Phytochemical characterisation of Phlomis linearis Boiss. & Bal and screening for anticholinesterase, antiamylase, antimicrobial, and cytotoxic properties

In the present work, essential oil and fatty acids and extracts obtained from aerial parts of Phlomis linearis Boiss. & Bal. were investigated for chemical composition and biological activities. The phytochemical analyses were conducted with gas chromatography-mass spectrometry/flame ionisation detector (GC-MS/FID) and liquid chromatography-mass spectromtetry (LC-MS/MS) techniques. The extracts and essential oil were studied for α-amylase and acetylcholinesterase activities with two different spectrophotometric methods. Antimicrobial activities of the extracts were investigated by microdilution. The extracts were evaluated in vitro for cytotoxic effects against cancer and normal cell lines by MTT assay. The essential oil (EO) contained α-pinene (12.5%) and β-caryophyllene (10.7%) as main compounds. Palmitic (26.5%) and nonadecanoic acids (26.6%) were determined as fatty acids. Phytochemical analysis of the extracts found phenolic acids, phlinosides, verbascoside, and flavonoids. The extracts and essential oil demonstrated poor α-amylase inhibitory activity. The best acetylcholinesterase inhibitory activity was obtained for diethly ether extract of P. linearis (67.2 ± 3.4%) at 10 mg /mL concentration. Ethyl acetate extract found to be effective against Staphlococcus aureus at a minimum inhibitory concentration (MIC) of 156.26 µg/mL. Diethyl ether extract of P. linearis was active on A549 cell lines with an IC50 = 316 ± 4.16 µg/mL when compared with cisplatin IC50 = 24.43 ± 0.14 µg/mL. To the best of our knowledge, the present work is the first comprehensive report on anti-acetylcholinesterase, anti-α-amylase, and antimicrobial activities, as well as cytotoxic effects of P. linearis.

and antiinflammatory activities or the EO of P. linearis has been investigated by Demirci et al. [22] on the chorioallantoic membrane.
To date, very little research has been carried out on phytochemistry and biological activity for P. linearis; there have been no attempts to examine the anti-acetylcholinesterase, anti-α-amylase, and antimicrobial activities, as well as cytotoxic effects against human lung cancer, human colon cancer, and mouse embryonic fibroblast cell lines for P. linearis. We aimed to investigate the phytochemical characterisation of EO, fatty acids and extracts from aerial parts of P. linearis, and biological activities of the extracts and EO.

Essential oil isolation
Plant material of P. linearis (40 g) was exposed to hydrodistillation (3 h) in the Clevenger apparatus to yield EO [23]. The EO yield was calculated based on dried plant material and stored in an amber vial at 4 °C up until the phytochemical and biological activity analyses.

Preparation of extracts
Plant material of P. linearis (40 g) were subjected to maceration with n-hexane, diethyl ether, ethyl acetate, and methanol (200 mL × 3), respectively for 24 h. The resulting extracts were collected, filtered, and concentrated in a rotary evaporator. The dried extracts were kept at 4°C.

Fatty acids analysis
The lipid extraction kit is used for the extraction of the total lipids from P. linearis. According to protocol, 0.15 g millground plant material was treated with a 3 mL solvent containing chloroform/MeOH (2:1). After homogenising and vortexing of mixture, 0.5 mL of an aqueous buffer of the kit was added, and the sample was mixed by a vortex again. Subsequently, the extraction solution was poured into a syringe system containing a filter. The eluted solvent contained the chloroform phase with total lipids. Then, 200 mL of aliquot of the total lipids dried under a stream of nitrogen for subsequent transesterification. After drying, 1 mL of BF 3 -MeOH solution and 0.3 mL of n-hexane were added. The mixture was heated at 95°C for 1 h under reflux. Then, 1 mL of n-hexane and 1 mL of distilled water were added. The mixture was vortexed and centrifuged at 500 × g for 5 min. The top hexane layer was transferred into a vial and then injected into the GC-MS and GC-FID system without solvent evaporation before injection.

Gas chromatographic analysis
GC-MS analysis was examined by an Agilent 6890N GC and Agilent 5975 GC/MSD systems (Agilent Technologies, SEM Ltd., Istanbul, Turkey). HP-Innowax FSC column (60 m × 0.25 mm, 0.25 μm film thickness (Agilent Technologies) was used with a helium carrier gas at 0.8 mL/min as reported previously [24].

