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УЧЕНЫЕ ЗАПИСКИ КАЗАНСКОГО УНИВЕРСИТЕТА. СЕРИЯ ЕСТЕСТВЕННЫЕ НАУКИ
2023, Т. 165, кн. 1 ISSN 2542-064X (Print)
С. 94-117 ISSN 2500-218X (Online)
ORIGINAL ARTICLE
UDC 543.5:543.8 doi: 10.26907/2542-064X.2023.1.94-117
EVALUATION OF THE ANTIOXIDANT PROPERTIES AND GC-MSD ANALYSIS OF COMMERCIAL ESSENTIAL OILS FROM PLANTS OF THE Lamiaceae FAMILY
A.D. Kalmykova a, E.N. Yakupova a'b, F.A. Bekmuratovab, I.M. Fitsevb,
G.K. Ziyatdinova a
aKazan Federal University, Kazan, 420008 Russia hFederal Center for Toxicological, Radiation, and Biological Safety, Kazan, 420075 Russia
Abstract
Plants of the Lamiaceae family have been used for thousands of years in cooking, as well as phyto- and aromatherapy. Their essential oils are characterized by high antioxidant and other types of biological activities. In our study, the phytochemical profile and quantification of the essential oil components of thyme, marjoram, and sage were analyzed by gas chromatography with mass-spectrometric detection (GC-MSD). The antioxidant properties of the samples were evaluated using total antioxidant parameters (total antioxidant capacity (TAC), ferric reducing power (FRP), antioxidant activity (AOA) towards 2,2-diphenyl-1-picrylhydrazyl (DPPff), and total phenolics by Folin-Ciocalteu method). The obtained FRP was 46-321-fold lower than TAC, which is consistent with the contents of phenolics identified in the samples. Terpenes, isopropyl-methylphenols, and eugenol turned out to be the major components of all essential oils and determined their TAC and AOA. The Folin-Ciocalteu method was applicable to the thyme essential oil only. Its FRP, which is based on the reaction of phenolic antioxidants with electrogenerated ferri-cyanide ions, agreed well with the total phenolic contents (329 ± 17 and 334 ± 15 mg of carvacrol per mL, respectively). The thyme essential oil had the highest antioxidant parameters, while sage showed the weakest antioxidant properties. Positive correlations (r = 0.8846-0.9964) of the anti-oxidant parameters were obtained.
Keywords: essential oils, total antioxidant capacity, ferric reducing power, total phenolics, coulometric titration, phytochemical profile, marjoram, thyme, sage
Introduction
The Lamiaceae family is one of the most representative in the plant kingdom. Owing to their essential oils, aromatic plants of this family, such as oregano, rosemary, thyme, and sage, are widely used in cooking, phyto- and aromatherapy [1], as well as for the extraction of various bioactive compounds. They also have potential as natural food preservatives and functional food additives, thus contributing to better human nutrition [1, 2]. Since essential oils are highly beneficial - they possess antioxidant, antimicrobial, antitumor, anti-inflammatory, antiviral, and other properties, their traditional application range is steadily expanding [3].
Of particular interest and practical utility are the antioxidant properties of essential oils. The latter are also very useful to characterize plant samples. However, it is important
to consider that the components of essential oils and their amounts are strongly affected by many factors: the type and geographic origin of the plant material, along with the conditions of its growth, harvesting, and storage, etc. [4-6]. These aspects thwart any efforts to unify the features of essential oils. Despite being heterogeneous, all essential oils contain terpenes (hydrocarbon and oxygenated mono- and sesquiterpenes, as well as diterpenes) [7, 8]. The presence of phenolic fragments and double bonds in the structure of terpenes enables them to react with reactive oxygen species, i.e., they exhibit antioxi-dant activity. Hence, essential oils exert a pronounced antioxidant effect due to the syn-ergistic action of terpenes and some individual compounds [9, 10].
Therefore, it is helpful to evaluate the total antioxidant parameters of essential oils in order to characterize the plant sample in general because this approach takes into account the possible mutual influence of the sample components and the effects they cause.
This paper focuses on thyme, marjoram, and sage essential oils as the samples under investigation. Our overview of the available literature demonstrates that marjoram and sage have been less studied than thyme, oregano, basil, and rosemary. The antioxidant properties of the essential oils of these plants have been characterized using various approaches. In many works, to assess the phytochemical profiles and quantify individual antioxidants, the method of gas chromatography with mass spectrometric detection (GC-MSD) has been applied [5, 11-13]. The total antioxidant parameters have been measured following standard spectrophotometry protocols. Typical examples are summarized in Table 1.
The most commonly studied parameter is the antioxidant activity (AOA) towards stable radicals like 2,2-diphenyl-1-picrylhydrazyl (DPPH^) [11, 13-21] or per-oxyl radicals obtained from 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+~) [13, 18, 19, 21]. Oxygen-centered radicals are used less often [15, 21-23] despite they rely on the processes that are more similar to those in living systems. Their application is probably limited by time-consuming procedure complicated by the addition of many reagents to evaluate the hydroxyl radical-scavenging activity [15, 22] and the need to use fluorescent detection for peroxyl radicals [21, 23]. In the P-carotene/lino-leic acid bleaching assay, the ability of antioxidants in essential oils to inhibit lipid pe-roxidation, which is similar to the processes that occur in living cell membranes in the presence of peroxyl radicals, is analyzed [25].
The ferric reducing properties of essential oils must be also taken into account to measure the reducing ability of antioxidants, but they apply only to certain antioxidants contributing to ferric reducing power (FRP) and ferric reducing antioxidant power (FRAP) [26]. Furthermore, the application of Fe2+ as a standard in FRP requires standardization because it is unstable and easily oxidized. Another disadvantage is that the results obtained are affected by the time needed to complete the analysis. The reaction between antioxidants and Fe3+ takes different amount of time (from several minutes to hours) and depends on the antioxidant nature [27]. For this reason, the resulting data can be controversial unless they reflect a complete reaction.
