Three new compounds from the seeds of Trachyspermum copticum

ABSTRACT

The effect of the application of nano-silicon (SiO₂; 1.5 and 3 mM) on components of essential oils of Trachyspermum copticum was evaluated. Essential oils extracted by hydrodistillation from Iranian T. copticum was characterized by means of gas chromatography-mass spectrometry and Nuclear Magnetic Resonance. Three new compounds, (1) 2, 5-Dihydroxy-4-methoxy-phenanthrene 2-O-b-Dglucopyranoside; (2) 4-Methoxy-2,5,7,9S tetrahydroxy-9,10-dihydrophenanthrene; and (3) trans-Ethyl cinnamate along with 12 known were isolated from the Eos T. copticum. The essential oil was also subjected to antimicrobial and antioxidant activities. The new compounds were particularly active against Bacillus cereus and Candida albicans, with the lowest minimum inhibitory concentration and minimum bactericidal concentration and minimum fungicidal concentration value. Three new compounds exhibited a higher activity in each antioxidant system with a special attention for β-carotene bleaching test, lipid peroxidation inhibition, and reducing power. The Thin-layer chromatography-bioautography screening and fractionation resulted in the separation of the main antioxidant compound which were identified as trans-Ethyl cinnamate (3). These results indicated that three new compounds might be applicable in natural medicine and healthy food.

KEYWORDS:

• Trachyspermum copticum
• Essential oil
• Nano silicon
• Antimicrobial and antioxidant activities

Introduction

There is an increased interest by consumers in new sources of aromas, flavors, and medicines. Recently, investigations of natural products for the discovery of active compounds with antimicrobial and antioxidant properties from plant origin that can be applied to the food industry has gained interest.^[1] Plant secondary metabolism is mostly related to defense mechanisms or interactions with the environment. Plants of the Apiaceae and Asteraceae families possess a range of compounds with many biological activities, such as the ability to induce apoptosis, and anti-bacterial, hypocholesteraemic, and cholinergic activities.^[2] Trachyspermum copticum is an aromatic annual plant grown in Iran. The seeds of T. copticum have several therapeutic effects, including diuretic, antivomiting, analgesic, antiasthma, and antidyspnea effects. In Persian folk medicine, the fruits of T. copticum were used as a diuretic, anti-vomiting, carminative, and antihelmentic agent.^[3] Mohagheghzadeh et al.^[4] showed that T. copticum has two chemotypes, thymol and carvacrol. The objectives of this work were to study the chemical composition and exploration of novel com-pounds from T. copticum, to explore antimicrobial and antioxidant activity (AA).

Materials and methods

Plant materials and nano-silicon (SiO²) treatments

The plant was identified by Mr. Esmaeili, and the voucher specimen was deposited at private herbarium of Dr F. Esmaeili (voucher no. 121). Seeds of T. copticum were sown in Jefe pot in experimental greenhouse of Ilam, Iran. Plants at flowering stage (2013–2014) were sprayed with distilled water as a control, and nano-silicon (SiO₂) at 1.5 and 3 mM. All spray solutions were sprayed to the point of run-off. The experiment was arranged in completely randomized block design with three replications for each treatment. At seed stage of T. copticum were harvested and air dried at ambient temperature in the shade.

Oil isolation and identification of the oil components

The T. copticum seeds were ground and the resulting powder was subjected to hydrodistillation for 3 h in an all glass Clevenger-type apparatus according to the method recommended by the European Pharmacopoeia.^[5] The obtained essential oils (EOs) were dried over anhydrous sodium sulphate and after filtration, stored at +4°C until tested and analyzed. The gas chromatography-mass spectrometry (GC-MS) analyses were executed on a Hewlett–Packard 5973N gas chromatograph equipped with a column HP-5MS (30 m length × 0.25 mm i.d., film thickness 0.25 lm) coupled with a Hewlett–Packard 5973N mass spectrometer. The column temperature was programmed at 50°C as an initial temperature, holding for 6 min, with 3°C increases per min to the temperature of 240°C, followed by a temperature enhancement of 15°C per min up to 300°C, holding at the mentioned temperature for 3 min. Injector port temperature was 290°C and helium used as carrier gas at a flow rate 1.5 mL/min. Ionization voltage of mass spectrometer in the EI-mode was equal to 70 eV and ionization source temperature was 250°C. Linear retention indices for all components were determined by coinjection of the samples with a solution containing homologous series of C8-C22 n-alkanes and comparing them and their mass spectra with those of authentic samples or with available library data of the GC-MS system (Wiley 2001 data software) and Adams libraries spectra.^[6] ₁H and ₁₃C NMR spectra were recorded on a 400 (100)-MHz Varian spectrometer, d in parts per million (ppm), using CDCl₃ as solvent and tetramethylsilane (TMS) as internal standard.

