flavonoids from the leaves of cyclanthera pedata: two new malonyl derivatives

210 P. MONTORO ET AL.

Copyright © 2005 John Wiley & Sons, Ltd. Phytochem. Anal. 16: 210–216 (2005)

PHYTOCHEMICAL ANALYSISPhytochem. Anal. 16, 210–216 (2005)Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002.pca.847

Copyright © 2005 John Wiley & Sons, Ltd.Received 5 August 2004

Accepted 20 January 2005

Flavonoids from the Leaves of Cyclantherapedata: Two New Malonyl Derivatives

Paola Montoro,1 Virginia Carbone2 and Cosimo Pizza1*1Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte don Melillo, 84084 Fisciano (SA), Italy2Centro di Spettrometria di Massa Proteomica e Biomolecolare, Istituto di Scienze dell’alimentazione—C.N.R., Via Roma 52a–c, 83100Avellino, Italy

Reversed-phase HPLC coupled with electrospray MS has been used for the simultaneous separation and deter-mination of flavonoid metabolites in leaves of Cyclanthera pedata, an edible Peruvian plant mainly used in SouthAmerica for its anti-inflammatory, hypoglycaemic and hypocholesterolaemic properties. The flavonoid contentof the leaves of C. pedata was compared qualitatively and quantitatively with that of the fruits. The isolationand structural characterisation by MS and NMR of two new minor components of the fruits, namely, 6-C-fucopyranosyl-(3-malonyl)-chrysin and 6-C-fucopyranosyl-(4-malonyl)-chrysin, are described. Copyright © 2005John Wiley & Sons, Ltd.Keywords: HPLC-MS; ESI/MS; flavonoids; Cyclanthera pedata.

INTRODUCTION

Fruits of Cyclanthera pedata Scrabs (Caigua), aPeruvian food plant belonging to the Cucurbitaceaefamily, are largely used in South America for their anti-inflammatory, hypoglycaemic and hypocholesterolaemicproperties. This latter activity is supported by clinicalstudies (Brank Egg, 1990) and, by virtue of this med-icinal use, the plant has a commercial interest in theherbal market of South America and, more recently, hasattracted attention in the European market.

In previous papers, the chemical compositions of thefruits and seeds of this plant have been investigated, andthe isolation and structural determination of cucurbitacinglycosides from the seeds, and triterpenoid saponins andflavone glycosides from the fruits, have been reported(De Tommasi et al., 1996, 1999; Montoro et al., 2001).Flavonoids are polyphenolic compounds with anti-oxidant properties (Cao et al., 1997; Rice-Evans et al.,1997), often correlated with hypocholesterolaemic act-ivity, occasioned by their ability to inhibit LDL oxida-tion (Viana et al., 1996; Naderi et al., 2003). The flavoneglycosides are the major constituents of the fruits of C.pedata and various have been isolated for the first timein this plant. For this reason, the flavone glycosides havebeen selected as marker compounds for the chemicalevaluation or standardisation of C. pedata fruits andcommercial preparations derived therefrom. A selective,specific and sensitive analytical HPLC-MS procedure forflavone glycosides from C. pedata was recently developedby Carbone et al. (2004). However, the leaves of C.pedata have yet to be studied with respect to theirphytochemical composition.

In the present paper, we report the qualitative andquantitative analysis of the flavonoids in leaves of C.pedata in comparison with the composition of the fruits.In addition we report the isolation and identification byMS and NMR of two new malonyl derivatives of fucosyl

chrysin that are present in the fruit as minor compounds,and the identification by MS of two new malonyl deriva-tives of fucosyl apigenin.

EXPERIMENTAL

Materials. Air dried and freeze-dried fruits and leavesof Cyclanthera pedata Scrabs were supplied by theUniversità di San Marco, Lima, Peru and were collectedin Peru in 1997. A voucher sample of the plant is depos-ited at the Herbario del Museo de Historia NaturalJ. Prado, Lima, Peru, and a further voucher sample isdeposited at the Dipartimento di Scienze Farmaceutiche,Università di Salerno, Salerno, Italy. Naringin, usedas internal standard, was purchased from Sigma (St.Louis, MO, USA). Pure standards of apigenin-6-C-fucopyranoside (1), chrysin-6-C-fucopyranoside (4) andisovitexin (7) were isolated as in a previous study(Montoro et al., 2002) and their structures elucidatedby NMR. Methanol (HPLC-grade), acetonitrile (HPLC-grade) and trifluoroacetic acid were purchased fromJ.T. Baker (Baker Mallinckrodt, Phillipsburg, NJ, USA).HPLC-grade water (18 mΩ) was prepared using aMillipore (Bedford, MA, USA) Milli-Q purificationsystem.

