Chemical, physical, and biological evaluation of hydro-distilled essential oil from leaves of Ethiopian thymus species

ABSTRACT

Physicochemical profile, phytochemical constituents, bioactivity, and thermal stability of the hydro-distilled essential oil (EO) for T.schimperi and T.serrulatus were investigated. EO yield for T.schimperi and T.serrulatus was 2.05± 0.09% and 1.2 ± 0.3% (w/w) respectively. The chemical profiles of the EO were analyzed using GC-MS (Gas Chromatography-Mass spectrometry).Twenty one (99.99 %) and Twenty (98.59%) EO components were identified from T. schimperi and T.serrulatus, respectively. The two stage mass losses, the first one (38%) was from 37°C to 164°C and (55%) from 46°C to 178°C for T.schimperi and T.serrulatus EO respectively and the second one was (11%) from 164°C to 220°C for T.schimperi and (8%) from 178°C to 223°C for T.Schimperi and T.serrulatus EO respectively were observed for TGA analysis.The essential oils in this study showed a strong antioxidant effect for different tests. IC50 value of 0.17 μg/mL on DPPH assay for both species; 73 ± 0.50 and 64 ± 3.4% inhibition for ABTS assay 4.6 ± 0.42 and 8.04 ± 0.9 mM Fe+2/mL percent in FRAP assay for T.schimperi and T.serrulatus respectively. Antimicrobial activities for both species EOs were investigated on Escherichia coli, Staphylococcus aureus, Salmonella typhi, and Streptococcus epidermidis. MIC values for E.coli and S.typhi were 0.5 and 1 μL/mL for T.schimperi, 2 and 4 μL/mL of T.serrulatus EO, for S.aureus and Streptococcus epidermidis were 0.2 and 4 μL/mL for T.schimperi, 2 and 0.5 μL/mL for T.serrulatus EO’s. MBC values for both E.coli and S.typhi were 2 μL/mL for T.schimperi and 4 μL/mL for T.serrulatus EO, While for S.aureus and Streptococcus epidermidis were 1 and 4 μl/ml for T.schimperi, 2 and 8 μL/mL for T.serrulatus EO.

KEYWORDS:

• Thymus schimperi
• T.serrulatus
• essential oil
• antibacterial activity
• antioxidant activity
• thermogravimetric analysis

Introduction

Dietary herbs have been utilized as flavorings and preservatives in food since ancient times because of the specific phytochemicals they contain that act as antioxidants.^[1,2] Some herbs are known to extend the storage life of foods in addition to adding distinctive flavors by avoiding rancidity^[3] and growth of microbes.^[4,5] There are various research activities undertaken on antioxidant, hypoglycemic,^[6] and anticancer activities of such herbs.^[7]

Thymus schimperi and Thymus serrulatus, both commonly referred to as “Tosign” in Ethiopia, are wild indigenous fragrant plants that grow in open grassland between bare rocks on mountain slopes. Thymus plants are one among 350 species, which are extensively spread in temperate regions.^[8] Thyme oil is utilized in food flavoring, preservation, fragrance, and medicinal industries. Investigation of thymus essential oils (EOs) from various species revealed the presence of a variety of constituents, predominantly high content of phenolic monoterpenes such as carvacrol, thymol, and γ–terpineol.^[9,10]

EO from thymus species is a clear, light-yellow liquid with a strong, warming aromatic scent. P-cymene, γ-terpinene, thymol, and carvacrol are the major components of the EOs.^[11] The plants are suggested as a local medicinal plant for the treatment of liver illness, colitis, dyspepsia, gastritis, cough, and sore throat, as well as respiratory issues (cough, bronchitis, sore throat).^[8] Although T. schimperi Ronniger is frequently employed in food flavoring and traditional medicine locally, little is known about their antioxidant^[3] and antimicrobial property.^[12] The extraction technique employed for available studies was focused more on solvent extraction as summarized in Table 1.

Table 1. Summary of previous studies done on T.schimperi and T.serrulatus extracts.

NO.	Activities	Plant species	Result	Reference
1.	Diuretic and antihypertensive activity of aqueous extract	T.schimperi	Showed positive result at 5h (500mg/Kg/day)	^[Citation13]
2	Diuretic activity of methanol crude extracts and its solvent fraction	T.serrulatus	Displayed a pronounced diuretic activity at a dose of 1000 mg/kg,	^[Citation14]
3	Antioxidant and α-amylase inhibition activities of solvent extracts	T.schimperi	The aqueous methanol extract showed the highest2, 2diphenylpicrylhydrazyl (DPPH) radical scavenging ability (IC50 = 11.0 ± 1.0 µg/ml), FRAP (60.1 ± 1.0 mg AAE/g), and TAC (1.1 ± 0.1 mg BHTE/g).	^[Citation8]
4	Acute oral toxicity study of EO	T.serrulatus and T.schimperi	The lethal dose (LD50) of each EO was between 2000 µL/kg and5000 µL/kg body weight.	^[Citation15]
5	Anti-diabetic activity of methanol extract and fractions.	T.schimperi	Positive test resulted and percentage reduction of blood glucose levels for 250, 500 and 750 mg/kg doses of extract 56.4, 49.16 and 46.98%, respectively.	^[Citation16]
6	Acute, sub-acute and skin irritation toxicity on essential oil.	T.schimperi	Did not cause any mortality up to the limit doses of 2000mg/kg.	^[Citation17]
7	Antimicrobial potential of the essential oil.	T.schimperi	MIC value ranged from 0.08 μl/ml to 0.31 μl/ml for Candida albicans, Aspegilusniger, Rhodotorularubra, Escherichia coli, Shigellaspp.,Bacillus spp. and Streptococci.	^[Citation12]
8	Composition and hepatoprotective activity of essential oils.	T.schimperi & T.serrulatus	The number of EO components ranged from 17 to 41. Hepatoprotective properties of the oils were demonstrated by reduced serum levels of hepatic marker enzymes.	^[Citation18]