Recently, coulometric titration with electrogenerated titrants (bromine and ferri-cyanide ions) has been introduced to evaluate the antioxidant properties of essential oils [28]. Based on the reactivity of individual antioxidants, electrogenerated bromine has been successfully applied to estimate the total antioxidant capacity (TAC) in the presence of phenolics and terpenes [28].
Table 1
Spectrophotometry evaluation of total antioxidant parameters of the thyme, marjoram, and sage essential oils
Antioxidant parameter Reagent Plant Units Ref.
Antioxidant activity DPPff Thymus algeriensis IC50, ^g mL-1 [111
Salvia officinalis L. [131
Thymus capitatus [14]
Origanum majorana L. from Albania [15]
Salvia tomentosa Miller [161
Origanum majorana L. from Nepal [17]
Thymus quinquecostatus Celak. [18]
Origanum majorana L. from Northwest Egypt Inhibition percentage [191
Origanum majorana, Thymus satureioides [20]
Thymus zygis and Thymus hyemalis from Spain ^mol TE mL [21]
ABTS+* Salvia officinalis L. IC50, ^g mL-1 [13]
Thymus quinquecostatus Celak. [18]
Origanum majorana, Thymus satureioides ^mol TE mg-1 [19]
Thymus zygis, Thymus hyemalis from Spain ^mol TE mL-1 [21]
Hydroxyl radical-scavenging activity •oh Origanum majorana L. from Albania IC50, ^g mL-1 [15]
Thymus caespititius, T. camphoratus, T. capitellatus, T. carnosus, T. pulegioides, T. zygis subsp. zygis, T. zygis subsp. sylvestris Inhibition percentage [22]
Oxygen radical absorption capacity ROO^ Thymus zygis, Thymus hyemalis from Spain ^mol TE g-1 [21]
Thymus mastichina L. from Murcia (Spain) mg TE [23]
P-carotene/ linoleic acid bleaching assay ß-carotene/ linoleic acid mixture Salvia tomentosa Miller Inhibition percentage [16]
Origanum majorana L. from Northwest Egypt [19]
Origanum majorana, Thymus satureioides [20]
Ferric reducing power Potassium ferricya-nide Salvia officinalis L. IC50, ^g mL-1 [13]
Thymus capitatus [14]
Origanum majorana L. from Nepal [17]
Origanum majorana, Thymus satureioides Percentage vs. BHA [20]
Thymus zygis, Thymus hyemalis from Spain ^M AAE** [21]
Ferric reducing antioxidant power Fe3+-2,4,6-tripyridyl-S-triazine complex Thymus caespititius, T. camphoratus, T. capitellatus, T. carnosus, T. pulegioides, T. zygis subsp. zygis, T. zygis subsp. sylvestris Inhibition percentage [22]
Thymus vulgaris, Thymbra spicata ^M of Fe+2/g [24]
Trolox equivalent. Ascorbic acid equivalents.
This behavior is defined by the properties of electrogenerated bromine: its ability to participate in oxidation reactions, electrophilic addition to multiple bonds, and electro-philic substitution in aromatic systems [29]. Electrogenerated ferricyanide ions react only with phenolic antioxidants and allow the evaluation of FRP reflecting the total phenolic contents [28, 30, 31]. Coulometric approaches are more simple compared to spectropho-tometry and can be used with antioxidants of various nature and with different mechanisms of action. Another plus is that an electron acts as a titrant, thereby making the use of standard antioxidants unnecessary. The method is absolute and is not affected by sample dilution; the possibility of miniaturization and automation is favorable in routine analysis [32].
A noteworthy detail is that previous studies on the antioxidant properties of the essential oils of marjoram, thyme, and sage have been based on the samples from wild or cultivated plants of different chemotypes and geographical origin. Another aspect of the majority of these investigations is that the impact of the extraction methods on the properties (antioxidant, antibacterial, etc.) of the final product was studied. Commercial essential oils should step out of the shade and have their phytochemical profile and antioxidant properties thoroughly inspected. Furthermore, total antioxidant parameters could be considered as potential markers of the quality of essential oils.
The purpose of this paper is to characterize the commercial essential oils of marjoram, thyme, and sage by analyzing their phytochemical profiles, quantifying their composition with GC-MSD, as well as assessing the total antioxidant parameters (TAC, FRP, AOA towards DPPH", and the total phenolic content) using the coulometric and spectro-photometric approaches. The relationship between the total antioxidant parameters and the phytochemical constituents of the essential oils is discussed.
1. Material and Methods
1.1. Samples and reagents. Commercially available essential oils of marjoram, thyme, and sage were studied. A tenfold dilution with ethanol was applied for the evaluation of antioxidant properties. Carvacrol (purity 98%) (Aldrich, Germany) was used as a standard for the evaluation of total phenolics. Its 100 mg L-1 stock solution was prepared by dissolving an accurately weighed portion in 5.0 mL of ethanol (rectificate). The exact dilution before measurements was used to get less concentrated solutions. A 0.10 mM solution of DPPH (Aldrich, Germany) was prepared in methanol (c.p.). The Folin-Ciocalteu reagent (Aldrich, Germany) was applied for the total phenolics determination. Other reagents were of chemical purity and used as received.
1.2. Phytochemical profile and analysis by GC-MSD. The identification and quantification of the essential oil components were performed by GC-MSD in the total ion current mode using a Crystal 5000.2 gas chromatograph with a quadrupole MSD and the Advanced Ion Source for the electron impact (EI) and chemical ionization (CI) (Chromatec, Russia), as well as a quartz capillary column CR-5MS ((5%-phenyl)-dime-thylpolysiloxane phase, 30 m x 0.25 mm x 0.25 ^m). Injection of 1 ^L of the essential oil was applied in 1:100 split mode. GC measurements were performed under the following conditions: injector temperature 280 °C, interface temperature 270 °C, ionic source temperature 250 °C. The column temperature was initially 60 °C for 1 min, then gradually increased to 210 °C at 5 °C min-1, raised again to 280 °C at 12 °C min-1, and
kept at 280 °C for 40 min. Helium with a constant flow rate of 0.9 mL min-1 was used as a carrier gas. Mass spectra in the positive ions mode were recorded in the range of m/z 50-550 after EI ionization at 70 eV. In the case of low intensity (< 1% rel.) of molecular ion [M+] peaks, the CI at 30 eV using methane as reagent gas (flow rate of 1.5 mL min-1) was applied to register more intensive peaks of protonated molecules [M + H]+. Mass spectra-based identification of the components was carried out using the following software: Chromatec Analytic (Chromatec, Russia); NIST MS Search Program V.2.3 (NIST, USA) and NIST 20 (NIST, Mass Spectra Libraries, USA); Wiley Registry of Mass Spectral Data, 12th ed. (Wiley Science Solutions, USA). In addition, the retention times and indices were compared with those reported in [33, 34] and presented in the databases mentioned above.