Instrumentation. A Bruker model DRX-600 spectro-meter (Spectroscopin, Fallanden, Switzerland), operat-ing at 599.19 MHz for 1H-NMR and at 150.858 MHz for13C-NMR, equipped with the UXNMR software pack-age, was employed for the NMR experiments. Sampleswere dissolved in deutero-methanol. 1H-13C heteronu-clear single quantum coherence (HSQC) (Bodenhausenet al., 1976; Bodenhausen and Ruben, 1986) andheteronuclear multiple bond correlation (HMBC)(Martin and Crouch, 1991) experiments were obtainedusing the conventional pulse sequences as describedin the literature.

Electrospray ionisation Mass spectrometry (ESI)/MSin the positive ion mode was performed using aFinnigan LCQ Deca ion trap mass spectrometer

* Correspondence to: C. Pizza, Dipartimento di Scienze Farmaceutiche,Università di Salerno, Via Ponte don Melillo, 84084 Fisciano (SA), Italy.Email: [email protected]

FLAVONOIDS FROM CYCLANTHERA PEDATA 211


(Thermo Finnigan, San Jose, CA, USA) equipped withXcalibur software. Samples were dissolved in methanoland infused in the ESI source using a syringe pump at aflow-rate of 3 µL/min: the capillary voltage was 5 V, thespray voltage was 5 kV, and the tube lens offset wasat 35 V; the capillary temperature was 220°C. Data wereacquired in the MS1 and MS/MS scanning modes.

HPLC-UV/PAD analyses were performed on anAgilent (Palo Alto, CA, USA) 1100 HPLC systemunder the chromatographic conditions described below.Extracts were analysed by HPLC-ESI/MS ‘on-line’using the mass spectrometer previously indicatedequipped with a Thermo Finnigan Spectra SystemHPLC. Flavonoids were separated using the chromato-graphic conditions reported below, and the eluate wasdirectly injected into the electrospray ion source underthe conditions previously described. Data were acquiredin the MS1 and MS/MS scanning modes and interpretedusing the software provided by the manufacturer.

Extraction of plant material and isolation of components.Freeze-dried leaves and fruits samples were powderedin a mortar whilst air-dried leaves and fruits were cut intosmall segments. For analytical purposes, a sample (1 g)of powdered air-dried leaves, freeze-dried leaves orfreeze-dried fruits, each containing naringin (2 mg) asinternal standard, was extracted at room temperaturewith 10 mL of methanol for 60 min with sonication. Theextraction mixture was then kept in the dark at roomtemperature overnight, after which the extract werefiltered and diluted 1:10 with methanol. An aliquot(20 µL) of each sample was injected in the Agilent HPLCsystem and separated on a Waters (Milford, MA, USA)Symmetry column (150 × 2.1 mm i.d.; 5 µm particle size).The mobile phase consisted of 0.05% trifluoroaceticacid in water (solvent A) and 0.05% trifluoroaceticacid in acetonitrile (solvent B). The elution programmewas 90:10 (A:B) held isocratically for 5 min, followedby a linear gradient to 60:40 in 40 min, and then to 35:65in 15 min: the flow rate was 0.3 mL/min.

For preparative purposes, a sample (100 g) of pow-dered leaves was defatted with 500 mL of n-hexane andthen extracted at room temperature with 500 mL ofmethanol for 60 min with sonication: the extractionmixture was then kept in the dark at room temperatureovernight. Fractionation of this extract was achieved byRP-HPLC on the Agilent HPLC system using a Watersµ-Bondapak C18 column (30 cm × 7.8 mm i.d.; 10 µmparticle size) with the gradient system described aboveand a flow rate of 2.5 mL/min. Pure samples of apigenin-6-C-fucopyranoside [1; retention time (Rt) 27.8 min],apigenin-6-C-fucopyranoside malonyl derivative (2; Rt

30.0 min), apigenin-6-C-fucopyranoside malonyl deriva-tive (3; Rt 32.1 min), chrysin-6-C-fucopyranoside (4; Rt

36.5 min), chrysin-4-malonyl-6-C-fucopyranoside (5; Rt

38.0 min), chrysin-3-malonyl-6-C-fucopyranoside (6; Rt

41.4 min) and a small amount of isovitexin (7; Rt

16.07 min) were obtained (Fig. 1).