Total phenolic determination

Total phenolic contents in seeds T. copticum were determined by Folin–Ciocalteu method.^[7] The total phenolic content was expressed as gallic acid equivalents (GAE; mg g^_1).

Total flavonoid determination

Total flavonoid contents in seeds T. copticum were measured as described previously.^[8] The total flavonoid content was calculated as rutin equivalents (mg g⁻¹).

Microorganisms

Gram-positive bacteria: Bacillus cereus (ATCC 10876), Enterococcus faecalis (ATCC 49452), Staphylococcus aureus (ATCC 25923). Gram-negative bacteria: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Proteus mirabilis (ATCC 35659), Salmonella typhimurium (ATCC 13311), Citrobacter freundii (ATCC 8090). Fungal strains: Candida albicans (ATCC 10231) and Aspergillus fumigatus (ATCC 46645). The bacteria species were maintained in Mueller Hinton agar and Tryptic soy agar. Strains of Candida spp. and Aspergillus spp. were maintained on Sabour and dextrose agar.

Antimicrobial activity

Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations/minimum fungicidal concentrations (MBC/MFC) were determined by microdilution method in 96-well microtitre plates, described by Douk et al.^[9] and EUCAST.^[10] Briefly, fresh overnight cultures of bacteria were adjusted with sterile saline to a concentration of 1.0 × 10⁵ colony forming units (CFU) per well, and 1.0 × 10⁴ CFU per well for fungi. EOs were added in TSB medium for bacteria, and sabouraud dextrose broth (SDB) medium for fungi. The microplates were incubated for 24 h at 37°C for bacteria, and 48 h at 37°C for fungi. The MIC was defined as the lowest concentration of EO inhibiting the visible growth of the test strain. However, the MIC/MBC values for bacteria and fungi were detected following the addition of 40 µL of piodonitrotetrazoliumviolet (INT) 0.2 mg/mL and incubation at 37°C for 30 min.^[11] The MBCs/MFCs were determined by serial subcultivations of 10 µL into microtiter plates containing100 µL of broth per well and further incubation for 24 h at 37°C. The lowest concentration with no visible growth was defined as the MFC, indicating 99.5% killing of the original inoculum. Following positive controls were used in both experiments: antibiotics (Streptomycin) and mycotic (Fluconazole). Each test was carried out in triplicates and repeated three times.

AA

The efficacy of the EOs to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was evaluated using a spectrophotometry method.^[12,13] On basis of bleaching of the bluish-red or purple color of DPPH solution as a reagent. Briefly, a 50 µL volume of various dilutions of each sample was mixed with 5 mL of 0.004% methanol solutions of DPPH followed by 30 min incubation at ambient temperature. Thereafter, the sample absorbance was recorded against control at 517 nm. The inhibition percentages were measured using Eq. (1). The antioxidants activity of the test samples in concentration providing 50% inhibition, were considered as IC50 (µg/mL).

(1)

Butylhydroxyanisole (BHA) and ascorbic acid were used as positive controls. All experiments were repeated three times and the average results and standard deviations (SD) calculated.

Rapid screening for antioxidants

For screening of antioxidant compounds in T. copticum EO, the Thin-layer chromatography-bioautography (TLC-bioautography) method was carried out.^[14,15] The diluted oil (1:20 in methanol) was spotted on silica gel sheets (silica gel 60 F254 TLC plates) and developed in n-hexane-ethyl acetate (9:1). Plates were sprayed with the methanolic solution of DPPH (0.2%). The active constituents were detected as yellow spots on a violet background. Only zones where their color turned from violet to yellow within the first 30 min (after spraying) were taken as positive results.