Characteristics of EOs includes determination of physicochemical properties of EOs like, specific gravity, refractive index, optical rotation, acid value, peroxide value, thermal and oxidative stabilities. These parameters can serve as indirect indicators of EOs quality.^[19] There is a very limited literature on a comprehensive investigation of the physicochemical characterization, phytochemicals, antioxidant, and antimicrobial properties, of hydro-distilled EO from the two Ethiopian Thymus species. Thus, this study was intended to fill the gaps in these areas.

Materials and methods

Sample collection

Fresh samples of T.schimperi (Accession No: Thymus schimperi: TDE001 and T.serrulatus (Thymus serrulatus: TDE002) with the collection code: Thymus schimperi: YTD001 and Thymus serrulatus: YTD002 were collected from North Shewa, Ankober (kundi) and Debresina, respectively, in the period between December to February 2021/2022. Plant specimens were transported to the laboratory using an ice bag. The species identification was made by a botanist at the Department of Biology, Debre Birhan University.

Sample preparation

Collected fresh samples were washed using distilled water to remove dirt and dried under open air by protecting from direct sunlight. Dried leaves of the plants were carefully separated from woody parts, placed in polyethylene bag and stored at room temperature until used.

Methanolic extracts of T.schimperi and T.serrulatus

Powdered leaves of T.schimperi and T.serrulatus (100 g each) were macerated with 375 mL of 80% methanol for 72 hours at room temperature. The mixtures were filtered using Whatmann No.1 filter paper. The filtrates were concentrated in vacuo at 40°C to give greenish-black colored sticky materials which were kept in a refrigerator until further use.

Essential oil extraction and chemical composition

EO from dried aerial part of the plants (300 g) was extracted by hydro-distillation. Volume of the oil obtained was measured, dried using anhydrous sodium sulfate, and stored in amber colored glass vials at 4°C until used. Identification of EO constituents was conducted using GC/MS. The EOs test samples were diluted in hexane to final concentration of l2.5 μL/mL. Analysis was carried out using Agilent 7890B GC coupled to an Agilent MSD5977A MS detector (Agilent Technologies, China). Auto sampler (G4513A) and a capillary column HP-5 MS Ul (30 m × 0.25 mm, 0.25 μm film thickness) were used. One microliter of the diluted sample was injected in split mode (1:50), the injector temperature was 280°C. Oven temperature of the GC was programmed to 120°C at a rate of 3°C/min, and 120–260°C at the rate of 10°C/min and held at 260°Cfor 8 min. The acquisition scan ranged from 50-550 m/z with an electron impact mass spectrum of 70eVionization energy (Mart, 2009). Constituents of EOs were identified based on their relative retention indices (RI) by comparing with the National Institute of Standards and Technology Library (NIST) MS data base.^[20]

Physicochemical characterization of the essential oil

Yield of EO: expressed as the ratio of the volume of oil to mass of sample in percentage.^[20] Specific gravity of the oil was determined following a standard method, by comparing the mass of 2 mL of EO to the mass of the same volume of distilled water at room temperature. Refractive index of the oil samples was determined using a refractometer (A.KURSS, Germany-DR6000-T).^[21] Two drops of EO was added on the prism with the help of a syringe, and the prism was firmly closed by tightening the screw head and allowed to stand for 1 min, and then the reading was recorded.

Optical rotation of EO was determined using ADP 410 Polari meter. Solutions of 40 g/L of the EO samples in analytical grade chloroform was used for the determination.^[22] Acid value of the EOs was determined following a standard procedure.^[21] The EO (2.04 g) was dissolved in 10 mL of 96% ethanol, and a few drops of 1% phenolphthalein were added to the mixture. KOH solution (0.1 N) was used to titrate this mixture until it turned pink. Peroxides present are determined by titration against sodium thiosulfate in the presence of KI using starch as an indicator.^[23]

Phytochemical screening of T.schimperi and T.serrulatus methanol extracts

Eighty percent methanolic extracts of T.schimperi and T.serrulatus were screened to test for the presence/absence of phytochemicals including, saponins, phenols, flavonoids, alkaloids, tannins, terpenoids, antraquinons, steroids, and/or phytosterols using standard procedures.^[24]

Testing oxidative stability of essential oils using rancimat method

Both T.schimperi and T.serrulatus EOs (3 g) were subjected to the Rancimat test following Metrohm Rancimat model 892 at 120°Cwithan airflow of 20 L/h. The effect of T.schimperi and T.serrulatus essential oil on the induction time was also determined by using 200ppm ascorbic acid as a positive control and Sunflower oil as a negative control. The protection factor (PF) of each test samples were expressed as follows;

(1) $\mathit{PF}=\frac{\mathit{t}\mathit{i}\mathit{ant}}{t{i}_{\text{o}}}$(1)

where ti ant is the induction time of the samples treated with antioxidant and ti_o is the induction time of the control system (without antioxidant).

