1.   Introduction

Aromatic and medicinal plants have been of great interest, as they have been the sources of natural products, commonly named as bioactive compounds with antioxidant activity and other beneficial properties to human health [1]. Specifically, the natural compounds from the Lamiaceae family (thyme, sage and rosemary) have been reported in several studies for their antioxidant, anti-inflammatory, antimicrobial, antidiabetic, antiaging and anti-carcinogenic activities [2-5]. Among the sources of these bioactive molecules, the Thymus genus is particularly noted for its long history of use for different food, pharmaceutical and medicinal purposes, which could be attributed to its polyphenolic compounds. It is also beneficial in treating respiratory and gastrointestinal tract disorders [6-10]. This genus, belonging to the Lamiaceae family, includes 350 species widespread around the world and their phytochemical composition has been studied earlier [11-13].

In Tunisian flora, the Thymus genus is mainly represented by Thymbra capitata L. Cav. (syn. Thymus capitatus (L.) Hoffman and Link) which has been commonly used as a spicy herb and is locally known under the common name “zaâtar” [14]. Tunisian Thymus capitatus (L.) Cav. is known to be an effective potential source of natural antioxidants, which confer benefits to human health [9, 15]. Their bioactive properties are mainly due to their polyphenolic compounds. The aerial parts of Thymus capitatus (L.) Cav. plants have a long history of use in food and traditional medicine. Because of its flavoring and seasoning properties, this plant has been widely used in the preparation of many foods and for the treatment of different kinds of disorders [16].

To the best of our knowledge, no previous research has investigated the effect of solvents on the variation of the phenolic content and antioxidant activity of Thyme aerial parts extracts. In this context, this research aims to determine the impact of different solvents (water, ethanol, and methanol) on the total polyphenolic and flavonoid contents and in vitro antioxidant activity of Thymus capitatus (L.) Cav, in order to promote this plant as a potential source of bioactive molecules with beneficial effects for human health.

2.   Materials and methods

2.1. Plant material and preparation of thyme extracts

The plant materials consisted of the aerial parts of thyme were acquired from a local supermarket (Gafsa, Tunisia). The dried aerial parts of the plant were crushed into a fine powder using an electric milling machine. Dried sample (1g) was macerated in 10 mL of distilled water, hydroethanolic (70%), and hydromethanolic (70%) solvents for 24 hours at room temperature [5]. The obtained thyme extracts were filtered using Whatman filter paper and dried in a forced-air dryer at 37 °C. The residue was redissolved in the same solvent and made up to 5 mL. The yield of the extracts was expressed in terms of milligrams of dry extract per gram of dry weight (mg DE/g DW). The final extract was kept in vials at 4°C until the corresponding analyses were conducted.

2.2. Estimation of total polyphenol content                  

The total phenolic content (TPC) in the thyme extracts was determined by the Folin-Ciocalteu reagent method [17] with slight modification. 20 μL of the different thyme extracts were added to 1155 μL of distilled water and 100 μL of Folin-Ciocalteu reagent (10%). A vigorous stirring was performed and 225 μL of sodium carbonate (10%) was added. After 30 min of incubation at 25 °C, the absorbance of the resulting blue-colored solution was measured at 765 nm. A standard curve was prepared by using different concentrations ranging from 0.1 to 1 mg/mL of gallic acid. The TPC was expressed as milligrams of gallic acid equivalents per gram of dry extract (mg GAE/g DE). All experiments were performed in triplicate.

2.3. Estimation of total flavonoid content

The total flavonoid content (TFC) was measured using an aluminum chloride colorimetric assay [18]. Briefly, 100 μL of the different thyme extracts were mixed with 900 μL of distilled water in a test tube. After 3 min, 500 μL of AlCl₃ (2%) was added and allowed to stand for another 15 min. The absorbance was measured against the blank at 510 nm. Quercetin was used to prepare a standard calibration curve with different concentrations ranging from 0.1 to 1 mg/mL of quercetin. The results were expressed as mg of quercetin equivalent per gram of dry extract (mg QE/g DE). All measurements were performed in triplicate.

2.4. Radical scavenging activity by DPPH

The scavenging activity of different thyme extracts was measured according to the method described by Brand-Williams et al. [19]. 500 μL of thyme extracts, at different concentrations (10, 20, 30, 40, 50 μL from the solution that has been diluted), were added to 1000 μL of DPPH^• solution (0.1 mM) and kept in the dark at room temperature for 30 min. The absorbance was measured at 517 nm against a control (500 μL of solvent and 1000 μL of DPPH solution). All the assays were conducted in triplicate. The radical scavenging activity (RSA) was calculated as a percentage of DPPH discoloration, using the following equation:

%I = [(Abs_control - Abs_sample ) / Abs_control] x 100

The results were expressed as the inhibitory concentration of the extract needed to decrease DPPH• absorbance by 50% (IC₅₀). Concentrations are expressed in micrograms of dry extract per milliliter of solvent (IC₅₀, μg/mL).

