Silylation of thymol extracted from Thymus migricus essential oil,improvement of lipophilicity properties,and investigation of its pharmacological properties

2.1. Characterization

Fourier transform infrared spectroscopy (FT-IR) analysis were recorded by a Bruker Vector-22 FT-IR spectrometer. Nuclear magnetic resonance (¹ H NMR) spectra of the compound was obtained in CDCl₃ using a FT-NMR Bruker (400 MHz) spectrometer. Mass spectroscopy (Agilent Technologies 5973 Network Mass Selective Detector, TOF LC/MS, UK), ion source, Electron Impact (EI) 70 eV and at the temperature of 230°C and Quadrupole analyzer at the temperature of 230°C was used to determine the molecular weight of the compounds. HPLC: A Jasco HPLC system, consisted of a PU-1580 isocratic pump, a Rheodyne 7725i injector with a 10–μL loop (Rheodyne, Cotati, CA, USA) and a UV-1575 spectrophotometric detector was used in the experiment. UV-Vis: Double-beam spectrophotometer (UV-Vis T80) company made in England PG1100-190 wavelength range with deuterium lamp background correction equipped with a tungsten lamp as a radiation source. Commercial TLC aluminum sheets were used (0.25-mm silica gel with fluorescent indicator, Alugram SIL G/UV254, Macherey- Nagel).

2.2 Materials

Chlorotri-ethyl silane, tert-butyl dimethyl silyl chloride, chloro vinyl dimethyl silane, sodium hydroxide, hydrochloric acid and tri-ethyl amine (Et₃ N) were purchased from Merck Co. DPPH (2, 2-diphenyl-1 picrylhydrazyl, free radicals), ferric iron, TPTZ (2, 3, 5-triphenyl-1,3,4- triaza-2-azoniacyclopenta-1,4-diene chloride) and methylen-blue agar (EMB) were purchased from Sigma- Aldrich Co. Acetonitrile, pentane, n-hexane, ethyl acetate, tetrahydrofuran (THF), methanol and all other solvents were obtained from Merck Co.

2.3 Collection and preparation of thyme plant

In August 2021, samples of Thymus migricus Klokov & Desj. -Shos were gathered in Arasbaran, Azarbaijan, Iran during the blossoming season. Thyme was allowed to air dry for 10 days at room temperature in a shaded area. The mechanical mill was then used to completely grind the plant sample. After the plant material had dried, it was used for the extraction of the essential oil.

2.4 EO extraction

Powdered aerial organs from the previous stage of the plant sample, including leaves and flowers, were obtained by hydrodistillation with the clevenger apparatus. For this purpose, 100 g of herbal sample powder and 400 ml of distilled water were placed in a round balloon, and the clevenger was installed on the balloon. After about 3–4 hours of distillation (until the amount of essential oil stayed constant), the essential oil was separated. The essential oils were derived before analysis by water-free sodium sulfate. After being transferred to the microtube, it was kept in the refrigerator.

2.5 EO high-performance liquid chromatography (HPLC) analysis

A volumetric flask containing 10 mg of essential oil was filled with the mixture of acetonitrile: water (80 : 20) (100 g/ml). Three injections were given to each of the six prepared samples. The experiment was conducted using a Jasco HPLC system, which included a PU-1580 isocratic pump, a Rheodyne 7725i injector with a 10-L loop (Rheodyne, Cotati, CA, USA), and a UV-1575 spectrophotometric detector. Using the LC-Net II/ADC interface, Jasco’s HSS-2000 was used to control the chromatographic system. The BORWIN programme was used to process the data (version 1.50). A Perfect Sil Target ODS-3 analytical column (MZ-Analyse ntechnik, Germany) with an ODS-3 pre column (104.0 mm I.D., 5-m), both of which were kept at room temperature, was used for separation. Acetonitrile and water, in a 50:50 v/v ratio, were the components of the isocratic mobile phase, which moved through the column at a constant flow rate of 1 ml/ min. UV detection at a wavelength of 274 nm was used to keep an eye on the effluent. A membrane-type GV filter with a 0.22 m pore size was used to filter the mobile phase (Millipore). The mobile phase was degassed using a 40 kHz, 138 W, temperature-controlled ultrasonic water bath (sonic bath type LBS2 - FALC instruments S.r.l. TREVIGLIO, Italy). A standard acetonitrile-water solution with a thymol content of 500 mg/l was made and kept at 4 °C.