Identification of compounds
The compounds were identified by comparison of their mass spectra with those in Wiley NIST Library (NY, USA), Mass Finder software 4.0 (Dr. Hochmuth Scientific Consulting, Hamburg, Germany), and Adams Library (digital library) 1,2 .
In addition, identification of compounds was confirmed by comparison of their RRI values with data reported for polar column. The relative percentage amounts of the separated compounds were determined from FID chromatograms. Confirmation was done using the in-house "Başer Library of Essential Oil Constituents" database, analysed with known pure compounds by chromatographic runs at the same conditions. 2.7. LC-MS/MS analysis LC-MS/MS analyses of extracts were carried out using the Applied Biosystems 3200 Q-Trap LC-MS/MS (Antes, İstanbul, Turkey) system equipped with an ESI source operating in negative ion mode. For the chromatographic separation, a GL Science Intersil ODS (Tokyo, Japan) 250 × 4.6 mm, i.d., 5 µm particle size, octadecyl silica gel analytical column operating at 40 °C was used. The solvent flow rate was maintained at 0.5 mL/min. Detection was carried out with PDA detector. The elution gradient consisted of mobile phases (A) acetonitrile:water:formic acid (10:89:1,v/v/v), and (B) acetonitrile:water:formic acid (89:10:1, v/v/v), respectively. The amount of B was increased from 10% to 100% in 40 min. LC-ESI-MS/MS data were collected, and processed by Analyst 1.6 software [25 -26].

Determination of α-amylase inhibition
The antidiabetic potential of P. linearis extracts and EO was determined upon inhibition of the α-amylase enzyme that is involved in hydrocarbon's metabolism. The iodine/potassium iodide (I/KI) method was used [27]. The concentration of samples was prepared with MeOH at 10 mg/mL, and the enzyme was prepared with 0.8 U/mL in 20 mM in sodium phosphate buffer pH (6.9). The control wells contained all the reagents without the sample (the solvents of the samples instead were added). Acarbose (inhibitor of α-amylase) prepared in concentration of 0.25 mg/mL was used as the positive control. The percentage of inhibition was calculated according to Equation (1): Abs control : the absorbance of control ; Abs control blank : the absorbance of blank Abs sample the absorbance of sample; Abs sample blank : the absorbance of blank 2.9. Determination of acetylcholinesterase inhibition Acetylcholinesterase (AChE) inhibition of the extracts and EO were evaluated according to Ellman's method [28] with a slight modification.

Minimum inhibitory concentration (MIC, µg/mL)
Antimicrobial activity performed for different extracts of P. linearis. The extracts were prepared within DMSO, and the standard antimicrobial powders were obtained from Sanovel Pharmaceutical Industry (İstanbul, Turkey). Ampicillin, cefuroxime, and fluconazole were used as reference drugs. The MIC values of the strains were evaluated with a slight modification of microdilution methods [29 -30]. The extracts were diluted 2-fold initially with a final concentration range of 2500 to 19.53 µg/mL. Ampicillin, cefuroxime, and fluconazole prepared at 64-0.125 µg/mL within DMSO and water. Bacterial suspensions were grown overnight in double strength broth and standardized to 10 5 CFU/mL for bacteria. Candida suspensions were standardized using a turbidimeter (McFarland densitometer, Biosan, Latvia, 0.5 density) to 5 x 10 3 CFU per well in RPMI medium under sterile conditions. 10 μL yeast inoculum was added to each well of the microplates.
After serial dilution of samples in 96 well, each microorganism suspension was pipetted into each well and incubated at 35 °C for 24 h. Positive growth controls (to assess the presence of turbidity) were performed in wells not containing antimicrobial agents. Microbial growth was observed by adding 20 µL of resazurin of 0.01% with minor modifications of CLSI standards. The experiment was done in triplicate and calculated the mean of MIC.

In vitro cytotoxicity assay
Human lung epithelial cell line (A549, ATCC CCL-185), human colon cancer cell line (HT-29, ATCC® HTB-38), and mouse embryonic fibroblast cell line (NIH/3T3, ATCC CRL-1658) were used for determining IC 50 of the extracts by MTT method according to previous studies [31][32][33]. Stock solutions of the extracts were prepared in ethanol. The tested extracts were added to the wells (1000-7.8125 µg/mL) in quadruplicates. Inhibition % was calculated for the extracts. Nonlinear regression analysis was used for IC 50 values. Calculations on the results were performed according to Equation (2): ( * ) × 100 2.11.1 Selectivity index Selectivity index (SI) was also calculated to compare the selectivity of the compounds according to a previous study [34] as follows: SI = IC 50 of compound in the NIH3T3 cells / IC 50 of the same compound in the cancer cells.
The sample collected by us in Sivas province was characterised with quit rather content of monoterpenes (14.9%) with predominance of α-pinene (12.5%). However, the plant sample collected in Kayseri province did not contain monoterpenes at all. In addition, acetophenone was also not mentioned in the previous report [22]. High percentage of acetophenone (7.5%) was found in EO from fresh leaves of P. umbrosa Turcz. [39].

