HPLC Quantification of Flavonoids and Biflavonoids in Cupressaceae Leaves

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HPLC Quantification of Flavonoids

and Biflavonoids in

Cupressaceae

Leaves

2002,

56,

4 6 9 - 4 7 4

A. Romani 1.

/

C. Galardi 1

/

P. Pinelli ~ / N. Mulinacci ~ / D.

HeimleP

1 Dipartimento di Scienze Farmaceutiche, Universitd~ degh Studi di Firenze, Via G. Capponi 9, 50121 Firenze, Italy;

E-Maih an nahsa.romani@u nifi.it

2 Dipartimento di Scienza del Suolo e Nutrizione della Pianta, Universitd~ degli Studi di Firenze, P. le delle Cascine 18, 50144 Firenze, Italy

Key Words

Column liquid chromatography - mass spectrometry

LC-diode array detection

Flavonols

Biapigenin

Cupressaceae

leaves

S u m m a r y

The aim of this investigation was to obtain qualitative and quantitative profiles of the flavonoid

and biflavonoid composition of six cypress species -

Cupressus funebris

L.,

Cupressus semper-

Wrens

L.,

Cupressus glabra

L.,

Cupressus arizonica

L.,

Cupressus goveniana

L., and

Cupressus

lusitanica

L. HPLC-diode-array detection (DAD), HPLC-MS, and HPTLC were used to identify the

individual compounds. A chromatographic method was optimized for identification and

quantification of the main flavonoid glycosides and biflavonoids. The flavonoids identified

and calibrated were: rutin, quercetin glucoside, quercetin rhamnoside, and kaempferol 3-0-

rhamnoside. The biflavonoids identified and calibrated were: cu pressuflavone, amentoflavone,

robustaflavone, hinokiflavone, methylrobustaflavone, methylamentoflavone, and dimethylcu-

pressuflavone.

Introduction

The

Cupressaceae

family comprises sev-

eral genera and many species. Cypress trees are found in the northern hemisphere only and are divided into three geographi- cal groups Afromediterranean, Ameri- can, and Asiatic. The best known and most widespread species in Italy is

Cupres-

sus sempervirens

L., an evergreen conifer

with small, scale-like leaves which can grow as high as 30 m. Cypress is usually cultivated as an ornamental tree to beauti- fy parks and cemeteries; in many areas,

Tuscany in particular, it constitutes an un- mistakable element of the landscape [1,2].

The aerial parts of the plant have been widely used in folk medicine. Nowadays they are sometimes used for suffumiga- tions, as antitussives, and in solution for washing and bandages for treatment of circulatory diseases [3]. As reported by Hegnauer [4], the main chemotaxonomic chemical components of the

Cupressus

genus are the biflavonoids. Since the first biflavone, gingetin, was isolated in 1929 more than one hundred biflavonoids have been identified in plants [5]. A wide variety

of biological activity has been ascribed to these molecules, e.g. peripheral vasodila- tation, hypoglycemic, antimicrobial, and antidiabetic effects, and inhibitory effects on lipid peroxidation [6 8]. Other more specific activity has also been reported, e.g. stimulation of R N A synthesis in rat epatocyte suspensions, cytotoxicity, inhi- bition of the expression of EBV virus gene, and anti-spasmogenic, antibradyki- nin, and hepatoprotective activity [9, 10]. Recent studies have found evidence that the molecules have antifungal and anti-in- flammatory activity [11 13], and espe- cially remarkable antiviral activity against HIV, adenovirus, HSV, HCMV, varicella zooster virus, and hepatitis B [14, 15].

Despite these properties, biflavones are a class of phenolic compounds which have rarely been studied. Several studies have been performed on the biflavonoid content of species such as

Ginkgo biloba

and

Hypericum perforatum,

which are also im-

portant in the pharmaceutical industry [16 20], but few data are available about their occurrence in the genus

Cupressus.

