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\begin{document}

\title{Investigation of the change in essential oil components with the
composition of fatty acids from Hypericum heterophyllum Vent., an
endemic species in Turkey}



\author[1]{Belgin COSGE ŞENKAL}%
\author[1]{Tansu Uskutoglu}%
\affil[1]{Yozgat Bozok University }%


\vspace{-1em}



  
  \date{May 28, 2024}


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\selectlanguage{english}
\begin{abstract}
Turkey is an important center in terms of Hypericum species and 49 of
the 119 taxa are endemic. Hypericum heterophyllum is one of these
endemics. This study was carried out in order to determine some chemical
properties of H. heterophyllum. In dry capsules, 14\% protein and 23\%
oil were detected. This oil has been found to be rich in unsaturated
fatty acids (oleic and linoleic). The highest essential oil rate
(0.09\%) in flowering dry herb was recorded in the period before
flowering. Also, germacrene-D, bicyclogermacrene, \selectlanguage{greek}δ-\selectlanguage{english}cadinene,
spathulenol, \selectlanguage{greek}α-\selectlanguage{english}guaiene, and valencene in herbage essential oil and
limonene, cis-ocimene, \selectlanguage{greek}β-\selectlanguage{english}myrcene, and \selectlanguage{greek}α, β-\selectlanguage{english}pinene in dry capsule were
determined as main components. It has been noted that both the essential
oil content and the components show variation depending on the
developmental stages of the plant and the part used.%
\end{abstract}%



\sloppy


\textbf{1. INTRODUCTION}

There are 482 \emph{Hypericum} species distributed in different
geographies of the world from the equatorial zone to the Nordic
countries in the north (Crockett \& Robson, 2011; C\selectlanguage{polish}ı\selectlanguage{english}rak \& Kurt, 2014).
Species of the genus \emph{Hypericum} have been used in many parts of
the world for many years because of their healing, bactericidal,
diuretic, anti-inflammatory and sedative effects (C\selectlanguage{polish}ı\selectlanguage{english}rak \& Kurt, 2014).
Especially, several extracts from \emph{H. perforatum} are used as a
drug in Europe (Brutovska et al., 2001). Turkey is an important center
in terms of \emph{Hypericum} species and 49 of the 119 taxa are endemic
(Guner et al., 2012). One of these endemics is \emph{H.
heterophyllum}which is a perennial, shrub form, and blooming in August.
Its habitat is reported as \emph{Pinus} woodlands (1200-1600 m altitude)
(TUBIVES, 2019). This species is known locally as ``Yaraotu'' in Turkey
(Guner et al., 2012).

Secondary metabolites such as essential oils, alkaloids, glycosides,
steroids, saponins, resins etc. are invaluable effects phytochemicals
(Baydar, 2013). \emph{Hypericum} species contain a large number of
secondary metabolites of at least 11 different classes, including
naphthydiantrons, flurogonol derivatives, flavonoids, organic acids,
essential oils, amino acids, xanthones, tannins, proxyanidins and other
water-soluble components (Greeson et al., 2001; Tanaka \& Takaishi,
2006). However, Patocka (2003) refers to the pharmacological effects
of\emph{Hypericum} extracts to hyperfine, flavonoids, and essential
oils, which are hyperfine and sedohipericin, and flurugonol derivatives,
with naphthydiantron pigments. Essential oils secreted by aromatic
plants are stored in droplets in some specific metabolic cells and
tissues such as secretion hairs, secretion channels and resin channels
(Baydar, 2013). They are obtained from different organs of the plant
such as leaves, flowers, and stalks. It is known that essential oils
have various biological activities. The essential oil isolated from
\emph{H. heterophyllum} exhibited antifungal activity (Cakir et al.,
2004). The aqueous extracts prepared from aerial parts of this species
shown clastogenic and genotoxic effects in human lymphocytes cultures
(Ocal \& Eroglu, 2012). Also, it was observed that \emph{H.
heterophyllum} had significant impact on several bacteria
(\emph{Bacillus} sp.,\emph{Esherichia coli} , \emph{Klebsiella} sp.,
\emph{Pseudomonas} sp.,\emph{Staphylococcus} sp., and \emph{Salmonella}
sp.) (Tanker et al., 1980; Akgoz, 2015). The amount of bioactive
substances composed in secondary metabolites (essential oils, saponins,
and tannins etc.) varies significantly according to the plant's organs
(morphogenetic variability), life cycles of the plant (ontogenetic
variability) and the time of collection of the plant (ontogenetic
variability) (Baydar, 2013; Ramakrishna \& Ravishankar, 2011).

