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Secondary metabolites of Hypericum species from the Drosanthe and
Olympia sections
C. Cirak
a,
⁎
, Jolita Radusiene
b
, Valdas Jakstas
c
, Liudas Ivanauskas
c
, Fatih Yayla
d
, Fatih Seyis
e
,NecdetCamas
a
a
Vocational High School of Bafra, Ondokuz Mayis University, Samsun, Turkey
b
Nature Research Centre, Institute of Botany, Vilnius, Lithuania
c
Medical Academy, Faculty of Pharmacy, Lithuanian University of Health Sciences, Kaunas, Lithuania
d
Faculty of Arts and Sciences, Department of Biology, Gaziantep University, Gaziantep, Turkey
e
Faculty of Agriculture and Natural Sciences, Department of Field Crops, Recep Tayyip Erdoğan University, Rize, Turkey
abstractarticle info
Article history:
Received 27 July 2015
Received in revised form 18 September 2015
Accepted 24 September 2015
Available online xxxx
Edited by AK Jäger
Eight Hypericum species native to Southern Turkey from Drosanthe and Olympia sections were investigated for
the presence of several bioactive compounds, namely, hypericin, pseudohypericin, hyperforin, adhyperforin,
the chlorogenic, neochlorogenic, caffeic and 2,4-dihydroxybenzoic acids, hyperoside, isoquercitrin, quercitrin,
quercetin, avicularin, rutin, (+)-catechin, (−)-epicatechin, mangiferin, I3, II8-biapigenin, and amentoflavone
for the first time. Plants were harvested at flowering, dried at room temperature, dissected into different tissues,
and assayed for chemical contents. HPLC analysis of methanolic fractions displayed similar chemical profile and
significant quantitative differences among the investigated taxa. The present results support the taxonomic value
of hypericins, rutin, and mangiferin at the sectional level and make an important contribution to our current
knowledge about Hypericum chemistry. Such kind of data could also be beneficial for explanation of the chemo-
taxonomic utility of the corresponding compounds as well as phytochemical evaluation of the species tested.
© 2016 SAAB. Published by Elsevier B.V. All rights reserved.
Keywords:
Chemotaxonomy
HPLC
Hypericum
Hypericins
Mangiferin
Rutin
1. Introduction
Hypericum is a genus, included by the plant family Hypericaceae and
consists of 484 species in forms of small trees, shrubs, and herbs, distrib-
uted in 36 taxonomic sections (Crockett and Robson 2011). These species
occur naturally in all temperate parts of the world but are absent in
habitats having extreme environmental conditions such as deserts and
poles. Turkey is an important center for the genus Hypericum,andaccord-
ing to the most recent count by Güner et al. (2012), there are a total of 96
Hypericum species in the flora of Turkey from 19 sections and 46 of them
are endemic. All Hypericum species have been traditionally used in
Turkish folk medicine under the names “kantaron, peygamber çiçeği,
kılıçotu, kanotu, kuzukıran, and binbirdelik otu” as sedatives, antiseptics,
and antispasmodics (Bingol et al. 2011). A number of Hypericum species
native to southern part of Anatolia assigned to the sections Drosanthe
Spach. and Olympia Spach. with 20 and 2 representatives in flora of
Turkey, respectively (Davis 1988).
Phytochemical investigations on the species from sect. Olympia,such
as H. polyphyllum Boiss. et Balansa and H. olympicum L. (Kitanov 2001),
and the species from sect. Drosanthe, such as H. olivieri (Spach) Boiss.,
H. scabrum L., H. lydium Boiss. (Cirak 2006; Cirak et al. 2 007a; Ayan
et al. 2008; Camas et al. 2014), H. helianthemoides (Spach) Boiss.
(Moein et al. 2011), and H. hyssopifolium Vill. (Smelcerovic et al.
2008), have revealed that these species are valuable sources of
naphthodianthrones, phloroglucinol derivatives, phenolic acids, flavonols,
and biflavonoids. In addition, alkanes, fatty acids, and essent ial oils
were id enti fied in some species of corresponding sections such as
H. olympicum (Stojanovic et al. 2003 ), H. salsolifolium Hand.-Mazz.,
H. retusum Aucher (Bagci and Yu ce 2011a), H. lydium (
Şerbet
çi et al.
2012), H. hyssopifolium, H. lysimachioides Boiss. & Noe (Toker et al.
2006), H. scabrum (Morteza-Semnani et al. 2006), H. capitatum
Choisy var. capitatum and var. luteum Robson (Bag ci and Yuce
2011b). The occurrence of these phytochemicals in Hypericum plants
is associated with the antidepressant (Stein et al. 2012), anti-inflamma-
tory (Crockett et al. 2008), antiproliferative (Schmidt et al. 2012), and
antibacterial (Saddiqe et al. 2010) activities of Hypericum extracts. On
the other hand, some chemotaxonomic significance has also been attrib-
uted to flavonoids hyperoside, quercetin, quercitrin (Cirak et al. 2010),
naphthodianthrones hypericins (Kitanov 2001), dimeric phloroglucinol
uliginosin B (Ferraz et al. 2002a), xanthone mangiferin (Nunes et al.
