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Evaluation of essential oil composition, total phenolics, total flavonoids and antioxidant activity of Malus sylvestris (l.) Mill. fruits.

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The principal aim of this study was to investigate the chemical composition of volatile and phenolic compounds obtained from ripe fruits of European wild apple (Malus sylvestris (L.) Mill.) originating from Kosovo, as well as their antioxidant activity. Gas chromatograph equipped with Flame Ionisation Detector (GC/FID) and Gas chromatograph coupled with Mass Spectrometer Detector (GC/MS) were used for the analysis of the volatile compounds whereas determination of total phenolic content (TPC) and total flavonoid content (TFC), as well as antioxidant activities was done spectrophotometrically (UV/VIS). Antioxidant activities of extracts were assessed using Ferric Reducing Antioxidant Power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. GC analysis of volatile compounds delivered totally 41 compounds were, sesquiterpenes being the principal constituents. In addition, sample analysis showed that European wild apple is reach in phenolic compounds. FRAP antioxidant capacity was 518.7±42.9 mg TE/g dry mass, and DPPH radical scavenging capacity, 56.7±10.2 DPPH mg TE/g dry mass or 53.6±9.7% inhibition.
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Research 2018, 23, 71-85
EVALUATION OF ESSENTIAL OIL COMPOSITION,
TOTAL PHENOLICS, TOTAL FLAVONOIDS AND
ANTIOXIDANT ACTIVITY OF MALUS
SYLVESTRIS (L.) MILL. FRUITS.
Behxhet Mustafaa, Dashnor Nebijab, Avni Hajdaria,
Abstract
The principal aim of this study was to investigate the chemical
composition of volatile and phenolic compounds obtained from ripe
fruits of European wild apple (Malus sylvestris (L.) Mill.) originating
from Kosovo, as well as their antioxidant activity. Gas chromatograph
equipped with Flame Ionisation Detector (GC/FID) and Gas
chromatograph coupled with Mass Spectrometer Detector (GC/MS)
were used for the analysis of the volatile compounds whereas
determination of total phenolic content (TPC) and total flavonoid
content (TFC), as well as antioxidant activities was done
spectrophotometrically (UV/VIS). Antioxidant activities of extracts
were assessed using Ferric Reducing Antioxidant Power (FRAP) and
2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. GC
analysis of volatile compounds delivered totally 41 compounds were,
sesquiterpenes being the principal constituents. In addition, sample
analysis showed that European wild apple is reach in phenolic
compounds. FRAP antioxidant capacity was 518.7±42.9 mg TE/g dry

a Department of Biology. Faculty of Mathematical and Natural Science. University
of Prishtina. Mother Theresa St. 10000 Prishtinë. Kosova
b Department of Pharmaceutical Chemistry, Faculty of Medicine, University of Prishtina,
Mother Theresa Street,10000 Prishtinë, Kosova.
* Corresponding author: avni.hajdari@uni-pr.edu
KDU 58:543
Research 23 72
mass, and DPPH radical scavenging capacity, 56.7±10.2 DPPH mg
TE/g dry mass or 53.6±9.7% inhibition.
Key words: European wild apple, GC/MS, volatile compounds,
phenolic compounds, antioxidant activity.
Introduction
European wild apple, also known as Crab Apple (Malus sylvestris
(L.) Mill.), Rosaceae, is a deciduous fruit tree, with expended crowns
often appearing like bushes. It growths as a native species, found in
broad range of latitudes, in European forests, especially in deciduous
and mixed forest habitats and on soil environmental conditions [1-3].
