Effect of Tannins on the Screening of Plants
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Transcript of Effect of Tannins on the Screening of Plants
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7/24/2019 Effect of Tannins on the Screening of Plants
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Phytomedicine VoL 3 3 , pp. 281-285 1996
1996 by Gustav Fischer Verlag, Stuttgart- Jena . New York
Effect
of
tannins on
screening of
plant
extracts
for
enzyme
inhibitory
activity
and
techniques
for
their
removal
M E WALL1 M C WANP D M BROWN
1
F FULLAS1
J B
OLWALD1 F F
JOSEPHSON
1
N M
THORNTON1
J M PEZZUT02 C W
W
BEECHER2 N
R FARNSWORTH2
G CORDELL2 and D KINGHORN2
Chemistry and Life Sciences Group, Research Triangle Institute, Research Triangle Park, USA.
Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medical Chemistry
and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois.
Summary
Ethyl acetate and aqueous extracts of tannin-containing topoisomerase inhibitory plant sam
ples were subjected to one or more of seven tannin removal procedures, and the resulting pro
ducts were subsequently evaluated for topoisomerase inhibitory activity. In most of the samples
investigated, the initial activity was lost after tannin removal.
was concluded that the activity
initially observed was primarily due to tannins. Procedures are presented for routinely obtaining
tannin-free organic and aqueous fractions.
Key words: tannin, topoisomerase I, topoisomerase II
Introduction
Tannins constitute a group of secondary metabolites
widely distributed in the Plant Kingdom Haslam, 1989 . In
addition to their original use in preparation of leather, the
tannins have recently become of interest for potential addi
tional use in a variety of disease states Okuda et al., 1989 .
The reactivity of such polyphenolic compounds toward
proteins is well known. In several well-documented in
stances, tannins have given positive results in certain assays
due primarily to their react ion with proteins of enzymes.
For example, in a study of plant extracts prepared by 50
aqueous ethanol treatment, a number of positive tumor in
hibition results were obtained in sarcoma 180 assays.
When the tannins were removed, these extracts then gave
negative results Wall et a1. 1969 . Tannins have been found
to interfere with assays for reverse transcriptase Tan et al.,
1991 Nakane et al. 1990 Nishizawa et al. 1989 , DNA
polymerase Nakane et al. 1990 Nishizawa, et al, 1989
Berry,et al. 1992 , and the enzymes topoisomerase I and II
Berry et al. 1992 Kashiwada et al. 1993 . We have been
making an extensive survey of plants for agents which me
diate DNA nicking Sugiyama et aL 1985 Chaudhuri, et
al., 1995 or which act as inhibitors of the enzymes to
poisomerase I T-I Berry et al., 1992 and topoisomerase
II T-II Kashawada et al., 1993 . Previously we found that
tannins, when present, gave false positive reactions in the
case of T-II assays.
Our
initial general screening methodol
ogy involved methanol extraction of the plant material and,
after concentration of this extract, partition of the residue
between water and ethyl acetate Fig. 1 . Unexpectedly, it
was found that the ethyl acetate extracts contained tannins
which were very difficult to completely remove by sequen
tial washing of ethyl acetate extracts with water. As a con
sequence, a study of various procedures for facile removal
of tannin from organic or aqueous extracts was initiated.
In the present communication, we report the application
of several tannin removal methodologies to the removal of
tannins from plant extracts and the effects of these proce
dures on T-I and T-II assays.
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282 M. E. Wall er al.
1. MeOH Percolation
2. Add 10 H
2
to make 90
MeOH
3. Partition between 90 MeOH-hexane
Organic fraction
submitted for
testing for T-1
and T-2 inhibition
90 M
eOH
Concentrate
Partition between
ethyl acerate-HrO
or
20 Me
OH-
CHCI
TH
Aqueous fraction
submitted for
testing for T-1
and T-2 inhibition
Hexane
(discard)
Fig. 1: Dried and Ground Plant Material (20 g).
teri ls nd methods
General Experimental Procedures
All plant materials examined in this investigation were
collected and authenticated by the plant collection program
of NCNPDDPG, a consortium comprised of the University
of Illinois at Chicago (UIC), Research Triangle Institute,
and Glaxo Group Research Ltd. Tannin test was conducted
on EtOAc and H
extracts by the procedure of Wall et al.
