Plant Foods for Human Nutrition

https://doi.org/10.1007/s11130-019-0716-3

ORIGINAL PAPER

Phytochemical Properties of Satureja kitaibelii, Potential Natural

Antioxidants: a New Insight
Kristina Gopčević 1 & Slavica Grujić 2 & Jelena Arsenijević 3 & Ivanka Karadžić 1 & Lidija Izrael-Živković 1 &
Zoran Maksimović 3

# Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
Satureja kitaibelii Wierzb. ex Heuff. has a great importance in Serbian ethnopharmacology/herbal traditional medicine, as well as
a flavoring food additive. Ethanol extract of aerial parts of Satureja kitaibelii analyzed by liquid chromatography-mass spec-
trometry revealed the presence of 18 compounds among which the most abundant were phenolic acids, flavonoids, jasmonic acid
derivatives and rosmanol. The extracts were rich in total phenolics and flavonoid contents, while rosmarinic acid was the
dominant compound (18.30–29.52 mg/g). As assessments of antioxidant properties of natural extracts are important because
of their growing use in medicine and food industry, antioxidant activity of ethanol extracts of Satureja kitaibelii was analyzed by
several assays. The half maximal scavenging capacity (SC50) of 2,2′-diphenyl-1-picrylhydrazyl ranging from 71.20 to 125.65 μg/
mL, the total antioxidant capacity from 272.37 to 714.12 mg ascorbic acid/g, and ferric ion reducing antioxidant power ranging
from 0.74 to 1.94 μmol Fe/mg, clearly imply a significant antioxidant potential of Satureja kitaibelii. The extracts inhibit growth
of Micrococcus luteus and Pseudomonas aeruginosa with inhibition zones 20–30 and 16–26 mm, respectively. Antioxidant and
antibacterial activity of compounds identified in extracts suggest a great potential for Satureja kitaibelii application as valuable
food ingredient.

Keywords S. Kitaibelii . Ethanol extracts . LC-PDA-MS analysis . Antioxidant activity . Natural antioxidants

Abbreviations Rt Retention time

DPPH 2,2-diphenyl-1-picrylhydrazyl TPC Total phenolic content
TPTZ 2,4,6-tris(2-pyridyl)-s-triazine TFC Total flavonoid content
LC-MS Liquid chromatography – mass spectrometry TAC Total antioxidant capacity
RA Rosmarinic acid GA Gallic acid
ESI Electron spray ionization AA Ascorbic acid
PDA Photo diode array detector FRAP Ferric ion reducing antioxidant power
UV Ultra violet

Electronic supplementary material The online version of this article

(https://doi.org/10.1007/s11130-019-0716-3) contains supplementary
material, which is available to authorized users.
* Kristina Gopčević
kristina.gopcevic@med.bg.ac.rs; kristinagopcevic@yahoo.com
1
Department of Chemistry Faculty of Medicine, University of
Belgrade, Belgrade 11000, Serbia
2
Institute of Botany and Botanical Garden Jevremovac, Faculty of
Biology, University of Belgrade, Belgrade 11000, Serbia
3
Department for Pharmacognosy, Faculty of Pharmacy, University of
Belgrade, Belgrade 11000, Serbia
Introduction
The growing interest in ingredients from natural sources made
attractive the use of raw herbal materials, especially herbal
extracts, as functional additives in foods, drinks, and cos-
metics. The aromatic plants, their extracts and essential oils
represent almost an ideal source for multi-purpose functional
use in food and drink industry [1]. Diverse biological activities
of herbal extracts, particularly their antioxidant and free radi-
cal scavenging activities [2] as well as antimicrobial [3], and
anticancer [4] capacities are due to the presence of complex
mixtures containing the phenol compounds and flavonoids

