Acta Bot. Croat.

79 (1), 78–86, 2020 CODEN: ABCRA 25

DOI: 10.37427/botcro-2020-010 ISSN 0365-0588
eISSN 1847-8476

Chemical and morphological diversity among wild

populations of Hypericum aviculariifolium Jaub.
et Spach subsp. depilatum (Freyn et Bornm.) N.
Robson var. depilatum
Cuneyt Cirak1*, Aysel Özcan2, Emine Yurteri2, Dursun Kurt1, Fatih Seyis2
1
Ondokuz Mayis University, Vocational High School of Bafra, Samsun, Turkey
2
Recep Tayyip Erdoğan University, Faculty of Agriculture and Natural Sciences, Department of Field Crops, Rize, Turkey

Abstract – In this study, the chemical and morphological diversity among eleven wild populations of Hypericum
aviculariifolium Jaub. et Spach subsp. depilatum (Freyn et Bornm.) N. Robson var. depilatum, an endemic Turkish
species was studied. These populations were investigated for their contents of hypericin, pseudohypericin, hyper-
forin, the chlorogenic, neochlorogenic, caffeic and 2,4-dihydroxybenzoic acids, hyperoside, quercitrin, isoquerci-
trin, avicularin, 13,118 biapigenin, (+)-catechin and (–)-epicatechin as well as for their morphological traits, in-
cluding density of leaf light and dark glands, leaf area, leaf length/width ratio and plant height. The top two-thirds
of the plants representing thirty individuals was harvested at full flowering from eleven sites and analyzed for the
content of bioactive compounds by high-performance liquid chromatography after being dried at room tempera-
ture. Morphological characterization of the wild populations was performed on twenty randomly selected indi-
viduals from each plant-growing locality. The content of the tested compounds, except for caffeic acid and avicu-
larin, and some morphological traits, namely, the density of leaf translucent glands and black nodules and leaf
area varied significantly with the investigated populations. It was observed that hypericin and pseudohypericin
contents were connected positively with leaf black nodule density, but negatively with leaf area and the contents
of hyperforin, quercitrin and 13,118-biapigenin were correlated positively with leaf translucent gland density.
Data presented here could be useful in determining future targets for further wide-ranging studies on this en-
demic species as well as in identifying superior germplasm in terms of high chemical content.

Keywords: 13,118-biapigenin, chemical and morphological diversity, hyperforin, hypericin, Hypericum aviculariifolium,
phenolic acids, pseudohypericin, quercitrin

Introduction
Hypericum genus (Hypericaceae) includes over 400 spe- traditionally been used for sedative, wound healing, disin-
cies with world-wide distribution and is one of the 100 larg- fectant and spasmolytic preparations in Turkish folk medi-
est genera including twenty two percentage of angiosperm cine with the local names of “sarıkantaron, askerotu, kılıç
variety (Carine and Christenhusz 2010). Among its mem- otu, kanlıot and kuzu kıran”. Turkey is a centre of great ex-
bers, only Hypericum perforatum L. has been investigated tensity for the Hypericum genus and according to Güner et
widely so far. This species, herbal preparations of which have al. (2012) there are a total of 96 Hypericum taxa in the Turk-
been utilized largely as a medicine in the treatment of mild ish flora, of which 46 are endemic. Hypericum aviculariifo-
to moderate depression, especially over the last three de- lium Jaub. et Spach subsp. depilatum (Freyn et Bornm.) N.
cades (Ng et al. 2017), is considered officinal. Hypericum spe- Robson var. depilatum [syn. Hypericum origanifolium var.
cies are well-known medicinal plants and have been used for depilatum (Freyn et Bornm.) N. Robson, sensu WFO 2019]
centuries as traditional healing agents owing to their large is one of these endemic species (Davis 1988), growing wild
number of pharmacological activities. All these species have in arid, stony and limy areas of Northern Turkey. The distri-

