Agastache Species (Lamiaceae) as a Valuable Source of Volatile Compounds: GC–MS Profiling and Investigation of In Vitro Antibacterial and Cytotoxic Activities

Nowadays, there is an increasing interest in the study of medicinal and aromatic plants, due to their therapeutic properties that correlate with the presence of different active compounds. Agastache species (sp.) are aromatic plants that belong to the Lamiaceae family, originating from North America and East Asia. The present study aimed to evaluate the composition of essential oils (EOs) obtained from different Romanian cultivated Agastache sp. and to investigate their antibacterial and cytotoxic activities. The gas chromatography-mass spectrometry (GC–MS) screening revealed that menthone was the dominant constituent of A. foeniculum (31.58%), A. rugosa (39.60%) and A. rugosa ‘After Eight’ (39.76%) EOs, while estragole was the major constituent of A. foeniculum “Aromat de Buzău” (63.27%) and A. mexicana (41.66%) EOs. The investigation of the antiproliferative effect showed that A. rugosa and A. foeniculum “Aromat de Buzău” EOs had significant cytotoxic activity on MDA-MB-231 and HEPG2 tumour cell lines, with the most promising effect on the MDA-MB-231 breast cancer cell line for A. foeniculum “Aromat de Buzău” EO (IC50 = 203.70 ± 0.24 μg/mL). Regarding the antibacterial activity, A. rugosa EO was most active against E. coli (8.91 ± 3.27 μL/mL) and S. aureus (10.80 ± 0.00 μL/mL). To the best of our knowledge, this is the first report on the cytotoxic effect of Agastache sp. EOs on MDA-MB-231, HCT116 and HEPG2 tumour cell lines. The results of our study provide new and promising information for the subsequent in vivo study of the pharmacological properties of Agastache sp. essential oils.


Introduction
Agastache species (sp.) are aromatic plants from the Lamiaceae family that originate from North America and East Asia.Known under the common name of giant hyssop, they are used empirically in traditional medicine as a remedy for pain, hypertension, gastrointestinal disorders and infections [1][2][3][4].They are also used as ornamental plants, spices and as an important source of essential oils (EOs) [1][2][3].Some of the most studied sp. of the genus are Agastache rugosa (Fisch.& C.A.Mey.)Kuntze, Agastache foeniculum (Pursh) Kuntze and Agastache mexicana (Kunth) Lint & Epling [2][3][4].
Several Agastache sp. have been successfully acclimatised and introduced in culture in Romania [18], raising our interest for their phytochemical and pharmacological investigation.Thus, the aim of our study was to evaluate the chemical composition through gas chromatography-mass spectrometry (GC-MS) analysis, one of the most used methods for the characterisation of essential oils, and to investigate the antibacterial and cytotoxic activities of EOs obtained from the aerial parts of different Romanian cultivated Agastache sp.: A. foeniculum (AF) and its cultivar "Aromat de Buzău" (AFB), A. rugosa (AR) and its cultivar 'After Eight' (ARA8) and A. mexicana (AM).The cytotoxic effect was tested against a panel of cancer cell lines, representative for triple-negative breast (MDA-MB-231), colon (HCT116), lung (A549) and liver (HEPG2) cancers.This is the first report on the cytotoxic effect of Agastache sp.EOs on MDA-MB-231, HCT116 and HEPG2 tumour cell lines.Moreover, AFB is a new Romanian cultivar, with only one study available describing its chemical composition and some pharmacological properties [16], and ARA8 is a cultivar [18] for which studies regarding its chemical composition or therapeutic effects are lacking.

Results
The EOs were obtained by hydrodistillation with different yields (Table 1); their chemical composition was evaluated using GC-MS analysis, and the in vitro antibacterial and cytotoxic properties were investigated.

