Pharmaceutical Biology

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High-Speed Extraction and HPLC Fingerprinting

of Medicinal Plants – I. Application to Passiflora
Flavonoids

Ehab A. Abourashed, John R. Vanderplank & Ikhlas A. Khan

To cite this article: Ehab A. Abourashed, John R. Vanderplank & Ikhlas A. Khan (2002) High-
Speed Extraction and HPLC Fingerprinting of Medicinal Plants – I. Application to Passiflora
Flavonoids, Pharmaceutical Biology, 40:2, 81-91, DOI: 10.1076/phbi.40.2.81.5844

To link to this article: https://doi.org/10.1076/phbi.40.2.81.5844

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2002, Vol. 40, No. 02, pp. 81–91 © Swets & Zeitlinger

High-Speed Extraction and HPLC Fingerprinting of Medicinal

Plants – I. Application to Passiflora Flavonoids

Ehab A. Abourashed1#, John R. Vanderplank2 and Ikhlas A. Khan1

1
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences and Department of
Pharmacognosy, School of Pharmacy, The University of Mississippi, MS, USA; 2National Collection of Passiflora,
Greenholm Nurseries Ltd., Kingston Seymour, Clevedon, North Somerset, UK

Abstract
An approach comprising accelerated solvent extraction fol-
lowed by quantitative HPLC analysis has been adopted in
fingerprinting 115 samples of different species of the
genus Passiflora. The C-flavonoid glycosides schaftoside (1),
isoschaftoside (2), isoorientin (3), orientin (4), isovitexin (5)
and vitexin (6) were chosen as analytical standards and their
overall prevalence in all samples was determined. The devel-
oped HPLC method utilizes gradient elution on an analyti-
cal Phenomenex Hypersil ODS column and UV detection
at 280 nm. The availability of unique fingerprints, as well
as quantitative data, for each species can provide a number
of benefits including, but not limited to (a) authentication
of samples, (b) determination of chemotaxonomic markers,
(c) identification of constituent patterns related to specific
geographical locations, (d) supportive data in genetic studies,
(e) identifying possible substitutes for Passiflora incarnata,
(f ) differentiating between closely related species, and (g)
relating biological activities to phytochemical profiles.
Keywords: Passion flower, passion fruit, Passifloraceae, C-
flavonoid glycosides, accelerated solvent extraction, HPLC.
Introduction
Plants of the genus Passiflora are indigenous to the tropical
and semi-tropical zones of North, Central and South
America, and are characterized by their flower structure. The
first passion flower to be described, Passiflora incarnata L.
(Passifloraceae), was recorded in the mid 16th century and
was brought to Europe by a Spanish missionary. Over 480
Passiflora species have been recognized with more to be pub-
lished in the future (Vanderplank, 1996). Out of all the
reported species, P. incarnata L. and P. edulis Sims stand out
as being the most extensively investigated for their chemical
composition and biological activities. Of the two, P. incar-
nata is the one that is commonly used in many sedative
herbal preparations, either alone or in combination with other
sedative ingredients, such as valerian, hawthorn and kava-
kava. Other indications for P. incarnata include the control
of abnormal cardiac arrhythmias and tension-related asthma,
and as an anxiolytic, muscle relaxant, antidepressant and/or
adaptogen (Leigh, 1998). The neuropharmacological activity
of P. incarnata extracts has been studied and the results
obtained support the use of that herb in sedative preparations
and in those intended for managing hypertension and ben-
zodiazepine withdrawal syndrome (Rasmussen, 1997).
However, the main active constituent(s) responsible for
such an activity is not yet determined and it is still contro-
versial whether flavonoids, alkaloids and/or other com-
pounds, such as maltol, are involved (Kimura et al., 1980;
Speroni et al., 1996). Phytochemical investigations of
P. incarnata show that it is rich in C-glycoside flavones of the
apigenin and luteolin type, mainly schaftoside (1), isoschafto-
side (2), isoorientin (3), orientin (4), isovitexin (5) and vitexin
(6) (Meier, 1995). Other chemical constituents of P. incarnata
include trace amounts of harman alkaloids, maltol and ethyl
maltol, as well as some essential oils (Aoyagi et al., 1974;
Buchbauer & Jirovetz, 1992; Rehwald et al., 1995). HPLC
methods for analysis and fingerprinting of crude and herbal