* Corresponding author e-mail: kalinor27@gmail.com

78 ACTA BOT. CROAT. 79 (1), 2020

 CHEMICAL DIVERSITY AMONG HYPERICUM AVICULARIIFOLIUM POPULATIONS

bution range of this endemic species is very localized by its quercitrin, quercetin and amentoflavone were also reported
exogenously dormant seeds (Cirak et al. 2007a). Its shoots significantly to promote the antidepressant (Tusevski et al.
are up to 30 cm in length with yellow inflorescence and typi- 2018), neuroprotective (Silva et al. 2008), antioxidant and
cal dark glands on all aerial parts (Fig. 1). Results of recent antimicrobial (Zorzetto et al. 2015) activities.
studies documenting the antibacterial (Gül et al. 2017) and A great number of Hypericum species have been subject-
antioxidant (Maltas et al. 2013) properties of H. aviculariifo- ed to studies, documenting their chemical content/compo-
lium subsp. depilatum var. depilatum indicate that this en- sition from Turkish flora as well as other growing localities
demic species can be a substitute for widely known H. per- of the world such as Brazil, Iran, Jordan, Serbia, Italy, Por-
foratum L. tugal, Tunisia, Peru and Lithuania (Cirak et al. 2016, and
Naphthodianthrones, principally represented by hyperi- references therein).
cin and psudohypericin, the phloroglucinol derivatives ad- Results from the former works revealed significant differ-
hyperforin and hyperforin, flavonoids such as rutin, hypero- ences attributed to concentrations of the ingredients among
side, quercetin and quercitrin, phenolic acids and essential the various Hypericum species from several taxa (Cirak et
oils with a wide range of bioactivities are considered to be al. 2016); diversified populations of the same species from
the principal constituents of Hypericum plant taxa (Zhao et various geographic regions (Nogueira et al. 2008), various
al. 2015). In the past, hypericins were indicated as the main phenological stages of the same species (Abreu et al. 2004)
chemicals responsible for the antidepressant activity of Hy- and between various shoots as well, regenerated from the
pericum extracts; however, recent studies have proved that same in vitro culture (Cellarova et al. 1994). However, the
antidepressant activity is revealed synergistically by both hy- precise pattern of bioactive compound accumulations inside
pericins and hyperforin (Nabavi et al. 2018). Hyperforin and and among members of Hypericum genus is not fully under-
its derivatives were also reported to induce antitumor, anti- stood. It is not explained to what extent the chemical content
angiogenic and neuroprotective activities (Ma et al. 2018). and composition bear upon specific genotypes within spe-
Although hyperforin and hypericin have been indicated as cies. It is also not clarified how far plant geographic origin
providing essential support to the pharmacological activ- affects the spectrum of phytochemicals.
ities of Hypericum-derived products, some other ingredi- In our previous works, we reported H. aviculariifolium
ents such as chlorogenic acid, quinic acid, hyperoside, rutin, subsp. depilatum var. depilatum to include hypericins, hyper-
forins, various flavonoids and phenolics as
hyperoside, quercetine, chlorogenic acid, ru-
tin, isoquercetine and quercitrine (Cirak et
al. 2007b, 2013). However, population vari-
ability of the chemical compounds as well
as of morphologic traits has not yet been
studied with respect to the endemic species.
Hence, in the present work, our intention has
been to specify for the first time the regional
variability in the content of hypericin, pseu-
dohypericin, hyperforin, the chlorogenic,
neochlorogenic, caffeic and 2,4-dihydroxy-
benzoic acids, hyperoside, quercitrin, iso-
quercitrin, avicularin, (+)-catechin, (–)-epi-
catechin and 13,118-biapigenin as well as
five morphological traits including light and
dark gland density on leaves, leaf area, leaf
length/width ratio and plant height as well
as the correlations between the chemical and
morphological data among H. aviculariifo-
lium subsp. depilatum var. depilatum popu-
lations from eleven localities in the Middle
Black Sea geographic region of Northern
Turkey. In addition, neochlorogenic, caffeic
and 2,4-dihydroxybenzoic acids, 13,118-bi-
apigenin, isoquercitrin, avicularin, (+)-cate-
chin and (–)-epicatechin were not detected
Fig. 1. Hypericum aviculariifolium subsp. depilatum var. depilatum plant flowering in
previously in this endemic species. Hereby,
its native habitat (a), and its aerial parts with typical dark glands, namely leaves and we also report the first occurrence of the cor-
stems (b), floral buds (c) and flowers (d, e). Dark and translucent glands on leaf under responding compounds in H. aviculariifoli-
dissecting microscope (f). Scale bars = 5 cm (a) and 1 cm (b–f). um subsp. depilatum var. depilatum.