The chemical structures of the main biflavones present in cypress species are reported in Figure 1.

Gadek and Quinn [21] first reported the presence of amentoflavone and cupressuflavone in the leaves of

Cupressus

sempervirens L., Cupressus lusitanica L.,

and

Cupressus glabra

L. Heimler and Pier-

oni [22] separated and identified flavonoid glycosides and biflavonoids from

Cupres-

sus sempervirens

L. by use of two different

thin-layer chromatographic methods. Al- though a variety of biflavonoids has been reported to be present in cypress tissues,

Original Chromatographia 2002,

56,

October (No. 7/8)

469

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OlI

H

O

~

O

/

~

lit)"

v

OH

Amentoflavone

OH

O

~ . j O t

Ho....~ . . . . / . o . ~ ~ ~

OH

O

Cupressuflavone

~

()H

Oil

Hinokiflavone

Figure

1. Structural formulae of the main biflavonoids detected in Cupressus leaves.

Table I. Linear solvent gradient used for analytical HPLC-DAD and HPLC-MS analysis.

H20 (%)

CH3CN (%) Time (min) 93.0 7.0 0.1 50.0 50.0 5.0 25.0 75.0 10.0 25.0 75.0 13.0 0.0 0.0 15.0 0.0 0.0 20.0

the identity of the compounds was usually inconclusive and quantification was not attempted [21,23].

In the investigation reported here the flavonoid and biflavonoid content of hydroalcoholic extracts of six cypress species

C. funebris L. (Asiatic group), C. sem-

pervirens L. (Afromediterranean group),

C. glabra L., C. arizonica L., C. goveniana

L., and C. lusitanica L. (American group) was determined by HPLC-DAD, HPLC-MS, and HPTLC. A chromatographic method was optimized for identification and quantification of the main flavonoid glycosides and, in particular, biflavonoids. To the best of our knowledge

this is the first report on quantification of the main individual polyphenols in Cu- pressus leaf tissues.

Experimental

Sample Preparation

and Extraction of Polyphenols

Green leaves of C. sempervirens, C. funeb- ris, C. glabra, C. goveniana, C. lusitanica,

and C. arizonica were analysed; all the samples were collected in October 2001. Leaf laminae were frozen rapidly in liquid nitrogen and stored at 80 ~ until analysis. Frozen leaf tissue was then ground under liquid nitrogen by use of a pestle and mortar. Fresh tissue (1 g) was extracted with 4 x 30 mL 70:30 (% v/v) EtOH- water adjusted to pH 2.0 by addition of HCOOH. The raw ethanolic extract was then evaporated to dryness under vacuum (Rotavapor 144 R; Biichi, Switzerland) at room temperature and then dissolved in 100 mL water at pH 2.0 (adjusted by addition of HCOOH). This solution was then

defatted, by extraction with 4 x 50 mL n- hexane, and the ethanolic extract was con- centrated under reduced pressure and fi- nally dissolved in pH 2 ethanol to a final volume of 5 mL. A sample (6 pL) of this solution was analysed by HPLC with diode-array detection (DAD) and HPLC- MS for qualitative and quantitative eva- luation.

Identification and Quantification

of Individual Flavonoids

and Biflavonoids

Identification of individual polyphenols was achieved by use of their retention times and both spectroscopic and spectro- metric data. Authentic standards of quercetin 3-O-glucoside (isoquercitrin), quercetin 3-O-rhamnoside (quercitrin), quercetin 3-O-rutinoside (rutin), hinokiflavone, and amentoflavone were purchased from Extrasynth&e (Lyon, Nord-Genay, France).

Individual polyphenols were quanti- fied by use of a five-point regression curve (r 2 > 0.999) in the range 0 30 pg on the basis of authentic standards; calibration was performed directly by HPLC-DAD at the wavelength of maximum absorbance (350 rim).

