Chemical composition, antioxidant, antibacterial and cytotoxic effects of Artemisia marschalliana Sprengel extract

Abstract The present study was to investigate the gas chromatography/mass spectrometry (GC/MS), in vitro antioxidant, antibacterial and anticancer activity of the ethanolic extract from aerial parts of Artemisia marschalliana Sprengel against human gastric carcinoma (AGS) and L929 cell lines. Phytochemical analysis of A. marschalliana Sprengel extract showed 22 major components and the most dominant compounds were trans-phytol (29.22%), α-Linolenic acid (13.47%) and n-Hexadecanoic acid (9.28%). In addition, the antioxidant and anticancer activity of A. marschalliana Sprengel extract were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods, respectively. Antibacterial activity against selected pathogenic bacteria was also determined. According to the present obtained results, it seems that this plant has potential uses for pharmaceutical industries and further studies of pharmaceutical importance were suggested to be performed on A. marschalliana Sprengel.


Introduction
Some plants have been used as popular folk medicines in different countries and their extracts are the main source of drugs (Giang et al. 2014). The genus Artemisia belongs to Asteraceae family which comprises over 500 species in the world and about 34 species in Iran (Sefidkon et al. 2002). Based on literature, this genus is rich in monoterpenoids, sesquiterpenoids, coumarins and flavonoids, and some Artemisia species have been reported to have antimalarial, anti-hepatotoxic, antibacterial, antifungal and antioxidant activity (Ornano et al. 2015). Artemisia annua is one of the most important plants that its derivatives, such as Artemisinin is a group of compounds used to treat malaria caused by Plasmodium falciparum (Abolaji et al. 2014). In addition, Artemisia diffusa is used in the traditional medicine as antimalarial agent which contains sesquiterpene lactones including Tehranolide (Rustaiyan et al. 2009). Artemisia marschalliana Sprengel is an Iranian evergreen or semi-evergreen sub-shrub of the genus Artemisia and it is grown in east Azerbaijan province, Arasbaran, Iran (emami et al. 2012). Previous studies have been carried out on the chemical composition, antioxidant and cytotoxicity effects of different species of Artemisia extracts (Sampietro et al. 2015;Ornano et al. 2016). However, majority of the species have not yet undergone comprehensive chemical and cytotoxicity studies to investigate their bioactive compounds. This study was conducted to study the phytocomponents of A. marschalliana Sprengel using GC-MS analysis, antioxidant, and biological studies.

Results and discussion
GC/MS analysis of the A. marschalliana extract ( Figure S1) revealed 22 peaks indicating the presence of phytochemical constituents. Based on the comparison of the mass spectra of the constituents with the NIST library, the 22 phytocompounds were identified (Table S1). The different phytochemicals with medicinal activities in A. marschalliana Sprengel are shown in Table S2. The mass spectra of all the phytochemicals identified in the whole plant ethanolic extract of A. marschalliana Sprengel are as shown in Figure S1. Of the 22 constituents identified, the most dominant compounds were trans-phytol (29.22%), α-Linolenic acid (13.47%), n-Hexadecanoic acid (9.28%), but Falcarinol, an alcoholic compound does not have any activity. To date, most studies have been conducted on GC/MS analysis of other Artemisia spp and this is the first study conducted on GC/MS analysis of A. marschalliana Sprengel extract.

MTT assay
MTT assay was performed to study the in vitro cytotoxic property of A. marschalliana Sprengel extract ( Figure S2). Both the AGS and L929 cells were treated with a concentration of 0.2-60 mg/ml of the A. marschalliana Sprengel extract for 24 h to determine the inhibitory percentage against cancer-treated and untreated cell lines. Our results showed that the cancer and normal cell viability was affected with A. marschalliana Sprengel extract. The relevant IC 50 values of the AGS cells and L929 cells were 36.10 mg/ml and 77.34 mg/ml for Artemisia quttensis extract, respectively. Figure S2 reveals that the cytotoxicity of A. marschalliana Sprengel extract was dose dependent and the highest cytotoxicity on AGS and L929 cell lines was observed at 60 mg/mL and it was significant as compared to the control (P < 0.001). In addition, the MTT assay results showed that 0.2 and 2 mg/mL concentrations had low cytotoxicity and it is not significant as compared to the control. In addition, the effect of extract on the growth of L929 cells did not exhibit significantly at the 0.2-20 mg/mL concentrations.

Antibacterial activity
The antibacterial effectiveness of the A. marschalliana Sprengel extract was measured by determining the MIC and MBC values. The results of this study indicated that the highest antibacterial action was observed against Pseudomonas aeroginosa (Table S3).

DPPH radical scavenging
The ethanolic extract of A. marschalliana Sprengel showed antioxidant activity in the DPPH with SC50 375 ± 0.08 μg/mL (Table S4). This value was found to be less than ascorbic acid as standard. In this study, the antioxidant properties of A. marschalliana Sprengel extract were exhibited and it is concluded that further studies were performed for A. marschalliana Sprengel pharmaceutical importance. Temraz et al. studied the antioxidant activity of Artemisia vulgaris extract and their plant extract exhibited scavenging potential with IC 50 value of 11.4 μg/ml for DPPH, the values were found to be close to those of the standard (10 μg/ml) (Temraz & el-Tantawy 2008).

Conclusion
As a result of the current study, the ethanolic extract of A. marschalliana Sprengel was observed to be an effective antioxidant and anticancer agent and can be proposed as a natural additive in food and pharmaceutical industries. Moreover, the in vivo works need to be investigated in mouse models prior to its possible pharmaceutical application, such as antitumour drug design.