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. A. CORDELL2 and A. D. KINGHORN2
I

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 samples were subjected to one or more of seven tannin removal procedures, and the resulting products were subsequently evaluated for topoisomerase inhibitory activity. In most of the samples
investigated, the initial activity was lost after tannin removal. It 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 additional 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 instances, tannins have given positive results in certain assays
due primarily to their reaction with proteins of enzymes.
For example, in a study of plant extracts prepared by 50%
aqueous ethanol treatment, a number of positive tumor inhibition 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 mediate DNA nicking (Sugiyama et aL 1985) (Chaudhuri, et
al., 1995) or which act as inhibitors of the enzymes topoisomerase 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 methodology 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 sequential washing of ethyl acetate extracts with water. As a consequence, 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 procedures on T-I and T-II assays.

282

M. E. Wall er al.
1. MeOH Percolation
2. Add 10% H 20 to make 90 % MeOH
3. Partit ion between 90% MeOH-hexane

90 % MeOH

Hexane
(discard)

I
Concent rate
Part ition between
ethyl acerate-H rO
or
20% MeOH-CHCI TH20

I
Organic fraction
subm itted for
testing for T-1
and T-2 inhibition

Aqueous fraction
submitted for
testing for T-1
and T-2 inhibiti on

Materials and methods

General Ex perimental Procedures
All plant mate rials examined in th is 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 Gla xo Group Research Ltd. Tannin test was conducted
on EtOAc and H 20 extracts by th e pr ocedure of Wall et al.
(Wall et al., 1954). In brief, EtOAc plant ext racts are concentrated to a solid residue and aqueous extracts freeze
dri ed. Either sample 10 mg was treated with hot distilled
and deionized H 20 (6 rnl) and filtered if necessary. The solut ion was divided into three parts. To the first 1% Na CI
solution is added, while to the second 1% NaCI solution
and 5 % gelatin are added. Form ation of a precipitate in the
second indicates the presence of tann ins, which is confirmed by the appearance of blue, blue-black or blue-green
color on addition of FeCl3 solution to the third portion.

were conc entrated, tested for tan nins and evalu ated for T-I1
inhibitor y activity.

Polyvinylpyrrolidine (PVPj Method

Samples (50 mg) were dissolved in H 20 (15 m!) for aqu eous extracts and in MeOH-H 20 (1:1, 15 ml ) for EtOAcsoluble extrac ts. PVP (1.25 g) was added and the resulting
mixture vortexed and centrifuged. Th e supern atant was
withdrawn and dried . It was th en tested for tannins and assayed 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
pro cedure repeated. The supernatants were combined,
dried and evaluated in T-I inhib ition assay.

Differential Solvent Triturat ion

Polyam ide Chromatography

The extract (50 mg) was dissolved in a minimum volume
of MeOH and appl ied to a column (1.1 cm, i.d. ) containing
2.5 g of pol yamide previou sly soaked in H 20 overnight.
The column was then eluted with a bout 100 ml MeOH or
until the eluate was clear. Th e MeOH-eluted fraction was
tested for tannins and evaluated in T-I1 inhib ition assay.

Fig. 1: Dried and Ground Plant Material (20 g).

Each aqueous sample (1 g) 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 20-soluble. Each portion was filtered off, concentrated and tested
for tannins. A total of thr ee comb ined fractions were obtain ed and these were then evaluated for T-I inhibition activity.

Sephadex LH-20 Chrom atography

Each extract (about 100 mg) was dissolved in 80 %
EtOHlH 20 (1.5 ml) and applied to a column (1. 1 em, i.d. )
conta ining 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 portions

Silica Gel Chromatography

The eth yl acetate-soluble sample (100 mg) was loaded on
a column (1.1 ern, i.d.) contain ing Si gel (5 g) in 5 %
MeOH/CHCl3 Elution was carried out by a stepwise gradient of 5 %, 8%, 12%, 15 %, 50 %, 75 % MeOH/CHCl 3
to

Effect of tannins on screening of plant extracts for enzyme inhibitory activity and techniques for the ir removal
and finally MeOH (100 ml each ). Fractions were then combined based on tannin test . The combined fract ions wer e
then dried and evaluated in T-Il inhibit io n assay.