Calibration for isoquercitrin, quercitrin, rutin, amentoflavone, and hinokiflavone was performed by use of the appro- priate pure standards. Calibration for cupressuflavone was performed by use of amentoflavone as reference compound. Calibration for kaempferol 3-O-glucoside was performed by use of kaempferol as reference compound after correcting for the specific molecular weight.

Analytical Techniques

and Equipment

HPL C-DAD AnaIysis

Analysis was performed by use of an HP l l00L liquid chromatograph equipped with a DAD (Agilent Technologies). The polyphenol compounds were separated at 26 ~ on a 150 mm x 3.0 mm i. d., 5-pm particle, Luna C18 (2) column (Chemtek analytica, Bologna) equipped with a 4 mm length x 3.0 mm i. d. ODS (Cis) precol- umn. The mobile phase was a four-step linear gradient prepared from water (adjusted to pH 3.2 by addition of H3PO4) and acetonitrile. The starting composition 470 Chromatographia 2002, 56, October (No. 7/8) Original

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mAU 2000 1500 1000- 5OO mAU 2000 1500 1000 500

Sig=350 nm Cupressus funebris

2//,

A, f,

1 0 11

2 4 ~ 8 10 12 14

Sig=350 nm Cupressus lusitanica

Time (rain)

2

3 7 10

g 6 8 10 12 14 Time (min)

Figure 2. Chromatographic profile acquired by use of HPLC-DAD at 350 nm from ethanolic extracts of

Cupressusjunebris

and

Cupressus lusitanica.

Peaks: 1 = rutin; 2 = quercetin 3-O-glucoside; 3 = quercetin 3-O-rhamnoside; 4 = kaempferol 3-0- rhamnoside; 5 = cupressuflavone; 6 = amentoflavone; 7 = robustaflavone; 8 = hinokiflavone; 9 = methylrobustaflavone; 10 = methylamentoflavone; 11 = dimethylcupressuflavone.

was 93:7 (%

v/v)

H20 CH3CN and the CH3CN content was increased to 75% over a 13-min period. The composition of the gradient is reported in Table I. The mobile phase flow rate was 0.6 mL min 1. UV- visible spectra were recorded in the range 190 450 nm and chromatograms were acquired at 240,280,330, and 350 nm.

HPLC-MS AnaJysis

HPLC-MS analysis was performed, as de- scribed elsewhere [24], by use of an HP 1100 MSD API-electrospray coupled to an HP l l 0 0 L liquid chromatograph equipped with a D A D (Agilent Technolo- gies). Positioning of the nebulizer ortho- gonal to the capillary inlet enabled the use of the same conditions as for HPLC- D A D analysis, although in this analysis the water was adjusted to pH 3.2 by addition of HCOOH.

HPTLC AnaJysis

Two-dimensional H P T L C was performed on 5 cm x 5 cm silica gel 60F254 plates (Merck) in a Desaga (Carlo Erba, Milan, Italy) chromatography chamber for hori- zontal development comprising a solvent- proof body (Teflon) with a tray for mobile phase and a tight-fitting glass lid. The mobile phase was transferred from the tray to

the layer by means of an exchangeable sin- tered-glass plate. The mobile phase for both runs was toluene-pyridine-formic acid, 100:20: 7 [22]. After development the plates were dried and sprayed with a 1% methanolic solution of the complex of di- phenylboric acid with ethanolamine, fol- lowed by 5% ethanolic poly(ethylene gly- col) 4000. Spots were identified by virtue of their fluorescence at 365 nm.

Results and Discussion

Identification of Individual

Flavonoids and Biflavonoids

The aim of this work was to develop a ra- pid H P L C method for identification and quantification of the main flavonoids and biflavonoids present in the leaf tissue of six species of

Cupressaceae.