ACTA BOT. CROAT. 79 (1), 2020 79

CIRAK C, ÖZCAN A, YURTERI E, KURT D, SEYIS F

Materials and methods Preparation of plant extracts

Air-dried plant material was mechanically ground us-
Plant materials
ing a laboratory mill to obtain a homogeneous drug pow-
The plant materials were described in our previous stud- der. Samples of about 0.1 g (weighed with 0.0001 g precision)
ies (see Cirak et al. 2013, Cirak and Bertoli 2013). The spe- were extracted in 10 mL of 100% methanol by ultrasonica-
cies were identified by Dr. Samim Kayikci, Mustafa Kemal tion at 40 °C for 60 min in an ultrasonic bath. The prepared
University, Faculty of Arts and Sciences, Department of Bi- extracts were filtered through a membrane filter with a pore
ology, Turkey. Voucher specimens were deposited in the size of 0.22 µm (Carl Roth GmbH, Karlsruhe, Germany) and
herbarium of Ondokuz Mayis University Vocational High kept in a refrigerator at 4 °C until analysis. The extracts for
School of Bafra and the numbers of the voucher specimens naphthodianthrones analyses were exposed to light under
are given in Tab. 1. xenon lamp (765 W/m2) for 8 min for the photoconversion
of protohypericins into hypericins.
Experimental procedures
HPLC Analyses and quantification
The aerial parts of H. aviculariifolium subsp. depilatum
Separation of the flavanoids and phenolic acids tested
var. depilatum plants exemplify 30 shoots were harvested at
was carried out by using an RP-18 (5 µm, 250  4.0 mm) col-
flowering stage from eleven localities in Middle Black Sea
umn in a Shimadzu LC-2030C-3D HPLC device equipped
geographic region of Northern Turkey (Tab. 1). The top two
with a DAD detector. The binary gradient elution method
thirds of the plants was reaped between 14:00 pm and 15:00
was used for detection of corresponding compounds. The
pm. Conditions on the day of collection were clear and sun-
mobile phase A consisted of water acidified with 0.3% phos-
ny at all sites and temperatures varied between 28 and 30 °C. phoric acid as eluent A and acetonitrile containing 0.3%
The plant materials were dried at room temperature (20 ± phosphoric acid as eluent B. The elution profile was used
2 °C), and subsequently analyzed for chemical in gredients as following: 0-10 min 10% B, 10-30 min 25% B, 30-38 min
by HPLC. 60% B, 38-45 60% B and 45-45.01 min 1% B. Flow rate was
Morphological characterization of plants was made, 0.6 mL min–1 at 25 °C column temperature. The extract in-
as described previously in our previous study (Cirak et al. jection volume was 10 µL. The calibration of components
2007b), on 20 randomly selected plants from each grow- was obtained at 203 – 280 – 320 – 360 nm wavelengths using
ing locality according to plant height, leaf dark and trans- 5, 10, 20, 50, 100 and 200 ppm standard solutions.
lucent gland density, leaf area, and leaf length/width ratio. For hypericin, pseudohypericin and hyperforin, the same
Plant height was measured from the flowering crown of the device, Shimadzu LC-2030C-3D HPLC equipped with a
primary stem to the base of the plant. Leaf area, leaf length/ DAD detector, was used. Separation of these chemicals was
width ratio and the number of dark and light spheroid nod- carried out using an RP-18 (5 µm, 250  4.0 mm) column.
ules, were measured on 10 leaves of each selected plant from The mobile phase of isocratic solution consisted of ethyl ac-
11 different sites. The number of leaf dark and translucent etate, aqueous 0.1 M sodium dihydrogen phosphate solution
glands was counted using a dissecting microscope (Fig. 1). was adjusted to pH 2.0 by using phosphoric acid and meth-
For leaf area and leaf length/width ratio calculations, leaves anol (39:41:160 v/v). The flow rate was 1 mL min–1 at 40 °C
were placed on aphotocopier, held flat and secure and cop- column temperature. The volume of extract injected was 20
ied onto an A3 sheet (at 1:1 ratio). Placom Digital Planim- µL. The calibration of components was obtained at wave-
eter (Sokkisha Planimeter Inc., Model KP-90) was utilized lengths 207 and 589 nm using 1, 5, 10, 20, 50 and 100 ppm
to measure the actual leaf area of the copy. Leaf width (cm) standard solutions. Analytical standards used for HPLC
was measured from tip-to-tip at the widest part of the lamina analysis and validation values of the method are shown in
and leaf length (cm) was measured from lamina tip to the On-line Suppl. Tab. 1. The standards are also described in
point of petiole intersection along the midrib. On-line Suppl. Tab. 2.