Most individuals grow up to 10 m. [4. European wild apple is an insect
pollinated species with flowering time from April to May and fruit
ripening time from September to October. [5] The most suitable
morphological characteristics to distinguish species wild apple from
domestic apple are considered to be the pubescence of leaves and
flowers, fruit size and color. [6] Wild apple fruits are rich in pectins and
can be consumed raw or cooked. They are used for the production of
the apple cider, vinegar, jellies, jams etc. Teas can be made from the
leaves and fruits. [3] Furthermore, Wild apple fruits have been
traditionally used against various ailments, principally owing to their
astringent and laxative properties. Due to the pectin content they are
used to obviate the constipation, and the crushed fruit pulp can be used
externally to heal inflammations or small flesh wounds. In addition,
they are used for respiratory problems (colds, flu), treatment of other
symptoms such as fever and headache and externally for the treatment
of wounds and sunburns. [7] In Kosovo traditional medicine, the fruits
of European wild apple are used to treat different disorders such: warts,
earache, skin infections, headache, hypertension, diarrhoea, caught,
hyper-cholesterol, diabetes, as expectorant and mucolytic. [8,9]
Furthermore, in Kosovo it is used to prepare: jams, compote (first sliced
and dried (ahaf), then boiled in water prior to drink (wintertime food)
and tea, as well. [10]
The role of apples and their constituents in human health has been
thoroughly reviewed. Different mechanisms such as antioxidant,
antiproliferative, and cell signalling effects could be responsible for
their health benefits such as prevention of cancer, cardiovascular
disease, asthma, Alzheimer's disease, improved outcomes related to
Research 23 73
cognitive impairment, diabetes, weight loss, bone health, pulmonary
function, and gastrointestinal protection. [11-23] It has been
documented that Malus wild species are rich sources of phenolic
compounds with high antioxidant activity. [24] Wild apple fruit contain
different components such as polyphenols, condensed tannins
(procyanidins), chlorogenic acid, and epicatechin. The most important
constituents are caffeic acid derivatives, p-coumaric acid derivatives,
flavan-3-ols, flavonols, dihydrochalcones. [25]
Polyphenolic profile of crab-apple fruits and its hybrids with
domestic apple showed characteristic polyphenol profile characteristic
for genus Malus, however low content of flavan-3-ols and derivatives
of cinnamic acid and high content of procyanidin B1, phloridzin,
anthocyanes, and quercetin glycosides. Procyanidin B2 was not found
in both peel and flesh of Siberian crabapple. [26] Chlorogenic acid was
the main polyphenol detected in the flesh, whereas phloridzin was the
main phenolic compound detected in seeds and steams of crab apple
pomace. [27] In the case of domestic apple Malus domestica Borkh,
experimental data revealed that the most abundant phenolic compounds
were quercetin glycosides, procyanidin B2, chlorogenic acid,
epicatechin, phloretin glycosides. Vitamin C and phenolic compounds
concentrations correlate with antioxidant capacity. [28] It was shown
that the peels of apples, in particular, contain high amount of phenolic
compounds. Due to the higher concentration of phenolics and
flavonoids, the peels have significantly higher total antioxidant
activities than the flesh of the apple varieties examined. [29] In the
study of Jakobek, et al. [30] flavanols, dihydrochalcones and phenolic
acids were identified in the flesh of apple varieties studied (including
the crab apple) and flavanols, dihydrochalcones, phenolic acids,
flavonols and anthocyanins were identified in peels. Comparing to other
apples, wild apple contained much higher amounts of flavanol and
phenolic acids in the flesh, while the amount of these compounds in
peel was similar. Antioxidant and antiproliferative activity of extracts
and juices obtained from crab apples was reported and experimental
data revealed that antiproliferative effect was more correlated to the
amount of polyphenol than of anthocyanins [31-32]. Polyphenols
profile and antioxidant activity of skin and pulp of Malus pumila Mill.,
originating from Italy was studied too. [33]
Literature data showed that only a small number of papers address
the composition of volatile compounds of European wild apple,
although, some studies of this nature have been reported for mature
Research 23 74
fruits (whole fruit, peel and flesh) and flowers of Malus pumila Mill
originating from Italy [34, 35], from fresh leaves of Malus domestica
growing in Western Himalaya (India) [36], from leaves of apple-tree
(Malus domestica Borkh.) growing in Lithuania [37]. Solid-phase
microextraction or liquid–liquid extraction coupled with GC-FID
and/or GC-MS analysis European wild apple fruit distillates revealed
complex volatile profile. The main classes of compounds were alcohols,
shikimate metabolites, esters, terpenes, aldehydes and acetals, fatty
acids and carotenoid-derived compounds. [38]
The principal aim of this study was to analyse the chemical
composition of volatile and phenolic compounds of wild apples
originating from Kosovo and to study their antioxidant and radical
scavenging activity.
