Seasonal Variation of Hypericin and Pseudohypericin Contents in Hypericum Scabrum L. Growing Wild in Turkey

Abstract

The present study was conducted to determine ontogenetic and morphogenetic variations of hypericin and pseudohypericin contents in Hypericum scabrum growing in Turkey. Plants were harvested at vegetative, floral budding, full flowering, fresh fruiting and mature fruiting stages and observed for the presence of dark glands. Subsequently, they were dissected into stem, leaf and reproductive tissues, which were dried separately, and subsequently assayed for hypericin and pseudohypericin contents by HPLC. No hypericins were detected in stem tissues, while leaves and reproductive parts accumulated both compounds at different levels depending on growth stages. In general, higher levels of hypericin and pseudohypericin accumulation were observed in reproductive parts. Content of both hypericin forms decreased with advancing of plant development and reached their highest levels at floral budding stage.

Full text

Seasonal Variation of Hypericin and Pseudohypericin
Contents in Hypericum scabrum L. Growing Wild in Turkey
Ali Kemal Ayana, Cüneyt Çırakb,* and Kerim Güneyc
aThe High School of Profession of Bafra, University of Ondokuz Mayıs, Samsun, Turkey
bFaculty of Agriculture, Department of Agronomy, University of Ondokuz Mayıs, Kurupelit,
Samsun, Turkey
cUniversity of Kastamonu, Faculty of Forestry, Department of Forestry Botany, Kastamonu, Turkey
cuneytc@omu.edu.tr
Received: October 24th, 2007; Accepted: December 3rd, 2007
The present study was conducted to determine ontogenetic and morphogenetic variations of hypericin and pseudohypericin
contents in Hypericum scabrum growing in Turkey. Plants were harvested at vegetative, floral budding, full flowering, fresh
fruiting and mature fruiting stages and observed for the presence of dark glands. Subsequently, they were dissected into stem,
leaf and reproductive tissues, which were dried separately, and subsequently assayed for hypericin and pseudohypericin
contents by HPLC. No hypericins were detected in stem tissues, while leaves and reproductive parts accumulated both
compounds at different levels depending on growth stages. In general, higher levels of hypericin and pseudohypericin
accumulation were observed in reproductive parts. Content of both hypericin forms decreased with advancing of plant
development and reached their highest levels at floral budding stage.
Keywords: Hypericum scabrum, hypericin, pseudohypericin, ontogenetic variation, infrageneric classification.
Hypericum scabrum L. is an herbaceous perennial
plant which grows in dry rocky slopes and open
woodland of Turkey and has been used by
Turkish folk for its antispasmodic, sedative and
anti-inflammatory properties. In Turkey, an ointment
prepared from the aerial parts of the plant is used in
folk medicine against psoriasis. This plant has
important pharmacological potential, with its well
documented antimicrobial activity [1].
Hypericins belong to a group of compounds known
as naphthodianthrones and the analytical method
utilized for Hypericum identification in botanical
preparations depends on the presence of the
naphthodianthrones, as marker compounds. The
photodynamic and photocytotoxic properties of
hypericins allow them to act as antiviral agents
indicating their possible use in the treatment of
human immune deficiency virus type 1 (HIV-1) and
cancer [2]. However, it should be noted that results
from recent studies have indicated that hyperforin
rather than the hypericins is the main chemical
responsible for the antidepressant effects of
Hypericum extracts. Although hyperforin is a major
component, occurring in concentrations of 2–4% of
the total extract of H. perforatum, hypericins remain
the popular marker substances for the standardization
of the herbal product, because of the instability of
hyperforin in the presence of oxygen and light [3].
The studies documenting hypericin content/variation
in H. scabrum are limited and the occurrence of this
compound in this species has long been a disputed
subject. No hypericin was detected in H. scabrum by
either Kitanov [4] or Ayan et al. [5], but Tanker [1]
reported it to contain both hypericin forms, although
Zevakova et al. [6] determined only hypericin, but
not pseudohypericin. To our knowledge, no study has
been undertaken on the variation of hypericins in
H. scabrum. The present study deals with the
determination of hypericin and pseudohypericin
contents and their variations in H. scabrum at
different stages of plant phenology.
NPC Natural Product Communications 2008
Vol. 3
No. 2
241 - 244