Ethyl acetate and methanol extracts composition of P. linearis
Phenolics acids, flavonoids, and phenylethanoid glycosides were identified for the ethyl acetate and methanol extracts via LC-MS/MS technique. The list of the compounds detected in P. linearis ethyl acetate and methanol extract with MS detector is summarised in Table 3. The results of phytochemical analyses of the extracts were examined with 5-caffeoylquinic acid, 3.5/1.5 dicaffeoylquinic acid, phlinosides, verbascoside, quercetin, and luteolin derivatives. Chromatographic profiles EAc and MeOH extracts of P. linearis obtained with liquid chromatography were given in Figures 1 and 2.

α-Amylase inhibitory activity
Diabetes mellitus is a metabolic disorder and characterised by hyperglycemia. α-amylase is a key enzyme that hydrolysis carbohydrates to disaccharides, and α-glucosidases hydrolysis disaccharides to monosaccharides like glucose. Therefore, inhibition of these enzyme systems helps to control hyperglycemia and digestion of carbohydrates to reduce blood glucose levels [60][61].
In this study, the extracts and EO of P. linearis were evaluated for in vitro α-amylase activity. As indicated in Table  4, the EO inhibited the enzyme's activity by 25.7% at concentration of 10 mg/mL. The following order of the extracts against α-amylase activity was observed at 10 mg/mL concentration: n-hexane (31.5 ± 2.6%), methanol (30.5 ± 1.4), diethyl ether (28.3 ± 4.0%) and ethyl acetate (24.8 ± 2.0%). It should be noted that acarbose displayed more potent inhibition of α-amylase (57.2%, concentration at 0.25 mg/mL) than all tested extracts and EO.
In this study, phenolics acids, flavonoids, and phenylethanoid glycosides were identified for the EAc and MeOH extracts. These compounds have been reported to have antidiabetic effects [62][63][64]. Twenty-one flavonoids were investigated for inhibitory activities against α-glucosidase and α-amylase. Luteolin inhibited α-glucosidase (36%, at 0.5 mg/mL) [63]. Six group of flavonoids were reported inhibitory activities against α-glucosidase and α-amylase. Among them, luteolin, myrcetin, and quercetin were found as potent inhibitors with IC 50 of 0.36, 0.38, and 0.50 mM, respectively against α-amylase enzyme [64]. Moreover, MeOH extract of Phlomis stewartii displayed α-glucosidase inhibitory activity (80.2% at 1.0 mg/ mL) [65]. The α-amylase inhibitory capacities of these extracts might be attributed to their phytochemicals contents. However, there have been no reports for EO and different extracts of P. linearis against α-amylase inhibitory activity. The α-amylase inhibitory activity of the extracts P. linearis was performed for the first time in this study.

Acetylcholinesterase inhibitory activity
The extracts and EO of P. linearis were in vitro evaluated for acetylcholinesterase enzyme inhibitory activity. AChE inhibition activity was represented as inhibition percentage and compared with galanthamine ( Table 4).
The EO inhibited AChE (39.5 %) at 10 mg/mL concentration. In this report, EOs of P. linearis consist of α-pinene (12.5%) and β-caryophyllene (10.7%) as major compounds. In a previous research, α-pinene, 1,8-cineole, and camphor were found to be uncompetitive reversible inhibitors of AChE [66]. According to recent study, the EO of P. kurdica was also reported for AChE (41.4%) and butyrylcholinesterase (BChE) (36.2%) inhibitory activity at 250 µg/mL concentration [67]. Additionally, EOs for Piper species demonstrated their acetylcholinesterase activities. EOs contain terpenes and phenylpropanoids as predominant compounds like P. linearis essential oil. [68]. Therefore, anti-AChE activity of the tested P. linearis EOs could be attributed the presence of the predominant compounds. However, it should be noted that essential oils are mixture of many chemical compounds, which may potentially modulate enzyme inhibitions. Therefore, main and minor compounds of the EO may attempt to enzyme inhibitions.
The extracts of P. linearis demonstrated anti-AChE activity ranged between 9.2% and 67.2% at 10 mg/mL concentrations. The best inhibitory activity was obtained for DE extract of P. linearis (67.2%) and followed with methanol (44.7%) and hexane (42.8%) extracts. The ethyl acetate extract demonstrated poor inhibitory activity (9.2%).
In this report, analysis of the extracts contains caffeoylquinic acid and its derivatives as well as flavonoids and phenylethanoid glycosides by LC-MS/MS. Previously a study reported anticholinesterase potentials of phenolic acids and various flavonoid derivatives [69]. In the current study, quercetin showed a considerable inhibition (76.2%) against AChE, while genistein (65.7%), luteolin-7-O-rutinoside (54.9%), and silibinin (51.4%) performed a moderate inhibition on BChE.
For this purpose, the AChE inhibitory activity of P. linearis extracts and essential oil has never been reported before, and obtained results indicate that P. linearis could serve as an inhibitor against AChE enzyme.

