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Effect of BAP on total hypericin production in

shoot cultures of Hypericum scabroides: An
endemic species in the Eastern Anatolia Region of
Turkey.

ARTICLE · NOVEMBER 2015

DOI: 10.5053/ejobios.2015.9.0.6

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Ahmet Onay
Dicle University
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 EurAsian Journal of BioSciences
Eurasia J Biosci 9, 46-51 (2015)
http://dx.doi.org/10.5053/ejobios.2015.9.0.6

Effect of BAP on total hypericin production in shoot

cultures of Hypericum scabroides: An endemic
species in the Eastern Anatolia Region of Turkey
Hilal Surmus Asan*, Hasan Cetin Ozen, Ahmet Onay, Nurettin Asan
Department of Biology, Faculty of Science, Dicle University, Diyarbakir, Turkey

*Corresponding author: hilalsuran@gmail.com

Research Note
Abstract
Background: Due to the therapeutic importance of hypericin, a number of Hypericum species are
being investigated. The aim of this study was to determine the effect of various BAP (6-
benzylaminopurine) concentrations on seed germination, shoot proliferation, and total hypericin in
a tissue culture of Hypericum scabroides which is endemic to the Eastern Anatolia region, Turkey.
Material and Methods: Hypericum scabroides specimens were collected from Eastern Anatolia
(Elazig, Turkey). The effects of various BAP concentrations (0.0 (control), 0.5, 1.0, and 2.0 mg/L) on
seed germination, shoot multiplication, and the accumulation of total hypericin were determined
using tissue cultures of H. scabroides. Murashige and Skoog (MS) medium was used for germination
and shoot cultures. Measurements of total hypericin were taken using a UV spectrophotometer.
Results: The best germination rate (59.2%) was obtained using hormone-free MS medium (control
group). Apical tips of freshly germinated seedlings were proliferated on the MS medium
supplemented with various BAP concentrations (0.5, 1.0, and 2.0 mg/L) and the control group
(without BAP). The highest number of shoots (42.7 shoot/explant) and longest shoot length (2.50
cm) were obtained on MS medium supplemented with 2.0 mg/L BAP. Total hypericin was found in
trace amounts and it was found that the total hypericin was not affected by the concentration of
BAP.
Conclusions: Our results showed that increasing concentrations of BAP stimulated shoot
multiplication but did not affect seed germination rates or total hypericin in in vitro cultures of H.
scabroides.
Keywords: Hypericum scabroides, hypericin, shoot culture, BAP.

Abbreviations: BAP: 6-Benzylaminopurine; DPPH: di(phenyl)-(2,4,6-trinitrophenyl)-iminoazanium; IAA: Indole-3-

acetic acid; IBA: Indole-3-butyric acid; Kin: Kinetin (6-Furfurylaminopurine); MS: Murashige and Skoog; NAA: 1-
Naphthaleneacetic acid; TDZ: Thidiazuron.

Asan HS, Ozen HC, Onay A, Asan N (2015) Effect of BAP on total hypericin production in shoot
cultures of Hypericum scabroides: An endemic species in the Eastern Anatolia Region of Turkey.
Eurasia J Biosci 9: 46-51.

http://dx.doi.org/10.5053/ejobios.2015.9.0.6 ©EurAsian Journal of BioSciences

acid, chlorogenic acid, hyperoside, quercetin and

INTRODUCTION
Plants of the genus Hypericum have been used as
traditional medicines in various parts of the world
(Yazaki and Okada 1994). These species are used in
Turkish folk medicine as sedatives, antiseptics and
antispasmodics (Baytop 1999). The Hypericum genus
of Guttiferae is represented in Turkey by 89 species
(Davis 1988) and Hypericum scabroides Robson &
Poulter is an endemic plant species to the Eastern
Anatolia region (Baytop 1984). In particular,
Hypericum perforatum L. has been studied to
determine the biological activity of its extracts
(Shelton 2009). H. perforatum extracts contain
bioactive substances such as hyperforin,
adhyperforin, hypericin, pseudohypericin, caffeic
rutin (Kasper et al. 2010). Among these, hypericins,
hyperforins and flavonoids possess a wide range of
biological properties (Patocka 2003). Hypericins
(hypericin and pseudohypericin) have been found
only in Hypericum species (Kitanov 2001). Hypericins
have antidepressive, antimicrobial, antiviral,
antitumor, and anti-inflammatory properties (Karioti
and Bilia 2010).
Plant tissue cultures enable the propagation of
plants that are rare, economically or medicinally
Received: March 2015
Received in revised form: August 2015
Accepted: October 2015
Printed : November 2015