Experimental Section
Collection and extraction of European wild apple samples
Plant material of European wild apple (skin and flesh fruits) was
procured from local markets in Prizren (Kosovo), from five different
sellers. In total 5 samples were distilled, samples were analysed
separately. Fruits were air-dried in shade at room temperature and cut
into small pieces (<0.5 cm). For distillation, 50 g of dry tissue were
placed into 0.5 liter of water in a 1 liter flask and distilled at a rate of 3
mL/min in a Clevenger apparatus for 3 h. The volatiles were collected
with n-Hexane and stored in the dark at -18°C in the freezer pending
further analysis.
GC and GC/MS analyses
Chromatographic analyses were made using an Agilent 7890A gas
chromatography system equipped with flam ionisation (FID) detector
(Agilent Technologies). The separation was conducted on a HP-5MS
column 30 m x 0.25 mm with 0.25 mm film thickness. Helium was used
as the carrier gas with an initial flow rate of 0.6 mL/min and subsequently
at a constant pressure of 16.6 psi. The front inlet was maintained at 250°C
in a split ratio of 50:1. The GC oven temperature increased from 60°C to
260°C at a rate of 5°C/min, and the FID operated at 250°C with an air
flow of 350 mL/min and a hydrogen flow of 35 mL/min. The injection
Research 23 75
volume was 1.0 µL. Gas chromatography/mass spectrometry analyses
were performed using an Agilent 7890A gas chromatograph system
coupled to a 5975C mass spectrometer detector (MSD) (Agilent
Technologies). The ionization energy was 70 eV with a mass range of 40
- 400 m/z. The separation was conducted with the same column and
temperature program as for the analytical GC.
Identification of each component of the essential oil was made by
comparing their Kovats retention indexes with those in literature [39].
The calculation of the Kovats index was made based on a linear
interpolation of the retention time of the homologous series of n-alkanes
(C9 - C28) under the same operating conditions. The components were
also identified by comparing the mass spectra of each constituent with
those stored in the MS library search (NIST 08.L and WILEY MS 9th)
and with mass spectra from the literature. [39]
Determination of Total Phenolics and Total Flavonoids.
For the analysis of total phenols, total flavonoids and antioxidant
activity (DPPH and FRAP), fruits were dried, ground and 150 mg of
dried fruits were extracted with 25 ml of methanol (50%) in water bath
for 90 minutes at 75°C and stored at -18°C in a freezer until further
analyses.
The total flavonoids in the extracts were determined using a
photometric method according to. [40] Catechin (0-10 mg/ml) was used
as a standard to establish the calibration curve. Absorbance was
measured at a wavelength of 510 nm. The total content of flavonoids
was expressed as mg catechin equivalent/g plant dry weight.
The total phenolic content in the extracts was determined using
the Folin-Ciocalteu method in an alkaline environment. [41] Caffeic
acid (0-25 mg/ml) was used as a standard to establish the calibration
curve, and absorbance was measured at 725 nm against the blank. The
results were expressed as mg caffeic acid equivalent/g plant dry weight.
Evaluation of Antioxidant Activity
The DPPH (2.2-diphenyl-l-picrylhydrazyl) radical scavenging
assay and Trolox (2.5 mM in methanol) were used as reference
substances following the protocol of [41]. Trolox (0-50 mg/ml) was
used to construct the calibration curve. The absorbance of the
decolorizing process was measured at 515 nm against the blank. The
Research 23 76
results are expressed as the percent scavenging of DPPH free radicals
and were measured using the following equation: % DPPH radical
scavenging = [(absorbance of control - absorbance of test sample) /
(absorbance of control)] x 100.