Accepted: September 13, 2001

Address correspondence to: Ikhlas A. Khan, National Center for Natural Products Research, Research Institute of Pharmaceutical
Sciences, School of Pharmacy, University of Mississippi, University, MS 38677, USA. Tel: (662) 915-7821, Fax: (662) 915-7062,
E-mail: rikhan@cotton.vislab.olemiss.edu
#Current address: Ehab A. Abourashed, GlaxoSmithKline CHRD, 1500 Littleton Road, Parsippany, NJ 07054, USA. Tel: (973) 889-2195,
Fax: (973) 889-2268, E-mail: ehab.a.abourashed@gsk.com
82 E.A. Abourashed et al.
formulations of P. incarnata for its flavonoidal content have
recently been published (Bokstaller & Schmidt, 1997; Pietta
et al., 1986, 1989; Rehwald et al., 1994).
The second common herb of the same genus, P. edulis,
also known as passion fruit or purple granadilla, has a fruit
that is eaten fresh or processed as a common ingredient in
many tropical beverages, fruit cocktails, jams and sweets.
Phytochemical investigation of P. edulis shows the presence
of harman alkaloids (Lutomski et al., 1975), flavonoidal
(Moraes et al., 1997) and cyanogenic (Chassagne et al.,
1996b) glycosides, sulfur-containing volatiles (Engel &
Tressl, 1991), essential oils (Arriaga et al., 1997), aroma pre-
cursors (Chassagne et al., 1996a) and triterpenes (Andreetti
et al., 1977). However, the use of P. edulis in phytopharma-
ceuticals is not as common as the use of passion flower,
P. incarnata. In addition to P. incarnata and P. edulis,
only about 40 other species have been phytochemically in-
vestigated since the late 1960s. The most common types of
compounds characterized in these species are mainly
C-flavonoids, harman alkaloids, cyanogenic glycosides and
volatile compounds.
With the large number of identified Passiflora species
and with the relatively small number investigated so far, a
closer inspection is justified. A chance for such a closer look
became available when accessibility was established to dried
voucher specimens (2–20 g) of ca. 100 identified species and
hybrids of Passiflora. An approach to extract and analyze
these samples in a simple, quick and efficient way was
designed. This approach is based on high speed extraction
combined with HPLC fingerprinting of each sample for such
compounds expected to be present in relatively high abun-
dance, mainly C-flavonoid glycosides. The utilization of this
analytical approach resulted in the generation of charac-
teristic chromatographic profiles in which the C-flavonoid
glycosides 1–6 were used as standard markers. Such markers
were selected based on the fact that they are all present in
the reference species P. incarnata, and that most of them are
commercially available. Results of our fingerprinting studies
would be useful in: (a) authentication of passion flower
preparations; (b) selecting species that are phytochemically
equivalent to P. incarnata for use as substituents in regions
were P. incarnata is not abundant; (c) differentiating between
species that are morphologically similar; (d) understanding
the chemical profiles resulting from hybridization; (e) iden-
tification of chemotaxonomic markers of genus Passiflora;
(f) propagating species that can produce high levels of spe-
cific markers, (g) studying the effect of seasonal and geo-
