Introduction
Scutellaria genus, as a member of the Lamiaceae family, has been distributed worldwide with over 350 species [1]. Twenty species and two hybrids of this genus exist in Iran [2]. S. orientalis is a perennial herb and medicinal plant which grows in Tabriz City, Iran. It grows on soils with little clay, at the height of 1650 m above sea level, and an annual rainfall of 250 to 300 mm. All parts of this plant, including leaves, fruits, roots as well as seeds, are widely used in Iranian traditional medicine to treat constipation, wounds, stress, neurological disorders, dermatitis, bronchitis, and inflammation [3]. Pharmacological properties of the Scutellaria genus are due to the presence of 4′-deoxyflavones such as chrysin, baicalein, wogonin, and their glycosides (baicalin, wogonoside) [4]. 
Baicalein, wogonin, chrysin, pinocembrin, baicalin, skullcapflavon II, and wogonoside are the main flavonoids of S. orientalis species [5, 6]. S. orientalis essential oil contains different classes of terpenes, such as monoterpenes and sesquiterpenes, which among them, oxygenated monoterpenes (49.8%) constitutes the major part [7]. Acteoside, verbascoside, allysonoside, and martynoside are the main phenylethanoid glycosides determined from S. orientalis root and shoot [6]. Identifying natural plant compounds has advanced from product quality assurance to fundamental research. Regardless of the considerable progress in analytical techniques in the past few decades, identifying unknown compounds has remained a complex problem. Analytical methods such as Gas Chromatography (GC) and Gas Chromatography/Mass Spectrometry (GC/MS) are frequently used to recognize plant extract components. Measurement of mass fragmentation ions (m/z) of chemical compounds and comparison with available mass spectrum collections of known compounds based on similar fragmentation patterns is the usual approach to confirm compound identification. This study aimed to collect S. orientalis from its habitat, including limited populations in Tabriz, Iran, and identify and quantify its phytochemical profiles by GC/MS analysis.

Materials and Methods
Plant material collection
S. orientalis roots and shoots were collected from Marand City, Iran, during the summer (July 2018). The plant was identified by Dr Talebpour (plant taxonomy department, University of Tabriz, Tabriz, Iran). A voucher specimen was deposited in the herbarium of the East-Azerbaijan Agricultural and Natural Resources Research and Education Center, Tabriz, Iran (No. 2488).

Preparation of methanolic extracts
Roots and shoots of S. orientalis were used for phytochemical studies. Approximately 10 g of plant roots and shoots was extracted using 30 mL of methanol solvent. The extraction was carried out at room temperature by the percolation method for 48 hours. The extract was subsequently filtered. The methanol solvent was concentrated to 1 mL. About 1 μL of the methanolic extract was used for GC/MS analysis to identify phytochemical compounds.

Gas Chromatography/Mass Spectrometry (GC/MS)
    The qualitative assessment of methanolic extracts was carried out by GC/MS. GC analysis was conducted in a Hewlett-Packard (HP, Palo Alto, CA) HP 7890A gas chromatograph system. Helium (99.999%) was utilized as the carrier gas at the flow rate of 1 mL/min. The injection port temperature was set at 240°C; column temperature was firstly held at 40°C for 1 min, and then slowly increases to 240°C at the rate of 3°C/min.

Identification and quantification of the compounds
The chemical components of the methanolic extracts were determined by comparing their mass spectra with those of the GC/MS library (WILEY 7n D. 04.00 and NIST). The relative percentage of methanol extract components was quantified based on GC peak areas.

Fourier Transform Infrared (FTIR) spectrophotometer 
The S. orientalis root and shoot methanolic extracts were subjected to Fourier Transform Infrared (FTIR) analysis to identify functional groups. FTIR spectra were recorded in the infrared region of 400 to 4000 cm-1 using a Bruker FT-IR spectrophotometer as KBr (Potassium Bromide) disks [8].

Results and Discussion
The extraction and analysis of plant compounds are crucial for developing, improving, and quality control of herbal formalization. Therefore this study was directed to determine the bioactive compounds of methanolic extracts of S. orientalis by GC/MS analysis. The bioactive constituents, along with their percentage (%), are presented in Tables 1 and 2.


GC/MS chromatograms of S. orientalis methanolic root and shoot extract are shown in Figure 1.