Calibration, quantification and statistical analysis. Stand-ard curves for each of the flavonoid standards wereprepared over a concentration range of 5–125 µg/mLemploying four different concentration levels andtriplicate injections at each level. Peak area ratiosbetween the area of each flavonoid standard and that ofnaringin, used as internal standard, were calculated and

Figure 1. (A) Structures of the flavonoid glycosides fromCyclanthera pedata leaves (Glc = glucose, Fuc = fucose, Rha= rhamnose, Ma = malonyl); (B) structures of the newflavonoids 5 and 6 isolated from C. pedata leaves.

plotted against the corresponding standard concentrationusing weighted linear regression to generate standardcurves.

RESULTS AND DISCUSSION

HPLC-UV, HPLC-ESI/MS and HPLC-ESI/MS/MSanalyses of the flavonoids in Cyclanthera pedata leaves

For qualitative purposes a preliminary HPLC analysiswas carried out on methanolic extracts of air-dried leavesand freeze-dried leaves of Cyclanthera pedata (Fig. 2).Flavonoids from the leaf extracts were identified bycomparison of Rt values and UV spectra with thoseof the reference standards, and subsequently byHPLC-MS and HPLC-MS/MS analysis. Apigenin-6-C-fucopyranoside (1), chrysin-6-C-fucopyranoside (4) andisovitexin (7) were the major components of the air-driedleaves. In the freeze-dried leaves, compound 4 andits corresponding malonyl derivatives, chrysin-4-malonyl-6-C-fucopyranoside (5) and chrysin-3-malonyl-6-C-fucopyranoside (6), were the main constituents,together with 1, apigenin-6-C-fucopyranoside malonylderivative (2) and apigenin-6-C-fucopyranoside malonylderivative (3) in moderate amounts, and 7 as a minorcomponent.

The total ion current chromatogram of an extractof freeze dried leaves of C. pedata obtained frompositive ion electrospray HPLC-MS analysis in thefull-scan mode is shown in Fig. 3. Reconstructed ionchromatograms were obtained for each value of m/z



Figure 2. HPLC-UV chromatograms detected at 350 nm of extracts of (A) air-dried leaves of Cyclantherapedata; and (B) freeze-dried leaves of C. pedata. For the key to peak identity see Table 2. (For chromato-graphic protocol see the Experimental section.)

Figure 3. (A) HPLC-MS total ion current chromatogram of an extract of freeze dried leaves of Cyclanthera pedata;(B) HPLC-UV chromatogram detected at 350 nm of an extract of freeze dried leaves of C. pedata. For the key to peakidentity see Table 2. (For chromatographic protocol see the Experimental section.)



observed for the standard compounds in order toimprove the separation and the identification of thesingle flavonoids. Using HPLC-MS experiments, allof the flavonoids in the extract were identified with-out time-consuming pre-purification steps or previousoptimisation of the chromatographic procedure. HPLC-MS analysis of the leaf extract revealed the presenceof two compounds, 2 and 3, not previously detectedin the fruit of this species. HPLC-MS showed for boththe compounds a pseudo-molecular ion at an m/z of 503.In order to characterise these compounds further, anHPLC-ESI/MS/MS product ion scan experiment wasperformed. The m/z values selected as precursor ionswere the following: m/z 503, in order to differentiatebetween compounds 2 and 3; and m/z 487, correspond-ing to fucosyl chrysin malonyl derivatives, 5 and 6,characterised by NMR data (see below) in order to com-pare the fragmentation patterns of these compoundswith those of compound 2 and 3.

The chromatographic profile obtained from theHPLC-ESI/MS/MS experiment for the [M+H]+ ion atm/z 487.0 revealed the presence of two major peaks atdifferent Rt values (see Fig. 4). Analysis of the tandemMS of the two different species, in the product ion

scan experiment, showed the same fragment ions but atdifferent intensities. In particular, the compound at Rt

35.9 min (compound 5) showed an initial loss of one (m/z 468.9) and two (m/z 450.9) molecules of water and aloss of malonic acid corresponding to the loss of a 104 Daneutral fragment. The fragment ion at m/z 468.9 wasmore intense than that at m/z 450.9, and the most intensefragment ion was that corresponding to the loss ofthe neutral malonyl unit subsequent to the loss of onemolecule of water (m/z 365.0). Compound 5, charac-terised fully by NMR data (see below), was esterifiedwith a malonyl moiety in position 3 of the sugar unit. Thecompound at Rt 39.3 min (compound 6) showed the sameinitial loss of one and two molecules of water (at m/zvalues of 468.9 and 450.9, respectively) and a subsequentloss of malonic acid corresponding to the loss of a 104Da fragment (m/z 365.0). The fragment ion at m/z 468.9was less intense than that at m/z 450.9, whilst the mostintense fragment ion was observed at m/z 347.1 corres-ponding to the loss of the malonyl neutral unit sub-sequent to the loss of two molecules of water. Compound6, characterised fully by NMR data (see below), wasesterified with a malonyl moiety in position 4 of the sugarunit.

