Phytochemical screening, phenolic compounds determination, and antioxidant activity of Thymus satureioides from two regions of southern Morocco EL OUAHMANI Nadia1*, EL GHAZOUANI Fatima1, ALEM Chakib2, ZEKHNINI Abderrahmane1, YACOUBI Bouchra1 1 Laboratory of Aquatic Systems, Faculty of Sciences, Agadir, Morocco. 2 Laboratory of Biochemistry of Natural Products, FST, Errachidia, Morocco [email protected] INTRODUCTION Thymus saturieioides is an endemic plant of Morocco and Algeria, belonging to the family of lamiaceae. This plant is very well known in Morocco by its therapeutic virtues, hence its wide use in traditional medicine, it is also used as a condiment in culinary preparations. Several studies have shown that Thymus saturieioides is endowed with several biological activities, such as antibacterial, antifungal and antioxidant activity, due to the many secondary metabolites it contains, namely flavonoids and tannins etc... The objective of this study is to compare the phytochemical composition and the antioxidant activity of Thymus saturieioides from two regions of south east Morocco. The plant material used is collected from two areas at different altitudes 1800 m for Rheriss and 1300 m for Er-rich. Both localities belong to the province of Errachidia known by a dry continental climate. Extraction The hydroalcoholic extraction was carried out by ethanol at 80% according to the following process : References • Amri, O., Zekhnini, A., Bouhaimi, A., Tahrouch, S., Hatimi, A., 2018. Anti-inflammatory activity of methanolic extract from Pistacia Atlantica Desf. leaves. Pharmacogn. J. 10, 71–76. https://doi.org/10.5530/pj.2018.1.14 • DOHOU, N., YAMNI, K., TAHROUCH, S., IDRISSI HASSANI, L., BADOC, A., GMIRA, N., 2003. Screening phytochimique d’une endémique ibéro-marocaine, Thymelaea lythroides. Bull. Soc. Pharm. Bord. 142, 61–78. • Karumi, Y., 2004. 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Systematic • Kingdom : Plantae • Subdomain : Tracheobionta • Division : Magnoliophyta • Class : Magnoliopsida • Subclass : Asteridae • Order : Lamiales • Family : Lamiaceae • Genus : Thymus • Species : Thymus saturieioides Photochemical screening The main families of secondary metabolites were determined by the qualitative method. The free quinones were determined with NaOH (DOHOU et al., 2003). Sterols were revealed by the Liebermann Buchard test. For polyphenols, FeCl3 was used for their revelation, while saponin was determined by persistent foam method (Koffi et al., 2009). The revelation of tannins was done by FeCl3 (Karumi, 2004). Determination of total polyphenols The content of total polyphenols was determined with the method of (Singleton and Rossi, 1965), with modification (Amri et al., 2018), using folin-ciocalteu gallic acid was used as standard and the results are expressed as mg AGE/ g dry matter. Flavonoid assay Flavonoid determination was performed with aluminum chloride (Amri et al., 2018). A standard range was performed with rutin and the results are expressed as μg rutin equivalent/mg dry matter. DPPH free radical scavenging activity The DPPH radical scavenging assay was determined according to the method reported by Masuda et al. (1999). The percentage inhibition of the DPPH radical was calculated according to the formula : % ℎ = − 100 Ferric reducing antioxidant power FRAP The FRAP technique consists in evaluating the antioxidant capacity of a sample through the reduction of a ferric complex into another ferrous one, this technique was carried out according to the Benzie and Strain 1996 method. The results are expressed in μmol ascorbic acid equivalent antioxidant capacities (AAEAC)/mg DW. Statistical analysis All the tests performed in this study were done in triplicate. The results of the different parameters studied are expressed as mean ± SE. The significant difference in the phytochemical study was carried out by Student's test. Using IBM SPSS software (base version 26). RESULTS Secondary metabolites Thymus saturieioides Er-rich Thymus saturieioides Rheriss Tanins +++ +++ Saponines ++ ++ sterols ++ + Polyphenols ++ ++ Free quinones ++++ ++++ Table 1 shows the results of the qualitative phytochemical screening, and demonstrates the presence of polyphenols, tannins, saponins and free quinones at the same intensity in both varieties of Thymus saturieioides, except for sterols where the Er-rich variety is more intense. Description Thymus saturieioides is a shrub of 10 to 30 cm, very aromatic perennial, with woody stem, growing in rather dry stony grounds. Thymus saturieioides Er-rich Thymus saturieioides Rheriss Polyphenols mg AGE/ g DW 70.75± 1.92 a 73.02± 4.8 a Flavonoïds μg RE/mg DW 184.4 ±9.7 a 155.5± 3.12 b MATERIALS AND METHODS The results of the polyphenol assay did not show any significant difference between the Er-rich and Rheriss harvests. The results Thymus saturieioides Er-rich is more concentrated in flavonoid.(Table 2). Thymus saturieioides Er-rich Thymus saturieioides Rheriss IC 50 DPPH mg/ml 0.81± 0.064 a 0.57 ± 0.053 b FRAP μmol (AAEAC)/mg DW 39.69± 1.43 a 47.05± 4.47 b Both varieties tested showed antioxidant activity. According to the statistical tests the antioxidant activity is more intense in Thymus saturieioides Rheriss (Table3). The results show that Thymus saturieioides is a plant of the lamiaceae family, containing high concentrations of secondary metabolites which explains its biological effects, especially the antioxidant activity. Thymus saturieioides Rheriss showed a more intense antioxidant activity, this can be explained by the difference in altitude which is greater for the latter. Table 1 : phytochemical screening of to region Er-rich and Rheriss Thymus saturieioides Table 2 : Polyphenols and Flavonoïds concentration of Thymus saturieioides of Er-rich and Rheriss CONCLUSION Table 3 : antioxydant activity of Er-rich and Rheriss Thymus saturieioides