Research Article

Received: 3 February 2011, Revised: 22 June 2011, Accepted: 27 June 2011 Published online in Wiley Online Library: 21 August 2011

(wileyonlinelibrary.com) DOI 10.1002/pca.1348

Analysis of C-glycosyl Flavonoids from South

American Passiflora Species by HPLC-DAD and
HPLC-MS
Silvana Maria Zucolotto,a Carize Fagundes,a Flávio Henrique Reginatto,a
Freddy A. Ramos,b Leonardo Castellanos,b Carmenza Duqueb and
Eloir Paulo Schenkela*
ABSTRACT:
Introduction – Leaves and fruits of Passiflora species are widely used around the world in popular medicine, mainly as seda-
tives and tranquilisers. C-glycosyl flavonoids are the main components of these species.
Objective – To investigate the constituent patterns and to develop a chromatographic method for the characterisation of the
C-glycosyl flavonoids profile of the extracts of the leaves and the pericarp of South American Passiflora species.
Methodology – The chemical composition of extracts from the leaves and the fruits’ pericarp of Passiflora edulis var. flavicarpa,
P. edulis var. edulis, Passiflora alata, Passiflora tripartita var. mollissima, Passiflora quadrangularis, Passiflora manicata and
Passiflora ligularis was evaluated for the presence of C-glycosyl flavonoids. Two separate HPLC methods were developed
suitable for a diode array detector (DAD) and a MS detector. Separation by HPLC-DAD was achieved on a Luna C-18 column,
using solvent A (tetrahydrofuran–isopropanol–acetonitrile) and solvent B (H3PO4 0.5%) in an isocratic elution mode. In the
HPLC-MS, the components were separated on a Luna RP-18A column by a gradient elution (water–acetonitrile–formic acid).
Results – The presence of C-glycosyl flavonoids was identified in leaves and pericarp of P. edulis var. flavicarpa, P. alata, P.
edulis var. edulis and P. tripartita var. molissima, but only in leaf extracts of P. quadrangularis and P. manicata and not at
all in P. ligularis. The different species and varieties showed different major constituents. The C-glycosyl flavonoids identified
more frequently were orientin, isoorientin, vitexin and isovitexin.
Conclusion – The methods established are simple and can be used as a tool for the characterisation and quality control of
pharmaceutical preparations containing these Passiflora extracts. Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: Chemical markers; flavonoids; Passiflora

Introduction Coleta et al., 2006; Zucolotto et al., 2009), saponins (Reginatto

et al., 2001) and alkaloids (Lutomski and Malek, 1975). Specifi-
The genus Passiflora, comprising around 500 species, is the most
cally in relation to C-glycosyl flavonoids, the presence of api-
important genus of the Passifloraceae family and is widely distributed
genin and luteolin glycosydes derivatives, such as orientin,
throughout Latin America (Sacco, 1980). In North America and
isoorient, vitexin and isovitexin, are commonly reported as the
Europe these species are popularly known as passion fruit or passion
major compounds (Abourashed et al., 2002; Dhawan et al.,
flower, but in South America they have different local names
2004). In addition, recent research has associated several phar-
(Table 1). Additionally, the leaves of several species are tradition-
macological effects with the presence of C-glycosyl flavonoids
ally used in many countries as sedative or tranquilliser and in treat-
(De-Paris et al., 2002; Coleta et al., 2006; Santos et al., 2006; Sena
ment for inflammatory problems (Pio Corrêa, 1978; Sacco, 1980).
et al., 2009; Zucolotto et al., 2009), and analysis of these constitu-
In Brazil, Passiflora alata and Passiflora edulis var. flavicarpa are
ents is proposed for the authentification of a sample or to char-
included in the Brazilian Pharmacopoeia (Farmacopeia Brasileira,
acterise closely related species (Abourashed et al., 2002).
1977; ANVISA, 2010). In addition, leaf extracts of these species
Considering the extensive utilisation of Passiflora species in
are included as an active component in many Brazilian regis-
the phytopharmaceutical industry it is necessary to develop
tered pharmaceutical preparations. In Colombia, Passiflora
tripartita var. mollissima, Passiflora edulis var. edulis, Passiflora
quadrangularis and Passiflora ligularis are widely used for juice
preparation. Further, extracts of the leaves of Passiflora tripartita * Correspondence to: E. P. Schenkel, Programa de Pós-Graduação em Farm-
ácia, Universidade Federal de Santa Catarina, Campus Universitário, Trin-
var. mollissima are accepted as sedative and hypnotic compo- dade, Florianópolis, SC, 88.049-900, Brasil. Email: eloirschenkel@gmail.com
nents in phytopharmaceutical preparations by the Colombian
a
Agency for the Control of Foods and Drugs (Invima, 2005). Programa de Pós-Graduação em Farmácia, Universidade Federal de Santa
The leaf extracts of P. incarnata, P. edulis and P. alata have Catarina, Campus Universitário, Trindade, Florianópolis, SC 88.049-900,
Brasil
been investigated most extensively. Studies have revealed the
presence of C-glycosyl flavonoids (Mareck et al., 1991; b
Departamento de Química, Universidad Nacional de Colombia, Carrera 30
Raffaelli et al., 1997; Abourashed et al., 2002; Muller et al., 2005;
232

