
tracted with water (1:20) in a waring Blendor for 3 minutes
and filtered with.suotion.

Precipitation, concentration, and dialysis: the pro-
cedure followed was the same as described under the prepar-
ation of fractions R-B 30, R-B 31, R-B 32, and RPB 33 on
page 33.

The extractive after ethanol treatment, condensation,
and dialysis was fractionated similarly to the previous
sample with.twc exceptions: the upper limit of the fraction-
ation was 65 per cent acetone instead of 70 per cent as in
R-B 32 and the supernatant of the 65 per cent acetone pre-
cipitate was not freed of acetone and tested for inhibition

as was R-B 33.

 

 

Results:
Single ‘7 ‘leight change
Sample dose Dose Result in g. Remarks
toxicity cc. Treated/Controls" ‘
R93 34 5.0 0.8 1 3 died
a-s :55 5.0 0.3 ..' -2,‘o 1 died
-O.5

R-B 56 3.0 0.4-0.2 2 4 died

 

Discussion: the inconclusive and negative results
obtained with.these fractions could not be interpreted as
confirmation of activity after the ethanol and subsequent

3'7

treatments prior to attempted fractionation with acetone :
however, the low toxicity of a single dose followed by the
cusmlative toxicity as observed with fraction R-B lo landed
support to the belief that the activity was still present.
The results seemed to indicate that acetone following ethanol
would not effect the desired separation. is of necessity,
the length of time required in the preparation of fractions
by this procedure was long, and the possibility that the
activity was lost or diminished on standing could not be
excluded in suggestions for modification of this method.

It was decided to shorten the time of concentration and
dialysis, and to attempt a single acetone precipitation
technique (after ethanol) at a concentration midway between
that of R-B 34 and R-B 36, is. ca. 57 per cent acetone.

Direct acetone precipitation (rapid method) (R-B 37, R-B as)

b Extraction and filtration: a 20 g. sample was extracted

with water (1:20) in a Waring Blendor for 3 minutes and

filtered with suction using No. 2 Whatman paper.
Precipitation and condensation: the extractive was

cooled to 7°c. and made so per cent ethanol and the mix-

ture allowed to stand overnight. The precipitate was

filtered off with suction and discarded. The filtrate was

condensed under vacuum using a dry ice trap. The time of
condensation was ca. 4 hours.

Dialysis and separation of fractions: the condensate
was dialyzed on the rotating apparatus for 3% hours against
two portions of 6 liters of distilled water. It was noted

that a precipitate formed after ca. 2 hours in the bag.
This pricipitate was collected by centrifugation, air dried,
and dissolved in 50 ml. of water for testing as R-B 37.

The supernatant was made 67 per cent acetone and allowed
to stand in the cold for ca. 6 hours. The precipitate formed
was collected by centrifugation and.was taken up in water
and redialyzed for 2 hours to attempt removal of pigment.

The pigment was non-dialyzable.

The volume of the solution in the dialyzing membrane
was measured as 90 ml. ‘An attempt to reprecipitate the
active principle was made by making this solution 57 per
cent acetone and allowing to stand overnight in the cold.
There was no apparent separation effected.by the second
precipitation. After the removal of the acetone by an air
current, the residue was diluted and bottled as R-B 38.

 

 

.Results:
Single Height change
Sample dose Dose Result in 3. Remarks
toxicity ace. . ‘Treated/Controls
3.8 37 5.0 0.5-00‘ - .430
-100

3.3 as . 3.0 0.5-0.25 : p 3 died

 

v—.._ —_—_

Discussion: the negative result of R-B 37 was inter-
preted as confirmation of the assumption that the precipi-
tates collected as R-B 26 and R-B 27 were not separated
portions of the active principle. The inconclusive result
.of £93 38 was attribued to the lethal effect on the treated
mice and could not confirm.the assumption that the active

39

principle was present in the treated sample prior to the
precipitation with acetone as carried out in this procedure.
The results of the use of acetone following ethanol tech!
niques as reported here, and consideration of the precipi-
tating characteristics of these two substances suggested
that a study be made utilizing acetone, without ethanol,

to attempt isolation of the active principle.

gigggt’aoetone precipitation agg.reprccipitation.(R-B 39,
863 40, 393 41, R-B 42, R-B 43, R-B 44) Fig. 5.