(Wall et al., 1954). In brief , EtOAc plant extracts are con
cent rated to a so lid residue and aqueous extracts freeze
dried. Either sample 10 mg was treated with hot distilled
and deionized H
(6 rnl) and filtered if necessary. The so
lut ion was divided into three parts. To the first 1 NaCI
solution is added, while t the second 1 NaCI solution
and 5 gelatin are added. Formation of a precipitate in the
second indicates the presence of tann ins, which is con
firmed by the appearance of blue, blue-black or blue-green
color on addition of FeCl
3
solution to the third portion.
Polyamide Chromatography
The extract (50 mg) was dissolved in a minimum volume
of
MeOH
and applied to a column (1.1 cm, i.d.) containing
2.5 g of polyamide prev iously soaked in H
overnight.
The column was then eluted with about 100 ml MeOH or
until the eluate was clear. The MeOH-eluted fraction was
tested for tannins and evaluated in T
I
inhib ition assay.
Sephadex 20 Chromatography
Each extract (abo ut 100 mg) was dissolved in 80
EtOHlH
(1.5 ml) and applied to a column (1.1 em, i.d.)
containing Sephadex LH-20 (10 g) pre-equilibrated with
EtOH. The column was then eluted with EtOH (100 ml),
followed by 50 ml of acetone-HyO (1:1). Both por tions
were concentrated, tested for tannins and evaluated for T I
inhibitory activity.
Polyvinylpyrrolidine PVPj Method
Samples (50 mg)were dissolved in H
(15 m ) for aque
ous extrac ts and in M
eOH-H
(1:1, 15 ml) for EtOAc
soluble extracts. PVP (1.25 g) was added and the resulting
mi
xtur
e vortexed and centrifuged . The supernatant was
withdrawn and dried.
t
was then tested for tannins and as
sayed for T-II inhibition.
Collagen Method
The extract was dissolved in
DMF
and diluted into 1:100
aqueous suspension of collagen, incubated for 15 minutes
and centrifuged. The supernatant was withdrawn and the
procedure repeated. The supernatants were combined,
dried and evaluated in T-I inhibition assay.
Dif ferential Solvent Trituration
Each aqueous sample (1g) was in turn stirred for 20 min
successively with 15 , 25 , 50 , 75
MeOH/CHCl
3
and finally with MeOH . The final residue was H
2 solu-
ble. Each portion was filtered off, concentrated and tested
for tannins. A total of three combined fractions were ob
tained and these were then evaluated for T-I inhibition ac
tivity.
Silica Gel Chromatography
The ethyl acetate-soluble sample (100 mg)was loaded on
to a column (1.1 ern, i.d .) contain ing Si gel (5 g) in 5
M
eOH/CHCl
3
Elution was carried out by a stepwise gra
dient o f 5 , 8 12 15 , 50 , 75 MeOH/CHCl
3
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Effect of tannins on screening of plant extracts for enzyme inhibitory activity and techniques for their removal 283
and
finally MeOH 00 ml each). Fractions were then com
bined based on tannin test . The combined fract ions were
then dried and evaluated in T-Il inhibition assay.
olvent artition
After partitioning of the residue from defatted methanol
between chloroform-methanol-water, the chloroform frac
t ion was washed with 1 NaCI to remove t
ann
ins Figure
1). After solvent concentration, the residual extracts were
evaluated for T-I and T I inhibition.
esults nd discussion
Polyamide column chromatography Wall et aI., 1969)
an et aI., 1991), Sephadex LH-20 Hagerman and
Butler,
1980), polyvinylpyrrol idone [PVP] Tan et aI., 1991) Loo
mis and Batta ile, 1966), and collagen have been used by
various workers to either remove tannins from plant ex
tracts or to isolate them.
In the present investigation, a total of 50 tannin-contain
ing Tfl- inhibirory plant extracts 32 ethyl acetate and 18
aqueous) were submitted to polyamide column chroma
tography Table la . On eluting the column with MeOH,
non-tannin constituents were washed off the column, while
tannins were retained on the column. In all of the extracts,
the MeOH-eluted portion accounted for ab
ou t
50 of the
initial weight applied to the column. The MeOH eluates
tested negative for tannins when the tann in test Wall et aI.,
1954 ) was administered, and all of them lost
T I
inhibito
ry activity. The activity of these
ex t
racts was therefore due
to
tann
ins.
Sephadex LH 20 column chromatography was appl ied to
a batch of eight T-I1inhibitory ethyl acetate extracts and six
T-I inhibitory aqueous extracts
d
Table l b). The chroma
tography involved eluting with EtOH to remove non-tannin
constituents, followed by acetone-water 1:1) wash, as de
scribed by Hagerman and Butler to purify tannins Hager
man and Butler, 1980). In the case of the aqueous e
xtra
cts,
four of the extracts retained their T-I inhibi tory activity,
both in the EtOH eluates and the acetone-water eluates.
