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Chemical compositions, antimicrobial effects, and cytotoxicity of Asia minor wormwood (Artemisia splendens Willd.) growing in Iran

Background Artemisia splendens from the Asteraceae family is a new source of biologically active compounds. The current study investigated to evaluate antimicrobial and cytotoxicity activity of methanolic extracts and their fractions obtained from aerial parts by agar disk diffusion and MTT methods, respectively. The active fractions were subjected to preparative HPLC for isolating the pure compounds, which were structurally elucidated, by 1H and 13C NMR. Results The results showed that the methanolic extract and its 60% SPE fraction have the anti-proliferative activity on A549 cell line in comparison with the control group. Meanwhile, the methanolic extract and its 40% SPE fraction can inhibit the growth of Gram-positive strains as anti-microbial activity. The 60% SPE fraction also illustrated anti-proliferative activity on the HT-29 cell line compared to the control group. Chromatographic separations via preparative HPLC yielded 5 flavonoids and three flavonoid glycosides. Conclusion Based on the results it can be concluded that A. splendens as a potential source of cytotoxic and antimicrobial compounds can be used in pharmaceutics.


Introduction
The genus Artemisia is one of the largest and most important genera of the Asteraceae family used in traditional medicine worldwide [1]. Biologically active secondary metabolites such as phenolic compounds (flavonoids, phenylpropanoids, and coumarins), and terpenoids (sesquiterpene lactones) [2], which were isolated from plants of this genus, exhibited various pharmacological activities such as antimalarial, antitumor, antioxidant [3], anti-microbial [4], and anti-inflammatory [5]. It is also a (14.5%), germacrene D (14.3%), α-pinene (11.3%) and bicyclogermacrene (11.3%) [7]. However, their chemical composition has not hitherto been described. Thus, the aim of this study was to analyze the chemical constituents of methanolic fractions obtained from the aerial parts of A. splendens to exert antimicrobial activity and cytotoxic effects on cancerous cells.

Plant material
Artemisia splendens aerial parts were collected from Arasbaran, at E: 31° 49′ 46″, N: 10° 49′ 38″ (altitude of 2470) East Azerbaijan Province, Iran in 2015. The plant material was identified by botanist Dr. Fatemeh Ebrahimi and the voucher specimen of the plant was deposited at the Herbarium of Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran, under the registry code Tbz-FPh 717.

Extraction
Air-dried aerial parts (100 g) were ground and extracted in a Soxhlet apparatus using MeOH. The extract was concentrated under the vacuum by a rotary evaporator at 45 °C and stored under refrigeration before analysis and biological evaluation.

Anti-proliferative activity Cell culture
HT-29 (colon carcinoma cell line) and A549 (adenocarcinoma human alveolar basal epithelial cells) cell lines were prepared and cultured according to described method by Afshar et al. [11]. At 75% confluence, the cells were washed by phosphate-buffered saline (PBS) and cultured in 96-well plates (Nunc, Denmark) for MTT experiment.

MTT assay
The anti-cancer effect of the MeOH extract of A. splendens and its fractions were evaluated by MTT reduction colorimetric assay. The cells were cultured in a 96-well plate at a density of 1 × 10 4 cells/well and incubated for 24 h. Cells were treated with different concentrations of extract and fractions which were prepared in dimethyl sulfoxide (DMSO). After 48 h of incubation, the medium was discarded and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/ml in PBS) was added to each well and incubated at 37 °C and 5% CO 2 for 4 h [12]. The medium was removed and then DMSO was added to dissolve purple insoluble formazan. The wells were shaken for 5 min and then the optical density of each well was determined at 570 nm wavelength by a microplate reader (ELISA plate reader, Bio Teck, Bad Friedrichshall, Germany). Each assay was accomplished three times for each cell line. Antiproliferative activity of extracts and their fractions were measured by relative viability according to the method described by Afshar et al. [11].