graphical variations on specific marker production; and (h)
correlating certain pharmacological activities with specific
chemical constituents.
Materials and methods
Samples and standards
Dried voucher specimens (2–20 g) of 115 samples of Passi-
flora species were obtained from the National Collection of
Passiflora, Clevedon, England. A locally grown P. incarnata
species was also used as a reference during method devel-
opment and analytical runs. The binomial names for all
samples are included in Table 2. A mixture of 1 and 2 was
kindly provided by Professor Ihsan Calis, Hacettepe Univer-
sity, Ankara, Turkey. Standards 3–6 were purchased from
Indofine (Somerville, NJ). All solvents were purchased from
Aldrich (Milwaukee, WI) and those used in the analysis were
of HPLC grade.
Sample preparation and extraction
Approximately 2–10 g of each sample was ground to a fine
powder in a coffee blender (Braun®) and stored in glass vials
at 4°C. Each powdered sample (2.0 g) was loaded into a
stainless steel cartridge and batches of 24 samples were
extracted in a Dionex® Accelerated Speed Extractor accord-
ing to the following program: solvent, 80% MeOH in H2O;
temperature, 40°C; pressure, 1000 psi; cycle duration, 10 min
with solvent flush; cycle repeats, 3. Each extract was accu-
rately divided into two equal portions. One portion was evap-
orated to dryness in a Speedvac® centrifuge and stored
at 4 °C for future use. The other portion was adjusted to
20.0 ml with 80% MeOH, filtered through a C18 Sep-pak®
cartridge and 10 mL of the filtrate was directly injected on the
HPLC column.
HPLC method
A Waters® system, composed of a 600E controller, a 996
photodiode array detector, an autoinjector, auto degasser, and
controlled by Millennium® Chromatography Manager 2010
running under MS Windows 3.1, was used for sample
analysis. Column: Phenomenex® Hypersil ODS 5 mm (150 ¥
4.6 mm) with a SecurityGuard® cartridge (C18, 4 mm ¥
3 mm). Mobile phase: 0.01% H3PO4 in H2O, pH 3.1 (solvent
A) and CH3CN/THF/Isopropanol, 80/80/20 v/v/v (solvent
B). Elution was run at a flow rate of 1 ml/min following a
gradient that began with A for 5 min, then A to B over
55 min. Each run was followed by 5 min wash with MeOH
then re-equilibration with A for 20 min. Peaks were detected
at 280 nm. Samples were analyzed in duplicates and the mean
concentration of each standard was calculated for each
sample. Peak identification and quatification was based on
calibration curves constructed from five concentrations of
each of the four standards used (Table 1). The capacity factor
(k¢) for each peak (Table 1) was calculated from the formula:
k¢ = (tR - to) / to; where tR is the peak retention time and to
is the solvent retention time (1.8 min). Peak resolution, R,
was calculated as: R = 2 (tR2 - tR1) / (W1 + W2), where tR1 is
the retention time of the first peak, tR2 is the retention time
of the second peak, and W1 + W2 are the widths of the first
and the second peak, respectively. Compounds 1 and 2 were
used as qualitative markers, with retention times of 27.0 min
(k¢ = 14.0) and 27.5 min (k¢ = 14.3), respectively, and their
concentrations were calculated as isovitexin.
 HPLC analysis of Passiflora flavonoids 83