Positive tannin tests were evident in both. It can be inferred
that
oth
er polyphenolics were presumably elu ted wi th
EtOH
in one extract,
and
one tannion-free sample retained
activity. In the case of the ethyl acetate-soluble fractions, the
T-I inhibition activity was absent in the
EtOH eluates
and
present in the tannin fraction eluted with aqueous acetone.
Another method adopted for removal of tannins was by
mixing solutions of three ethyl acetate and three aqueous
soluble extracts all T I inhibitors) with insoluble polyvi
nylpyrrolidone PVP) Table l c). In all of the samples tan
nins were removed as judged by negative tannin tests. As
noted in Table l c,
T I
inhibition activity was retained in
two of the aqueous extracts.
In an
oth
er procedure, collagen was used to remove tan
nins from three T-l- inhib iting aqueous samples Table t d).
Tannins were effectively removed and the T-I inhibition ac
tivity was absent in the tannin-free
extr
acts. It must be not
ed, however, that the PVP and collagen procedures can on
ly be used for screening purposes
and
appear to be unsuit
able for large sample sizes.
Tri turat ion of six tannin-containing aqueous extracts
with successive porti on s of 15 , 25 , 50 ,
75
MeOH/CHCl
j
and MeOH and filtration after each stage,
d id not provide tannin-f ree samples Table Ie ). The T-I
inhibitory activity was also retained. This technique was
therefore not effective in removing t
ann
ins, nor in concen
trating the activity. In another pilot test, five ethyl acetate
soluble samples were in
turn chromato
graphed on a small
Si gel column, eluting with a gradient of
MeOH-CHCI
J
- 10
0
Table
If).
As expected, the activity in each case
was concentrated into the more polar fractions. This obser
vation was consistent with the presence of tannins in these
fractions.
A furth er method of tannin removal involved solvent par
titioning Table 19). In this procedure, five T-Il inhibitory
ethyl acetate extracts were concentrated in vacuo and the
residues were partitioned between 20
MeOHlCHCl
j
and
H
2
0 .
In all o these samples, tannins were removed and the
activity was lost. The same procedure was applied to four
T-I inhibitory ethyl acetate extracts. In all of these samples,
tannins were removed
and
the activity was lost. Alterna
tively, an
oth
er batch of 15 T-I1inhibitory ethyl acetate ex
tracts were partitioned between CHCl
j
and H
2
0 ,
followed
by 1 saline wash of the chloroform portion. This proce
dure also effectively removed tannins from the organic ex
tract. In allcases, no T-II inhibition activity was noted after
tannin was removed.
Of the various proc
edur
es discussed
abo
ve for removing
tannins from organic solvent fractions, the simplest method
which can be applied to small or large scale plant extracts
is based entirely on solvent partitioning methods.
Methan
ol
extracts of plant samples, after defatting with hexane Fig.
1), are concentrated in vacuo. The residue was partitioned
between chloroform
and
water,
and
the chloroform frac
tion washed free of any residual tannin with 1 NaCI Fig.
1). If for any reason the tannin fraction is of interest, the Se
phadex procedure can be applied to both aqueous and or
ganic solvent fractions as described above see Experimen
tal Section ).
As demonstrated in this investigation, tannins occurring
in plants appear to be easilyextracted into ethyl acetate and
to display T-l, and TI inhibitory activity. Subsequently, it
was found that a DNA nicking, nonenzymatic assay Su
giyama et aI., 1985) Chaudhuri et aI., 1995), gave false
positive results if tannins were present. these bioassays
are to be utilized to guide fract ionation to discover non
t
ann
in antitumor leads from plants, then it would be pref
erable to use 20 MeOHlCHCl
j
or
CHCliH
2
0
partition,
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7/24/2019 Effect of Tannins on the Screening of Plants
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284 M. E. Wall et al.
Table 1. Samples Subjected to Tannin Removal Treatments'
Table 1a. Samples Subjected to Polyamide Treatment.
1. Ethyl acetate extracts
Table
lb .
Samples Subjected
to
Sephade x
LH20
Tretament.
1.
Ethyl acetate extracts
oronia inornata
Turca (Rutaceae) ep
ucida macrostachys
Standley (Cornbret aceae) sb
Diplorhynchus condylocarpon (Muell. Arg.)