Tab. 1. Geographical data and annual climatic conditions of Hypericum aviculariifolium subsp. depilatum var. depilatum-growing locali-
ties from Northern Turkey. BMYO stands for “Bafra Meslek Yüksekokulu”, Vocational High School of Bafra, Turkey; Popul. - population;
Latit - latitude; Long - longitude Elev - elevation, T - mean annual temperature; P - mean annual precipitation
Collection Voucher
Popul. Latit (N) Long (E) Elev (m) T (°C) P (mm) Habitat
date no.
1 June 03, 2018 BMYO # 27/1 40° 54΄ 35° 25΄ 1053 08.78 765 Rocky and open slopes
2 June 03, 2018 BMYO # 27/2 40° 54΄ 35° 38΄ 1075 08.52 782 Rocky and open slopes
3 June 03, 2018 BMYO # 27/3 40° 55΄ 35° 25΄ 1293 08.07 821 Rocky and open slopes
4 June 03, 2018 BMYO # 27/4 40° 55΄ 35° 24΄ 1452 07.52 875 Rocky and open slopes
5 June 03, 2018 BMYO # 27/5 40° 50΄ 35° 09΄ 952 09.29 922 Igneous slopes and rock ledges
6 June 04, 2018 BMYO # 27/6 40° 50΄ 35° 10΄ 882 11.53 937 Pinus woodland
7 June 04, 2018 BMYO # 27/7 40° 49΄ 35° 09΄ 989 10.64 932 Arid pasturelands
8 June 04, 2018 BMYO # 27/8 40° 45΄ 35° 08΄ 1243 09.11 856 Stony riverside
9 June 04, 2018 BMYO # 27/9 40° 45΄ 35° 07΄ 1373 08.77 872 Stony riverside
10 June 04, 2018 BMYO # 27/10 40° 45΄ 35° 08΄ 1262 08.92 727 Stony riverside
11 June 04, 2018 BMYO # 27/11 41° 25΄ 36° 58΄ 441 12.64 982 Igneous slopes and rock ledges

80 ACTA BOT. CROAT. 79 (1), 2020

 CHEMICAL DIVERSITY AMONG HYPERICUM AVICULARIIFOLIUM POPULATIONS

Tab. 2. Mean contents (mg g–1 DM) of different compounds: hypericin (a), pseudohypericin (b), hyperforin (c), chlorogenic acid (d),
neochlorogenic acid (e), caffeic acid (f), 2,4-dihydroxybenzoic acid (g), 13,118-biapigenin (h), hyperoside (i), isoquercitrin (j), querci-
trin (k), avicularin (l), (+)-catechin (m), (–)-epicatechin (n) in Hypericum aviculariifolium subsp. depilatum var. depilatum populations
(Popul.) located in Northern Turkey. Values are means of three replications and those, followed by different small letters in each column
are significantly different (P < 0.01) according to Duncan’s Multiple Range test. Se = standard errors
Compounds
Popul.
a b c d e f g h i j k l m n
1 0.14 d 1.17 d 0.25 c 12.13 b 0.47 c 0.25 0.26 b 1.30 b 0.11 c 0.51 a 2.53 e 0.65 1.38 b 1.05 b
2 0.28 c 1.93 c 0.21 c   9.91 c 0.69 b 0.55 0.42 a 1.46 b 0.24 b 0.63 a 2.97 e 0.65 1.26 b 0.79 c
3 0.39 b 2.10 c 0.07 de   7.64 c 0.45 c 0.26 0.09 c 1.29 b 0.73 a 0.38 b 3.65 d 0.65 1.00 bc 0.69 c
4 0.31 c 3.53 b 0.11 d   2.11 e 0.19 e 0.32 0.14 c 1.09 b 0.01 c 0.15 d 2.16 e 0.64 0.47 c 0.21 d
5 0.27 c 1.67 d 0.15 d   3.45 de 0.27 d 0.23 0.11 c 1.58 b 0.01 c 0.16 d 2.34 e 0.64 0.62 c 0.11 d
6 0.30 c 2.16 c 0.03 e   4.47 d 0.23 de 0.25 0.09 c 1.21 b 0.36 b 0.29 c 5.93 c 0.65 0.68 c 0.19 d
7 0.44 b 2.64 c 0.01 e   4.56 d 0.31 d 0.27 0.09 c 0.98 c 0.01 c 0.24 c 5.00 c 0.64 0.69 c 0.12 d
8 0.53 a 4.16 a 0.27 c 11.81 b 0.38 c 0.24 0.58 a 1.59 b 0.38 b 0.42 b 5.42 c 0.66 1.65 b 1.94 a
9 0.55 a 4.85 a 0.34 c   3.31 d 0.18 e 0.26 0.12 c 2.01 a 0.13 c 0.22 c 3.85 d 0.66 0.68 c 0.21 d
10 0.58 a 4.89 a 0.65 b   8.86 c 0.42 c 0.28 0.29 b 1.97 a 0.44 b 0.41 b 6.57 b 0.66 1.10 b 1.11 b
11 0.31 c 2.16 c 1.63 a 21.71 a 0.85 a 0.27 0.22 b 2.09 a 0.29 b 0.45 b 7.10 a 0.66 2.42 a 1.82 a
Se 0.042 0.393 0.140   1.720 0.063 0.027 0.048 0.179 0.068 0.046 0.534 0.002 0.173 0.202

