Phytochemical composition and biological activities of Artemisia quettensis Podlech ethanolic extract

Abstract The present study aim to investigate the phytochemical composition, antibacterial, antioxidant and anticancer activities of the ethanolic extract from aerial parts of Artemisia quettensis Podlech. The aerial part of A. quettensis Podlech extract was used for Gas chromatography–mass spectrometry (GC-MS) analysis, antioxidant, antibacterial and anticancer activities. GC/MS analysis of extract from this plant showed 23 major components and the most dominant components were acetic acid, [4-(1-hydroxy-1-methylethyl) cyclohex-1-enyl] methyl ester (13.88%), trans-Phytol (10.06%) and 2,6-Dimethyl-2,6-octadiene-1,8-diol diacetate (6.8%). The extract had significant antibacterial and anticancer effects. The highest percentage of antioxidant activity was 78.46% at 2 mg/mL concentration of extract. Moreover, the highest antibacterial effects of extract were against to gram-positive bacteria and the IC50 cell cytotoxicity value on HT29 cell line in 24 h, 48 h and 72 h were 31.54, 6.08 and 2.96 mg/mL, respectively. From this study, A. quettensis Podlech could be considered as a promising source for novel drug compounds.


Introduction
Recently, traditional medicinal plants are considered as alternative medicines and most researchers have focused on their biological effects. So far, a variety of medicinal plants were studied for their biological properties (Sabourian et al. 2016). Among these, the Artemisia genus, with the common Persian name of Dermaneh, are playing an important role in human health. The Artemisia genus is the important member of the Asteraceae family that is mostly distributed in the northern temperate regions of the world, such as Asia, Europe, North America and South Africa (Tan et al. 1998;Kazemi et al. 2010). Moreover, this genus comprises more than 500 species in the world and about 34 species in Iran (Azizi et al. 2016). Traditionally, Artemisia is a promising natural source of phytochemicals with potent antipyretic, antiseptic antimalarial, antiviral, antitumour, antihemorrhagic, anticoagulant, antianginal, antioxidant, antihepatitis, antiulcerogenic, antispasmodic, anticomplementary properties (Bora & Sharma 2011). Based on the literature, this genus is rich in monoterpenoids (Gunawardena et al. 2002), sesquiterpenoids (Wen et al. 2010) and coumarins (Jung et al. 2012). However, the secondary metabolite are often affected by environmental conditions (Ghlissi et al. 2016). The Artemisinin is one of the bioactive compounds that has been successfully isolated from Artemisia genus (Weathers et al. 2011). Artemisinin has antibacterial, antifungal and anticancer effects (Abolaji et al. 2014). So far, numerous studies showed the extracts constituents using gas chromatography-mass spectrometry (GC-MS) from Artemisia species including A. marschalliana Sprengel (Salehi et al. 2016), A. alba Turra (Peron et al. 2016), A. caerulescens (Ornano et al. 2016) and A. proceriformis (Sampietro et al. 2016). Moreover, it has been reported that antibacterial compounds, which are widely distributed in most of the plants, could be very beneficial to human health (Ríos & Recio 2005). Several interesting studies using Artemisia spp. showed a series of antimicrobial activities (Sanna et al. 2015;Guetat et al. 2017;Rashid et al. 2017) and few secondary metabolites were successfully isolated and used in pharmaceutical industry as antibacterial agents (Efferth et al. 2011). Overall, the determination of potential antimicrobial activity of Artemisia extracts could be more informative for the future use in clinical treatment as natural antimicrobial agents.
Moreover, free radicals are chemically unstable atoms that destroy the lipid, proteins and DNA structures due to generation of reactive oxygen species (ROS). They are known to be the underlying cause of oxidative stress which is implicated in various diseases, such as cancer. These radicals can be scavenged by natural antioxidant agents that isolated from some medicinal plants (Ameziane-El-Hassani & Dupuy 2017). The use of natural antioxidant has gained much attention from consumers because they are considered safer than synthetic antioxidants (Wojcik et al. 2010). However, majority of medicinal plants have not been investigated for their antioxidant potency. In this study, Artemisia qeuttensis podlech, formerly referred to as Seirphidium qeuttense (Podlech) Y.R. Ling, was investigated for its chemical composition and biological activities. Previous studies have been carried out on chemical composition, antioxidant and cytotoxicity of several species of Artemisia extract (Hwang et al. 2016). However, more species are yet to undergo comprehensive chemical and cytotoxicity assay to justify the activity of their bioactive compounds. Based on our knowledge, to date, no literature has reported the gas chromatography/mass spectrometry (GC/MS) analysis and biological activities of A. quettensis Podlech extract. The present study aim to investigate the phytochemical composition, antioxidant, antibacterial and cytotoxic potential of the ethanolic extract from aerial parts of A. quettensis Podlech.