Thermal stability of essential oil

Simultaneous thermogravimetry (TG)-differential thermal analysis (DTA): Simultaneous TG/derivative thermogravimetry (TG) and DTA were performed in air and nitrogen in the temperature range 20–600°C at increasing rate of 20°C per min on TGA (Beijing, HCT-1) using alumina crucibles (for a sample size 10 mg)as sample holder.

Fourier transform infrared spectroscopy (FTIR)

The FT-IR spectra of EO samples were recorded using iS50 ABX, Model AUP 1,600,388 spectrophotometer equipped with ATR cell, on the 1000–4000 cm⁻¹ wavelength range. The results were processed using Origin Pro 2022 software.

Antioxidant activity of thyme essential oil

DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical scavenging assay

The free radical scavenging activity of T.serrulatus and T.schimperi EO was measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH).^[25] Absorbance of the samples was measured (PerkinElmer, Lambda 950) at 517 nm. The positive control vitamin C was used to calculate the Vitamin C equivalent antioxidant capacity (VCEAC). Percentage reduction (inhibition) of the DPPH radical scavenging activity was calculated using the following formula:

(2) $\mathrm{\%}\mathit{inhibition}=\frac{\mathit{AB}-\mathit{AA}}{\mathit{AB}}\ast 100$(2)

where AB – absorption of blank sample, AA – absorption of tested sample solution, IC₅₀, the amount of dried matters required to give 50% inhibition, was calculated by plotting the concentration against the % inhibition.

Ferric reducing/antioxidant power assay (FRAP)

Ferric reducing antioxidant power (FRAP) was determined following the method described by.^[26] Absorbance of the samples was measured (PerkinElmer, Lambda 950) at 593 nm. The antioxidant capacity was calculated as mM Fe (II)/mL from the calibration curve drawn using iron (II) sulfate heptahydrate (FeSO₄.7 H₂O).

ABTS⁺ radical cation assay

Antioxidant activity of thyme EOs as per ABTS⁺ assay was measured.^[27] Absorbance of the samples was measured (PerkinElmer, Lambda 950) at 734 nm. The positive control (vitamin C) was used to calculate vitamin C equivalent antioxidant capacity (VCEAC). The ABTS⁺ radical scavenging activity was calculated as the percentage reduction (inhibition) as follows;

(3) $\mathrm{\%}\mathit{inhibition}=\frac{\mathit{AB}-\mathit{AA}}{\mathit{AB}}$(3)

Where, AB = absorption of blank sample and AA = absorption of test sample solution.

Antimicrobial activity

Disk diffusion assay

The bacterial strains, used in this study, were obtained from Debire Birhan University, Microbiology laboratory. EOs from T.schimperi and T.serrulatus were tested against two strains of Gram positive (Staphylococcus aureus ATCC 25923, Streptococcus epidermis ATCC 12228) and two strains of Gram-negative bacteria (Escherichia coli ATCC 25922 and Salmonella typhi ATCC 13311). The microbial susceptibility of each microorganism was determined according to.^[28] Stock strain was suspended in Nutrient broth and incubated at 37°C for 24 h. The cultures were allowed to stand overnight and adjusted to a density of 100 CFU/ml (0.5 McFarland turbidity standards). The suspensions were spread on solidified Muller Hinton agar (Hemidia). Paper disks (6 mm diameter) were soaked in 10μL of EO diluted with DMSO (Dimethylsulfoxide) to a final concentration of 150 μl/ml. The impregnated discs were placed on the inoculated plates. Content of the plates was then incubated at 37°C for 24 hr. Dimethyl sulfoxide (DMSO) was used as a negative control and gentamicin (Oxoide) as a positive control. Zones of inhibition were measured in millimeters.^[29]

Minimum inhibitory concentration (MIC)

was determined using agar dilution method.^[12] The test was performed at seven concentrations of each EO (8 μL/mL, 4 μL/mL, 2 μL/mL, 1 μL/mL, 0.5 μL/mL, 0.2 μL/mL, and 0.1 μL/mL) employing doubling serial dilutions of EO in Muller Hinton agar up to the seventh dilution. Overnight incubated suspension of the test organisms in nutrient broth was prepared and 50 mL was added to all the test tubes containing the EO at different concentration. Mixtures were incubated at 37^∘C for 24 hours. After incubation, using a sterile wire loop, suspension of each tube was inoculated on Muller Hinton agar to see if bacterial growth was inhibited. Growth of bacteria on solid media indicated that a particular concentration of EO was unable to inhibit the bacteria.

Minimum bactericidal concentration (MBC)

Determination of MBC values was performed in line with the MIC experiment. The contents of test tubes above or equal to MIC cutoff points were subjected for aseptic sub culturing in Muller Hinton agar without antimicrobial disks and incubated at 37°C for 24 hours. Following incubation, the lowest concentration of the EO at which no bacterial growth occurred on agar plates was treated as the MBC cutoff point.