Extraction.and filtration: a 15 3. sample was ex-
tracted with water (1:25) in a waring Blendor for 3 minutes
and filtered twice on a Buchner funnel.

Dialysis: the extractive was dialyzed for 2% hours
against 6 liters of distilled.water changed once at 1} hours.
No precipitation was observed. I

Precipitation: the extractive after dialysis was
placed in the cold and made 31 per cent acetone and allowed
to stand overnight in the cold. It was assumed that the
character of the precipitate would‘be similar to that
obtained with ethanol at 50 per cent concentration. The
precipitate was collected by centrifugation, the supernatant
was treated as described below. The precipitate was air
.dried and taken up in physiological saline and.dialyzed for
ca. 1%- hours against 1 liter of distilled water changed
once. The suspension of the precipitate in the dialyzing
bag was centrifuged, and the precipitate was taken up in

Figure 5

3—3 39, 2-3 ho, 3-5 hl and R—B h2
Crude Katerial

1. EXtracted with 320 (1:25) in a Waring Blendor
2. Extractive dialyzed 2} hours at ca. 21°C

3. Extractive made 31% acetone at 7°C

h. Stood overnight at 7°C

5. Centrifuged

 

 

R-Z 3'? _
m at... ,i —»+
Preci itate :17 ‘r” d‘”
1. TakenPup in saline 1. Made Elk acetone at 7°C
2 Dialyzed ca 2 hours 2. Centrifuged (Supernatant R-B h3)
. . 3. Ppt. dissolved in H20
Inhibition test results (R-B 39) h' Dialyzeg 3 h°ur5 O
toxicity _ no report 5. Made 51h acetone at 7 C
Inhibition _ (1—) 6. StOOd 3% hours at 70C
7. Ce trifuged
- T as ho,
Supernatant Precipitate
1. Kade 55% acetone at 7°C ‘ ' l. Dissolved in H O
2. Stood h hours at 7°C Inhibition test results Rafi hO)
3. Centrifuged Toxicity - no report

Inhibition - (-)

:jfgigg I . R—B hz
._1

 

 

Precipitate ' ' Supergatant
1. Lissolved in H90 1. bade 75p acetone at 7°C
~ 2. Stood overnight at 7°C
Inhibition test results (3.3 t1) 3- Centrifused
Toxicity — no report R;B hp I
Inhibition - (1*) f t
Precipitate Supernatant discarded

1. Dissolved in H20

Inhibition test results (Rea h2)
Toxicity - no report
Inhibition - (s+)

4O

physiological saline for testing as R-B 39.

Fractionation above 31 per cent acetone (R-B 40,
R-B 41, R-B 42, R-B 4:5, R-B 44)

The supernatant from the 31 per cent precipitation

was further treated with.acetone to a concentration of 57
per cent and allowed to stand overnight in the cold. The
precipitate was collected by centrifugation and taken up
in water for subsequent fractionation and the supernatant
collected and treated as described below under R-B 43.

The 57 per cent acetone precipitate after being disper-
sed in water was dialyzed for 3 hours and fractionation
attempted as follows:

The solution was made 51 per cent acetone and allowed
to stand in the cold for 3% hours. The precipitate was
collected by centrifugation and taken up in water for test-
ing as R-B 40. The supernatant from.R~B 40 was made 55 per
cent acetone and the precipitate collected after 4 hours
of standing in the cold. The precipitate was taken up in
35 ml. of water for testing as R-B 41. The supernatant
from.R-B 41 was further treated to 75 per cent acetone and
allowed to stand overnight in the cold. It was noted that
the supernatant of the 75 per cent treatment was colorless
after standing overnight and it was assumed that the
separation of the precipitatable material was complete
within practical limits. The precipitate was taken up in
50 ml. of water as R-B 42.

41

The supernatant from R-B 40, after the 57 per cent
acetone precipitate was removed, was condensed.under an
air currect to a volume of 240 m1. and then made 68 per
cent acetone in the cold. After ca. 3 hours the precipi-
tate was collected by centrifugation and taken up in water
for testing as R-B 43. The supernatant from R-B 43 was
then.made 75 per cent acetone and allowed to stand in the
cold for ca. 3 hours. The precipitate was collected.by
centrifugation and taken up in water for testing as R-B 44.