As examples the chromatographic profiles obtained from

C. funebris

and

C. lusitanica

extracts, recorded at 350 nm, are presented in Fig- ure 2. The figure reveals both the qualitative composition of the cypress leaves and the efficiency of the chromatographic method used. The flavonoids rutin, isoquercitrin, quercitrin, and kaempferol 3- O-rhamnoside, with retention times between 0.0 and 6.5 min, were identified, as were the biflavonoids amentoflavone, cu-

pressuflavone, robustaflavone, hinokiflavone, and other biflavonoids, with retention times between 7.0 and 11.0 min.

For better characterization of both flavonoid glycosides and biflavonoids, H P L C - D A D was combined with HPLC- MS operating in the negative ion mode with modulated fragmentation patterns. Isoquercitrin, quercitrin, rutin, and amentoflavone were identified by comparison of retention times and UV-visible spectra of leaf extracts with those from authentic standards.

Kaempferol-3-O-rhamoside was detected by HPLC-MS; the fragmentation pattern contains signals at

m/z

431 and 285, corresponding, respectively, to the quasi-molecular ion [M Hi and to the fragment obtained by loss of rhamnose [M 146] . The position of the substituent was confirmed by comparing results from the cypress extract with those from a grape extract in which this compound had previously been detected [23].

The mass spectrum and chemical structure of cupressuflavone, the most representative biflavone in all the samples analysed, are shown in Figure 3. The most important peaks are those at

m/z

537 and 375, which correspond to the quasi-molecular ion [M Hi and to the fragment [M 162] .

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1 0 0 8 0 6 0 4 0 2o i

A P I - E S N e g a t i v e

[ M - 1 6 2 1

375.2

537.3

I M - H ] -

5 0 0 m/z'

Figure 3. Negative-ion mass spectrum of cupressuflavone, and the corresponding chemical structure. The spectrum was acquired by use of API-electrospray HPLC-MS analysis at a negative frag- mentor potential of 180 V. MSD1 API-ES Negative ,=,,, ,' I 400000 [ ', f': i , 20000 ,~ ~ / 2.5 715 1'0

125 1'5

#5

2o

2~5

MSD1 537 API-E$ Negative 100000 i 'l 75000 I'=j, 50600 i , , , & 215 . . . . 5 . . . 7,5 10 12.5 1'5 ' 17.5 2'0 (A) 22.5 MSD1 551 API-ES Negative 2 ,~ ii I,,. 215 5 7 1 5 1'0 12.5 15 17.5 20

(a)

2 2 . 5 ' MSD1 565 API-ES Negative 3 O 0 0 2 0 O 0 1 , i , , i J , 2 . 5 I = , , ] . . . . i . , i , 5 7.5 li0 112.5 1~5 17.5 2~0

(c)

22.5 T i m e

( m i n )

Figure 4. Extracted ion-current chromatograms obtained from the hydroalcoholic extract of

CupressusJimebris

leaves. The spectrum was acquired by use of API-electrospray HPLC-MS analysis operating in negative ionization mode at 180 eV. Chromatograms were recorded at

m/z

537, 551, and 565,

whichc•rresp•ndt•them/z•fcupressu•av•ne,ament••av•ne,hin•ki•av•ne,andr•busta•av•ne([M

H] = 537) (A); their methyl derivatives ([M

H] = 551) (B), and their dimethyl derivatives ([M H] = 565) (C), respectively. G a d e k and Quinn [21] reported the oc-

currence of variable amounts of hinokiflavone and its derivatives in

Cupressus

species. Two-dimensional H P T L C was used to verify the presence of these molecules in the cypress samples analysed. The accuracy and reproducibility of H P T L C m e t h o d are g o o d when compared with those of H P L C . The fluorescence characteristics (at 365 nm) and

RF

of

compo-

nents of the cypress extracts were compared with those of authentic standards of amentoflavone and hinokiflavone. This revealed the presence of trace amounts of hinokiflavone in two species

C. funebris

and

C. arizonica.

This techni-

que was used because fluorescence detection is more than two orders of magnitude m o r e sensitive than H P L C - D A D , as reported elsewhere [25].