Pincho (Apocynaceae) If
Garcinia pinnata Elmer (Gurriferae) sd
Man
gifera sylvatica
L. (Anacardiaceae) br
Oz
oroa
insignis
Delile (Anacardiaceae) sb
Sandoricum koetjape (Meliaceae) tw
Terminalia
mucro
nata
Craib
Hutchinson
If
(Combretaceae)
Table 1
f.
Samples Subjected to Silica Gel Chromatography.
ucida macrostachys
Standley (Combretac eae ) sb
Coriaria ruscifolia
L. (Coriariaceae) st
Duabanga
grandiflora
Walp. (Sonneratiaceae) If
Sap ium
baccatum Roxb. (Euphorbiaccae) If
Term inalia mucronata Craib Hutchinson
(Combretaceae) If
Table Le Samples Subjected
to
Solvent Trituration Treatment.
osciasalicifolia
Oliv. (Capparidaceae) sb
Careya sphaerica Roxb. (Lecythidaceae) If
Eugenia glaucicalyx Merrill (Myrta ceae) sb
Ficus sur Forsk. (Moraceae) If
Schima wallichii Chais y (Theaceae) st
Te
rmin
alia alata
Roth (Combretaceae) lf.sr-
Plant Partb
tw
br
sw*
st,
w
sb
sb
sb
If
st
If d
s
sb
d
If
fse
sb
ep
rb, rw,
C
rt
tw
st
rt
If,tw,st,tb'
If
sr.tb
rb, cw,
.sw '
rt
sw
Plant Name
Table 1 c. Samples Subjected to PVPTreatment.
1. Ethyl acetate extracts
Chukrasia velutina
Roem (Meliaceae)
Mang
ifera sylvatica
L. (Anacardiaceae)
Terminal ia sericea
DC (Combretaceae)
2. Aqueous extracts
lak ea woodsonii
Gleason ex. Woods
&
Siab
(Melastomataceae)
ucida macrostacbysStandley (Combretaceae)
Lagerstroemia villosa Wall (Lythraceae)
2. Aqueous extracts
oscia salicifoliaOliv. (Cappar idaceae)
Careya sphaerica
Roxb. (Lecythidacea)
Eugenia glaucicalyx
Merrill (Myrraceae)
Ficus sur
Forsk. (Moraceae)
S
chima
wallichii
Chais y (Theaceae)
Terminalia alata Roth
(Combretaceae)
Table I d. Samples Subjected to Collagen Treatment.
Eugenia glaucicalyx
Merrill (Myrtaceae)
Ficus sur
Forsk (Moraceae)
Terminalia alata
Roth (Combretaceae)
Table I g. Samples Subjected to Solvent Partitioning.
1.
20 MeOHlCHCI
3
and H
2
0 ;
Topo
I
na
cardium occidentale L.
(Anacardiaceae)
arringtonia sp (Lecythidaceae)
Carallia brachiata Merrill (Rhizophoraceae)
Grewia a
cuminata Roxb. (Tiliaceae)
2. 20
MeOHlCHCI
3
and H
2
0 ;
Topo
II
Combretum apiculatum
Sond. (Combretaceae)
C
ombretum
erythrophy llum
Sond. (Combretaceae)
Maytenus heterophylla
(Eckl. Zeyh.) N.
Rob
so
(Celastraceae)
Term inalia sericea
DC (Combretadeze)
3. CHCI
3
and
H
2
0
oronia inornataTurcz. (Rutaceae)
Combretum apiculatum Sond. (Combretaceae)
Combretum
erythrophyllum Sond. (Combretaceae)
Connarus cochinchinensis
Pierre (Connaraceae)
Coriaria ruscifolia
L. (Coriariaceae)
Dipl
orhynchus condylocarpon
(Muell. Arg.)
Pincho (Apocynaceae)
If
If,sb, sw'
st,tw
If
lf.tw
fr
st
If
Plant Part
sb
I f
If
sd
sb
sb
br
sw
ep
sb
tw
fr,lf,tw
bk
rb,*rw,
:sw*c
sb.sw
sb.sw
tw
If
If
bk
rb,rw,sw'
If
st
If
If
If
lf.sb
sb
If,sb,sw
sb
Combretum erythrophyllum Sond.
(Combretaceae)
Connarus cochinchinensis
Pierre (Connaraceae)
onnarus comosus Planch (Connaraceae)
Coriaria ruscifolia
L. (Coriariaceae)
Dalb
ergia cana
R. Grah. (Leguminosae)
Diplorhynchus
condylocarpon (Muell. Arg.)
