GC-MS analysis and in vitro antioxidant and enzyme inhibitory activities of essential oil from aerial parts of endemic Thymus spathulifolius Hausskn. et Velen

Abstract

We investigated the antioxidant and enzyme inhibitory activities and chemical composition of the hydro-distilled essential oil (0.35% yield) from aerial parts of Thymus spathulifolius. Antioxidant capacity of the oil was assessed by different methods including free radical scavenging (DPPH and ABTS), reducing power (FRAP and CUPRAC) and phosphomolybdenum assay. Inhibitory activities were analyzed against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α-amylase, α-glucosidase, and tyrosinase. Twenty-one constituents were identified representing 97.2% of the total oil with thymol (50.5%), borneol (16.7%) and carvacrol (7.7%) as the major components. The essential oil exhibited good antioxidant activity with IC₅₀ values of 3.82 and 0.22 mg/mL determined by free radical scavenging DPPH and ABTS, respectively. EC₅₀ values of FRAP and CUPRAC were found to be 0.12 and 0.34 mg/mL, respectively. The results of the present study support the uses of T. spathulifolius essential oil as a source of natural antioxidants and bioactivities for functional foods and phytomedicines.

• Antioxidant
• enzyme inhibition
• thymol
• Thymus spathulifolius

Introduction

Currently, there is global interest in finding novel, safely and natural agents from medicinal plants to prevent major health problems linked to oxidative stress. These problems including Alzheimer’s diseases and diabetes mellitus affect a large portion of the world population. From this point, the development of new treatment strategies for the diseases is one of the most remarkable topics in the scientific area.

Nowadays, the inhibition of key enzymes related to the pathology of the diseases is considered as one of the most effective strategies for the treatment and management of the diseases1–2. For example, inhibition of acetylcholinesterase (AChE), which is involved in the acetylcholine hydrolysis, is the most accepted strategy for the treatment of Alzheimer’s disease3^,4. Once more, inhibition of α-amylase and α-glucosidase are accepted as an attractive strategy for the management of blood glucose level in the diabetes mellitus5^,6. Many compounds (e.g. galantamine and tacrine for Alzheimer’s disease and acarbose for diabetes mellitus) are synthetically developed in drug industry, but they show adverse effects including gastrointestinal diseases and hepatotoxicity7–9. In this context, enzyme inhibitory properties of medicinal plants or plant-derived products are of great value in drug development era since their possible uses as natural enzyme inhibitors emerged from growing tendency to replace synthetic inhibitors by natural ones.

The genus Thymus (Lamiaceae), generally called thyme, belongs to economically and medicinally valuable plant species that are widely distributed in the Mediterranean region of Africa, Europe and Asia10. In Turkey, the genus includes 41 species, 24 of which are endemic11–13. Thymus species have multiple biological and pharmacological properties including antioxidant, antibacterial, antiviral, spasmolytic, anti-parasites and insecticidal10. Also, the aerial parts of Thymus species are commonly used as an herbal tea. They also act as sedative tonic, antiseptic, digestive and carminative10^,14. Moreover, Thymus species have been used to relieve stomachache, respiratory tract problems and flu in Turkish folk medicine15–18.

The uses of these species in traditional medicine could be causes of the high number of studies on members of the genus10^,19. Thymus spathulifolius is growing in Central Anatolia region of Turkey (narrowly distributed in Sivas) and endemic to Turkey11. In a previous study, essential oil composition, antibacterial and antioxidant properties of T. spathulifolius was investigated20. According to this study, the essential oil of T. spathulifolius had great potential of antioxidant and antimicrobial activities against bacteria, moulds and yeast species tested.

To the best of our knowledge, no investigation has been carried out on enzyme inhibitory activities and detailed antioxidant capacity of T. spathulifolius. The aim of the present work was to evaluate the yield, chemical composition, antioxidant and enzyme inhibitory properties of the hydro-distilled essential oil from aerial parts of T. spathulifolius, which is wildly grown on calcareous slopes in Erzincan. Antioxidant capacity were evaluated by different chemical assays, including free radical scavenging (DPPH and ABTS assays), reducing power (CUPRAC and FRAP assays) and phosphomolybdenum assays. Enzyme inhibitory activities were investigated against cholinesterase (AChE and BChE), tyrosinase, α-amylase and α-glucosidase. The obtained results may be valuable for preparing new foods supplements and models for drug formulations.