46
EurAsian Journal of BioSciences 9: 46-51 (2015)
important, and can be used for the production of
plant secondary metabolites by changing the
nutrient and hormonal media culture conditions
(Collin 2001). Recent studies have demonstrated
that in-vitro culturing is an option for the
multiplication and production of secondary
metabolites of Hypericum species such as H.
perforatum var. angustifolium DC (Mulinacci et. al.
2008), H. perforatum (Franklin and Dias 2011),
Hypericum sampsonii Hance and H. perforatum (Liu et
al. 2007), Hypericum hirsutum L. and Hypericum
maculatum Crantz (Coste et al. 2011), Hypericum
undulatum Schousb. ex Willd. (Rainha et al. 2012),
Hypericum rumeliacum Boiss. (Danova et al. 2010)
and Hypericum retusum Aucher (Namli et al. 2010).
Several studies have been conducted on H.
scabroides. Mansour et al. (2014) reported that H.
scabroides contained trace amounts of
pseudohypericin, hypericin, chlorogenic acid, rutin,
hyperoside, isoquercitrin, and kaempferol, and
additionally determined its anti-inflammatory
activity. Kizil et al. (2008) found that extracts of H.
scabroides exhibit radical scavenging and
antimicrobial activity (Kizil et al. 2004). In addition,
the chemical composition of fatty acid methyl esters
(FAMEs) of the flowering tips of H. scabroides was
identified by Özen and Bashan (2003). However, the
in-vitro culturing of H. scabroides has not been
reported thus far.
The aim of this study was to develop, for the first
time, a multiple-shoot production procedure and to
determine the hypericin content in shoot cultures of
H. scabroides, which is endemic to the Eastern
Anatolia region.
MATERIALS AND METHODS
Plant material
Specimens of Hypericum scabroides Robson &
Poulter (Clusiaceae) were collected from Eastern
Anatolia (Elazığ/Turkey) (38° N, 39° E and 1.269 m
above sea level ) in June 2010 and were identified by
Prof. Dr. A. Selcuk Ertekin (Dicle University). Voucher
specimens (No: DUF-2512) were deposited in the
herbarium of the Department of Biology, Faculty of
Sciences, Dicle University, Diyarbakır (Turkey).
47
Asan et al.
Seed germination and shoot proliferation
To initiate in-vitro cultures, seeds of H. scabroides
were washed with tap water for 5-10 min followed
rinsed with 70% (w/v) ethanol for 30 s. for pre-
sterilization. Then, the seeds sterilized via
immersion in a 5% (w/v) commercial bleach solution
(NaOCI) for 10 min and three times rinsed with
sterile distilled water for 5 min to remove the
artifacts of NaOCI. The sterilized seeds were
germinated on a 50 mL hormone-free MS medium in
Magenta vessels. The medium was adjusted to pH
5.8 before autoclaving (25 min at 121°C, 1 atm). The
seeds were germinated under a photoperiod of
(16/8 h) light, at a light intensity of 36 μmol s-1 m-2 in
a growth chamber at 25±2°C.
The effect of various BAP treatments (0.5, 1.0,
and 2.0 mg/L) and a control group (without BAP)
were determined on the germination of H. scabroides
seeds. For the establishment of auxenic cultures, the
apical tips of the germinated seedlings were
cultured to shoot proliferation a containing various
BAP concentrations (0.5, 1.0, and 2.0 mg/L) 30 g/L of
sucrose and 5.5% agar. After five w, the regenerated
shoots were cut into segments and cultured to new
proliferation medium including 2.0 mg/L, 30 g/L of
sucrose and 5.5% agar.
Percentages of seed germination, length of
shoots and the number of new shoots were recorded
after 5 w of culture.
Determination of total hypericin
At the end of the 5th w of culture, in vitro grown
shoots were washed with distilled water in order to
remove the residues of agar. Then, they were air-
dried for three d. For hypericin extraction, ground
samples (0.2 g) were extracted with 10 mL methanol,
then sonicated (Sanyo MSE-Soniprep 150, U.K.) for 5
min. to remove their chlorophyll contents. The
process was repeated twice.
After extraction, the solvent was removed in
vacuo. After removing chloroform, the samples were
re-extracted with methanol (10 mL) in the sonicator.
With methanol extraction process was repeated
three times and the methanol extracts were
combined and then evaporated (IKA, RV 10 DS 99).
The final samples were dissolved in methanol. Then,
supernatant (1 mL) was placed in a test tube.
EurAsian Journal of BioSciences 9: 46-51 (2015)
Absorbance was measured at 589 nm using a UV
spectrophotometer (UV-160, Shimadzu, Kyoto,
Japan). The hypericin content was calculated and
expressed as the percentage hypericin content in a
given amount of dry material. Three measurements
were made for each sample and the mean value was
calculated. Total hypericin was calculated and
expressed as the percentage of total hypericin in a
given amount of dry material.
Statistical analysis
All the analyses were performed using SPSS 15.0
for Windows. The values of different percentages
were expressed as the mean values. Significance was
determined by analysis of variance (ANOVA) and
comparisons for two groups were made with
Duncan’s multiple range test. The level of
significance was defined as P≤0.05.
RESULTS
Seed germination and shoot proliferation
The surface sterilized seeds were germinated in
the MS media were supplemented with different
BAP concentrations and the control group within 7-
10 d. The best germination rate (59.2%) was
obtained on MS medium with the control group. The
germination rate decreased to 13.9, 19.7 and 29.5%
at concentrations of 0.5, 1.0, and 2.0 mg/L BAP,
respectively (Table 1). The shoot tips and nodal bad
explants were cultured on MS medium containing 1
mg/L BAP and 3% sucrose for the production of
stock cultures.
The effect of BAP concentrations on shoot
proliferation were presented in Table 1.
Multiplication of the seedlings of H. scabroides was
successfully performed in all treatments, including
the three BAP concentrations, within 2-3 w of
culturing. The maximum number of shoots (42.7
shoot/explant) and the longest shoot length (2.50
cm) were achieved on the MS medium supplemented
with 2.0 mg/L BAP (Table 1). The other treatments
studied produced significantly lower shoot number
and shoot lengths.
Hypericin content
The shoots proliferated with the use of MS
medium containing BAP (0.5, 1.0, and 2.0 mg/L) and
Asan et al.
the control group (without BAP) were analyzed to
determine their total hypericin. Very low levels of
hypericin were found when using the MS medium
supplemented with BAP and hypericin was not
detected in the control group.
DUSCUSSION
The cell, tissue and organ culture systems are
used for in-vitro growth of plants and production of
medicinal phytochemicals (Verpoorte et al. 2002).
Through in-vitro systems, medicinally important
plants can be grown in sterile, standardized
conditions and are free from contamination. In this
study, the best germination rate (59.2%) was
obtained on the cultures of the MS medium without
BAP. Likewise, the seed germination rates of H.
retusum Aucher (Namli et al. 2010) and Hypericum
triquetrifolium Turra (Karakas et al. 2009) were found
to be higher (~90.0 and 69.2%, respectively) on the
MS medium without BAP. Among the cytokinins (BAP
and Kin), BAP was more efficient than Kin in
promoting shoot formation (Ćellàrovà et al. 1992).
The same effect was observed when leaves (Murch
et al. 2002) or excised seedling parts (Pretto and
Santarem 2000) were used as explants for the in-
vitro culture of H. perforatum. The formation of new
shoots was observed in all MS media supplemented
with various concentrations of BAP, except in the
hormone-free medium, which indicates that H.
scabroides is responsive to plant growth regulators
after two subcultures. Our results showed that
increasing the concentration of BAP stimulated
shoot proliferation. The treatment of shoots with
BAP altered the morphology of shoots: resulting in
internode shortening, differentiation of smaller
leaves, and suppression of rooting; however, it also
increased the proliferation rate (Coste et al. 2011).
Acemi et al. (2012) and Karakas et al. (2009) obtained
the highest shoot number of H. triquetrifolium Turra
and Amsonia orientalis Decne. on the MS medium
including BAP (0.5 and 2.0 mg/L). Organ or cell
cultures have emerged as a viable route for
biosynthesising phytochemicals when limited time
and space is an available ( Jeong et al. 2009). There
are several reports suggesting that the culturing of
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EurAsian Journal of BioSciences 9: 46-51 (2015) Asan et al.