2010), and to several volatile constituents as non-terpenes and sesquiter -
penes (Smelcerovic et al. 2007).
South African Journal of Botany 104 (2016) 82–90
⁎ Corresponding author at: The Vocational High School of Bafra, University of Ondokuz
Mayıs, 55400, Bafra, Samsun, Turkey. Tel.: +90 362 5426763; fax: +90 362 5426761.
E-mail address: cuneytc@omu.edu.tr (C. Cirak).
http://dx.doi.org/10.1016/j.sajb.2015.09.022
0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
South African Journal of Botany
journal homepage: www.elsevier.com/locate/sajb
DespitethelargenumberofHypericum species, only H. perforatum L.
has been investigated intensively throughout the world both chemically
and pharmacologically. It is a commercialized species, and its extracts
are now widely used in Europe as a drug for the treatment of mild to mod-
erate depression (Fiebich et al. 2011). When compared to H. perforatum,
few studies have been undertaken on other members of this genus
although their proven pharmacological importance (Stojanovic et al.
2013) and the chemical profile of approximately three-quarters of
Hypericum species has not yet been surveyed (Karioti and Bilia 2010).
Considering the pharmacological potential of Hypericum species and the
lack of chemical inform ation on Hypericum genus, we aimed to present
chem ical evaluation of eig ht Hypericum species from Drosanthe and
Olympia sections according to the content of naphthodianthrones
hypericin and pseudohypericin, phloroglucinol derivatives hyperforin
and adhyperforin, phenolic acids as chlorogenic, neochlorogenic, caffeic
and 2,4-dihydroxybenzoic acid s, flavonol glycosid es hyperoside,
isoquercitrin, quercitrin, quercetin, avicularin, rutin, flavanols (+)-
catechin and (−)-epicatechin, xanthone mangiferin, and biflavonoids
I3,II8-biapigenin and amentoflavone.
2. Materials and method
2.1. Brief description of plant materials
Sect. Drosanthe includes herbaceous, perennial, sometimes woody
at the base plants; flowers with petals and stamens, petals sometimes
red-veined or red tinged, usually unguiculate, black glands located
along to sepal and petal margins, rarely superficial, or at leaf apices.
Flowering plants of evaluated 6 species from this section are shown in
Fig. 1.
The species from sect. Olympia are perennial herbs, often shrubby
at the base, glabrous, stems usually without axillary shoots, petals, and
stamens are persistent; black glands present on anthers and sometimes
on leaves, sepals and petals, and petals on times with superficial black
glands (Davis 1988). Flowering plants of evaluated two species from
sect. Olympia are shown in Fig. 2.
The aerial parts of Hypericum plants from both sections representing
a total of 30 individuals for each species were collected at full flowering
from Southern Turkey in June 2013. The species names, their voucher
numbers, and geographical data of collection sites are shown in Table 1.
Species were identified by Dr. Fatih Yayla, Gaziantep University, Faculty
of Arts and Sciences, Department of Biology, Turkey. Voucher specimens
were deposited in th e herbarium of Ondokuz Mayis University Agri-
cultural Faculty. The top of 2/3 plants was harvested be tween
11:00 am and 13: 00 pm. Condi tions on the da y of collection were
clear and sunny at all sites and temperatures ranged from 28 °C to
35 °C. The plant materials were dried at room temperature (20 ±
2
°C) and, after separated into different tissues, were subsequently
assayed for chemical contents by HPLC.
2.2. Preparation of plant extracts
Air-dried plant material was mechanically ground with a laboratory
mill to obtain a homogenous drug powder. Samples of approximately
0.1 g were extracted in 10 μL of methanol by ultrasonication at 40 °C
for 30 min. The prepared extracts were filtered through a 0.22 μm
membrane filter and stored at 4 °C until analysis. The extracts for
naphthodianthrones analysis were exposure to light under xenon lamp
(765 W/m
2
) for 8 min. Due to the photoconversion of protohypericins
into hypericins.
2.3. HPLC conditions, analysis, and quantification
A Waters Alliance 2695 (Waters, Milford, USA) separation module
system equipped with Waters 2487 UV/Vis and Waters 996 PDA
diode-array detectors was used for HPLC analysis. Data were analyzed
using Empower Software chromatographic manager system (Waters
Corporation, Milford, USA).
The separation of flavonoids, epicatechin, and hyp erforin was
carri ed out according to the pharmacopoeial met hod (Pharm . Eur.,
2012) on an ODS hypersil column (3 μm, 150 mm × 4.6 mm i.d., Thermo
Fisher scientific Inc. USA) with 10 mm guard-precolumn. The binary
gradient elution method was used for detection of corresponding
Fig. 1. Flowering plants of H. capitatum var. capitatum (A), H. capitatum var. luteum (B), H. retusum (C), H. spectabile (D), H. elongatum var. elongatum (E), and H. salsolifolium (F) from sect.