[5]. Considering health benefits that have been associated with
polyphenolic consumption, medicinal and aromatic plants
could appear as a good source of these groups of phytochem-
icals [6]. The genus Satureja L. (Lamiaceae) comprises about
200 species of herbs and shrubs, often aromatic, widely dis-
tributed in Mediterranean area, Asia and boreal America [7].
Satureja kitaibelii Wierzb. ex Heuff. is an annual, aromatic
species which inhabits arid limestone areas in eastern Serbia.
Aerial parts of this species have been used as remedy in
Serbian traditional medicine, as well as a flavoring and pre-
servative additive in food due to antimicrobial, antidiuretic,
digestive activities [8]. In addition, polyphenols and flavo-
noids present in the species of this genus exhibit a protective
role against the reactive oxygen species induced by stresses,
which have been discussed in detail in [9, 10]. While biolog-
ical activity such as antimicrobial [11] and cytotoxic [12] of
essential oils [11] and polyphenolic compounds [5] of
S. kitaibelii has been investigated, data about the chemical
composition and the antioxidant activities of extracts of
S. kitaibelii are scarce and lacking except in [13]. In the pres-
ent study the chemical composition of ethanol extracts of ae-
rial plant parts of S. kitaibelii was investigated by liquid
chromatography-mass spectrometry (LC-MS) and related to
their functional antioxidative and antimicrobial activities.
Material and Methods
Chemicals and Plant Samples
2,2′-diphenyl-1-picrylhydrazyl (DPPH) was purchased from
Merck (Germany). Sodium carbonate anhydrous, potassium
acetate, potassium peroxidisulfate and L(+)-ascorbic acid (vi-
tamin C) were AnalaR NORMAPUR® grade chemicals
(VWR Chemicals, Leuven, Belgium). Aluminum nitrate
nonahydrate and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) were
purchased from FlukaChemie AG (Buchs, Switzerland). All
other reagents were purchased from Sigma-Aldrich (St. Louis,
USA) and were of analytical purity. Solvents used for liquid
chromatography – mass spectrometry (LC-MS) analysis were
of LC-MS grade. Reference compounds were obtained from
Carl Roth (Karlsruhe, Germany) (rosmarinic acid) and Sigma
Aldrich (Isoquercitrin, Luteolin 7-O-glucuronide, luteolin 7-
O-rutinoside and diosmin). Aerial parts of S. kitaibelii were
collected in August 2015 from Rtanj Mountain in Serbia.
Description of the collecting area and plant material are pro-
vided in Supplementary data S1 (description of the collecting
area and plant material). Voucher specimen has been deposit-
ed in the Herbarium of the Institute of Botany and Botanical
Garden Jevremovac, Faculty of Biology, University of
Belgrade, Serbia (No. 17141 BEOU).
Plant Foods Hum Nutr
Preparation of the Extracts
Air dried plant material was separated into two sub-samples:
(1) stem, leaves and flowers and (2) leaves plus flowers (i.e.,
aerial parts without stem), and then powdered. Extracts were
prepared by bimaceration and ultrasound extraction proce-
dures (using an ultrasonic bath SB-1.5 L, S, frequency
40 kHz (MiaVab, Serbia), of 10 g plant material with
100 mL of ethanol (96%, v/v, 2 × 24 h), and ultrasound
assisted extraction was done during the first and the last hour
within the 24 h, in the dark at room temperature. After filtra-
tion (paper filter, Whatman No. 1), solvent was evaporated to
dryness under reduced pressure. The dried bimacerated ex-
tract: E1 (stem, leaves and flowers), E2 (leaves plus flowers)
as well as ultrasound assisted extract E3 (stem, leaves and
flowers) and E4 (leaves plus flowers) were refrigerated at
4 °C in dark glass bottles for further investigation. Extract
were used in concentration 1 mg/mL in ethanol.
LC-PDA-MS Analyses of Phenolic Constituents
The separation and quantification of flavonoids and phenolic
acids was performed on Liquid Chromatograph Agilent 1260
(US) on a Zorbax SB-aq column at 27 °C, coupled with a mass
detector Agilent MSD 6100 (US). Extract solutions were
injected while the binary pump was operating at a flow rate
of 0.3 mL/min. Gradient elution was performed. Identification
of constituents was performed by comparison of ultraviolet
(UV), mass spectrometry (MS) and retention time (Rt) data
with those obtained for the standard compounds, as well as
tentatively, by comparison with the literature reports. The con-
tents of rosmarinic acid (RA) and clinopodic acid O,
expressed as RA, were determined at 320 nm by the same
procedure and by applying the external calibration method
using RA as the standard (See Supplementary data S2:
Validation parameters for determination of rosmarinic acid
and clinopodic acid O by LC-PDA/MS).