2.6 Thymol isolation

1 g of the EO was dissolved in 5 ml of pentane to obtain a fraction of the thymol phenolic component, and the mixture was then extracted with a 20% sodium hydroxide in water solution. The pentane layer’s phenolic components were removed in this way after being treated to this procedure. The hydrochloric acid solution that 10% diluted in water was employed to neutralize the aqueous phase, which contained dissolved phenolic compounds sodium salts. In the end, the thymol was separated out using an extraction procedure that used pentane (5 ml). The efficiency of this separation procedure was verified using thin-layer chromatography (TLC) on silica gel plates (mobile phase: n-hexane: ethyl acetate 85:15 v/v), and the validation of the phenolic compound fraction’s high level of purity was obtained as a consequence.

2.7 Synthesis of silyl derivatives of thymol

Triethyl silyl thymol (S2), tert-butyl dimethyl silyl thymol (S3), and vinyl dimethyl silyl thymol (S4) were made in separate experiments. A solution of 2 ml (13 mmol) thymol (S1) was stirred for 24 hours at room temperature separately with 1.84 ml triethylchlorosilane, 1.96 ml tert-butyldimethylchlorosilane, and 1.79 ml vinyldimethylchlorosilane in 1.81 ml Triethylamine base and 30 ml dry THF solvent under Argon (Ar) atmosphere. The progress of the reaction was monitored by TLC. Thin layer chromatography with n-hexane and ethyl acetate solvent systems separated the products after 24 hours. The pure yellow liquid product was obtained by drying the product under vacuum.

2.8 Pharmacological properties of thymol and silyl derivatives

2.8.1 Antioxidant activity

Many and common methods for testing the antioxidant activity of samples have been presented, each of which provides information for the antioxidant activity and power of the samples based on their desired parameters, and in this study, the antioxidant activity of thymol and its silylated derivatives was investigated using the DPPH and FRAP methods.

2.8.1.1. 2, 2’-Diphenyl-1-picrylhydrazyl (DPPH) assay. The bleaching of a purple DPPH solution in methanol served as a gauge for the hydrogen atoms or electron donating capacities of the related samples. This test was conducted using the same methodology that has been described elsewhere (19, 20). To a methanol solution of DPPH (2, 2-diphenyl-1 picrylhydrazyl, free radicals), various volumes of samples dissolved in methanol were added until the final volume was 1 ml. After an incubation time of 30 minutes at room temperature, the absorbance was measured at 517 nm in comparison to a blank. The absorbance of the control reaction, which contained all reagents except the test compound, and the absorbance of the test compound are used to calculate the inhibition free radical DPPH, expressed as a percentage (I%): I%  = (A control - A sample/A control) 100. The samples were prepared in various concentrations and subjected to a DPPH assay. These samples included thymol (S1), triethyl silyl thymol (S2), tert-butyl dimethyl silyl thymol (S3), and vinyl dimethyl silyl thymol (S4). (Table 1). Microsoft Excel was utilized for the computation of these values.

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Table 1

Different concentrations of samples (v/v) stored at 4–6 °C

Volume of sample (μl)	Volume of DPPH solution (μl)	Concentration (V/V)
40	960	% 4
60	940	% 6
100	900	% 10

2.8.1.2. Ferric Reducing/Antioxidant Power (FRAP) assay. By using this technique, antioxidants’ capacity to scavenge ferric iron is measured. Its foundation is the reduction of the 2, 3, 5-triphenyl-1,3, 4-triaza-2-azoniacyclopenta-1,4-diene chloride (TPTZ) and ferric iron complex to the ferrous form at low pH. Using a diode-array spectrophotometer, the change in absorption at 593 nm is measured to track this reduction. The FRAP values were computed using the ferrous sulphate standard curve (Fig. 1). The antioxidant activity of thymol along with three synthesized silylated derivatives was investigated using FRAP method.

