Effect of Tannins on the Screening of Plants

7/24/2019 Effect of Tannins on the Screening of Plants

1/5

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.


2/5

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


3/5

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,


4/5

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)


5/5

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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Documents

Effect of Tannins on the Screening of Plants