Table 1. Calibration data of the quantitative standards of passion flower.

Ret. Time Capacity Regression Standard

Standard (min) Factor (k¢) Equation R2 Error

Isoorientin (3) 31.5 16.5 y = 2.59e + 06 x 0.9980 4.3029e + 04

Orientin (4) 35.9 18.9 y = 2.41e + 06 x 0.9980 3.9744e + 04
Isovitexin (5) 38.3 20.3 y = 2.82e + 06 x 0.9974 5.6066e + 04
Vitexin (6) 41.2 21.9 y = 2.59e + 06 x 0.9992 2.7036e + 04

OH
R2
R1 R2 R3
HO O Schaftoside (1) glc ara H
R3 Isoschaftoside (2) ara glc H
Isoorientin (3) glc H OH
Orientin (4) H glc OH
R1 Isovitexin (5) glc H H
Vitexin (6) H glc H
OH O

Figure 1. Chemical structures of the C-flavone glycoside markers. glc = b-D-glucopyranosyl, ara = a-L-arabinopyranosyl.

Results and discussion of the originally collected samples. These locations are the
tropics (Latin America), North America (United States and
The use of a fast, efficient and automated procedure enabled
Mexico), Europe (United Kingdom), and Australia. Of these
the speedy extraction of 115 samples in about 96 h. The
groups, the Latin American group contained the highest
extractor utilizes programmed high pressure and temperature
number of samples (66) and the Australian one contained the
control to extract powdered herbal material loaded in spe-
least (4). As shown in Table 2, all analyzed samples showed
cially designed stainless steel cartridges. Such a design allows
a wide diversity in both the number and concentration of
for complete exhaustion of a single sample by subjecting it
the marker flavonoids. The chromatographic profiles ranged
to 3 consecutive extractions in 1 h. Efficiency of extraction
from simple (containing as little as one or two components)
was determined by analyzing four consecutive extracts of
to complex (containing more than ten components). In all
the same sample of P. incarnata whereby the fourth extract
such profiles the markers were totally present, partially
showed no trace of the flavonoids of interest as compared to
present or totally absent. In addition to the marker flavonoids,
the preceding extraction batches. The HPLC method applied
many other peaks could be detected in a number of species,
is a modification of that reported by Rehwald et al. (1994) for
such as P. ampullacea, P. anfracta, and P. foetida var. hibis-
the analysis of P. incarnata flavones. In the present study, an
cifolia. Such a diversity in composition resulted in the gen-
initial 5-min isocratic stage followed by a gradient elution
eration of HPLC fingerprints that were unique for almost
over 55 min produced acceptably resolved peaks within a total
every one of the analyzed species. In the next discussion we
run time of 60 min. The six C-flavonoid glycosides 1–6 were
will cite some examples, extracted from the generated data,
chosen as analytical standards (Fig. 1). The sequence of peak
that demonstrate the usefulness of our general procedure in
elution of the P. incarnata sample was comparable with that
some applications that may benefit from the availability of
reported earlier (Rehwald et al., 1994) but peak shape was
these HPLC fingerprints.
enhanced especially for the more retained components, 5 and
6 (Fig. 2A). The standards were adjusted to elute in a 14 min
window within the mid third of the analytical run, thus allow-
Authentication of passion flower preparations
ing for the observation of any additional fast or slow eluting
peaks that may appear in any of the analyzed samples. All Two P. incarnata samples were available for analysis.
marker peaks were well resolved (R > 1.0) except for 1 and The superimposed chromatograms of these samples were
2, which were partially resolved (R = 0.50). Also, since 1 and almost qualitatively identical (Fig. 2B). Although the charac-
2 were supplied as a mixture in a minute amount (less than 1 teristic components were present at different concentration
mg), no calibration curve could be generated for them. There- levels, the fingerprint charactersitics were conserved. This
fore, they were used as qualitative standards for matching same pattern is very similar to that reported earlier in 14
peaks in the analyzed samples and their combined total con- different samples of P. incarnata (Rehwald et al., 1994).
centration was calculated as isovitexin. The HPLC method can thus be used to validate the quality
The analyzed samples were mostly from cultivated plants and authenticity of phytopharmaceuticals containing passion
and were grouped according to the geographical locations flower extract.
84 E.A. Abourashed et al.

Figure 2. HPLC fingerprint of Passiflora incarnata extracts; peak 1: schaftoside, 2: isoschaftoside, 3: isoorientin, 4: orientin, 5: isovitexin,
6: vitexin; (A) Sequence of elution and intermediate positioning of the reference standards; (B) Superimposable fingerprints of two different
samples.

Taxonomical differentiation between samples with
similar morphological characteristics
In certain occasions, identification of the right species may
be challenging to less-experienced collectors. This is clearly
apparent with such species as P. capsularis and P. rubra, P.
vitifolia and P. quadrifaria, P. xiikzodz and P. coriacea whose
morphological similarities may lead to confusing one with
the other, especially when flowers or fruits are absent
(Vanderplank, 1996). Even in the presence of flowers, certain
situations may be confusing, such as the case with P. caerulea
and P. allantophylla. These two species are similar in their
basic morphology except for the size of their flowers, where
P. allantophylla has markedly smaller flowers than those of
 HPLC analysis of Passiflora flavonoids 85

Table 2. Percent concentrations of marker flavonoids in 115 samples of Passiflora.