Materials and methods

Plant material

Aerial parts from T. spathulifolius Hausskn. et Velen plant were collected from Kemah (Yahsiler village, calcareous slopes), Erzincan, Turkey during July 2014. Taxonomic identification of the plant material was confirmed by a senior taxonomist Dr. Ali Kandemir, in the Department of Biology, Erzincan University, Erzincan, Turkey. The voucher specimens were deposited at the Herbarium of the Department of Biology, Erzincan University, Erzincan, Turkey (Voucher No.: Kandemir 6735).

Isolation and analysis of the essential oil

The shade-dried and ground plant material (100 g) was hydro-distilled for 5 h using a British-type Clevenger apparatus (ILDAM Ltd., Ankara, Turkey) (yield 0.35% v/w). The isolated essential oil was dried over anhydrous sodium sulphate and after filtration, stored at +4 °C until tested and analyzed.

The essential oil was analyzed by GC-FID and GC-MS techniques. The GC-MS analysis was carried out with an Agilent 5975 GC-MSD system coupled to an Agilent 7890A GC (Agilent Technologies Inc., Santa Clara, CA). HP-Innowax FSC column (60 × 0.25 mm, 0.25 μm film thickness) was used with helium (purity 99.99%) as carrier gas (1.2 mL/min). The GC oven temperature was kept at 60 °C for 10 min and programmed to 220 °C at a rate of 4 °C/min, and kept constant at 220 °C for 10 min and then raised to 240 °C at a rate of 1 °C/min. The split ratio was used at 40:1. The injector temperature was at 250 °C. Mass spectra were recorded at 70 eV. Mass range was from 35 to 450 m/z. GC-FID analysis was carried out by simultaneous auto-injection using Agilent 7693A series autosampler; 1 μL injections were used.

The GC analysis was carried out using an Agilent 7890A GC system. In order to obtain the same elution order with GC/MS, simultaneous triplicate injections were done by using the same column and same operational conditions. The FID temperature was 300 °C. The identification of constituents was achieved on the basis of retention index determined by co-injection with reference to a homologous series of n-alkanes (C₈–C₃₀), under same experimental conditions. Further identification was carried out by comparison of their mass spectra with those from NIST 05 and Wiley 8th version and home-made MS library built up from pure substances and components of known essential oils, as well as by comparison of their retention indices with literature values21.

Radical scavenging activity

Free radical scavenging activity (DPPH)

The effect of the sample on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was estimated according to the procedure described by Sarikurkcu22. Sample solution (1 mL) was added to a 4 mL of a 0.004% methanol solution of DPPH. The sample absorbance was read at 517 nm after 30 min incubation at room temperature in the dark. Inhibition of free radical DPPH in percent (I%) was calculated in following way: where A_control is the absorbance of the control reaction (containing all reagents except the test compound) and A_sample is the absorbance of the test compound. Fifty percent of free radical inhibition (IC₅₀) of samples was calculated. The lower the IC₅₀ value indicates high antioxidant capacity.

ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] radical cation scavenging activity

The scavenging activity against ABTS cation radical was measured according to the method of Re et al.23 with slight modification. Briefly, ABTS^.+ radical cation was produced directly by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate and allowing the mixture to stand for 12–16 h in dark at the room temperature. Prior to the assay, ABTS solution was diluted with methanol to an absorbance of 0.700 ± 0.02 at 734 nm. Sample solution (1 mL) was added to ABTS solution (2 mL) and mixed. The sample absorbance was read at 734 nm after 30 min incubation at room temperature. The results were reported as IC₅₀.

Reducing power methods

Cupric ion reducing (CUPRAC) method

The cupric ion reducing activity (CUPRAC) was determined according to the method of Apak et al.24. Sample solution (0.5 mL) was added to premixed reaction mixture containing CuCl₂ (1 mL, 10 mM), neocuproine (1 mL, 7.5 mM) and NH₄Ac buffer (1 mL, 1 M, pH 7.0). Similarly, a blank was prepared by adding sample solution (0.5 mL) to premixed reaction mixture (3 mL) without CuCl₂. Then, the absorbance of the sample and blank was read at 450 nm after 30 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample. The EC₅₀ value (the effective concentration at which the absorbance was 0.5) was calculated for each sample and standard.