Activity guided fractionation of the EO for antioxidants

For the isolation and identification of the active compounds in the EO, TLC was performed using the conditions previously described.^[15] The regions showing DPPH scavenging activity were scrapped off then, they were eluted with chloroform. All resulting constituents were analyzed by GC-MS and also tested for their antioxidant activities.

β-carotene-linoleic acid model system (β-CLAMS)

The β-CLAMS method by the peroxides generated during the oxidation of linoleic acid at elevated temperature.^[16] The AA of the extracts was evaluated in term of β-carotene blanching using the following formula:

where A₀ is the absorbance of the control at 0 min, and A₁ is the absorbance of the sample at 120 min. The results are expressed as IC₅₀ values (µg/mL). All samples were prepared and analyzed in triplicate.

Reducing power and lipid peroxidation inhibition

The ability of the extracts to reduce Fe³⁺ was assayed by the method of Oyaizu.^[17] One milliliter of T. copticum EO were mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% K₃Fe (CN) 6. After incubation at 50°C for 25 min, 2.5 mL of 10% trichloroacetic acid was added and the mixture was centrifuged at 650 g for 10 min. Finally, 2.5 mL of the upper layer was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% aqueous FeCl₃. The absorbance was measured at 700 nm. The mean of absorbance values were plotted against concentration and a linear regression analysis was carried out. Increase absorbance of the reaction mixture indicated increased reducing power. EC₅₀ value (µg/mL) is the effective concentration at which the absorbance was 0.5 for reducing power. Ascorbic acid was used as positive control. Lipid peroxidation inhibition was determined by Shirwaikar et al.^[18] Ascorbic acid and Trolox was used for comparison.

Statistical analysis

The results are presented as mean ± SD and statistically analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s test.

Results and discussion

Identification of new compounds

C₁

Isolated compounds were identified by ultraviolet (UV), MS, and Nuclear Magnetic Resonance (NMR) instruments. Identification of 2,5-Dihydroxy-4-methoxy-phenanthrene 2-O-b-Dglucopyranoside: Colorless colloidal solid; mp 166–169°C; [a] 25 D _{_52} (c 0.8,MeOH); UV (MeOH) kmax (log ^e) 214.5, 254, 283.5, and 313.5 nm; IR (KBr) mmax 3427, 1614, 1384, 1261, 1073, 819, and 595 cm²1; for 1H NMR and 13C spectroscopic data NMR; HRESI- MS m/z 425.1208 (calcd for C21H22O8Na, 425.1208).

C₂

Identification of 4-Methoxy-2,5,7,9S-tetrahydroxy-9,10-dihydrophenanthrene: yellowish powder; mp 167–172°C; [a] 25^D ₊₆₂ (c 0.15, MeOH); UV (MeOH) max (loge) 211.5, 274.5 and 306.0 nm; IR (KBr) mmax 3439, 1636, 1615, 1438, 1161, 871, and 617 cm²1; for ₁H NMR and ₁₃C NMR spectroscopic data, HR-ESI-MS m/z 297.0736 (calcd for C15H14O5Na, 297.0736).

C₃

Identification of trans-Ethyl cinnamate: trans-Ethyl cinnamate displayed the following spectroscopic data: d 1H NMR 1.06 (3H, t, J = 6.9 Hz, CH2CH3), 3.98 (2H, q, J = 6.9 Hz, CH2CH3), 6.18 (1H, d, J = 17.6 Hz, H-2), 7.08 (3H, m, H-30, 40, and 50), 7.23 (2H, m H-20, and H-60), and 7.44 (d, J = 19.4 Hz, H-3); MS m/z 176 [M]+, (base peak), C₁₁H₁₂O₂.

