The genus Satureja contains medicinal plants that belong to the family Lamiaceae. Medicinal plants of this genus contain about 0.5% of essential oil [1-2]. Studies show that there are differences in essential oils between species and subspecies of this plant genus . During recent years, antiviral , anti-nociceptive and anti-inflammatory , anti-bacterial and anti-fungal [6-8], anti-spasmodic and remedy of diarrhea  and vasodilatory  effects have been reported for species of Satureja.
Medicinal plants of the genus Satureja usually grow in the northern and western regions of Iran. Two important species of Satureja are used as culinary plants. Important ingredients of Satureja essential oil include carvacrol and thymol, p-cymene, β-caryophyllene, linalool and other terpenoids .
Satureja rechingeri is one of the exclusive species of savory in Iran that grows in dry, sunny and calcareous rocky soils of southwestern Iran in Lorestan, Ilam and Khuzestan provinces. Due to the limited distribution of this plant species "Rechingeri savory" in natural habitats and the expansion of demand for plant raw materials, it is necessary to create high quality and homogeneous cultivars for cultivation in the agricultural system and supply plant raw materials required by the pharmaceutical industry. This study aimed to investigate phytochemical compounds of methanolic extract and essential oil of safflower and to investigate and determine its antioxidant effect.
Material and methods
Green plant material was collected from around Khorramabad area, Lorestan, Iran, and identified at the herbarium of the Lorestan University (Figure 1) (Voucher specimen (57587)). Fresh green plant parts were dried in the shade. The dried plant was pulverized by a milling machine.
Isolation of essential oil
The essential oil of the plant was extracted by the Clevenger apparatus using the Hydrodistillation method for 6 hours. The extracted essential oil was stored in the dark at 4 ° C.
Figure1: Satureja rechingeri plant
Preparation of extracts
10 grams of dried powder from plant was extracted by refluxing in 50 ml of %95 methanol three times for 72 hours, filtered off, and concentrated using a rotary evaporator under reduced pressure at 40°C. The crude extract was placed in a dark bottle and stored at -20°C prior to further use.
GC-MS analysis of essential oil
Gas chromatography was performed with a Shimadzu model GC-17A (Kyoto, Japan) instrument equipped with a Shimadzu Quadropole-MS (QMS) model QP5050 detector. The present phytochemical study was performed based on the methods of Adams (2007), Baher et al. (2002), Jennings and Shibamoto (1998) and Adams (2001) [13-16]. Component relative concentrations were calculated based on GC peak areas without using correction factors.
Helium, 99.999%, used as carrier gas, was purchased from Roham Gas Company (Tehran, Iran). The alkane mixture consisting of the C8-C26 alkanes (concentration of 40 mg/mL in hexane) was purchased from Fluka. All other chemicals were of the highest purity available from Merck or Fluka. Doubly distilled deionized water was used.
Determination of total phenols and flavonoids in extracts
Total phenolic content was developed by Chang et al. (2001) based on the Folin-Ciocalteu method .
DPPH radical scavenging assay
The radical scavenging activity of the extracts was performed with a few changes in the method of Abe et al. (1998) and Bozin et al. (2006) [19, 20].
Result and Disscusion
GC-MS analysis of essential oil
Identification of essential oil compounds of Satureja rechingeri leaves
Hydro distillation of S. rechingeri leaves produced greenish yellow essential oil. 25 compounds were identified in the essential oil of S. rechingeri, which accounted for 100% of the total essential oil. Chemical composition of the essential oil was determined by gas chromatography and mass spectrometry (Table 1). Among these compounds, the main compounds were carvacrol (83.90%), gamma-terpinene (4.57%) and paracymene (2.92%). The results of this table also show that mono terpenes occupy more than 99% of the volume of S. rechingeri essential oil, and among these, cavacrol as a phenolic mono-ring monoterpene with nearly 84% is the most significant composition of this essential oil. With the exception of mono terpenes, sesquiterpenes such as farnesene as a linear sesquiterpene and bisabolone as a single-ring sesquiterpene form a small fraction (less than one percent) of S. rechingeri essential oil.
As for essential oil compounds of flower S. rechingeri, according to Table 2, 25 compounds were identified in the essential oil of flower S. rechingeri, which constituted 100% of the total essential oil. Among these compounds, the main compounds were carvacrol (83.11%), gamma-terpinene (4.66%) and paracymene (3.1%). The results of this table show that most of the essential oil of this plant is composed of carvacrol.