In Vitro Antibacterial Activity of Agastache Species Essential Oils
The results of the antibacterial assay are displayed in Table 7.The following standard strains were tested: Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076, Staphylococcus aureus ATCC 6538P, Listeria monocytogenes ATCC 19114.AR EO was most active against E. coli (8.91 ± 3.27 µL/mL) and S. aureus (10.80 ± 0.00 µL/mL) and less active against S. enteritidis (22.6 ± 0.00 µL/mL) and L. monocytogenes (18.72 ± 6.86 µL/mL).The same pattern was observed for all the tested EOs (AF, AM, ARA8).E. coli was most susceptible to the inhibitory activity of AR EO, while S. enteritidis was most susceptible to ARA8 EO. S. aureus was most susceptible to AM EO.

In Vitro Cytotoxic Activity of Agastache Species Essential Oils
The findings of the MTT assay are displayed in Figure 1.AR EO and AFB EO were used to investigate the in vitro cytotoxic effect, as they displayed the highest concentration of menthone (39.60%) and estragole (63.27%), respectively.In 96-well plates, cells were cultured and treated with full media containing serial dilutions of EO (3.91-1000 µg/mL).Every plate contained a blank control (full media only) and a positive control (100% DMSO).
The results of the study showed that AR EO had a significant cytotoxic effect on MDA-MB-231 and HEPG2 tumour cell lines, reducing cell viability below 50% at a concentration of 1 mg/mL and a 24 h exposure (Figure 1a).In contrast, AR EO had no significant cytotoxic effect on lung and colon tumour cell lines, cell viability percentage being above 50% for all tested concentrations.No important decrease in cell viability was observed for HUVEC and LX-2 normal cell lines at concentrations below 1 mg/mL.
AFB EO showed superior in vitro cytotoxic activity compared to AR EO on all investigated tumour lines (Figure 1b).AFB EO reduced cell viability below 50% for MDA-MB-231, HEPG2 and A549 tumour cell lines with statistically significant differences relative to the negative control.The most promising effect was on the MDA-MB-231 cancer cell line (IC 50 = 203.70± 0.24 µg/mL), with a well-represented dose-effect relationship.No marked decrease in cell viability was observed for normal cell lines, indicating selectivity over tumour cells.

Discussion
In the present study, we investigated the chemical composition, as well as antibacterial and cytotoxic effects of different Agastache sp.aerial parts' EOs.The vola

