quercetinanticanceririsgermanica

Introduction

The use of underground parts of several species of

Iris

(Iridaceae)

was well established in traditional European folk medicine for cen-

turies. Peeled and dried rhizomes of

Iris germanica

L.,

I. florentina

(syn.

I. germanica

var.

florentina

auct.vix.L.),or

I. pallida

Lam. (Iri-

daceae),collectively known as

Rhizoma iridis

,enjoyed popularity

due to their emetic,cathartic,diuretic,stimulant,expectorant and

errhine properties. In addition,powdered dry rhizomes of several

Iris

species,but in particular of

I. germanica

,were used as ingredi-

ents of toothpowders. As a specialty drug,

Rhizoma iridis pro

infantibus

,being composed of small,selected rhizome pieces,

provided pain relief as a masticatory for teething children [1],[2].

Cancer chemoprevention aims to halt or reverse the develop-

ment and progression of cancer cells through use of non-toxic

Cancer Chemopreventive

in vitro

Activities of

Isoflavones Isolated from

Iris germanica

Eckhard Wollenweber

Jan Frederik Stevens

2, 4

Karin Klimo

Jutta Knauft

Norbert Frank

Clarissa Gerhäuser

Affiliation

Institut für Botanik,Technische Universität,Darmstadt,Germany

Department of Chemistry,Oregon State University,Corvallis,OR,USA

AG Chemoprävention (C0202),Deutsches Krebsforschungszentrum,Heidelberg,Germany

Present address: Institut für Pflanzenbiochemie,Halle/Saale,Germany

Correspondence

Prof. Dr. E. Wollenweber ́ Institut für Botanik ́ Technische Universität Darmstadt ́ Schnittspahnstr. 4 ́

D-64287 Darmstadt ́ Germany ́ E-mail: wollenweber@bio.tu-darmstadt.de ́ Fax: +49 6151 16 4630

Received

May 3,2002 ́

Accepted

August 13,2002

Bibliography

Planta Med 2003; 69: 15±20 ́ Georg Thieme Verlag Stuttgart ́ New York ́ ISSN 0032-0943

Abstract

Six known isoflavones were isolated from the rhizomes of

Iris

germanica

,and were established by UV,MS and NMR techniques

as irisolidone (

),irisolidone 7-

-glucoside (

),irigenin

(

),irilone (

),iriflogenin (

),and iriskashmirianin (

). These

compounds were examined for their cancer chemopreventive

potential. They were shown to be potent inhibitors of cyto-

chrome P450 1A activity with IC

values in the range 0.25 ±

4.9

M. The isoflavones

and

displayed moderate activity

as inducers of NAD(P)H:quinone reductase (QR) in cultured

mouse Hepa 1c1c7 cells,with CD values (concentration requir-

ed to double the specific activity of QR) of 3.5 ± 16.7

whereas weak activity was observed with compounds

and

in the radical (DPPH) scavenging bioassay (IC

values 89.6 and

120.3

M,respectively). With respect to anti-tumor promoting

potential based on anti-inflammatory mechanisms,none of the

compounds demonstrated significant activity in the concentra-

tion range tested.

Keywords

Iris germanica

́ Iridaceae ́ isoflavones ́ cancer chemopreven-

tion ́ carcinogen metabolism

Abbreviations

APCI: atmospheric pressure chemical ionization

CD: concentration required for doubling of the specific

activity of NAD(P)H:quinone reductase

Cox: cyclooxygenase

Cyp: cytochrome P450

DAD: diode-array detector

DPPH: 1,1-diphenyl-2-picrylhydrazyl

HMBC: heteronuclear multiple bond correlation

: half-maximal inhibitory concentration

iNOS: inducible nitric oxide synthase

NF: naphthoflavone

QR: NAD(P)H:quinone reductase

: half-maximal scavenging concentration

OriginalPaper

nutrients and/or pharmacological agents [3],[4]. Thus,the

identification,mechanistic investigation,validation and utili-

zation of dietary components,natural products or their syn-

thetic analogous as potential cancer chemopreventive agents

has become an important issue in current public health-relat-

ed research [5],[6]. To this end,we have fractionated an extract

I. germanica

rhizomes in order to determine the cancer che-

mopreventive potential of

Iris

constituents in a series of

vitro

bioassays relevant for the inhibition of carcinogenesis

vivo

MaterialandMethods

Generalexperimentalprocedures

TLCs were run on polyamide (DC 11,Machery-Nagel,Düren,Ger-

many) using solvents A (toluene-petrol

10 0 ± 140

-MeCOEt-MeOH,

12 : 6:2 : 1) and B (toluene-dioxane-MeOH,8 : 1:1),and on silica

gel using solvent C (toluene-MeCOEt,9 : 1). Chromatograms

were viewed under UV light (366 nm) before and after spraying

with `Naturstoffreagenz A' (a 1 % methanolic solution of diphe-

nylboric acid-ethanolamine complex); CC: Sephadex LH-20

(Pharmacia,Peaback,NJ,USA ) and silica gel 60 (40 ± 63

Merck,Darmstadt,Germany).