S. orientalis methanolic root extract components
GC/MS chromatogram of S. orientalis methanolic root extract is shown in Figure 1a. Sixty-three components including flavonoids (15.3%), esters (12.5%), fatty acids (10.5%), glycoside (7.37%), ketones (5.9%), alcohols (3.44%), steroids (3.4%), acetates (2.25%), amides (0.46%), aldehydes (0.27%), ethers (0.24%), amino acids (0.15%), and alkaloids (0.08%) were determined in the methanolic root extract of S. orientalis (Table 1).
By comparative inspection, the main components in terms of their relative abundance were wogonin; propanoic acid,2-oxo-,methyl ester; n-hexadecanoic acid; 5-hydroxymethylfurfural; 2-propanone; 1-hydroxy-,ethyl iso-allocholate; 2-methoxy-4-vinylphenol; ethanethioic acid; and s-(dihydro-2,5-dioxo-3-furanyl) ester, corresponding to 18.1%, 12.6%, 4.75%, 4.2%, 3.55%, 3.44% 3.39%, 3.2% and 2.55%, respectively. A molecular docking and dynamics study by Malathi et al. revealed that ethyl iso-allocholate isolated from a medicinal rice variety, Karungkavuni, can serve as a potent inhibitor for dihydropteroate synthase activity in Escherichia coli [9]. Figure 2 shows the mass spectrum of some root-specific biologically active compounds determined in S. orientalis methanolic extract.

In the same context described above, the minor components were within a range of 0.05% to 2%. Specifically, 1-nitropyrrolidine, γ-chlorobutyrophenone, and 1,2-cyclohexanedione represented the components with the lowest amounts in methanolic extract of S. orientalis root. The obtained results revealed the presence of several bioactive components. 

S. orientalis shoot methanolic extract components
GC/MS chromatogram of S. orientalis methanolic shoot extract is shown in Figure 1b. GC/MS analysis of S. orientalis methanolic shoot extract identified 79 components. These components belonged to multiple chemical groups, including fatty acids (21.6%) with palmitic acid (5.38%) as the main acid, esters (9.16%) with 9,12,15-octadecatrienoic acid, 2,3-dihydroxypropyl ester, (Z,Z,Z)- (3.3%) as the main ester, ketones (6.7%) with methylglyoxal (2.13%) as the main ketone, aldehyde (3.9%), steroids (3.33%), alcohols (3%), glycosides (4.94%), acetates (1.12%), amides (2.4%), sesquiterpenes (1.98%), diterpenes (phytol; 1%), carotenoid (0.42%) and vitamins (0.2%) (Table 2).

The main components in terms of relative abundance were palmitic acid, dibenz(a,c)cyclohexane, 2,4,7-trimethoxy, ethyl iso-allocholate, 9,12,15- octadecatrienoic acid, 2,3-dihydroxypropyl ester, (Z,Z,Z)-, 9,12-octadecadienoic acid (Z,Z)-, 2-methoxy-4-vinylphenol and o-guaiacol, corresponding to 5.38%, 3.56%, 3.33%, 3.27%, 3.2%, 3.12% and 3.07%, respectively. 
Methylglyoxal was identified as the most influential antibacterial component of manuka honey, derived from the Manuka tree (Leptospermum scoparium) in New Zealand [9]. Methylglyoxal has antibacterial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. Hayashi et al. reported the effect of methylglyoxal against Multidrug-Resistant Pseudomonas aeruginosa (MDRP) using 53 clinically isolated strains [10]. They also assessed the impact of omitting the five multidrug efflux systems in P. aeruginosa, as well as the efflux systems in Escherichia coli and Salmonella enterica serovar Typhimurium, on minimum inhibitory concentrations of methylglyoxal. Their results revealed that methylglyoxal inhibits the growth of MDRP at concentrations of 128–512 μg/mL (1.7–7.1 mM) and was not recognized by drug efflux systems [10]. Figure 3 shows the mass spectrum of some shoot-specific biologically active compounds recognized in S. orientalis methanolic extract.