Determination of Total Phenolics Content
and Flavonoid Content
Total phenolic content (TPC) was assessed spectrophotomet-
rically at 765 nm, using UV-2600 (Shimadzu, Japan), by the
Folin-Ciocalteu (FC) phenol reagent method [14]. Total fla-
vonoid content (TFC) was determined using method with
Al(III)-nitrate [13].
Total Antioxidant Capacity and DPPH Free Radical
Scavenging Activity
Total antioxidant capacity (TAC) was evaluated by the
phosphomolybdenum method [15].
Plant Foods Hum Nutr
Free radical-scavenging activity was determined by meth-
od of Takao et al. [16].
Ferric Ion Reducing Power Assay
Ferric ion reducing power assay (FRAP) was determined by
method of Szőllősi et al. [17].
Antimicrobial Activity
It was examined by the agar diffusion method [18].
Statistical Analysis
All the results are presented as mean ± standard deviations of
three determinations. Statistical analyses were performed
using Student’s t test and one way analysis of variance. IC50
values were calculated determined by nonlinear regression
analysis from the sigmoid dose-response inhibition curves.
Statistica 6.0 software (2001, Stat Soft, Tulsa, OK, USA)
was used to evaluate the p < 0.05 level (regarded as signifi-
cant) and p < 0.01 (very significant).
Results and Discussion
Chemical Composition Results obtained by LC-PDA-MS anal-
ysis are presented in Table 1, Suppl. Table S1 (peak assign-
ments, UV and mass spectral data (from the negative mode) of
phenolic constituents in the extracts of Satureja kitaibelii obtain-
ed by LC-PDA-MS (ESI-Single quad)) and Suppl. Fig. S1
(chromatograms of Satureja kitaibelii ethanol extracts (E1-E4)
recorded at 350 nm (a)). Overlapped and magnified chromato-
grams of all extracts obtained between 12th and 16th minute of
separation (b). Chromatograms show that bimacerated extracts
Table 1 Identified constituents in
the extracts of Satureja kitaibelii Phenolic acids
obtained by LC-PDA-MS
Phenolic acidb
Rosmarinic acid
hexosideb
(iso)Salvianolic acid A
isomerb
Rosmarinic acida
Clinopodic acid Ib
Clinopodic acid Ob
Clinopodic acid Kb
a
identified using commercial reference compounds;
b
tentatively identified by comparison of UV and MS data with literature reports
of: stem, leaves and flowers (E1) and leaves plus flowers (E2)
and ultrasound assisted extracts of: stem, leaves and flowers
(E3) and leaves plus flowers (E4) have similar qualitative and
quantitative composition (Suppl. Fig. S1 a). Except the com-
pound 1 present in E1 and E2, compound 3 present only in
E3, compound 4 present in E1, E3 and E4, compound 17 pres-
ent in E1, E2 and E4, all other compounds are present in all
extracts. Main difference in composition of analyzed extracts
was found between 12th and 16th min of separation (Suppl.
Fig. S1 b). Analysis by LC-PDA-MS of the S. kitaibelii extracts
revealed the presence of phenolic acids and flavonoids, as well
as jasmonic acid derivatives and the diterpene, rosmanol
(Table 1, and Suppl. Fig. S1). The extracts were rich in phenolic
acids, namely oligomers of caffeic acid (CA) (peaks 5, 10, 12,
13, 16, 17). The most abundant compound in all the analyzed
extracts was RA (Suppl. Fig. S1). The amount of all other con-
stituents was substantially lower. Other CA derivatives that were
present in the extracts were tentatively identified, based on their
UVand MS spectral features [19]. Among them, clinopodic acid
O, a CA hexamer (peak 16), was present in significant amounts
in all the extracts. Several flavonoid heterosides were detected in
the extracts, mainly heterosides of the flavones luteolin and
apigenin and their methyl derivatives. It is worthy of noting that
the amount of luteolin 7-O-rutinoside (peak 7) was higher in the
extracts of all the aerial parts (which included the stems) than in
the extracts of leaves plus flowers, regardless of whether soni-
cation was applied or not. In addition, a heteroside of flavanone,
eriodyctyol, and a flavonol heteroside, isoquercitrin, were also
present. Three jasmonic acid derivatives were detected in the
extracts, all of them showing the characteristic ion at m/z 387,
corresponding to the deprotonated hexosyl-jasmonate moiety.
Similar results were previously obtained for Calamintha nepeta
(L) Savi. and Thymus longicaulis C. Presl. [20, 21], but until
now not for S. kitaibelii. Previously analyzed extracts of
Satureja species were also rich in phenolic constituents. RA
Flavonoids Jasmonic acid derivatives Diterpene
Eriodictyol 12-O-hexosyljasmonateb Rosmanolb
rutinosideb
Luteolin 12-O-(caffeoylhexosyl)jasmonateb
7-O-rutinosidea
Isoquercitrina 12-O-((methylcaffeoyl)hexosyl)jasmonateb
Luteolin
7-O-glucuronidea
Apigenin rutinosideb
Diosmina
Methyl-apigenin
rutinosideb