número 45-03, Bogotá, Colombia

Phytochem. Anal. 2012, 23, 232–239 Copyright © 2011 John Wiley & Sons, Ltd.
Phytochem. Anal. 2012, 23, 232–239
Table 1. Passiflora species selected for the study, local names, parts of the plant and origin of the material investigated
Species
P. edulis Sims var. flavicarpa
Degener
P. edulis Sims var. edulis
P. alata Curtis
P. tripartita var. mollissima
Holm-Nielsen
& Møller Jørgensen
P. quadrangularis Linneaus
P. ligularis Juss.
Copyright © 2011 John Wiley & Sons, Ltd.
P. manicata Juss.
wileyonlinelibrary.com/journal/pca
233
Local name
Maracujá-amarelo (Brazil)
Maracuyá (Colombia)
Maracujá-roxo (Brazil)
Gulupa (Colombia)
Maracujá-doce
Curuba de castilla
Badea
Granadilla
Curuba de monte, diablito
Parts of the plant Location Coordinates Identification
Leaves and pericarp Antonio Carlos/Santa 27
310 0000 S Dr Daniel de Barcellos Falkenberg/ Herbarium
Catarina, Brasil 48
460 0300 W of Universidade Federal de Santa Catarina
(FLOR 33886)
Leaves and pericarp Santa Sofia/Boyacá, 05
430 0100 N Prof. Luis Carlos Jimenez/ Instituto Nacional de
Colombia 73
360 2000 W Ciencias, – Universidad Nacional de Colombia
(COL 530661)
Leaves and pericarp Nova Santa Rita/Rio 29
510 2500 S Dr Branca Severo/ Universidade de Passo Fundo
Analysis of C-glycosyl Flavonoids from South American Passifloras
Grande do Sul, Brasil 51
160 2600 W (RSPF 7232)
Leaves and pericarp Santa Sofía/Boyacá, 05
430 0100 N Prof. Luis Carlos Jimenez/ Instituto Nacional de
Colombia 73
360 2000 W Ciencias, – Universidad Nacional de Colombia
(COL 530663)
Leaves and pericarp Neiva/Huila, Colombia 2
590 5500 N Prof. Luis Carlos Jimenez/ Instituto Nacional de
75
180 1600 W Ciencias, – Universidad Nacional de Colombia
Leaves and pericarp Bogotá/Cundinamarca, 4
350 6000 N Prof. Luis Carlos Jimenez/ Instituto Nacional de
Colombia 74
40 6000 W Ciencias, – Universidad Nacional de Colombia
(COL 530660)
Leaves Villa de Leyva/Boyacá, 5
380 000 N Prof. Luis Carlos Jimenez/ Instituto Nacional de
Colombia 73
320 000 W Ciencias, – Universidad Nacional de Colombia
(COL 530662)
 S. M. Zucolotto et al.