 

 

Results:
Single ‘leight change
Sample dose Dose Results in . Remarks
toxicity cc. Treats Controls - '
R-B 39 0.3-0.1 . if -2.5
~2.0
R-B 40 0.3 - -2:O
-2.0
R-B 41 0.5-0.1 1+ ~5,c 2 died
.. "2.0
R-B 42 e.s-c.1 s.” :32 2 died
-2.0
R-B 43 5.0 0.6-0.3 - ‘:;g§
.200

R-B 44 Samplelost after shipment

 

Discussion: the result of R-B 39 indicated that a
portion of the active principle was precipitated by 31 per
cent acetone, which.was unexpected by the slight differences
between acetone and ethanol as precipitating agents. The
negative result of R-B 40 may have been due to an insufficient

42

amount of the active principle in the precipitate which in
itself was slight. The demonstration of activity in samples
R-B 41 and R-B 42 supported evidence that acetone could not
be used as a fractionating agent in this procedure for
separating the active principle from.the toxic materials.
The negative result of R-B 43 suggested that the precipi-
tation of the active principle was complete below an.acetone
concentration of 68 per cent in the extractive. Experience
prompted a serious objection to this particular method of
isolation. The length of time during which the extractive
was kept in the presence of high.concentrations of acetone,
even.in the cold, may have altered the structure of the
active principle and possibly its activity. The differences
in concentration of acetone at which.fractions were collected
did not seem to be far enough apart to be suitable in a
generalized procedure involving the use of acetone due to
its high.volatility even at the temperature of the cold room
used (7°C.).

After consideration of the results described here, it
was considered advisable to modify this method of fraction-
ation in an effort to shorten the 1ength.of time during
which.the extractive was held in the presence of high.acetone
concentrations. The method suggested was to employ a
process involving only two precipitations from a single
sample. This method was employed in the preparation of
fractions R-B 46, R-B 47, R-B 48, and R-B 49.

43

Detoxification technique (Pancreatin digestion) (R-B 45).

Extraction and filtration: a 15 g. sample was extracted
with.water (1:20) in a waring Blendor for 3 minutes and
filtered with suction.

Ethanol precipitation: the extractive was made 50 per
cent .thnnol'end allowed to stand overnight in the cold.
The precipitate was centrifuged off and discarded and the
supernatant treated as follows:

Concentration: the supernatant was condensed under
vacuum.using a dry ice trap to remove the alcohol. Time
ca. 5 hours.

. Dialysis: the condensate was dialyzed for 4% hours
against 6% liters of distilled water with.two changes. No
precipitation was noted.

Acetone fractionation: the residue in the dialyzing
membrane was made 48 per cent acetone and allowed to stand
overnight in the cold. The precipitate was collected.by
centrifugation and stored in a refrigerator for ca. 24 hours.
The supernatant was treated with additional acetone to a
concentration of 57 per cent and allowed to stand in the
cold for ca. 24 hours. The precipitate was collected by
centrifugation and combined with the precipitate collected
at 48 per cent acetone. This combined.material was taken
up in 45 ml. of water and treated as follows:

Pancreatin digestion: the solution of precipitates

was allowed to stand 18 hours at room temperature after the

addition of 0.5 per cent pancreatin? and mixing. After
18 hours, the mixture was filtered with.suotion and the
filtrate collected. The volume of the filtrate was 64 ml.
and was bottled for testing as R-B 45 without dilution.

 

Results:
Single weight change
Sample dose Dose Result in g. Remarks
toxicity cc. Treated/Controls

 

R-B 45 3e0 0e4-003 1 3 died

Discussion: this inconclusive result could not be
interpreted as indicating that the panoreatin digestion
was effective in detoxifying the active fraction, but rather
suggested that either the pancreatin in solution was toxic
to the mice or that possibly bacterial toxins were produced
in the medium during the period of digestion.**

‘Acetone fractionation (2 step method) (R-B 46, R-B 47,
R-B 48, R-B 49) Fig. 6.

Extraction and filtration: three separate 15 3.
samples were extracted with.water (1:20) in a Faring Blendor
for 3 minutes and filtered on separate Buchner funnels
using No. 2 Whatman paper. The three filtrates were
treated individually as follows:

 

4* Panel-satin (ms-P. XI). Line. by Difoo Laboratories.

ii The use of enzymatic digestion as a means of detoxi-
fication was suggested by Dr. C. 0. Stock of the
Division of Experimental Chemotherapy, Sloan-Kettering
Institue for Cancer Research.