Pincho (Apocynaceae)
Duabanga
grandifiora
Walp. (Sonneratiaceae)
Garcinia pinnata Elmer (Gutt iferae)
Lecaniodiscus [raxinifolius Bak. (Sapindaceae)
Luehea speciosa Willd. (Tiliaceae)
Mang
era syluatica
L. (Anacardiaceae)
Mayt
enus heteroph
ylla
(Eckl.
&
Zeyh.)
N. Robso (Celastraceae)
O zoroa insignis
Delile. (Anacardiaceae)
Peltophorum
africanum Sond. (Leguminosae)
Sandoricum ko etiape
(Meliaceae)
Sapium baccatum Roxb . (Euphorbiaceae)
Terminalia bellirica Wall (Combretaceae)
Terminalia
mu
cronata Craib Hutchinson
(Combretaceae)
Terminalia sericea
DC (Combretaceae)
lakeawo
odsonii Gleason ex. Woods Siab
(Melastomataceae)
ucida
macrostachys
Standley (Combretaceae)
Combretodendron m ro rpus
Beariv,
(Lecythidaceae)
ombretum apiculatum Sond. (Combretaceae)
Combretum
erythrophyllum Sond. (Combretaceae)
Coriaria ruscifolia L.
(Coriariaceae)
Dillenia paruiflora
Griff. (Dilleniaceae)
Duabanga grandiflora Walp. (Sonnera tiaceae)
Grias neuberthii Macbride (Lecythidaceae)
Lagerstroemia villosa
Wall (Lythraceae)
Sand
ori
cum
kd
etjape
Merrill (Meliaceae)
Terminalia sericea
DC (Combretaceae)
Zi
ziphus
mauritiana
Laro. (TRhamnaceae)
Plant Name
oronla inornataTurcz. (Rutaceae)
ucidamacrostachys Standley (Cornbreraceae)
hukra
sia velutina
Roem (Meliaceae)
Coccoloba
h
ondur
ensis Lundell (Polygona ceae)
Combretodendron m croc rpus
Beativ
(Lecythidaceae)
Combretum
apiculatum Sond. (Cornbreraceae)
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Effect of tannins on screening of plant extracts for enzyme inhibitory activity and techniques for their removal 285
Acknowledgments
This investigation was suppor ted by NCI Gran t U01-CA
52956.
a The majority of these samples were devoid of cytotoxis activity
in a broad range of human tumor cell lines in the init ial screen.
b Plant part abbrevia tions: bk - bark; br - branches; ep - ent ire
plant; fr - fruit; If- leaf;rb - roorbark;
- root; rw - roorwood;
sb - stembark; sd - seed; st - stem; sw - s temwood ; tb
- t runkbark; rw - twig .
c Plant parts listed were ana lyzed separately.
d Sample retained activity after treatment.
,. Plants with broad cytotoxic activity.
instead of EtOAc with cons
tant
m
onit
oring of tannins by a
qualitative test. The Sephadex procedure (
Hagerman
and
Butler, 1980) is the best method for removing tannins from
aqueous extracts .
Several thousand extracts, organic and aqueous, were
screened for tannins. From these, 56 tannin-free orga nic
solvent fractions were obtained with activity in DNA relat
ed assays. Of these, 26 were active in the DNA nicking as
say (Sugiyama et al., 1985) (Chaudhuri er a l., 1995), 21
were active in the T-I inhibition assay (Berry et al.,
1992
),
and 9 in the T
I
inhibition assay (Kashawada et al., 1993).
Man
y of the plant extracts listed in the tables were inactive
in the UIC broad cytotoxicity screen (Likhitwitayawuid et
al., 1993 ). A number of the plant extracts, however,
showed broad, strong activity in this screen. These are indi
cated by an asterisk in Tables
la 1g
. Tannins do not give
false positive results in cytotoxicity assays because they
cannot pass through cell wa lls.
Plant Name
Garcinia pinnata Elmer (Guttiferae)
May tenus beterophylla (Eckl.
Zeyh.) N. Robso
(Celastraceae)
Oz oroa insignis
Delile. (Anacardiaceae)
Sandoricum k oetjape (Meliaceae)
Term inalia sercea
DC (Combretaceae)
Plant Part
sd sw
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M. E. Wall, Chemistry and Life Sciences
Grou
p Research
Triangle Institute, P.O. Box
1219
4 Research Triangle Park,
NC 277
09, USA.