Result and discussion

Chemical composition

The EOs of T.schimperi and T.serrulatus identified using GC/MS were presented in Table 2. Twenty-one compounds representing 98.2% of the total oil from T.schimperi was identified. Similarly 20 compounds with 97.5% the total oil was identified from T.serrulatus. The EO components listed in Table 2 are classified in various categories on the basis of their chemical structures including aromatic oxygenated monoterpenes, monoterpene hydrocarbons, oxygenated monoterpenes, and Sesquiterpene group … .etc.

Table 2. Compounds detected in the essential oils of T. schimperi and T.serrulatus.

No.	T.schimperi		T.serrulatus
	Compounds	% area	Compounds	% area
Monoterppene hydrocarbons		26.6		18.9
1	β-Myrcene	1.2	β-Myrcene	0.4
2	p-Cymene	17.5	m-Cymene	10.8
3	γ-Terpinene	6.8	α-Terpinen	0.9
4	trans-4-thujanol	1.1	γ-Terpinene	6.4
5	-	1.5	Geranyl acetate	0.4
Oxygenated monoterpenes		3.9		2.5
6	Linalool	1.4	trans-Sabinene hydrate	0.8
7	Terpinen-4-ol	0.6	Terpinen-4-ol	0.6
8	α-Terpineol	1.9	α-Terpineol	1.1
Sesquiterpene hydrocarbons		4.7		5.4
9	β-Caryophyllene	2.8	β-Caryophyllene	0.9
10	γ-Cadinene	0.8	β-levantenolide	4.5
11	D-Germacrene	0.5	-
12	Spathulenol	0.6	-
13	E-Nerolidol	0.8	-
Oxygenated Sesquiterpene hydrocarbons		1.6		0.4
14	Caryophyllene oxide	1.6	Caryophyllene oxide	0.4
Aromatic Oxygenated Monoterpenes		59.4		68.7
15	Thymol	10.1	Thymol	34.8
16	Carvacrol	47.2	Carvacrol	31.6
17	Carvacryl acetate	1.5	o-Acetylthymol	0.7
18	-		5-Isopropyl-2-methylphenyl acetate	0.8
19	p-Cymene-2-ol methyl ether	0.6	p-Cymene-2-ol methyl ether	0.4
20	-		p-Cymene-2,5-diol	0.4
Other		2.0%		1.6%
21	1-Octen-3-ol	0.5	-
22	3-Octanone	0.9	3-Octanone	1.2
23	3-Octanol	0.6	3-Octanol	0.4
Total		98.2		97.5%

EOs obtained from Thymus schimperi primarily composed of aromatic oxygenated monoterpenes (59.4%) followed by monoterpene hydrocarbons (26.6%). Aromatic oxygenated monoterpenes comprised of a dominant carvacrol (47.2%) followed by thymol (10.1%) and carvacryl acetate (1.5%), while monoterpene hydrocarbons contain p-cymene (17.5%), γ-terpinene (6.8%), β-myrcene (1.2%), and trans-4-thujanol (1.1%) as the main constituents of T.schimperi EO. Also, α-terpineol (1.9%) and linalool (1.4%) were categorized in oxygenated monoterpenes and β-caryophyllene (2.8%), and carvacrol methyl were identified as Sesquiterpene groups in considerable amounts (Table 2).

In the EO extracted from Thymus serrulatus, aromatic oxygenated monoterpenes were elucidated as the major class of compounds (68.7%). Thymol was dominant (34.8%), followed by carvacrol (31.6%). Considerable amount of monoterpene hydrocarbons (18.9%), Sesquiterpenes (5.4%) and oxygenated monoterpenes (2.5%), with m-cymene (10.8%), γ-terpinene (6.4%), α-terpineol (1.1%), β-levantenolide (4.5%), 3-octanone (1.2%), and other compounds were determined as the major substances.

There are both qualitative and quantitative differences between the oils of the two species. T.schimperi oil is rich in monoterpene hydrocarbons (26.6%) than T.serrulatus oil (18.9%) while, aromatic oxygenated monoterpene is significantly lower (59.4%) than T. serrulatus EO (68.8%). The EOs of Thymus schimperi from different localities (Tarmaber, Butajira and Bale)^[18] recorded the predominance of thymol, carvacrol, and p-cymene as the main components (Table 3). This result was significant different from the result obtained in the current study identified EO as major components for the same thymus species. However, other additional components (γ-terpinene, caryophyllene, α-terpineol, carvacryl acetate, linalool, β-myrcene, and trans-4-thujanol) were identified in the current study. The same study,^[18] has also shown qualitative and quantitative differences in the EO content of T.serrulatus (Table 3).

Table 3. Major components of T.schimperi and T.serrulatus essential oil from different localities (Damtie et al., 2018).