K r a u z e - B a r a n o w s k a et al. isolated the biflavonoids 4'- O-methylcupressuflavone, 7'-O-methylamentoflavone, and 4 ' " - 0 - methylamentoflavone f r o m the leaves of

Cupressocyparis leylandii,

a m e m b e r of

the subfamily

Cupressaceae,

and charac- terized the c o m p o u n d s by use of b o t h mass spectrometry and N M R [26]. It has previously been suggested that methylamentoflavone, methylhinokiflavone, and 4 7 2 C h r o m a t o g r a p h i a 2002,

56,

October (No. 7/8) Original

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I I methyl amentoflavone I methyl amentoflavone ,__,' ' A m e n t o f l a v o n e A ,' A m e n t o f l a v o n e A

f1

i ! , / " \, / . . .

/

?

00 , /

',i

0

2;0 aoo 3;0 400 4~0 sO0 550 o 2so aoo aso 400 4;0 soo ~so or~

Figure

5 A. First derivative spectra of amentoflavone and methylamentoflavone. B. Second derivative spectra of amentoflavone and methylamentoflavone.

Figure

6. Total polyphenol content of the hydroalcoholic extracts of leaf tissue from six cypress species (CFUN = CupressusJunebris; CSEMP = Cupressus sempervirens; CGLAB = Cupressus glabra;

CGOV = Cupressus goveniana; CARIZ = Cupressus arizonica; CLUSIT = Cupressus lusitanica).

Quantitative data are expressed as mg g 1 fresh weight.

rivative was f o u n d in C. funebris a n d C.

goveniana only. Because the f r a g m e n t a - t i o n p a t t e r n , w h i c h shows the quasi-molecular ion [M H] a n d f r a g m e n t s indicative of the loss o f one or two m e t h y l units, is similar for these molecules, t h e i r chemical structures are n o t easily d e t e r m i n e d . M o r e detailed i n f o r m a t i o n a b o u t these molecules was o b t a i n e d by a p p l i c a t i o n of the derivative f u n c t i o n to UV-visible spectra derivative spectra reveal m o r e specific details t h a n the original spectra w h e n different c o m p o u n d s are b e i n g c o m p a r e d . Small differences b e t w e e n spectra are m u c h m o r e obvious, a n d easier to identify visually. O v e r l a i d derivative spectra of a m e n t o f l a v o n e a n d its m e t h y l derivative are r e p o r t e d in F i g u r e 5. T h e small differ- ence b e t w e e n the spectra was indicative of c o r r e l a t i o n b e t w e e n t h e m , a n d so the presence of a m e t h y l derivative of a m e n t o f l a - v o n e was confirmed. W h e n the same m a t h e m a t i c a l f u n c t i o n was applied to the c o m p o u n d spectra o f all the leaf extracts a m e t h y l derivative o f r o b u s t a f l a v o n e a n d a d i m e t h y l derivative o f c u p r e s s u f l a v o n e were identified. O u r findings are in agree- m e n t w i t h p r e v i o u s results showing the occurrence of b i f l a v o n o i d m o n o m e t h y l derivatives as m i n o r c o n s t i t u e n t s o f Cu- pressaceae [26, 27]; this is, however, the first r e p o r t o f the presence o f the m o r e highly m e t h y l a t e d biflavones.

Figure

7. Different polyphenol subclasses present in the hydroalcoholic extracts of leaf tissue from six Cupressus species. (CFUN = Cupressusjunebris; CSEMP = Cupressus sempervirens; CGLAB =

Cupressus glabra; CGOV = Cupressus goveniana; CARIZ = Cupressus arizonica; CLUSIT = Cupres- sus lusitanica; TB = total biflavonoids; TMBD = total methyl biflavonoid derivatives; TFG = total flavonoid glycosides). Data are expressed as mg g 1 fresh weight.