Semi-preparative HPLC separations were achieved on a 10

Econosil RP-18 (250 ” 22 mm) column (Alltech,Deerfield,IL,

USA). Linear gradient elution was used starting from 40 % to

100 % MeCN in 1 % aq. HCOOH over 30 min at a flow rate of

11.2 ml/min. The UV trace was recorded at 280 nm. Peak fractions

were collected manually,concentrated on a rotavapor and then

lyophilized. Partially purified extracts and isolated isoflavonoids

were analyzed by analytical HPLC with a 5

m C-18e LiChrospher

column (250 ” 4.6 mm; Merck,Darmstadt,Germany) using a lin-

ear solvent gradient from 10 % to 95 % MeCN in 1 % aq. HCOOH

over 30 min at 1.0 ml/min. On-line UV spectra of isoflavonoids

were recorded with a diode-array detector (Merck Hitachi L-

4500A,Darmstadt,Germany).

H-NMR spectra were recorded at 300 and 500 MHz on Varian

instruments (Varian Gemini 300 and Varian Unity 500) and at

600 MHz on a Bruker DRX 600 spectrometer.

C-NMR spectra

were taken at 75 MHz (Varian Gemini 300) and at 150 MHz (Bru-

ker DRX 600).

H-

C HMBC experiments were carried out on the

Bruker DRX 600 instrument using standard Bruker pulse sequen-

ces. All NMR experiments were run in DMSO-

at room tem-

perature.

Atmospheric pressure chemical ionization (APCI) and electro-

spray mass spectra were recorded on a PE Sciex API

III-

plus triple

quadrupole instrument (PE Sciex,Thornhill,Ontario,Canada).

For APCI-MS,samples were dissolved in MeCN-H

O (1 : 1,v/v)

and introduced into the mass spectrometer by loop injection at

a flow rate of 0.5 ml/min using MeCN-H

O (1 : 1,v/v) as carrier

solvent. The heated nebulizer interface was set at 400

C. Ioniza-

tion of the analyte vapour mixture was initiated by a corona dis-

charge needle set at ca 8 kV and a discharge current of ca 3

The orifice plate voltage was + 55 V (positive ion mode). Electro-

spray MS was carried out by continuous flow injection at 5

min and a needle voltage of + 4.5 kV.

Chemicals

All cell culture material was obtained from GIBCO BRL Life Tech-

nologies (Eggenstein,Germany). Fetal bovine serum was from

Greiner Labortechnik GmbH,(Frickenhausen,Germany). Calcein

AM,3-cyano-7-ethoxycoumarin (CEC),and 3-cyano-7-hydroxy-

coumarin (CHC) were purchased from Molecular Probes (Mobi-

tec,Göttingen,Germany). All other chemicals were purchased

from Sigma Chemical Co. (Deisenhofen,Germany).

Plantmaterial

Rhizomes of

Iris germanica

L. (Iridaceae) (identified by Dr. S.

Schneckenburger) were collected in March,2001,in the Botani-

scher Garten der TU Darmstadt. A voucher specimen (no 40 032)

is deposited in the herbarium of the Institut für Botanik in Darm-

stadt.

Extractionandisolation

After cleaning and removal of side roots and bad spots,240 g of

fresh rhizomes of

I. germanica

were cut into pieces and extracted

in a blender with 1.7 l of MeOH. The homogenized mixture was

filtered,and the filtrate concentrated and passed through a col-

umn of Sephadex LH-20 (36 ” 5 cm) in order to remove large

amounts of resinous material. Flavonoid containing fractions

were pooled and evaporated to yield a crude flavonoid mixture.

A portion of this mixture was submitted to column chromatogra-

phy on silica gel,using toluene-MeCOEt-MeOH mixtures of in-

creasing polarity as eluents. Fractions were collected and moni-

tored by TLC on silica gel and polyamide. Flavonoids detected in

mixed fractions were purified to homogeneity by preparative

TLC on silica gel (solvent: toluene-MeCOEt,9 : 1). A final purifica-

tion step was carried out by semi-preparative HPLC which yield-

ed the known isoflavones,irisolidone (

,2 mg),its 7-

-glucoside

(

,1 mg), irigenin (

,15 mg), irilone (

,10 mg), iriflogenin (

mg) and iriskashmirianin (

,1.5 mg). These materials were ob-

tained as white-yellow powders after lypophilization. Their pur-

ity was 95+% by analytical HPLC and

H-NMR.

Another portion of the crude flavonoid mixture was hydrolyzed

by boiling it,after addition of some drops of 25 % sulphuric acid,

for 30 minutes. Isoflavonoid aglycones were recovered from the

hydrolyzate by extraction into ethyl acetate and compared with

the original flavonoid mixture and flavonoid standards.

Isoflavonoidsisolated

5,7-Dihydroxy-6,4

-dimethoxyisoflavone

(Irisolidone,

). HPLC:

= 21.4 min,DAD-UV

max

= 265 nm; APCI-MS,

m/z

=315

[M+H]

(100).