Discussion
In the present study, we investigated the chemical composition, as well as the antibacterial and cytotoxic effects of different Agastache sp.aerial parts' EOs.The volatile profiles were evaluated using the GC-MS analysis.The results showed differences in the phytochemical profile depending on species.Menthone, a monoterpene ketone, was the major compound in the EOs of AF, AR and ARA8.In contrast, the major constituent of AFB and AM EOs was estragole, a phenylpropanoid derivative.In AR and ARA8 EOs, estragole was found in a lower concentration (between 4.97% and 8.95%).Moreover, pulegone was found in high amounts in AR and ARA8 EOs (between 11.05 and 27.06%), while AFB EO contained only 0.33-0.46%pulegone.Thus, we identified several categories regarding the chemical composition of Agastache sp.EOs, considering the first two major compounds: menthone + estragole (AF), menthone + pulegone (AR, ARA8), estragole + limonene (AFB), estragole + pulegone (AM).Considering the variation in the chemical composition of the essential oils, the following compounds could be considered as markers for the species cultivated in Romania: menthone for AR, ARA8 and AF EOs and estragole for AFB and AM EOs.
Compared to our results, most studies that evaluated the composition of AF EO showed that estragole was the major volatile compound [19][20][21][22][23]. Regarding AR, some studies suggested that estragole was the main component of the EO [24][25][26], while others found methyl eugenol [27], pulegone, menthone [7] or limonene [28] as major compounds.Considering the variability of the chemical composition of the EOs within the same species, several chemotypes have been identified for AR.According to Chae et al. [29], there are five main chemotypes: menthone, menthone + pulegone, methyl eugenol, methyl eugenol + limonene and estragole.Furthermore, two studies pointed out that estragole was the major compound in AM EO [30,31], in accordance with the results of our experiment.
We identified only one study referring to the chemical composition of EO from AFB, a new Romanian cultivar.Thus, our results are consistent with those reported by Bălănescu et al. [16], who found estragole as the major compound (94.89%),followed by limonene (2.91%) and caryophyllene (0.74%).Other compounds were represented by eugenol, methyl isoeugenol, methyl eugenol and germacrene D, compounds also identified in our study, except for methyl eugenol.In our study, 48 different compounds were identified in AFB EO, of which 7 were also reported by Bălănescu et al. [16], the rest of the compounds being reported for the first time.Regarding ARA8 EO, this is the first study to investigate its chemical composition, identifying menthone as the major compound.
EOs are well known for their antibacterial properties.Our study showed that the bacterial strains most sensitive to the action of Agastache sp.EOs were E. coli ATCC 25922 and S. aureus ATCC 6538P, E. coli being the most sensitive to the action of AR EO (MIC: 8.91 ± 3.27 µL/mL) and S. aureus being the most inhibited by AM EO (MIC: 5.14 ± 0.00 µL/mL).In the literature, there are few reports regarding the antibacterial activity of Agastache sp.EOs.This is the first study that investigates the antibacterial potential of the EOs from AM, ARA8 and AFB.
Regarding the antibacterial activity of AR, our results are in accordance with those obtained by Haiyan et al. [7], who found that the EO extracted from AR flowers and leaves showed a moderate to high level of antibacterial activity.The EO from flowers had a significant in vitro inhibitory effect against S. aureus (MIC: 21 µg/mL) and E. coli (MIC: 21 µg/mL), but the EO from leaves had a higher inhibitory effect against E. coli (MIC: 9.4 µg/mL).Another study validated the EO's antibacterial activity against S. aureus and E. coli, while also reporting antimicrobial activity against two other Gram-negative bacteria (Salmonella enteritidis and Pseudomonas aeruginosa) [32].Compared to our results, other researchers obtained superior inhibitory activity for AF EO on bacterial strains of L. monocytogenes (MIC: 1.25 µL/mL) and Salmonella sp.(MIC: 1.25 µL/mL) and, in addition, found antibacterial action on other Gram-positive (Curtobacterium flaccumfaciens, Bacillus subtilis) and Gram-negative (Proteus vulgaris, Klebsiella pneumoniae) bacteria, with MICs ranging from 0.157 to 2.50 µL/mL [33].
Studies have also shown antibacterial properties for the major compounds identified in Agastache sp.EOs.Batista et al. found that estragole had antibacterial activity against S. aures and E. coli, as well as a synergistic effect in combination with gentamicin [34].
Moreover, menthone displayed an antibacterial effect against MRSA, with a minimal inhibitory concentration and minimal bactericidal concentration of 3.54 and 7.08 µg/mL, respectively [35].
Another aim of our study was to investigate the in vitro cytotoxic effect of AFB and AR EOs using the MTT assay.The most promising cytotoxic effect was recorded on MDA-MB-231 and HEPG2 cell lines for both EOs.This is the first study to evaluate the in vitro antiproliferative effect of AR and AFB EOs on MDA-MB-231 and HEPG2 cell lines.Other studies demonstrated that AR and AFB EOs were cytotoxic against MCF-7 hormonaldependent breast cancer cell line [11,16], while our study showed antiproliferative effects on a non-hormonal cancer cell line, the MDA-MB-231 triple-negative breast cancer cell line.MDA-MB-231 is a highly aggressive, invasive and poorly differentiated cell line, lacking the expression of estrogen and progesterone receptors and showing overexpression of HER2 receptors [36].Hormonal therapy or targeted therapy cannot be used in triplenegative breast cancer [36]; thus, new therapeutic agents are needed for the treatment of this aggressive pathology.
Few studies have investigated the antiproliferative potential of Agastache sp.EOs, and, therefore, information regarding their mechanism of action is limited.However, there are studies that have demonstrated the antitumour effect and mechanism of action of estragole and pulegone, two of the primary constituents of Agastache sp.EOs.According to Lashkari et al. (2020) [17], estragole was cytotoxic against the MCF-7 breast cancer line, inducing apoptotic morphological changes, including nuclear fragmentation, chromatin condensation and destruction of cell membrane integrity.Furthermore, estragole and pulegone had antiproliferative effects on the SGC-7901 stomach cancer line at concentrations ranging from 12.5 to 100 µg/mL [15].
Some studies have shown that high doses of estragole (600 mg/kg body weight) can cause liver cancer in rodents, leading some regulatory agencies to classify it as a possible human carcinogen [39].However, our study revealed that Agastache sp.EOs containing estragole did not display significant cytotoxic activity in vitro against HUVEC and LX-2 normal cell lines, indicating selectivity over tumour cells.
In our study, AFB EO showed superior cytotoxic activity compared to AR EO on all investigated tumour lines.The differences in the chemical composition could explain the variability of the cytotoxic effect.AFB EO contains 63.27% estragole, a phenylpropanoid derivative that demonstrated antiproliferative effects in vitro on MCF-7 [17] and SGC-7901 [15] tumour cell lines.On the other hand, menthone is the major compound (39.60%) of AR EO, followed by pulegone (24.72%) and limonene (8.36%), with a much lower concentration of estragole (4.97%).However, the chemical composition of the EOs varies significantly.Consequently, the cytotoxic activity may be influenced by the synergistic effect of the active compounds.Further studies are needed to explain the mechanism of action involved in the manifestation of the cytotoxic effect, including morphological analysis and clonogenic cell survival assay.