1.3. Evaluation of TAC and FRP. TAC and FRP were evaluated using coulo-metric titration of the samples with electrogenerated bromine and ferricyanide ions, respectively [28], using the coulometric analyzer Expert-006 (Econix-Expert, Russia) supplied with a glassy electrochemical cell with four electrodes. Two electrodes (working and auxiliary) formed a generating circuit. The working electrode was a platinum wire with 0.5 cm2 surface area. The auxiliary electrode (platinum wire) was separated from the anodic compartment of the cell with the semipermeable membrane to avoid side reactions. The other two needle platinum electrodes were polarized with a potential of 200 mV and used as an indicator circuit. Electrogeneration of bromine and ferricyanide ions was carried out from a solution of 0.2 M KBr in 0.1 M H2SO4 and 0.1 M K4Fe(CN)6 in 2 M NaOH, respectively, at a current density of 5 mA cm-2, providing 100% yield of the titrants. The volume of the solution in the electrochemical cell was 20 mL. Coulometric titration was carried out in the following way: the titrant was electrogenerated to the indicator current of 40 ^A, an aliquot portion (10 ^L) of the 10-fold diluted essential oil was added to the cell, and the timer was started simultaneously. The titration end point was registered at the moment when the indicator current reached the value of 40 ^A. TAC and FRP were expressed as the quantity of electricity spent on the titration of the sample and recalculated per 1 mL of the essential oil.
1.4. AOA towards DPPH\ The standard procedure was applied for the estimation of AOA using DPPH as a reagent [35]. Briefly, 3 mL of 0.10 mM DPPH solution were mixed with 4 ^L of the essential oil (10-fold diluted with ethanol) and incubated in the dark for 20 min. Then, the absorption was read at 515 nm using methanol containing 4 ^L of the sample as a blank on the spectrophotometer PE-5300 (NPO Ecros, Russia). Control DPPH* absorption was measured vs. methanol. The AOA of the essential oil was expressed as a relative decrease in the DPPH absorption.
1.5. Total phenolics determination. Total phenolic contents were evaluated by the Folin-Ciocalteu method [36] with slight modifications. 0.5 mL of the 10 000-fold diluted thyme essential oil and 1000-fold diluted marjoram and sage essential oils or the standard solution of carvacrol (10, 25, 50, 75, and 100 mg L-1) were placed in a 5.0 mL volumetric flask. Then, 2.5 mL of the diluted Folin-Ciocalteu reagent (1:10 (v/v)) were added and thoroughly mixed. After 4 min, 2.5 mL of 7.5% Na2CO3 solution were added, mixed, and incubated for 1 h. The absorbance of the solution was measured at 765 nm in a 0.5 cm cuvette. The blank solution contained all the reagents excluding
essential oil, which was replaced with 0.5 mL of ethanol. Total phe-nolics were expressed in carvacrol equivalents recalculated per 1 mL of the essential oil. Carvacrol calibration graph parameters (Equation 1) were used:
A [a.u.] = (0.007 ± 0.005) + (32.3 ± 0.8)-10-4 ccarvacrol [mg L-1]. (1)
1.6. Statistical and correlation analysis. The antioxidant parameters were evaluated as an average value of five (for coulometric titration) or three (for spectrophotometry) parallel measurements. GC-MSD was run in three replications. Statistical treatment of the data obtained was performed at a significance level of 5%. The results were presented as an average value ± coverage interval. A random error was reflected by the relative standard deviation (RSD).
Correlation analysis was performed in the OriginPro 8.1 software (OriginLab, USA).
2. Results and Discussion
2.1. Phytochemical profile of the essential oils. The phytochemical profile of the essential oils was studied by GC-MSD (Figs. 1-3). Identification and quantification data are summarized in Table 2. Components with © > 0.04% are shown.
The terpene components were relatively similar for the marjoram, thyme, and sage essential oils (Table 2). The major terpenes (© > 2%) for the marjoram essential oils were terpinene-4-ol (24.7% and 28.0%), isoterpinene (15.53% and 17.5%), y-terpinene (14.2% and 2.42%), linalyl acetate (8.82% and 10%), a-terpineol (8.35% and 9.5%), p-caryophyllene (4.6% and 4.89%), o- or p-cymene (5.27% and 4.16%, respectively), a-pinene (3.45% and 3.5%), a-phellandrene (2.98% and 3.3%), and c/5-sabinene hydrate (1.72% and 2.32%). Limonene (2.49%) was found only in marjoram sample 2. o-Cymene (12.0%), p-caryophyllene (4.26%), y-terpinene (4.1%), linal-ool (3.3%), and p-myrcene (2.01%) were the major terpenes of the thyme essential oil. Lower levels of a-terpinene (1.86%), terpinen-4-ol (1.67%), and a-pinene (1.66%) were also found. Sage was characterized by the high contents of eucalyptol (19.5%), camphor (17.6%), borneol (9.6%), thujone (6.0%), a-pinene (5.92%), isoborneol (5.8%), linalyl anthranilate (5.7%), camphene (5.2%), p-pinene (4.4%), linalool (3.5%), p-caryophyllene (2.82%), a-terpineol (2.48%), p-myrcene (2.3%) p-cymene (2.3%), and a-humulene (2.01%). Other terpenes were present in amounts less than 2%.