Drosanthe.
83C. Cirak et al. / South African Journal of Botany 104 (2016) 82–90
compounds. The mobile phase consisted of water Milli-Q acidified with
0.3% phosphoric acid as eluent A and acetonitrile containing 0.3%
phosphoric acid as eluent B. The elution program was used as follows:
16% B at 0–12 min, from 16% to 53% B at 12–18 min, from 53% to 97% B
at 18–18.1 min, 97% at 18.1–29 min, and from 97% to 16% at 29–30 min.
Flow rate was 0.6 mL/min at 0–19 min and was changed to 0.8 μL/min
at 19–30min.Thecolumntemperaturewas25°C.Thevolumeofextract
injected was 10 μL. Peaks were detected at a wavelength range of
270–360 n m.
The ACE C18 column (5.0 μm, 250 × 4.6 mm i.d.; MAC-MOD Analytical,
Inc) with guard-precolumn was used for separation of phenolic acids,
catechin, and mangiferin. The binary gradient elution of eluent A—water
acidified with 0.5% glacial acetic acid and eluent B—100% methanol was
used as a mobile phase for the detection of the compounds. The separa-
tion was fixed as following program: from 5% to 35% B at 0–30 min,
from 35% to 90% B at 30–36 min, and from 90% to 5% B at 36–37 min.
The flow rate was 1.0 μL/min, and the column temperature was 25 °C.
Detection was monitored at a wavelength of 275–360 nm.
Naphthodiantrones were analyzed according to a little modi fied
pharmacopoeial HPLC method (Pharm. Eur., 2012) using the ACE C18
column ( 5.0 μm, 150 mm × 4.6 mm i.d. (MAC-MOD Analytical, Inc)
with guard-precolumn. The mobile phase of isocratic elution consisted
of ethyl acetate, aqueous 0.1 M sodium dihydrogen phosphate solution
adjusted to pH 2.0 using phosphoric acid and methanol (16:17:67% v/v).
The flow rate was 1.0 μL/min at 40 °C column temperature. The volume
of extract injected was 20 μL. Detection was performed at 560 nm wave
length.
Chromatographic peaks were identified by comparing retention
times and spectral characteristics of the eluting peaks to those o f
authentic reference standards using HPLC-PDA.
The quantification of the compounds was carried out by the external
standard method. Standards stock solutions were prepared freshly in
methanol and diluted to six different concentrations to obtain a set of
concentration ranges. Three injections per concentration were performed
to determine linearity. A calibration curve for each of the compounds was
constructed by plotting peak area against the known concentration of
standard solution. A linear regression equation was calculated by the
least squares method.Theregressioncoef
ficients
of all calibration curves
were R
2
≥ 0.999, confirming the linearity of the concentration ranges. The
results are reported in terms of RSD. The retention time, linear range,
regression equation, correlation coeffi cient, and RSD values of each
analyte are summarized in Table 2. The concentration of compounds
was expressed as mg/g dry mass (DM).
Solvents used were of HPLC grade and purchased from Roth GmbH
(Karlsruhe, Germany). Water was filtered through the Millipore HPLC
grade water preparation cartridge (Millipore, Bedford, USA). Reference
substances were purchased from ChromaDex (Santa Ana, USA), Sigma-
Aldrich (Saint Louis, USA), HW I ANALYTIK GmbH, and Roth GmbH
(Karlsruhe, Germany).
2.4. Data analysis
Principal compone nt analysis (PCA) was carried o ut usi ng the
statistical software package SPSS Version 20.0. This analysis is the
two-dimensional visualization of the position of investigated exemplars
relative to each other. The principal components represent the a xes
which are the orthogonal projections for the values representing the
highest possible variances in this case of PC1 and PC2.
The obtained data were used to create scatter plot diagra ms
(Backhaus et al. 1989). Therefore, a factor analysis was performed,
whereby each variable was used to calculate relation ships between
varia ble and investigated factors. Ba sed on the obtained data, also a
dendogram (cluster) was created (Backhaus et al. 1989) showing the
Fig. 2. Flowering plants of H. olympicum (A) and H. polyphyllum (B) from sect. Olympia.
Table 1
Collection sites and habitat of the Hypericum species examined.
Species
a
Voucher numbers Collection site Latitude (N) Longitude (E) Elevation (m) Habitat
H. capitatum var. capitatum OMUZF # 122 Yeniyazı 37° 04′ N 37° 42′ E 620 Arid pasturelands
H. capitatum var. luteum OMUZF # 123 Yeniyazı 37° 04′ N 37° 42′ E 620 Pinus woodland
H. elongatum var. elongatum OMUZF # 114 Nizip 37° 00′ N 37° 52′ E 440 Rocky and open slopes
H. olympicum OMUZF # 135 Nizip 37° 00′ N 37° 52′ E 440 Igneous slopes and rock ledges
H. polyphyllum OMUZF # 136 Kocatepe village 37° 02′ N 37° 41′ E 780 Arid pasturelands
H. retusum OMUZF # 141 Hamo hill/İslahiye 36°57′ N 36° 30′ E 1200 Igneous slopes and rock ledges
H. salsolifolium OMUZF # 122 Huzurlu Plateau/İslahiye 36° 59′ N 36° 26′ E 1400 Igneous slopes and rock ledges
H. spectabile OMUZF # 144 Hamo hill/İslahiye 36° 57′ N 36° 30′ E 800 Rocky and open slopes
a
Species are listed in alphabetically.