No.	T.schimperi				T.serrulatus
	Compound	Tarmaber %	Butajira %	Bal %	Ofla %	Alamata %	Yilmana densa %
1	p-cymene	1.9	3.8	3.2	3.1	4.8	3.7
2	thymol methyl ether	—	—	—	2.7	6.6	1.3
3	thymol	48.8	15.8	53.6	49.6	65.6	6.5
4	carvacrol	42.1	71.8	34.6	36.3	6.7	80.8
5	g-cadinene	—	—	—	1.6	1.5	—
6	caryophyllene oxide	—	0.9	—	1.1	2.2	—

Note: Tar; Tarmaber, But; Butajira, Ofl; Ofla, Alam; Alamata &Yilm; Yilmanadiensa

However, the current study of T.schimperi was qualitatively consistent (Carvacrol chemotype) of the study of^[30] resulted rich in Carvacrol (66.2%) followed by γ-Terpinene (13.2%), 3-Octanol (4.5%), Thymol (3.6%), α-Terpinene (3.4%). But, quantitatively, Carvacrol (47.2%) of the current study was lower than the study reported by.^[30] In the study reported,^[31] thymol (36.5%), Carvacrol (29.8%) as the major constituents of the oil of T. spathulifolius which is somewhat similar with T.serrulatus oil with the exception of p-Cymene has not been detected in the current study. It is noteworthy to point out that the composition of any plant EO studied is depend on several factors, such as geographic location, climatic conditions, seasonal of pant collection, genetics/species of the plant and experimental conditions.^[31] In general, the oils of the two investigated species are rich in aromatic oxygenated monoterpene (especially, thymol and carvacrol) and due to this high phenol contents, they can be used as better preservative active substances.^[32]

Physicochemical characterization of essential oil

In this study, oils from the two thymus species prepared by hydro-distillation were evaluated for their physicochemical characteristics and data is presented in Table 4. The extracted oils were light yellow in color, transparent and had pleasant odor. The EO yield from dried T.schimperi and T.serrulatus was 2.1 ± 0.0% and 1.2 ± 0.3% (w/w), respectively. EO yield of T.schimperi in the present study was higher than the value reported earlier^[13] [1.02% (v/w) for semidried and 1.48% (v/w) from fresh leaves]. The yield of EO from T.serrulatus in the present study is low compared to an earlier report^[11] from fresh leaves collected in the flowering season from Tigray region (1.83% w/w). This difference may be due to geographical variation and/or age of the plant (plant collected after flowering season). A significant differences was observed on the yield of EOs between the two species (p < .05).

Table 4. Physicochemical characteristics T.schimperi and T.serrulatus EO.

Physicochemical characteristics	T.schimperi	T.serrulatus
Yield	2.1 ± 0.1%(w/w)	1.2 ± 0.3
Refractive index	1.5 ± 0.0	1.5 ± 0.0
Specific Gravity	0.9 ± 0.0	0.9 ± 0.0
Specific Rotation	9.4 ± 1.2	8.0 ± 2.0
Acid Value	2.9 ± 0.0	3.9 ± 0.4
Peroxide Value	3.6 ± 0.9	3.9 ± 0.3

Refractive index of the oils found in this study was 1.5 ± 0.0 and 1.5 ± 0.0 for T.schimperi and T.serrulatus species, respectively. These results were consistent with previous works^[33]on T.zygis (1.50 ± 0.05) but differed from the value for T. willdenowii EO (1.33 ± 0.04) collected in May, 2019. As can be seen from Table 3, there were no significant differences on the refractive index of the two species (T.schimperi and T.serrulatus).

In the present analysis, oils from T.schimperi and T.serrulatus displayed specific gravity values of 0.9 ± 0.0and 0.9 ± 0.0 at room temperature respectively. There was no significant difference in specific gravity between the oil of the two species as shown in Table 4. It is slightly different in the report of^[34] on Thymus serpyllum L essential with value of ranged in 0.95 to 0.94 collected from different altitudes. The difference may be due to geographic location/altitude of plant growth and species of the plant.

Acid value (2.9 ± 0.0 and 3.9 ± 0.4), optical rotation (+9.4 ± 1.2 and + 8.0 ± 2.0), and peroxide value (3.6 ± 0.9 and 3.9 ± 0.3) were obtained for oils of T.schimperi and T.serrulatus respectively. Statistical comparison between the findings for the two species showed a significant difference on the acid value but insignificant difference on the specific rotation and peroxide value between T.schimperi and T.serrulatus EO. There is no report from previous work on these physicochemical characteristics for T.schimperi and T.serrulatus EO to compare with the findings from the current study.

Qualitative analysis of phytochemicals

Results of the phytochemical analysis of T.schimperi and T.serrulatus 80% methanol leaf extracts is presented in Table 5. Phytochemical screening of T.schimperi and T.serrulatus revealed the presence of alkaloids, flavonoids, phenols, saponins, tannins, steroids, and terpenoids. Antraquinons were not detected in both species. The present study reports the presence of diverse phytochemicals in T.serrulatus (like terpenoids and flavonoids)than earlier reports by.^[14] The phytochemical report presented here for T.schimperi methanolic extract is the first one.

Table 5. Phytochemical constituents of Methanolic extract of T.schimperi and T.serrulatus leaf powder.