Identification of known compounds

The constituents of the obtained Eos of T. copticum treated with nano silicon are presented in Table 1. Twelve components were identified in nano silicon-treated plants (Table 1). The differences were supposed to be the effects of nano silicon on chemical composition of T.copticum EO. Thymol was increased with nano silicon-treatment (Table 1). The yield of the T. copticum oil was 2.35% (1.5 mM) and 3.05% (3 mM). Nano silicon significantly increased the yield of EO (Table 1). Khajeh et al.^[19] showed that hydrodistilled oil of the plant contained eight main compounds, including thymol (49%), q-cymene (15.7%), c-terpinene (30.8%), and b-pinene (2.1%), but supercritical carbon dioxide extraction (SFE) of the EO revealed only three compounds (thymol, q-cymene, and c-terpinene), and the content of each depended on SFE conditions. Kobraee et al.^[20] reported that nano iron foliar application enhanced soybean yield by influencing number of seeds per plant and seed weight. Therefore, iron deficiency in soils could be a restricting factor of yield and extremely decrease crop yield quality. Application of nano-iron oxide at 0.75 g/L compare to other treatments had maximum effect on dry pod weight. It seems that the use of iron nano-particles causes increasing in pod and dry leaf weight and finally will increase total yield.^[21] Lu et al.^[22] have shown that application of nano fertilizers could increase the nitrate reductase enzyme in soybean (Glycine max L.), increase its abilities of absorbing and utilizing water and fertilizer, promote its antioxidant system, and, in fact, accelerate its germination and growth.

Table 1. New compounds and known from the seeds of trachyspermum copticum.

		^aT. copticum EO (%)
Components		SiO2 (1.5 mM) (%)	SiO2 (3 mM; %)	^bRetention index	Identification methods
New compounds
1	2, 5–Dihydroxy–4–methoxy–phenanthrene 2–O–b–Dglucopyranoside	8.36	18.65	–	NMR
2	4–Methoxy–2,5,7,9S–tetrahydroxy–9,10–dihydrophenanthrene	3.47	10.25	–	NMR
3	Trans–Ethyl cinnamate	9.02	13.14	–	NMR
Known compounds
1	α–pinene	2	1.33	855	MS, RI
2	β–pinene	1.00	1.23	190	MS, RI
3	β–myrcene	0.34	0.41	920	MS, RI
4	P–cymene	8.04	6.45	950	MS, RI
5	Limonene	0.57	0.87	960	MS, RI
6	γ– terpinene	26.54	10.17	980	MS, RI
7	4–terpineol	0.98	2.36	1.63	MS, RI
8	Cis limonene oxide	1.58	2.00	1085	MS, RI
9	Thymol	17.65	22.58	1208	MS, RI
10	Ethylene methacrylate	1.68	1.74	1235	MS, RI
11	Nonadecane	0.98	0.36	1293	MS, RI
12	Carvacrol	5.65	7.68	1306	MS, RI
Total	87.86	99.22
Yield	2.35%	3.05%

^aPercentage composition determined on column HP 5.

^bThe retention Kovats indices were determined on HP 5 capillary column in reference to n-alkanes.

MS: mass spectroscopy; RI: retention index.

Extraction yield, total phenolic contents, and total flavonoid contents

The AA of plant extracts has been correlated to their total phenolic content due to their property of scavenging free radicals.^[23] It is well-known that phenolic compounds contribute to quality and nutritional value in terms of modifying color, taste, aroma, and flavor and also in providing health-beneficial effects. They also serve in plant defense mechanisms to prevent damage by microorganisms, insects, and herbivores.^[24] Moreover, a few studies on the T. copticum revealed that they are good dietary sources of antioxidants. Thus, we determined the total phenolic and flavonoid contents of the methanol extracts of T. copticum wild vegetables. As shown in Table 2, the extraction yield of T. copticum ranged from lowest 78.00 ± 23 mgg^_1 (control) to highest 131.80 ± 75 mgg^_1 (nano silicon [3 mM]). Among the three T. copticum extracts, T. copticum treated with nano silicon at 3 mM showed the highest total phenolic content (245.08 ± 87 mgg^_1) and showed the highest total flavonoid content (221.45 ± 47 mg g^_1). These results showed that the total phenolic and total flavonoid contents have an obvious variation in various concentrations.

Table 2. Effect of nano silicon (SiO₂) on extraction yields, total phenolic contents, and total flavonoid contents of T. copticum extracts.

	Extract	Extraction yield^a	Total phenolic^b	Total flavonoid^c
1	Control	78.00 ± 23	85.42 ± 38	90.12 ± 87
2	SiO2 (1.5 mM)	97.12 ± 58	118.75 ± 38	105.87 ± 05
3	SiO2 (3 mM)	131.80 ± 75	245.08 ± 87	221.45 ± 47

The data are expressed as mean ± SD.