The essential oils from Lamiaceae plants are a rich source of natural phenolics (oxygenated terpenes), which is confirmed by the GS-MS data for the essential oils of the marjoram, thyme, and sage samples. Isopropylmethylphenols were the major phe-nolics. Their contents varied significantly depending on the plant material. The highest contents of both carvacrol and thymol were found in the thyme essential oil (61.5% and 1.50%, respectively). The marjoram essential oils contained carvacrol (0.18% and 0.20%) and trace thymol. Only trace thymol was identified in sage. Furthermore, the thyme essential oil contained eugenol (0.080%), and the marjoram essential oils contained anethole (0.22% and 0.23% in samples 1 and 2, respectively).
In general, the major components identified are similar to those reported for the marjoram [12, 15, 17, 37-41] and thyme [1, 21, 22, 41-45] essential oils. The slight differences were observed in minor components.
The major component of the marjoram essential oils turned out to be terpinene-4-ol. This fits very well with most studies [12, 15, 17, 37-40, 46-48], in which its content
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Table 2
GC-MS identification and quantification of the marjoram, thyme and sage essential oils (n = 3,p = 0.95)
No.* in (min)** RI*** Component m/z and peak relative intensity (%) CO (%)
Marjoram 1 Marjoram 2 Thyme Sage
1 5.84 764 Methyl 3-methyl-butanoate 117 ([M + H]+, 42%), 74 (100%), 43 (30%) 0.070 ± 0.004
2 10.20 833 4-Ethenyl-l,5,5-trimethylcyclopentene 136 ([IVf], 28%), 93 (100%), 121 (58%) 0.060 ± 0.005 0.11 ±0.04
3 10.26 926 ±2 P-Thujene 137 ([M + H]+, 55%), 93 (100%), 77 (36%) 0.13 ±0.06
10.25 0.65 ±0.08
4 10.27 930 3-Thujene 136 ([N'T], 11%), 93 (100%), 91 (52%) 0.16 ±0.03
5 10.60 933 ±7 a-Pinene 137 ([M + H]+, 18%), 93 (100%), 91 (41%) 3.45 ±0.01
10.58 3.5 ± 0.1
10.53 1.66 ±0.05
10.63 5.92 ±0.06
6 11.09 946 ±8 Camphene 137 ([M + H]+, 15%), 93 (100%), 121 (58%) 0.70 ± 0.05
11.08 0.49 ±0.02
11.07 0.25 ±0.06
11.17 5.2 ±0.1
7 11.80 967 ±2 Sabinene 136 ([N'T], 18%), 93 (100%), 91 (40%) 0.59 ±0.06
11.79 0.65 ±0.08
11.81 0.12 ±0.03
8 11.85 971 Oct-l-en-3-ol 129 (fM + Hf, 31%), 57 (100%), 43 (21%) 0.25 ±0.09
9 12.00 974 ±5 P-Pinene 137 ([M + H]+, 38%), 41 (61%), 77 (28%) 0.60 ±0.08
11.99 0.60 ±0.06
11.97 0.36 ±0.08
12.06 4.4 ±0.1
10 12.28 983 ±3 P-Myrcene 137 ([M + H]+, 86%), 69 (80%), 39 (30%) 1.39 ±0.06
12.27 1.7 ±0.2
12.25 2.01 ±0.06
12.30 2.3 ±0.1
11 12.41 987 Octan-3-ol 131 ([M + H]+, 44%), 59 (100%), 55 (69%) 0.060 ± 0.004
12 12.93 998 ±8 a-Phellandrene 136 ([M+],16%), 93 (100%), 91 (33%) 2.98 ±0.04
12.91 3.3 ±0.4
12.85 0.23 ±0.09
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13 12.97 1010 (Z)-P-ocimene 137 ([M + H]+, 22%), 93 (100%), 91 (50%) 0.12 ±0.06
14 13.15 1024 ±2 Isocineole 154 ([M1"], 21%), 43 (100%), 111 (73%) 0.94 ±0.03
13.16 1.84 ±0.05
15 13.23 1030 ±2 a-Terpinene 136 ([M1"], 43%), 121 (100%), 93 (85%) 1.83 ±0.05
13.24 0.050 ±0.003
16 13.27 1033 cw-Sabinene hydrate 154 (fM+1, 5.3%), 43 (100%), 93 (93%) 1.72 ±0.04 2.32 ±0.08
17 13.53 1050 ±7 />-Cymene 134 ([M1"], 25%), 119 (100%), 91 (16%) 4.16 ±0.05
13.62 2.3 ±0.2
18 13.53 1060 ±3 o-Cymene 134 ([M1"], 26%), 119 (100%), 91 (39%) 5.27 ±0.05
13.56 12.0 ±0.3
19 13.70 1065 Limonene 136 ([M+], 16%), 68 (100%), 93 (50%) 2.49 ±0.04
20 13.71 1067 2-Bornene 136 ([M1!, 32%), 93 (100%), 121 (90%) 2.34 ±0.06
21 13.73 1072 ±12 Eucalyptol 154 ([M+], 36%), 43 (100%), 81 (65%) 0.70 ± 0.04
13.77 1.02 ±0.04 1.70 ±0.06
13.88 19.5 ±0.4
22 14.11 1085 (E)-|3-Ocimene 137 ([M + Hf, 28%), 93 (100%), 41 (36%) 0.070 ± 0.004
23 14.57 1090 ±14 y-Terpinene 136 ([M+], 36%), 93 (100%), 91 (37%) 4.1 ±0.1 0.66 ±0.08
14.73 14.2 ±0.1
14.76 2.42 ±0.01
24 14.88 1105 trans- Sabinene hydrate 155 ([M + H]+, 37%), 93 (100%), 43 (50%) 0.33 ±0.06
25 14.97 1108±2 4-Thujanol 155 ([M + H]+, 43%), 93 (80%), 43 (75%) 0.87 ±0.08
14.96 0.92 ±0.06
26 15.48 1111 ra-Cymenene 132 (fM+1, 100%), 117 (96%), 115 (67%) 0.080 ±0.003
27 15.41 1113 ±20 Isoterpinene 136 ([M1"], 82%), 93 (100%), 121 (76%) 0.18 ±0.05
15.44 0.15 ±0.04
15.66 17.5 ±0.2
15.68 15.53 ±0.06
28 15.80 1135 ±12 Linalool 155 ([M + H]+, 18%), 71 (58%), 93 (42%) 3.3 ±0.3
15.97 3.5 ± 0.1