Data Analysis hydroxybenzoic acid and quercitrin were accumulated at sig-

Data of secondary metabolites contents and morphologi- nificantly higher levels by plants of population-8 (0.58 and
cal characters of plant material were subjected to one-way 7.10 mg g–1 DM, respectively). The highest accumulation level
analysis of variance (ANOVA) and significant differences of hyperoside and isoquercitrin was reached in plants of pop-
among mean values were tested with the Duncan Multiple ulation 3 and population 2 (0.73 and 0.63 mg g–1 DM, respec-
Range Test (P < 0.01). Correlation analysis was performed tively) (Tab. 2). The present results also indicate that H. avicu-
to clarify the relationship between the chemical and mor- lariifolium subsp. depilatum var. depilatum accumulates lower
phological data, and principal component analysis (PCA) concentrations of hyperforin, hypericin, psedohypericin, neo-
was carried out to elucidate the relationship of investigated chlorogenic acid, hyperoside, isoquercitrin, (+)-catechin and
populations regarding the chemical and morphological di- (-)-epicatechin, comparable concentrations of avicularin and
versity they exhibited by using the statistical software pack- 13,118-biapigenin and higher concentrations of chlorogenic
age XLSTAT2010 Trial Version. PCA analysis is the two- acid, caffeic acid, 2,4-dihydroxybenzoic acid and quercitrin
dimensional visualization of the position of investigated when compared to H. perforatum, a well known commercial
accessions relative to each other. The principal components source of the compounds examined (Tab. 3).
represent the axes which are the orthogonal projections for Significant variations (P < 0.01) were also observed in
the values representing the highest possible variances in the mean values of leaf dark and translucent gland density and
case of PC1 and PC2. The obtained data were used to cre- leaf area among the investigated populations; however, leaf
ate scatter plot diagrams (Backhaus et al. 1989). Therefore, length/width ratio and plant height did not vary with plant
a factor analysis was performed, whereby each variable was growing localities (Tab. 4). Results of correlation analysis in-
used to calculate relationships between variable and inves- dicated an evident connection between leaf dark gland densi-
tigated factors. Based on the obtained data the cluster den- ty/leaf area and hypericin/pseudohypericin contents of plants
drogram was created.
and leaf translucent gland density and hyperforin, quercitrin
and 13,118-biapigenin contents of plants. No significant cor-
Results relation was determined among the rest of the morphologi-
cal traits and secondary metabolites tested (On-line Suppl.
Results of the present study indicate that the contents of
Tab. 3).
hypericin, pseudohypericin, hyperforin, the chlorogenic, ne-
ochlorogenic and 2,4-dihydroxybenzoic acids, hyperoside, The number of translucent glands and black nodules on
quercitrin, isoquercitrin, (+)-catechin, (-)-epicatechin and leaf and leaf area varied considerably with the investigated
13,118-biapigenin in plants differed greatly by populations (P populations. Leaf dark gland density was significantly high-
< 0.01) whereas caffeic acid and avicularin were accumulated er in plants of the populations 10, 9 and 8 whose hypericin
at similar levels in all growing localities. Plants from popula- and pseudoypericin contents were also found to be signifi-
tion-11 supplied the highest accumulation level of hyperfor- cantly higher. In a similar way, population 11, which accu-
in, chlorogenic acid, neochlorogenic acid, 13,118-biapigenin, mulated the highest hyperforin, quercitrin and 13,118-biapi-
(+)-catechin and (–)-epicatechin (1.63, 21.71, 0.85, 2.09, 2.42 genin contents, was found to be superior to the others with
and 1.82 mg g–1 DM, respectively) whereas hypericin and respect to leaf translucent gland density. Positive and signifi-
pseudohypericin were yielded in the highest level by plants of cant relationships were determined between leaf dark gland
population-10 (0.58 and 4.89 mg g–1 DM, respectively). 2,4-di- density and hypericin (r2 = 0.86, P < 0.01) / pseudohypercin
ACTA BOT. CROAT. 79 (1), 2020 81
CIRAK C, ÖZCAN A, YURTERI E, KURT D, SEYIS F