Figure 4. HPLC-MS/MS analysis of an extract of freeze dried leaves of Cyclanthera pedata leaves showing: (A) thereconstructed ion chromatogram of the profile for m/z 487.0; (B) the reconstructed ion chromatogram of the profilefor m/z 503.0; and (C) the MS/MS spectra of compounds 2, 3, 5 and 6.



Although the loss of the first molecule of water thatoccurs during the fragmentation of C-glycosyl flavonoidsis well detailed in the literature (Li et al., 1992) andappears to involve the OH group at position 2 of thesugar moiety, the rearrangement and proton transfermechanism giving rise to the second loss of water isuncertain. Nevertheless, the evidence that compound 5,esterified in position 3 of the sugar unit, is more reluct-ant to release the second water molecule leads us tosuggest an involvement of the hydroxyl in position 3 ofthe sugar moiety in the rearrangement mechanism thatgives rise to the loss of the second water molecule.

The fragmentation data related to the knownesterification positions, confirmed by NMR data, helpedus to hypothesise the structures of the other two com-pounds present in small amounts. The chromatographicprofile obtained from the HPLC-ESI/MS experimentfor the [M+H]+ ion at m/z 503.0, revealed the presence

of two peaks (Rt values 28.4 and 30.12 min). Analysis ofthe tandem MS of the two species in the simultaneousHPLC-ESI/MS/MS experiment showed for the com-pound (2) at the shorter Rt a behaviour similar to that of5, whereas the compound (3) at the longer Rt presenteda fragmentation pattern similar to that of 6. From thisevidence we suppose that compounds 2 and 3 are themalonyl derivatives of fucosyl apigenin, with malonylresidues being substituted, respectively, at positions 3and 4 of the fucosyl moiety.

Identification of flavonoids from the leaves of C. pedata

Compound 5: C24H22O11; obtained as a yellow amorphouspowder; UV (MeOH) λmax, 330, 275 and 210 nm; ESI/MS, m/z 487 [M+H]+, 509 [M+Na]+, and 365 [M+H-122]+;1H- and 13C-NMR data, see Table 1.

Table 1. 1H- and 13C-NMR data for chrysin-6-C-fucopyranoside (4), chrysin-4-malonyl-6-C-fucopyranoside (5) and chrysin-3-malonyl-6-C-fucopyranoside (6)

Compound

4 5 6

1H-NMR data (δ)3 6.85 s 6.85 s 6.85 s8 7.09 s 7.09 s 7.09 s2′ 8.06 br d (8.0) 8.06 br d (8.0) 8.06 br d (8.0)3′ 7.61 m 7.61 m 7.61 m4′ 7.59 m 7.59 m 7.59 m5′ 7.61 m 7.61 m 7.61 m6′ 8.06 br d (8.0) 8.06 br d (8.0) 8.06 br d (8.0)Fucose1 4.88 d (8.0) 5.03 d (8.0) 4.94 d (8.0)2 4.65 br t (9.0) 4.50 br t (9.0) 4.24 br t (9.0)3 3.57 dd (9.0; 3.5) 4.94 dd (9.0; 3.5) 3.81 dd (9.0; 3.5)4 3.75 m 4.03 m 5.32 m5 3.81 m 3.93 m 3.97 m6 1.28 d(6.5) 1.22 d 1.26 d(6.5)CH2 (malonyl) — 3.39 s 3.39 s13C-NMR data (δ)2 166.2 166.2 166.23 107.0 107.0 107.04 185.0 185.0 185.05 162.1 162.1 162.16 111.9 111.9 111.97 164.5 164.5 164.58 96.5 96.5 96.59 158.7 158.7 158.710 107.0 107.0 107.01′ 132.0 132.0 132.02′ 128.2 128.2 128.23′ 133.4 133.4 133.44′ 131.0 131.0 131.05′ 133.4 133.4 133.46′ 128.2 128.2 128.2Fucose1 76.12 75.3 75.82 71.1 68.0 70.03 77.0 80.0 74.94 74.1 70.4 76.45 76.7 75.7 75.36 17.6 17.3 16.7CH2 (malonyl) — 47.2 47.2COOH — 170.3 170.3COO- — 168.7 168.7