Schaftoside/
Species Isoschaftoside Isoorientin Orientin Isovitexin Vitexin Location

Tropics:
adenopoda DC. 0.02 – – – – various
allantophylla Mast. 0.58 0.09 0.04 0.01 0.09 Guatemala
alata Curtis – – – – –
alata “Shannon” – – – – –
ambigua Hemsl. – – 0.68 – 0.01 Ecuador
ampullacea (Mast.) 0.16 0.05 – 0.1 0.05 Columbia
Harms
ampullacea (2) 0.20 0.01 – 0.04 0.02 Ecuador
anfracta Mast.ex – 0.26 0.2 – – Ecuador
André
auriculata Kunth 0.12 – – – – French Guyana
biflora Lam. – – – - 0.15 Trinidad
biflora (2) – 0.05 – 0.01 0.18 Costa Rica
boenderi J.M. 0.05 – – 0.04 – Costa Rica
MacDougal
caerulea L. 0.1 0.03 – 0.37 – Brazil
caerulea (2) 0.17 0.17 – 0.19 0.03 Brazil
capsularis L. – 0.04 0.15 – – Costa Rica
colinvauxii Wiggins 0.1 0.05 0.02 0.07 – Galapagos Islands
coriacea Juss. – 0.1 – – 0.5 Costa Rica
cuneata Willd. 0.02 0.25 0.3 – – Venezuela
cuprea L. 0.02 0.07 – – 0.02 Bahamas
discophora P. Jørg. 0.94 0.02 – 0.02 – Ecuador
& Lawesson
filipes Benth. 1.15a 0.66 0.02 0.03 – Venezuela
foetida L. 0.15 0.03 0.14 0.17 0.03 Central, South A.
foetida L. subsp. 0.17 0.09 0.01 0.11 – Venezuela
onocensis Killip in
L.H. Bailey
giberti N.E. Br. – – – – – Venezuela
Gilbertiana J.M. 0.03 0.33 0.65 0.24 – Costa Rica
MacDougal
gracilis J. Jaq. ex 0.07 – – 1.62 0.31 Venezuela
Link
hahnii (Fourn.) – – – – – Costa Rica
Mast.
holosericea L. – – 0.02 – 0.81 Honduras, Mexico
holosericea (2) 0.03 0.02 0.04 0.01 0.47 Costa Rica
laurifolia L. – – 1.17 – 0.03 Venezuela
ligularis Juss. – – – – – Venezuela
lobata (Killip) 0.04 0.04 – 0.09 – Costa Rica
Hutch. ex J.M.
MacDougal
macrophylla Mast. 0.04 – – – – Ecuador
macrophylla (2) 0.39 0.01 – 0.02 – Costa Rica
maliformis L. 0.47 – – – – Venezuela
manicata (Juss.) – 0.23 0.06 1.78 0.39 Venezuela
Pers.
murucuja L. 0.03 0.05 0.27 0.05 – Dominica
mayarum J.M. – – – – – Guatemala
MacDougal
mayurum (2)b 0.07 0.01 – – –
menispermifolia – – – – – Venezuela
Kunth
86 E.A. Abourashed et al.

Table 2. Continued

Schaftoside/
Species Isoschaftoside Isoorientin Orientin Isovitexin Vitexin Location