Tab. 3. Comparison of the chemical content (mg g–1 DM) in Hypericum aviculariifolium subsp. depilatum var. depilatum (in the present
study) and Hypericum perforatum, globally known commercial species of Hypericum genus (compiled from various relevant sources).
H. aviculariifolium
Compound subsp. depilatum H. perforatum References
var. depilatum*
Hyperforin 0.21–1.63 8.35–11.50 Greeson et al. 2001, Maggi et al. 2004, Couceiro et al. 2006
Hypericin 0.14–0.58 0.01–2.77 Sirvent et al. 2002, Southwell and Bourke 2001, Bagdonaite et al. 2010
Psedohypericin 1.17–4.89 0.05–6.75 Ayan and Cirak 2008, Bagdonaite et al. 2010, Büter and Büter 2002,
Bagdonaite et al. 2012
Chlorogenic acid 2.11–21.71 1.11–2.19 Maggi et al. 2004, Cirak et al. 2007b, c
Neochlorogenic acid 0.42–0.85 3.34–4.25 Jürgenliemk and Nahrstedt 2002
Caffeic acid 0.23–0.55 <0.01 Patocka 2003, Nahrstedt and Butterweck 1997
2,4–dihydroxybenzoic acid 0.09–0.58 trace Jürgenliemk and Nahrstedt 2002
Hyperoside 0.01–0.73 2.07–7.69 Maggi et al. 2004, Bagdonaite et al. 2012
Quercitrin 2.16–7.10 0.05–4.77 Martonfi and Repcak 1994, Radusiene et al. 2004
Isoquercitrin 0.22–0.63 3.19–6.99 Jürgenliemk and Nahrstedt 2002
Avicularin 0.64–0.66 0.32–0.96 Wu et al. 2002, Wei et al. 2009
13,118–biapigenin 1.21–2.09 1.78–2.65 Jürgenliemk and Nahrstedt 2002, Nahrstedt and Butterweck 1997
(+)–catechin 0.62–2.42 1.41–8.70 Ploss et al. 2001, Kalogeropoulos et al. 2010
(–)–epicatechin 0.11–1.94 20.6–118.9 Ploss et al. 2001, Kalogeropoulos et al. 2010
*The lowest and highest contents of the corresponding compounds, observed in the present study.

(r2 = 0.92, P < 0.01) contents and leaf translucent gland density A two-dimensional (2D) visualization of the relative
and hyperforin (r2 = 0.75, P < 0.05), quercitrin (r2 = 0.71, P < position of the phytochemicals tested was created by using
0.05) and 13,118-biapigenin (r2 = 0.77, P < 0.05) contents. As the values of the principal components (the bioactive com-
for the leaf area, the highest and lowest values were detected pounds examined here) relative to the investigated popu-
in population-1 and population-10 (11.38 and 4.82 cm2, re- lations. This was provided by utilizing the principal com-
spectively) yielding the highest and lowest levels of hyperi- ponent analysis (PCA). A biplot was created to see the
cin and pseudohypericin accumulations. Likewise, the popu- correlations between samples and the investigated traits. Re-
lations producing higher amounts of hyperforin, quercitrin sults of biplot analysis revealed that the investigated popula-
and 13,118-biapigenin had lower values of leaf area. Leaf area tions could clearly be differentiated according to their chem-
was found to be negatively correlated with the hypericin (r2 = ical contents and morphological traits. Populations 3, 4, 5, 6,
0.81, P < 0.01) and pseudohypericin (r2 = 0.86, P < 0.01) con- 7 and 9 were different in plant height and populations 1 and
tents of plant material. 2 were different with regards to caffeic acid content and leaf

Fig. 2. Principal component analysis biplot showing populations (1–11) and vectors of the chemicals and morphological traits based on
20 samples for each population.

82 ACTA BOT. CROAT. 79 (1), 2020

 CHEMICAL DIVERSITY AMONG HYPERICUM AVICULARIIFOLIUM POPULATIONS

Tab. 4. Mean values of the morphological characters evaluated in Hypericum aviculariifolium subsp. depilatum var. depilatum popula-
tions located in Northern Turkey. Values are means of three replications and those, followed by different small letters in each column are
significantly different (P < 0.01) according to Duncan’s Multiple Range test. Se = standard errors.
Population Dark glands (per mm2) Light glands (per mm2) Leaf area (cm2) Leaf length/width Plant height (cm)
1 0.25 e 5.81 c 11.38 a 2.20 35
2 0.30 e 5.01 c 10.39 b 2.71 40
3 0.32 e 4.20 d 8.33 c 2.27 37
4 0.48 c 4.83 d 6.35 e 2.29 38
5 0.30 d 5.07 c 10.62 b 2.11 36
6 0.48 c 4.34 d 7.99 c 2.36 42
7 0.47 c 3.42 e 6.88 e 2.33 41
8 0.64 b 6.42 b 7.59 d 2.04 35
9 0.66 b 6.51 b 5.67 f 2.36 42
10 0.84 a 6.84 b 4.82 g 2.04 39
11 0.49 c 7.30 a 7.69 d 2.30 34
Se 0.055 0.371 0.631 0.056 0.878

