Free Radical Scavenging Activity of Essential Oil of Eugenia caryophylata from Amboina Island and Derivatives of Eugenol

Article abstract of DOI:10.1515/chem-2019-0047

Essential oil from Eugenia caryophylata was normally used to heal many different deseaces. Various chemical compositions of essential oil distilled and steamed of Moluccas Eugenia caryophylata has been investigated by many different researchers. Even though an intensive research has been carried out of the local chemotypes, a very detail study has not been fully investigated to find out the complete chemical compounds from the plant essential oil and its content associated with their biological activities. In present paper, we assess the free radical scavenging of E. caryophylata collected from Moluccas islands, Indonesia. Essential oil was extracted from leaves, buds, and stems of plant by steam distillation and analyzed using GC-FID and GC-MS. The result showed that free radical activity of essential oil, main constituent and its derivatives were analized using in vitro method. Essential oil activity from stem obtained as (0.82±0.15 μg/mL) was higher than that from bud and leaf possessing both 1,1-diphenyl-2-picrylhydrazyl (DPPH) and (2,2'-azino-bis-3-ethylbenzthizoline-6-sulphonic acid (ABTS) radical scavenging assays by sinergism of eugenol, eugenyl acetate, β-caryophylene and humulene. The activity of isoeugenol (2) (3.59±0.54 μM) and (5.0±0.53 μM) scavenging DPPH and ABTS, respectively, as derivatives eugenol was higher than (3), (4) and (5). Although (6) was active originally, it was inactive after conversion of the ester. While the change of the double bond of location to conjungation structure caused more activity scavenging radicals than the starting molecule.

Full text of DOI:10.1515/chem-2019-0047

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Open Chem., 2019; 17: 422–428
Research Article
Hanoch Julianus Sohilait*, Healthy Kainama
Free Radical Scavenging Activity of Essential Oil
of Eugenia caryophylata from Amboina Island and
Derivatives of Eugenol
https://doi.org/10.1515/chem-2019-0047
Keywords: Amboina island; free radical scavenging;
received October 23, 2018; accepted January 27, 2019.
isomethyl eugenol; methyl eugenol hydroxy.
Abstract: Essential oil from Eugenia caryophylata was
normally used to heal many different deseaces. Various
chemical compositions of essential oil distilled and 1 Introduction
steamed of Moluccas Eugenia caryophylata has been
investigated by many different researchers. Even though Natural product compounds, including essential oils (EOs)
an intensive research has been carried out of the local distilled from Eugenia caryophylata have various chemical
chemotypes, a very detail study has not been fully contents depending on local distribution of the plants.
investigated to find out the complete chemical compounds There are three parts of clove tree in famous Moluccas
from the plant essential oil and its content associated islands of Indonesia such as buds, leaves, and stems can
with their biological activities. In present paper, we assess produce clove oil by using steam distillation. Indonesia,
the free radical scavenging of E. caryophylata collected Tanzania, Sri Lanka, Madagascar are world largest clove-
from Moluccas islands, Indonesia. Essential oil was producing countries on earth. While, India had a limited
extracted from leaves, buds, and stems of plant by steam scale of clove product [1]. The primary contents of clove
distillation and analyzed using GC-FID and GC-MS. The oil are eugenol, β-caryophylene, α-humulene, eugenyl
result showed that free radical activity of essential oil, acetate, and humulene epoxide [2-3]. Even though there
main constituent and its derivatives were analized using were many compounds found in clove oil closely related
in vitro method. Essential oil activity from stem obtained to geographical location, and the plant tissue, eugenol
as (0.82 0.15 μg/mL) was higher than that from bud and remains the primary constituent of clove oil [4-5]. In 2018
leaf possessing both 1,1-diphenyl-2-picrylhydrazyl (DPPH) [6], Da Silva et al., found that by modifiying the molecular
and (2,2’-azino-bis-3-ethylbenzthizoline-6-sulphonic acid structure of an active subtance it can be used as an anti
(ABTS) radical scavenging assays by sinergism of eugenol, tumor and anti infective drug.
eugenyl acetate, β-caryophylene and humulene. The
Eugenol, a major compound inside clove oil, is
activity of isoeugenol (2) (3.59 0.54 μM) and (5.0 0.53 μM) generally isolated by using a distillation process with a
scavenging DPPH and ABTS, respectively, as derivatives reduced pressure. Moreover, the eugenol hydroxyl and
eugenol was higher than (3), (4) and (5). Although (6) was allyl groups can be changed to other functional groups.