misera Kunth 0.21 0.03 0.44 0.01 – Panama

mollissima (Kunth) – – – 0.04 – Venezuela
Bailey
mucronata Lam. – 0.02 – – – Brazil
naviculata Griseb. – – – – – Bolivia
oerstedii Mast. – – 0.01 – – Venezuela
oerstedii var. choconiana – – – – – Venezuela
(S. Watson) Killip
penduliflora Bertero 0.07 – – 0.01 – Jamaica
ex DC.
perfoliata L. – – – 0.03 – Jamaica
phoenicia Lindl. – – 0.18 – –
pilosicorona Sacco – – 0.15 – 0.27 Bolivia
pinnatistipula Cav. 0.19 – 0.04 0.10 0.47 Chile
platyloba Killip 0.37 0.11 0.02 0.13 – Venezuela
punctata L. 0.17 0.13 0.41 0.11 – Colombia
quadrifaria R.J.R. – – – 0.27 – Brazil
Vanderplank
rovirosae Killip 0.12 0.07 0.26 0.03 0.03 Guatemala
rubra L. – 0.01 0.04 – – Trinidad
rufa Feuillet – – – 0.02 0.01 French Guyana
serratifolia L. 0.04 0.05 0.15 – – Costa Rica
tenuifila Killip 0.07 0.06 – 0.1 0.06 Brazil
tenuifila (2) 0.15 0.09 0.02 0.12 0.08 Paraguay
tricuspis Mast. – 0.39 – 0.18 – Bolivia
tryphostemmatoides 0.04 – – – 0.06 Ecuador
Harms
trisecta Mast. 0.02 – – – – Bolivia
tuberosa Jacq. – – 0.11 0.02 – Trinidad
vitifolia Kunth 0.03 – 0.74 – 0.14 Brazil
xiikzodz J.M. 0.06 – – – – Guatemala, Mexico
MacDougal
Europe:
“Adularia” – 0.06 0.12 – – U.K.
“Amethyst” 0.11 – – 0.09 – U.K.
x belotii Pepin – – 0.04 0.02 0.11 U.K.
coriacea Juss. 0.09 – – – 0.06 U.K.
“Curiosa” – – – – – U.K.
“Debby” 0.55 0.10 0.06 0.27 0.05 U.K.
x decaisneana – – 0.06 – – U.K.
Gontier ex Planchon
x exconiensis 0.14 – 0.01 – 0.23 U.K.
foetida L. var. 0.15 – 0.05 0.07 0.13 U.K.
hibiscifolia (Lam.)
Killip
garaynglia – – – – 0.02 U.K.
jorullensis Kunth – 0.06 0.48 1.97 – U.K.
x kewensis Hort. 0.01 – – – 0.05 U.K.
morifolia Mast. 0.1 0.01 – 0.15 – U.K.
x piresae R.J.R. – – – – – U.K.
Vanderplank
quinquangularis S. – – 0.3 – – U.K.
Calderon
racemosa Brot. 0.22 – – – – U.K.
 HPLC analysis of Passiflora flavonoids 87

Table 2. Continued

Schaftoside/
Species Isoschaftoside Isoorientin Orientin Isovitexin Vitexin Location

sanguinolenta Mast. – 0.12 0.75 – – U.K.

& Linden
“Sapphire” 0.26 0.12 – 0.33 – U.K.
serratifolia L. – – 0.22 – – U.K.
“Star of Clevedon” 0.02 – – – 0.16 U.K.
“St. Rule” – 0.01 – 0.01 – U.K.
“Sunburst” – 0.71 0.16 1.24 – U.K.
tetrandra Banks ex 0.08 0.01 – 0.01 – U.K.
DC.
urbaniana Killip – 0.1 0.04 0.13 – U.K.
x violacea Loiseleur- 0.07 – – 0.05 – U.K.
Deslongchamps
North America:
actinia Hook. 0.08 – – 0.16 – U.S.A.
cincinnata Mast. – 0.02 0.05 0.12 – U.S.A.
cincinnata (2) – 0.03 – 0.02 – U.S.A.
conzattiana Killip – – – – – U.S.A.
foetida var. 0.23 0.06 – 0.21 – Mexico
gossipifolia (Desv.
ex Hamilton) Mast.
helleri Peyr. – – – 0.45 – U.S.A.
incarnata L. 0.13 0.06 0.01 0.42 0.06 Mississippi
incarnata (2) 0.33 0.02 0.06 0.11 0.06 Florida
“Incense” 0.62 0.04 0.07 0.19 – Florida
juliana J.M. – – 0.04 – – Mexico
MacDougal
lutea L. 0.02 0.19 – – 0.03 Missouri
multiflora L. 0.13 0.02 – 0.04 0.01 Florida
pilosicorona Sacco – – 0.1 0.23 0.09 U.S.A.
platyloba Killip 0.8 – – – – U.S.A.
resticulata Mast. & – – – 0.03 – U.S.A.
Andre
sexflora Juss. 0.22 0.07 – 0.01 – Florida
suberosa L. – – 0.06 – – Florida
subpeltata Ortega 0.01 0.05 – 0.03 – Mexico
vitifolia Kunth 0.05 – 0.23 0.01 – U.S.A.
yucatanensis Killip – 0.03 0.15 – 0.06 Mexico
Australia:
edulis Sims – – – – – Wild
edulis “Golden 0.12 0.10 0.01 0.07 – Cultivated
nugget”
herbertiana Ker – – 0.03 – – Wild
Gawl.
obtusifolia Sesse & – – 0.04 – –
Moc.

a
Maximum detected concentration of each marker is represented in bold face; b Not fully identified but closely related to P. suberosa; (–) indi-
cates undetectable levels.
88 E.A. Abourashed et al.