a m e t h y l r o b u s t a f l a v o n e occur in cypress leaves, a l t h o u g h their chemical structure was n o t fully clarified [21, 27]. A l t h o u g h n o d a t a are available o n the occurrence o f m o r e highly m e t h y l a t e d b i f l a v o n o i d s in these species, the presence of such derivatives was assessed by i n v e s t i g a t i o n o f H P L C - M S e x t r a c t e d - i o n profiles a n d the c o r r e s p o n d i n g m a s s spectra. As a n exam- ple, Figure 4 shows the extracted ion chro- m a t o g r a m s o b t a i n e d f r o m the extract o f

the leaf tissue o f C. funebris. These chro- m a t o g r a m s were r e c o r d e d in the negative- ion m o d e at the m/z o f b i f l a v o n o i d s (m/z

537), the m/z o f b i f l a v o n o i d m e t h y l derivatives (m/z 551), a n d the m/z of d i m e t h y l derivatives (m/z 565). This f u r n i s h e d evidence for the presence of four biflavonoids, two m e t h y l derivatives a n d a di- m e t h y l derivative in this species.

M e t h y l derivatives were identified in all the species analysed, b u t a d i m e t h y l de-

Quantification of Flavonoids

and Biflavonoids in Cupressus

Leaf Tissues

The a m o u n t s of p o l y p h e n o l s d e t e r m i n e d in the samples, expressed in m g g 1 fresh weight, are listed in T a b l e II; all the d a t a are averages f r o m three analyses; stan- d a r d d e v i a t i o n s were < 2 % . The t o t a l a m o u n t o f p o l y p h e n o l s varies f r o m 2.91 to 7.63 m g g ] fresh weight, as s h o w n in f o r m of a h i s t o g r a m in F i g u r e 6. T h e lar- gest a m o u n t was f o u n d in C. funebris a n d the lowest in C. arizonica. The p o l y p h e n o l c o n t e n t of the o t h e r species was similar, r a n g i n g f r o m 3.69 to 4.48 m g g 1 fresh weight. T h e results r e p o r t e d in T a b l e II in- dicate t h a t the b i f l a v o n o i d s are the m o s t representative c o m p o u n d s in the extracts, a n d they seem to be the only p o l y p h e n o l s present in C. glabra.

C u p r e s s u f l a v o n e was the m o s t a b u n - d a n t b i f l a v o n o i d , levels r a n g e d f r o m 2.02 to 2.55 m g g 1 fresh weight a n d a c c o u n t e d for between 43 a n d 66% of t o t a l biflavonoids except for C. arizonica, in w h i c h it Original C h r o m a t o g r a p h i a 2002, 56, O c t o b e r (No. 7/8) 4 7 3

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Table II. Qualitative and quantitative results from HPLC-DAD analysis of flavonoids and biflavonoids in different Cupressaceae leaves. Data are

averages from three analyses and are expressed as mg g 1 fresh weight.

C F U N CSEMP CGLAB CGOV CARIZ CLUSIT Rutin 0.11• 3 0.01• 4 nd 0.01• 4 nd nd

Quercetin glucoside 0.09 • 1.610 3 0.18 • 3.210 3 nd 0.01 • 1.910 4 nd 0.09 • 1.510 3 Quercetinrhamnoside 0.46• 3 0.15• 3 Trace 0.01• 4 0.06• 4 0.05• 3 Kaempferol3-O-rhamnoside 0.12• 1.810 3 Trace nd nd nd Trace Cupressuflavone 2.02• 2 2.12• 2 2.55• 2 2.38• 2 2.38• 2 2.39• 2 Amentoflavone 0.64• 3 1.50• 2 1.72• 2 0.86• 3 0.18• 3 1.33• 2 Robustaflavone 0.23• 3 0.03• 4 0.05• 4 0.04• 4 0.07• 3 0.01• 4 Hinokiflavone 0.04• 8.910 4 nd nd nd 0.05 • 4 nd Methylrobustaflavone 0.42• 3 nd 0.16• 3 0.19• 3 0.17• 3 nd Methylamentoflavone 2.74• 3.210 2 0.01 • 1.910 4 0.00 0.13 • 3 nd 0.31 • 5.610 3 Dimethylcupressuflavone 0.76• 1.210 3 nd Trace 0.06• 1.110 3 nd nd