29 16.01 1148 ±2 iraMs-Sabinene hydroxide 155 ([M + H]+, 30%), 43 (50%), 91 (44%) 0.42 ± 0.07
30 16.19 1155 ±2 Thujone 153 ([M+ H]+, 33%), 110 (100%), 81 (88%) 6.0 ±0.5
31 15.92 1160 ±20 cw-p-Menth-2-en-1 -ol 155 ([M + H]+, 37%), 43 (100%), 139 (51%) 0.090 ± 0.004
16.64 0.22 ±0.08
16.65 0.19 ±0.05
32 16.67 1182 Fenchol 155 ([M + H]+, 66%), 81 (100%), 80 (53%) 0.22 ± 0.06
33 16.95 1195 P-Terpineol 155 ([M+ H]+,42%), 43 (100%), 71 (53%) 0.070 ± 0.004 0.20 ±0.03
34 17.07 1200 1,2-Dihydro-linalool 157 ([M + H]+, 31%), 73 (100%), 41 (58%) 0.050 ±0.003
35 17.60 1238 Camphor 153 ([M + H]+, 30%), 95 (100%), 41 (79%) 17.6 ±0.6
36 18.00 1262 Isoborneol 155 ([M + H]+, 55%), 95 (100%), 41 (20%) 5.8 ±0.2
37 18.33 1270 Borneol 155 ([M + Hf, 40%), 95 (100%), 110 (22%) 9.6 ±0.4
38 18.33 1273 ± 20 Terpinen-4-ol 154 ([M+],15%), 71 (100%), 111 (50%) 1.67 ±0.06
18.64 28.0 ±0.5
18.71 24.7 ±0.2
39 18.85 1295 ±18 a-Terpineol 155 ([M + H]+, 32%), 121 (73%), 136 (44%) 2.48 ±0.08
18.96 8.35 ±0.09
18.98 0.14 ±0.05
19.19 9.5 ±0.2
40 19.22 1305 ±2 trans-Piperitol 155 ([M + H]+, 25%), 84 (100%), 41 (27%) 0.14 ±0.06
19.23 0.12 ±0.08
41 19.48 1315 a-Fenchyl acetate 197 ([M+], 20%), 81 (100%), 43 (74%) 0.090 ±0.005
42 19.93 1333 Carvacrol methyl ether 164 ([M+], 31%), 149 (100%), 91 (25%) 0.18 ±0.03
43 20.30 1362 Linalyl anthranilate 274 ([M + Hf, 63%), 137 (100%), 119 (70%) 5.7 ± 0.1
44 20.38 1370 ±2 Linalyl acetate 197 ([M + H]+,16%), 80 (69%), 55 (23%) 10.0 ±0.2
20.39 8.82 ±0.05
45 20.64 1388 P-Citral 153 ([M + H]+, 40%), 41 (100%), 69 (85%) 0.050 ±0.002
46 20.74 1392 a-Citral 153 ([M + H]+, 18%), 41 (100%), 69 (96%) 0.090 ±0.003
47 20.78 1395 Dihydrolinalool 157 ([M + Hf, 31%), 73 (100%), 41 (58%) 0.050 ±0.003
48 21.32 1412 ± 4 Anethole 148 ([M+], 100%), 147 (54%), 117 (33%) 0.23 ±0.04
21.34 0.22 ± 0.05
49 21.36 1418 Bornyl acetate 197 ([M + H]+, 43%), 95 (100%), 43 (70%) 1.99 ±0.09
50 21.39 1422 Thymol 150 ([IVf], 25%), 135 (100%), 91 (16%) 1.50 ±0.06
51 21.44 1425 ±2 fraws-Ascaridol glycol 171 ([M + H]+, 17%), 109 (100%), 127 (55%) 0.10 ±0.03
21.45 0.070 ± 0.008
52 21.58 1430 ±20 Carvacrol 150 ([M+], 27%), 135 (100%), 150 (27%) 0.20 ± 0.02
21.59 0.18 ±0.06
21.89 61.5 ±0.4
53 22.30 1463 2-Methyl-5-(pro-pan-2-ylidene)cyclo-hexane-l,4-diol 171 ([M + H]+, 56%), 74 (100%), 109 (43%) 0.22 ± 0.06
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54 22.45 1476 trans- 3 -p-Men-then-1,2-diol 170 ([M1"], 15%), 112 (100%), 97 (74%) 0.050 ±0.003
55 22.96 1494 ±4 a-Terpinyl acetate 197 ([M + H]+, 37%), 121 (92%), 68 (31%) 0.16 ±0.04
22.97 0.36 ±0.05
22.98 0.34 ±0.04
56 23.15 1508 Eugenol 164 ([M+], 100%), 103 (36%), 77 (35%) 0.080 ± 0.004
57 23.16 1510 Nerol acetate 197 ([M + H]+, 32%), 69 (100%), 93 (51%) 0.21 ±0.06
58 23.42 1531 Carvacryl acetate 192 ([M+], 7%), 135 (100%), 150 (61%) 0.17 ±0.02
59 23.66 1547 ±4 Geraniol acetate 197 ([M + H]+, 31%), 69 (100%), 43 (62%) 0.58 ±0.08 0.42 ±0.05
23.68 0.51 ±0.05
60 23.93 1578 Cadina-3,5-diene 204 ([M+], 25%), 161 (100%), 105 (66%) 0.090 ± 0.006
61 24.72 1621 Isocaryophyllene 205 (fM + Hf, 16%), 41 (100%), 93 (78%) 0.050 ± 0.002
62 25.16 1655 ±4 ß-Caryophy llene 205 ([M + H]+, 72 %), 93 (100 %), 91 (85 %) 4.26 ± 0.04
25.17 2.82 ±0.08
25.18 4.89 ±0.04
25.19 4.6 ±0.1
63 25.99 1701 ±5 a-Humulene 205 ([M + H]+, 20%), 93 (100%), 80 (28%) 0.13 ±0.06
26.04 2.01 ±0.06
64 26.95 1747 ±4 Bicyclogermacrene 205 ([M + H]+, 41%), 93 (100%), 107 (57%) 0.10 ±0.06
26.97 0.10 ±0.04
65 27.10 1763 ß-Bisabolene 204 ([M+], 25%), 69 (100%), 93 (69%) 0.51 ±0.05
66 27.84 1788 fraws-a-Bisabo-lene 205 ([M + H]+, 15%), 93 (100%), 119 (35%) 0.17 ±0.06
67 28.83 1826 Spathulenol 221 (fM + Hf, 48%), 43 (100%), 41 (63%) 0.070 ± 0.004 0.090 ± 0.004
68 28.99 1835 ± 2 ß-Caryophyllene oxide 221 ([M + H]+, 27%), 43 (100%), 41 (93%) 0.28 ±0.08 0.88 ±0.07 0.23 ±0.04
29.00 0.15 ±0.05
69 29.24 1861 Viridiflorol 223 ([M + H]+, 24%),109 (100%), 43 (76%) 0.37 ±0.05
70 29.54 1899 Humulene-1,2-epoxide 221 ([M+ H]+, 17%), 109 (100%), 67 (84%) 0.13 ±0.04
71 30.57 1946 14-Hydroxy-caryophy llene 220 ([M+], 5%), 91 (100%), 41 (92%) 0.090 ±0.005
72 32.71 1967 ±2 Isopropyl myristate 271 ([M + H]+, 14%), 102 (67%), 55 (39%) 0.090 ±0.003
32.72 0.16 ±0.08
73 34.36 1992 ±2 »г-Camphorene 273 ([M + H]+, 35%), 69 (100%), 91 (26%) 0.050 ±0.003 0.11 ±0.06
34.37 0.050 ±0.003