H-NMR (DMSO-

,300 MHz):

= 13.0 (br s,OH-

5),8.34 (1H,s,H-2),7.48 (2H,d,

= 8.5 Hz,H-2

and H-6

),6.99

(2H,d,

= 8.5 Hz,H-3

and H-5

),6.45 (1H,s,H-8),3.78 and 3.73

(each 3H,2 ” OCH

5,7-Dihydroxy-6,4

-dimethoxyisoflavone-7-

O-

glucopyranoside

(Irisolidone-7-

-glucoside,

). HPLC: R

= 15.3 min,DAD-UV

max

= 265 nm; Electrospray-MS:

m/z

= 499 [M+Na]

(100),477

[M+H]

(53),315 [Aglycone+H]

(16);

H-NMR (DMSO-

,300

MHz):

= 13.9 (br s,OH-5),8.48 (1H,s,H-2),7.51 (2H,d,

=8.8

Hz,H-2

and H-6

),7.00 (2H,d,

= 8.8 Hz,H-3

and H-5

),6.89 (1H,

s,H-8),5.45 (1H,d,

= 3.8 Hz,H-1 gluc),3.78 and 3.76 (each 3H,

2 ” OCH

C-NMR (DMSO-

,75 MHz):

= 180.4 (C-4),159.0 (C-

),156.5 (C-7),154.8 (C-2),152.7 and 152.3 (C-5 and C-9),132.3

Wollenweber E et al. Cancer Chemopreventive in vitro¼ Planta Med 2003; 69: 15±20

OriginalPaper

(C-6),130.0 (C-2

and C-6

),122.6 (C-3),121.6 (C-1

),113.6 (C-3

and

C-5

),106.3 (C-10),100.0 (C-1 gluc), 94.0 (C-8), 77.2 (C-5 gluc), 76.6

(C-3 gluc),73.1 (C-2 gluc),69.6 (C-4 gluc),60.6 (C-6 gluc),60.3 and

55.2 (2 ” O

5,7,3

-Trihydroxy-6,4

-trimethoxyisoflavone

(Irigenin,

). HPLC:

= 17.1 min,DAD-UV:

max

= 265 nm; APCI-MS:

m/z

=361

[M+H]

(100);

H-NMR (DMSO-

,600 MHz):

= 13.03 (br s,

OH-5),10.8 and 9.27 (both br s, OH-7 and OH-3

),8.38 (1H,s,H-

2),6.71 and 6.66 (1H each,d,

= 1.9 Hz,H-2

and H-6

),6.51 (1H,

s,H-8),3.79,3.75 and 3.69 (3H each,s,3 ” OCH

5,4

-Dihydroxy-6,7-methylenedioxyisoflavone

(Irilone,

). HPLC:

= 19.7 min,DAD-UV:

max

= 271 nm; UV (MeOH)

max

= 273

nm; APCI-MS:

m/z

= 299 [M+H]

(100);

H-NMR (DMSO-

,600

MHz):

= 12.92 (br s,OH-5),9.62 (br s,OH-4

),8.43 (1H,s,H-2),

7.39 (2H,d,

= 8.5 Hz,H-2

and H-6

),6.89 (1H,s,H-8),6.82 (2H,

= 8.5 Hz,H-3

and H-5

),6.18 (2H,s,O-C

-O).

5,4

-Dihydroxy-3

-methoxy-6,7-methylenedioxyisoflavone

(Iriflo-

genin

). HPLC: R

= 19.9 min,DAD-UV:

max

= 273 nm; UV

(MeOH)

max

= 273 nm; APCI-MS:

m/z

= 329 [M+H]

(100);

H-

NMR (DMSO-

,600 MHz):

= 12.95 (br s,OH-5),9.18 (br s,OH-

),8.47 (1H,s,H-2),7.15 (1H,d,

= 2.1 Hz,H-2

),7.00 (1H,dd,

= 8.2,2.1 Hz,H-6

),6.90 (1H,s,H-8),6.83 (1H,d,

= 8.2 Hz,H-

),6.18 (2H,s,O-C

-O),3.80 (3H,s,3

-OCH

);

C-NMR (DMSO-

,150 MHz):

= 180.9 (C-4),154.7 (C-2),154.0 (C-7),152.9 (C-

9),147.3 (C-3

),146.8 (C-4

),141.4 (C-5),129.6 (C-6),122.2 (C-3),

121.8 (C-6

),121.3 (C-1

),115.3 (C-5

),113.3 (C-2

),107.4 (C-10),

102.9 (O-

-O),89.5 (C-8),55.7 (3

-O

-Hydroxy-5,3

-dimethoxy-6,7-methylenedioxyisoflavone

(Iris-

kashmirianin

). HPLC: R

= 17.3 min,DAD-UV:

max

= 265 nm;

APCI-MS:

m/z

= 343 [M+H]

(100);

H-NMR (DMSO-

,500

MHz):