Phytochemicals	Methods	T.schimperi	T.serrulatus
Saponine	Froth test	+ve	+ve
Phenol	Ellagic acid test	+ve	+ve
Flavonoid	FeCl3 test	+ve	+ve
Alkaloid	Wagner’s test	+ve	+ve
Tannin	Braemer’s Test	+ve	+ve
Terpinoid	Liebermann Burchard-	+ve	+ve
Antraquinon	Borntrager’s test	_ve	_ve
Steroid & Phytosteroid	Libermann-Buchard’s test	+ve	+ve

Note: SE- solvent extract, the sign of – indicates the absence of phytoconstituent in test sample and + sign the presence of phytoconstituent on the test sample

Oxidative stability of the essential oils

Oxidative stability of T.schimperi and T.serrulatus EO and their activities on the oxidative stability of sun flower oil were evaluated based on induction time (ti) measurement by Rancimat analysis as shown in Figure 1. T.schimperi showed lower induction time compared to that of T.serrulatus EO. This may be due to the difference in the type and amount of compounds in the oils, especially the most effective compound carvacrol with broad spectrum antioxidant activity, whose concentration is determined to be 47.2% in T.schimperi and 34.8% in T.serrulatus EO. T.schimperi EO was observed to have better oxidative stability effect on sun flower oil than that of T.serrulatus EO. Protection factors for T.schimperi and T.serrulatus were 1.4 and 0.97 respectively. Protection factor (Pf) greater than one indicates the antioxidative potential of additives while Protection factor less than one indicates prooxidative potential of additives.^[35] This factor expresses the effectiveness of antioxidants and represents the possible blockage of radical chain process by interacting with the peroxyl radicals.^[36] The antioxidant addition increased the induction time (t_i) of sun flower oil as shown in the Figure 1. Pf value obtained in the current study for T.schimperi EO indicates that it can serve as a potential antioxidant additive while the EO obtained from T.serrulatus indicates its prooxidative character.

Figure 1. Oxidative stability of T.schimperi and T.serrulatus EO and their effect compared with ascorbic acid on sunflower oil stability.

Note: A; T.schimperi, B; T.serrulatus, C; Control (sun flower oil alone), A & SFO; T.schimperi with sun flower oil, B & SFO; T.serrulatus with sun flower oil, Asc& SFO; Ascorbic acid with sun flower oil.

Thermal analysis of essential oils

There are vast and diverse applications of thermal analysis in industrial activities, such as chemical, pharmaceutical, cosmetics, food and petrochemicals, among others. Thus, thermal characterization of EOs determine their applications as ingredients in food formulations and other industries. Thermogravimeteric analysis method (TGA) was used to analyze the thermal properties of the essential oils produced from the two species (T.schimperi and T.serrulatus) and the TGA result of the EOs is presented in Figure 2. As can be observed in the figures, there are two weight loss stages for the two EOs. The first one (38%) is from 51°C to 164°C and (55%) from 61°C to 178°C for T.schimperi and T.serrulatus EOs respectively. This may be related to the degradation of low boiling compounds (such as 1-Octen-3-ol and Octanone for T.schimperi and 3-Octanone and β-Myrcene of T.serrulatus EO. The second one (11%) is from 164°C to 226°C for T.schimperi and (8%) from 178°C to 220°C for T.serrulatus EO which corresponds to the aromatic compounds with high boiling point such as thymol, carvacrol, p-Cymene, and γ-Terpinene.^[37] The remaining (37%) of mass from T.schimperi and (51%) from T.serrulatus was mass residue stable up to a temperature of 600°C. The TG profile of thyme oil of the current study was nearly in agreement with the previous report^[37] which showed that mass loss of commercial thyme EO begins at temperatures around 50°C. In general, a thermal property of EOs from thymus plants of current study is unstable/most of the active components evaporated/degraded at high temperature. This indicates that the oil could not be used for baking food items as a preservative (antimicrobial agent).

Figure 2. TGA curves of T.schimperi and T.serrulatus essential oils.

Fourier-transform infrared (FTIR) spectroscopy

Thyme oil’s FT-IR analysis revealed the presence of various functional groups like alkyl, aromatic and carbonyl group containing compounds. In Figure 3(a,b), the results of the FT-IR spectra for T.schimperi and T.serrulatus oil are displayed. The result of the current study (T.serrulatus and T.schimperi) of these EOs display O-H functional group at intense peaks at a wave number of 3385–3378 cm⁻¹; C-H group at 2960–2869 cm^−1, C=O group at a wave number of 1699 cm⁻¹, C=C also displayed at wave number of 1619 cm⁻¹ and N-O stretching at 1585–1513 cm⁻¹ for both species of EO which could be from the alkaloidal constituents. The present study is in agreement with previous reports.^[38,39]

Figure 3. FT-IR Results of T.schimperi and T.serrulatus essential oil.

Antioxidant activity of thyme essential oils

DPPH scavenging antioxidant assay

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity is the most commonly used antioxidant test.^[20] Results of the antioxidant activities of T. schimperi and T.serrulatus EO and ascorbic acid are shown in Figure 4. From the result, it is observed that, the antiradical power of T.schimperi and T.serrulatus EO has been increased with increasing of oil concentrations. T.schimperi EO is as effective as Ascorbic acid at 40–140 μg/ml concentration. T.serrulatus EO has lower DPPH inhibiting capacity compared to T.schimperi EO. This difference may be the variation of amount monoterpene hydrocarbon (26.6%) in T.schimperi and (18.9%) in T.serrulatus oil. There is also the amount and type of major component differences (Carvacrol (47.2%) dominant compound in T.schimperi than T.serrulatus EO (31.6%). This leads to T.schimperi has a better radical scavenging activity than T.serrulatus EO. This may be confirmed by the study reported of.^[39] In the other hand, m-cymene and β-Levantenolide which was detected in T.serrulatus have less potent antiradical inhibition than p-cymene and caryophyllene found only in T.schimperi oil as a major component. However, both species have significant DPPH scavenging capacity.