84 C. Cirak et al. / South African Journal of Botany 104 (2016) 82–90
relationship of investigated samples regarding their chemical
composition.
3. Results
In the present study, eight species of Hypericum native to Southern
Turkey were analyzed for the presence and quan tity of 19 bio active
compounds. HPLC analysis of methanolic fractions displaye d similar
chemical profile and signific ant quantitative differences among the
investigated taxa. No caffeic acid accumulation was observed in plants
from sect. Drosanthe while plants of sect. Ol ympia did not produce
hyperforin and adhyperforin. Generally, lower accumulation level of
the chemicals was observed in stems. Flowers were found to be superior
over leaves with respect to hypericin, ps eudohypericin, hyperforin,
adhyperforin, caffeic acid, quercetin, I3,II8-biapigenin, amentoflavone,
mangiferin, and (+)-catechin accumulations while chlorogenic acid,
neochlorogenic acid, and isoquercitrin were mainly accumulated
in leaves in both sections. The accumulation pattern of the tested
compounds in flowers and leaves varied with sections. For example,
hyperoside, quercitrin, rutin, and (−)-epicatechin accumulations were
the highest in flowers of the species from sect. Drosanthe but in leaves
of the species from sect. Olympia. Accordingly, leaves of the species from
sect. Drosanthe accumulated the highest level of 2,4-dihydroxybenzoic
acid and avicularin while flowers of species from the sect. Olympia
dominated with the highest content of c orresponding compo unds
(Tables 3 and 4).
Results of PCA illuminated the accumulation pattern of the inves-
tigated compounds in different plant parts more deeply. The calculated
principal component (PC) values for the tested compounds are shown
in Table 5.
The score plots for the first two PCs explained 26.72% and 20.85%
(totally 40.81%) of the total variance of the chemical data. The obtained
scatter plot using PC1 and PC2 is s hown in Figs. 3 and 4. The results
indicated that the stems of all the investigated Hypericum speci es
display nearly similar chemical profile, while flowers of H. capitatum
var. luteum, H. capitatum var. capitatum, H. elongatum var. elongatum,
H. spectabile, H. polyphyllum, H. retusum, and the leaves of H. spectabile
differed significantly according to their chemical composition.
Table 2
The retention time, linear range, regression equation, correlation coefficient, and precision of each detected analytes of HPLC analysis on examined Hypericum species.
Analytes Processing wavelength, nm Retention time, min Linearity range, μg/μL R
2
Regression equation RSD (%)
2,4-Dihydroxybenzoic acid 290 13.3 0.31–9.80 0.9995 Y = 2.01·10
4
X + 1.56·10
3
3.08
Neochlorogenic acid 324 15.0 0.61–196.00 0.9999 Y = 3.43·10
4
X − 5.32·10
3
0.51
(+)-Catechin 275 19.3 0.30–95.00 0.9999 Y = 1.20·10
4
X + 3.96·10
3
3.19
Chlorogenic acid 324 21.4 0.30–194.00 0.9999 Y = 3.05·10
4
X + 4.43·10
3
0.31
Caffeic acid 324 24.4 0.31–49.00 0.9999 Y = 5.25·10
4
X + 7.17·10
3
0.18
Mangiferin 360 29.0 2.19–280.80 0.9997 Y = 1.82·10
4
X 1.00
(−)-Epicatechin 277 7.2 0.30–195.60 0.9999 Y = 1.08·10
4
X 2.50
Rutin 360 14.8 0.28–178.42 0.9990 Y = 2.73·10
4
X 2.73
Hyperoside 360 15.2 0.29–187.02 0.9996 Y = 4.75·10
4
X 4.67
Isoquercitrin 360 15.5 0.29–188.30 0.9984 Y = 3.30·10
4
X 8.60
Avicularin 360 16.2 0.15–19.16 0.9999 Y = 4.53·10
4
X 1.58
Quercitrin 360 16.6 0.31–196.76 0.9991 Y = 3.53·10
4
X 6.92
Quercetin 360 19.2 0.31–196.00 0.9990 Y = 4.59·10
4
X 7.07
13,II8-Biapigenin
a
360 20.5 0.28–181.80 0.9990 Y = 4.26·10
4
X 7.09
Amentoflavone 360 20.9 0.28–181.80 0.9990 Y = 4.26·10
4
X 7.09
Hyperforin 270 25.5 3.11–199.00 0.9999 Y = 2.42·10
4
X 0.83
Adhyperforin 270 26.0 1.02–65.00 0.9999 Y = 2.42·10
4
X 0.51
Pseudohypericin 590 2.9 0.38–96.20 0.9998 Y = 6.86·10
4
X 2.13
Hypericin 590 8.4 0.37–95.10 0.9997 Y = 1.00·10
5
X 2.52
a
Processing of 13,II8-biapigenin peaks was performed by using calibration curve of amentoflavone reference substance.