Solvent Partition
After partitioning of th e residue from defatted methanol
between chlorofo rm-metha nol-water, the chloroform fraction was wa shed with 1% N aCI to remove tannins (Figure
1). After solvent concentrat ion , the residu al extracts were
evaluated for T-I and T-I1 inh ibition.

Results and discussion

Polyamide column chromatography (Wall et aI., 1969)
(Tan et aI., 1991), Sephadex LH-20 (Hagerman and Butler,
1980), pol yvinylpyrrol idone [PVP] (Tan et aI., 1991 ) (Loo mis and Battaile, 1966), and collagen have been used by
various workers to eith er remove tannins from plant extracts or to isolate them.
In the pre sent investigation , a total of 50 tannin-containing Tfl-inhibiror y plant ext racts (32 ethyl aceta te and 18
aqueo us) were sub mitted to pol yam ide column ch rom atography (Table la). On elut ing 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 M eOH-eluted portion accounted for ab out 50 % of the
initial weight applied to th e column. Th e MeOH eluates
tested negat ive for tannins when the tann in test (Wall et aI.,
1954 ) wa s administered, and all of them lost T-I1 inhibitory act ivity. Th e activity of these extracts was therefore due
to tannins.
Sephadex LH 20 column chro matogra phy was appl ied to
a batch of eight T-I1inh ibitory eth yl acetat e extr acts and six
T-I inhibitory aqueo us extracts (d. Table l b). The chromatography involved eluting with EtOH to remove non-tannin
constituents, followed by aceto ne-water (1:1) wash , as described by Hagerman and Butler to purify tan nins (Hagerman and Butler, 1980). In the case of the aqueo us extracts,
four of the extracts retained their T-I inhib itor y acti vity,
both in the EtOH eluates and the acet on e-wate r eluates.
Positive tannin tests were evident in both . It can be inferr ed
that other polyphenolics were pre sum abl y eluted with
EtOH in one extract, and one tannion-free sample retained
activity. In the case of the eth yl acetate-soluble fractions, th e
T-I inhibition activity was abs ent in the EtOH eluates and
present in the ta nnin fraction eluted with aqueous acetone.
Another method adopted for removal of tann ins wa s by
mixing solutio ns of thr ee ethyl acetate and thre e aqu eou ssoluble extracts (all T-I1 inh ibitors) with insoluble polyvinylpyrrolidon e (PVP) (Table l c). In all of the samples tannins were remo ved as judged by negati ve tannin tests. As
noted in Table l c, T-I1 inhibition activity was retained in
two of the aqueous extracts.