methodologies to ensure the quality control of these extracts. In
this sense, the development of chromatographic identification
methods based on the chemical constituents may therefore con-
tribute significantly to the standardisation of the crude drugs. In
the present work we have therefore investigated the C-glycosyl
flavonoids fingerprint of aqueous extracts from the leaves and
pericarp of two species widely used in Brazil and of five species
from the Andean region of Colombia (Table 1), using HPLC-DAD
(diode array detector) and HPLC-MS.
Experimental
Chemicals and reagents
Acetonitrile, formic acid, tetrahydrofuran and isopropanol HPLC
grade were purchased from TediaW. The LC-MS solvents were
purchased from MerckW. Other reagents of analytical grade were
purchased from NuclearW. The reference standards orientin (≥ 98%)
and isoorientin (≥ 98%) were purchased from ExtrasynthèseW and
vitexin (≥ 96%) and isovitexin (≥ 98%) from FlukaW. The compound
6,8-di-C-glycosylchrysin isolated from Lychnophora ericoides
leaves was provided by Dr Norberto Peporine Lopes. Spinosin
and swertisin were obtained from Wilbrandea ebracteata roots
(Santos et al., 1996). Vicenin-2 was isolated from P. edulis var.
flavicarpa leaves, identified by NMR spectral data and its purity
was confirmed by the HPLC-DAD (Zucolotto et al., 2009).
Plant material
The Passiflora species selected for the study are listed in
Table 1 together with the parts of the plants used and their
geographical location.
Preparation of extracts and samples
The leaves of all species were air-dried at 40
C for 7days in an
airflow oven, powdered and extracted using hot water (90
C)
by infusion (plant:water, 1:10, w/v) for 10min. After cooling, the
aqueous extracts were filtered using WHATMANW Grade 1 qual-
ity filter paper, concentrated under reduced pressure to 500mL
and partitioned with ethyl acetate (3300mL) and n-butanol
(3300mL), yielding the ethyl acetate and butanol fractions
for the leaves.
The fruits were purchased at a mature stage. The pulp (edible
part) was removed and the pericarp cut into small parts and
extracted by infusion (90
C – plant:water, 1:3, w/v) for 10min, during
which the infusion was occasionally stirred. Thereafter, the extract
was filtered as described above, concentrated under reduced pres-
sure to 500mL and partitioned with n-butanol (3300mL), yield-
ing the butanol fractions for the pericarps.
For HPLC analysis, each extract was previously filtered through
a 0.45mm membrane and 10mL of each sample (extracts=1mg/
mL and reference standards=100mg/mL) was injected into the
HPLC.
HPLC-DAD
The HPLC-DAD analysis were carried out using the Perkin Elmer
(Series 200 High-Performance Liquid Chromatography – HPLC)
system, equipped with an EP Diode Array Detector (DAD), quater-
nary pump, on-line degasser and automatic sampler. The chro-
matographic analysis were performed using a Luna C-18 column
(1504.6mm, 5mm). The isocratic mobile phase consisted of
solvent A (tetrahydrofuran:isopropanol:acetonitrile, 10:2:3, v/v/v)
and solvent B (H3PO4 0.5%]) (12% A in B). The detection wave-
length and the flow rate were 340nm and 1.0mL/min, respectively.