= 9.12 (br s,OH-4

),8.24 (1H,s,H-2),7.11 (1H,d,

= 2.0 Hz,H-2

),7.02 (1H,s,H-8),6.93 (1H,dd,

= 8.3,2.0 Hz,

H-6

),6.80 (1H,d,

= 8.1 Hz,H-5

),6.19 (2H,s,O-C

-O),3.90

(3H,s,5-OCH

),3.79 (3H,s,3

-OCH

Invitro

cancerchemopreventiveassays

Inhibition of Cyp1A activity

was measured according to Crespi et

al. [7] with minor modification,using cell homogenates of H4IIE

rat hepatoma cells (American Type Culture Collection,Manassas,

VA,USA) cultured for 38 h with 10

-naphthoflavone (

-NF)

in MEME containing 100 units/ml penicillin G sodium,100 units/

ml streptomycin sulfate and 250 ng/ml amphotericin B,supple-

mented with 10 % fetal bovine serum at 37

Cina5%CO

atmo-

sphere as a source of Cyp1A [8]. Activity was measured in 96-

well plates by the rate of dealkylation of 3-cyano-7-ethoxycou-

marin (CEC,5

M in PBS,100

l per well) to 3-cyano-7-hydroxy-

coumarin for 40 min at 37

C in a Cytofluor 4000 fluorescence

reader (PE Applied Biosystems,Wiesbaden,Germany,excitation

wavelength Ex 408/20 nm,emission wavelength Em 460/40 nm).

The protein content of cell homogenates was determined accord-

ing to Smith [9] using bicinchoninic acid (BCA) reagent and bo-

vine serum albumin as a standard (Sigma,Deisenhofen,Germa-

ny). Activity of

-NF-induced controls: 16.9 4.5 nmol/min/mg

of protein (

= 4).

-Naphthoflavone,a known Cyp1A inhibitor,

was employed as a positive control; its IC

value was 0.016

0.005

= 4).

Induction of NAD(P)H:quinone reductase (QR) activity

was deter-

mined in Hepa 1c1c7 mouse hepatoma cells (provided by Dr. J.P.

Whitlock,Jr.,Stanford University) as described earlier [8],[10].

Cells were grown in

-MEM containing 100 units/ml penicillin

G sodium,100 units/ml streptomycin sulfate and 250 ng/ml

amphotericin B supplemented with 10 % fetal bovine serum at

Cina5%CO

atmosphere. Enzymatic activities of com-

pound-treated cells were measured and compared to the activity

of solvent-treated cells to calculate relative induction. The pro-

tein content was estimated by crystal violet staining of an identi-

cal set of test plates. Specific activities of untreated controls:

103.2 6.7 nmol/min/mg protein (

= 5).

-NF with a CD value

(concentration required to double the specific activity of QR) of

0.025 0.007

= 5) was used as a positive control.

Radical-scavenging activity

was determined photometrically by

reaction with 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals

at 515 nm using a microplate reader (Spectramax 340,Molecular

Devices,Ismaning,Germany). Briefly,test compounds dissolved

in DMSO were treated with a solution of 100

M DPPH in ethanol

for 30 min at 37

C. Scavenging potential was compared with a

solvent control (0 % radical scavenging) and ascorbic acid (250

M final concentration,100 % radical scavenging,used as a

blank),and the half-maximal scavenging concentration SC

was generated from the data obtained with 8 serial two-fold di-

lutions of test compounds tested in duplicates ([8],modified

from [11]). The SC

value obtained with (±)-epigallocatechin

gallate from green tea used as a positive control substance was

4.0 0.8

= 2).

Inhibition of cyclooxygenase 1 (Cox-1) activity

using 0.2 U Cox-1-

containing microsomal fractions derived from ram seminal vesi-

cles (specific activity 0.2 ± 1 U/mg protein) was measured at

C by monitoring oxygen consumption using a Clark-type O

electrode (Hansatech Ltd.,Kings Lynn,Great Britain) [8],[12].

Piroxicam,a nonsteroidal anti-inflammatory drug,was used as

a control with an IC

value of 0.35 0.05

= 2).

Inhibition of lipopolysaccharide-mediated inducible nitric oxide

synthase (iNOS) induction

in murine Raw 264.7 macrophages

(American Type Culture Collection,Manassas,VA,USA) was deter-

mined

via

nitrite levels in culture supernatants by the Griess reac-

tion and effects on cell growth estimated by sulforhodamin B

(SRB) staining were measured as described previously [8],[13]. Ni-

trite levels in supernatants of unstimulated controls were 2.7 0.3

nmol nitrite/mg protein,after lipopolysaccharide-stimulation 86.6

7.7 nmol nitrite/mg protein (

=4).CurcuminwithanIC

value

of 13.7 1.3

= 4) was used as a positive control.

ResultsandDiscussion

IdentificationofIrisisoflavonoids

The crude flavonoid mixture,obtained from the rhizomes of

germanica

,was compared chromatographically with a hydro-

lyzed aliquot of the same mixture. This comparison indicated

that the larger portion of the flavonoid material was present in

glycosidic form. Because the hydrolysate did not contain addi-

tional aglycones,it was decided to focus on the aglycones of the

crude flavonoid mixture.