283

In another pro cedure, coll agen was used to remo ve tannin s from th ree T-l- inhib iting aq ueo us samples (Table t d).
Tannins were effectively removed and th e T-I inhibition activity was absent in the tannin-free extracts. It must be noted, however, that the PVP and collagen procedures can only be used for screening purposes and appear to be un suit able for large sample sizes.
Triturat ion of six tannin-containing aqueou s extract s
with successive porti on s of 15% , 25 %, 50 % , 75%
MeOH/CH Cl j and M eOH and filtrati on after each stage,
did not pro vide tannin-free samples (Table Ie ). Th e T-I
inhibitory activity was also retained. Thi s technique was
therefore not effecti ve in removing tannins, no r in concentrating the activity. In another pilot test, five ethyl aceta tesoluble sampl es were in turn chromatographed on a small
Si gel column, eluting with a gradient of MeOH-CHCI J
(5- 100%) (Table If) . As exp ected, the activ ity in each case
was concentrated into the more polar fraction s. This observation was co nsistent with the presence of tannins in the se
fractions.
A furth er meth od of tannin removal involved solvent partit ioning (Table 19). In thi s procedure, five T-Il inhib itor y
eth yl aceta te extracts were co ncentrated in vacuo and the
residues were partitioned between 20% MeOHlCHCl j and
H 2 0 . In all "of these samples, tannins wer e removed and the
activity was lost. The same procedure was applied to four
T-I inhibitor y ethyl acetate extracts. In all of these sampl es,
tannins were remo ved and the activity was lost. Altern atively, another batch of 15 T-I1 inhibitory eth yl acetate extracts were pa rtitioned betw een CHCl j and H 20 , followed
by 1% saline wash of th e chloroform po rtion. Thi s pro cedure also effectively removed ta nn ins from the organ ic extract. In a ll cases, no T-II inhibitio n activity was noted after
tann in was remo ved.
Of the vari ou s proc edures discussed above for remo ving
tannins from organic solvent fractions, th e simplest method
which can be applied to sma ll or large scale plant extracts
is based enti rely on solvent partitioning meth od s. Methanol
extracts of plant sa mples, after defatting with hexane (Fig.
1), are concentra ted in vacuo . The residu e was partitioned
betw een chloroform and wat er, and the chloro for m fraction washed free of an y residual tannin with 1% NaCI (Fig.
1). If for any reaso n the ta nn in fraction is of int erest, the Sephadex pro cedure can be applied to both aqueo us and organic solvent fractions as descr ibed above (see Experimental Section ).
As demonstrated in this investigation, tannins occurring
in plants app ear to be easily ext racted into ethyl acetate and
to displa y T-l, and T-I1 inhibitory acti vity. Subsequently, it
was found th at a DNA nicking, nonenzymatic assay (Sugiyama et aI., 1985 ) (Chau d huri et aI., 1995 ), gave false
positive results if tannins were present. If these bioa ssays
are to be ut ilized to gu ide fractionation to discover nontannin antitum or leads from plants, then it would be preferable to use 20% MeOHlCHCl j or CHCliH 2 0 partition,

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
Plant Name

Plant Part"

Bor on la inornata Turcz . (Rutaceae)

Bu cida macrostachys Standl ey (Cornbreraceae)
Chukrasia velutina Roem (M eliaceae)
Co ccoloba hondurensis Lundell (Polygonaceae)
Co m breto de ndro n mac rocarpus Beativ
(Lecythidaceae)
Co m b retum api culatum Sond . (Cornbreraceae)

ep
sb
tw
fr,lf,tw
bk
rb ,*rw,
'!:sw*c

Co m bretum erythrophyllum Sond.

(Combretaceae)
Connarus co chinchinensis Pierr e (Co nnaraceae)
Connarus comosus Planch (Connaraceae)
Coria ria ruscifolia L. (Coriariaceae)
Dalbergia cana R. Grah. (Legumin osae)
D iplorhynchus condylocarpon (Muell. Arg. )
Pincho (Apocynaceae)
Duabanga grandifiora Walp. (Sonneratiaceae)
Garcinia pinnata Elmer (Gutt iferae)
Lecaniodiscus [raxinifolius Bak. (Sapind acea e)
Lu eh ea sp eciosa Willd. (Tiliaceae)
Mangifera syluatica L. (Anacardiaceae)
Ma ytenus heterophylla (Eckl. & Z eyh. )
N. Rob so (Celastraceae)
O zoroa insignis Delile. (Anacardiaceae)
Peltophorum africanum Sond. (Legumino sae)
Sandoricu m k oetiape (Meliaceae)
Sapium baccatum Roxb. (Euphor biacea e)
Term inalia bellirica Wall (Combretaceae)
Termi nalia mucronata Craib & Hutchinson
(Combretaceae)
Terminalia sericea DC (Combretaceae)
Blakea woodsonii Gleason ex. Woods & Siab
(M elastomataceae)
Bucida macrostachys Standley (Combretaceae)
Combret odendron macrocarpus Beariv,
(Lecythidaceae)
Combretum apiculatum Sond. (Combretaceae)
Co m bretum erythrophyllum Sond. (Combretaceae)
Coria ria ruscifolia L. (Coriariaceae)
Dillenia paruiflora Griff. (Dilleniaceae)
Duabanga grandiflora Walp. (Sonneratiaceae)
Grias neuberthii Macbride (Lecythid aceae)
Lag erst roemia villosa Wall (Lythraceae)
Sandori cum kdetjape Merrill (Meliac eae )
Terminalia sericea DC (Combretaceae)
Ziziphus mauritiana Laro. (TR hamnaceae)