Compound 6: C24H22O11; obtained as a yellow amor-phous powder; UV (MeOH) λmax, 330, 275 and 210 nm;ESI/MS, m/z 487 [M+H]+, 509 [M+Na]+, and 347 [M+H-140]+; 1H- and 13C-NMR data, see Table 1.

The molecular formula of C24H22O11 for 5 was obtainedby ESI/MS and 13C-NMR data. MS, 13C-NMR and 13C-DEPT NMR analyses indicated the flavonoid nature ofthe compound and, in particular, 15 carbon atoms ascrib-able to the aglycone, six to the sugar moiety and threeto an esterifying residue. Comparison of the 1H-NMRspectrum of 5 with that of 4 (6-C-fucopyranosyl-chrysin)confirmed that the sugar moiety was a fucose unitesterified at position 3. In fact, the proton H-3fuc wasshifted downfield at δ 4.94 in comparison with the valueof 3.57 observed for the non-esterified compound. Thechemical shift of C-3fuc was observed at δ 80.0, comparedwith δ 77.0 in 4; up-field γ effects on C-2fuc and C-4fuc werealso observed (Table 1). In addition a singlet centred atδ 3.39 corresponding to two protons was attributed to theCH2 of a malonyl moiety.

An HSQC experiment correlated all proton reson-ances in 5 with those of the corresponding carbon atoms(Table 1). The HSQC experiment confirmed the pres-ence of a malonyl residue, by observing the signals dueto the protons of CH2 occurring at δ 3.39, correlated tothe carbon signal centred at δ 47.2. An HMBC long-range proton–carbon experiment showed correlationbetween one of the carboxyl groups of the malonylresidue and the proton H-3 of fucose unit. Compound 5was thus assigned the structure of 6-C-fucopyranosyl-(3-malonyl)-chrysin.

The molecular formula of C24H22O11 for 6 was obtainedby ESI/MS and 13C-NMR data. MS, 13C-NMR and 13C-DEPT NMR analyses indicated the flavonoid nature ofthe compound and, in particular, 15 carbon atoms ascrib-able to the aglycone, six to the sugar moiety and three toan esterifying residue. Comparison of 1H-NMR spectrumof 6 with that of 4 confirmed that the sugar moiety was afucose unit esterified at position 4. The proton H-4fuc wasshifted downfield at δ 5.32 in comparison with the valueof 3.75 observed for the non-esterified compound. Thechemical shift of C-4fuc was δ 76.4 instead of δ 74.1 asobserved for 4; γ effects on C-2fuc and C-4fuc were also

observed (Table 1). In addition a singlet centred at δ 3.39corresponding to two protons was attributed to the CH2

of a malonyl moiety.An HSQC experiment correlated all proton reson-

ances in 6 with those of the corresponding carbon atoms(Table 1). The HSQC experiment confirmed the pres-ence of a malonyl residue, by observing the signals dueto the protons of CH2 occurring at δ 3.39, correlated toa signal of a carbon centred at δ 47.2. An HMBC long-range proton–carbon experiment showed correlationbetween one of the carboxyl groups of the malonylresidue and proton H-4 of fucose unit. Compound 6 wasthus assigned the structure of 6-C-fucopyranosyl-(4-malonyl)-chrysin.

Quantitative analysis

Quantitative determinations of the flavonoid content offreeze-dried fruit and freeze-dried leaves of C. pedatawere performed by HPLC-MS. The reconstructed ionchromatograms were used to improve the componentseparation for each compound in the mixture. The cali-bration graphs were obtained by plotting the area ratiobetween the external and internal standard versus theknown concentration of each compound in the range 5–125 µg/mL for flavonoids 1 and 4–7. Quantitation of 2and 3 was realised using the calibration graph of 1 since2 and 3 are derivatives of this compound. Five aliquotsof the crude extract of C. pedata fruits were also analysedin order to quantify the flavonoid content.