was commonly present in large quantities, and often was the
most abundant compound [19–23]. Higher CA oligomers (tet-
ramers, hexamers), on the other hand, were rarely detected.
Tetramers of CA (i.e., RA dimers), such as clinopodic acid I,
were previously detected in the extracts of S. biflora,
S. hortensis, S. montana, and S. spicigera [19, 22]. CA
hexamers (e.g., clinopodic acids O) were previously found only
in an extract of S. biflora [19]. The contents of total phenols
(TPC), total flavonoids (TFC), rosmarinic acid (RA) and
clinopodic acid O (CO) in the ethanol extracts of S. kitaibelii
are presented in Table 2. The TPC has been measured in
S. kitaibelii for the first time in this work. The TPC in the inves-
tigated extracts ranged between 105 and 195 mg GA/g, whereas
TFC in the extracts varied from 29.18 to 44.00 mg Qu/g. The
TPC was higher in the samples obtained by ultrasound extrac-
tion (158.85–195.95 mg GA/g) compared to the TPC of
bimacerated extracts (105.06–158.54 mg GA/g). The highest
TPC was found in ethanol extract from leaves plus flowers
extracted by the ultrasound procedure (195.95 mg GA/g).
Obviously, the method of extraction affects the amount of ob-
tained phenols [23]. Our measured TPC values were higher than
obtained by Lopez Cobo et al. [5] (25.82 mg GA/g) and Hajdari
et al. [24] (68.1–102.6 mg GA/g), but lower than the values for
methanol extracts of S. montana in Zeljković Ćavar et al. [25]
(614.7 mg GA/g), and similar to the aqueous extract of
S. montana in Serrano et al. [26] (111.18 mg GAE/g). The
TFC was very similar in all our investigated extracts (41.5–
44.0 mg Qu/g). The highest TFC was determined in the ethanol
extract of leaves plus flowers obtained by bimaceration
(44.0 mg Qu/g). The lowest amount of flavonoids was in the
ethanol extract of all plant parts obtained by our ultrasound
extraction procedure (29.18 mg Qu/g). Literature data on flavo-
noid contents of S. kitaibelii are lacking, but could be compared
with the TFC of methanol extract of S. montana reported to be
75.62 mg Qu/g [23] and 38.3–67.0 mg Qu/g [22]. Ethanol ex-
tract of the complete aerial parts of S. kitaibelii obtained by
ultrasound procedure (E3) contained the highest amount of
Table 2 The contents of total phenols (TPC), total flavonoids (TFC), rosmarinic acid (RA) and clinopodic acid O (CO) and antioxidant activity and
DPPH radical scavenging activity in the ethanol extracts of S. kitaibelii
Sample TPC TFC RA
(mg GA/g) (mg Qu/g) (mg /g)
E1 105.06 ± 11.32 41.5 ± 6.23 18.66 ± 0.76
E2 158.54 ± 14.15 44.0 ± 8.15 18.30 ± 0.17
E3 158.85 ± 15.02 29.18 ± 4.35 29.52 ± 0.06
E4 195.95 ± 21.35 41.60 ± 7.05 21.15 ± 0.02
FRAP of AA = 7.41 ± 0.35; and BHA = 1.83 ± 0.41. DPPH of AA =5.50 ± 0.05, GA = 2.18 ± 0.02 and BHA = 5.34 ± 0.03. Results are presented as
mean ± SD (n = 3)
E1 plant extract (stem, leaves and flowers) obtained by bimaceration, E2 plant extract (leaves and flowers) obtained by bimaceration; E3 plant extract
(stem, leaves and flowers) obtained by ultrasound extraction; E4 plant extract (leaves and flowers) obtained by ultrasound extraction. AA ascorbic acid,
GA gallic acid, BHA buthylated hydroxyl anysol
Plant Foods Hum Nutr
RA (29.52 mg/g) and clinopodic acid O (8.55 mg/g, calculated
as RA) in comparison to the other extracts (1.88–4.48 mg/g,
calculated as RA).
Antioxidant Activity The antioxidant activity of S. kitaibelii
extracts was evaluated by TAC, FRAP and DPPH and related