Table 3
Hypericin (1), pseudohypericin (2), hyperforin (3), adhyperforin (4), chlorogenic acid (5), neochlorogenic acid (6), caffeic acid (7), 2,4-dihydroxybenzoic acid (8), hyperoside (9),
isoquercitrin (10), quercitrin (11), quercetin (12), avicularin (13), rutin (14), I3, II8-biapigenin (15), amentoflavone (16), mangiferin (17), (+)-catechin (18), and (−)-epicatechin
(19) contents (mg/g DM) in different plant parts of some Hypericum species from sect. Drosanthe.
Species
a
Plant parts Compounds
12345 6 789 10111213141516171819
H. capitatum var. capitatum
b
Leaf 0.03 0.01 ––0.12 9.68 – 0.10 1.98 2.07 9.91 0.35 1.69 0.98 0.07 –––0.89
Flower 0.11 0.12 0.01 0.01 0.08 2.71 – 0.07 2.25 5.22 12.03 1.01 0.95 2.06 0.73 0.02 – 0.32 2.69
Stem ––––0.06 0.21 – 0.05 0.69 1.41 1.84 0.04 0.42 –––––0.46
H. capitatum var. luteum
b
Leaf 0.02 0.01 0.02 0.02 0.11 11.23 – 0.16 3.12 3.18 9.49 0.21 3.46 0.64 0.08 ––0.03 1.12
Flower 0.04 0.06 0.04 0.04 0.05 4.30 – 0.06 6.39 4.03 10.88 0.76 1.36 3.23 0.78 0.02 – 0.09 3.34
Stem ––––– 0.45 – 0.04 1.79 2.35 2.50 0.03 0.92 ––––0.04 1.43
H. retusum Leaf 1.04 1.32 ––12.39 8.72 – 0.17 6.61 12.22 0.96 0.25 0.36 5.11 0.21 – 0.04 – 1.49
Flower 1.18 2.41 0.03 0.03 10.62 4.71 – 0.07 12.02 10.14 1.45 0.74 0.18 15.66 2.16 0.07 0.09 0.11 2.02
Stem 0.02 0.03 ––0.40 0.17 – 0.03 0.63 1.51 0.06 0.06 0.02 0.75 0.01 –––1.31
H. spectabile
b
Leaf 0.60 1.60 ––88.37 2.46 – 0.08 – 0.80 1.27 0.01 0.07 23.73 0.01 ––0.04 0.31
Flower 1.35 2.33 ––16.73 1.02 – 0.03 0.34 0.27 5.52 0.07 – 63.94 2.48 0.12 2.18 0.41 1.50
Stem – 0.01 ––3.25 0.03 – 0.04 0.03 0.15 0.54 ––2.03 ––0.08 0.18 0.66
H. elongatum var. elongatum Leaf 0.02 – 0.01 0.01 0.04 0.12 – 0.19 1.14 1.91 8.52 0.11 0.14 23.54 0.12 –––0.44
Flower 0.03 – 0.04 0.04 0.03 0.04 – 0.07 1.29 0.91 10.81 0.60 0.06 27.51 1.07 0.04 0.05 – 1.03
Stem ––––0.03 0.02 – 0.09 0.33 0.87 7.40 0.10 – 2.78 0.01 –––0.27
H. salsolifolium
b
Leaf 0.01 0.02 ––0.69 5.01 – 0.09 1.98 7.74 0.40 0.13 0.94 0.33 0.07 ––0.18 3.73
Flower 0.14 0.39 ––0.44 1.61 – 0.05 6.11 1.80 0.53 0.46 0.52 1.56 0.75 0.05 – 0.25 3.95
Stem ––––0.40 0.26 – 0.05 1.56 0.92 0.42 0.09 0.09 0.24 0.01 ––0.01 2.17
a
Species are listed in accordance with the classification by Davis (1988).
b
Endemic.
85C. Cirak et al. / South African Journal of Botany 104 (2016) 82–90
Regarding the quantitative amount of tested compounds, hypercin,
and pseudohypericin concentrations varied from 0.01 mg/g DM in
leaves of H. salsolifolium to 1.3 5 mg/g DM in flowers of H. spectabile
and from 0.01 in leaves of H. capitatum var. capitatum and var. luteum
to 2.41 mg/g DM in flowers of H. retusum. H. spectabile, H. salsolifolium,
H. olymp icum, and H. polyphyl um did not accumulate hyperforin and
adhyp erforin and those compounds were detected only in flowers
and/or le aves of the rest species at low amounts (0.01–0.04 mg/g
DM). As for the phenolic acids, an extreme variation was noticed in
accumulation levels of chlorogenic and neochlorogenic acids. Yields
for correspon ding compounds ranged from 0.03 mg/g DM in flowers
and stems of H. elo ngatum var. elongatum to 88.37 mg/g DM in lea ves
of H. spectabile for chlorogenic acid and from 0.02 mg/g DM in stems of
H. elongatum var. elongatum to 11.23 mg/g DM in leaves of H. capitatum
var. luteum for neochlorogenic acid. 2,4-Dihydroxybenzoic acid was accu-
mulated at quite low amounts in all tested species and its accumulation
levels varied between 0.03 and 0.19 mg/g DM, depending on species
and plant parts. Caff eic acid was detected only in species from sect.