Disc diffusion test
Activated microorganisms were transferred into Muller-Hinton broth medium (Merck, Germany) and incubated overnight at 37 °C. For providing an optical density of 0.5 McFarland (10 8 CFU/mL as a standard optical density) of bacterial concentration, the centrifuged pellets (3000 rpm for 15 min) were washed twice and suspended in saline solution. The ultimate concentration of inocula was set up to 10 6 CFU/mL with sterile saline solution. To achieve unified microbial growth, 10 mL of prepared inoculum suspensions were seeded in the autoclaved Muller-Hinton agar medium. Sterile discs (Whatman paper no. 6 mm diameter) located on the surface of media, were impregnated with 50 µL of different concentrations of extracts (1:1, 1:5, 1:10) which were dissolved in 50% aqueous DMSO. Then they were imbued with a 0.5 McFarland's standard of mentioned bacteria covering 3 disks for test samples, one disk was used for negative control (including antimicrobial extracts and culture medium) and one disk was used for the positive control (including 0.5 McFarlend of the pathogen and culture medium). Afterward, for incubation and analysis, 100 µL of test solutions were poured into respective wells (200 mg/mL). The plates were transferred into the refrigerator to allow the diffusion of extracts approximately for 30 min, and then petri dishes were incubated at 37 °C for 24 h. Finally, the diameter of the inhibition zones obtained nearby each well (excluding well diameter), showing no bacterial growth, was measured with venire caliper. Doublet plates were prepared for each sample. Extracts and fractions displaying considerable antimicrobial effects were further evaluated for their minimum inhibitory concentration (MIC), which is the lowest concentration of each product for completely inhibition of the bacterial growth [13]. Successively, two-fold dilutions of MeOH extract and its fractions were utilized in broth. The pure sterile nutrient broth was used as control. MIC values were calculated after 24 h incubating the plates at 37 °C [14].

Statistical analysis
All statistical analyses were done using Graph Pad Prism 8.01 and SigmaPlot 11 software. One Way ANOVA with a post hoc Tukey test was used for evaluating significant differences between groups. The experiments were performed in triplicates (n = 3) and all data were presented as mean ± S.D. P values < 0.05 which are regarded as significant.

Results
In the present study, the pure compounds [1][2][3][4][5][6][7]9] obtained from SPE fractions have been assayed. Moreover, anti-proliferative and anti-microbial activities of the MeOH extract and its different SPE fractions from aerial parts of A. splendens were determined and the results are indicated in Tables 1, 2. (NMR spectroscopic data of compounds 1-8 (CD 3

OD, chemical shift δ in ppm, coupling constant J in Hz in parentheses):
Compound 1: 1 HNMR and 13 CNMR of the isorhamnetin-3-O-rutinoside was as follows: 1     Compound 8: This compound was named as Narcisin. The NMR data was the same as compound 1.

Cytotoxic assay
In our study, the MeOH extract of A. splendens and its fractions were investigated for their cell growth inhibitory effect in human tumor cell lines such as HT-29 and A549 by MTT assay. The IC 50 values of corresponding extract and fractions are reported in Table 1. The results indicated that the methanolic extract had weak to moderate anti-proliferative activity on the A549 cell line in comparison with DMSO control (P < 0.001) with an IC 50 value of 617.72 ± 292.90 μg/mL. Whereas (10:90), (20:80), (40:60) MeOH: water SPE fractions did not affect the growth of A549 cells, 60% fraction inhibited cell growth of A549 cell line compared with control (P < 0.001) with an IC 50 value of 98.63 μg/mL. In terms of colon cancer cell line, although, MeOH extract, could not decrease the amounts of cancer cells, its 60% SPE fraction significantly indicated anti-proliferative activity (P < 0.001).

Anti-microbial activity
Among all the strains, only inhibition zones were observed against gram-positive S. epidermidis and S. aureus ( Table 2). The MIC values for S. epidermidis was 200 mg/mL and for S. aureus 100 mg/mL which considered as very weak activity compared to the control by MeOH extract and 40% SPE fraction.