peak number; retention time; retention indice; [M+] (EI at 70 eV) and [M + H]+ (CI at 30 eV).
varies in a wide range (21.3-38.4%). Isoterpinene (terpinolene) has been identified in two experiments [17, 41]: their results show that its contents vary significantly (2.5% [17] and 17.1% [41]). These values correspond to those found in our marjoram essential oil samples (15.53 and 17.50%). The detected contents of y-terpinene, linalyl acetate, and a-terpineol are comparable with the earlier ones [17, 41]. Other studies demonstrate either significantly lower [49] or higher [50] amounts of linalyl acetate. P-Caryophyllene contents in our samples were approximately twice as high as those in [17, 41]. Another characteristic component was sabinene hydrate (both cis- and trans -isomers), the contents of which were significantly lower than in [12, 15, 17, 37, 39, 40, 51]. According to [41, 52], carvacrol is usually present in trace amounts or absent. However, we found 0.20% carvacrol in the marjoram essential oils.
Carvacrol was the primary component of the thyme essential oil samples. This obviously differs from the data in [21, 41, 43-45] suggesting the prevalence of thymol. Interestingly, in the essential oils extracted from Portuguese thyme species (Thymus caespiti-tius Brot., Thymus zygis Loefl. ex L. subsp. sylvestris, and Thymus zygis Loefl. ex L. subsp. zygis), carvacrol ranked as a major component. Its content range was 31.861.9% [22], and these values are close to our data. o-Cymene prevailed among the ter-penes in our samples, while other researchers [21, 41, 44, 45, 53] distinguished p-cymene as the main terpene. P-Caryophyllene and y-terpinene contents were in line with those given for the essential oils of Thymus vulgaris L. obtained by hydrodistillation using a Dering-type apparatus [41] and commercial samples [44].
The principal components of the sage essential oil were consistent with the reports on the essential oils of Salvia officinalis growing in Sudan [54] and other sage plants at various phenological stages [13, 55]. Eucalyptol as a major component and its contents are also in line with these results. The content of camphor was similar to that for the essential oils from Algeria [56].
Generally, the phytochemical profile of essential oils and the content of their main components are determined by a number of factors, such as variations in the chemotypes of plant species [57], place of their origin, seasonal climate variations, as well as the conditions and method of essential oil production [4, 5, 58, 59].
Thus, the identified components of the essential oils are indicative of their antioxidant properties.
2.2. Total antioxidant parameters of the essential oils. TAC and FRP were evaluated based on the reactions of the essential oil components with electrogenerat-ed bromine and ferricyanide ions, respectively (Fig. 4). In our data, TAC was significantly higher than FRP (46-321-fold difference), which is due to the presence of hydrocarbon terpenes that are reactive towards electrogenerated bromine but do not undergo oxidation by ferricyanide ions [28].
The thyme essential oil had the highest TAC and FRP values (1540 ± 20 and 4.8 ± 0.2 C mL-1, respectively) among the studied samples, which is consistent with the phytochemical profile of this sample. Carvacrol was the major contributor to both TAC and FRP. Terpenes (P-caryophyllene, linalool, P-myrcene, a- and P-pinenes, camphene) defined the TAC value of the thyme essential oil. Thymol was also found to be reactive towards both titrants and had an impact on TAC and FRP.
Fig. 4. Total antioxidant parameters of the marjoram (samples 1 and 2), thyme (sample 3), and sage (sample 4) essential oils based on the coulometric titration data: (a) TAC, (b) FRP
<
o <
40-
10
2 3
Essential oil samples
Fig. 5. AOA towards DPPH' of the marjoram (samples 1 and 2), thyme (sample 3), and sage (sample 4) essential oils
Statistically significant differences in TAC and FRP were observed for the marjoram essential oil samples. Both these parameters changed in a similar way. The difference in FRP can be explained by the carvacrol contents in the samples. The trace amounts of thymol contained in the samples also contributed to FRP.