Wollenweber E et al. Cancer Chemopreventive in vitro¼ Planta Med 2003; 69: 15±20

OriginalPaper

Introduction

The use of underground parts of several species of

Iris

(Iridaceae)

was well established in traditional European folk medicine for cen-

turies. Peeled and dried rhizomes of

Iris germanica

L.,

I. florentina

(syn.

I. germanica

var.

florentina

auct.vix.L.),or

I. pallida

Lam. (Iri-

daceae),collectively known as

Rhizoma iridis

,enjoyed popularity

due to their emetic,cathartic,diuretic,stimulant,expectorant and

errhine properties. In addition,powdered dry rhizomes of several

Iris

species,but in particular of

I. germanica

,were used as ingredi-

ents of toothpowders. As a specialty drug,

Rhizoma iridis pro

infantibus

,being composed of small,selected rhizome pieces,

provided pain relief as a masticatory for teething children [1],[2].

Cancer chemoprevention aims to halt or reverse the develop-

ment and progression of cancer cells through use of non-toxic

Cancer Chemopreventive

in vitro

Activities of

Isoflavones Isolated from

Iris germanica

Eckhard Wollenweber

Jan Frederik Stevens

2, 4

Karin Klimo

Jutta Knauft

Norbert Frank

Clarissa Gerhäuser

Affiliation

Institut für Botanik,Technische Universität,Darmstadt,Germany

Department of Chemistry,Oregon State University,Corvallis,OR,USA

AG Chemoprävention (C0202),Deutsches Krebsforschungszentrum,Heidelberg,Germany

Present address: Institut für Pflanzenbiochemie,Halle/Saale,Germany

Correspondence

Prof. Dr. E. Wollenweber ́ Institut für Botanik ́ Technische Universität Darmstadt ́ Schnittspahnstr. 4 ́

D-64287 Darmstadt ́ Germany ́ E-mail: wollenweber@bio.tu-darmstadt.de ́ Fax: +49 6151 16 4630

Received

May 3,2002 ́

Accepted

August 13,2002

Bibliography

Planta Med 2003; 69: 15±20 ́ Georg Thieme Verlag Stuttgart ́ New York ́ ISSN 0032-0943

Abstract

Six known isoflavones were isolated from the rhizomes of

Iris

germanica

,and were established by UV,MS and NMR techniques

as irisolidone (

),irisolidone 7-

-glucoside (

),irigenin

(

),irilone (

),iriflogenin (

),and iriskashmirianin (

). These

compounds were examined for their cancer chemopreventive

potential. They were shown to be potent inhibitors of cyto-

chrome P450 1A activity with IC

values in the range 0.25 ±

4.9

M. The isoflavones

and

displayed moderate activity

as inducers of NAD(P)H:quinone reductase (QR) in cultured

mouse Hepa 1c1c7 cells,with CD values (concentration requir-

ed to double the specific activity of QR) of 3.5 ± 16.7

whereas weak activity was observed with compounds

and

in the radical (DPPH) scavenging bioassay (IC

values 89.6 and

120.3

M,respectively). With respect to anti-tumor promoting

potential based on anti-inflammatory mechanisms,none of the

compounds demonstrated significant activity in the concentra-

tion range tested.

Keywords

Iris germanica

́ Iridaceae ́ isoflavones ́ cancer chemopreven-

tion ́ carcinogen metabolism

Abbreviations

APCI: atmospheric pressure chemical ionization

CD: concentration required for doubling of the specific

activity of NAD(P)H:quinone reductase

Cox: cyclooxygenase

Cyp: cytochrome P450

DAD: diode-array detector

DPPH: 1,1-diphenyl-2-picrylhydrazyl

HMBC: heteronuclear multiple bond correlation

: half-maximal inhibitory concentration

iNOS: inducible nitric oxide synthase

NF: naphthoflavone

QR: NAD(P)H:quinone reductase

: half-maximal scavenging concentration

OriginalPaper

nutrients and/or pharmacological agents [3],[4]. Thus,the

identification,mechanistic investigation,validation and utili-

zation of dietary components,natural products or their syn-

thetic analogous as potential cancer chemopreventive agents

has become an important issue in current public health-relat-

ed research [5],[6]. To this end,we have fractionated an extract

I. germanica

rhizomes in order to determine the cancer che-

mopreventive potential of

Iris

constituents in a series of

vitro

bioassays relevant for the inhibition of carcinogenesis

vivo

MaterialandMethods

Generalexperimentalprocedures

TLCs were run on polyamide (DC 11,Machery-Nagel,Düren,Ger-

many) using solvents A (toluene-petrol

10 0 ± 140

-MeCOEt-MeOH,

12 : 6:2 : 1) and B (toluene-dioxane-MeOH,8 : 1:1),and on silica

gel using solvent C (toluene-MeCOEt,9 : 1). Chromatograms

were viewed under UV light (366 nm) before and after spraying

with `Naturstoffreagenz A' (a 1 % methanolic solution of diphe-

nylboric acid-ethanolamine complex); CC: Sephadex LH-20

(Pharmacia,Peaback,NJ,USA ) and silica gel 60 (40 ± 63

Merck,Darmstadt,Germany).