to its composition (Table 2). Since our results are the first
related to the TAC, FRAP and DPPH•+ in this plant, there is
no available literature data for comparison. Extract E4 had the
highest TAC of 714.12 mg AA/g, while extracts E3, E2 and
E1 possessed lower TACs: 365.57, 338.07 and 272.37 mg
AA/g, respectively. The scavenging effect of extracts of
S. kitaibelii measured on the DPPH had the highest value of
125.65 μg/mL for E4 and the lowest of 71.20 μg/mL for E3.
Comparing to SC50 values of the standard antioxidants: 5.50
for AA, 2.18 for GA and 5.34 μg/mL for BHA, a very high
free radical scavenging capacity of extract of S. kitaibelii was
found. DPPH•+ values for aqueous infusion of S. montana–
116.36 μg/mL, [13] and 9.75 and 27.92 μg/mL for hot and
cold infusions, respectively, and its ethanol extract,
108.79 μg/mL [26] shows that our results are extremely good
and linked to the scavenging activity of RA [27] and presence
of large amounts of polyphenolic compounds [1]. The results
obtained are in agreement with the high level of phenol com-
pounds found, and were in order with the value found in the
related plant, S. montana L., by Serrano et al. [26]. Indeed, the
DPPH•+ value was highly correlated with total phenol com-
pounds by HPLC (r = 0.9999; p < 0.01). The highest value of
FRAP for S. kitaibelii (Table 3.) was obtained for E3
1.94 μmol Fe/mg. FRAP values of S. montana were 221.74
and 271.88 μmol Fe/g for cold and hot aqueous extracts re-
spectively and for an ethanol extract, 93.60 μmol Fe/g [26]. It
is well known that FRAP, TAC and DPPH•+ assays can detect
compounds with reduction potential lower than, respectively:
0.77, 0.43 and 0.34 V [27, 28]. A good correlation for antiox-
idative assays and reduction potentials [28, 29] gives a possi-
bility to explain differences obtained for antioxidant reactivity
CO* FRAP TAC DPPH
(mg /g) (μmol Fe/g) (mg AA/g) SC50 (μg/mL)
1.88 ± 0.37 1.16 ± 0.02 272.37 ± 19.25 102.24 ± 15.35
3.74 ± 0.12 0.74 ± 0.01 338.07 ± 25.70 106.94 ± 15.80
8.55 ± 0.00 1.94 ± 0.04 365.57 ± 27.20 71.20 ± 9.55
4.48 ± 0.05 1.41 ± 0.03 714.12 ± 51.30 125.65 ± 16.45
Plant Foods Hum Nutr
Table 3 Antimicrobial activity of
ethanol extracts of Satureja S. kitaibelii ethanol extractsa inhibition zone (mm)
kitaibelii
Microorganism E1
S. aureus ATCC 25923 na
M. luteus ATCC 4698 26
E. coli ATCC 25922 na
P.aeruginosa ATCC 27853 26
C. albicans ATCC 10231 na
AMP ampicillin, applied as a commercial disc, na no activity. E1 extract of stem, leaves and flowers obtained by
bimaceration, E2 extract of leaves and flowers obtained by bimaceration, E3 extract of stem, leaves and flowers
obtained by ultrasound extraction, E4 extract of leaves and flowers obtained by ultrasound extraction
a
Extracts were prepared in DMSO in concentrations of 10 mg/mL and applied in volumes of 100 μL in agar plate wells
of S.kitaibeli extracts, measured by different tests. As Tables 2
and 3 show E3 has a lower content of TFC, higher of RA and
CO, resulting in a high FRAP. Flavonoids with potential, Eap
lower than 0.4 were found to be strong antioxidant in FRAP
assay, while flavonoids with potentials, Eap higher than 0.79
showed no activity [30]. Apigenin with Eap of 0.9 which was
detected in our study undoubtedly makes TFC low active
against FRAP. On the other hand, a high content of RA whose
potentials are low (Eap1/2 are 0.17 and 0.22 V) allows for a
high FRAP. Low value of DPPH for E3 implies a low content