Although there is a great deal of research on the chemical composition
of \emph{Hypericum} species, there are limited studies on the
distribution of fatty acids. In studies on fatty acid composition, it
has been observed that the oil obtained from different parts of some
species in this genus are rich in palmitic, linoleic and linoleic fatty
acids (Ozen \& Bashan, 2003; Ozen et al., 2004).

This study was carried out in order to determine some chemical
properties of \emph{H. heterophyllum} , which is an endemic species.

\textbf{2.EXPERIMENTAL PROCEDURES}

\textbf{2.1. Location information}

The aerial parts and dry capsules of \emph{H. heterophyllum} were
collected from natural area (Study Area: Inside the Yozgat Bozok
University Campus Area; Altitude: 1340 m; Locality:
9\textsuperscript{o}46\selectlanguage{ngerman}\textsuperscript{´}48,04\textsuperscript{´´}N-34\textsuperscript{o}48\textsuperscript{´}02,34\textsuperscript{´´}
E) in Yozgat/Turkey. According to the climate data of the area where
plant samples were collected for many years, total precipitation was
562.5 mm, average temperature was 9.1 \textsuperscript{o}C, average
highest temperature was 14.6 \textsuperscript{o}C, average lowest
temperature was 4 \textsuperscript{o}C, average sunshine time was 82.0
hours and average number of rainy days was 113.5 (TSMS, 2020).
Identification of plants collected was performed in Biology Laboratory
at Yozgat Bozok University/Turkey.

\textbf{2.2. Plant material}

The aerial parts were collected in four different stages as before
flowering (BF1, in May), beginning flowering (BF2, in June), 50\% of
flowering (50\%F, in July), and full flowering (FF, in July). Dry
capsules were collected in October. In the laboratory, seeds were
removed from dry capsules.

\textbf{2.3. Determination of Chemical Properties}

Within the scope of the study, the following chemical properties were
examined:

\textbf{1-} Capsule oil content and fatty acids composition

\textbf{2-} Capsule protein content

\textbf{3-} Capsule and herbage essential oil contents and chemical
compositions

\textbf{2.3.1 Capsule oil content and fatty acids composition}

For the oil content of the capsule, dry capsules were ground and 2 grams
of sample were extracted in a Soxhlet apparatus containing 80 mL of
petroleum ether as a solvent. The solvent was then evaporated and the
oil content of the sample was determined gravimetrically (AOAC, 2005).
0.1 g oil sample was dissolved in 5 ml hexane. 0.5 ml 0.2N KOH (prepared
in methanol) was added. After shaking well, it was kept in the dark for
a while. Centrifugation was applied. 1 ml was taken from the upper phase
and placed in the vials. Finally, the device was injected with a
syringe. The fatty acid compositions of the oil were detected with
GC/MS-QP 2010 (Schimadzu) in the Science and Technology Application and
Research Center of Yozgat Bozok University (Turkey). The chromatographic
method is given in Table 1.

\textbf{2.3.2 Capsule protein content}

Total nitrogen was determined in 0.5 grams ground dry capsule according
to Kjeldahl method and the percentage (\%) nitrogen ratio was multiplied
by 6.25 coefficient and the crude protein content (\%) was determined
according to Kacar \& Inal (2008) principles. Essential oil, oil and
protein analyses were performed in triplicate.

\textbf{2.3.3 Capsule and herbage essential oil contents and chemical
compositions}

The amount of essential oil in drug herbage and dry capsule were
determined by hydro Clevenger distillation device. 100 grams of sample
for drug herbage and 50 grams of sample for dry capsule were used. The
samples were ground in the blender and 10 times pure water was added,
and distilled for 3h. Essential oil values (\%, v/w) were calculated by
volume over dry mater. The obtained essential oils were taken into dark
colored flasks and stocked at 4\textsuperscript{o}C in a refrigerator
until they were analyzed (Damyanova et al., 2016).

The chemical components of the essential oil samples from four
collection times were defined by gas chromatography (GC) and gas
chromatography-mass spectrometry (GC/MS) analyses. The analyses were
performed using a Shimadzu GC-2010 Plus and Shimadzu GC/MS-QP2010 Ultra
instruments. The 0. 1mI oil sample was dissolved in 10ml hexane and
shaken vigorously. It was kept in the dark for 1-2 hours. The sample was
taken into vials and given to the device. The information about the
chromatographic method is given below:

Column: RXI-5MS (0.25um X 30m X 0.25mm)

Scan range: 35-600 m/z

Split ratio: 30

Oven temperature: 60\textsuperscript{o}C for 1 min followed by a
temperature rise at a 4 \textsuperscript{o}C/min rate to
250\textsuperscript{o}C (held for 4 min).