^aExpressed as mg of extract per gram dry material.

^bExpressed as milligram of gallic acid per gram dry extract.

^cExpressed as milligram of rutin per gram dry extract.

Antimicrobial activity

The in vitro antimicrobial activities of new compounds (C₁-C₃) and thymol against the studied microorganisms were assessed by the MIC and MBC/MFC (Table 3). According to the results given in Table 3, new compounds (C₁-C₃) and thymol exhibited significant antimicrobial activity against all tested strains. Inhibition values were in the following range: MIC 3.5 ± 0.08 (Bacillus cereus; c₃) to 25 ± 0.95 µg/mL (Staphylococcus aureus; c₂) and MBC 3.5 ± 0.32 µg/mL (Escherichia coli; c₃) to 20 ± 0.74 µg/mL (Pseudomonas aeruginosa; c₁) for bacteria, and MIC 2 ± 0.21 (Candida albicans; c₃) to 5.5 ± 0.35 µg/mL (Aspergillus fumigatus; c₂) and MFC 2 ± 0.65 (A. fumigatus; c₃) to 5.5 ± 0.67 µg/mL (C.albicans; Thymol) for fungi. Results obtained from MIC and MBC/MFC indicated that the antimicrobial activity of the three isolated compounds against B. cereus and C. albicans was greater than those of thymol. Among the individual constituents of EOs, carvacrol, isoeugenol, nerol, citral, and sabinene exhibited potent anti-H. pylori effects.^[25] The major components of thyme and oregano EOs, thymol and carvacrol, inhibited pathogenic bacterial strains, such as E. coli, Salmonella enteritidis, S.choleraesuis, and S. typhimurium.^[26] Eugenol, terpenen-4-ol, and carvacrol showed an inhibitory effect against the growth of four strains of E. coli O157:H7 and L. monocytogenes, but obtained results indicated that three isolated compounds was greater than those of monoterpenes (benzyl alcohol, camphor, cinnamaldehyde, borneol, and cineole) but weaker than those of monoterpenes (thymol, carvacrol, carveol, and geraniol).^[27] Comparing the results of compounds C₁-C₃ with that of standard, streptomycin, and fluconazole, it was concluded that the oils possesses more potent anti-oral-pathogen activity. The compounds C₁-C₃ expressed higher antibacterial activity than thymol and both antibiotics tested. In our study, most of the antimicrobial activity in EOs from T. copticum appears to be associated with compounds C₁-C₃ and thymol (Table 3).

Table 3. Antimicrobial activity of the new compounds and thymol (µg /mL) from T. copticum using minimum inhibitory (MIC) and minimum bactericidal/fungicidal (MBC/MFC) test.