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Fig. 1

Ferrous sulphate standard curve.

2.8.2 Antibacterial activity

Thymol is a pure natural compound extracted from the Thymus vulgaris, as well as tert-Butyl dimethyl silyl thymol and tri-ethyl silyl thymol, which were both synthesised in the lab. Numerous gram-positive and gram-negative bacteria, including Proteus, Klebsiella, Escherichia coli, and Staphylococcus aureus, were cultured separately in Eosin Methylen-Blue Agar medium. Assays for antibacterial activity were performed using the disc diffusion method. The middle of the inoculated plates was placed with four discs (6 mm in diameter), each of which had been individually impregnated with S1, S2, S3 and S4 (Fig. 2). Inhibition zones were measured in diameter (mm) all the way around the discs after the bacterial cultures were incubated at 37°C for 48 hours.

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Fig. 2

Bacterial culture and loaded disks.

3.1 Isolation procedure

The extraction of essential oil from the thyme plant was done several times with a clevenger, and after collecting the essential oil for dehydrating and obtaining pure essential oil, sodium sulfate was used for dehydrating it in such a way that the addition of sodium sulfate to the essential oil was done slowly until the addition of sodium Sulfate should be made into a powder form of essential oil and should not be lumped. The pure essential oil obtained was stored at 4°C.

Then the natural composition of thymol was extracted from the essential oil, the extracted thymol was identified by HPLC, and its silyl derivatives were synthesized. Silyl ethers are a group of chemical compounds which contain a silicon atom covalently bonded to an alkoxy group. The general structure is R₁R₂R₃Si–O–R₄ where R₄ is an alkyl group or an aryl group. Silyl ethers are usually used as protecting groups for alcohols in organic synthesis. One of the main features of the silicon-containing material is a dramatic increase in their lipophilic properties. Thymol is a crucial phytochemical, as was already discussed in the previous section, but it also happens to be one of the compounds with hydrophilic properties. Some qualities are anticipated to be improved by the compounds’ increased lipophilicity, which is provided by the addition of silicon-containing groups. The qualities and effects of the medicine are multiplied by increasing its lipophilicity since it passes through the lipid barrier more quickly. Chemical hydrolysis affects the cleavage of the silicon-drug bond and is less prone to interpatient variability than the metabolic mechanism needed to activate conventional prodrugs [43–45]. Pyridyl-substituted 5-Si-soxazolines have been studied for their neurotropic and antitumor properties in other works, and it has been found that these compounds are more effective than their analogues at protecting against hypoxia [46]. Additionally, silatranes are tris(2-hydroxy-alkyl) amine esters of intracomplex organosilicon that have demonstrated a wide range of biological activity [47, 48]. In addition to these compounds other organosilicion compounds were synthesised and their biological activities were studied [49, 50]. Because of this, we synthesise some silyl ethers of thymol (scheme 1).

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Scheme 1

Synthesis reaction of silylated thymol derivatives.

The thymol, natural component in the EO of Thymus vulgaris was validated by HPLC analysis. In this analysis, at a wavelength of 277 nm the absorption peak intensity 22860 inhibitions time at 20.47 reveals that exactly fits the peak of the standard material (Fig. 3a). HPLC analysis was also performed on silylated thymol derivatives. In the results of this analysis, it can be seen that the inhibition time of silylated derivatives is significantly increased compared to thymol, considering that the HPLC column is of the C18 type and non-polar, so substances with a non-polar nature interact more effectively with the column substrate and have a high inhibition time. Based on the results of this analysis, the inhibition time of silylated derivatives is longer than that of thymol, which is due to the increase in lipophilicity of thymol after its silylation. This increase in non-polarity has increased the inhibition time, and this is a confirmation of the correct synthesis of silylated compounds and increase in their property is lipophilic (Fig. 3b).

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Fig. 3

HPLC analysis of (a) extracted thymol and thymol standards and (b) thymol and silyl derivatives of thymol.