Flow rate: 1.50 ml/min

The essential oil components were identified by comparing their mass
spectra, retention times, retention indices, and relative to
C\textsubscript{5}-C\textsubscript{40} n-alkanes, the FFNSC 1.2 and
W9N11.1 mass spectral library, and the literature (Babushok et al.,
2011).

\textbf{2.4. Data analysis}

The numerical data were stated as means ± standard error of the mean.
The amount of essential oil was made with three replications. Analysis
of variance was performed using TARIST package program, and the means
were compared using LSD test (\emph{p} \selectlanguage{english}[?]0.05) (Acikgoz et al., 2004).

\textbf{3. Results and Discussion}

The investigated chemical properties of \emph{H. heterophyllum} are
summarized in Figure 1 and Figure 2. In dry capsules, 14\% protein and
23\% oil were detected.

\textbf{3.1. Fatty acid compositions}

The data related to fatty acid compositions of oil obtained from capsule
of \emph{H. heterophyllum} are presented in Table 2. This oil consists
of 12\% saturated and 87\% unsaturated fatty acids. In the oil, 13
saturated fatty acids were determined, of which the values of the
others, except C\textsubscript{16:0} and C\textsubscript{18:0} acids,
were recorded below 1\%. On the other hand, monounsaturated fatty acids
constituted 48\% of the oil and C\textsubscript{18:1} acid had a share
of 48\% in the six identified fatty acids. As the polyunsaturated fatty
acid, only C\textsubscript{18:2} acid was determined (Figure 3).

The limited research is available on the fatty acid composition
of\emph{Hypericum} taxa. Some research results related to the subject
are summarized in Table 3 and Table 4. It is seen that the fatty acid
composition of the oil obtained from \emph{Hypericum} taxa varies
according to the plant and the part of the plant used. For example,
C\textsubscript{10:0} acid was 20.74\% in the aerial part of the\emph{H.
ericoides} ssp. \emph{roberti} taxon, 1.34\% in the aerial part of the
\emph{H.tomentosa} species (Hosni et al.,2017), and 3.62\% in the
flowers of the \emph{H. uniglandulosum} species (Turkoglu et al., 2015).
C\textsubscript{8:0}, C\textsubscript{10:0}, C\textsubscript{11:0},
C\textsubscript{12:0}, C\textsubscript{14:0}, C\textsubscript{16:0},
C\textsubscript{18:0}, C\textsubscript{20:0}, C\textsubscript{22:0}, and
C\textsubscript{24:0} acids were determined as saturated fatty acids in
oils obtained from \emph{Hypericum} taxa. However, it was observed that
the ratio of C\textsubscript{16:0} was higher in these (Table 3). The
oil obtained from different parts of \emph{Hypericum} taxon has been
found to contain both mono (C\textsubscript{16:1},
C\textsubscript{18:1}, C\textsubscript{22:1}, and C\textsubscript{24:1})
and polyunsaturated (C\textsubscript{18:2}, C\textsubscript{18:3}, and
C\textsubscript{20:3}) fatty acids, and the amount of
C\textsubscript{18: 1}, C\textsubscript{18: 2}, and C\textsubscript{13:
3} acids was higher (Table 4).

Oil was obtained from aerial parts, leaves, seeds and flowers
of\emph{Hypericum} taxa, but no studies on the fatty acid composition of
the fruit capsule were found among the studied literature. The fatty
acid composition of oils is the most important factor that determines
the commercial uses. Fatty acids and their chemical derivatives play an
important role in almost every area of modern life. The main fatty acids
found in oils and fats are grouped into saturated fatty acids,
monounsaturated fatty acids and polyunsaturated fatty acids (Mariod et
al., 2011). The prevailing fatty acids in vegetable oils are unsaturated
fatty acids, and also saturated fatty acid ratio is less than 20\%.
Vegetable oils are divided into six groups as ``C\textsubscript{12:0},
C\textsubscript{16:0}, C\textsubscript{18:1}, C\textsubscript{18:2},
C\textsubscript{18:3}, and C\textsubscript{22:1}'' considering the
dominant fatty acid. Oleic-linoleic acid group is the largest and most
important group among the oils (Nas et al., 2001). According to our
results, \emph{H. heterophyllum} capsule oil is in the oleic-linoleic
acid group. Oleic and linoleic acids have important uses in our daily
lives (Table 5). Generally, C\textsubscript{16:0} acid is the most
common saturated fatty acid found in all vegetable oils and animal fats.
It is relatively common in C\textsubscript{18:0} acid. Similar results
were taken from \emph{H. heterophyllum} capsule oil (Table 2).