area. With the first two principal components, 68.19% of the heterogeneity of Hypericum plants from different origins is
present variation could be explained (Fig. 2). reported to influence the pharmacological activity of plant
Further, the investigated populations were differentiated extracts significantly and to pose a great risk to the standard-
into two main groups namely, group A including popula- ization of final Hypericum-derived products (Costa et al.
tions 1, 2, 3, 4, 5, 6, 7, 9 and group B consisting of popula- 2016). Hence, there have been many investigations regard-
tions 8, 10, 11, with respect to their chemical contents and ing population variability of bioactive compounds from Hy-
morphological traits on the dendogram, created by biplot pericum species. In H. perforatum L., the most common and
analysis. As shown in Fig. 3, populations 1 and 2 and popula- commercially recognized species of the genus, wild popula-
tions 4 and 7 from group A were found to be similar chemi- tions of Turkey (Cirak et al. 2007c), Canada, Australia, Ar-
cally and morphologically. menia and Lithuania (Bagdonaite et al. 2010, and references
therein) are shown to yield significantly different amounts
of hyperforin, pseudohypericin and hyperforin. Essential oil
composition is reported to differ significantly in accordance
with the geographic origin of wild accessions of H. pulchrum
L., H. humifusum L., H. perfoliatum L. and H. linarifolium
Vahl. (Nogueira et al. 2008).
Considerable differences were determined in concentra-
tions of hypericins, hyperforin and various phenolics such as
rutin, hyperoside, amentoflavone and quercetin in the four
wild accessions of H. triquetrifolium Turra from Turkey. In a
similar way, eleven populations of H. orientale L. and five wild
populations of H. montbretii Spach and H. lydium Boiss. are
reported to yield different quantities of hypericins, hyperfo-
rins, phenolic acids and several flavonoids such as hypero-
side, quercetin, amentoflavone, rutin, avicularin isoquerci-
trin and quercitrin (Cirak et al. 2015, and references therein).
Fig. 3. Dendrogram showing the differentiated groups of Hyperi- Results of previous studies indicated the geographic ori-
cum aviculariifolium subsp. depilatum var. depilatum populations gin of plants as a distinct factor influencing the observed
(Group A represents populations 1, 2, 3, 4, 5, 6, 7 and 9; Group chemical variation among wild Hypericum populations. In a
B represents populations 8, 10 and 11) regarding their chemical similar way, we observed significant differences in accumula-
contents and morphological traits.
tion levels of 14 bioactive compounds among H. aviculariifo-
lium subsp. depilatum var. depilatum from eleven geographic
Discussion origins in the present work. Two populations of this endemic
species are also reported to yield quantitatively and qualita-
Among the factors contributing to the variations in the tively different amounts of essential oil (Cirak and Bertoli
phytochemical accumulation in the Hypericum genus, the 2013). The investigated populations varied with the main
geographic origin of plants is of considerable importance as environmental factors creating different growing conditions
the main environmental factors of a plant-growing habitat as they were located in different places of Northern Turkey
such as altitude, temperature, soil etc. influencing synthesis as shown in Table 1. The wide variation observed in accu-
and accumulation of a given bioactive compound were di- mulation levels of the bioactive compounds tested among
verse mainly according to the growing sites. The chemical the populations could somewhat be attributed to adaptive
ACTA BOT. CROAT. 79 (1), 2020 83
CIRAK C, ÖZCAN A, YURTERI E, KURT D, SEYIS F