The results of the quantitative analyses (Table 2)showed that in the freeze dried leaves compounds 4,5 and 6 (i.e. fucosyl-chrysin and its derivatives withmalonic acid) were the major components, whilst inthe freeze dried fruits 4 and 8 were the most abund-ant. This evidence is particularly important becausethese compounds are characteristic for the species.Chrysin-7-O-glucopyranosyl-6-C-fucopyranoside (8),chrysin-6-C-glucopyranoside (9), and chrysin-7-O-glucopyranosyl-(1-4)-l-rhamnopyranoside (10) werepresent in moderate amounts in the fruit but wereabsent in the leaves.

Table 2. Quantitative comparison of the flavonoid content of extracts of freeze dried fruit and freeze dried leaves of Cyclantherapedata

Compound Freeze-dried fruit extracta Freeze-dried leaf extracta

1 Apigenin-6-C-fucopyranoside 0.542 ± 0.06 0.7952 Apigenin-6-C-fucopyranoside malonyl derivative n.d.b 0.1223 Apigenin-6-C-fucopyranoside malonyl derivative n.d. 0.1854 Chrysin-6-C-fucopyranoside 1.132 ± 0.08 1.0955 Chrysin-4-malonyl-6-C-fucopyranoside 0.981 ± 0.03 1.2016 Chrysin-3-malonyl-6-C-fucopyranoside 1.132 ± 0..05 1.1567 Isovitexin 0.662 ± 0.05 0.2108 Chrysin-7-O-glucopyranosyl-6-C-fucopyranoside 1.894 ± 0.02 09 Chrysin-6-C-glucopyranoside 0.411 ± 0.05 0

10 Chrysin-7-O-glucopyranosyl-(1-4)-L-rhamnopyranoside 0.210 ± 0.01 0

a Mean values ± standard error (n = 3); mg/g of dried plant material.b n.d., none detectable.



REFERENCES

Bodenhausen E, Ruben DJ. 1986. Unexpected cross peaksin two-dimensional homonuclear-relayed coherencetransfer spectroscopy of a peptide. J Magn Reson 69:185–186.

Bodenhausen E, Freeman R, Turner DL. 1976. Two-dimensional spectroscopy proton coupled carbon-13NMR. J Chem Phys 65: 839–840.

Brank Egg A. 1990. Dicionario Enciclopedico de Plantas utilesdel Perú. CBC: Cuzco; 171–172.

Cao G, Sofic E, Prior RL. 1997. Antioxidant and prooxidantbehavior of flavonoids: structure-activity relationships.Free Rad Biol Med 22: 749–760.

Carbone V, Montoro P, de Tommasi N, Pizza C. 2004.Analysis of flavonoids from Cyclanthera pedata byliquid chromatography/electrospray mass spectrometry.J Pharm Biomed Anal 34: 295–304.

De Tommasi N, De Simone F, Pizza C. 1996. Studies onthe constituents of Cyclanthera pedata (Caigua) seeds:Isolation and characterization of six new cucurbitacinglycosides. J Agric Food Chem 44: 2020–2025.

De Tommasi N, De Simone F, Speranza G, Pizza C. 1999.Studies on the constituents of Cyclanthera pedatafruits: isolation and structure elucidation of new

triterpenoid saponins. J Agric Food Chem 47: 4512–4519.

Li QM, Van de Heuvel H, Dillen L, Claeys M. 1992. Differentia-tion of 6-C and 8-C glycosidic flavonoids by positive ionfast atom bombardment and tandem mass spectrometryBiol Mass Spectrom 21: 213–221.

Martin GE, Crouch RC. 1991. Inverse detected two dimen-sional NMR methods: application in natural productschemistry. J Nat Prod 54: 1–70.

Montoro P, Carbone V, De Simone F, Pizza C, De Tommasi N.2001. Studies on the constituents of Cyclanthera pedatafruits: isolation and structure elucidation of new flavonoidglycosides and their antioxidant activity. J Agric FoodChem 49: 5156–5160.

Naderi GA, Asgary S, Sarraf-Zadegan N, Shirvany H. 2003.Anti-oxidant effect of flavonoids on the susceptibility ofLDL oxidation Mol Cell Biochem 246: 193–196.

Rice-Evans CA, Miller NJ, Paganga G. 1997. Antioxidantproperties of phenolic compounds Trends Plant Sci 2:152–159.

Viana M, Barbas C, Bonet B, Bonet MV, Castro M, Fraile MV.1996. In vitro effects of a flavonoid-rich extract on LDLoxidation Atherosclerosis 123: 83–91.

flavonoids from the leaves of cyclanthera pedata: two new malonyl derivatives

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