In order to determine the mass-to-charge ratio (m/z) of one or more molecules found in a sample, mass spectrometry is a useful analytical tool. The precise molecular weight of the sample’s constituent parts can frequently be determined using these measures as well. Thymol’s natural compound (A) has a 150.28 molecular mass. The fragment of the molecule [A-CH₃] that was observed at m/z = 135 and the fragment of the molecule [A-isopropyl group] that was observed at m/z = 107 are connected. The fragment molecule Phenyl is related to the fragments of the molecule’s benzyl carbocation (m/z = 91), phenyl cation (m/z = 77) and cyclopentadienylium (m/z = 65). (Fig. 4).

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Fig. 4

Thymol mass spectrometry analysis.

Thymol’s broad peak in the 3392 cm^- 1 region of the FT-IR is connected to the stretching vibrations of O-H phenol. The aromatic C = C stretching vibrations are related to the aromatic C-H stretching vibration peak at 3020 cm^- 1, the asymmetric stretching vibrations of aliphatic C-H at 2927 cm^- 1, the symmetric stretching vibrations of aliphatic C-H at 2869 cm^- 1, and the as harp peak at 1620 cm^- 1 (Fig. 5a). The peak around 3040 cm^- 1 in the FT-IR spectrum of thymol silyl derivatives corresponds to aromatic C-H stretching vibrations; the peak at 2932 cm^- 1 corresponds to aliphatic C-H asymmetric stretching vibrations; and the peak at 2885 cm^- 1 corresponds to aliphatic C-H symmetric stretching vibrations. The peak related to Si-C stretching vibrations is at 1270 cm^- 1; the peak related to Si-C bending vibrations is observed at 819 cm^- 1; the sharp peak at 1612 cm^- 1 is related to aromatic C = C stretching vibrations; and the peak at 1056 cm^- 1 is related to Si-O stretching vibrations. Finally, the absence of the peak related to thymol O-H in the region of 3392 cm^- 1 indicates the synthesis of the desired compounds. Due to the very close similarity of the FT-IR spectra of the derivatives, only one spectrum is given (Fig. 5b).

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Fig. 5

FT-IR of a) thymol, b) thymol silyl derivatives.

Binary peak in the 1.3 ppm range of the ¹HNMR spectrum of thymol is connected to the 6 protons of the methyl group with isopropyl bound to the aromatic ring. 3 protons from the methyl group linked to the aromatic ring were responsible for a single branch peak in the 2.3 ppm range. Peak at 2.9 ppm is associated with the isopropyl group’s single proton, which is divided into 7 branches by the methyl group’s 6 protons. A single proton of the hydroxyl group attached to the aromatic ring causes a broad peak with single branches at 5.8 ppm, which is related. Single branches peak at 6.7 ppm in relation to the aromatic ring’s attached proton. One proton attached to the aromatic ring that undergoes a binary split with an adjacent proton causes the peak in the 6.8 ppm spectrum. Peak at 7.1 ppm is caused by a binary split of an aromatic ring proton by an adjacent proton (Fig. 6a).

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Fig. 6

¹HNMR of a) thymol, b) triethylsilyl thymol, c) tertiobutyl dimethylsilyl thymol, d) vinyl dimethylsilyl thymol.

In the ¹HNMR spectrum of triethylsilyl thymol, the peak at 0.8 ppm corresponds to 6 methylene protons attached to silicon, which are split into 4 by three adjacent methyl protons, and the peak at 1.1 ppm corresponds to 9 methyl protons, which are the side methylene groups are split into 3. The peak in the region of 1.3 ppm corresponds to 6 protons of methyl isopropyl groups attached to the aromatic ring, which is split into 2 by a single proton of isopropyl. The peak of the single branch at 2.25 ppm corresponds to the 3 protons of the methyl group, which is located on the aromatic ring. The 2.9 ppm peak corresponds to an isopropyl group proton, which is split into 7 by 6 methyl protons of the isopropyl group. The single peak in the region of 6.73 ppm, 6.8 ppm, and 7.1 ppm corresponds to the protons of the aromatic ring (Fig. 6b).