active originally, it was inactive after conversion of the On the other hand, eugenol as one of phenolic compounds
ester. While the change of the double bond of location to can easily be converted into methyl eugenol by the use of
conjungation structure caused more activity scavenging a methylation reaction of eugenol with DMS [7-8]. While
radicals than the starting molecule.
another method to produce methyl eugenol is by the
process of eugenol with dimehyl carbonate (DMC) with the
help of Phase transfer catalyst (PTC) [9-14]. The reaction
of isoeugenol with acetic anhydride forms isoeugenol
acetate [15-16]. Isomerization of alkenyl-alkoxybenzenes,
especially, linked to a migration process of a double
bond in the alkenyl group of alkenyl-alkoxybenzenes
from a not conjugated position with the benzene ring to
a conjugated position with the benzene ring by the use of
*Corresponding author: Hanoch Julianus Sohilait, Department
of Chemistry Faculty of Mathematic and Natural Science, Pattimura
University, Ambon, 97233, Indonesia, Email: nokesohilait@yahoo.com
Healthy Kainama: Departement of Chemistry Education, Faculty
of Training and Education, Pattimura University, Ambon, 97233,
Indonesia
Open Access. © 2019 Hanoch Julianus Sohilait, Healthy Kainama, published by De Gruyter.
Creative Commons Attribution alone 4.0 License.
This work is licensed under the
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Free Radical Scavenging Activity of Essential Oil of Eugenia caryophylata from Amboina Island...
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Scheme 1: Derivatives of Eugenol.
a base catalyst. Eugenol and methyleugenol, which have heaters, evaporators and tools of Buchii laboratory
an allyl group can be converted into an isomer by using glassware. The second ones were spectroscopy
KOH-alcoholic, or without the use of an alcoholic solvent instruments of Spectrophotometer PD-303S, Apel, Japan,
in order to yield isoeugenol and isomethyl eugenol, Infra Red spectrophotometer (FTIR-8400S, Shimadzu),
1
respectively [17-19]. While the synthesis of secondary and H-NMR Spectrophotometer (JEOL-MY 400). Finally,
alcohols that changes the double bond to be a hydroxyl Gas Chromatography 2010 Shimadzu (GC), and Mass
group shown in scheme 1 can be conducted using few Spectrophotometer QP 2010 Plus (MS), Shimadzu (GC-MS)
types of techniques of oxymercuration-demercuration of were employed to identify the compounds.
olefin by adding mercury acetate and NaBH4 based on in
situ according to ref. [20-21]. The other method to produce
a secondary alcohol was by changing the allyl group of 2.2 Materials
methyl eugenol through additional formic acid, and then
followed by hydrolysis treatment [22].
There were bud, leaf, stem as parts of E.caryophylata taken
Free radicals existance produced in plants are very from local garden in Amboina Island as row materials.
important to initiate chain reactions that heal damage
cells, and cause a death cell alive. In general, many deathly
diseases such as cardiovascular and neurodegenerative 2.3 The Essential Oil Isolation and Synthesis
diseases were because of damage cells. The free radicals in
plants can fight against the such effects due to antioxidant
Derivatives of Eugenol
subtances [23]. Eugenol as one of antioxidant compounds The volatile oils of clove were obtained by conventional
is powerful to fight oxidative stress for a better health. distillation [24]. The dried materials (1.00 kg) of bud,
Therefore, the broad spectra of biological activities of leaf (1.00 kg) and stem (0.60 kg) were treated by steam
eugenol in this research work was to find out its derivatives distillation for 6 hours. The composition of essential oil
by chemical proceses of isomerization, subtitution and was analysed using GC. The isolation of eugenol (1) from
addition reactions, respectively. We present this novel stem clove oil was done by using a sodium hydroxide
work that connects between the specific compounds (NaOH) solution to obtain sodium eugenolate, followed by
found in E. caryophylata from Amboina island, and its acidification with 20% hydrochloric acid (HCl), and then
EOs.
distilled under pressure reduction at 1200oC/10 mmHg
yielding eugenol (1) as large as 74.09% [19,24]. The
spectral data corresponded with the earlier reports [17,18].