Ferric reducing antioxidant power (FRAP) method

The FRAP assay was carried out as described by Aktumsek et al.25 with slight modifications. Sample solution (0.1 mL) was added to premixed FRAP reagent (2 mL) containing acetate buffer (0.3 M, pH 3.6), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) (10 mM) in 40 mM HCl and ferric chloride (20 mM) in a ratio of 10:1:1 (v/v/v). Then, the sample absorbance was read at 593 nm after 30 min incubation at room temperature. The results were evaluated by EC₅₀ values.

Total antioxidant activity for phosphomolybdenum method

The total antioxidant activity of the oil sample was evaluated by phosphomolybdenum method according to Berk et al.26 with slight modifications. Sample solution (0.3 mL) was mixed with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The sample absorbance was read at 695 nm after 90 min incubation at 95 °C.

Enzyme inhibitory activity

Cholinesterase inhibition

Cholinesterase (ChE) inhibitory activity was measured using Ellman’s method, as previously reported7. Sample solution (50 µL) was mixed with DTNB (125 µL) and AChE (or BuChE) solution (25 µL) in Tris–HCl buffer (pH 8.0) in a 96-well microplate and incubated for 15 min at 25 °C. The reaction was then initiated with the addition of acetylthiocholine iodide (ATCI) or butyrylthiocholine chloride (BTCl) (25 µL). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (AChE or BChE) solution. The absorbance of the sample and blank were read at 405 nm after 10 min incubation at 25 °C. The absorbance of the blank was subtracted from that of the sample and the results were reported as IC₅₀.

α-Amylase inhibition

α-Amylase inhibitory activity was performed using the Caraway–Somogyi iodine/potassium iodide (IKI) method7. Sample solution (25 µL) was mixed with α-amylase solution (50 µL) in phosphate buffer (pH 6.9 with 6 mM sodium chloride) in a 96-well microplate and incubated for 10 min at 37 °C. After pre-incubation, the reaction was initiated with the addition of starch solution (50 µL, 0.05%). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-amylase) solution. The reaction mixture was incubated for 10 min at 37 °C. The reaction was then stopped with the addition of HCl (25 µL, 1 M). This was followed by the addition of iodine/potassium iodide solution (100 µL). The absorbance of the sample or blank was recorded at 630 nm. The absorbance of the blank was subtracted from that of the sample and the results were reported as IC₅₀.

α-Glucosidase inhibition

α-Glucosidase inhibitory activity was performed by the previous method7. Sample solution (50 µL) was mixed with glutathione (50 µL), α-glucosidase solution (50 µL) in phosphate buffer (pH 6.8) and PNPG (4-nitrophenyl-α-d-glucopyranoside) (50 µL) in a 96-well microplate and incubated for 15 min at 37 °C. Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-glucosidase) solution. The reaction was then stopped with the addition of sodium carbonate (50 µL, 0.2 M). The absorbance of the sample and blank were read at 400 nm. The absorbance of the blank was subtracted from that of the sample and the results were reported as IC₅₀.

Tyrosinase inhibition

Tyrosinase inhibitory activity was measured using the modified dopachrome method with l-DOPA as substrate, as previously reported27 with slight modifications. Sample solution (25 µL) was mixed with tyrosinase solution (40 µL) and phosphate buffer (100 µL, pH 6.8) in a 96-well microplate and incubated for 15 min at 25 °C. The reaction was then initiated with the addition of l-DOPA (40 µL). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (tyrosinase) solution. The absorbance of the sample or blank was read at 492 nm after 10 min of incubation at 25 °C. The absorbance of the blank was subtracted from that of the sample and the results were reported as IC₅₀.

Results and discussion

Yield and chemical composition of the essential oil

The yield of the essential oil from aerial parts of T. spathulifolius was 0.35%. Sokmen et al.20 and Celen et al.28 reported 3.74 and 2.3% from T. spathulifolius growing in Sivas, respectively. Variations in the yield of T. spathulifolius might be attributed to the varied agro-climatic conditions of the regions of Turkey. Moreover, rainfall, soil texture, average temperature and humidity in the selected region can affect the yield of the essential oil. According to reports there is a strong relationship between climatic conditions and the yield of several essential oils obtained from same species29–32. This obtained value is comparable to the values reported in the literature for other Thymus species such as T. capitatus [6% reported by Bounatirou et al.33], T. linearis [11.2% reported by Hussain et al.19], T. saturejoides [2.40% reported by Kasrati et al.34] and T. migricus [0.29% reported by Kucukbay et al.35].