As for the constituents of the essential oil of the stem of S. rechingeri, according to Table 3, 18 compounds were identified in the essential oil of safflower stalk, which constituted 100% of the total essential oil. Among these compounds, the main compounds were carvacrol (95.70%), gamma-terpinene (0.66%) and paracymene (0.74%). The results of Table 3 show that most of the essential oil of this plant is composed of carvacrol and the percentage of carvacrol essential oil of stem is higher than that of leaves and flowers.
According to the results of Tables 1, 2, 3, the most important constituents of essential oils of different organs were carvacrol, gamma terpinene and parasymen . The essential oil yield of S. rechingeri aerial parts collected from the habitat at the beginning of flowering by water distillation was reported to be 4.72%. At the full flowering stage, the essential oil yield of the same sample in different distillation methods was reported between 2.46% and 4.24%. The highest essential oil yield was obtained by water distillation and the lowest yield was obtained by steam distillation .
At the beginning of flowering, 43 compounds in the essential oil were identified as carvacrol (56.1%) and para-cement (14%) as major components. Also, in the full flowering stage, 23 compounds were identified in the essential oil, of which carvacrol (84.3% -89.3%) was the most important essential oil compound. Research has shown that the essential oil of this species has a strong antimicrobial effect [23, 24].
Total phenols and flavonoids
Phenolic compounds have been reported to exhibit various biological activities like antioxidant, antimicrobial, and etc. Total phenolic compounds in extracts were determined by Folin-Ciocalteu method and expressed as Gallic acid equivalents (GAEs). Many studies have revealed that the phenolic contents in the plants are associated with their antioxidant activities probably due to their redox properties, which allow them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers .
Folin-Ciocaltue was used as a reagent to measure phenolic compounds. In this method, gallic acid was used as a standard and methanolic extract of different organs was prepared at concentrations of 2000 μg/ml. It was then prepared and diluted in 100 μl of extracts with 1500 μl of Folin-Ciocaltue 0.1% and with 1000 μl Mix distilled water; after 1 minute, 1500 μl of 20% sodium carbonate solution was added, the samples were transferred to a dark place at room temperature for two hours and then absorbed at 760 nm. The microplate reader was read.
To measure flavonoid compounds, methanolic solutions with a concentration of 2000 μg / ml were prepared, then 500 μl of the extract was mixed with 1500 μl of methanol, 100 μl of 10% aluminum chloride, 100 μl of 1 M potassium acetate and 2800 μl of distilled water. The samples were kept at room temperature for 40 minutes and their absorbance was read at 420 nm using a microplate reader. Quercetin was used as the standard.
The results of comparing the mean values of phenolic compounds showed that the leaves of S. rechingeri with 223.71 μg of gallic acid per mg of extract had the highest phenol content and the stem with 158.95 μg of gallic acid per mg of extract containing the lowest number of phenolic compounds. Therefore, the phenolic content of different organs of savory sage is in the form of stem
The results of comparing the mean values of flavonoid compounds showed that the flower of Satureja rechingeri with 76.08 μg quercetin per mg of extract had the highest phenol content and the stem with 30.48 μg quercetin per mg of extract contained the lowest number of compounds, e.g. phenolic. Therefore, the phenolic content of different organs of S. rechingeri is in the form of stem
Evaluation of antioxidant activity of the studied samples by beta-carotene linoleic acid
Unsaturated fatty acids such as linoleic acid are very sensitive to the oxidation process; therefore, inhibition of oxidation of this substance is used as a valuable method in determining antioxidant activity. In this method, antioxidant activity is measured by inhibiting the oxidation of linoleic acid and preventing the formation of volatile compounds and conjugated hydroperoxides. First, stock solution was prepared from beta-carotene linoleic acid solution, by dissolving 0.5 mg of beta-carotene in 1 ml of chloroform, then 25 μl of linoleic acid and 200 mg of tween forty were added. Next, the chloroform was completely evaporated and then 100 ml of oxygen-saturated distilled water was added with stirring. Methanolic extracts of different organs were prepared with methanolic solutions at concentrations of 500 μg / ml. To evaluate the antioxidant properties, 50 μl was added to 2500 μl of beta-carotene linoleic acid solution. The absorbance of the samples was read at zero moment and also after two hours of incubation at 50 ° C using the microplate reader at 470 nm; the amount of antioxidant activity of the extracts was calculated.
