Jordan Journal of Chemistry Vol. 11 No.2, 2016, pp. 108-119 108 JJC Salvia ceratophylla from Jordan: Volatile Organic Compounds, Essential oil composition and antioxidant activity Hala I. Al Jaber Department of Physics and Basic Sciences, Faculty of Engineering Technology, Al-Balqa Applied University, Amman, Jordan Received on April 3, 2016 Accepted on June 5, 2016 Abstract This work aimed at evaluating the chemical composition of the hydro-distilled essential oils of fresh and air-dried Salvia ceratophylla growing wild in Jordan by GC/MS in addition to evaluating the actual composition of the volatile organic compounds (VOC’s) extracted by SPME (Solid Phase Micro-Extraction) of S. certaphylla fresh roots and flowers at the pre-, full and post- flowering stages. Different classes of terpenoids at varying concentrations were detected. The hydro-distilled oil of fresh flowers was dominated by sesquiterpene hydrocarbons (27.26%) while the air dried flowers contained oxygenated monoterpenes as the main constituents (58.28%). The VOC’s profile of the roots and the flowers during the pre-, full and post-flowering stages contained monoterpene hydrocarbons as the main constituents (60.14%; 80.49%; 92.28%; 59.66%, respectively). In addition, the hydro-alcoholic extracts obtained from the aerial flowering parts of S. ceratophylla showed interesting antioxidant activity on ABTS •+ assay (IC50=0.09 (mg/mL) ± 2.91) as compared to that of the DPPH assay (IC50=0.11 (mg/mL) ± 1.85). Keywords: Salvia ceratophylla; Essential oil; GC/MS; SPME; Terpenoids; Antioxidant activity. Introduction The genus Salvia belongs to the mentheae tribe within the Nepetoideae subfamily of the family Lamiaceae (formerly referred to as Labiateae). [1] This genus, represented by more than 1000 species distributed worldwide, is considered as the most important genus of the Lamiaceae family. Plants belonging to this family are generally recognized for their various pharmacological properties including analgesic, anti-inflammatory, [2] antioxidant, antibacterial, [3] anti-tumoral and central nervous system depressant activities. [4] The flora of Jordan includes about 19 species of the genus Salvia. [5] Generally, Salvia plants are used as herbal remedies for the treatment of many ailments, including abdominal pain, fever and bronchitis. Many species belonging to this genus are known for their carminative, spasmolytic and antibacterial activities. Also, Corresponding author: e-mail: [email protected]
Transcript
Jordan Journal of Chemistry Vol. 11 No.2, 2016, pp. 108-119
108
JJC
Salvia ceratophylla from Jordan: Volatile Organic Compounds, Essential oil composition and antioxidant activity
Hala I. Al Jaber
Department of Physics and Basic Sciences, Faculty of Engineering Technology, Al-Balqa Applied University, Amman, Jordan
Received on April 3, 2016 Accepted on June 5, 2016
Abstract This work aimed at evaluating the chemical composition of the hydro-distilled essential
oils of fresh and air-dried Salvia ceratophylla growing wild in Jordan by GC/MS in addition to
evaluating the actual composition of the volatile organic compounds (VOC’s) extracted by SPME
(Solid Phase Micro-Extraction) of S. certaphylla fresh roots and flowers at the pre-, full and post-
flowering stages. Different classes of terpenoids at varying concentrations were detected. The
hydro-distilled oil of fresh flowers was dominated by sesquiterpene hydrocarbons (27.26%) while
the air dried flowers contained oxygenated monoterpenes as the main constituents (58.28%).
The VOC’s profile of the roots and the flowers during the pre-, full and post-flowering stages
contained monoterpene hydrocarbons as the main constituents (60.14%; 80.49%; 92.28%;
59.66%, respectively). In addition, the hydro-alcoholic extracts obtained from the aerial flowering
parts of S. ceratophylla showed interesting antioxidant activity on ABTS•+ assay (IC50=0.09
(mg/mL) ± 2.91) as compared to that of the DPPH assay (IC50=0.11 (mg/mL) ± 1.85).