The results obtained by GC-MS analysis of the essential oil of T. spathulifolius are shown in Table 1. Twenty-one components were identified representing the 97.2% of the total content and the major components were found to be thymol (50.5%), borneol (16.7%) and carvacrol (7.7%). Sokmen et al.20 reported thymol (36.5%), carvacrol (29.8%) and p-cymene (10%) as the major constituents of the oil of T. spathulifolius collected from Sivas province of Turkey. Similarly, according to Celen et al.28, carvacrol (49%), thymol (17.8) and borneol (3.5%) were found to be the main compounds of the T. spathulifolius oil.

Table 1. Chemical composition of the essential oil of aerial parts of T. spathulifolius.

No.	RI^a	Components	(%)
1	1212	1,8-Cineole	4.2
2	1250	γ-Terpinene	0.4
3	1259	3-Octanone	0.3
4	1276	p-Cymene	0.3
5	1452	1-Octen-3-ol	0.1
6	1536	Camphor	0.6
7	1594	Bornylacetate	1.2
8	1614	Terpinen-4-ol	1.7
9	1618	β-Caryophyllene	6.1
10	1691	α-Humulene	0.4
11	1709	α-Terpineol	1.3
12	1718	Borneol	16.7
13	1749	β-Bisabolone	0.9
14	1786	(E)-α-Bisabolone	0.6
15	1808	Cumin aldehyde	0.1
16	1862	p-Cymene-8-ol	0.5
17	1868	Thymyl acetate	0.4
18	2018	Caryophyllene oxide	3.0
19	2122	Cumin alcohol	0.2
20	2200	Thymol	50.5
21	2229	Carvacrol	7.7
		Total identified	97.2
		Essential oil yield	0.35

^aRetention index relative to n-alkanes on HP-innowax capillary column.

Generally, the chemical composition of the tested oil was consistent with those investigated previously by others. In several studies, thymol and carvacrol were identified as the major components of many Thymus essential oils such as T. linearis [thymol (36.5%) and carvacrol (9.5%)]19, T. eriocalyx [thymol (42.6%) and carvacrol (32.3%)]36, T. eigii [thymol (30.6%) and carvacrol (26.1%)]37 and T. ciliatus [thymol (17.3%) and carvacrol (26.2%)]38. A comparison of the major constituents and antioxidant activities of the essential oil from different species of Thymus is depicted in Table 2. Borneol and p-cymene were found to be the principal components followed by thymol and carvacrol in some Thymus species such as T. saturejoides [borneol (19.7%)]34, T. fallax [p-cymene (7.1%)]35 and T. boveii [p-cymene (19.80%)]39. The compositional variations in Thymus essential oils might be linked to several factors, including climatic, seasonal and geographical. Similar observations were noted for some Lamiaceae species by several researchers40^,41.

Table 2. Comparison of the major components and antioxidant activities of essential oils from different Thymus species.