Olympia and the highest con tent was ac cumulated in flowers of
H. polyphyllum (0.06 mg/g DM). Hyperoside, isoquercitrin, quercitrin,
quercetin, avicularin, ruti n, 13,II8-biapigenin, and (−)-epicatechin
were detected generally in all parts of Hypericum plan ts studied.
Flowers and leaves o f H. retusum pr oduced the highes t level of
hyperoside and isoquercitrin (12.02 and 12.22 mg/g DM, respectively),
while the lowest amounts of the corresponding compounds were
observed in stems of H. s pectabile and H. olympicum (0.03 and
0.01 mg/g DM, respectively). Flowers of H. spectabile yielded the
highest lev el of rutin and 13,II8- biapigenin (63.94 and 2.48 mg/g
DM, respectively), and these compounds were absent in stems of
H. capitatum var. capitatum and var. luteum. The highest level of quercitrin
and avicularin was detected in flowers and leaves of H. capitatum var.
capitatum (12.03 and 1.69 mg/g DM, respecti vely), and ste ms of
H. retusum yielded the lowest amount of both compounds (0.06
and 0.02 mg/g DM, respe ctivel y). (−
)-Epicatechin contents ranged
bet
ween 3.95 mg/g DM in H. salsolifolium flowers and 0. 01 mg/g
DM in H. polyphyllum stems. Querc etin was accumulated at moderate
levels (1.01–0.02 mg/g DM depending on species and plant parts)
when compared to the other tested flavonols. H. elongatum var.
elongatum did not produce (+)-cate chin while this compound was
detected in different tissues of the rest species at low amounts. No
mangiferin accumulation was observed in species from sect. Olympia.
This compound was detected only in different parts of H. retusum,
H. spectabile, and H. elongatum var. elongatum and reached its highest
accumulation level in flowe rs of H. spectabil e (2.18 mg/g DM).
Amentoflavo ne was detec tab le in all s peci es but only in flowers. Its
accumulation levels varied between 0.02 and 0.12 mg/g DM depending
on the species (Tables 3 and 4).
4. Discussion
The taxonomy of the genus Hypericum is largely based on morphology
(Crockett and Robson 2011). However, using only the morphological
characteristics has caused uncertainties in taxonomical division of this
genus. First, some sections closely resemble to each other and few mor-
phological characteristics serve to differentiate and identify them. Besides,
identifying the separate plants merely based on morphological characters
is hard and can lead to some mistake as mentioned by Davis (1988) to
indicate the morphological parallelism among members of the sections
of Adenosepalum Spach. and Origanifolia Stef. Thus, studies on qualifying
chemical profileofspeciescanserveasanadditionaltoolintaxonomic
analysis of Hypericum genus (Camas et al. 2014).
In a previous paper, Kitanov (2001) reported H. olympicum and
H. polyphyllum to contain hypericin and pseudohypericin, but these
species were not investigated so far for the presence of other Hypericum
chemicals. Besides, to the best of our knowledge, there is no previous
report on polar chemistry of the investigated species of sect. Drosanthe.
Thus, it is the first time we have reported the presence of 19 compounds
in H. capitatum var. capitatum and var. luteum, H. retusum, H. spectabile,
H. elongatum
var. el
ongatum, and H. salsolifolium as well as in
H. olympicum and H. polyphyllum.AsshowninTables 3 and 4, chemical
profiles of the specie s from the same section closely resemble each
other despite the distinct quantitative variation in chemical content of
plant material. Six species from sect. Drosanthe yielded all the tested
compounds at various levels excluding caffeic acid, absent in all species;
hyperforins, absent in H. spectabile and H. salsolifolium; and mangiferin,
absent in H. salsolifolium and both varieties of H. capitatum. In our previous
study, we described chemical constituents of three other Hypericum
species from sect. Drosanthe,namely,H. olivieri, H. scabrum,and
H. lydium ( Camas et al. 2014). The comparison between present
and previous results revealed that all members of sect. Drosanthe include
Table 4
Hypericin (1), pseudohypericin (2), hyperforin (3), adhyperforin (4), chlorogenic acid (5), neochlorogenic acid (6), caffeic acid (7), 2,4-dihydroxybenzoic acid (8), hyperoside (9),
isoquercitrin (10), quercitrin (11), quercetin (12), avicularin (13), rutin (14), I3, II8-biapigenin (15), amentoflavone (16), mangiferin (17), (+)-catechin (18), and (−)-epicatechin
(19) contents (mg/g DM) in different plant parts of some Hypericum species from sect. Olympia.