Essential Oil Obtaining Procedure
The essential oils were obtained from dried aerial parts by hydrodistillation, using a Neo-Clevenger type apparatus.Then, 100 g powdered plant material was boiled in 1 L water, for 3 h [7,40].

GC-MS Profiling
A GC-MS Shimadzu QP 2010 PLUS Mass Spectrometer coupled with Gas Chromatograph (Shimadzu equipped with an AOC-20i+s injector, and a ZB-5MS Plus capillary column (30 m × 0.25 mm, 0.25 µm film thickness, Phenomenex) was used to perform the analysis of the volatile compounds.The MS transfer line and injector temperatures were adjusted to 250 • C. The oven was programmed at 60 • C for 1 min; then, it was set to rise from 60 • C to 120 • C at a rate of 30 • C per minute.It was then programmed to hold this temperature for 5 min, then to increase from 120 • C to 250 • C at a rate of 5 • C per minute, and from 250 • C to 300 • C at a rate of 20 • C per minute.Helium (99.99990% from Linde Hungary) was used as the carrier gas, flowing at a steady 0.8 mL/min.One µL of injection was made at 250 • C in split mode (20:1).The acquisition range (m/z) of the detector was adjusted at 35 to 800 in scan mode, with the electron impact mode (EI, 70 eV) selected, at an acquisition rate of 500 ms [41].
The resulting compounds were identified by comparing their mass spectra to the NIST (NIST 27, 147 libraries), WILEY library database (>90% match), and by calculating their retention indices (RIs) in relation to n-alkanes (C6-C20).Each volatile compound's relative percentage was calculated as a fraction of its integrated ion area relative to the 100% overall area of the ion chromatogram.For twenty-four hours, each strain was cultured in a test tube with 10 mL of sterile nutritional broth (Oxoid Ltd., Basingstoke, Hampshire, UK) at 37 • C. A loopful of the inoculum was transferred onto selective media, which included Palcam agar (Oxoid Ltd., Basingstoke, Hampshire, UK) for L. monocytogenes, Baird-Parker agar base supplemented with Egg Yolk Tellurite Emulsion for S. aureus, XLD agar for S. enteritidis and TBX agar for E. coli [42].The plates were incubated at 37 • C for 24 h.Sterile saline solution (8.5 g/L) was used to transfer multiple colonies of standard cultures that were grown on Mueller-Hinton agar at 37 • C for 24 h.The colonies were adjusted to meet the turbidity of the McFarland 0.5 standard (1.5 × 108 CFU/mL) (McFARLAND).Optical microscopy was used to confirm the bacterial morphology [43].