If"
lf.twfr
st
If

If'
If

sd"
sb
sb
br
sw
sb.sw"
sb.sw"
tw
If
If
If
If,sb, "sw'"
st,tw
sb
bk
rb,rw,sw'
If"
st
If
If
If

lf.sb"
sb
If,sb,sw"
sb

Table lb. Samples Subjected to Sephade x LH20 Tretament.

1. Ethyl acetate extracts
Bor onia inornata Turca (Rutaceae)
ep
Bu cida macrostachys Standley (Cornbretaceae)
sb
D ipl orhynchus condylocarpon (M uell. Arg.)
Pincho (Apocynaceae)
If"
Ga rcinia pinnata Elmer (Gurriferae)
sd "
Mangifera sylvatica L. (Anaca rdiaceae)
br "
Ozoroa insignis Delile (Anacardiaceae)
sb
Sandoricum koetjape (M eliaceae)
tw
Terminalia mucronata Craib & Hutchinson
If
(Combretaceae)

Plant N ame
2. Aqueous extracts
Boscia salicifolia Ol iv. (Capparidaceae)
Careya sphaerica Roxb. (Lecythidacea )
Eugenia glauc icalyx Merrill (M yrraceae)
Ficus sur Forsk. (M oraceae)
Schima wallichii Chais y (Theaceae)
Term inalia alata Roth (Combretaceae)
Table 1 c. Samples Subjected to PVP Treatment.
1. Ethyl acetate extracts
Chu k rasia velutina Roem (Meliaceae)
Mangifera sylvatica L. (Anacardiaceae)
Terminal ia sericea DC (Combretaceae)
2. Aqueous extracts
Blak ea w oodsonii Gleason ex. Woods & Siab
(Melastomataceae)
Bu cida macrostacbys Standley (Combretaceae)
Lagerstroemia villosa Wall (Lythrac eae)

Plant Part b
sb
sb
sb
If
st

If,d sec

tw
br
sw*
st,

rw"

Table I d. Samples Subjected to Collagen Treatment.

Eugeni a g lauc icalyx Merrill (My rt aceae)
sb d
Ficus sur Forsk (M oraceae)
If
Terminalia alata Roth (Combretaceae)
If,se<
Table Le, Samples Subjected to Solvent Trituration Treatment.
Bos cia salicifolia Oliv. (Capparidace ae)
sb
Careya sphaerica Roxb. (Lecythidaceae)
If
Eugenia glaucicalyx Merrill (Myrta ceae )
sb
Ficus sur For sk. (M oraceae)
If
Schima wallichii Chais y (Theaceae)
st
Terminalia alata Roth (Combretaceae)
lf.srTable 1 f. Samples Subjected to Silica Gel Chromatography.
Bucida macrostach ys Standley (Combretac eae )
sb
Co riaria ruscifolia L. (Coriariaceae)
st
Duabanga grandiflora Walp. (Sonneratia ceae)
If
Sap ium baccatum Roxb. (Euphorbiaccae)
If
Term inalia mucronata Craib & Hutchinson
(Combretaceae)
If
Table I g. Samples Subjected to Solvent Partitioning.
1. 20% MeOHlCHCI 3 and H 2 0 ; Topo I
Anacardium occidentale L. (Anacardiacea e)
Barr ingt onia sp. (Lecythidaceae)
Carallia brachiata Merrill (Rhizophoraceae)
G rew ia acuminata Roxb. (Tiliaceae)
2. 20 % MeOHlCHCI 3 and H 20 ; Topo II
Co m b retum apiculatum Sond. (Combreta ceae)
Combretum ery th rophy llum Sond. (Combretaceae)
Maytenus he terophylla (Eckl. & Z eyh. ) N. Robso
(Celastraceae)
Term inalia seri cea DC (Combretadeze)
3. CHCI 3 and H 2 0
Bor on ia inornata Turcz . (Rutaceae)
Co m b retum apiculatum Sond. (Co mbretaceae)
Com b retum erythrophyllum Sond. (Combretaceae)
Co nnarus cochinchinensis Pierre (Conna raceae)
Coriaria ruscifolia L. (Coriariaceae)
Diplorhynchus condylocarpon (Muell. Arg.)
Pincho (Apocynaceae)