Thymus species	Major components (%)	DPPH	ABTS	FRAP	CUPRAC	Phosphomolybdenum	References
T. spathulifolius	Carvacrol (49.0), thymol (17.8)	77.31% (at 1 mg/mL concentration)	–	–	–	–	Celen et al.28
T. kotchyanus	Thymol (39.7), γ-terpinene (11.4)	278 ± 1.9	–	–	–	–	Amiri36
T. daenensis subsp. lancifolius	Carvacrol (52.3) and Thymol(16.4)	99.6 ± 0.5	–	–	–	–	Amiri36
T. eriocalyx	Thymol (42.6) and carvacrol (32.3)	198 ± 1.4	–	–	–	–	Amiri36
T. hirtus subsp. algeriensis	Terpinen-4-ol (33.34) and 1.8 cineole (19.96)	85% (at 50 µg/mL concentration)	16% (at 50 µg/mL concentration)	–	–	–	Fatma et al.42
T. migricus	α-terpineol (30.6) and thymol (20.7)	45.36% (at 750 µg/mL)	–	–	–	–	Kucukbay et al.35
T. fallax	Carvacrol (66.1) and p-cymene (7.1)	65.96% (at 750 µg/mL)	–	–	–	–	Kucukbay et al.35
T. pubescens var. pubescens	Cis-carveol (29.6) and α-terpineol (10.8)	42.29% (at 750 µg/mL)	–	–	–	–	Kucukbay et al.35
T. capitatus	Carvacrol (65.9) and p-cymene (16.7)	21.1% (at 1 mg/mL)	–	–	–	–	Bounatirou et al.33
T. saturejoides	Carvacrol (25.3) and borneol (19.7)	184.00 ± 0.01	–	–	–	–	Kasrati et al.34
T. maroccanus	Carvacrol (72.6) and γ-terpinene (6.0)	353.01 ± 6.75	–	–	–	–	Jamali et al.38
T. linearis	Thymol (36.5) and carvacrol (9.5)	42.9 ± 1.4	–	–	–	–	Hussain et al.19
T. serpyllum	Carvacrol (44.4) and α-terpineol	34.8 ± 1.9	–	–	–	–	Hussain et al.19
T. longicaulis subsp. longicaulis  var. longicaulis	γ-terpinene (27.80) and thymol (27.65)	63.26% (at 0.5 mg/mL)	–	–	–	–	Sarikurkcu et al.43
T. eigii	Thymol (30.6) and carvacrol (26.1)	225.00 ± 5.25	–	–	–	–	Tepe et al.37
T. daenensis	Carvacrol (76.8) and linalool (6.9)	70.9%	1.5 mmolTE/µL oil	5.5 mmolFe2^+/µL oil	–	–	Saidi47
T. capitata	Carvacrol (88.98) and linalool (1.57)	44.16 ± 0.81	99.98 ± 0.01	–	–	–	El-Abed et al.44
T. ciliatus	Carvacrol (26.2) and thymol (17.3)	206.57 ± 10.48	–	184.05 ± 1.12	–	–	Jamali et al.38
T. boveii	Carvacrol (41.34) and p-cymene (19.80)	82.75% (at 0.5 mg/mL)	–	–	–	–	Tepe et al.39

Antioxidant capacity

The results of different antioxidant assays are given in Table 3.

Table 3. Radical scavenging activity (IC₅₀: mg/mL) and reducing power (EC₅₀: mg/mL) of BHT, trolox, thymol and essential oil of T. spathulifolius.

	Free radical s cavenging activities*		Reducing power activities*
Samples	DPPH assay	ABTS assay	CUPRAC assay	FRAP assay
T. spathulifolius	3.82 ± 0.16	0.22 ± 0.01	0.12 ± 0.01	0.34 ± 0.01
BHT	0.33 ± 0.02	0.35 ± 0.01	0.05 ± 0.01	0.14 ± 0.01
Trolox	0.06 ± 0.01	0.20 ± 0.01	0.07 ± 0.01	0.02 ± 0.01
Thymol	0.90 ± 0.03	0.14 ± 0.01	0.08 ± 0.01	0.17 ± 0.01

*Values expressed are mean ± SD of three parallel measurements.

The reduction ability of ABTS and DPPH radicals was determined by the decrease in their absorbance at 734 and 517 nm induced by antioxidants, respectively.

The free radical scavenging assays reflected hydrogen-donating abilities of antioxidants. IC₅₀ values for DPPH and ABTS were found to be 3.82 and 0.22 mg/mL, respectively. DPPH radical scavenging activity of the essential oil, BHT (butylated hydroxyltoluene), trolox and thymol (the principal component of the tested oil) decreased in order of trolox > BHT > thymol > essential oil. Interestingly, thymol has the highest radical scavenging activity in ABTS assay, followed by trolox, essential oil and BHT. At this point, the higher ABTS radical scavenging activity of essential oil can be explained due to the presence of thymol. On the other hand, DPPH scavenging activity of thymol was found to be higher than that of essential oil. As possible explanation of this biological behavior, the mixture of major components and other compounds may exert an antagonistic effect. Similar trends were reported for some essential oils from Calamintha origanifolia and Artemisia annua by Formisano et al.45 and Cavar et al.46, respectively.