Semi-preparative HPLC separations were achieved on a 10

Econosil RP-18 (250 ” 22 mm) column (Alltech,Deerfield,IL,

USA). Linear gradient elution was used starting from 40 % to

100 % MeCN in 1 % aq. HCOOH over 30 min at a flow rate of

11.2 ml/min. The UV trace was recorded at 280 nm. Peak fractions

were collected manually,concentrated on a rotavapor and then

lyophilized. Partially purified extracts and isolated isoflavonoids

were analyzed by analytical HPLC with a 5

m C-18e LiChrospher

column (250 ” 4.6 mm; Merck,Darmstadt,Germany) using a lin-

ear solvent gradient from 10 % to 95 % MeCN in 1 % aq. HCOOH

over 30 min at 1.0 ml/min. On-line UV spectra of isoflavonoids

were recorded with a diode-array detector (Merck Hitachi L-

4500A,Darmstadt,Germany).

H-NMR spectra were recorded at 300 and 500 MHz on Varian

instruments (Varian Gemini 300 and Varian Unity 500) and at

600 MHz on a Bruker DRX 600 spectrometer.

C-NMR spectra

were taken at 75 MHz (Varian Gemini 300) and at 150 MHz (Bru-

ker DRX 600).

H-

C HMBC experiments were carried out on the

Bruker DRX 600 instrument using standard Bruker pulse sequen-

ces. All NMR experiments were run in DMSO-

at room tem-

perature.

Atmospheric pressure chemical ionization (APCI) and electro-

spray mass spectra were recorded on a PE Sciex API

III-

plus triple

quadrupole instrument (PE Sciex,Thornhill,Ontario,Canada).

For APCI-MS,samples were dissolved in MeCN-H

O (1 : 1,v/v)

and introduced into the mass spectrometer by loop injection at

a flow rate of 0.5 ml/min using MeCN-H

O (1 : 1,v/v) as carrier

solvent. The heated nebulizer interface was set at 400

C. Ioniza-

tion of the analyte vapour mixture was initiated by a corona dis-

charge needle set at ca 8 kV and a discharge current of ca 3

The orifice plate voltage was + 55 V (positive ion mode). Electro-

spray MS was carried out by continuous flow injection at 5

min and a needle voltage of + 4.5 kV.

Chemicals

All cell culture material was obtained from GIBCO BRL Life Tech-

nologies (Eggenstein,Germany). Fetal bovine serum was from

Greiner Labortechnik GmbH,(Frickenhausen,Germany). Calcein

AM,3-cyano-7-ethoxycoumarin (CEC),and 3-cyano-7-hydroxy-

coumarin (CHC) were purchased from Molecular Probes (Mobi-

tec,Göttingen,Germany). All other chemicals were purchased

from Sigma Chemical Co. (Deisenhofen,Germany).

Plantmaterial

Rhizomes of

Iris germanica

L. (Iridaceae) (identified by Dr. S.

Schneckenburger) were collected in March,2001,in the Botani-

scher Garten der TU Darmstadt. A voucher specimen (no 40 032)

is deposited in the herbarium of the Institut für Botanik in Darm-

stadt.

Extractionandisolation

After cleaning and removal of side roots and bad spots,240 g of

fresh rhizomes of

I. germanica

were cut into pieces and extracted

in a blender with 1.7 l of MeOH. The homogenized mixture was

filtered,and the filtrate concentrated and passed through a col-

umn of Sephadex LH-20 (36 ” 5 cm) in order to remove large

amounts of resinous material. Flavonoid containing fractions

were pooled and evaporated to yield a crude flavonoid mixture.

A portion of this mixture was submitted to column chromatogra-

phy on silica gel,using toluene-MeCOEt-MeOH mixtures of in-

creasing polarity as eluents. Fractions were collected and moni-

tored by TLC on silica gel and polyamide. Flavonoids detected in

mixed fractions were purified to homogeneity by preparative

TLC on silica gel (solvent: toluene-MeCOEt,9 : 1). A final purifica-

tion step was carried out by semi-preparative HPLC which yield-

ed the known isoflavones,irisolidone (

,2 mg),its 7-

-glucoside

(

,1 mg), irigenin (

,15 mg), irilone (

,10 mg), iriflogenin (

mg) and iriskashmirianin (

,1.5 mg). These materials were ob-

tained as white-yellow powders after lypophilization. Their pur-

ity was 95+% by analytical HPLC and

H-NMR.

Another portion of the crude flavonoid mixture was hydrolyzed

by boiling it,after addition of some drops of 25 % sulphuric acid,

for 30 minutes. Isoflavonoid aglycones were recovered from the

hydrolyzate by extraction into ethyl acetate and compared with

the original flavonoid mixture and flavonoid standards.

Isoflavonoidsisolated

5,7-Dihydroxy-6,4

-dimethoxyisoflavone

(Irisolidone,

). HPLC:

= 21.4 min,DAD-UV

max

= 265 nm; APCI-MS,

m/z

=315

[M+H]

(100).