 

 

 

figure 6
1".) ‘ L7 3.116. 11-; L3
Srudc Hatcrial
{fronted in duplicate)
1. Extract with HaO (l: G) in tiring blender
2. ziltc red th suction'
a. holid r~cuducf discarded
(fixtructivcs treated separately A and *)
It
1. Extractive made 553 acetone at 7°C
2. Stood 5: hours at 7°C
3. Centrifugcd
”_fi LE 1
Precipitate Gupcrna tant
1. Air dried i. Laue 60 acetone at 7Ou
2. Suspended in 320 2. ctood overnight at 7°C6
Inhibition test results \t-- 36) 3. Centrifuged
Toxicity — (0.5} 7—3 Q7 1
lnhioition -<-) '—* M" ‘ I
irocipitdte Supern;tant discarded
1. Air dried
2. hissolvcc in H60
InLiuition test results (fi-B h?)
Soxicity - (3.0)
B Inhinition - (1‘)
1. Extractive made 63% acetone at 7°C
2. Stood Sg hours it 70;
3. Jcntrifugcd
Precioitate dis co rdcd Lun':rn tart

55

l. AVILUL -—/\J
I)

L.
3.

24:2

Precipitatc
Air dried
his solved in }.(O
InhiJition test results (Y-B h8)
Toxicity - (5.0)
Inhibition — (i-)

17

4

l.
?

acetone at 7°C

Stood*cvc1night at 703
Centrifgped

i

Supernitint.discardcd

45

A. (R-B 46 and R-B 47): the extractive was made
55 per cent acetone and allowed to stand in the cold for
5% hours, the precipitate collected by centrifugation,
air dried, and taken up in 80 ml. of water for testing as
R-B 4d. The supernatant volume was measured and additional
acetone added to a concentration or do per cent and the
solution allowed to stand in the cold overnight. The pre-
cipitate was collected‘by centrifugation, air dried, and
taken up in 80 ml. or water for testing as R-B 47.

B. (R-B 48): a second extractive was treated with
acetone directly to a concentration of do per cent and
allowed to stand in the cold for 5} hours. ’The precipi-
tate was centrifuged down and discarded. The supernatant
was then.made.65 per cent acetone and allowed to stand
overnight in the cold. The precipitate was collected by
centrifugation, air dried, and taken up in 80 ml. or water
for testing as R—B 48.

c. (R-B 49): the third extractive was treated with
acetone directly to a concentration of 65 per cent and
allowed to stand in the cold for 5% hours.- The precipitate
was centrifuged down and discarded. The supernatant was
then.made 70 per cent acetone and allowed to stand overnight
in the cold. The precipitate was collected by centrifugation,
air dried, and taken up in 80 ml. or water for testing as
R-B 49.

46

 

 

Results:
Single ‘Weight change
Sample dose Dose Result in 3. Remarks
toxicity cc. Treated/Controls
R-B 46 0.5 0.6-0.5 1" -3.0 2 died
dil. 1:5 -1,o
R-B 46 (retest) 0.6 1* -:5.0
dile 1:5 ~2.0
R-B 47 3.0 0.5-0.2 xf’ -5.5 1 died
-100
R-B 47 (retest) -0.5-0.25 '1’ -5,0
.290
R-B 48 5.0 0.6-0e3 :- .5” 2 6.1“
-1.0
R-B 49 5.0 0.6 1' -3.5 1 died
-l.0

 

Discussion: the results could not be interpreted as
a confirmation of the ability of acetone as a fractionating
agent for the active principle within this range of concen-
tration. :

The apparently conflicting toxicity results were inter-
preted as confirming the extreme toxicity of the “crude
protein" precipitated at relatively low acetone concentrations
and suggested that the cunmlative toxicity could be attrib-
uted to the active principle. That the activity was appar-
ently evenly distributed in the 55 - 60 per cent, 60 - 65
per cent, and 65 - 70 per cent precipitations and that a
comparatively lower toxicity was observed with these frac-
tions supported the belief that the two step method could
be modified to obtain the major portion of the active prin-

47

ciple in the extractive relatively free from.the highly
toxic “crude protein“. The modifications suggested by
these considerations were two in number: (1) that the
range of acetone concentrations over which.a single pre-
cipitate be taken be widened using 55 per cent acetone as
a lower limit and 75 per cent as an upper limit, and (2)
that rather than utilize different acetone concentrations
as criteria for fractionations, allow time to be the vari-

able with a constant concentration of acetone.