DPPH radical scavenging activity
DPPH assay has been extensively used for screening plant extracts because many samples can be accommodated in short period and are sensitive enough to detect active ingredients at low concentrations .
In this method, DPPH was used as a reagent and the activity of hydrogen atoms and electrons of the extracts was measured by discoloration of DPPH solid purple solution. From the dried extracts of different organs, concentrated solutions (0.01, 0.008, 0.006, 0.004 and 0.002) mg / ml and 0.004% solution of DPPH were prepared, then for each sample. Plant organs were tested three times with 50 μl of the extract and 1 ml of DPPH solution, then the samples were read at room temperature at 517 nm. In this method, BHT was used as a positive control.
The results of antioxidant activity in this method showed that stem essential oil with 90.25% had higher antioxidant power than leaves and flowers (78.63% and 77.44%, respectively), but the antioxidant power of BHT was slightly lower. In this study, the antioxidant activity of flowers was higher than that of stems and the order of antioxidant power of essential oils of different organs was in the form of leaves
Figure 4: Free radical scavenging of leaves, flowers and stems metanolic extracts of S. rechingeri compared with BHT by DPPH assays
Beta-carotene linoleic acid method
The results of comparing the means showed that in this method the leaf essential oil of S. rechingeri had significant antioxidant power (87.12%) so its antioxidant power was higher than BHT (83.75%). The antioxidant power of the stem was also greater than that of the leaf (stem
Figure 5: Antioxidant activity of leaves, flowers and stems metanolic extracts of S. rechingeri compared with BHT by β-carotene-linoleic acid assays
Antioxidant activity of methanolic extract
The results of antioxidant activity in this method showed that methanolic extract of S. rechingeri leaf had higher antioxidant power than stem and flower but its antioxidant power was less than BHT. The antioxidant power of the flower was higher than the stem, so according to Figure 6, the antioxidant power of different organs of the plant S. rechingeri and the BHT standard was in the form of stem
Beta-carotene linoleic acid method
The results of comparing the means showed that in this method, methanolic extract of flower had a very high antioxidant power, so its antioxidant activity was higher than BHT. According to Figure 7-9, the order of antioxidant power of different organs of Satureja rechingeri plant and BHT standard is in the form of leaf
Figure 6: Phenolic content of different organs methanolic extracts of S. rechingeri
Figure 7: Flavonoid content of different organs methanolic extracts of S. rechingeri
Figure 8: Free radical scavenging of leaves, flowers and stems essential oils of Satureja rechingeri compared with BHT by DPPH assays
Studies have shown that medicinal plants, due to their phytochemical, active ingredients and medicinal and antioxidant compounds, have beneficial effects on human health and have a therapeutic effect on various organs of the body and various diseases [27-37].
Nature is a rich source of phytochemicals compounds, some of which are found in medicinal plants. Phytochemical compounds are found in various organs of medicinal plants and have biological and medicinal activities. Phytochemicals are biologically active compounds that are rich in phenols, flavonoids, tannins, anthocyanin, minerals and antioxidants and can be used for medicinal and health purposes [38-40].
Figure 9: Antioxidant activity of leaves, flowers and stems essential oils of S. rechingeri compared with BHT by β-carotene-linoleic acid assays
Extract of S. rechingeri possesses remarkable antioxidant activity compared with its essential oil. Thus, the extract of Satureja rechingeri can prove beneficial in food and pharmaceutical industry.
This study was conducted as part of an M.Sc. thesis. The author would like to thank Khorramabad Branch of Islamic Azad University, Iran.
This research did not receive any specific grant from Khorramabad Branch, Islamic Azad University, Iran.
All authors contributed toward data analysis, drafting and revising the paper and agreed to be responsible for all the aspects of this work.
Conflict of Interest
We have no conflicts of interest to disclose.
HOW TO CITE THIS ARTICLE
Malieh Noormohammadi, Shabnam Shamaei. Phytochemical Composition and the Evaluation of Antioxidant Activity of Methanolic Extract and Essential Oil Of Satureja Rechingeri Extract and Essential Oil, J. Med. Chem. Sci., 2022, 5(1) 65-75