The ferric reducing antioxidant power (FRAP) assay measures
the ability of antioxidants to reduce the ferric 2,4,6-tripyridyl-s-triazine
complex [Fe(III)]3+ to the intensely blue-colored ferrous complex
[Fe(II)]2+ in acidic medium. The FRAP assay was performed as
described by (Chizzola 2008). The calibration curve was constructed
using calibration standards of Trolox from (0 to 400 mg/ml) in ethanol,
and absorbance was measured at a wavelength of 593 nm. The results
were estimated as mg Trolox equivalent/g plant dry weight.
All spectrophotometric measurements in the following analyses
were performed using a UV-Vis (ultra-violet/visible) spectrophotometer
(Thermo Scientific™ GENESYS 10S UV-Vis spectrophotometer), and
the results represent the average of 5 measurements.
Results and discussion
Experimental results, using GC and GC/MS analysis,
documented that volatile oil obtained from European wild apple is a
complex mixture of constituents, composed by various volatile
compounds classes. There were identified totally 41 compounds, and,
as presented in the Table 1, the principal classes of chemical
constituents were sesquiterpenes and oxygenated sesuiterpenes, with
50.6% and 30.7%, respectively. The concentration of (E)-spiroether
(en-yn-dicycloether) was 9%. Other classes of compounds such
monoterpenes, hydrocarbons, phenylpropanoids etc. were present at
lower percentages. In the Figure 1 chemical formulas of the most
prominent compounds in the volatile oils of European wild apple are
presented.
E-ȕ-Farnesene (35.03%), E-Caryophyllene (7.17%) and
Germacrene D (5.76%) were the principal sesquiterpenes, whereas Į-
Bisabolol oxide B (4.82 %), Spathulenol (4.78%), Į-Eudesmol
(4.52%), Caryophyllene oxide (4.46%), 2Z, 6 E-Farnesol (4.34%) and
Z-dihydro-Apofarnesol (2.22) were the most prominent sesquiterpene
oxides. In lower concentrations appeared hydrocarbons (2.01%)
monoterpenes (1.04%) and other compounds, including fatty acids and
phenylpropanoids etc. (1.09%).
Research 23 77
Table 1. Chemical composition of volatile compounds of European wild apple
1 Rt Compounds KI %
2 6,45 cis-Cr
y
santhen
y
l acetate 1265 0.25
3 8,27 Carvacrol eth
y
l ether 1298 0.16
4 8,99 Eu
g
enol 1359 0.35
5 10,99 Decanoic acid 1366 0.20
6 11,48 ȕ-Patchoulene 1381 0.29
7 13,31 Į-Isocomene 1388 0.29
8 19,90 ȕ-Isocomene 1408 0.40
9 22,89 E-Car
y
oph
y
llene 1419 7.17
10 24,89 E-ȕ-Farnesene 1456 35.03
11 25,70 deh
y
dro-Sesquicineole 1471 1.33
12 25,84 Germacrene D 1481 5.76
13 26,14 ȕ-Selinene 1490 1.00
14 26,64 Z-dih
y
dro-Apofarnesal 1498 0.30
15 26,38 Bic
clo
ermacrene 1500 0.21
16 26,85 Piperon
y
l acetate 1503 0.40
17 27,33 E,E-Į-Farnesene 1505 0.05
18 27,57 10-epi-Cubebol 1535 0.30
19 28,53 Geran
y
l butanoate 1564 0.63
20 29,45 Z-dih
y
dro-Apofarnesol 1572 2.22
21 29,61 Spathulenol 1578 4.78
22 30,24 Car
y
oph
y
llene oxide 1583 4.46
23 30,62 Isolon
g
ifolan-7-Į-ol 1619 0.44
24 30,81 ȕ-Acorenol 1636 0.41
25 31,41 Į-Muurolol 1646 1.26
26 33,08 Į-Eudesmol 1653 4.52
27 36,52 Į-Bisabolol oxide B 1658 4.82
28 41,29 Unknown 1 1671 0.75
29 43,46 Khusinol 1680 0.42
30 45,56 Elemol acetate 1680 1.57
31 47,57 Į-Bisabolene oxide A 1685 1.75
32 48,19 2Z,6Z-Farnesol 1698 0.23
33 49,51 2Z,6E-Farnesol 1723 4.34
34 50,97 Unknown 2 1733 0.45