Figure 4. Percent of DPPH inhibition of T.schimperi, T.serrulatus and Ascorbic acid.

The antioxidant activity of Thymus EOs has been reported previously^[40] and this activity has been responsible mainly to the content of phenolic components, especially thymol and carvacrol. According to,^[39] in vitro antioxidant activity of the EOs of several thymus species collected in June,2008 were reported with the radical scavenging capacity for T. migricus T. fallax and T. pubescens var. pubescens as 45.4 ± 0.8%, 65.9 ± 0.1%, and 42.3 ± 0.6%, respectively, at 100 μg/ml. Those results have lower DPPH inhibition capacity in compared to T.schimperi and T.serrulatus (89% and 76% respectively) as shown in Figure 4. On the other study,^[41] the DPPH scavenging capacity of Thymus capitatus oil were recorded as 27 ± 2.0, 30 ± 0.7 and 40 ± 0.3%, for concentrations of 12.0; 15.0; 25 and 62.5 μg/mL), however, the current study has shown better scavenging capacity than the aforementioned study (Figure 4). The differences between current results and the previous report may be attributed to the differences in the sources of the samples, geographical location and/or season of plant collection.

The IC₅₀ values of T.schimperi, T.serrulatus and ascorbic acid was 0.2 μg/mL which indicating that the two plant species has comparable antioxidant potential to that of standard ascorbic acid. The T.schimperi and T.serrulatus EO IC₅₀ value of the DPPH assay in the current study is much higher (0.2 μg/mL) than the one obtained in^[42] from Omani thyme EO collected in September, 2018 with the concentration 23.64 μg/mL. In accordance with,^[43] IC₅₀ value of thymus vulgaris L.) and wild thyme (Thymus serpyllum L.) EOs slightly lower (0.3 ± 0.0 and 0.4 ± 0.1 g/L respectively) than the current study. In the other study,^[44] the IC₅₀ value of Thymus vulgaris L., Lamiaceae EO also consistent (0.2 μg/mL) with the current study both T.scimperi and T.serrulatus (0.2 μg/mL). The high content of carvacrol and/or thymol in wild and cultivated T. maroccanus and T. broussonetii oils have strong antioxidant activity.^[40] In the study of^[44] the isolated compound thymol and carvacrol possess lower scavenging activities than thyme and oregano essentials. Therefore, the antioxidant activity of EOs is the product of additive, synergistic and/or antagonistic effects, as they are complex mixtures of several classes of compounds.

Ferric reducing antioxidant power (FRAP)

Results of the ferric reducing antioxidant power (FRAP) are shown in Table 6. The EOs of T.serrulatus have higher FRAP values (8.0 ± 0.9 mMFe²⁺/ml oil) in comparison with the EO of T.schimperi (4.6 ± 0.4 mMFe²⁺/ml oil). In accordance with^[20] report, the FRAP value of T.vulgaris EO was 3.3 ± 0.02 μM Fe (II)/g. In another study, FRAP value of 310.4 ± 13.9 mM Fe (II)/mL for T.vulgaris was reported.^[26] The result of the current study has higher ferric reducing antioxidant power than the one reported by,^[20] but less ferric reducing antioxidant power^[26] with FRAP value of 4.6 ± 0.4 for T.schimperi and 8.04 ± 0.9 mM Fe²⁺/mL sample for T.serrulatus. This variation may be due to the variation of aromatic oxygenated monoterpenes and Sesquiterpene.

Table 6. Ferric reducing antioxidant power and % of inhibition of ABTS.

Plant species	Antioxidant assay
	FRAP (mMFe^2+/ml oil	ABTS (%)
T.schimperi	4.6 ± 0.4	72.7 ± 0.5
T.serrulatus	8.0 ± 0.9	63.7 ± 3.4

2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) antioxidant assay

The ABTS assay is based on the reduction of ABTS⁺ radicals by antioxidants of the sample tested and is better to assess the antiradical capacity of both hydrophilic and lipophilic compounds. It can be used in both organic and aqueous solvent system as compared to other antioxidant assay methods such as DPPH. T.schimperi and T.serrulatus EO extracted by hydro-distillation were evaluated for their ABTS radical cation scavenging activity. As shown in Table 6, both extracts displayed strong radical scavenging capacity. T.schimperi showed better ABTS⁺ reducing capacity (72.7 ± 0.5%) compared to T.serrulatus (63.7 ± 3.4%). This variation may be due to the variation of major chemical components which have a potent antiradical scavenging activity. To the best of our knowledge there are no previous reports on antioxidant activities T.schimperi and T.serrulatus EO. However, when compared with other species of previous reports, the current study EO’s demonstrated better ABTS⁺ reduction capacity than example is Thymus hirtus sp. algeriensis (16 ± 0.1%).^[45]

Antibacterial activity

Disc diffusion assay

The antibacterial activity of T.schimperi and T.serrulatus EO was assessed by recording the zone of inhibition (mm) against Staphylococcus aureus, Streptococcus epidermidis, Salmonella typhi, and E.coli at 150 μL/mL concentrations with an amount of 10 μL/Disk and 10 μg/Disk gentamicin as positive and DMSO as negative controls. As shown in Figure 5, the EO’s showed antibacterial activity against Gram +ve and Gram⁻ve bacteria. T.schimperi EO was more pronounced on growth inhibitory effect on E coli. ATCC 25,922, S.typhi ATCC 13,311, and S.epidermidis ATCC 12,228, but T.serrulatus EO has more effective on the growth of S.aureus ATCC 25,923 than T.schimperi EO and even the standard drug gentamicin as shown in Figure 5. According to these results, the amounts of carvacrol and thymol contents were varying between the two plant species. These results indicated that, genetically variation caused changed in the main components in T. schimperi and T.serrulatus EOs, and these components can be effect in antibacterial activities of the plants.