The isomerization of eugenol (1) with KOH in a solvent
ethylene glycol monomethyl ether was refluxed at 140oC
for 4 hours, followed by distillation under the pressure
of 10 mmHg at 120-122oC yielding isoeugenol (2) with the
content of 69.72% [18]. The methylation of eugenol (1) was
2 Experimental
2.1 Equipment
The equipment used in this research consisted of carried out using DMS at the 103oC for 90 minutes, followed
three groups of apparatuses. The first group was from by distilling under pressure at 140oC/20 mmHg which
fractional distillation under reduced pressure, electric yielded methyl eugenol with the amount of 62.08% [8,22].
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Hanoch Julianus Sohilait, Healthy Kainama
The spectral data was fitted with that given in previous [26]. By oxidating ABTS with potasium persulfate, the
reports [8,16,17,22]. The isomerization of methyleugenol (3) ABTS radial was obtained. In order to form ABTS cation
with KOH, and without any solvent at the 140oC for 4 hours solution, 5 mL ABTS (7 mm) was mixed with 100 μL of
yields isomethyleugenol (4) (66.68%). The compound was potassium persulphate, and then incubated for 12 hours.
then identified using GC yielding cis-isomethyleugenol Furthermore, the working solution was prepared with a
(9.80%) and trans-isomethyleugenol (90.20%) [16-17]. The certain amount of the former solution following by adding
oxymercuration-demercuration of methyl eugenol (3) was the phosphate buffer saline solution until the absorbance
carried out with Hg(OAc)2-NaBH4, followed by distillation as large as (0.70 0.001) at 734 nm. The freshly prepared
processwithreducedpressurereductionat146oC/10mmHg working solution was then kept in the dark for 30 minutes.
to obtain methyleugenyl alcohol (5) (78.03%) [20-21]. The Avolumeof1mLofdifferentconcentrationsofplantextract
spectral data was comparable with that in ref. [8,22]. The was mixed thoroughly with 1 mL of working solution, and
isoeugenyl acetate was synthesized from isoeugenol (2) kept in the dark for 10 minutes. The absorbance of the
and acetate anhydrous using sodium acetate as a catalyst. test sample was measured at the same wave length of 734
The mixture was refluxed at 120oC for 90 min, followed by nm. of the control sample. The percentages of inhibition
recrystallization in methanol to obtain isoeugenyl acetate were calculated using Eq. (1) in which the Abscontrol was
(6) with yield as high as 63.08% at b.p 65oC [15].
absorbance of blank, while the Abstest were absorbance
of all the samples. A lower absorbance in the sample
was indicated as a higher ABTS scavenging activity. The
2.4 DPPH and ABTS Free Radical Scavenging concentration dependence of IC50 value was determined
from EOs and its derivatives prepared with the ABTS
Assays
radical to 50% of the control. Ascorbic acid was used as
2.4.1 DPPH free radical scavenging assay
a standard.
Ethical approval: The conducted research is not
The DPPH activity was investigated with the method related to either human or animal use.
described in Ref. [25]. The test sample (10 mg) was
dissolved in 1 mL methanol. The reaction mixture
consisted of 1 mL of DPPH 6 x 10-5 M and 33 μL sample
3 Results and Discussion
solution in methanol. After 20 min incubation at 37°C, the
absorbance values of the sample caused by the reaction in
the prepared mixture were measured at a wavelength of 3.1 Chemistry
515 nm using a spectrophotometer PD-303S, Apel, Japan.
The blank samples comprised of 33 mL of methanol in a The starting material eugenol (1) was isolated from clove
DPPH solution was also measured at the same wavelength. stem oil. The isomerization of (1) with KOH in ethylene
The experiment was performed in triplicate. While the glycol monomethyl ether produced (2). Methylation
percentage of inhibition was calculated using equation:
of eugenol (1) with dimethyl sulphate (DMS) produces
methyleugenol (3) and isomerization of (3) with KOH
Percentage of inhibition = - Abstest/ Abscontrol x 100 (1) without solvent produced isomethyleugenol (4).