Detoxification techniquo‘(Trypsin) (R-B 50, R-B 51)

Extraction and filtration: duplicate 15 g. samples
were extracted with.water (1:20) in a Waring Blendor for
5 minutes and filtered with suction. The extractives‘were
treated as duplicates in the fractionation procedure
followed below.

The extractives were made 55 per cent acetone and
allowed to stand in the cold for 5% hours, the precipitates
centrifuged down and discarded. The supernatants vere
further treated with.acetone to 75 per cent and allowed to
stand overnight. The precipitates were centrifuged, air
dried, and taken up in 80 ml. of distilled water for the
tryptic treatment. These samples were numbered as R-B 50
and R-B 51 and shipped to Dr. C. 0. Stock, Division of
Experimental Chemotherapy, SloanAKettering Institute for
Cancer Research, New York, New York, for further treatment

with crystalline trypsin.

48

The technique of the tryptic digest was not reported
for these samples, but the inhibition test results were

 

 

reported.
Results:
Single 'Weight change
Sample dose Dose Result in 3. Remarks
- toxicity cc. Treated/Controls
R-B 5O dil. 1:10 1.0 - -230
-105
R-B 51
1 control for trypsin _
1.0 ' .2'e5‘
-155
2 control for trypsin :-
l.O ~4,o
-l.5

 

Discussion: these results could not be interpreted as
confirming the ability of crystalline trypsin to detoxify
the active principle, bum suggested further study.

_5_§ £93; 9322 acetone precipitation (single stop method)
(R-B 52, R-B 53, R-B 54).

The result of a retest of fraction.R-B 46 indicated
that although the single dose toxicity was extremely high
’ (o.5) no mice died during the inhibition test. It was re-
quested by Dr. Stock that a similar preparation be sent to
be tested for anti-tumor activity against other tumors
(spectrum).

For these preparations, 15 3. samples were treated
in triplicate following the procedure described under

R-B 46 (page 45). ,

The triplicate samples of 80 ml. were numbered R~B 52,
R-B 53, and R-B '54 respectively and shipped for testing. The
results of the spectrum.had not been reported at the date
of this writing, but the results of the sarcoma 180 test

were reported.

 

 

Results:
Single ‘Weight change
Sample dose Dose Result in . Remarks
toxicity oc." “ ‘ ‘Treated Controls
R-B 52 1.0 1.0-0.5 1 4 died

d11e 185,

 

Discussion: this result indicated that the toxicity
of the "crude protein" in this preparation was apparently
responsible for the inconclusiveness of the inhibition test.

leetone fractionation (variable time method) (R-B 55, R-B 56).
is was suggested in the discussion of fractions R-B 46
- R-B 49 a study was made of the effect of’time on the charac-
teristics of the material precipitated by acetone from the
extractive. Excessive acetone (90 per cent) was selected
as the "constant“ factor in this procedure and two fractions
were obtained: the first collected about 10 minutes after
the addition of the acetone to the extractive, and the
second after allowing the mixture to stand overnight in the
cold.
Extraction and filtration: a 15 g. sample was extracted
with.water (1:20) in a waring Blendor and filtered with

49

suction.

Fractionation: the extractive volume was measured as
250 m1. and placed in the cold (7°C.). After reaching
thermal equilibrium.sufficient cold acetone (1,950 mi.) to
be excessive (90 per cent) was added to the extractive and
mixed by swirling. Immediately after mixing, the solution
was filtered with suction as rapidly as possible (ca. 10
min.) on two 6 inch.Buchner funnels using No. 50 Whatman
paper. The precipitate was dissolved with.difficulty from
the filter paper in ca. 94 ml. of water and bottled for
testing as R-B 55. The filtrate was placed in the cold and
allowed to stand for 12% hours. The precipitate was
collected on a Buchner funnel using No. 50'lhatman paper
and was dissolved (readily) in so ml. of water and bottled
as R-B 56 for testing.