Figure 5. Antibacterial activities of T.schimperi, T.serrulatus essential oil and gentamicin.

The results of this study highlighted the ability of T.schimperi and T.serrulatus EO to inhibit bacterial growth and their potential as a powerful antibacterial agent and food preservative. There is no enough previous works on antimicrobial activities of concentrated T.schimperi and T.serrulatus EO against four bacterial strains (Escherichia coli, S. aureus, S.typhi, and S.epidermis). However, there was one study reported by^[46] on the aforementioned bacteria of concentrated T.schimperi EO with the zones of inhibition observed were 12.0 ± 0.4, 23.5 ± 1.5, 27.3 ± 1.1, and 33 ± 1.5 mm, respectively. From this, result zone of inhibition for Escherichia coli and Streptococcus epidermidis was much higher than the result of current study.

Minimum inhibitory concentration and bacterial concentration (MIC and MBC)

MIC values of the EOs of T. schimperi and T.serrulatus were evaluated with agar dilution assay. As can be seen from Table 7, EO of the two plants showed different antimicrobial activity against the tested bacteria. MIC values on E.coli and S.typhi was 0.5 and 1 μL/mL for T.schimperi, 2 and 4 μL/mL of T.serrulatus EO, respectively. MIC value for S.aureus and S.epidermidis was 0.2 and 4 μL/mL for T.schimperi, 2 and 0.5 μL/mL of T.serrulatus EO’s respectively. This is may be due to EOs rich in phenolic compounds, such as carvacrol, are widely reported to possess high levels of antimicrobial activity.^[47] Therefore, T.schimperi oil was rich in carvacrol content and has better antibacterial effect than T.serrulatus EO. Compared to an earlier finding^[48] on Thymus marschallianus Will and Thymus proximus Serg collected on June which was in the range of 1.81 to 4.52 μL/mL, respectively.

Table 7. MIC and MBC T.schimperi and T.serrulatus essential oil.

S.NO.	Bacterial strain	MIC (μL/mL)		MBC(μL/mL)
		T.schimperi	T.serrulatus	T.schimperi	T.serrulatus
1	E. coli	0.5	2.0	2.0	4.0
2	S. typhi	1.0	4.0	2.0	4.0
3	S. aureus	0.2	2.0	1.0	2.0
4	S.epidermidis	4.0	0.5	4.0	8.0

On the other hand MBC was confirmed by absence of growth of the test bacterial strains streaked form their lowest clear MIC. T.schimperi and T.serrulatus EO showed bactericidal activity against the tested pathogenic organisms (E.coli, S.typhi, and S.aureus and S.epidermidis) with MBC values as shown in Table 7. EO from T.schimperi was more effective against all tested bacterial strains (2 μL/mL both E.coli ATCC 25,922 and S.typhi ATCC 13,311; 1 μL/mL for S.aureus ATCC 25,923 and 4 μL/mL for S.epidermidis ATCC 12,228) compared to T.serrulatus EO (E.coli ATCC 25,922 and S.typhi ATCC 13,311 4 μL/mL, and 2 μL/mL for S.aureus ATCC 25,923 and 8 μL/mL for S.epidermidis ATCC 12,228). Escherichia coli and S.typhi showed the same sensitivity to T.schimperi EO with MBC value of 2 μL/mL and that of T.serrulatus EO was 4 μL/mL. The value of MIC and MBC against S.epidermidis was the same for T.schimperi EO. S.aureus has also the same MIC and MBC value of 2 μL/mL for T.serrulatus EO. However, S.epidermidis was resistant to T.serrulatus EO up to 8 μL/mL. The antibacterial nature of the EO studied is apparently related to their high aromatic oxygenated monoterpene, particularly carvacrol and thymol and this finding is in agreement with a previous report.^[31]

Conclusion

The EOs investigated in this study showed comparable and, in some cases, higher percentage yield than that for plants in the genus reported in literature. The antibacterial and antioxidant activity of the EOs in this study revealed that T.schimperi and T.serrulatus EOs have a potential to inhibit oxidation and growth of bacteria. EO from T.schimperi and T.serrulatus were observed to be sensitive to oxidation but have a high potential as antioxidant and antibacterial effect that can inhibit oxidation and growth of bacteria. This property could be the reason for their traditional use as a healthy and safe natural food preservative.

Acknowledgments

The authors are thankful to the Department of Food Process Engineering Laboratory of Addis Ababa Science and Technology University for providing laboratory facilities. The authors also acknowledge Addis Ababa University Food Science and Nutrition Research Center and Debire Berhan University for additional laboratory services.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This research is funded by Ministry of Higher Education (MOHE), Debre Berhan University.