Keywords: Salvia ceratophylla; Essential oil; GC/MS; SPME; Terpenoids; Antioxidant
activity.
Introduction The genus Salvia belongs to the mentheae tribe within the Nepetoideae
subfamily of the family Lamiaceae (formerly referred to as Labiateae).[1] This genus,
represented by more than 1000 species distributed worldwide, is considered as the
most important genus of the Lamiaceae family. Plants belonging to this family are
generally recognized for their various pharmacological properties including analgesic,
anti-inflammatory,[2] antioxidant, antibacterial,[3] anti-tumoral and central nervous
system depressant activities.[4]
The flora of Jordan includes about 19 species of the genus Salvia.[5] Generally,
Salvia plants are used as herbal remedies for the treatment of many ailments,
including abdominal pain, fever and bronchitis. Many species belonging to this genus
are known for their carminative, spasmolytic and antibacterial activities. Also,
Aldrich) were used as reference substances in GC/MS analysis.
Plant material
S. ceratophylla L. was collected from Abu-Hamid village at Abu-Nseir region,
located about 15 km to the north of the capital Amman, Jordan, during its full flowering
period (March – April, 2015). The identity of the plant was confirmed by Prof. Dr.
Barakat E. Abu-Irmaileh, Department of Plant Protection, Faculty of Agriculture, The
University of Jordan. A voucher specimen (No: BAU/03/LS1003) has been kept in the
Department of Physics and Basic Sciences, Faculty of Engineering Technology, Al-
Balqa Applied University, Marka-Amman, Jordan. The plant material, was air dried in
shade until constant weight was achieved, and then subsequently assayed for
chemical and antioxidant activity evaluation.
Hydro-distillation and Solid Phase Micro-Extraction (SPME) of the volatile constituents
from S. ceratophylla and preparation of the hydroalcoholic extracts
The two experiments were performed according to the procedure described in
the literature.[10, 11] The extractions and analytical experiments were repeated twice.
For the preparation of hydroalcoholic extracts, a 10 g sample of plant material
was refluxed in 100 ml of solvent (70% ethanol-water (v/v)). The solvent was then
evaporated affording 1.2 g residue that was then assayed for its antioxidant activity.
GC/MS and GC/FID analysis
Quantitative analysis of the essential oil constituents was performed according
to the procedure mentioned in the literature.[11] The instrument used was Hewlett-
Packard HP-8590 gas chromatograph equipped with a Flame Ionization Detector
(FID). Split-splitless injector (split ratio 1:50) and an optima-5 fused silica capillary
column (30 m × 0.25 mm, 0.25 film thickness) was used. The temperatures of the
injector and detector were maintained at 250 ºC and 300º C, respectively. The analysis
of the essential oils was performed under linear temperature conditions, the
temperature was programmed from 60 ºC to 250 ºC at a rate of 10 ºC/min, the
temperature was then held constant at 250 ºC for 5 min. In the analysis of the hydro-
distilled volatile oils obtained from fresh and dried aerial parts of S. ceratophylla, the
relative peak area of each component of the oil was measured. The relative peak
areas of the oil components were used for the concentration calculation of the detected
compounds. Gas chromatography -Mass spectrometry (GC-MS) analysis of the essential oil
was performed on a Varian chrompack CP-3800 GC/MS/MS-200 (Saturn,
Netherlands) GC/MS equipped with DB-5 (5% diphenyl, 95% dimethyl polysiloxane)
GC capillary column (30 m ×0.25 mm i.d., 0.25 μm film thicknesses), using a sample of
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about 1 μL of each oil diluted in GC grade n-hexane. The temperature of the MS
source reached approximately 180 ºC and the ionization voltage was 70 eV. The
column was kept at 60 ºC for 1 min (isothermal). The temperature was then ramped to
246 ºC at a rate of 3 ºC/min and kept constant at 246 ºC for 3 minutes (isothermal).
