1. Introduction

In today's crowded world, one of the smartest ways to stay healthy and fight disease is to use the potential of medicinal plants [1]. This group of low-cost plants can replace the use of a large number of drugs, fungicides and other chemical compounds [2]. One of the valuable medicinal plants is Satureja sahendica. This plant is from the Lamiaceae family and is the exclusive owner of Iran [3]. The antioxidant properties of foods are crucial in nutrition. Over the last decade, extractable and non-extractable compounds have become key in the evaluation/determination of the antioxidant properties of food matrices because of their relevance in human health [4]. Compounds such as phenol and flavonoids are valuable phytochemicals with antioxidant properties that can protect the human body against many diseases [5-10], for more information please see [11]. The purpose of this research is to investigate the antioxidant and phytochemical activity of S. sahendica extract as an endemic plant in Iran.

 

2. Materials and methods

2.1 Plant materials

Miandoab is one of the cities of the West Azerbaijan province in Iran, which has a classically semi-arid climate at the altitude of 1352 m above the sea level, Latitude: 38°190´ N; and Longitude: 45°180´ E. This region (Miandoab) is one of the habitats of S. sahendica in Iran. The aerial parts of S. sahendica plants were collected from Miandoab city during the flowering stage in 2019 and the plant sample was dried in the form of dry shade at room temperature (25 °C). The voucher herbarium sample of the collected S. sahendica species has been deposited at herbarium of the Urmia University of Medical Sciences.

2.2. Preparation of methanol extract

The powdered aerial parts (2 g) were extracted with MeOH (80%, v/v) by ultrasound (Elmasonic, Singen, Germany) process at 120 Hz power at 25 ºС for 30 min. The recovered extract after centrifugation at 3000 x g for 5 min (centrifuge ROTANTA 380/380R, Hettich, Germany) was kept at 4 °C in dark glass, until analysis.

2.3. Antioxidant activity

2.3.1. Antioxidant activity by DPPH assay

The electron donation ability of the obtained methanolic extract was measured by bleaching of the purple-coloured solution of DPPH according to the literature [12]. Extracts (25, 50, and 75 µL) were added to 2 mL of 0.1 M DPPH methanolic solution. After an incubation period of 30 min at room temperature, the absorbance was determined against a blank at 517 nm. Percentage inhibition of free radical DPPH (PI%) was calculated as follows:

 

2.3.2. Antioxidant activity by FRAP assay

To measure the antioxidant activity by the FRAP method, the samples along with 3 mL of FRAP fresh reagent [2.5 mL of a 10 mM TPTZ solution in 40 mM HCl, 2.5 mL of 20 mM FeCl₃.6H₂O and 25 mL of 300 mM acetate buffer (pH 3.6)] were mixed together. The resulting mixture was put in a warm water bath (37 ˚C) and its absorption was read on the 593 nm wavelength by using spectrophotometer (Unico 2100UV Single Beam UV/Vis, Shanghai, China). The FeSO₄·7H₂O was used to draw the standard curve. Moreover, the obtained results were quantified based on mM Fe⁺⁺/100g DW plant [13].

2.4. Phytochemical compounds

2.4.1. Total phenol content (TPC)

Phenolic content was measured using Folin–Ciocalteu reagent. An aliquot of 100 µL of extract was added to 180 µL of deionised water and 1200 µL of the F–C reagent. After shaking, the mixture was incubated for 5 min at room temperature and then (7.5%) sodium carbonate was added. The samples were kept in the dark within the room temperature for 30-45 minutes. Finally, absorption was measured at 760 nm wavelength by spectrophotometer [14]. The deionized water was used as the control while gallic acid was used as standard. The standard curve was drawn according to gallic acid, and the results were reported in mg of gallic acid equivalents per 100 g of dry weight.

2.4.2. Total flavonoid content (TFC)

To assay the total flavonoid content, 150 μL of 5% sodium nitrite, 300 µL of aluminum chloride (10 %) and 1000 µL of acetate solutions (of 1 mM NaOH) were added to a specific amount of each extract. The volume obtained was adjusted to 5mL using distilled water. The absorbance of the solution was measured at 380 nm against a blank. Additionally, to draw the standard curve, quercetin was used [15]. The amount of total flavonoids in all the extracts was reported as mg of quercetin equivalents per 100 g of dry weight.

2.4.3. Total carotenoids and chlorophylls A and B

In order to measure the content of carotenoids and chlorophylls, 0.05 g of the fresh plant tissues were homogenized with 5 mL acetones in ice bath. Then, 1 g of anhydrous sodium sulfate was added to the homogenate and filtered using the filter paper. The volume of the filtered was adjusted to 10 mL with acetone and then it was centrifuged at 4000 rounds for 10 minutes. The absorption of the upper phase solution was measured at the 662, 645, 470 nm wavelengths using acetone as control. The amount of carotenoid and chlorophylls A and B for each extract was measured as reported in the literature [16].