Reducing power assays are often used as an indicator of electron-donating activity, which is an important mechanism of antioxidant compounds. Therefore, FRAP and CUPRAC methods were applied to evaluate reducing power potentials of T. spathulifolius essential oil. Lower EC₅₀ value indicates higher reducing potential. In FRAP and CUPRAC assays, essential oil and its major component, thymol, exhibited weaker reducing power than that of BHT and trolox.

The order of ferric reducing power activity was: trolox > BHT > thymol > essential oil. However, BHT exerted greater antioxidant activity than that of trolox in CUPRAC assay. Literature is scarce about reducing potentials of Thymus species hence we could not compare the results of present experiments. In some recent studies, ferric reducing powers of T. ciliatus and T. daenensis were reported by Jamali et al.38 and Saidi47, respectively.

Phosphomolybdenum assay is based on the reduction of Mo(VI) to Mo(V) by the antioxidants at acidic pH and subsequent formation of green phosphate/Mo(V) complex which is measured spectrophotometrically at 695 nm. The essential oil exhibited concentration-dependent activity in this assay and the absorbance values are depicted in Table 4. Higher absorbance indicated that the phosphomolybdenum activity is increased. The phosphomolybdenum reduction capacity of the essential oil at the concentration of 2, 1 and 0.5 mg/mL were 0.789 ± 0.025, 0.502 ± 0.007 and 0.289 ± 0.005, respectively. Thymol demonstrated poor phosphomolybdenum reducing capacity compared to essential oil, BHT and trolox at 0.5 mg/mL concentration. In this direction, the high activity of essential oil can be explained by a synergistic effect of minor and major volatile components. Our findings are in agreements with those of Dawidowicz and Olszowy48 and Polatoglu et al.49 who also reported the synergetic effects of essential oil components in some plant species. As far as we know, there is no study on the phosphomolybdenum reducing capacity of the members of Thymus.

Table 4. Phosphomolybdenum activities of BHT, trolox, thymol and essential oil of T. spathulifolius [at different concentration (0.5, 1 and 2 mg/mL) (absorbance values at 695 nm)].

	Concentration (mg/mL)*
Samples	2	1	0.5
T. spathulifolius	0.789 ± 0.025	0.502 ± 0.007	0.289 ± 0.005
BHT	–	–	1.735 ± 0.031
Trolox	–	–	0.870 ± 0.007
Thymol	–	–	0.111 ± 0.010

*Values expressed are mean ± SD of three parallel measurements.

Enzyme inhibitory activities

ChE inhibition

Table 5 depicts the enzyme inhibitory activities of T. spathulifolius essential oil and standard compounds and the results were expressed as IC₅₀ (mg/mL). The essential oil has weak anti-AChE (0.95 mg/mL) and anti-butyrylcholinesterase (BChE) (1.05 mg/mL) activity as compared to galantamine (1.51 µg/mL for AChE and 1.77 µg/mL for BChE). According to these results, galantamine was excellent an inhibitor on both AChE and BChE. This value is comparable to those reported in the literature for other Thymus species such as T. lotocephalus (0.90 mg/mL for AChE and 0.50 mg/mL for BChE), as reported by Costa et al.50 and T. vulgaris (0.217 mg/mL for AChE) as reported by Aazza et al.51. Probably, the AChE and BChE inhibitory activity may due to the presence of thymol and carvacrol. A research on T. vulgaris essential oil also indicates its neuroprotective effects52. Volatile components of essential oils are likely to readily cross the blood–brain barrier because of their small molecular size and lipophilicity53. From this point, Thymus species could be useful as a source of natural inhibitors for the management of Alzheimer’s diseases.

Table 5. Enzyme inhibitory activity (IC₅₀: mg/mL) of reference drugs and the essential oil from T. spathulifolius.

	Anti-ChE activity*		Skin-care effect*	Anti-diabetic activity*
Samples	AChE	BChE	Tyrosinase	α-Amylase	α-Glucosidase
T. spathulifolius	0.95 ± 0.01	1.05 ± 0.01	11.03 ± 2.82	7.19 ± 0.14	1.08 ± 0.05
Galantamine†	1.51 ± 0.05	1.77 ± 0.01	–	–	–
Kojic acid	–	–	0.14 ± 0.01	–	–
Acarbose	–	–	–	0.98 ± 0.05	6.67 ± 0.01

*Values expressed are mean ± SD of three parallel measurements.