	Thymol	C1	C2	C3	Antibiotic (streptomycin)
	MIC	MIC	MIC	MIC	MIC
Microorganisms	MBC	MBC	MBC	MBC	MBC
Bacillus cereus	5.5 ± 0.65	4 ± 0.22	4 ± 0.25	3.5 ± 0.08	10 ± 0.70
	5 ± 0.57	4 ± 0.31	4 ± 0.52	4.5 ± 0.04	15 ± 0.32
Enterococcus faecalis	5 ± 0.47	5 ± 0.40	4.5 ± 0.38	4 ± 027	10 ± 0.59
	5 ± 0.07	5 ± 0.50	5 ± 0.04	4 ± 0.27	10 ± 0.52
Staphylococcus aureus	15 ± 0.23	15 ± 0.21	25 ± 0.95	10 ± 0.35	250 ± 0.0
	15 ± 0.74	15 ± 0.20	15 ± 0.42	10 ± 0.24	250 ± 0.0
Escherichia coli	9 ± 0.23	8.5 ± 0.24	9 ± 0.85	8.5 ± 0.86	25 ± 0.5
	9 ± 0.10	8.5 ± 0.98	9 ± 0.27	3.5 ± 0.32	25 ± 0.5
Pseudomonas aeruginosa	20 ± 0.65	20 ± 0.95	20 ± 0.19	10 ± 0.38	100 ± 0.34
	20 ± 0.28	20 ± 0.74	20 ± 0.37	10.5 ± 0.27	150 ± 0.87
Proteus mirabilis	7 ± 0.74	7.5 ± 0.75	7.5 ± 0.45	7 ± 0.65	20 ± 0.54
	9 ± 0.08	8.5 ± 0.38	7.5 ± 0.00	7 ± 0.35	15 ± 0.5
Salmonella typhimurium	5.5 ± 0.35	5.5 ± 0.75	5 ± 0.65	5 ± 0.38	15 ± 0.05
	6.5 ± 0.45	5.5 ± 0.21	6 ± 0.25	5 ± 0.11	15 ± 0.02
Citrobacter freundii	5.5 ± 0.5	5 ± 0.84	5.5 ± 0.48	5 ± 0.65	15 ± 0.87
	6.5 ± 0.05	6 ± 0.35	6.5 ± 0.31	5 ± 0.12	20 ± 0.56
Microorganisms	thymol	C1	C2	C3	mycotic (Fluconazole)
	MIC	MIC	MIC	MIC	MIC
	MFC	MFC	MFC	MFC	MFC
Candida albicans	5.5 ± 0.25	5.5 ± 0.00	4.5 ± 0.35	2 ± 0.21	4.5 ± 0.00
	5.5 ± 0.67	5.5 ± 0.35	4.5 ± 0.21	2.5 ± 0.32	5 ± 0.00
Aspergillus fumigatus	5 ± 0.12	5.5 ± 0.35	4 ± 0.75	2 ± 0.35	5 ± 0.9
	5.5 ± 0.27	5 ± 0.14	4 ± 0.23	2 ± 0.65	3.5 ± 0.5

All tests were performed in triplicate, repeated three times twice (means ± SD). Statistically, the differences were significant at p ≤ 0.01 or at least at p ≤ 0.05 (in few cases), using Duncan’s test.

C₁: 2, 5-Dihydroxy-4-methoxy-phenanthrene 2-O-b-Dglucopyranoside.

C₂: 4-Methoxy-2,5,7,9S-tetrahydroxy-9,10-dihydrophenanthrene.

C₃: trans-Ethyl cinnamate.

AA

AA is a complex process usually occurring through several mechanisms. Due to its complexity, the evaluation of the AA for pure compounds or extracts should be carried out by more than one test method.^[28] The lower IC₅₀ value indicates a stronger ability of the extract to act as a DPPH scavenger while the higher IC₅₀ value indicates a lower scavenging activity of the scavengers as more scavengers were required to achieve 50% scavenging reaction. The results presented in Table 4 revealed that compounds C₁-C₃ and thymol exhibited a remarkable activity. In particular, C₃ exhibited clearly a higher activity (11.27 ± 0.68 µg/mL) followed by C₂ (12.01 ± 0.37 µg/mL), C₁ (12.04 ± 0.35 µg/mL), and T. copticum EO (12.04 ± 0.34 µg/mL) (Table 4).The positive controls BHT and ascorbic acid exhibited IC50 values equal to 12.78 ± 0.68 µg/mL and 13.98 ± 0.35 µg/mL, respectively. Table 4 depicts the inhibition of β-carotene bleaching by the new compounds (C₁-C₃) and thymol. The IC₅₀ value was 12.02 ± 0.68, 12.00 ± 0.17, and 11.00 ± 0.08 µg/mL, respectively. As shown in Table 4, the reducing power of new compounds (C₁-C₃), expressed as CE50, was clearly more significant than that of the positive BHA and AA. Because of high antioxidant and free radical-scavenging activities of new compounds (C₁-C₃), further investigation was carried out to identify its active constituents. Therefore, a preliminary screening was initially carried out using the dot-blot DPPH stainingmethod on TLC. As the new compounds (C₁-C₃) and thymol presented a significant AA in the assays and bioautography test, it was subjected to the TLC for isolation of the active compounds. Components identified and their AA relative percentages have been shown in Table 5. The major compound found in the active band was c3 (34.14%; Table 5). Many aroma components of EOs, such as terpenes and terpenoids, were proposed to contribute to the AA of EOs; including thymol and eugenol, linalool and 1,8-cineole, results obtained indicated that three isolated compounds was greater than thymol. According to these results, there is a relationship between total phenolic contents and AA.