Determination of the Minimum Inhibitory Concentration (MIC)
A microtiter-plate-based antibacterial test with resazurin was used to determine the MIC.Stock solutions of the EOs were created by mixing 50% ethanol and Tween 80 (8:1) [41].The first well of a 96-well microtiter plate contained 100 µL of sterile nutrition broth and 100 µL of the sample.To perform serial 11-fold dilutions, 100 µL was moved from one well to another, and 100 µL from the last well in the row was eliminated.Ten µL of inoculum (1.5 × 108 CFU/mL) was added to each well.Gentamicin (0.04 mg/mL in saline solution) was used as a positive control, whereas the negative control was a combination of saline solution, 50% ethanol, and Tween80 (1:8:1).The microplates were incubated at 37 • C for 20 to 22 h.After adding 20 µL of resazurin aqueous solution (0.2 mg/mL) to each well, the microplates were incubated for two hours at 37 • C. The minimum inhibitory concentration (MIC) was the concentration that prevented the blue colour from turning pink.Three replicates were performed for each sample [44].

Determination of the Minimum Bactericidal Concentration (MBC)
The MBC was evaluated by plating 10 µL of aliquot on the Mueller-Hinton medium (Oxoid Ltd., Basingstoke, Hampshire, UK) from the last four wells that showed inhibitory activity against the growth of bacteria during MIC testing.After incubation for 24 h at 37 • C, MBC was determined as the lowest concentration that inhibited bacterial growth.The experiment was performed in triplicate [41,45].4.5.In Vitro Cytotoxic Activity 4.5.1.Cell Lines A panel consisting of 4 cancer cell lines and 2 normal cell lines was chosen for in vitro testing of essential oils: triple-negative breast cancer (MDA-MB-231), lung cancer (A549), liver cancer (HEPG2), colon cancer (HCT116).The HUVEC endothelial cell line and LX-2 hepatic stellate cell line was chosen as normal cell lines.All cell lines were maintained in a humidified chamber at 37 • C and 5% CO 2 .All reagents required for cell culture were purchased from Gibco (Dublin, Ireland).Cell lines included in this study were purchased from American Type Culture Collection (ATCC) and provided by the laboratory biobank [46].

Essential Oil Stock Solution Preparation
The essential oils were dehydrated over anhydrous sodium sulfate and stored at −80 • C until the treatment was applied to the cells.The stock solution was prepared at a concentration of 1 mg/mL by dissolving the required amount of volatile oil in the culture medium for each cell line.

MTT Assay
The cytotoxic effect was studied using the MTT test.Cells were cultivated at a density of 1 × 10 4 cells/well in a 96-well flat-bottom plate, with three duplicates for each treatment.After 24 h of incubation, cells were treated with varying doses of EO (3.91-1000 µg/mL) for 24 h.To remove extract residues that may interfere with the MTT assay, a wash step with phosphate-buffered saline 1× (PBS) was performed.Cell viability was then measured after 5 h of incubation with 100 µL 1 mg/mL MTT.After removing the solution, 150 µL of DMSO was added to dissolve the crystals.The absorbance of viable cells was measured at λ = 570 nm using a SPARK10M multiplate reader (Tecan, Männedorf, Switzerland).Each plate contained a blank control (medium alone) and a positive control (DMSO 100%) [46].

Statistical Analysis
The program Graphpad Prism (version 8) (GraphPad Software, San Diego, CA, USA) was used to conduct the statistical analysis.Cell viability was reported as mean ± SEM and expressed as a percentage of the control group (set at 100%).One-way ANOVA was used to evaluate the data, and then, Dunnett's multiple-comparison test was performed.