H-NMR (DMSO-

,300 MHz):

= 13.0 (br s,OH-

5),8.34 (1H,s,H-2),7.48 (2H,d,

= 8.5 Hz,H-2

and H-6

),6.99

(2H,d,

= 8.5 Hz,H-3

and H-5

),6.45 (1H,s,H-8),3.78 and 3.73

(each 3H,2 ” OCH

5,7-Dihydroxy-6,4

-dimethoxyisoflavone-7-

O-

glucopyranoside

(Irisolidone-7-

-glucoside,

). HPLC: R

= 15.3 min,DAD-UV

max

= 265 nm; Electrospray-MS:

m/z

= 499 [M+Na]

(100),477

[M+H]

(53),315 [Aglycone+H]

(16);

H-NMR (DMSO-

,300

MHz):

= 13.9 (br s,OH-5),8.48 (1H,s,H-2),7.51 (2H,d,

=8.8

Hz,H-2

and H-6

),7.00 (2H,d,

= 8.8 Hz,H-3

and H-5

),6.89 (1H,

s,H-8),5.45 (1H,d,

= 3.8 Hz,H-1 gluc),3.78 and 3.76 (each 3H,

2 ” OCH

C-NMR (DMSO-

,75 MHz):

= 180.4 (C-4),159.0 (C-

),156.5 (C-7),154.8 (C-2),152.7 and 152.3 (C-5 and C-9),132.3

Wollenweber E et al. Cancer Chemopreventive in vitro¼ Planta Med 2003; 69: 15±20

OriginalPaper

(C-6),130.0 (C-2

and C-6

),122.6 (C-3),121.6 (C-1

),113.6 (C-3

and

C-5

),106.3 (C-10),100.0 (C-1 gluc), 94.0 (C-8), 77.2 (C-5 gluc), 76.6

(C-3 gluc),73.1 (C-2 gluc),69.6 (C-4 gluc),60.6 (C-6 gluc),60.3 and

55.2 (2 ” O

5,7,3

-Trihydroxy-6,4

-trimethoxyisoflavone

(Irigenin,

). HPLC:

= 17.1 min,DAD-UV:

max

= 265 nm; APCI-MS:

m/z

=361

[M+H]

(100);

H-NMR (DMSO-

,600 MHz):

= 13.03 (br s,

OH-5),10.8 and 9.27 (both br s, OH-7 and OH-3

),8.38 (1H,s,H-

2),6.71 and 6.66 (1H each,d,

= 1.9 Hz,H-2

and H-6

),6.51 (1H,

s,H-8),3.79,3.75 and 3.69 (3H each,s,3 ” OCH

5,4

-Dihydroxy-6,7-methylenedioxyisoflavone

(Irilone,

). HPLC:

= 19.7 min,DAD-UV:

max

= 271 nm; UV (MeOH)

max

= 273

nm; APCI-MS:

m/z

= 299 [M+H]

(100);

H-NMR (DMSO-

,600

MHz):

= 12.92 (br s,OH-5),9.62 (br s,OH-4

),8.43 (1H,s,H-2),

7.39 (2H,d,

= 8.5 Hz,H-2

and H-6

),6.89 (1H,s,H-8),6.82 (2H,

= 8.5 Hz,H-3

and H-5

),6.18 (2H,s,O-C

-O).

5,4

-Dihydroxy-3

-methoxy-6,7-methylenedioxyisoflavone

(Iriflo-

genin

). HPLC: R

= 19.9 min,DAD-UV:

max

= 273 nm; UV

(MeOH)

max

= 273 nm; APCI-MS:

m/z

= 329 [M+H]

(100);

H-

NMR (DMSO-

,600 MHz):

= 12.95 (br s,OH-5),9.18 (br s,OH-

),8.47 (1H,s,H-2),7.15 (1H,d,

= 2.1 Hz,H-2

),7.00 (1H,dd,

= 8.2,2.1 Hz,H-6

),6.90 (1H,s,H-8),6.83 (1H,d,

= 8.2 Hz,H-

),6.18 (2H,s,O-C

-O),3.80 (3H,s,3

-OCH

);

C-NMR (DMSO-

,150 MHz):

= 180.9 (C-4),154.7 (C-2),154.0 (C-7),152.9 (C-

9),147.3 (C-3

),146.8 (C-4

),141.4 (C-5),129.6 (C-6),122.2 (C-3),

121.8 (C-6

),121.3 (C-1

),115.3 (C-5

),113.3 (C-2

),107.4 (C-10),

102.9 (O-

-O),89.5 (C-8),55.7 (3

-O

-Hydroxy-5,3

-dimethoxy-6,7-methylenedioxyisoflavone

(Iris-

kashmirianin

). HPLC: R

= 17.3 min,DAD-UV:

max

= 265 nm;

APCI-MS:

m/z

= 343 [M+H]

(100);

H-NMR (DMSO-

,500

MHz):