In the ¹HNMR spectrum of tert-butyldimethylsilyl thymol, a single peak in the 0.22 ppm region corresponds to 6 protons of two methyl groups attached to silicon. The single branch peak in the 1 ppm area corresponds to 9 protons of the tert-butyl group attached to silicon. The double peak in the 1.25 ppm area corresponds to 6 protons of the methyl isopropyl group attached to the aromatic ring. The peak of the single branch in the region of 2.2 ppm corresponds to 3 protons of the methyl group attached to the aromatic ring. The multiple peaks in the region of 2.8 ppm corresponds to single proton of the isopropyl group. The single peak in the area of 6.62 ppm, 6.72 ppm and 7.03 ppm correspond to the aromatic ring protons (Fig. 6c).

In the ¹HNMR spectrum of vinyl dimethylsilyl thymol, the peak in the region of 0.5 ppm corresponds to 6 protons of two methyl groups attached to silicon. The double peak in the region of 1.35 ppm corresponds to the 6 protons of the methyl isopropyl group attached to the aromatic ring. The peak in the region of 2.3 ppm corresponds to 3 protons of the methyl group attached to the aromatic ring. The peak in the region of 2.9 ppm corresponds to a proton of the isopropyl group, which is split into a heptad by 6 methyl protons of the same group. The peak in the area of 6 ppm, 6.2 ppm, and 6.4 ppm corresponds to protons of the vinyl group. The peaks at 6.85 ppm, 6.9 ppm, and 7.15 ppm are related to aromatic ring protons (Fig. 6d).

One of the most potent plant essential oil constituents with antimicrobial effects is thymol, which belongs to the monoterpene phenols class. These positional isomers of isopropyl cresol, which have a broad range of bioactive properties, reduce ergosterol content, impair membrane permeability, block efflux pumps, and restore fluconazole susceptibility to antifungal treatment in resistant Candida strains. Exposure to these natural compounds induces a cascade of stress responses, which are important to comprehend their microbicidal mechanisms. The antioxidant properties of thymol have been well documented in various preclinical studies including cell lines and animal models. At high-rate constants, it effectively scavenged the hydroxyl free radicals thereby producing major transient species named phenoxyl radicals. The generated adducts from the phenoxyl radicals undergo dehydration which can be accelerated by an alkaline medium. The addition of hydroxyl radicals at the ortho position (C6 atom) of the phenolic group yields the phenoxyl radical after dehydration. The attack at the ortho position is more favourable energetically while the attack at the para position is also expected to occur. Furthermore, additions at the ortho positions occur without any precomplex formation. The non-toxicity and redox potential of the thymol /thymol couple makes it a promising antioxidant (Venu et al., 2013).

In this study, the DPPH method and the FRAP method were used to assess the antioxidant activity of thymol and its derivatives.

3.6.1 DPPH assay

Each sample was generated at various concentrations (Table 1), and the antioxidant activity of the samples was assessed using the DPPH technique. Results show that thymol has greater free radical scavenging capacity than silyl derivatives (Fig. 7). The hydroxyl group of thymol has been substituted with silyl groups, which is the only structural difference between thymol and silyl derivatives. These results demonstrated that the substitution of a hydrophobic group for a hydroxyl group significantly reduced the antioxidant activity of thymol. The findings amply supported the theory that thymol’s antioxidant properties are primarily dependent on the hydroxyl functional group, which is in this case protected by a silyl group.

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Fig. 7

Antioxidant activity results from the DPPH method.

3.6.2 FRAP assay

The antioxidant activity of the samples was measured using the FRAP method. The results of the FRAP assay also revealed that thymol has stronger antioxidant properties than its derivatives and that the hydroxyl functional group is largely responsible for this (Fig. 8). Results from the two techniques, DPPH and FRAP, demonstrated that thymol had greater antioxidant activity than silyl derivatives. We can also infer that the hydroxyl functional group is responsible for thymol’s antioxidant properties.

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Fig. 8

Antioxidant activity results from the FRAP method.