Species
a
Plant parts Compounds
12345 678910111213141516171819
H. olympicum Leaf 0.24 1.56 ––35.16 9.94 0.01 0.11 0.45 0.21 0.17 0.03 0.01 24.74 0.05 ––0.09 0.81
Flower 0.31 1.86 ––11.80 3.01 0.03 0.14 0.32 0.09 0.13 0.21 0.05 5.63 0.81 0.04 – 0.19 0.09
Stem – 0.04 –– 3.76 0.77 – 0.06 0.09 0.01 0.06 0.03 0.03 2.63 0.01 –––0.40
H. polyphyllum Leaf 0.47 1.79 ––16.21 6.31 0.03 0.03 0.45 0.43 0.69 0.02 0.01 52.57 0.06 ––0.01 0.70
Flower 0.56 2.01 ––15.78 5.01 0.06 0.06 0.04 0.23 0.50 0.13 0.05 17.91 1.09 0.07 – 0.28 0.25
Stem 0.01 0.02 –– 2.79 0.57 – 0.03 0.17 0.74 0.23 0.05 0.01 6.23 0.01 ––0.01 0.01
a
Species are listed in accordance with the classification by Davis (1988).
Table 5
Cumulative values of calculated principal components for 8 Hypericum species.
Principal component Total Cumulative
1 25.672 25.672
2 20.846 46.518
3 12.682 59.200
4 9.125 68.325
5 7.861 76.186
6 7.472 83.659
7 4.493 88.152
8 3.477 91.629
9 3.727 94.356
10 1.719 96.074
11 1.607 97.681
12 0.906 98.586
13 0.556 99.143
14 0.416 99.559
15 0.216 99.774
13 0.123 99.897
17 0.074 99.971
18 0.022 99.993
19 0.070 100.00
86 C. Cirak et al. / South African Journal of Botany 104 (2016) 82–90
hypericin, pseudohypericin, chlorogenic acid, neochlorogenic acid,
2,4-dihydroxybenzoic acid, amentoflavone, hyperoside, isoquercitrin,
quercitrin, quercetin, avicularin, rutin, and (+)-catechin and (−)-
epicatechin and hav e similar chemical profile. In a nalogy to sect.
Drosanthe, all the tested chemicals were detected in the two presented
species of sect. Olympia except for hyperforins and mangiferin.
Fig. 3. Scatter plot of investigated Hypericum species.
Fig. 4. Dendogram of investigated plant parts in eight Hypericum species (HCVC = H. capitatum var. capitatum,HCVL=H. capitatum var. luteum,HEVE=H. elongatum var. elongatum,
HO = H. olimpicum,HP=H. polphyllum;HR=H. retusum, Has = H. salsilifolium,HSp=H. spectabile,S=Stem,F=flower, L = leaf).
87C. Cirak et al. / South African Journal of Botany 104 (2016) 82–90
Among the bioactive compounds, hypericins were reported to have
an authenticated taxonomic value for the infrageneric classification of
the genus Hypericum (Robson 1977). Kitanov (2001) did not detect
hyper icin and pseudohypericin in the taxa of the primitive sections
but identified these compounds only in the species of the most phyloge-
netically advanced sections. Moreover, hypericins were notified to be
included only in the species of Hypericum whose aerial parts bear dark
glands (Lu et al. 2001). In our previous studies, we reported a positive
correlation between dark gland number and hypericin content of leaf
in H. perforatum (Cirak et al. 2007b), H. aviculariifolium Jaup. and
Spach subsp. depilatum (Freyn and Bornm.) Robson var. depilatum and
H. pruinatum Boiss. and Bal. (Cirak et al. 2006). Several authors also
reported that the absence of dark glands in Hypericum plants is con-
cerned with the lack of both hypricin forms in these species (Ferraz
et al. 2002b; Nor et al. 2008; Crockett and Robson 2011). In the present
study, we observed that detection of hypericins is consistent with the
presence of dark glands in all aerial parts of the investigated species as
shown in Fig. (5).
Rutin, a flavonol making an important contribution to the antide-
pressant activity of Hypericum extract (Noldner and Schotz 2002)was
detected in all investigated species in the present study. This compound
was also detected in H. olivieri, H. scabrum,andH. lydium from sect.
Drosanthe; H. confertum Choisy, H. t hymifol ium Banks and Sol.,
H. linarioides Bosse, and H. pruinatum from s ect. Taeniocarpium
(Camas et al. 2014); H. origanifolium Willd. (Cirak e t al. 2007c)and
H. aviculariifolium subsp. depilatum var. depilatum (Cirak. et al.
2013)fromsect.Origanifolia; H. perf or atum (Dogrukol et al. 2001)
and H.
triquetrifolium (Hosni et al. 2011) from sect. Hypericum;
H. adenotrichum Spach (Cirak et al. 2009)andH. orientale L. (Cirak et al.
2012)fromsect.Crossophyllum, but not detected in 13 Hypericum species,
native to Southern Brazil from sect. Trigynobrathys (Nunes et al. 2010)and
some chemotypes of H. perforatum (Martonfi et al. 2001). The results
indicate that the compound may have some chemotaxon omic utility at
the sectional or subsectional level.