Table 2. Chemical structure, retention time (tR) and molecular weight of C-glycosyl flavonoids used as chemical markers

C-glycosyl flavonoid tR (min) Molecular weight [M-H] m/z Aglycone R1 R2 R3 R4 R5

Vicenin-2 (1) 6.5 594 593 Apigenin Glu Glu H OH H
6,8-di-C-glycosylchrysin (2) 8.0 578 577 Chrysin Glu Glu H H H
Spinosin (3) 9.2 608 607 Apigenin Glu-O-glu (1!2) H H OH CH3
Isoorientin (4) 16.5 448 447 Luteolin Glu H OH OH H
Swertisin (5) 19.7 446 445 Apigenin Glu H H OH CH3
Isovitexin (6) 26.0 432 431 Apigenin Glu H H OH H
Vitexin-2’’-O-rhamnoside (7) 27.0 578 577 Apigenin H Glu-O-rham H OH H
Orientin (8) 27.0 448 447 Luteolin H Glu OH OH H
Vitexin (9) 38.0 432 431 Apigenin H Glu H OH H
Glu = glucose; rham = rhamnose.
234

wileyonlinelibrary.com/journal/pca Copyright © 2011 John Wiley & Sons, Ltd. Phytochem. Anal. 2012, 23, 232–239
Analysis of C-glycosyl Flavonoids from South American Passifloras

HPLC-MS
The analysis were performed on a Shimadzu LC-10A coupled to a
selective mass detector (LCMS-2010EV). A Phenomenex Luna
RP-18 (2) column (1502mm, 5mm) was used, and the com-
pounds were separated using a mobile phase consisting of A
(acetonitrile:water:formic acid, 3:87:10, v/v/v) and B (acetonitrile:
water:formic acid, 40:50:10, v/v/v) in a gradient elution (0–30min –
90% A until 50% A; 30–35min – isocratic 50% A). The UV detection
was at 340nm. Electrospray ion source (ESI)-MS spectra were
acquired in both positive and negative ion modes, and the
interface and MS detector parameters were as follows: detec-
tor voltage, 1.5kV; curved desolvation line (CDL) temperature, 300
C
and 150V. For full scan MS analysis, the spectra were recorded in
the range of m/z100–1000, using nitrogen as the nebulising gas
(1L/min). In all HPLC-DAD and HPLC-MS analyses the following

AU AU 272
0.01 0.01
m/z = 593 271 334
mAU
317
65.00
A 1
0.005 0.005
60.00
m/z = 447
55.00
4 0.0 1 0.0 2
50.00
200 300 400 200 300 400
45.00
40.00 m/z = 607
AU
35.00 2 0.015
m/z = 431 349
269
30.00 6 256

25.00 0.0070
m/z = 577
20.00 3 m/z = 447
8 m/z = 431 0.0 4
15.00
9 200 300 400
10.00

0 5 10 15 20 25 30 35 40 45 50 55
Time (min)
mAU
65.00 B
60.00
m/z = 607
55.00
50.00
45.00 m/z = 577
40.00
35.00
30.00 m/z = 593 4
25.00 1
m/z = 447
20.00 2 3
15.00
10.00
5.00
0.00
0 5 10 15 20 25 30 35 40 45 50 55
Time (min)
mAU
65.00 C
60.00
55.00
50.00
45.00
40.00
35.00 4
30.00
m/z = 447
25.00
20.00
15.00
10.00
5.00
0.00
0 5 10 15 20 25 30 35 40 45 50 55
Time (min)

Figure 1. Chromatograms of extracts from P. edulis var. flavicarpa: (a) leaves, (b) pericarp (sample from Brazil) and (c) pericarp (sample from Colombia),
with diode array detection at 340nm. For chromatographic conditions, see ‘Experimental’ section. Pseudomolecular ions (M  H) obtained by LC-ESI-
MS are indicated for the labelled peaks.
235

Phytochem. Anal. 2012, 23, 232–239 Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/pca
 S. M. Zucolotto et al.