Helium was used as a carrier gas at a flow rate of 0.9 ml/min. A homologous C8-C20 n-
alkanes hydrocarbon mixture was analyzed separately by GC/MS under the same
chromatographic conditions using the same DB-5 column.
The components of the essential oil were identified using built in libraries (Nist
Co and Wiley Co, USA) and by comparing their calculated retention indices relative to
the homologous C8-C20 n-alkanes hydrocarbon mixture, with their literature values
measured with columns of identical polarity[12] or with authentic samples.
DPPH• scavenging Activity Assay
The radical scavenging capacity of the hydro-alcoholic extract obtained from the
air dried S. ceratophylla was determined and compared to the values obtained for the
positive controls, ascorbic acid and α-tocopherol, based on the procedure listed in the
literature for their reaction with 2,2-diphenyl-2-picrylhydrazyl radical (DPPH•).[13] Briefly,
a solution of DPPH• (0.1 mM) was prepared in methanol and 1.0 mL sample of various
concentrations (0.005 - 0.600 mg/mL) of the hydroalcoholic extract in methanol was
added to 2 mL of the DPPH• solution. The solutions were allowed to stand at room
temperature in the dark for 30 min, then their absorbances were measured at 517 nm
(against blank samples) using a UV-visible spectrophotometer. All determinations were
performed in triplicate. The ability to scavenge the DPPH• was calculated using the
following equation:
퐷푃푃퐻• 푟푎푑푖푐푎푙 푠푐푎푣푒푛푔푖푛푔 푎푐푡푖푣푖푡푦 = 퐴 − 퐴
퐴 × 100%
The antioxidant activity was expressed as IC50, which was defined as the
concentration (in mg/mL) of extract required to inhibit the formation of DPPH radicals
by 50%.
ABTS•+ scavenging Activity Assay
The total antioxidant activity was determined by 2,2'-azino-bis(3-ethylbenzo-
thiazoline-6-sulphonic acid radical cation (ABTS•+) decolorization assay according to
the method described by Re et al (1999)[14] with some modifications. The ABTS•+
solution was prepared by combining equal amounts of 7 mM of ABTS•+ and 2.4 mM of
potassium persulfate (K2S2O8) solution which were allowed to react for 16 hour at room
temperature in the dark. Before use, this solution was diluted with methanol to get an
absorbance of 0.75 ± 0.02 at 734 nm. The reaction mixture comprised 3 mL of ABTS•+
solution and 1 mL of the hydro-alcoholic extract at various concentrations (0.005 -
0.600 mg/mL). The absorbance of the resulting mixture was measured at 734 nm
using UV- visible spectrophotometer. The blank was run in each assay and all
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measurements were done after at least 5 min. Ascorbic acid and α- tocopherol were
used as positive controls. The percentage inhibition was calculated according to the
equation:
퐴퐵푇푆 푟푎푑푖푐푎푙 푠푐푎푣푒푛푔푖푛푔 푎푐푡푖푣푖푡푦 = 퐴 − 퐴
퐴 × 100%
The ABTS•+ scavenging ability was expressed as IC50 (mg/mL).
Results and Discussion Hydro-distillation of the fresh and air dried aerial flowering parts of S.
ceratophylla afforded pale yellow oil with a yield of 0.5% and 0.1% (w/w, fresh and dry,
respectively). In total, 43 components were identified in the oil obtained from the fresh
plant material amounting to 94.53% of the total oil content (Table 1). This oil was
dominated by 10 sesquiterpene hydrocarbons, of which germacrene D had the main
contribution (18.10%). The second major compound detected in the essential oil of the
fresh plant was the monoterpene hydrocarbon limonene, which amounted to 17.75% of
the total oil content. Other classes of terpenoids were detected in appreciable amounts
including oxygenated monoterpenes (9.73%), oxygenated sesquiterpenes (8.50%),
diterpene hydrocarbons (0.75%), oxygenated diterpenes (1.66%) and phenyl-
propanoids (1.54%). Moreover, several aliphatic hydrocarbons, aldehydes, ketones
and esters were detected in the essential oil obtained from the fresh plant, amounting
in total to 24.60% of the total oil content.