rt
If,tw,st,tb'
If
sr.tb"
rb, "cw,
* .sw '"
rt"
sw

sb"
ep
rb, "rw,
sw C
rt"
tw
st

Effect of tannins on screening of plant ext rac ts for enzyme inhibito ry act ivity an d techniqu es for their remova l
Plant Name

Plant Part"

Garcinia pinnata Elmer (Guttiferae)

May tenus beter ophylla (Eckl. & Zeyh.) N. Robs o
(Celastrace ae )
Ozoroa insignis Delile. (Anacardiaceae)
Sandoricum k oetjape (Meliaceae)
Term inalia sercea DC (Combretaceae)

sd"<
sw
sb
tw
If,sb,'>sw''

Th e majority of these sample s were devoid of cytotoxis activ ity

in a broad range of human tumor cell lines in th e initial screen .
b Plant part abbreviations: bk - bark; br - branches; ep - entire
plant; fr - fruit; If - leaf; rb - ro orbark; rt - root; rw - roorwood;
sb - stembark; sd - seed; st - stem ; sw - stemwood; tb
- trunkbark; rw - twig.
c Plant parts listed were ana lyzed sepa ra tely.
d Sample ret ained activity after treatm ent.
,. Plants with broad cytot oxic activity.

instead of EtOAc with constant monito ring of ta nnins by a

qualitative test. The Sephadex proce dure (Hagerman and
Butler, 1980) is the best meth od for removing ta nnins fro m
aq ueo us extracts .
Severa l tho usa nd extracts, orga nic an d aq ueo us, were
screened for tannins. From these, 56 tannin-free orga nic
solvent fractions were obtained with activity in DNA related assays. Of th ese, 26 were active in the DNA nickin g assay (Sugiyam a et al., 19 85) (Chaudhuri er al., 1995), 21
were active in the T-I inh ibit ion assay (Berr y et al., 1992),
and 9 in the T-I1 inhi bition assay (Kashawada et al., 1993).
Many of the plant extracts listed in the ta bles were inactive
in the UIC broad cyto toxicity screen (Likhitwitayawuid et
al., 1993). A number of the plant extracts, however,
showed bro ad, strong act ivity in this screen. Th ese are ind icate d by an asteris k in Tab les la-1g. Tannins do not give
false positive results in cytotoxicity assays because they
cannot pass through cell wa lls.
Acknowl edgments
This investigation was supported by NCI Grant U01-CA52956.