Oxymercuration-demercuration of methyleugenol with
In Eq. (1), the Abscontrol was absorbance of blank, while Hg(OAc)2-NaBH4, produced methyleugenyl alcohol (5)
the Abstest were absorbance of all the samples. A lower and esterification of (2) with anhydrous acetate produces
absorbance in the sample was indicated as a higher DPPH isoeugenyl acetate (6) as shown in Scheme 1.
scavenging activity. The concentration dependence of
IC50 value was determined from EOs and its derivatives
prepared with the DPPH radical to 50% of the control. 3.2 Free Radical Scavenging Activities
Ascorbic acid was used as a standard.
To evaluate the antioxidant properties of essential oil,
eugenol and derivatives, two methods of DPPH and ABTS
2.4.2 ABTS free radical scavenging assay
were employed. The possitive control used in this research
was ascorbic acid. The inhibition concentration, IC50 was
The ABTS radical scavenging assay was carried out measured for the antioxidant activity and the compounds,
according to the method described by Fitriana et al. respectively. The results of scavenging activities are
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Free Radical Scavenging Activity of Essential Oil of Eugenia caryophylata from Amboina Island...
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Figure 1: Scavenging effect of different essential oils and ascorbic acid (A) DPPH, (B) ABTS assays at concentration 319.45 µg/mL and 99.00
µg/mL. Mean values are significantly average SD n =3.
Table 1: IC50 value free radical scavenging of essential oil.
mL. The percentage of inhibition of ABTS from bud
essential oil was high (93.53 0.68%), followed by leaf
DPPH (µg/mL) ABTS (µg/mL)
(92.33 0.34%) and stem (91.94 0.68%). The value of IC50
showing high activity of ABTS was (0.54 0.77), (0.66 0.67)
and (0.81 0.16), for bud, leaf, stem, respectively.
The higher antioxidant activity of oil of E. caryophylata
EOS could be related to the synergistic property
associated with the high content of eugenol (97.75%)
with other constituents viz., β-caryophylene (0.90%),
Essential oil from stem (EOS)
Essential oil from bud (EOB)
Essential oil from leaf (EOL)
Ascorbic acid (positive control)
0.82 0.15
1.18 0.56
1.16 0.74
1.38 0.65
0.81 0.16
0.54 0.77
0.66 0.67
0.74 0.54
listed in Table 1. The sample amount needed in order to eugenyl acetate (0.76%) and caryophylene oxide (0.59%)
decrease the absorption of DPPH and ABTS by 50% was reported previously (Sohilait et al, 2018). The presence
then calculated graphically by plotting % of inhibition of eugenyl acetate (20.93%) synergic with eugenol
against the concentration in μg/mL.
(75.30%), β-caryophylene (3.0%), humulene (0.37%)
The free radical scavenging activity of the essential and caryophylene oxide (0.40%) in EOB makes its ABTS
oil on the DPPH scavenging was observed at 319.45 μg/ activity higher than EOL’s. On the other hand, the absence
mL to 9.98 μg/mL concentration. All of the essential oils, of eugenyl acetate in EOB could cause higher antioxidant
regardless of distilation steam method, showed DPPH activity of scavenging ABTS than EOL’s. Humulene in
scavenging activity that increased in a concentration EOS had no indicator of the presence of antioxidant
dependent manner. As shown in Figure 1A, all the activity, which may be due to the absence of participating
essential oils, including the reference ascorbic acid (AA), electron delocalization group or protons. The presence
exhibit intense DPPH radical scavenging activity at low of β-caryophylene, caryophylene oxide and humulene
concentration 9.98 μg/mL. The percentage of inhibition from the EOs could show no effect of antioxidant activity
of DPPH from stem essential oil was high (99.00 1.05%), [27-28]. The essential oils of eugenol were compared to
followed by bud (98.80 0.96%), and leaf (98.10 0.76%). a known synthetic antioxidant ascorbic acid (Table 1).
The value of IC50 value showing high activity of DPPH was The oil of E. caryophylata showed better antioxidant
(0.82 0.15), (1.18 0.56) and (1.16 0.65), for stem, bud, leaf, property than pure eugenol. The important facts found
respectively.
are the nonspecific responses and synergistic effects
As shown in Figure 1B, the free radical scavenging of the compounds from essential oil. For this reason, a
activity of the essential oil on the ABTS scavenging were bioassay-direct fractionation of an active extract was not
observed at concentration of 99.00 μg/mL to 0.77 μg/ always contribute to the isolation of active compouds. An
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