Conclusions
This study reported a comparative characterisation of the chemical composition of the EOs obtained from different Agastache sp.cultivated in Romania.The GC-MS profile of the EOs revealed differences in terms of their major classes of compounds depending on species.AR, ARA8 and AF EOs were dominated by oxygenated monoterpenes, whilst AM and AFB EOs were dominated by phenylpropanoids.Menthone was identified as the most abundant compound in AR, ARA8 and AF, whilst estragole was identified as the major compound in AM and AFB.The evaluation of the antiproliferative activity showed that AR and AFB EOs display significant cytotoxic activity against the MDA-MB-231 and HEPG2 tumour cell lines.Regarding the antibacterial activity, the tested EOs were most active against E. coli and S. aureus.
This study indicated that the EOs isolated from different Romanian cultivated Agastache sp.display important phytochemical diversity, showing in vitro antibacterial and cytotoxic potential against different tumour cell lines.These results provide valuable preliminary data regarding the therapeutic potential of Agastache sp.EOs, which warrants further investigation of the mechanism of action involved in the manifestation of the antibacterial and cytotoxic properties and, also, the investigation of these properties in vivo using animal models.

Figure 1 .
Figure 1.The effect of essential oils on cell viability: (a) A. rugosa (AR) EO, (b) A. foeniculum "Aro de Buzău" (AFB) EO.The panel of cell lines includes the triple-negative breast (MDA-MB-231), l (A549), colon (HCT116) and liver (HEPG2) cancer cell lines and normal cell lines represented by HUVEC (human umbilical endothelial cell) line and LX-2 (hepatic stellate cells).Cells were expo to the indicated concentrations of EOs for 24 h.Untreated cells (blank) were exposed only to cul medium, while the positive control was represented by cells treated with 100% DMSO.Cell viab was expressed relative to the negative control (considered 100%) and represented as the mean determinations.Data were analyzed using one-way ANOVA followed by Dunnett's mult comparison test.Asterisks *, **, *** and **** indicate significant differences at p < 0.05, p < 0.01 0.001 and p < 0.0001, respectively, relative to the corresponding control.

Figure 1 .
Figure 1.The effect of essential oils on cell viability: (a) A. rugosa (AR) EO, (b) A. foeniculum "Aromat de Buzău" (AFB) EO.The panel of cell lines includes the triple-negative breast (MDA-MB-231), lung (A549), colon (HCT116) and liver (HEPG2) cancer cell lines and normal cell lines represented by the HUVEC (human umbilical endothelial cell) line and LX-2 (hepatic stellate cells).Cells were exposed to the indicated concentrations of EOs for 24 h.Untreated cells (blank) were exposed only to culture medium, while the positive control was represented by cells treated with 100% DMSO.Cell viability was expressed relative to the negative control (considered 100%) and represented as the mean of 3 determinations.Data were analyzed using one-way ANOVA followed by Dunnett's multiple comparison test.Asterisks *, **, *** and **** indicate significant differences at p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively, relative to the corresponding control.

4. 4 .
In Vitro Antibacterial Activity 4.4.1.Preparation of Microbial Strains The antibacterial activity was tested against Staphylococcus aureus ATCC 6538P, Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076 and Listeria monocytogenes ATCC 19114.Every KWIK-STIK lyophilised microorganism pellet was acquired from Microbiologics, 200 Cooper Avenue North St Cloud, Mn 56303.KWIK-STIKs are widely used for all types of testing of microbiological control.

Table 2 .
Volatile profile of AF EO (expressed as Area %).

Table 3 .
Volatile profile of AFB EO (expressed as Area %).

Table 4 .
Volatile profile of AM EO (expressed as Area %).

Table 5 .
Volatile profile of AR EO (expressed as Area %).

Table 7 .
Antibacterial activity of Agastache species essential oils.