Table 4. Antioxidant activity new compounds and thymol: scavenging activity (expressed as IC₅₀ values: µg/mL), and β-carotene bleaching test. Reducing power was expressed as EC50 values (µg/mL). Butylhydroxyanisole (BHA) and ascorbic acid were used as positive controls.

Tested compounds	IC50 (μg/mL)
T. copticum EO	12.04 ± 0.34 µg/mL
c1	12.04 ± 0.35 µg/mL
c2	12.01 ± 0.37 µg/mL
c3	11.27 ± 0.68 µg/mL
thymol	12.11 ± 0.60 µg/mL
T. copticum EO (β-Carotenes IC50 µg/mL)	12.47 ± 0.28 µg/mL
c1	12.02 ± 0.68 µg/mL
c2	12.00 ± 0.17 µg/mL
c3	11.00 ± 0.08 µg/mL
T. copticum EO (PR EC50 µg/mL)	12.21 ± 0.08 µg/mL
c1	12.03 ± 0.68 µg/mL
c2	12.00 ± 0.07 µg/mL
c3	11.07 ± 0.68 µg/mL
BHA	12.78 ± 0.68 µg/mL
AA	13.98 ± 0.35 µg/mL

Values are mean ± SD of three replications.

*IC₅₀ values have been presented with their respective 95% confidence limits.

Table 5. Components identified and their antioxidant activity relative percentages.

Compounds	%
c1	20.17
c2	22.14
c3	24.14
Thymol	15.11
Other components	18.44

Results are expressed as a percentage of antioxidant activity relative. Experiments were carried out in triplicate.

Lipid peroxidation inhibition

According to the results obtained, new compounds (C₁-C₃) and thymol significantly inhibited the formation of TBARS in brain homogenates in a concentration-dependent manner (Table 6). The suppressive power on the lipid peroxidation of new compounds (C1-C3) were found to be the most potent (C3: 83.51 ± 0.74,C2: 79.35 ± 0.78 and C1: 78.68 ± 0.68 µg/mL), followed by thymol (79.45 ± 0.35 µg/mL). Ascorbic acid and Trolox showed significant suppressive power on lipid peroxidation in mice brain homogenate with IC₅₀ value of 77.14 ± 0.65 and 70.25 ± 0.65 µg/mL (Table 6). Phenolic compounds exist in most plant tissues as secondary metabolites, i.e., they are not essential for growth, development or reproduction but may play roles as antioxidants and in interactions between the plant and its biological environment. Phenolics are also important components of the human diet due to their potential AA, their capacity to diminish oxidative stress induced tissue damage resulted from chronic diseases and their potentially important properties such as anticancer activities.^[29] These results indicated that Ca. copticum EO and its main component displayed significant AA in inhibiting peroxidation of rat brain homogenate in vitro.

Table 6. Lipid peroxidation inhibition of new components thymol (expressed as IC₅₀ values: µg/mL). Trolox and ascorbic acid (AA) were used as positive controls.

Tested compounds	IC50 (μg/mL)
T. copticum EO	79.35± 0.14 µg/mL
C1	78.68± 0.68 µg/mL
C2	79.35± 0.78 µg/mL
C3	83.51± 0.74 µg/mL
Thymol	79.45± 0.35 µg/mL
Trolox	70.25± 0.65 µg/mL
AA	77.14± 0.65 µg/mL

Experiments were carried out in triplicate and the results are expressed as mean ± SD.

Conclusion

EOs showed variations in their chemical components and physicochemical profiles. The present study is the first report describing the antimicrobial and AA of T. copticum EO. In addition, a high correlation between the AA and the total phenolic content was found. Considering that T. copticum showed a medium yield in EO, which confers an industrial potential interest, and its volatile oil has a pleasant smell and is safe in bioactive concentrations, the results presented here emphasize its potential use as an antioxidant and anti-inflammatory for the food, cosmetic and nutraceutic industries. Also, the high content of thymol makes this species an important natural source of this compound. Our data indicate that the new compounds (C1-C3) extracted from T. copticum exhibits potent biological activities, which support their use in traditional medicine. There was a good correlation between new compounds (C1-C3) and antimicrobial and antioxidant capacity of the extracts. In conclusion, T. copticum extracts appear to contain new compounds (C1-C3) with antimicrobial and antioxidant activities.