P. caerulea (Vanderplank, 1996). In all such cases, the fin-
gerprints of the species under debate were considerably dif-
ferent and easily distinguishable from each other. The utility
of HPLC fingerprinting is illustrated in Figure 3, which
shows the difference between the phytochemical profile of
P. xiikzodz and P. coriacea.
Hybridization studies
Seven groups were selected where each group included three
samples: a hybrid, its male and its female cross-breeders.
HPLC fingerprinting of two groups (P. “Sunburst”, P. gilber-
tiana, P. jorullensis and P. “Sapphire”, P. edulis, P. quadri-

Figure 3. HPLC fingerprints of two morphologically similar Passiflora species. (A) P. xiikzodz, (B) P. coriacea. Peak 1: schaftoside; 2:
isoschaftoside; 3: isoorientin; 6: vitexin.
 HPLC analysis of Passiflora flavonoids 89

faria) showed that the hybrids P. “Sunburst” and P. “Sap- glia and P. serratifolia, there was a common unidentified
phire” had close qualitative resemblance to their parent peak eluting at 40.0 min. Such a peak had characteristic UV
species. Two other groups (P. x belotii, P. alata, P. caerulea absorbance maxima at 267 and 337 nm suggesting a closely
and P. x violacea, P. caerulea, P. racemosa) showed partial related analog of the chosen markers. Other major uniden-
resemblance of the hybrids P. x belotii and P. x violacea to tified peaks were also detected as single components in
their respective parents. In the remaining three groups, (P. x P. hahnii (42.7 min), P. edulis (53.8 min), P. “Curiosa”
piresae, P. quadrifaria, P. vitifolia; P. “Curiosa”, P. coriacea, (34.1 min), P. naviculata (58.4 min), P. conzattiana (25.9
P. suberosa; and P. “St. Rule”, P. subpeltata, P. x decaisneana) min). Selection and propagation of any of these species can
the hybrids P. x piresae, P. “Curiosa” and P. “St. Rule” provide a rich source for the specific major components
did not exhibit any of the markers originally present in detected in their simple fingerprints. In an attempt to find
their respective parents (Fig. 4). The inconsistency of the some clues to the identity of the unknown peaks, a literature
hybrid/parent correlation data may be attributed to the vari- search showed that previous phytochemical investigation of
ation in sample sourcing. A possible approach to overcome the flavonoid content has been conducted on three species of
this problem is to fingerprint the parents and the hybrid by those mentioned above. P. alata was reported to have only
HPLC analysis at preset time points over the entire course of one flavonoid, saponarin, which might be the one detected at
the hybridization study. 40.0 min in our HPLC fingerprint (Ulubelen et al., 1982). A
United States-cultivated P. serratifolia was reported to
contain five C-glycosylflavones, three of which are vitexin,
Species rich in one major component
orientin and isovitexin (Ulubelen & Mabry, 1980). It is note-
A number of samples showed only one major peak corre- worthy that two samples of P. serratifolia were analyzed in
sponding to one of the marker standards or to unidentified our pool and that the Latin American sample showed more
components. Orientin was the main component found in P. than one component, in contrast to the European sample
ambigua, P. laurifolia, P. quinquangularis, and P. vitifolia at which had only one (Table 2). In P. edulis var. flavicarpa, the
concentrations ranging from 0.23 to 1.17%; vitexin was the presence of two luteolin 6-C-chinovosyl- and fucosylflavones
major one in P. holosericea (2) at 0.81%; and isovitexin was has been reported (Mareck et al., 1991). However, such a
major in “Star of Clevedon” and P. quadrifaria at 0.16 and report may not be significantly relevant because the flavi-
0.23%, respectively. In P. alata, P. x decaisneana, P. garayn- carpa forma is not identical to the original P. edulis.