Discussion
Cancer is one of the diseases with the highest mortality rate in developed countries and the second leading cause of death in developing countries [15]. Among all cancer types, lung and colon cancers are the most commonly diagnosed cancers in both genders [16]. Furthermore, due to the adverse effects of anti-microbial chemical drugs, the use of plants with anti-microbial effects is recommended to reduce side effects. Different studies have revealed that herbal medicines have anti-proliferative and anti-microbial activities through different functional mechanisms [17]. In the current study, the cytotoxic and anti-microbial effects of MeOH extract and its SPE fractions of A. splendens on A549 and HT-29 cells and different microorganisms have been investigated.
The results showed that 60% SPE fraction of MeOH extract had weak to moderate cytotoxic effect on both tumor cells in comparison with other fractions and MeOH extract as well as control (P < 0.001). It is worth mentioning that even though HT-29 was not influenced by MeOH extract, its 60% SPE fraction inhibited the growth of cells (P < 0.001). It can be concluded that the main anti-cancer ingredients of this extract have been accumulated in this fraction. Chromatographic separation of this fraction afforded 5 known flavonoids, namely isorhamnetin-3-O-rutinoside, quercetin, luteolin, kaemferol and genkwanin which are reported in this species for the first time. The anti-proliferative activity of various species of Artemisia has been evaluated by several researchers. These extracts inhibited the growth of different cell lines such as T47D, MCF-7 by inducing apoptosis [18]. Furthermore, previous studies have shown that quercetin and luteolin have cytotoxic effects on A549 with an IC 50 value of 10.0 and 3.1 µM, respectively [19]. Luteolin induces apoptosis through the downregulation of several apoptotic proteins in HT-29 [20]. It has been reported that kaempferol treatment promotes apoptosis of HT-29 human colon cancer cells through both extrinsic and intrinsic pathways [21]. Additionally, the IC 50 value of genkwanin on A549 was determined as 9 µM [22]. A combination of solid-phase-extraction (SPE) and reversed-phase prep-HPLC analysis of the aerial parts of the SPE fraction (60%) afforded five flavonoids, which were isorhamnetin-3-O-rutinoside, quercetin, luteolin, kaempferol, and genkwanin. Structural identification of isolated phytochemicals was carried out by UV and NMR analysis and comparison of the data with the reported values. According to our knowledge, none of these phenolic compounds were isolated from A. splendens. Although in prior studies some compounds such as melilotoside have been reported from this plant [23].
In terms of compound 2 (quercetin), the UV spectrum of this compound indicated the presence of OH-4′ and 3 moieties due to bathochromic shift of band I and destruction of it after 5 min. The addition of AlCl 3 reagent and observing a bathochromic shift in band I has been proved the presence of sensitive and resistant moieties of this structure. Subsequently, by adding HCL, the hypsochromic shift compared to the AlCl 3 graph as well as a bathochromic shift to the MeOH graph illustrated the existence of both sensitive moieties (ortho dihydroxy of 3′, 4′) and resistant ones (free 3-OH and 5-OH). Furthermore, 1 H and 13 C-NMR spectrum have been proved this structure as well. All of the mentioned data are consistent with many previous investigations as well [26]. Spectrum data of compound 4, completely are in line with compound 2, except little difference in B ring with a symmetric structure in which the spectrum data are in line with published papers [27]. In the case of compounds (3) and (5), UV spectra were identical with flavone structure, (which was supported by 1 H-NMR and 13 C-NMR spectrums, showing characteristic signals appeared at δ 6.54 (1H, s, H-3), 6.66 (1H, s, H-3), luteolin, and genkwanin, respectively [26,28].
MeOH extract and 40% SPE fraction of this plant showed inhibition zones against S. epidermidis and S. aureus. Although the MIC values for these pathogens are 100 and 200 mg/ml which considered as weak to inactive. It seems that it can be achieved potent anti-microbial agents via more isolating compounds from MeOH extract and 40% SPE fraction. Furthermore, in extract and fractions due to there are broad ranges of compounds, different biological activity might be observed (because the compounds have synergist or antagonist activity with each other). Previous studies showed MeOH extract of A. nilagirica indicated inhibitory activity against Gram-positive, as well as Gram-negative with DIZ value of 12 mm [29]. Furthermore, the MeOH extract of A. nilagirica was active against M. smegmatis [30]. Buffered methanol (80% methanol and 20% PBS) and acetone extracted substances from A. absinthium were active against E. coli, S. infantis, S. aureus, and L. monocytogenes [31]. A. capillaris Thunb. and A. caruifolia Buch. demonstrated inhibitory activity against B. cereus and L. monocytogenes [32]. A. annua and A. vulgaris L. illustrated the most prominent anti-bacterial activity against S. mutans [33].