†µg/mL.

These results are in agreement with those reported by earlier studies which also proved strong ChE inhibitory activity of thymol-rich essential oils from several Thymus species54^,55. Surprisingly, carvacrol exerted stronger ChE inhibitory activity than thymol in these studies. The difference may be related to position of the hydroxyl group in the molecular structure of thymol and carvacrol. Also, 1,8-cineol was reported as effective ChE inhibitor56. Again, several Lamiaceae species were reported as natural inhibitors against ChE in the literature56^,57. For example, several Salvia species have the higher level of monoterpenes and exhibited significantly anti-ChE activities58.

Tyrosinase inhibition

Tyrosinase is a copper-containing enzyme and involved in melanin synthesis. From this point, tyrosinase inhibitors have become increasingly important in medical and cosmetics industry to prevent hyperpigmentation59. As can be seen from Table 5, the tyrosinase inhibitory capacity (IC₅₀) of essential oil was found to be 11.03 mg/mL. However, kojic acid was an excellent inhibitor with IC₅₀ of 0.14 mg/mL.

To our knowledge, there are no published studies that have evaluated the tyrosinase inhibitory effect of Thymus species. However, Satooka and Kubo60 reported that thymol was an important inhibitor against tyrosinase in melanogenesis. In this direction, the high level of thymol in the studied essential oil could be responsible of tyrosinase inhibitory activity of the essential oil.

α-Amylase and α-glucosidase inhibition

Many studies on the treatment of diabetes mellitus have focused on the potential use of plant constituents. Several phytochemicals have been implicated in carbohydrate metabolism related enzymes inhibition (especially α-amylase and α-glucosidase)61. However, thus far, no scientific information on the antidiabetic activity of the essential oil tested is available. Therefore, we investigated the effect of the essential oil on the activity of α-amylase and α-glucosidase, which are key enzymes involved in the breakdown of carbohydrates and intestinal absorption, respectively.

The inhibitory effects of tested essential oil on α-amylase and α-glucosidase are presented in Table 5. IC₅₀ values for α-amylase and α-glucosidase were found to be 7.19 and 1.08 mg/mL, respectively. Apparently, the essential oil exhibited stronger α-glucosidase inhibitory activity than acarbose (6.67 mg/mL). This is in agreement with the reported by Li et al.62 and Luyen et al.63 that refer several plant constituents have stronger inhibitory effects than synthetics. On the other hand, acarbose (0.98 mg/mL) was found to exhibit superior α-amylase inhibitory activity as compared to studied essential oil.

According to the best of our knowledge, there are rarely studies reported on the anti-amylase and anti-glucosidase properties of essential oil or extracts obtained from Thymus species64^,65. In these studies, the extracts from T. quinquecostatus and T. capitatus had higher level of thymol and they exhibited excellent anti-amylase and anti-glucosidase effects. As observed in anti-ChE and anti-tyrosinase assays, the anti-diabetic activity of the essential oil could be attributed to the presence of thymol or carvacrol. Similar observations were reported earlier by Bejaoui et al.66 and Kamrani et al.67. In consistent with these studies, thymol or Thymus species (T. serpyllum) exhibit great potential as an anti-hyperglycemic agent in in vivo assays68^,69. From these findings, Thymus species may be valuable to control postprandial hyperglycaemia for type 2 diabetes management. Again, several Lamiaceae species were reported as strong anti-diabetic agents in in vivo and in vitro studies70^,71.

Conclusion

In this study, we analyzed the detailed composition of volatile components, antioxidant and enzyme inhibitory activities of Thymus spathulifolius essential oil. Thymol (50.5%), borneol (16.7%) and carvacrol (7.7%) were identified as the main components in the tested oil. In fact the oil can be explored as rich source of thymol. Generally, the essential oil was exhibited moderate antioxidant and enzyme inhibitory activities. Our study is the first report of comprehensive antioxidant and enzyme inhibitory effects of this oil. T. spathulifolius could be considered as a natural source for isolation of active constituents for food supplements and therapeutic applications. Further in vivo studies on the appraisal of biological effects of T. spathulifolius essential oil are recommended.

Acknowledgements

The authors thank to the Research Foundation of Selcuk University (BAP) for providing facilities and encouragement.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.