35 51,19 Camazulene 1731 0.42
36 51,37 Į-Bisabolol oxide A 1754 1.61
37 52,02 Unknown 3 1769 0.29
38 52,80 E-Spiroether 1890 8.99
Research 23 78
39 53,18 Oleic aci
d
2142 0.14
40 53,64 n-Docosane 2200 0.05
41 54,28 n-Tricosane 2300 0.06
42 54,48 n-Tetracosane 2400 0.41
43 54,64 n-Pentacosane 2500 0.04
44 54,92 Hexacosane 2600 1.45
Total identi
f
ied (%) 98.51
Sesquiterpenes 50.62
Oxygenated
sesquiterpenes 34.76
Spiroketals 8.99
H
y
drocarbons 2.01
Monoterpenes 1.04
Others 1.09
Fig. 1. Chemical formulas of the principal volatile compounds
HH
H
OH
H H
H
H
OH
HH
O
H
OH HO
H
H
OH
H
H
OH
H
O
O
(+)-(E)-Caryophyllene (E)-E-Farnesene Germacrene D
(Z)-Dihydro-apofarnesol Spathulenol Caryophyllene oxide
D-Eudesmol D-Bisabolol oxide (2Z,6E)-Farnesol (E)-Spiroether
Research 23 79
Comparing to our data, the study of volatile compound in mature
fruits (whole fruit, peel and flesh) of Malus pumila Mill. originating in
Italy, revealed that there were identified 30 compounds and terpenes
were the main volatiles, from whole fruits and peels, whereas aliphatic
esters were the most abundant constituents of flesh. The most prominent
constituent in all samples was sesquiterpene (E,E)-Į-farnesene. Other
monoterpenes and typical esters were present in smaller amounts [34].
On the other hand, in flower samples of other cultivar of red Italian apple,
linalool was the most abundant constituent, followed by (E, E)-Į-
farnesene [36]. In extracts obtained from fresh leaves of Malus domestica
growing in Western Himalaya (India) volatile oils were mostly composed
by mono-, sesqui-, di-terpenes, phenolics, and aliphatic hydrocarbons.
Seventeen compounds of the oil irere characterized and principal
compounds of the oil were eucalyptol (43.7%), phytol (11.5%), Į-
farnesene (9.6%), and pentacosane (7.6%) [36]. Sesquiterpenoids,
monoterpenoids, diterpenes and aliphatic hydrocarbons were the main
constituents of essential oils obtained from leaves of apple-tree (Malus
domestica Borkh.) growing in Lithuania. The most prominent
constituents were (iso)phytol (27.3%), (E, E)-Į-farnesene (22.0%), n-
hexyl benzoate (13.5%), tridecane (11.0%), and Į-cadinol (10.5%) [37].
In contrast to others studies, our samples were reach in (E)-spiroether
(9%), which was not previously reported to be present in fruits of
European wild apple. (E)-spiroether were reported to be present in other
plant species, such Matricaria recutita L. which shows that inhibited the
production of mycotoxins AFG1 (produced by Aspergillus parasiticus)
and 3-ADON (Fusarium graminearum) [45]. Thus, presence of (E)-
spiroether will reduce the mycotoxins contamination of European wild
apple fruits and reduce the human health risk caused by those toxins.
Except volatile compounds, total phenolics and total flavonoid
concentration, as well as the antioxidant activity (FRAP and DPPH) of
methanolic extracts were evaluated too. The concentration of total
phenolics was 63.5±6.7 mg caffeic acid equivalent/g of plant dry mass,
while the total flavonoid was 40.3±5.8 mg catechin equivalent/g of
plant dry mass. Regarding antioxidant activity, the FRAP antioxidant
capacity were 518.7±42.9 mg TE/g dm, while DPPH radical scavenging
capacity was 56.7±10.2 DPPH mg TE/g dm or 53.6±9.7% inhibition.