strategies of wild plants to changing environmental factors. glands of leaves in H. stellatum, H. annulatum Moris, H. an-
It is also possible to evaluate the observed chemodiversity drosaemum, H. kouytchense, H. monogynum, H. kalmianum
among populations as a result of genetic distinctness, but da- L., H. balearicum L. and H. canariense (Kuchariková et al.
ta on the connection between the genetic and phytochemical 2016b). However, no attempt has been undertaken so far to
structures in Hypericum spp. is scant and results are typically investigate the correlation between number of translucent
contradictory. For example, He and Wang (2013) reported glands and content of hyperforin. We report here for the first
only a partial correlation between chlorogenic acid, querce- time the significant and positive relationship between leaf
tin, rutin and hyperoside concentrations and genetic data translucent gland number and hyperforin accumulation lev-
of 12 wild H. perforatum populations from China. Howev- els which can be useful to explain sites of hyperforin synthe-
er, Tonk et al. (2011) detect significant connections between sis and function of the bioactive compound within the genus
hypericin content and random-amplified polymorphic DNA Hypericum. As opposed to hypericins and hyperforin, it may
(RAPD) data for 19 field-grown H. perforatum clones indi- not be feasible to ascertain a pattern of localization for flavo-
cating the necessity for further chemical and molecular re- noids as data on the localization of them on the aerial parts
searches on the genus Hypericum to differentiate exactly the are discrepant and vary with species. Hyperoside, isoquer-
genetic and environmental effects on the monitored chemi- citrin, quercetin and quercitrin were accumulated mainly
cal variation among wild populations. in leaf dark glands of H. olympicum L., H. perforatum, and
The comparison of eleven wild populations of H. avicu- H. rumeliacum Boiss. and rutin was accumulated only in
lariifolium subsp. depilatum var. depilatum revealed an intra- leaf black nodules of H. maculatum Crantz and H. erectum
specific diversity in the distribution of light and dark glands Thunb. However quercetin, the most prevalent flavonoid is
corresponding with the accumulation of hypericins, hyper- reported to localize mainly in leaf translucent glands of H.
forin, quercitrin and 13,118-biapigenin in the present study. rumeliacum (Kusari et al. 2015, Kuchariková et al. 2016a).
Hypericum plants are categorized generally by three types Hyperoside and isoquercitrin, interestingly, are also report-
of secretory structures namely, translucent glands, black or ed to accumulate mainly in leaf translucent glands in H. kal-
dark nodules and secretory canals (Kimáková et al. 2018). mianum (Kuchariková et al. 2016a). By contrast, hyperoside,
Among Hypericum chemicals, hypericins are reported to isoquercitrin and quercitrin are accumulated primarily in
accumulate most extensively in the black nodules of aerial both dark and translucent glands in H. humifusum leaves
parts (Kornfeld et al. 2007) and previous results have proved and quercetin and quercitin were accumulated principally
the localization of hypericin and pseudohypericin in the in translucent glands and non-secretory structures in leaves
dark glands of plant aerial parts in all species producing hy- of the species, namely, H. androsaemum, H. kouytchense, H.
pericins (Kusari et al. 2015, Kuchariková et al. 2016a, b). Be- monogynum, H. stellatum (Kuchariková et al. 2016a). In the
sides, the absence of dark glands in aerial parts is described present study, we detected a positive and significant connec-
as an accurate indication of the absence of hypericins in sev- tion between leaf translucent gland density and the content
eral species of Hypericum such as H. brasiliense Choisy, H. of quercitrin and 13,118-biapigenin indicating translucent
caprifoliatum Cham. et Schltdl., H. carinatum Griseb. (Fer- glands as the main site for the accumulation of correspond-
raz et al. 2002), H. androsaemum L., H. kouytchense Levl., ing compounds in H. aviculariifolium subsp. depilatum var.
H. monogynum L., H. stellatum N. Robson, and H. canar- depilatum.
iense L. (Kuchariková et al. 2016a). In previous researches, Principal component and cluster analyses are favored
a close relationship is observed between dark gland number means for characterization of genotypes and their grouping
of leaf and total hypericin content of plants in H. perfora- on similarity. PCA is a beneficial statistical tool for the differ-
tum (Southwell and Campbell, 1991) and H. lydium (Cirak entiation of plant materials, giving information on the varia-
2006). In accordance with the previous results, we observed tion in chemical content/composition of several species. A
a positive and significant relationship with high r2 values be- combination of the two statistical tools provides broad in-
tween leaf dark gland density and the plant content of hy- formation of the traits making significant contributions to
pericin and pseudohypercin in the present work. We also genetic diversity in crops. Biplot is another widely utilized
detected that plant content of hypericins is negatively cor- procedure for graphical display of accession groups with the
related with leaf area, as reported by Cirak et al. (2007c) for aim of searching for the relationships among agro-morpho-
H. perforatum. It may be speculated that in the enlargement logical characters in several cultivars (Malik et al. 2014). In
of leaf area concludes in a decrease of dark gland density the present study, we used the above mentioned statistical
and that the inverse connection between leaf area and the tools to evaluate the chemical and morphological data of
content of hypericins might be attributed to this decrease. eleven H. aviculariifolium subsp. depilatum var. depilatum
As for hyperforin, Soelberg et al. (2007) report that concen- populations from Turkish flora.
trations of the bioactive compound in translucent glands of In conclusion, chemical and morphologic characteriza-
leaves surpassed that of original leaves by more than 100% tions of wild plant populations seem to be first step to de-
in H. perforatum. Based on these results, the authors indicate fine superior germplasm and to provide improved chemi-
translucent glands as the main site of hyperforin accumula- cal profiles. Results of the present study indicate substantial
tion as confirmed latterly by Kusari et al. (2015). Hyperforin, correlations between hypericin, pseudohypericin, hyperfo-
besides, is reported to accumulate primarily in translucent rin, quercitrin and 13,118-biapigenin contents and densi-
84 ACTA BOT. CROAT. 79 (1), 2020
 CHEMICAL DIVERSITY AMONG HYPERICUM AVICULARIIFOLIUM POPULATIONS

ty of leaf dark and translucent glands and leaf area. Thus, Data presented here could also be useful in determining the
these morphological characters could be utilized as selec- forthcoming goals for further wide-ranging studies on this
tion criteria in identifying germplasm that is superior with endemic species as well as enriching data of current litera-
regard to high contents of the corresponding compounds. ture on Hypericum chemistry.

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