Conversely, the minor components among the entire components of S. orientalis shoot methanolic extract were those with a relative abundance range of 0.052%-2.28%, representing 1-nitropyrrolidine, γ-chlorobutyrophenone, and 1,2-cyclohexanedione as the lowest ones. GC-MS analysis of S. orientalis shoot extract identified several bioactive components.
The amount of volatile oil compounds, including sesquiterpenes, was comparatively less than other bioactive compounds (Table 2). However, Gharari et al. study on phytochemical screening of the methanolic root extract of Scutellaria orientalis subsp. bornmuelleri revealed the presence of several groups of terpenes, including sesquiterpenes and monoterpenes, which among them sesquiterpene hydrocarbons (12.66%) were more abundant [11]. Among different examined extracts of Scutellaria orientalis subsp. bornmuelleri, including n-hexane, CH2Cl2, EtOAc fractions, and aqueous and methanol extracts, the methanol extract had the most antiproliferative activity against HCT-116 and SW-480 cells at 48 h with IC50 values of 614.5 and 592.3 µg/mL, respectively [11]. Some of the identified compounds such as methylglyoxal (2.130%), phytol (1.008%), neocurdione (0.599%), spathulenol (0.48%), β-copaene (0.647%) and vanillin (1.322%) have strong pharmacological activities. Phytol is diterpene alcohol from chlorophyll. Phytol and its derivative, phytanic acid, exert various biological properties. It is an important essential oil used as a food additive and in medicinal fields as a potential candidate for an extended range of applications in the pharmaceutical industry. There is a wealth of evidence that phytanic acid has a critical function in developing pathophysiological states. Recent investigations with phytol indicated antinociceptive, antioxidant, anxiolytic, cytotoxic, metabolism-modulating, autophagy-inducing, apoptosis-inducing, immune-modulating, anti-inflammatory, and antimicrobial effects [12, 13]. PPARs- and NF-κB-mediated activities are responsible mechanisms for some of the bioactivities of phytol [14]. De Moraes et al.’s research on the application of phytol against neglected tropical disease schistosomiasis revealed its promising antischistosomal properties in vitro and a mouse model of Schistosomiasis mansoni [15]. Several studies demonstrated that spathulenol has antioxidant, anti-inflammatory, antiproliferative, and antimycobacterial activities [16]. Vanillin, another S. orientalis shoot methanolic extract compound, is a phenolic aldehyde with anti-carcinogenic [17], antimutagenic [18], and antioxidant [19] activities. It also has antifungal [20] and cytotoxic [21] properties.
One of the main differences between root and shoot content of S. orientalis methanolic extract was the presence of flavonoids (15.3%) and glycosides (monosaccharides and disaccharides; 7.37%) with high percentages in the root (Table 3).

Wogonin, the major bioactive flavonoid of the Scutellaria genus, was found in root methanolic extract with a high amount (12.6%). Wogonin is an effective anti-inflammatory, antiviral, anticancer, and antibacterial compound [22]. In addition, it has shown protective effects against Alzheimer disease [23].cardiovascular disease [24], and ethanol-induced gastric mucosal damage in the rat model [25].

FTIR spectra of the plant root and shoot
FTIR analysis of dry methanolic root and shoot extract of S. orientalis demonstrated the presence of normal polymeric O-H stretch, aliphatic alkenes, aromatic alkenes, amides, ammonium ions, alcohols, ethers, carboxylic acids, esters, and amines with major peaks at 3404, 2923, 2851, 1664, 1407, 1286, and 1046 (Tables 3, 4), respectively in the structure of compounds.

The FTIR spectrum of the S. orientalis roots and shoot extracts in the form of a KBr pallet is shown in Figure 4.

The medium-strong bands at 3416.01 and 3404.07 cm-1 in FTIR spectra belong to –NH and OH stretching in primary amines and amides [26]. The weak bands at 2924.25 and 2923.15 cm-1 attributed to the stretching vibration of –CH3 and –CH2 groups in alkanes, which indicates the presence of some alkane compounds, including chlorophyll groups [27] in medicinal plants [28]. The band at 2119 cm-1 was assigned to the cyanide -CN and alkyne groups [29]. The strong bands around 1664.05 and 1663.81 cm-1 are due to the amide, aldehyde, and ketone regions that are characteristic of proteins, enzymes, and sugars. The strong bands at 1616.37 and 1611.69 cm-1 showed alkene C=C stretch. The band at 1412.44 and 1407.88 cm-1 showed CH2 and CH3 bending vibrations.
The weak bands at 1289.81 and 1286.3 cm-1 represent the stretching vibrations of C-O due to the presence of alcohol and ether. Also, the C-N (aryl) stretching was assigned to peaks observed at 1289.81 and 1286.3 cm–1. The strong 1053.67 and 1046.43 cm–1 bands predict the presence of amines glycoside/C–OH bands in the polysaccharide/protein structures. The presence of Si-O- and aromatic CH bonds has been detected by means of the low-intensity band located at 920 cm-1 [30]. The IR spectra of methanolic root extract exhibited C-X (X=F, Cl, S) locations in the spectral region of 866.56-815 cm-1 [31]. The FTIR analysis of methanolic root and shoot extracts revealed the presence of functional groups in the structure of biologically active compounds.

Conclusions
In conclusion, the presence of different biologically active components in S. orientalis methanolic extracts indicates its great potential for future pharmaceutical investigations.

Ethical Considerations
Compliance with ethical guidelines
There were no ethical considerations to be considered in this research.

Funding
The paper was extracted from the thesis PhD Dissertation of the first author at Department of Plant production and Genetics, Faculty of Agriculture, University of Zanjan.

Authors' contributions
Conceptualization and Supervision: Khadijeh Bagheri and Ali Sharafi; Methodology: Zahra Gharari; Investigation, Writing – original draft, and Writing – review & editing: All authors; Data collection: Zahra Gharari; Data analysis: Zahra Gharari; Funding acquisition and Resources: Khadijeh Bagheri, Ali Sharafi.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors would like to thank the authorities of the School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran, for their support.

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