28 5

D., Isolat ion and Structu re Identification of an Active DNA

Strand-Sci ssion
Agent,
(+ )-3,4-Dihydroxy-8,9-methylenediox ypterocarpan.]. Nat. Prod . 58 : 1966-1969, 1995 .
H aslam, E., Plant Polyphen ols: Vegetab le Tannins Revisited, Cambrid ge University Press: Cambridge, 19 89.
Hagerman, A. E., Butler, L. G., Tanin Content of Cowpeas, Chickpeas, Pigeon Peas and M ung Beans. ]. Agri c. Food. Cbem. 28
(2): 459-461, 1980.
Kashiwada, Y., Nonaka, G.-I., Nishika, 1., Bori, 1., Fukushima, Y.,
Bastow, K., Lee, K.-H ., Tannins as Potent Inhibitors of DNA Topoisomerase II. In Vitro.]. Pharm. Sci. 82 (5): 48 7-492, 1993.
Loomis, W. D., Battai le, J ., Plant Phenol ic Compounds and the Isolation of Plant Enzymes. Phytochemistry 5: 42 3-438, 196 6.
Likhirwitayawui d, K., Angerh ofer, C. K., Cordell , G. A., Pezzuto,
J. M. , Ruangrungsi, N. , Cytotoxic and Antima laria l Bisbenzylisoquinoline Alkaloids from Stephania erecta. ]. Nat. Prod. 56:
30-38, 1993.
Nakane, H ., Fukishima, M ., Ono, K., Differential Inhibition of
Reverse Tran scriptase and Various DNA Polymera ses by Digallie Acid and its Derivatives.]. Nat. Prod. 53 : 1234-1240, 1990.
Nis hizawa , M ., Yamagishi, T., Dutsc hman, G. E., Parker, W. B.,
Bodner, A. J., Kilkuskie, R. E., Cheng, Y.-c., Lee, K.-H., AntiAIDS Agents, 1. Isolat ion and Cha racterization of Four New
Tetraga lloylquin ic Acids as a New Class of HIV Reverse
Transcriptase Inhibitors from Tannin Acid. [ . Nat. Prod: 52 :
762-768, 1989.
Ok uda, T., Yoshida, T., H at ano, T., Chemistr y and Biological Activity of Tannins in Medicinal Plants. In Economic and Medicinal Plant Research, Wagner, H .; Farnsworth, N . R., Eds; Academic Press: New York, NY, 130- 165, 1991. Presented at 34th
Annu al Meeting of Th e American Society of Pharmacognosy,
San Diego, CA, J uly 1993.
Sugiyama, H. , Ehrenfeld , G. M. , Shipley, J. B., Kilkuskie, R. E.,
Cha ng, 1. H ., H echt, S. M ., DNA Strand Scission by Bleomycin
Group Antibiotics.]. Nat. Prod 48: 869-877, 1985 .
Tan, G. T., Pezzuro, J. M ., Kingho rn, A. D., H ughes, S. H ., Evaluation of Natural Product s as Inhibito rs of Hu man Immunodeficiency Virus Type 1 (H IV-1) Reverse Transcriptase.]. Na t. Prod.
54 : 143-154,1991.
Wall, M. E., Taylor, H ., Ambrosio, L., Davis, K., Plant Antitumor
Agents. III. A Convenient Separa tion of Tannins from Other
Plant Cons tituents. ]. Pharm. Sci. 58: 839- 841 , 196 9.
Wall, M. E., Eddy, G. R., Willam an, J. J., Correll , D. S., Schubert,
B. G., Gentr y, H. S., Steroidal Sapog enins . XII. Survey of Plants
for Steroid al Sapoge nins and O ther Constitue nts.]. Am. Pharm .
Asso c. (Sci. Ed.) 43: 503-505, 1954 .

References
Berry, D. E., MacKenzie, L., Shultis , E. A., Chan, I. A., H echt, S.
M., Naturally Occurring Inhibitors of Topoisomerase I Mediated DNA Relaxation. ]. Org . Chem. 57: 420 -422, 1992.
Chaudhuri, S. K., H uang, L., Fullas, E, Brown, D. M., Wani, M.
c., Wall, M. E., Tucker, J. c, Beecher, C. W. W., Kingho rn , A.

Address
M. E. Wall, Chemistry and Life Sciences Group Research
Tri angle Institute, P.O. Box 12194 Resear ch Triangle Park,
NC 27709, USA.