DPPH free radical scavenging assay
In this study, the effect of various concentrations of A. quettensis Podlech extract on DPPH radical scavenging activity is investigated. The DPPH results are presented in Table S3, whereas the IC 50 value of extract was 1.3 mg/mL. This value was found to be less than ascorbic acid as standard (11.6 μg/mL). One of the reasons for its antioxidant activity may be correlated to the bioactive chemical constituents in extract. As seen in Table S5, the highest percentage of antioxidant activity was 78.46% comparing to ascorbic acid (94.11%). Similar studies showed the antioxidant activity of Artemisia extract. Lee in 2014 have reported that the Artemisia japonica extract had 91.2% DPPH radical scavenging activity.

Evaluation of antibacterial activity
The antibacterial activity assay showed that the extract was most effective on S. aureus with higher inhibition zone (Table S4). The results of disc diffusion assay indicated that the antibacterial effect of extract was dose-dependent. Moreover, the MIC assay was perform to determine the lowest concentration that inhibits the bacterial growth. The highest and lowest MIC values were belonged to S. aureus and K. pnemoniea, respectively (Table S5). In conclusion, the results of disc diffusion and MIC methods revealed that extract most effective on gram-positive than gram-negative bacteria. Hence, we conclude that the antibacterial activity of A. quettensis Podlech extract could be associated with aromatic constituent, such as Terpineol, cis-β, 1,7-Octadien-3-ol, 2,6-dimethyl, trans-Phytol (Radulović et al. 2006) and phenolics, alkaloids, flavonoids, terpenoids and polyacetylenes compounds (Cowan 1999) (Table S2). Abdul et al. indicated that the methanolic leaf extracts of Artemisia nilagirica had a higher inhibition activity against Escherichia coli, Yersinia enterocolitica, Salmonella typhi, Enterobacter aerogenes, Proteus vulgaris, Pseudomonas aeruginosa, Bacillus subtilis and Shigella flaxneri (Abdul & Waheeta 2010).

MTT assay
The cytotoxicity of A. quettensis Podlech extract on HT29 cell line at various concentration after 24, 48 and 72 h were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. As indicated in Figure S2, after 24, 48 and 72 h incubation, the cytotoxicity effects of the extract was time-dependent and highest activity was recorded at 72 h as compared to control (p < 0.05) ( Figure S2). Moreover, the IC 50 cell cytotoxicity value on HT29 cell line in 24 h, 48 h and 72 h were 31.54, 6.08 and 2.96 mg/mL, respectively. More studies have shown the cytotoxic potential of different species of Artemisia extracts. Behbahani et al. showed the cytotoxic activity of five Artemisia species extracts (Behbahani et al. 2014). In conclusion, the cytotoxicity of the A. quettensis Podlech extract might be caused by aromatic and alcoholic compounds (Table S2), but, more studies are needed to confirm the accurate cytotoxicity mechanisms.

Conclusion
For the first time, the results of this study show that the ethanolic extract of A. quettensis Podlech possess antibacterial, antioxidant and antitumour activities on HT29 cell line. Based on the data obtained, this plant could be used for more pharmaceutical studies as good alternative in medicinal and therapeutic applications. However, further analytical studies are necessary to fully identify the chemical responsible for its biological activities.