Table 1. Effects of BAP on seed germination, average number and length of shoots and total hypericins of shoots in
Hypericum scabroides.

Values followed by different small letters in columns are significantly different (P≤0.01) according to Duncan's multiple range test
(Mean±SE).
N.D.: Not detected

shoots in MS medium supplemented with BAP
enhances the production of hypericins (Coste et al.
2011), the total phenolic and flavonoid contents
(Danova et al. 2010), and hyperforin (Charchoglyan
et al. 2007). In addition, it has also been reported
that H. perforatum plantlets produce hypericin and
pseudohypericin in the presence of indole-3-butyric
acid (IBA) and indole-3-acetic acid (IAA) (Gadzovska
et al. 2005). In our study, the spectrophotometric
results showed that total hypericin was not
enhanced when BAP concentration was increased.
Total hypericin was detected in trace amounts at the
BAP concentrations tested and was not detected at
all in the control group. The spectrophotometric
analysis of H. scabroides showed that total hypericin
was not enhanced when the BAP concentration was
increased. Mansour et al. (2014) also reported that H.
scabroides contained trace amounts of hypericin. The
best germination rate (59.2%) was obtained using
hormone-free MS medium (control group). Apical
tips of freshly germinated seedlings were
proliferated on the MS medium supplemented with
various BAP concentrations (0.5, 1.0, and 2.0 mg/L)
and the control group (without BAP). The highest
number of shoots (42.7 shoot/explant) and longest
shoot length (2.50 cm) were obtained on MS medium
supplemented with 2.0 mg/L BAP. Total hypericin was
found in trace amounts and it was found that the
total hypericin was not affected by the
concentration of BAP.
ACKNOWLEDGMENTS
This study was supported by research grants from
the Dicle University Research Council (DUAPK,
project number 05-FF-22).

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