242 Natural Product Communications Vol. 3 (2) 2008 Ayan et al.
In contrast to the leaves and floral parts, no dark
glands were observed in stems. The leaves and
reproductive parts accumulated hypericin and
pseudohypericin at different levels depending on the
development stage, but the stems did not produce
either compound during the course of ontogenesis.
Reproductive tissues were found to be superior
to leaves with regard to content of both hypericin
forms. Ontogenetic changes in hypericin and
pseudohypericin content of leaves and reproductive
parts followed a similar trend. The content of both
compounds decreased significantly with advancing
plant development (Figures 1 and 2). The highest
level of hypericin and pseudohypericin in leaves and
reproductive parts was reached at the floral budding
stage (0.095 mg/g dry wt hypericin and 0.12 mg/g
dry wt pseudohypericin for leaves; 0.18 mg/g dry wt
hypericin and 0.19 mg/g dry wt pseudohypericin for
reproductive parts).
Figure 1: Ontogenetic changes in hypericin content of leaves and
reproductive tissues in H. scabrum (Values with different small letters
within columns for each tissue differ significantly at the level of P<0.01).
Figure 2: Ontogenetic changes in pseudohypericin content of leaves and
reproductive tissues in H. scabrum (Values with different small letters
within columns for each tissue differ significantly at the level of P<0.01).
Methods available for determination of hypericin
usually involve solvent extraction followed by either
spectrophotometric assay for total hypericins [7] or
HPLC determination of individual constituents [8].
By TLC it is also possible to determine the presence
of hypericins in ethanolic extracts, but it is not likely
to be able to quantify the different forms [9].
Secondary metabolite content of a given plant tissue
can exhibit significant variation depending on the
method by which chemical analysis is performed. As
mentioned above, previous reports on the occurrence
of hypericin in H. scabrum were not consistent.
Different methods for hypericin determination
have been used in preceding works. Kitanov [4]
and Ayan et al. [5] used the methods involving
spectrophotometric assay for total hypericins, while
Tanker [1] separated the hypericins in ethanolic
extracts of aerial parts of H. scabrum by TLC. In our
case, we used the HPLC method for hypericin and
pseudohypericin determination, as adopted by
Zevakova et al. [6]. In addition, the biosynthesis of
secondary metabolites, like hypericin, may be
influenced by genetic, metabolic and environmental
parameters. Environmental factors, including climate,
topography and stage of plant development are
thought to affect or modulate the production of
hypericins. The difference between present and
previous results may also be due to the aforesaid
factors.
Morphologically, Hypericum plants are characterized
by the presence of different kinds of secretory
structures, including light glands, dark glands and
secretory canals. Dark glands are also known as
‘nodules’ or ‘black nodules’. These glands are the
most important secretory structures in Hypericum
plants. Recent investigations on the anatomy and
content of the secretory structures of Hypericum
species have confirmed that at least hypericins are
sequestered within the dark glands and the
occurrence of dark glands in an organ is regarded as
an accurate index of the presence of hypericins [10].
For example, in a previous study we found a close
relationship between dark gland density of leaves and
hypericin content in H. aviculariifolium Jaup. and
Spach subsp. depilatum (Freyn and Bornm.) Robson
var. depilatum, H. perforatum L. and H. pruinatum
Boiss. and Bal. [11]. The dark glands on the
vegetative organs of many species of Hypericum
have also been used by botanists as an important
distinguishing mark for the classification of this
genus [4]. In the present study, we observed that
leaves, sepals and petals, but not stems of H. scabrum
were covered by dark glands, indicating the presence
of hypericins and the absence of the dark glands in
stems should be the reason why neither hypericin nor
pseudohypericin were detected in that tissue. The
infrageneric section to which H. scabrum belongs is
the section Hirtella Stef. [4, 12] and the members of
c
a
a
b
c
b
bc
0
0,04
0,08
0,12
0,16
0,2
Leaf Reproductive
Pseudoypericin content (mg/g DW)
Vegetative
Floral budding
Full flower ing
Fresh fruiting
Mature fruiting
d
a
ab
bc
cd
0
0,04
0,08
0,12
0,16
0,2
Leaf Re productive
Hypericin content (mg/g DW)
Vegetative
Floral budding
Full flo wering
Fresh fruiting
Mature fruiting