Total phenolics and total flavonoid concentration as well as the
antioxidant activity (FRAP and AOA) of the wild apple fruit extracts
originated from Serbia were evaluated too, thus total phenolics ranged
from 172.91–1556.99mg GAE/100g dry weight; total flavonoid ranged
Research 23 80
from 3.97 to 182.22 mg RE/100g d.w., while FRAP value ranged from
2.13 to 7.65mM Fe2+ and %AOA from 57.73% to 95.32 %AOA [42].
In fruit extracts originated from Malaysia the total content of phenolic
was 216.11± 7.72 (expressed as mg GAE/g extract weight); EC50 was
0.606 ± 0.008mg/ml (DPPH assay) and 14.48 ± 1.423 mM/100g (FRAP
value) [43]. Total flavonoid content of aqueous extracts of the fruits
peel of European wild apple was 44.62 ± 1.39 mg (expressed as mg
Quercetin equivalent per gram extract weight (mg QE/g extract weight);
total phenolic content 199.26 ± 8.15(mg GAE/g EW); DPPH
antioxidant scavenging capacity was 0.665 ± 0.032 EC50 (mg/ml) and
FRAP reducing capacities was13.91 ± 2.135 (mM/100g) [44].
Conclusion
In this study, the chemical composition of volatile constituents
obtained from fruits of European wild apple, growing in Kosovo has been
elucidated. Furthermore the total flavonoides and phenolic compounds
were assessed and their antioxidant and radical scavenging capacities have
been determined. Concerning the volatile compounds, the sesquiterpene
hydrocarbons were dominant constituents. In addition this study revealed
that European wild apple is very reach source of bioactive phenols and
flavonoids and these related data may encourage new studies in the future.
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Vlerësimi i përbërjes së vajit esencial, fenolet dhe flavonidet e
tërësishme dhe aktiviteti antioksidant i mollës së egër
Behxhet Mustafa, Dashnor Nebija, Avni Hajdari
Përmbledhje
Qëllimi kryesor i këtij studimi ishte analizimi i përbërjes kimike
komponimeve avullueshme, komponimeve fenolike, si dhe
vlerësimi i aktiviteti antioksidues i ekstrakteve të përftuara nga frutat e
pjekura të mollës së egër (Malus sylvestris (L.) Mill.) me origjinë nga
Kosova. Kromatografi i gaztë i shoqëruar me detektor jonizues me flakë
(GC/FID) dhe kromatografi i gaztë i shoqëruar me spektrometër
masës (GC/MS) janë përdorur për analizimin e komponimeve
avullueshme, ndërsa përcaktimi i përqendrimit të fenoleve totale (TPC)
dhe flavonoideve totale (TFC), si dhe vlerësimi i aktivitetit antioksidues
është bërë me spektrofotometër UV/VIS. Aktiviteti antioksidues i
ekstrakteve është bërë duke përdorur kapacitetin reduktues joneve
ferrike (FRAP) dhe aktivitetin zbërthyes të 2,2-difenil-1-pikrilhidrazil
(DPPH). Analizat nga kromatografi i gaztë ka rezultuar në identifikimin
e 41 komponimeve të avullueshme në total, prej të cilave seskuiterpenet
ishin përbërësit kryesorë. Përpos komponimeve të avullueshme, te
frutat e mollës egër është analizuar edhe përqendrimi i fenoleve totale
(63.5±6.7 mg acid kafeik/g në masën e thatë të frutave), flavonoideve
totale (40.3±5.8 mg katekinës/g masën e thatë frutave), si dhe
aktiviteti antioksidues FRAP (518.7 ± 42.9 mg TE /g në masën e thatë
frutave), dhe kapaciteti zbërthyes i DPPH (56.7 ± 10.2 DPPH mg
TE/g në masën e thatë të frutave ose 53.6 ± 9.7% inhibimit).
ResearchGate has not been able to resolve any citations for this publication.
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