2.5. Statistical analysis

All experiments were performed in triplicate (n = 3) and data were reported as means ± standard deviation (SD). A General Linear Model procedure was carried out to assess for significant differences (significant model was accepted for a p-value < 0.05) using the Variance analysis (ANOVA)

3.   Results and discussion

3.1. Extraction yield

Thyme extract yields varied depending on the type of solvent used (Fig. 1). The highest yield was obtained from hydroethanolic extract with a value of 9.4 mg DE/ g DW, followed by hydromethanolic extracts (8.2 mg DE/ g DW) and aqueous extract (7.42 mg DE/g DW). Extract yields were considerably impacted by the extraction solvents. In fact, the variation in extraction yield is due to the polar ability of solvents to dissolve extractable components, resulting in varied chemical compositions of plants [10, 20]. Indeed, the combined use of water and organic solvent improved the extraction of phenolic compounds. Variations in thyme extract yields based on the plant's geographical origin, extraction methods and type of solvent have been documented in the scientific literature [9, 21, 22].

Figure 1. Thyme aerial parts extract yields (mg DE/g DW).

(TAE: Thyme aqueous extract, TEE: Thyme hydroethanolic extract, TME: Thyme hydromethanolic extract, mg DE/ g DW: milligrams of dry extract per grams of dry weight).

3.2. Total phenolic and total flavonoid contents

The total phenolic content (TPC) of the aqueous, hydroethanolic and hydromethanolic extracts of thyme reached the values ​​of 30.20, 32.14 and 30.31 mg gallic acid equivalent/g dry extract (mg GAE/g DE), respectively. On the other hand, the total flavonoid content ranged from 4.18 to 16.20 mg quercetin equivalent/gram dry extract (mg QE/g DE) (Table 1). These values demonstrate that Thymbra capitata (L.) Cav. is rich in polyphenolic compounds. When comparing our results with previous studies, notable differences emerged. In fact, the total phenolic content reported by Msaada et al. [9] in methanolic extract of Tunisian thyme (18.40 mg GAE/g DE) was lower than our values. Phenolic compounds are well-known bioactive agents that have been extensively reviewed and have attracted considerable attention owing to their benefits to human health and in curing and averting numerous illnesses [23, 24]. In this context, several studies prove the richness of thyme in total polyphenolic content and variation of its amount depending on the solvent used for extraction [12, 25].

Table 1. Total phenolic and flavonoid contents of thyme aqueous, ethanolic, and methanolic extracts. 

In addition, the amount of TPC and TFC depends on the part, species of plant, method, solvent extraction, region of collection. Several studies showed that solvents significantly affect the yield of thyme crude extract and bioactive compound solubility and proved that methanol was the most efficient solvent to recover thyme polyphenol, followed by ethanol [15, 22, 24, 26, 27].  Similarly, Tamma et al. [28] proved the richness of thyme in flavonoid. They have been reported to be effective antioxidants, antibacterial, anticancer, cardioprotective, anti-inflammation, immune system promoting agents, skin protectors, and are therefore outstanding candidates for pharmaceutical and medical purposes [13, 29].

3.3. Antiradical scavenging activity

The antiradical activity of aqueous, ethanolic, and methanolic extracts of thyme, assessed through the DPPH assay, is presented in Fig. 2. All extracts of thyme have strong antioxidant activity. The hydromethanolic extract is the richest in antioxidants with IC₅₀ value of 15.44 µg/mL followed by the hydroethanolic extract with a value of 18.43 µg/mL followed by the aqueous extract (15.44 µg/mL). These results show that plants with high antioxidant capacity are characterized by high levels of total polyphenolic content [5, 15, 26, 30]. In fact, polyphenolic extracts of different parts of various plant sources reveal significant antioxidant activity, playing an essential role in mitigating oxidative stress-related diseases. Due to their potent bioactive molecules, these extracts are increasingly utilized as natural preservatives and functional ingredients in food products [26, 31]. The results obtained in different tests of antioxidant activity confirmed that the phenolic constituents are responsible for the antioxidant activity of thyme.

Figure 2. DPPH free radical scavenging capacity (IC₅₀ value) of Thyme aerial parts extracts.

(Values are expressed as means ± standard deviations (SD) of triplicate experiments. Means with different letters within the same column are significantly different at 5% using the Duncun test (P < 0.05). TAE: Thyme aqueous extract, TEE: Thyme hydroethanolic extract and TME: Thyme hydromethanolic extract).

4.   Conclusions

The present study has investigated the effect of solvents on extract yield, total phenolic content, total flavonoid content and antioxidant activity of Tunisian Thymbra capitata (L.) Cav. aerial parts. The thyme aqueous, hydroethanolic and hydromethanolic extracts showed a high amount of total phenolic and flavonoid contents.  The evaluation of the antioxidant activity of different solvents of thyme extract showed a strong antioxidant activity. This highlight confirmed that thyme aerial parts have the potential effect as natural antioxidants due to their significant antioxidant activity and seem to be useful in pharmaceutical, cosmetics, and food industries with beneficial properties for human health. Therefore, supplementing a balanced diet with herbs may have beneficial health effects. Further, in vitro and in vivo investigations are needed to fully understand its biological properties.

Authors’ contributions

Conceptualization, I.T., K.H.; Methodology, I.T., K.H, A.A., M.BZ.; Formal analyses, I.T., K.H., F.Z., A.F., R.A., A.A.; Investigation, I.T., K.H., A.A.; Resources, K.H.; Writing-original draft preparation, I.T., K.H.; Writing-review and editing, Supervision, K.H., S.S.E.

Acknowledgements

The authors thank the Faculty of Sciences of Gafsa for doing the work on their laboratories. This work was supported by the Tunisian Ministry of Higher Education and Scientific Research.

Funding

This research received no external funding.

Availability of data and materials

All data will be made available on request according to the journal policy.

Conflicts of interest

The authors declare that they have no financial and commercial conflicts of interest.

References