Table 1: Chemical composition of the hydro-distilled oil of fresh and air dried flowering parts and SPME volatile components of the roots and flowers at different flowering stages of S. ceratophylla growing wild in Jordan.
Total identified (%) 94.53 97.19 99.80 98.59 99.62 98.24 SC1: hydro-distilled oil from fresh flowering parts; SC2:hydro-distilled air dried flowering parts; SC3: fresh pre-flower SPME, SC4: fresh full flowering SPME; SC5: post-flowering SPME; SC6: fresh roots SPME.
The GC/MS analysis of the hydro-distilled oil obtained from the air dried aerial
parts of S. ceratophylla resulted in the identification of 35 volatile constituents which
accounted for 97.19% of the total oil content. In comparison to the results obtained
from the oil of the fresh samples, qualitative and quantitative variations were noticed.
The essential oil of the dry plant was dominated by oxygenated terpenoids. Linalool
(55.55%) was the main oxygenated monoterpene component detected in the oil
followed by the oxygenated diterpenoid manoyl oxide which accounted for 7.29% of
the total oil content. Oxygenated sesquiterpenoids amounted to 19.91% of the total oil
content; spathulenol (6.00%) and -eudesmol (3.88%) were detected as the main
components of this fraction. The content of monoterpene hydrocarbons,
phenylpropanoids and aliphatic hydrocarbons decreased greatly as compared to their
content in the oil obtained from the fresh plant. Figure 1 shows the variations in the
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chemical composition of the essential oil obtained from fresh and air dried aerial parts
of S. ceratophylla growing wild in Jordan.
Figure 1: Variations in the main classes of compounds detected in the hydro-distilled essential oil of fresh and air dried flowering parts of S. ceratophylla growing wild in Jordan.
The volatile organic compounds (VOC’s) emitted from the fresh roots and the
fresh flowers at the pre-flowering, full flowering and post flowering stages – extracted
by SPME, resulted in the identification of 28, 32, 25, and 26 components, acounting for
98.24%, 99.80%, 98.59% and 99.62% of total extract, respectively (Figure 2). The
VOC’s emitted by these fresh organs were characterized by having monoterpene
hydrocarbons as the main constituents, amounting to 60.14%, 89.49%, 92.28%, and
59.66% of the total composition. -pinene amounted to 22.93% of the total volatile
principles emitted by S. ceratophylla fresh roots, while -thujene was the main
component detected in the volatile vapor of the flowering organ at the pre-flowering
(40.61 %), fully expanded flower (40.92 %) and post flowering (35.29 %) stages. These
results, as compared to those obtained from hydrodistillation, clearly indicate the effect
of drying and heat exposure on the oxidation process (Table 1).
The chemical constituents of the hydro-distilled essential oil obtained from S.
ceratophylla has been investigated in Turkey. Gürsoy et al. (2012) evaluated the
chemical composition of the hydro-distilled oil of dried S. ceratophylla collected in 2009
from the B5 Kayseri-Incesu highway area and the oil was distilled using a clavenger
type apparatus.[15] The oil contained -muurolene (11.4%) and -pinene (7.6%) as the
main constituents, both, however, were detected at much lower concentrations as
compared to the current findings. Recently, geographical variations
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Figure 2: Variations in chemical composition of SPME extracted volatile principles of fresh flowers (different stages) and roots of S. ceratophylla growing wild in Jordan.