Variation of hypericins in Hypericum scabrum Natural Product Communications Vol. 3 (2) 2008 243
this section are distinguished by the presence of dark
glands in leaves and floral parts. It is important
to note that two species from this section, H.
hyssopifolium L. [13] and H. lydium Boiss. [14]
have already been reported to contain hypericin.
Hence, detection of hypericins in different tissues of
H. scabrum supports the taxonomic position of the
section Hirtella Stef. within the genus Hypericum and
indicates the naphthodianthrones as chemical markers
of the phylogenetically more advanced sections of the
genus Hypericum.
Investigations of ontogenetic variation of secondary
metabolites from different plant species have
been carried out over several decades. In the present
study, ontogenetic changes in hypericin and
pseudohypericin content of leaves and reproductive
parts were found to be significant (P<0.01) and the
highest levels of both compounds were reached at the
floral budding stage. The results are in accordance
with those of Kazlauskas and Bagdonaite [15], who
reported that the highest accumulation of hypericin,
as well as rutin, quercetin and isoquercetin, was
observed during the development of flowering buds
and at flowering time in Lithuanian populations of
H. perforatum. Similarly, the highest content of
hypericin in stem, leaf and reproductive parts of H.
perforatum, H. pruinatum and H. aviculariifolium
was determined during flower ontogenesis [11].
In the present study, floral parts had the highest level
of hypericin and pseudohypericin in H. scabrum.
Likewise, in all earlier reports, the floral parts had the
highest hypericin concentrations in H. perforatum
[16], H. pruinatum and H. aviculariifolium [11].
Previous works report hypericin concentrations in H.
perforatum ranging from 0.01 to 3.87 mg/g, dry wt
from USA, Canada [16], Australia [7] and Turkey
[11]. Our values ranged from 0.015-0.18 mg/g, dry
wt for hypericin and 0.03-0.19 mg/g, dry wt for
pseudohypericin, depending on ontogenetic and
morphogenetic sampling. It can be concluded that
H. scabrum produces low/moderate quantities of both
hypericin forms when compared to H. perforatum, a
well known and commercial source of hypericins.
Experimental
Plant material: H. scabrum L. was identified by Dr
Hasan Korkmaz, Faculty of Science and Art,
Department of Biology, University of 19 Mayis,
Samsun-Turkey. A voucher specimen was deposited
in the herbarium of Ondokuz Mayis University
Agricultural Faculty (OMUZF # 64). Plants were
collected in the Maçka district of Trabzon province,
Turkey (40° 49΄ N; 39° 37΄ E; 270 m sea level)
between April and October 2005, at different stages
of plant development. The sampling site was not
grazed or mown during the sampling periods. The top
two-thirds of the plants was collected between 12:00
am and 13:00 pm. Sampling from this wild
population involved a randomized collection of 30
individuals at each phenological stage, and all aerial
parts were observed, using a light microscope, for the
presence of dark glands.
Shoots with leaves were harvested at the vegetative
stage. For the floral budding stage, only shoots with
floral buds were selected. At the full flowering stage,
only shoots with fully opened flowers were sampled.
At the fresh fruiting stage, the shoots that had green
capsules were collected. At the mature fruiting stage,
the shoots with dark brown capsules were harvested.
After collection, the plant materials were dissected
into reproductive, leaf and stem tissues and dried at
room temperature (20 ± 2°C). The dried materials
were bulked and subsequently assayed for hypericin
and pseudohypericin contents. It should be noted that
there were no leaves on the plants at the mature
fruiting stage.
Chemicals: Reference standards of hypericin and
pseudohypericin were purchased from ChromaDex
Inc., Laguna Hills, CA, USA. HPLC-grade
acetonitrile, acetone and methanol were purchased
form Caledon, Mississauga, ON, Canada.
Triethylammonium acetate was from Sigma-Aldrich
Canada, Oakville, ON, Canada.
Extraction and high performance liquid
chromatography (HPLC) analysis of hypericin and
pseudohypericin: The isolation and analytical
method used for hypericin and pseudohypericin was
that previously published by Murch et al. [17].
Briefly, the plant tissues were ground into fine
powder with a laboratory mill, and approximately
100 mg was transferred into an amber-colored 20 mL
vial. Extracts were prepared in 5 mL
acetone:methanol (50:50, v:v) with 30 min
sonification (Ultra-sonic FS-14 Sonicator; Fisher
Scientific, Nepean, ON, Canada). Samples were
centrifuged at 3000 rpm for 10 min (GS-6 series
centrifuge, Beckman Instruments Inc, Palo Alto, CA,
USA) to precipitate particulate matter, filtered using a
0.2 µm nylon syringe filter (Waters Chromatography
Inc., Mississauga, ON, Canada), and a 500 µL aliquot

244 Natural Product Communications Vol. 3 (2) 2008 Ayan et al.
of each sample was transferred into a clear glass
auto-sampler vial, which was sealed with a Teflon
coated aluminum lid to minimize contamination with
air. The clear glass vials were exposed to a light
source (15 cm distant from a 100W tungsten light)
for 30 min to complete the conversion of the proto
forms of hypericin and pseudohypericin, before
analysis. A 20 µL sample of the extract for each
hypericin form was injected into a Shimadzu 10AD
HPLC system consisting of an SCL-10A system
controller, SIL-10A auto injector, SPD-M 10AV
photodiode array detector at 588 nm, and a CTO-10A
column oven (Shimadzu, Canada) with separation on
a Phenomenex Hypersil C18 column (3.0 µm; 4.6 x
100 mm) with a C18 guard column (4 x 3 mm)
(Phenomenex, Torrance, CA, USA). The analytes
were separated isocraticaly using a mobile phase of
0.1 M triethylammonium acetate and acetonitrile
(33:67, v:v) at a flow rate of 1 mL min-1. Calibration
curves (r2 > 0.989) were used for quantification of
each compound. The limit of detection of hypericin
and pseudohypericin was 0.1µg/mL. Recovery of
hypericin was above 91% and pseudohypericin
recovery was consistently greater than 65%.
Concentrations of hypericin and pseudohypericin
were expressed as µg g-1 dry weight.
Data analysis: Data for hypericin and
pseudohypericin content of each plant tissue were
subjected to ANOVA, and significant differences
among mean values were evaluated using the Duncan
Multiple Range Test with MSTAT statistical software
(P<0.01).
Acknowledgement - Authors are grateful to Dr PK.
Saxena and Dr A. Alan, Department of Plant
Agriculture, University of Guelph, Guelph, Ontario
N1G 2W1, Canada for technical assistance.
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