AU
0.04

351
mAU 257 267
19,00 0.02

18,00 A
17,00
0.0
16,00
200 300 400
15,00
14,00
13,00
12,00
11,00
10,00
9,00
8,00
7,00
6,00
5,00
0 5 10 15 20 25 30 35 40 45 50 55 Time (min)

mAU B
28,00
26,00
24,00
22,00
20,00
18,00
16,00
14,00
12,00
10,00
8,00
6,00
4,00
2,00
0,00
0 5 10 15 20 25 30 35 40 45 50 55 Time (min)

Figure 2. Chromatograms of extracts from P. edulis var. edulis: (a) leaves and (b) pericarp with diode array detection at 340nm. For chromatographic
conditions, see ‘Experimental’ section.

authentic samples were used: vicenin-2, spinosin, 6,8-di-C-
glycosylchrysin, swertisin, orientin, isoorientin, vitexin, isovitexin
and vitexin-2’’-O-rhamnoside (Table 2). The analysis were
performed in triplicate.
Results and Discussion
In order to select the appropriate HPLC conditions to analyse the
samples, relevant literature was consulted, and several systems
for C-glycosyl flavonoids analysis in Passiflora species were
found (Rehwald et al., 1994; Raffaelli et al., 1997, Abourashed
et al., 2002; Muller et al., 2005; Pereira et al., 2005). Specifically
related to the analysis of juice fruit composition, only one
work was appropriate (Mareck et al., 1991). The analytical
HPLC-DAD conditions (method I) used in this work were similar
to those proposed by Rehwald et al. (1994), with minor
modifications.
Nine C-glycosyl flavonoids (1–9) were used as authentic sam-
ples and their retention times are shown in Table 2. As an addi-
tional criterion to confirm the identity of the compounds, a
second experimental design was utilised using ESI-MS (method
II). The negative ion mode provided better sensitivity and the in-
terpretation of the spectra was found to be easier, as every
236
component gave a single ion, the [M  H] quasi-molecular ion,
under these conditions. The m/z value of the C-glycosyl flavo-
noids 1–9 are presented in Table 2 and in Figs. 1–3. In contrast
to O-glycosyl flavonoids, the fragmentation of the sugar moiety
could not be observed in our analysis.
On the basis of the comparison of UV spectra, retention time,
mass spectra and co-injection of the sample with standards, it
was possible to identify some C-glycosyl flavonoids in P. edulis
var. flavicarpa, P. edulis var. edulis, P. alata, P. tripartita var.
mollissima, P. quadrangularis and P. manicata.
The C-glycosyl flavonoids identified in the butanol fractions
from all investigated Passiflora species are shown in Table 3. In
P. edulis var. flavicarpa leaves (Fig. 1A), the UV spectra of the ma-
jor compounds (peaks 1 and 4), showed the typical UV absorp-
tion of apigenin and luteolin derivatives, respectively. The
identities of the above compounds were confirmed by the ESI-
MS data analysis and the major compounds were identified as
vicenin-2 (peak 1) and isoorientin (peak 4), followed by isovi-
texin (peak 6), orientin (peak 8), vitexin (peak 9), spinosin (peak
3) and 6,8-di-C-glycosylchrysin (peak 2). The butanol fraction
from the pericarp of P. edulis var. flavicarpa showed a similar
chromatographic profile to that of the leaves with isoorientin
as the major compound (Fig. 1B). These compounds have been

wileyonlinelibrary.com/journal/pca Copyright © 2011 John Wiley & Sons, Ltd. Phytochem. Anal. 2012, 23, 232–239
Analysis of C-glycosyl Flavonoids from South American Passifloras

AU
0.05

mAU 349
255 267
65,00 A 0.025

60,00 m/z= 445

55,00
8
0.0
50,00 200 300 400

45,00 I AU
II
40,00 0.05
350
35,00 m/z= 593 1 m/z= 447 m/z= 447 255 269
30,00
4 8 0.025
5 m/z= 431
25,00 9 4
20,00 0.0
200 300 400
15,00 6
10,00
5,00 m/z= 431
0,00
0 5 10 15 20 25 30 35 40 45 50 55
Time (min)
mAU
B AU
70,00 349
0.03
268
257
60,00
0.015
50,00
4
0.0
40,00 m/z= 447
200 300 400
4
30,00 m/z= 447
8
20,00
6
10,00

m/z= 431
0,00
0 5 10 15 20 25 30 35 40 45 50 55
Time (min)

Figure 3. Chromatograms of extracts from P. tripartita var. mollissima: (a) leaves and (b) pericarp with diode array detection at 340nm. For chromato-
graphic conditions, see ‘Experimental’ section.