Antimicrobial activity (MIC, µg/mL)
In this study, antimicrobial activity was evaluated for different extracts of P. linearis against E. coli ATCC 8739, S. enterica ATCC 14028, B.subtilis subsp spizizeni ATCC 6633, S. aureus ATCC 6538, and C. albicans ATCC 10231. Antimicrobial activity of the extracts was compared to cefuroxime, ampicillin, and fluconazole as the standard drugs as given in Table 5 by microdilution method.
The most effective extract was found to be EtOAc extract against S.aureus ATCC 6538 strain with MIC value 156.25 µg/mL. All of the extracts had the same MIC values with 625 μg/mL against E. coli ATCC 8739 and C. albicans ATCC 10231 strains. All of the extracts generally were found to be most effective against S. aureus ATCC 6538 range of MIC = 156.25-312.5 μg/mL in our study.
Generally, antimicrobial acitivity have been focused on EO extracted from Phlomis species in the literature. The EOs of P. ferruginea Ten [70] P. bovei De Noe subsp. bovei [71] P. bracteosa Royle ex Benth. [72] P. floccosa D. Don [73], P. kurdica Rech. fil. [67] and isolated a few compounds as, forsythoside B, phlinoside C and verbascoside from P. lanceolata [74] showed antimicrobial activities, while the EO of P. linearis has been reported only in antiangiogenic and antiinflammatory activity [22]. In addition to EO, methanol extracts of P. olivieri, P. bruguieri, and P.herba-venti were investigated in terms of their antibacterial effects against some bacteria pathogens [46]. To the best of our knowledge, this paper represents the first report on the antimicrobial activities of P. linearis Boiss. & Bal on different extracts.

Cytotoxicity assay
MTT test was evaluated to see cytotoxic activity of the extracts of P. linearis against A549 and HT-29 cancer cell lines. Also, the cytotoxic activities of extracts were studied against NIH3T3 cells, and to determine the selectivity of the extracts towards carcinogenic cell lines. The IC 50 values of the extracts were determined against cell lines in Table 6. The compounds should be nontoxic on healthy cell lines and show cytotoxic effect in cancer cell lines as anticancer drug candidates. Hence, cytotoxic effect of the extracts against NIH3T3 cell line was tested. Cytotoxic activity was found to be EAc (IC 50  On the basis of previous investigations, Phlomis species have shown cytotoxic activity against various cancer cell lines. Cytotoxic activity of P. lanceolata displayed against HT29, Caco2, T47D, and NIH3T3 cell lines. Petroleum ether extract was found to be the most active against all four cell lines [75]. In another study, cytotoxic activity of the 80% MeOH extracts  fallowing namely, P. kurdica, P. bruguieri, P. caucasica, P. olivieri, P. anisodontea, and P. persica were assessed against on HepG2, MCF7, HT29, and A549 cancer and one normal cell lines MDBK [76]. The present results have indicated aqueous anticancer effects of the of P. russeliana extract against Caco-2 cell lines [77]. To date, P. linearis has not been investigated for any cytotoxic assay. To the best of our knowledge, this is the first report on cytotoxic activity of P. linearis against two cancer cell lines and one normal cell line by MTT assay.

Conclusion
The present work is the first investigation on essential oil and extracts from aerial parts of P. linearis to include on anti-acetylcholinesterase, anti-α-amylase, and antimicrobial activities, as well as cytotoxic effects. The phytochemical characterisation of essential oil, fatty acids, and extracts from aerial parts of P. linearis were represented using GC-MS / FID and LC/MS-MS techniques. The essential oils and extracts of P. linearis were found to have valuable phytochemicals with biological activities. The results showed that ethyl acetate extract of P. linearis possess high antibacterial activity against S. aureus ATCC 6538. Therefore, P. linearis could be recommended for the combination with antimicrobial drugs for drug industry, especially against S. aureus. Furthermore, diethyl ether extract indicated cytotoxic effect against human lung cancer cell lines. The best acetylcholinesterase inhibitory activity was obtained for diethyl ether extract of P. linearis.
Finally, P. linearis could be evaluated for isolation of active components for therapeutical applications. However, it needs further in vivo studies for safety and efficacy in the aforementioned applications.