The essential oil of sage was characterized by the lowest TAC value because its major terpenes (eucalyptol, camphor, borneol, thujone, and isoborneol) do not react with electrogenerated bromine. The absence or trace contents of thymol resulted in a low TAC value as well. This is confirmed by the zero value of FRP for the sage essential oil.
The TAC and FRP values agree well with the phytochemical profile of the essential oils and the contents of hydrocarbon and oxygenated terpenes.
The DPPH' test was carried out as a standard procedure to describe the ability of the essential oils under study to react with free radicals. All samples showed AOA towards DPPH' (Fig. 5).
The average values of the investigated parameters differ significantly among the essential oils from Lamiaceae plants that were analyzed. The data obtained are in line with TAC and FRP. The highest inhibition of DPPH* was observed for the thyme essential oil containing the largest amounts of phenolics (carvacrol and thymol), which are the major contributors to AOA. The sage essential oil had the lowest AOA value and contained almost no phenolics (only trace thymol was identified by GC-MSD),
which is an indirect proof of the above finding. The marjoram essential oils demonstrate a 5.7-fold difference in the AOA values, even though their carvacrol contents were comparable. This confirms that other components of the studied essential oils also react with DPPH\ The analysis of the resulting phytochemical profile of the essential oils under consideration supports the conclusion that only terpenes can influence the AOA parameter. This assumption complies with the published data on the reactivity of terpenes towards DPPH* [60-62]. For example, limonene, P-myrcene [60], spathulenol [61], and other monoterpenes [62] show AOA in reactions with DPPH\ Furthermore, the DPPH* inhibition values are significantly lower than those for phenolic compounds because the H-atom transfer proceeds more easily from the H-O bond than from the H-C bonds (the dissociation energies are 364 kJ mol-1 for the allylic C-H bond, 410 kJ mol-1 for the al-kylic C-H bond, and 452 kJ mol-1 for the vinylic C-H bond [62] vs. 243-314 kJ mol-1 for the O-H bonds in natural phenolics [63]). Therefore, it is impossible to apply the parameter IC50, which corresponds to the concentration of terpene that causes 50% inhibition of DPPH*, because this value cannot be reached even at the highest concentration that this method allows [64]. In this case, relative DPPH* inhibition is usually used. Thus, it is more informative for the studied essential oils to apply AOA towards DPPH* expressed as a percentage of inhibited DPPH*.
A similar trend in AOA towards DPPH* has been detected for the thyme, marjoram, and sage essential oils according to [6, 41, 45]. AOA values are also considered with regard to the contribution of phenolics (mostly thymol and carvacrol) as the major components [41]. As known [65], high levels of phenolic constituents remarkably accelerate the reaction with DPPH*. The AOA values of the marjoram essential oils in our study were significantly lower than those reported for the Origanum majorana L. leaves essential oil from northwest Egypt [19], which can be attributed to the high contents of sabinene and terpinenes.
The total phenolic content of the studied essential oils was measured using the Fo-lin-Ciocalteu method. After a 10 000-fold dilution, the only essential oil which could be studied was that of thyme, while the essential oils of marjoram and sage became turbid after the addition of the photometric reagents. This is caused by the chemical composition of these essential oils. The phenolic content was negligible as compared to terpenes, which are insoluble in water media.
The phenolics of the thyme essential oil were mainly carvacrol (61.5%), thymol (1.50%), and trace eugenol, all being well-soluble in ethanol used in our study for sample dilution and not affected by water media used in subsequent determination of the total phenolic contents.
The total phenolic contents were expressed as equivalents of carvacrol, a major component of the thyme essential oil in our samples. Its average value was 334 ± 15 mg mL-1 with the RSD of 1.9%, which agrees well with the FRP value of 329 ± 17 mg mL-1 (RSD = 4.2%) obtained by coulometric titration and recalculated as carvacrol equivalents using its stoichiometric coefficient in the reaction with ferricyanide ions.
Gallic acid is usually used as a standard in the determination of the total phenolic content [20, 66-68]. In our opinion, this approach is not useful because the studied essential oils do not contain gallic acid and its reaction with the Folin-Ciocalteu reagent differs from that of carvacrol and/or thymol.
Table 3
Correlation of the essential oils' antioxidant parameters
Antioxidant parameter r *
AOAdpph (%) TAC (C mL-1)
TAC (C mL-1) 0.9964 -
FRP (C mL-1) 0.8846 0.8846
*
r crit = 0.950.
The antioxidant parameters obtained by coulometric titration and spectropho-tometry were compared. Positive correlations were revealed, and the corresponding r-values are given in Table 3.
The correlation is significant (p < 0.01 and r > rcrit) in the case of TAC vs. AOADPPH. . Other parameters showed statistically insignificant correlations at p > 0.1 and r < rcrit. However, the strong correlations of TAC with FRP and FRP with AOAdpph. were seen from the Chaddock scale [69] based on r-values.
The correlation data obtained testify that TAC is the most informative and comparable with AOAdpph. . It is applicable to a wide range of antioxidants in the essential oils of Lamiaceae plants.
Conclusions
The phytochemical profile and total antioxidant parameters of the commercial essential oils from the most commonly used Lamiaceae plants (thyme, marjoram, and sage) were studied. The basic composition of the samples was similar to that from previous works. Their antioxidant effects were determined by phenolic constituents. Ter-penes also showed a noticeable yet less pronounced antioxidant effect in the electron transfer reactions with electrogenerated bromine and DPPH\ The synergetic effect of hydrocarbon and oxygenated terpenes defined the antioxidant properties of thyme and marjoram. Sage, which does not contain oxygenated terpenes (phenolics), turned out to show weak antioxidant properties. Based on the total antioxidant parameters, essential oils can be characterized in general with respect to the mutual effects of their phyto-chemical constituents. Therefore, antioxidant parameters can be considered as markers for the primary screening of the essential oils from Lamiaceae plants.
Acknowledgments. This study was supported by the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030).