Table 3. C-glycosyl flavonoids identified in South American Passiflora species

Species Part of Vicenin-2 6,8-di-c Spinosin Swertisin Vitexin-2’’-O- Isoorientin Orientin Isovitexin Vitexin
plant glycosylchrysin rhamnoside
P. edulis var. Leaves ++ + +b – – ++ + + +
flavicarpa Pericarp + + +b – – ++ – – –
P. edulis var. edulis Leaves – – – – – – – –
Pericarp – – – – – – – – –
P. alata Leaves – – – – ++ + + + –
Pericarp – – – – + – – – –
P. tripartita var. Leaves + – – +b – + + + +
mollissima Pericarp – – – – – + + + –
P. quadrangularis Leaves – – – – + – – – –
Pericarp – – – – – – – – –
P. ligularis Leaves – – – – – – – – –
Pericarp – – – – – – – – –
P. manicataa Leaves – – – – + + + ++ +
a
This species does not have edible fruits.
b
Compound identified by co-injection, but without confirmation by MS.
+, identified compound; ++, major compound; – compound could not be identified.
237

Phytochem. Anal. 2012, 23, 232–239 Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/pca
 S. M. Zucolotto et al.

described previously by our group in the leaf (Zucolotto et al.,
2009) and pericarp (Sena et al., 2009) extracts of P. edulis var.
flavicarpa. It should be noted that P. edulis var. flavicarpa peri-
carp extracts obtained from two different sources (in Brazil and
in Colombia) were compared and both extracts showed
isoorientin as the major compound (Fig. 1B and C).
One important observation is that the HPLC C-glycosyl flavo-
noid profile of the leaves of P. edulis Sims var. edulis (Fig. 2A)
and P. edulis var. flavicarpa (Fig. 1A) were markedly different,
while the extracts of leaves and pericarp of P. edulis var. edulis
(Fig. 2A and B, respectively) showed great similarity. The main
compound from the leaves could not be identified (Fig. 2A, tR =
25.1min), but it showed UV absorption similar to that of luteolin
derivatives. It is difficult to compare our results concerning
the flavonoids composition of the leaves of var. edulis, since
some previous reports could not distinguish which P. edulis
variety was used. Specifically regarding the pericarp, it was
previously reported that luteolin and luteolin-6-C-glucoside
(Ichimura et al., 2006), quercetin-3-O-glucoside, luteolin-8-C-
neohesperoside, luteolin-8-C-digitoxoside and quercetin (Zibaldi
et al., 2007) are present.
Our results suggest that P. edulis varieties (yellow and purple
fruits) have different chemical composition, justifying the impor-
tance of studies aiming for the chemical characterisation of
these two varieties. It is important to remark that some of the
chemical and pharmacological studies reported for this species
(Maluf et al., 1991; Dhawan et al., 2001; Ichimura et al., 2006)
did not declare the variety employed in the investigation.
Among the samples analysed here, P. tripartita leaf
extract (Fig. 3A) displayed the greatest diversity of flavonoids.
The presence of isovitexin (Abourashed et al., 2002) and 40 -
methoxyluteolin-8-C-6’’acetylglucopyranoside (Ramos et al., 2010)
has been reported previously for the leaves of this species. In
our investigations, orientin (peak 8), isoorientin (peak 4), vitexin
(peak 9), isovitexin (peak 6), swertisin (peak 5) and vicenin-2
(peak 1) were identified (Fig. 3A). However, the two major com-
pounds in the extracts (peaks I and II, with tR =19.0 and 28.8min,
respectively; Fig. 3A) could not be identified.
As far as we are aware, there are no reports concerning the
presence of C-glycosyl flavonoids in the pericarp of P. tripartita.
In our analysis (Fig. 3B) isoorientin (peak 4), orientin (peak 8)
and isovitexin (peak 6) were identified.
In the analysis of P. alata leaf extract (Fig. 4A) only one major
peak was observed, and identified as vitexin-2’’-O-rhamnoside
(peak 7) according to the UV spectra and co-injection with
authentic sample, together with isoorientin (peak 4), isovitexin
(peak 6) and orientin (peak 8) as minor constituents. All these
C-glycosyl flavonoids were previously reported for P. alata