 

 

Results: ‘
Single __ weight change
Sample dose Dose Result in . Remarks
toxicity" cc. Treated/gcntrols
R-B 55 . 1.0 1.0-0.3 - ~535
dil. 1:5 -1,5
R-B 56 5.0 0.25-0.15 t“ -l.0
’ -105

 

Discussion: these results indicated that the method
described in this procedure was the most successful one
carried out in this study in an attempt to separate the
active principle from the toxic materials. The fact that
the treated.mice lost less weight than did the controls

50

suggested that a study be made to determine whether an
increase in the dose level or an increase in the concen-
tration of the active principle in the sample tested would
result in a corresponding greater inhibition of the growth
of the tumors.

SUMMARY

l. The lability of’an.anti-tumor growth substance from
Boletus edulis in acid or alkaline medium was demonstrated.

2. a procedure for the concentration of the active prin-
ciple from.water extractives of Boletus edulis samples was

tested and modified.

3. A procedure for the separation of the active principle
from the toxic factors in water extractives of Boletum

edulis was apparently successful.

CONCLUSIONS

Several attempts have been.made to determine a prac-
tical means of concentrating an anti-tumor growth factor
from.Boletus edulis. The use of acetone as a precipitating
agent for the concentration of the factor from crude extrac-
tives is to be preferred for it has been shown not to de-
stroy tne tumor-growth.inhlcitory activity at concentrations
as high.as 90 per cent. Admittedly, the quantitative
separation of the active principle was not realized in the
course of this study and.mmst necessarily await further
investigation.

It is suggested that the procedure to be employed for
the separation of the active principle from the toxic
materials in the crude extractives be the method as de-
scribed under R-B 55 and R-B 56 (page 48). The removal of
the initial precipitate immediately after treatment of the
extractive with excessive acetone appears to be the critical
step in this method and deserves further study.

That the active principle when quantitatively iso-
lated will be inherently non-toxic to the test animals may
not be concluded from this study and further study of de-
toxification techniques is suggested.

Although.the use of the biological assay method for
testing the inhibitory ability of the samples was conven-

55

ient, the results reported here suggest that a study be
attempted to determine a method of testing which will give
quantitative results without the interference of the toxic
effects of the preparations. This test, if developed,
should be a chemical test involving an analytical method
and must await the isolation and characterization of the

active factor.

l.

2.

5.

4.

5.

6.

'7.

3.

9.

BIBLIOGRAPHY

Buckley, S. M., C. C. Stock, M. L. Crossley and C.

P. Rhoads. Scientific proceedings. Cancer Research,
10:208-209, 1950.

Gye, W. E. Problem of cancer: retrospect and prospect.
Lancet, 2:1515-1317, 1955.

Laszlo, D. Studies on tumor growth inhibitory sub-
stances. Approaches 32 2919.3 Chemotherapy, A.A.A.S.,
pp. 148-156, 1947.

Leuchtenberger, C. Results of treatment of spontaneous
tumors with "folic acid' and allied substances.
Approaches to W Chemotherapy, A.A.a.S., pp. 157-
162, 1947.

Lewisohn, R. Review of the work of the laboratory 1937-
1945.. Approaches 333 m ChemOtherapl, A.A.A.S., pp.
159-147, 1947. ‘

Lucas, E. H. Unpublished data.

Hatsushita, T. New cancer remedy, carcinolysin. Deut.
Med. Wchnschr., 50: 15-14, 1924.

Reilly, H. C. and C. C. Stock. Studies on a tumor-
retarding agent produced by Aspergillus ftunigatus.
Cancer Research, 11:566-569, 1951.

Stock, 0. C. Aspects of approaches in experimental
cancer chemotherapy. The American Journal of Medicine,

I“

’1

e
D
H
D
O
O
Q .,
D
e s
Q
0 D

[I

55

8:658-674, 1950.

10. Sugiura, K. Effect of intravenous injection of yeast
and barley extracts and 9.9. page; factor upon spon-
taneous mammary adenocarcinoma in mice. Approaches
to Tumor Chemotherapy, A.A.A.S., pp. 208-215, 1947.

11. Torrey, J. c. and M. c. Kahn. Treatment of Flexner-
Jobling rat carcinomas with bacterial ferments.
Jour. Cancer Research, 11:554-576, 1927.

JUN 3

 

 

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