To the best of our knowledge, no data were available on the antimicrobial activity of A. splendens MeOH extract. 40% SPE fraction for bearing moderate inhibitory effect on microorganisms was selected for further investigation using prep-HPLC afforded three main compounds as following structure. The above-mentioned data suggesting the presence of a flavonoid structure with a para pattern in the B ring. In addition, the 1 H-NMR spectra of compound 6 confirmed the presence of an overlapping signal for five protons (δ 3-4 ppm) and an anomeric proton (δ 5.45, 1H, d, J = 7.48 Hz) corresponding to a glucosyl moiety with β configuration. The UV spectrum of compound 1 with MeOH as a solvent is characteristic of flavonols derivatives. Studying UV spectra data after the addition of NaOMe and production of 60 nm band I bathochromic shift is indicative of 4′-OH. Remaining of band I after 5 min indicated that C-3 has substitution except for OH. The addition of AlCl 3 /HCl did not cause any hypsochromic shift in the AlCl 3 spectrum. Therefore, it can be concluded there is no ortho-dihydroxy group (3′ and 4′-OH) in the B ring. Furthermore, it leads to the presence of OH in C-5. Addition of NaOAc and production of band II bathochromic shift of 7 nm indicative of free 7-OH. In studying the 13 C-NMR, the presence of four signals at δ 60-75, confirms the presence of glucose in the composition. Moreover, signals at δ 130.93 and 114.81 corresponded to C-2′, C-6′ and C-3′, C-5′, respectively. Thus, compound 6 was identified as Astragalin. All spectroscopic data were in good agreement with the literature data of respective compounds [34,35]. While Astragalin [6] was previously reported from A. transiliensis [36] and A. annua, [3] this is the first report on the presence of Astragalin in the A. splendens. There was also a methoxy signal (δH 3.95, 3H, s) that is indicative of 3′-OCH 3 . Although an anomeric proton (δ 5.43, 1H, d) and some of the glycosidic protons overlapped (δ 3-4 ppm) in the 1 H-NMR spectrum, in studying the 13 C-NMR, the presence of five signals at δ 60-75 and a resonance at δ 102.1 (anomeric carbon of glucose), confirms the presence of glucose in the composition. The presence of a signal at δ 55.3 ppm in the 13 C-NMR spectrum indicating a methoxy group which is confirmed by the presence of a singlet resonance at δ 3.95 (3H) in the 1 H-NMR spectrum. In 13 C -NMR, signals at δ 178.80, 164.60, 163.50, 160.00, 158.80, 130.90, 103.90, 98.40 and 93.30 corresponded to C-4, C-7, C-5, C-9, C-2, C-3, C-10, C-6 and C-8, respectively. Furthermore, signals at δ 151.90, 146.80, 122.30, 121.10, 114.60 and 112.9 corresponded to C-3′, C-4′, C-6′, C-1′, C-5′ and C-2′, respectively. Thus, compound 7 was identified unequivocally as isorhamnetin-3-O-beta-d-glucoside. Spectroscopic data were in good agreement with the literature data of the respective compound [37]. While isorhamnetin-3-O-beta-d-glucoside was previously reported from A. halodendron, [38] this is the first report for reporting this compound from A. splendens. Compound 8 was also isolated as a light yellowish powder from a 60% fraction as well. FABMS analyses (negative and positive ion modes) of compound 8 revealed the pseudo-molecular ions at m/z 623 [M−H] − and 647 [M+Na] + , which was in agreement with the molecular formula C 28 H 31 O 16 and C 28 H 32 O 16 Na, respectively. The 1 H-NMR and 13 C-NMR spectra of compound 8 were similar to those of compound 7, with the exception that one more rhamnose moiety has been regarded to this compound with four protons signals (δ 3.2-3.6), doublet resonance at δ 1.11 (3H, d, J = 6.04 Hz) and singlet resonance at δ 4.54 (1H, s), which is attributed to anomeric proton of H-1‴ of rhamnose moiety. Furthermore, the 1 H-NMR spectra of compound 8 verified the existence of one isorhamnetin nucleus. Our claim has been supported by methoxy group shift (δ 3.95, 3H, s) in 1 H-NMR as well as methoxy carbon signal (δ 55.30) at 13  The 1H-NMR spectrum of compound 3 showed the presence of one glucosyl moieties with characteristic doublet resonance at δ 5.24 (1H, d, J = 7.18 Hz) for the anomeric proton of β-glucose. In addition, the 1 H-NMR spectra of compound 1 confirmed the presence of overlapping signals for four protons (δ 3.2-3.6 ppm), an anomeric proton (δ 4.54, 1H, s) and δ 1.11 (3H, d, 6.04) (methyl group of rhamnose), corresponding to a rhamnose moiety in the composition. In studying the 13 C-NMR spectrum, the presence of five signals at δ 60-75 and a resonance at δ 102.7 (anomeric carbon of glucose), confirms the presence of glucose in the composition as well as the presence of four signals at δ 60-75, presence of a signal at δ 101.2 (anomeric carbon of rhamnose) and δ 16.5 (methyl group of rhamnose), was confirmed the presence of rhamnose in the composition. Furthermore, signals at δ 177.50, 164.60, 162.10, 157.30, 156.80, 134.30, 104.80, 98.10 and 93.50 corresponded to C-4, C-7, C-5, C-9, C-2, C-3, C-10, C-6 and C-8, respectively; as well as, signals at δ 149.80, 146.80, 122.70, 121.30, 115.30 and 113.40 corresponded to C-3′, C-4′, C-6′, C-1′, C-2′ and C-5′, respectively. On the basis of these data, the structure of 8 was elucidated as narcissin. Finding a literature review of previous works was compatible with our results [39]. While isorhamnetin-3-O-beta-D-glucoside was previously reported from A. sublessingiana, A. absinthium, A. vulgaris [40] and A. incana, [41] to the best of our knowledge, the respective compound was detected in this specie for the first time.