\textbf{3.2. Essential oils}

The aerial parts of \emph{H. heterophyllum} were recorded to contain
0.09+-0.01\%, 0.087+-0.006\%, 0.05+-0.00\% and 0.077+-0.02\% essential oil
for BF1, BF2, 50\%F, and FF stages, respectively (Figure 4). Essential
oil contents recorded in the BF1, BF2 and FF stages did not show
statistically (\emph{p} \selectlanguage{english}[?]0.05) significant differences. However, the
highest and lowest essential oil content was obtained from BF1 and 50\%F
stage, respectively. In the previous study, it was stated that
hydrodistillation of the dried aerial parts of \emph{H.
heterophyllum}yielded 0.09\% of the essential oil (Cakir et al., 2004).
The amount of the essential oil from aerial parts of \emph{H. aucheri} ,
\emph{H. montbretii} , \emph{H. kazdaghensis} , and \emph{H. perforatum}
was 0.28\%, 0.22\%, 0.17\% and 0.23\% in the before flowering, 0.27\%,
0.20\%, 0.24\% and 0.33\% in the beginning of flowering, 0.33\%,
0.23\%,0.26\% and 0.37\% in the full flowering, 0.02\%, 0.03\%, 0.02\%,
and 0.05\% in the capsule during, respectively (Pasa, 2013). In the
four\emph{Hypericum} species, the highest essential oil ratio was
obtained from the plants harvested in full flowering period. In our
study, the highest rate of essential oil was recorded in BF2, followed
by FF.

The chemical components of \emph{H. heterophyllum} essential oils from
four different development stages was given in Table 6 The total of 15,
15, 13, and 16 components representing 92.19\%, 80.08\%, 90.13\%, and
84.24\% of the total essential oils were detected in the BF1, BF2,
50\%F, and FF stages, respectively.

In this study, germacrene-D, bicyclogermacrene, \selectlanguage{greek}δ-\selectlanguage{english}cadinene, spathulenol,
\selectlanguage{greek}α-\selectlanguage{english}guaiene, and valencene having significant biological activities were
found to be main components of essential oils obtained from different
ontogenetic stages of \emph{H. heterophyllum} (Table 6 and Table 8). In
the study carried out by Cakir et al (2004), in the essential oil
of\emph{H. heterophyllum} , 35 compounds, representing 99.4\% of the
total essential oil, were determined, and isocaryophyllene (17.1\%),
\selectlanguage{greek}α-\selectlanguage{english}pinene (11.6\%), \selectlanguage{greek}δ-\selectlanguage{english}cadinene (9.5\%), \selectlanguage{greek}γ-\selectlanguage{english}muurolene (8.2\%), \selectlanguage{greek}γ-\selectlanguage{english}cadinene
(5.5\%), n-decane (5.8\%), and \selectlanguage{greek}β-\selectlanguage{english}Caryophyllene (4.5\%) were recorded as
major compounds in this essential oil. Although there is similarity
between these findings and present study, there are some differences.
Essential oil components have been reported to be affected by many
intrinsic (genetic, plant origin, type of plant part, stage of
development or seasonal sampling period etc.) and extrinsic factors
(environmental factors such as climate and habitat conditions, sowing
date, cultivation conditions, and postharvest techniques such as drying
methods and extractions, distillation time, and conditions of analysis)
(Moghaddam \& Mehdizadeh, 2017).

The variation of the main components has been given in Figure 5. The
highest concentrations of germacrene-D were recorded in the BF1, 50\%F,
and BF2 stages, respectively. This component showed a significant
decrease by average 3 times in the FF stage. Bicyclogermacrene reached
the maximum concentration in the BF1 stage. The amount of this compound
reduced approximately to half in the 50\%F and BF2 stages, and it was
not detected in the FF stage. Although the highest amount of \selectlanguage{greek}δ-\selectlanguage{english}cadinene
was recorded in the FF period, similar rates were obtained in BF1 and
50\%F stages. But a decrease of about 7\% was observed in the BF2 stage.
The highest ratio of spathulenol was recorded in the 50\%F stage,
followed by the BF2 stage. The lowest ratio was obtained from BF1 stage.
The amount of valencene being among the minor components in the BF1, BF2
and 50\%F stages was found to be 9.76 \% in the FF stage. \selectlanguage{greek}α-\selectlanguage{english}Guaiene was
detected only in the essential oil in the FF stage (Table 6).
Significant differences in the concentrations of the main components
of\emph{H. heterophyllum} essential oil were determined according to the
developmental stages. A similar situation was observed in the minor
components of the essential oil such as \selectlanguage{greek}β-\selectlanguage{english}caryophyllene, \selectlanguage{greek}α-\selectlanguage{english}humulene,
aromadendrene, viridiflorol, globulol, salvial-4(14)-en-1-one,
isospathulenol, tau-muurolol, \selectlanguage{greek}α-\selectlanguage{english}amorphene, and \selectlanguage{greek}α-\selectlanguage{english}cadinol (Table 6).