C F U N = Cupressusjunebris L.; CSEMP = Cupressus sempervirens L.; CGLAB = Cupressus glabra L.; CGOV = Cupressus goveniana L.; CARIZ = Cu- pressus arizonica L.; CLUSIT = Cupressus lusitanica L. n d = not detected.

a c c o u n t e d for 81.8% of t o t a l biflavonoids. A n o t h e r i m p o r t a n t c o m p o u n d is a m e n t o - flavone; a m o u n t s o f this c o m p o u n d were in the r a n g e 0.18 1 . 7 2 m g g 1 fresh weight. R o b u s t a f l a v o n e was < 4% of t o t a l biflavones, a n d h i n o k i f l a v o n e , present in

C. funebris a n d C. arizonica only, ac-

c o u n t e d for a p p r o x i m a t e l y 2% o f t o t a l biflavones.

These d a t a show t h a t C. funebris is dif-

ferent f r o m the o t h e r species analysed, because o f its h i g h e r m e t h y l a n d d i m e t h y l bi- f l a v o n o i d c o n t e n t in this species they ac- c o u n t for 51.37% o f t o t a l biflavones whereas the a m o u n t s in the o t h e r species v a r y f r o m 0.25% (C. sempervirens) to

10.3% (C. goveniana).

These d a t a show t h a t the a m o u n t s o f f l a v o n o i d s are very low the a m o u n t s o f these c o m p o u n d s in the samples a n a l y s e d v a r y f r o m 0 to 10.2%, w i t h the C. funebris

extract b e i n g richest in flavonols. The m o s t c o m m o n l y o c c u r r i n g f l a v o n o i d is quercitrin, except in C. lusitanica.

T h e m a i n p o l y p h e n o l subclasses present in cypress leaf extracts are s h o w n in F i g u r e 7. T h e d a t a reveal the peculiar q u a n t i t a t i v e c o m p o s i t i o n o f C. funebris.

This sample c o n t a i n s m o r e of all the poly- p h e n o l subclasses, in p a r t i c u l a r m e t h y l a n d d i m e t h y l derivatives, t h a n the o t h e r species, in w h i c h the m o s t a b u n d a n t com- p o u n d s are the biapigenins. F l a v o n o i d s are present as m i n o r a m o u n t s relative to the o t h e r sub-classes, a l t h o u g h the a m o u n t s p r e s e n t in C. funebris a n d C. sempervirens are quite high. Because bifla-

v o n o i d p a t t e r n s h a v e b e e n r e g a r d e d as va- luable characteristics in the t a x o n o m y o f some species [26], these findings m i g h t p r o v i d e a basis for the h y p o t h e s i s t h a t Cu- pressusfunebris belongs to the genus Cha- maecyparis r a t h e r t h a n the genus Cupres- sus, in a g r e e m e n t w i t h literature d a t a [28

3O].

The results o f o u r w o r k h a v e revealed significant differences between the bifla- v o n o i d c o n t e n t o f these species, for exam- ple the large a m o u n t s o f m e t h y l a n d di- m e t h y l derivatives in C. funebris (Asiatic

g r o u p ) a n d the presence o f h i n o k i f l a v o n e in C. funebris a n d C. arizonica only. The

b i f l a v o n o i d c o n t e n t of the A f r o m e d i t e r r a - n e a n g r o u p (C. sempervirens) a n d the

A m e r i c a n g r o u p ( C. goveniana, C. arizona ca, C. glabra, C. lusitanica) are n o t signifi-

cantly different.

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Received: Apr 22, 2002 Revised manuscript received: Jun 4, 2002 Accepted: Jun 24, 2002