of antioxidant compounds whose potentials is significantly
lower than 0.34. Our results clearly indicate that a single meth-
od for antioxidant capacity determination cannot show a com-
plex antioxidant potential of natural mixture.
Antimicrobial Activity As in Table 3 showed all extracts were
active against M. luteus and P. aeruginosa, while E3 and E4
were also active against S. aureus. Cetkovic et al. [13] report-
ed activity of extracts of S. montana L. subsp. kitaibelii against
S. aureus, ranging from 10.0–16.3 mm, while our E3 and E4
extracts showed 20 mm zones of inhibition. Activity of the
extracts against P. aeruginosa was greater than against other
microorganisms with 26, 18, 25 and 16 mm zones of inhibi-
tion for E1, E2, E3 and E4, respectively, while Cetkovic et al.
[13] obtained lower inhibition zone of 16.7 mm. As strains of
P. aeruginosa are multidrug resistant and associated with se-
rious illnesses, particularly hospital-acquired infections such
as ventilator-associated pneumonia and various sepsis syn-
dromes, our data clearly signify a potential of ethanol extracts
of S. kitaibelii to be applied in treatment of infections caused
by P. aeruginosa.
Conclusions
This study provides new insights on chemical composition,
antioxidant and antimicrobial capacity of Satureja kitaibelii
Standard antibiotic
inhibition zone (mm)
E2 E3 E4 AMP
na 20 20 35
20 30 24 35
na na na 20
18 25 16 na
na na na na
ethanol extracts. The extracts were rich source of polyphenolic
compounds: rosmarinic acid was the most abundant and it was
accompanied by lower amounts of caffeic acid hexamers
(clinopodic acid O); and tetramers identified in this plant for
the first time. In addition to phenolic acids and flavones,
jasmonic acid derivatives and compounds belonging to clas-
ses of flavonols and flavanones were also present in the ex-
tracts. Regarding the contents of total phenolics, rosmarinic
acid and clinopodic acid O, sonication was a more efficient
procedure for preparation of the extracts than was
bimaceration. The results indicate that all extracts have
marked antioxidant activities against free radicals, which is
in correlation with the high contents of phenolic compounds,
especially rosmarinic acid. They possessed pronounced free
radical scavenging activity, suggesting S. kitaibelii would be a
good starting material for the development of functional anti-
oxidants. All extracts were observed to possess considerable
antibacterial activity with pronounced activity against
Pseudomonas aeruginosa. TPC, RA, CO, antioxidant activity
measured as TAC and FRAP, in addition to antimicrobial ac-
tivity suggest combination of sonication and bimaceration as a
more efficient method for preparation of ethanol extract of
S. kitaibelii. All results highlight the potential of S. kitaibelii
as a valuable new source of natural antioxidants, suggesting
its potential to be used as an ingredient in functional food.
Acknowledgements This work was supported by the Ministry of
Education, Science and Technological Development of the Republic of
Serbia (Grants No.175056, 173029, ON 173021 and III43004).
Compliance with Ethical Standards
Conflict of Interest The authors declare no conflict of interest.
Human and Animal Studies This article does not contain any studies
with human or animal subjects.
Publisher’s Note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.

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