In terms of the effect of ontogenetic variability on essential oil
components, the full flowering stage was more effective in \emph{H.
perforatum} and \emph{H. aucheri} species (Pasa, 2013). The amount of
essential oil and the changes in its chemical composition during
ontogenesis are specific to each taxon (N\selectlanguage{ngerman}émeth, 2005). The findings from
previous studies showed that there may be similarities and differences
in the its chemical composition and amount of essential oil of various
species at different phonological stages or harvesting time
in\emph{Menhta aquatic} L. (Andro et al., 2013), \emph{Origanum
vulgare}L. (Chauhan et al., 2013), \emph{Ocimum basilicum} L.
(Lemberkovics et al., 1998), \emph{Cuminum cyminum} L. (Moghaddam et
al., 2015), and\emph{Thymus} \emph{capitatus} L. (Casiglia et al.,
2015).

The timing of the harvest or collection of the herbal crops is one of
the most important factors affecting the quality of the essential oils
obtained from them. The therapeutic properties of herbal drugs are
related to the bioactive substances they contain. The amount of
bioactive substances composed of secondary metabolites shows significant
changes according to the development stage of plant. For this reason,
the drug producer must first of all know the bioactive substance
exchange of the medicinal and aromatic plant very well and gather the
drug which is the richest of the active substances (Baydar, 2013).
Essential oils are the most important of other volatile secondary
metabolites derived from medicinal and aromatic plants. Therefore,
obtaining high essential oil yields with the most desirable chemical
compounds is very important for industrial purposes. The selection of
appropriate phonological stage can be help researchers to fulfill this
requirement (Afshari \& Rahimmalek, 2018).

In the dry capsules, 0.087±0.015\% essential oil was detected. In the
obtained essential oil, 23 components were determined, which make up
99.50\% of the essential oil. \selectlanguage{greek}α-\selectlanguage{english}Pinene had the highest value with
33.28\% among these components. This component was followed by \selectlanguage{greek}β-\selectlanguage{english}pinene
(17.80\%), \selectlanguage{greek}β-\selectlanguage{english}myrcene (9.30\%), limonene (5.95\%), and cis-ocimene
(5.45\%), respectively. In addition, trans-caryophyllene, \selectlanguage{greek}α-\selectlanguage{english}terpineol,
carvacrol, copaene, verbenone and trans- \selectlanguage{greek}β -\selectlanguage{english}ocimene have been recorded
as other important components (Table 7). No studies on the capsule
essential oil content and composition of \emph{H. heterophyllum} have
been found in the literature review. The essential oils examined were
found to be rich in the major components that exhibit various biological
activities (Table 8).

\textbf{4. Conclusion}

The oil obtained from dry capsules has been found to be rich in
unsaturated fatty acids (oleic and linoleic). Considering the usage
areas of these two fatty acids, the oil obtained from Hypericum capsules
has the potential to be evaluated in different areas. Significant
effects of different development periods on herbage essential oil rate
and composition were determined. The chemical composition of essential
oil in medicinal and aromatic plants is an important factor that
determines quality. The amount and composition of essential oil
contained in plants is an indicator of the economic value of that
product. Considering the change of bioactive substances in plants, it
should be well known in which development period the plant will be
collected/harvested. According to result of the literature searches,
germacrene-D, bicyclogermacrene,\selectlanguage{greek}\emph{δ} -\selectlanguage{english}cadinene, spathulenol,
\selectlanguage{greek}\emph{α} -\selectlanguage{english}guaiene, and valencene in herbage essential oil and limonene,
\emph{cis} -ocimene,\selectlanguage{greek}\emph{β} -\selectlanguage{english}myrcene, and \selectlanguage{greek}α, \selectlanguage{english}\selectlanguage{greek}\emph{β} -\selectlanguage{english}pinene in dry
capsule were determined that they exhibited important biological
activities. A lot of information obtained from this study, in which some
plant and chemical properties of \emph{H. heterophyllum} , which is an
endemic plant, is examined, is the first. Therefore, the findings
obtained will form an important basis for future studies.

\textbf{Conflict of Interest} The authors declare that they have no
conflict of interest.

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