Vitexin
Isovitexin
2.5 Orientin
X Isoorientin
Sch/IsoSch
2
conc. (%)

1.5

0.5

X X
X X X
0
caerulea 'Wild Brazil'

caerulea 'Wild Brazil'

x decaisneana
quadrifaria

"Sunburst"

gilbertiana

quadrifaria
jorullensis

subpeltata
vitifolia

Sapphire'
x belotii

x violaceae

suberosa

St. Rule'

edulis (2)
racemosa

coriaceae
x piresii

alata

Curiosa'

species

Figure 4. Parent/hybrid marker distribution in representative samples of Passiflora. Each group is composed of two parents and a
hybrid (X).
90 E.A. Abourashed et al.

Selection of phytochemically equivalent species (1.15% schaftoside/isoschaftoside), P. laurifolia (1.17%

(alternative sourcing)
Out of all of those analyzed, only the seven species: P. allan-
tophylla, P. foetida, P. holosericea, P. incarnata, P. rovirosae,
P. tenuifila, and the hybrid P. “Debby”, contained the six
marker flavonoids (Fig. 3). The relative concentrations of
the markers, however, varied considerably and the chro-
matograms were made more complicated by the presence of
other components. Although such a varied pattern gave each
species its unique fingerprint, it would be rational to select
them as potential candidates for sedative activity evaluation
and, hence, as possible local alternatives to P. incarnata in
regions where the species is scarce or absent.
Determination of taxonomic markers
The 115 analyzed samples represented 95 Passiflora species.
For some species there was more than one sample collected
at different times and/or locations. Of the six markers
selected, the schaftoside/isoschaftoside mixture was the most
abundant, detected in 61 samples (53%), and vitexin was the
least, detected in 38 (33%). Orientin, isoorientin and isovi-
texin were detected in 51 (44%), 55 (48%) and 60 (52%)
samples, respectively (Fig. 5). The species that had the
maximum concentration of each marker were: P. filipes
orientin), P. “Sunburst” (0.71% isoorientin), P. jorullensis
(1.97% isovitexin) and P. holosericea (0.81% vitexin). The
overall prevalence of the selected markers and their relative
chemical stability, imparted by their nature as C-flavonoids,
are good criteria to support their utilization in chemotaxo-
nomic studies by expanding the pool and variability of inves-
tigated species belonging to the family Passifloraceae.
Effect of geographical variation on
marker concentration
Examination of Table 2 shows that the overall levels of
marker concentration were markedly higher in samples origi-
nating from Latin America as compared to those originating
from North America, Europe and Australia. In some Latin
American species, such as P. filipes, P. laurifolia and P.
manicata, the concentration of certain individual markers
exceeded 1.00%.
Conclusion
The described extraction procedure and the compiled chro-
matographic data illustrate that quick and efficient extraction,
followed by reliable HPLC analysis, can be performed on a

100

90

80

70
% Prevalence in Pool

60
53 52
48
50
44

40
33

30

20

10

0
Sch/IsoSch Isoorientin orinetin Isovitexin vitexin

Figure 5. Marker prevalence in the analyzed Passiflora pool (115 samples).

 HPLC analysis of Passiflora flavonoids
large pool of samples to generate unique fingerprints that
would provide a general overview on its patterns and trends.
The generated data can offer solid support for different ap-
plications, such as chemotaxonomy, breeding, secondary
metabolite production and optimization, biological evalua-
tion and alternative sourcing. Through the same procedure,
the authenticity of phytopharmaceuticals containing P. incar-
nata may also be tested. With appropriate markers, unique
fingerprints, and relevant bioassays, a correlation may
eventually be established between the sedative activity of
passion flower and its chemical constituents. Moreover,
the accelerated solvent extraction/HPLC fingerprinting
procedure described in this work can be used as a model
that is applicable to similar flavonoid compounds in other
plant genera, or to other compounds in genus Passi-
flora. Further testing and validation of this model on the
harman alkaloids of Passiflora, as representatives of another
class of natural products, will be reported in a separate
publication.
Acknowledgement
This work was supported in part by the United States Depart-
ment of Agriculture, Agricultural Research Service Specific
Cooperative Agreement No. 58-6408-7-012.
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