AU
0.1
mAU 338
65,00 A 268

60,00 0.05

55,00
8
50,00 0.0
200 300 400
45,00
AU
40,00 m/z= 577 m/z= 447 0.05
7,8
35,00 347
255 269
30,00
0.025
25,00 m/z= 447
4
20,00 m/z= 431 7
15,00 6 0.0
200 300 400
10,00
5,00
0,00
0 5 10 15 20 25 30 35 40 45 50 55
Time (min)

mAU
B
30,00

25,00

20,00

15,00
m/z= 577
10,00 7

5,00

0,00
0 5 10 15 20 25 30 35 40 45 50 55
Time (min)

Figure 4. Chromatograms of extracts from P. alata: (a) leaves and (b) pericarp with diode array detection at 340nm. For chromatographic conditions,
see ‘Experimental’ section.
238

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Analysis of C-glycosyl Flavonoids from South American Passifloras
extracts (Ulubelen et al., 1982; Doyama et al., 2005). Specifically
with regard to P. alata pericarp, there was no previous report
concerning the flavonoids composition. In this work, the analysis
suggests the presence of vitexin-2’’-O-rhamnoside as a minor
component (Fig. 4B, peak 7).
To the best of our knowledge there is no previous report
concerning the flavonoids composition of leaves and pericarp of
P. quadrangularis species. In P. quadrangularis leaf extract, the
presence of vitexin-2’’-O-rhamnoside could be characterised as a
minor constituent (data not shown). On the other hand, the
two main components with tR of 19.5 and 30.5min, respectively,
could not be identified. These compounds showed UV maxima at
336 and 269nm, suggesting the presence of flavonoids with the
apigenin nucleus. In the pericarp extract it was not possible to
characterise the presence of the compounds used as standard.
A high accumulation of C-glycosyl flavonoids could be ob-
served in the extract of P. manicata leaves, with isovitexin as
the main constituent. The presence of orientin, isoorientin,
vitexin, isovitexin and vitexin-2’’-O-rhamnoside could also be
identified, but as minor constituents. Previous work on the flavo-
noids composition of P. manicata leaves reported the presence
of isovitexin, vitexin, orientin and isoorientin (Abourashed
et al., 2002). Additionaly, we observed the presence of vitexin-
2’’-O-rhamnoside (data not shown). The composition of the
pericarp was not investigated because this specie does not
produce edible fruits.
For P. ligularis leaf and pericarp extracts we could not detect
any compound similar to the reference standards used, but the
TLC analysis (data not shown) pointed to the presence of spots
with characteristics of the C-glycosyl flavonoids.
Considering the sum of our results, with the exception of
P. ligularis, the presence of C-glycosyl flavonoids could be
detected in all extracts from the leaves and pericarp of South
American Passiflora species and all of them showed different
HPLC profiles. Therefore, the prevalence of these C-glycosyl
flavonoids can be used as chemical markers for authentication
of samples, detection of adulterations, and differentiation among
closely related species.
Acknowledgements
This work was performed with financial support from CNPq,
CAPES and DIB-Universidad Nacional de Colombia. The authors
F.H. Reginatto and E.P Schenkel are grateful to CNPq for their re-
search fellowships. S.M. Zucolotto is also grateful to CAPES for
their PhD fellowship. We are also grateful to Liliana A. Santacruz
C. and César Andres Lopez (Departamento de Química-
Universidad Nacional de Colombia) for the LC-MS analysis.
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