Mangiferin is a widely distributed xanthone in the species of
Hypericum (Bennett and Lee 1989). Kitanov and Nedialkov (1998)
found this compound in 26 of 36 analyzed taxa and reported that
it seems to be specific only for the taxa of two tribes Hypericeae
and Cratoxyleae and thus has little chemotaxonomic significance for
infrageneric classification of the genus. We did not detect mangiferin
in species of sect. Olympia as Kitanov and Nedialkov (1998) but detected
in three species of sect. Drosanthe. This compound was not also detected
in 19 Brazilian species of Hypericum from sect. Trigynobrathys (Nunes
et al. 2010).
The monomeric phl oroglucinol derivatives like hyperforin was
reported to accumulate in several species of Hypericum from different
sections such as H. perforatum (Smelcerovic et al. 2008), H. montbretii
(Cirak and Radusiene 2007), H. lydium, H. pruinatum, H. confertum
(Camas et al. 2014), H. aviculariifolium subsp. depilatum var. depilatum,
and H. orientale (
Cirak. et al. 2013).
There is no evidence that monomeric
phloroglucinol derivatives have chemotaxonomic value unlike to dimeric
ones, which were reported to exhibit taxonomic evidence at the sectional
level for the species from sect. Brathys and Trigynobrathys (Barros et al.
2013). Hence, detection of hypericins, rutin, mangiferin as well as
hyperforins, and t he other tested compounds in Hypericum species
investigated in the pr esent study s upports the taxonomic pos ition
of the sect. Dros anth e and Olympia within the genus Hypericum.
However, it should be noted that several species of Hypericum from
other sections were previously reported to have the above mentioned
compounds. Thus, it may not be reasonable to assign them as a clear
taxonomic pattern at the infragener ic level (Barros et al. 2013). It is
interesting to note that no caffeic acid accumulation was observed
in se ct. Drosanthe, but both sp ecies of sect . Olympia y ielded this
compound which was also detected previously in four species from
sect. Taeinocarpium (Camas et al. 2014). Similarly, mangiferi n was
not accumulated in sect. Olympia but occurred in three species from
sect. Drosanthe, suggesting that the pattern for occurrence of caffeic
acid and mangiferin may be related to the evolution of the different
taxonomical groups of Hypericum.
Regarding the proven bioactivities of Hypericum chemicals, especially
the antimicrobial (Nogueira et al. 2013), antiviral (Schmittetal.2001),
hepatoprotective (Wang et al. 2008), and antidepressant (Stein et al.
2012) ones, the results presented here have also a pharmacological
value and may be helpful in the evaluation and selection of species as
new sources of valuable bioactive chemicals.
As shown in Fig. 3, results of PCA, an useful statistical analysis for the
differentiation of plant mate rial regarding their chemical profile
(Smelcerovic et al. 2008; Bertoli et al. 2011), indicated a considerable
variation in chemical accumulation among different plant parts of the
tested species. In the present paper, we observed that all the detected
chemicals were deposited in the same organs of species from the
same section. However, in some instances, the same compound was
accumulated mainly in leaves in sect. Olympia but in flowers in sect.
Drosanthe or vice versa. Light glands, dark glands, and secretory canals
were reported to be secretory structures of Hypericum plants and
main sites of synthesis and/or accumulation of Hypericum chemicals
(Ciccarelli et al. 2001). The localization of these secretory structures in
plant tissues varies depending on species (Nürk et al. 2013; Maggi
Fig. 5. Dark glands as an example in flowers of H. capitatum var. capitatum (A) and H. olympicum (B). Arrows indicate the glands.
88 C. Cirak et al. / South African Journal of Botany 104 (2016) 82–90
et al. 2004). The distinct chemical diversity among different plant parts
of the tested species can be attributed to the variation in localization of
secretory structures among the species from different sections.
5. Conclusions
Characterization of naphthodianthrones, monomeric phloroglucinol
derivatives, phenolic acids, flavonols, flavanols, biflavonoids, and
xanthones in the Hypericum species, native to Southern parts of Turkey,
has reconfirmed the value of Hypericum genus as a source of bioactive
compounds. In a chemotaxonomical point of view, the resemblance in
chemical profile of the species from the same section as well as the occur-
rence of hypericin, pseudohypericin, rutin, and absence of caffeic acid and
mangiferin in some tested species has indicated some taxonomic value
for the corresponding compounds with the requirement of further studies
to make more substantial inferences. Considering the fact that secondary
chemistry of an estimated 60% of Hypericum speciesisstilllargely
unknown (Crockett and Robson 2011), the present data have also
made an important contribution to our current know ledge about
chemistry of Hypericum genus.
Acknowledgments
Authors are grateful to Metropolitan Municipality of Gaziantep,
Turkey, for the valuable help in sampling the wild plant material.
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