= 9.12 (br s,OH-4

),8.24 (1H,s,H-2),7.11 (1H,d,

= 2.0 Hz,H-2

),7.02 (1H,s,H-8),6.93 (1H,dd,

= 8.3,2.0 Hz,

H-6

),6.80 (1H,d,

= 8.1 Hz,H-5

),6.19 (2H,s,O-C

-O),3.90

(3H,s,5-OCH

),3.79 (3H,s,3

-OCH

Invitro

cancerchemopreventiveassays

Inhibition of Cyp1A activity

was measured according to Crespi et

al. [7] with minor modification,using cell homogenates of H4IIE

rat hepatoma cells (American Type Culture Collection,Manassas,

VA,USA) cultured for 38 h with 10

-naphthoflavone (

-NF)

in MEME containing 100 units/ml penicillin G sodium,100 units/

ml streptomycin sulfate and 250 ng/ml amphotericin B,supple-

mented with 10 % fetal bovine serum at 37

Cina5%CO

atmo-

sphere as a source of Cyp1A [8]. Activity was measured in 96-

well plates by the rate of dealkylation of 3-cyano-7-ethoxycou-

marin (CEC,5

M in PBS,100

l per well) to 3-cyano-7-hydroxy-

coumarin for 40 min at 37

C in a Cytofluor 4000 fluorescence

reader (PE Applied Biosystems,Wiesbaden,Germany,excitation

wavelength Ex 408/20 nm,emission wavelength Em 460/40 nm).

The protein content of cell homogenates was determined accord-

ing to Smith [9] using bicinchoninic acid (BCA) reagent and bo-

vine serum albumin as a standard (Sigma,Deisenhofen,Germa-

ny). Activity of

-NF-induced controls: 16.9 4.5 nmol/min/mg

of protein (

= 4).

-Naphthoflavone,a known Cyp1A inhibitor,

was employed as a positive control; its IC

value was 0.016

0.005

= 4).

Induction of NAD(P)H:quinone reductase (QR) activity

was deter-

mined in Hepa 1c1c7 mouse hepatoma cells (provided by Dr. J.P.

Whitlock,Jr.,Stanford University) as described earlier [8],[10].

Cells were grown in

-MEM containing 100 units/ml penicillin

G sodium,100 units/ml streptomycin sulfate and 250 ng/ml

amphotericin B supplemented with 10 % fetal bovine serum at

Cina5%CO

atmosphere. Enzymatic activities of com-

pound-treated cells were measured and compared to the activity

of solvent-treated cells to calculate relative induction. The pro-

tein content was estimated by crystal violet staining of an identi-

cal set of test plates. Specific activities of untreated controls:

103.2 6.7 nmol/min/mg protein (

= 5).

-NF with a CD value

(concentration required to double the specific activity of QR) of

0.025 0.007

= 5) was used as a positive control.

Radical-scavenging activity

was determined photometrically by

reaction with 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals

at 515 nm using a microplate reader (Spectramax 340,Molecular

Devices,Ismaning,Germany). Briefly,test compounds dissolved

in DMSO were treated with a solution of 100

M DPPH in ethanol

for 30 min at 37

C. Scavenging potential was compared with a

solvent control (0 % radical scavenging) and ascorbic acid (250

M final concentration,100 % radical scavenging,used as a

blank),and the half-maximal scavenging concentration SC

was generated from the data obtained with 8 serial two-fold di-

lutions of test compounds tested in duplicates ([8],modified

from [11]). The SC

value obtained with (±)-epigallocatechin

gallate from green tea used as a positive control substance was

4.0 0.8

= 2).

Inhibition of cyclooxygenase 1 (Cox-1) activity

using 0.2 U Cox-1-

containing microsomal fractions derived from ram seminal vesi-

cles (specific activity 0.2 ± 1 U/mg protein) was measured at

C by monitoring oxygen consumption using a Clark-type O

electrode (Hansatech Ltd.,Kings Lynn,Great Britain) [8],[12].

Piroxicam,a nonsteroidal anti-inflammatory drug,was used as

a control with an IC

value of 0.35 0.05

= 2).

Inhibition of lipopolysaccharide-mediated inducible nitric oxide

synthase (iNOS) induction

in murine Raw 264.7 macrophages

(American Type Culture Collection,Manassas,VA,USA) was deter-

mined

via

nitrite levels in culture supernatants by the Griess reac-

tion and effects on cell growth estimated by sulforhodamin B

(SRB) staining were measured as described previously [8],[13]. Ni-

trite levels in supernatants of unstimulated controls were 2.7 0.3

nmol nitrite/mg protein,after lipopolysaccharide-stimulation 86.6

7.7 nmol nitrite/mg protein (

=4).CurcuminwithanIC

value

of 13.7 1.3

= 4) was used as a positive control.

ResultsandDiscussion

IdentificationofIrisisoflavonoids

The crude flavonoid mixture,obtained from the rhizomes of

germanica

,was compared chromatographically with a hydro-

lyzed aliquot of the same mixture. This comparison indicated

that the larger portion of the flavonoid material was present in

glycosidic form. Because the hydrolysate did not contain addi-

tional aglycones,it was decided to focus on the aglycones of the

crude flavonoid mixture.

Wollenweber E et al. Cancer Chemopreventive in vitro¼ Planta Med 2003; 69: 15±20

OriginalPaper