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Received January 25, 2023 Accepted February 2, 2023
Kalmykova Alena Denisovna, Student of A.M. Butlerov Institute of Chemistry
Kazan Federal University
ul. Kremlevskaya, 18, Kazan, 420008 Russia E-mail: alena. kalmykova.pnb. 2000@mail. ru
Yakupova Elvira Nailevna, PhD Student of A.M. Butlerov Institute of Chemistry; Engineer
Kazan Federal University
ul. Kremlevskaya, 18, Kazan, 420008 Russia Federal Center for Toxicological, Radiation, and Biological Safety
ul. Nauchny Gorodok-2, Kazan, 420075 Russia E-mail: [email protected]
Bekmuratova Feruzakhon Altmishevna, Junior Research Fellow
Federal Center for Toxicological, Radiation, and Biological Safety
ul. Nauchny Gorodok-2, Kazan, 420075 Russia E-mail: [email protected]
Fitsev Igor Mikhailovich, PhD in Chemistry, Leading Research Fellow Federal Center for Toxicological, Radiation, and Biological Safety
ul. Nauchny Gorodok-2, Kazan, 420075 Russia E-mail: [email protected]
Ziyatdinova Guzel Kamilevna, Doctor of Chemical Sciences, Professor, Department of Analytical
Chemistry
Kazan Federal University
ul. Kremlevskaya, 18, Kazan, 420008 Russia E-mail: [email protected]
ОРИГИНАЛЬНАЯ СТАТЬЯ
УДК 543.5:543.8 ао1: 10.26907/2542-064Х.2023.1.94-117
Оценка антиоксидантных свойств и ГХ-МСД анализ коммерческих эфирных масел из растений семейства Lamiaceae
А.Д. Калмыкова\ Э.Н. Якупова1'2, Ф.А. Бекмуратова2, И.М. Фицев2, Г.К. Зиятдинова1
1 Казанский (Приволжский) федеральный университет, г. Казань, 420008, Россия
2Федеральный центр токсикологической, радиационной и биологической безопасности,
г. Казань, 420075, Россия
Аннотация
Растения семейства Lamiaceae уже много тысячелетий широко используются в кулинарии, а также фито- и ароматерапии. Их эфирные масла обладают высокой антиоксидантной и другими видами биологической активности. Изучен фитохимический профиль и компонентный состав эфирных масел тимьяна, майорана и шалфея методом газовой хроматографии с масс-спектрометрическим детектированием (ГХ-МСД). Антиоксидантные свойства всех образцов оценивали по суммарным антиоксидантным параметрам (интегральной антиоксидантной емкости (АОЕ), железовосстанавливающей способности (ЖВС), антиоксидантной активности (АОА) по отношению к 2,2-дифенил-1-пикрилгидразилу (ДФПЛ) и общему содержанию фенольных соединений по методу Фолина - Чокальтеу). Полученные значения ЖВС были в 46-321 раз меньше, чем АОЕ, что согласуется с содержанием фенольных соединений в образцах. Выявлено, что основными компонентами исследуемых эфирных масел являются терпены, изопропилме-тилфенолы и эвгенол, вносящие вклад в АОЕ и АОА. Метод Фолина - Чокальтеу оказался применим только к эфирному маслу тимьяна. Его ЖВС, основанная на реакции фенольных антиок-сидантов с электрогенерированными феррицианид-ионами, хорошо согласуется с общим содержанием фенолов (329 ± 17 и 334 ± 15 мг карвакрола на мл соответственно). Эфирное масло тимьяна характеризовалось наиболее высокими антиоксидантными показателями, а шалфея - самыми низкими. По результатам проведенного анализа установлены положительные корреляции (г = 0.8846-0.9964) антиоксидантных параметров.
Ключевые слова: эфирные масла, интегральная антиоксидантная емкость, железовосста-навливающая способность, общее содержание фенольных соединений, кулонометрическое титрование, фитохимический профиль, майоран, тимьян, шалфей
Благодарности. Работа выполнена за счет средств Программы стратегического академического лидерства Казанского (Приволжского) федерального университета (ПРИОРИТЕТ-2030).
Поступила в редакцию 25.01.2023 Принята к публикации 09.02.2023
Калмыкова Алена Денисовна, студент Химического института им. А.М. Бутлерова
Казанский (Приволжский) федеральный университет ул. Кремлевская, д. 18, г. Казань, 420008, Россия E-mail: [email protected]
Якупова Эльвира Наилевна, аспирант Химического института им. А.М. Бутлерова; инженер
Казанский (Приволжский) федеральный университет ул. Кремлевская, д. 18, г. Казань, 420008, Россия Федеральный центр токсикологической, радиационной и биологической безопасности
ул. Научный городок-2, г. Казань, 420075, Россия E-mail: elviraeakupova96@mail. ru
Бекмуратова Ферузахон Алтмишевна, младший научный сотрудник
Федеральный центр токсикологической, радиационной и биологической безопасности
ул. Научный городок-2, г. Казань, 420075, Россия E-mail: [email protected]
Фицев Игорь Михайлович, кандидат химических наук, ведущий научный сотрудник
Федеральный центр токсикологической, радиационной и биологической безопасности
ул. Научный городок-2, г. Казань, 420075, Россия E-mail: [email protected]
Зиятдинова Гузель Камилевна, доктор химических наук, профессор кафедры аналитической химии
Казанский (Приволжский) федеральный университет ул. Кремлевская, д. 18, г. Казань, 420008, Россия E-mail: [email protected]
<For citation: Kalmykova A.D., Yakupova E.N., Bekmuratova F.A., Fitsev I.M., Ziyatdi-nova G.K. Evaluation of the antioxidant properties and GC-MSD analysis of commercial essential oils from plants of the Lamiaceae family. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2023, vol. 165, no. 1, pp. 94-117. doi: 10.26907/2542-064X.2023.1.94-117.
<Для цитирования: KalmykovaA.D., Yakupova E.N., Bekmuratova F.A., Fitsev I.M., Ziyatdi-nova G.K. Evaluation of the antioxidant properties and GC-MSD analysis of commercial essential oils from plants of the Lamiaceae family // Учен. зап. Казан. ун-та. Сер. Естеств. науки. -2023. - Т. 165, кн. 1. - С. 94-117. - doi: 10.26907/2542-064X.2023.1.94-117.