 

C_a = 11.75 A₆₆₂ – 2.350 A₆₄₅                            (1)

C_b = 18.61 A₆₄₅ – 3.960 A₆₆₂                            (2)

C_x+c = 1000 A₄₇₀ – 2.270 C_a – 81.4 C_b/227      (3)

 

3. Results and discussion

3.1 Antioxidant activity

Health-related uses of medicinal and aromatic plants have long been known. The compounds derived from these plants have antifungal, antimicrobial and antioxidant effects [17]. Reactive oxygen species (ROS) are highly reactive chemical molecules formed due to the electron acceptability of O₂. Examples of ROS include peroxides, superoxide, hydroxyl radical, singlet oxygen and alpha-oxygen [18]. Antioxidants are compounds that inhibit oxidation. Oxidation is a chemical reaction that can produce ROS, thereby leading to chain reactions that may damage the cells of organisms [19]. Antioxidants have two sources: synthetic and natural. Medicinal plants, including S. sahendica, are valuable sources of natural antioxidants [20]. The results of the antioxidant activity of S. sahendica extract by DPPH method showed that it was raising the concentration of the extract and time increases the amount of antioxidant activity. So that, the highest antioxidant activity was observed in concentration of 75 μL at 30 minutes (84.29 %) (Fig. 1A). The study of the simple effect of time on the amount of antioxidant activity showed that the increase in the amount of antioxidant activity between different treatments (time 15 and 30 mines) was significant. The highest antioxidant activity was observed at 30 minutes (64.87 %) (Fig. 1B). Furthermore, the study of the simple effect of concentration on the level of antioxidant activity showed that there was a statistically significant difference between different treatments (concentrations 25, 50 and 75 μL). The highest antioxidant activity was observed in concentration of 75 μL (81.93 %) (Fig. 1C). For DPPH test, the lowest IC₅₀ value was observed for time 15 min (30.37 μL) and the highest IC₅₀ value (39.49 μL) was recorded for the time 30 min (Fig. 1A). In addition, the results of measuring the antioxidant activity by FRAP method showed a value of 148.36±1.17 (mm Fe⁺⁺/100g DW) in S. sahendica extract (Table 1). The Ferric Reducing Antioxidant Power, well-known as FRAP assay and (DPPH) are valuable method for evaluation of total antioxidant power of the herbal extracts [21]. The antioxidant properties of medicinal plants may be due to the presence of phenolic compounds or other polar compounds such as terpenoid-glycosides in their aqueous extract, which are traditionally used as a decoction or infusion [22, 23].

Figure 1. Antioxidant activity of different concentration of methanol extraction during studied time. (A) Interaction, (B) Main effect (Time) and (C) Main effect (concentration).

3.2. Phytochemical content

3.2.1. Phenol and flavonoid content

A review of articles shows that medicinal plants, especially the large family Lamiaceae, have been used to treat patients since ancient times because they contain active medicinal and biologically active compounds [24]. Polyphenols and flavonoids are the common antioxidant natural products found in medicinal plants [21]. Previous research shows that both internal and external factors of genotype and environment along with plant growth stage and extraction method play an important role in the amount of phenolic compounds in plants [25]. Based on the measurements, the amount of phenol and flavonoids in the whole samples was equal to 95.85±0.50 (mg GAE/100g DW) and 19.33±0.30 (mg QUE/100g DW) respectively (Table 1). Extracts from some species of the Lamiaceae family may be recommended for the treatment of complications of diabetes due to their polyphenolic compounds [26]. It has been reported that phenolic compounds have antimicrobial and antioxidant properties, helping plants prevent infections from pathogens and pathogenic microorganisms [27]. Additionally, their presence in plant tissues protects them against the toxic effects of reactive oxygen species [28].

  Table 1. Biochemical composition and antioxidant activities of S. sahendica extract

Characteristics	Content
Antioxidant activity (FRAP)	148.36±1.17 (mm Fe++/100g DW)
Total Phenolic Content	95.85±0.50 (mg GAE/100g DW)
Total Flavonoid Content	19.33±0.30 (mg QUE/100g DW)
Total Carotenoid Content	1161.90±13.60 (mg/100g DW)
Chlorophyll A	7.80±0.02 (mg/100g DW)
Chlorophyll B	2.20±0.06 (mg/100g DW)

3.2.2. Plant pigments

Plant pigments protect plants from damage caused by UV and visible light [29]. Photosynthetic pigments (chlorophyll and carotenoids) play an important role in photosynthesis, chlorophyll is the most important pigment needed for photosynthesis and resistance to environmental stresses, chlorophylls are complexed with proteins that serve to absorb light and transfer energy to the photochemical center [30]. Carotenoids can absorb sunlight and are also essential as a pigment for photosynthetic function [31, 32]. Carotenoids have good effects on human health. They are precursors of vitamin A in the body and their antioxidant, anti-obesity and anti-cancer properties have been proven, they also have anabolic effects on bone components. Nowadays, carotenoids are used as food additives, animal feed supplements, and natural food coloring and dietary supplements. More recently, they have been used commercially as nutrients for cosmetic and medicinal purposes [33]. S. sahendica is a valuable source of plant pigments which S. sahendica plant extract contained 1161.90 ± 13.60 (mg/100 g DW) carotenoid pigment, 7.80±0.02 (mg/100g DW) chlorophyll A and 2.20 ± 0.06 (mg/100g DW) chlorophyll B pigment (Table 1).

 

4. Conclusions

The present study showed that the S. sahendica plant has a high TPC (95.85±0.50 mg GAE/100g DW) and TFC (19.33±0.30 mg QUE/100g DW). This has caused a very strong antioxidant activity (DPPH: 84.29% and FRAP: 148.36±1.17 mm Fe⁺⁺/100g DW). Overall, the results of the present study showed that S. sahendica due to its phytochemical compounds can be a good candidate for natural antioxidants and a good alternative to synthetic antioxidants. Also, the various pigments in this plant are a good source for use in the food and pharmaceutical industries. However, additional studies are needed to find out the other potentials of this plant.

 

Authors’ contributions

Data curation, formal analysis, validation, writing – original draft preparation and funding acquisition, G.G.; Methodology, project administration, software, writing – review and editing, S.R.

 

Acknowledgements

This research was supported by the Urmia University, Urmia, Iran.

 

Funding

Funding has been provided by Urmia University.

 

 

Availability of data and materials

All relevant data are within the paper and its supporting information files. Additional data will be made available on request according to the journal policy.

 

Conflicts of interest

Authors declare that there is no conflict of interest.

 

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