in the essential oil composition of S. ceratophylla collected from three different
locations in Turkey have been evaluated where micro-distillation was applied to extract
the essential oil of the plant.[9] The results of that investigation revealed that the three
oil samples corresponding to the three different locations were rich in -pinene (23.7 -
27.0%). As compared to the results of our study, linalool, the main constituent detected
in S. ceratophylla from Jordanian origin, was found in much lower concentrations (2.8 -
4.0%).[9] Many air dried Salvia species were reported to contain large amounts of
linalool including S. verbenaca (30.72 %),[11] S. palaestina (8.6%),[16] and S. aethiopis
(9.2%).[17] On the other hand, S. palaestina from Jordanian origin was poor in linalool
but was found to be rich in germacrene D (26.02%).[18] Other Salvia species from
Jordanian origin including S. lanigera, S. spinosa, and S. syriaca contained linalool in
small concentrations (1.5%, 0.4% and 0.38%, respectively).[19] To conclude, variations
in chemotypes and genotypes,[20] in addition to geographical, environmental, and
experimental factors can affect the chemical composition of the essential oils and
volatile principles. Moreover, time of harvest, drying and extraction methods, part of
plant being investigated are also other important factors known to affect greatly the
composition of the hydro-distilled and volatile principles of the plant.
Antioxidant activity of the hydro-alcoholic extract of S. ceratophylla
Many mechanisms are known to account for antioxidant activity. These include
the decomposition of peroxides, prevention of chain initiation, prevention of continued
hydrogen abstraction, free radical scavenging, reducing capacity and binding of
transition metal ion catalyst.[14] In the current investigation, the antioxidant activity of
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the hydro-alcoholic extract of the air dried aerial parts of S. ceratophylla using 2,2-
diphenyl-2-picrylhydrazyl radical (DPPH•) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-
sulphonic acid (ABTS•+) assays was determined. Ascorbic acid and -tocopherol were
used as positive controls. The results of the DPPH radical inhibition for the hydro-
alcoholic extract of S. ceratophylla are shown in Table 2.
The reduction ability of DPPH radical formation is determined by the decrease in
its absorbance at 517 nm induced by antioxidants.[15] This could be attributed to the
hydrogen donating ability of the antioxidants. Generally, the hydro-alcoholic extract
showed a concentration-dependent activity profile (Table 2). The strongest DPPH
radical scavenging activity was found to be at 0.6 mg/mL (89.33 % ± 0.47 mg/mL) with
an IC50 value of 0.11 ± 1.85 mg/mL. The DPPH radical scavenging activity of the
hydroalcoholic extract of the plant was lower than those observed for tocopherol and
ascorbic acid.
Table 2: Antioxidant activity and IC50 values of the hydro-alcoholic extract obtained from air-dried S. ceratophylla compared to positive controls (ascorbic acid and α-tocopherol) on DPPH• assay.
The ABTS•+ radical scavenging activity of the hydro-alcoholic extract of S.
ceratophylla increased steadily with increasing the concentration of the extract. The
data indicate that the hydro-alcoholic extract had interesting antioxidant activity that is
slightly lower than those observed for the positive controls employed in this
investigation (Table 3). Interstingly, at 0.6 mg/mL, the hydro-alcoholic extract revealed
higher inhibition activity for the ABTS•+ assay as compared to that observed for the
DPPH• method (98.760.38; 89.33 0.47, respectively). This is also confirmed by the
lower IC50 value (0.09 mg/mL ± 2.91) compared to that observed for the DPPH• assay
(0.11 mg/mL ± 1.85). This result indicates that the plant can be used as a source of
natural antioxidants.
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Table 3: Antioxidant activity and IC50 of the hydro-alcoholic extract obtained from air-dried S. ceratophylla and positive controls (ascorbic acid and α-tocopherol) on ABTS•+ assay.
[12] Adams, R.P., Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy, Carol Strea, IL: Allured Publishing, 2001.
[13] Al-Qudah, M.A.; Al-Ghoul, A.M.; Trawenh, I.N.; Al-Jaber, H.I.; Al Shboul, T.M.; Abu Zarga, M.H.; Abu Orabi, S.T., TBAP, 2014, 4, 52 – 61.
[14] Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Free Radical Biol. Med., 1999, 26, 1231-7.
