Isolation and LC-QToF Characterization of Secondary Metabolites from an Endemic Plant Artemisia heptapotamica Poljak

Phytochemical investigation of the aerial parts of Artemisia heptapotamica Poljak led to the isolation of ten known compounds, including four alkyl p-coumarates: octadecyl trans-p-coumarate (1), icosy trans-p-coumarate (2), docosyl trans-p-coumarate (3), and tetracosyl trans-p-coumarate (4), one sesquiterpene lactone: santonin (5), four flavonoids; axillarin (6), quercetin 3-O-methyl ether (7), luteolin (8), and quercetin (9), and one phenolic acid derivative: p-coumaric acid (10). The structures of the isolated compounds were identified by various spectroscopic analyses. Additionally, the antimicrobial activity of the total extract and different fractions was screened, and they exhibited no inhibition of the growth of Candida albicans, C. neoformans, Aspergillus fumigatus, methicillin-resistant Staphylococcus aureus (MRS), E. coli, Pseudomonas aeruginosa, Klebsiella pneumonia, and Vancomycin-resistant Enterococci (VRE) at the tested concentrations ranging from 8 to 200 μg/mL. The identification and tentative characterization of the secondary metabolites were conducted using LC-QToF analysis. This method helps in the putative characterization of sesquiterpene lactones, flavonoids, coumarate derivatives, and aliphatic compounds. The developed method identified 43 compounds, of which the majority were sesquiterpene lactones, such as eudesmanolides, germacranolides, and guaianolide derivatives, followed by flavonoids. The proposed LC-QToF method helps develop dereplication strategies and understand the major class of chemicals before proceeding with the isolation of compounds.


Introduction
Artemisia L. is the largest genus belonging to the family Asteraceae [1]. This genus is known for its essential oils with aromatic and medicinal properties, which are used in traditional medicine as well as in modern scientific medicinal practices [2,3]. Wormwood is widespread and widely found across geographical areas: in the temperate zone of Eurasia, North and South Africa, Europe, the Middle East, Afghanistan, Pakistan, China, Korea, Japan, and India (Himalayas). Plenty of species are found in Russia (174 species), mainly in Yakutia (22), Siberia (70), and Buryatia (46), and also in China (200) [4]. Approximately 500 species of wormwood are known worldwide, and 81 species grow in Kazakhstan. However, only 30 of Kazakhstan's wormwood species have been studied from various biological, ecological, and chemical perspectives. [5]. Extracts of Artemisia species improve digestion, stimulate appetite, and are used to treat dyspepsia, acid gastritis, gastrointestinal tract diseases, liver diseases, gall bladder problems, insomnia, malaria, influenza, and upper respiratory tract ailments. They have also been used to treat bronchial asthma, rheumatism, eczema, dysentery, anemia, jaundice, obesity, meteorism, migraine, hypertension, and tuberculosis. Artemisia species have been found to have various pharmacological activities, such as anthelmintic, antimicrobial, anti-inflammatory, antitumor, antioxidant, cytostatic, antifungal, antimalarial, antileishmaniasis, antinociceptive, immunomodulatory, and antipyretic activity, as well as potent inhibitory activity against FPTase [6]. One of the endemic Artemisia plants is Artemisia heptapotamica Poljak. There are few chemical studies on this plant, and a recent study showed the presence of methyl ether of quercetin [7], in addition to monomeric and dimeric sesquiterpene lactones from this plant. Most isolated monomeric sesquiterpenes showed strong inhibition of the lipopolysaccharide (LPS)-induced NF-κB activation in a THP1-Dual cell model [8]. This study aimed to isolate the compounds from the whole plant and characterize them using NMR and LC-QToF analysis, in addition to evaluation of the antimicrobial activity of the total extract and different fractions.

Antimicrobial Activity
The antibacterial and antifungal activities of the total extract, as well as n-hexane and EtOAc fractions of the aerial parts of A. heptapotamica, were studied. They exhibited no inhibition of the growth of Candida albicans, C. neoformans, Aspergillus fumigatus, methicillinresistant Staphylococcus aureus (MRS), E. coli, Pseudomonas aeruginosa, Klebsiella pneumonia, and Vancomycin-resistant Enterococci (VRE) at the tested concentration ranging from 8 to 200 µg/mL. Although different studies have shown that quercetin, its derivatives, and luteolin had broad-spectrum antibacterial and antifungal properties [17,18], the methanolic extract and different fractions displayed no activity on the tested microorganisms due to the small concentration used in the assay.

Identification and Tentative Characterization of Secondary Metabolites Using LC-QToF
The secondary metabolites from aerial parts of A. heptapotamica (methanolic extract) were separated using liquid chromatography, followed by their characterization using timeof-flight mass spectrometry. The mass accuracy for the putatively identified compounds was less than 4 ppm error. The identified compounds presented in Table 1 consisted of sesquiterpene lactones and flavonoids in the majority. In addition, coumarate derivatives and p-coumaric acid were also identified. Sesquiterpene lactones were detected in an ESI-positive ionization mode with [M + NH 4 ] + and [M + Na] + adduct precursor ions. The tentative characterization of compounds was processed based on the molecular features, such as accurate mass, fragment ions (neutral ions), and precursor ion molecular formula. The representative base peak chromatograms (BPC) in negative and positive modes, along with LC-DAD profiles at 210 nm, are shown in Figure 2.  [19]. Furthermore, based on the high-resolution mass spectrometric data, corresponding fragment ions of different types of sesquiterpene backbone skeletons were observed, i.e., eudesmanolides, germacranolides, and guianolide derivatives. The molecular features depicted in Table 1 provided further confirmation of the various sesquiterpene moieties. A total of twenty-two compounds of sesquiterpene lactones were tentatively characterized. The identified compounds were reported in various Artemisia species. The m/z values of the tentatively characterized sesquiterpenes are shown in Table 1 under the guidance of reported mass fragments and isolation reports (from the dictionary of natural products) [20][21][22][23][24]. Based on the fragmentation pattern of santonin, remaining sesquiterpene lactones were identified and tentatively characterized.

Flavonoids (23-36)
Using the isolated reference compounds and exact mass measurements, fourteen flavonoid compounds were identified. Most of the compounds are flavone derivatives. There are two quercetin derivatives, along with quercetin and luteolin. Flavonoids have established mass fragmentation patterns based on the literature previously reported [20,[22][23][24][25].
The flavone derivatives showed that the fragmentation starts with the loss of functional groups, such as water, methoxy group, and -glc. The aglycone molecular weight helps in understanding the backbone skeleton.  [26], respectively. In addition, non-polar compounds (compounds 40-43) were matched with isolated standards. The corresponding adduct ions in the negative mode, along with their retention times, are listed in Table 1.   The isolated compounds, as well as chromatographic peaks with distinctive fragment ions, were confirmed based on the reported literature studies whose corresponding references are listed in Table 1. Furthermore, compounds whose fragment ions were not observed in this study were tentatively identified based on their molecular features and database searches such as from the dictionary of natural products and other literature of isolated compounds from Artemisia species.

Plant Material
The whole plant of A. heptapotamica was collected in October 2021 from Kokpek village, Almaty, Kazakhstan. Identification and authentication were performed by Dr. Danilov Mikhail Petrovich. The sample was stored with the voucher number 0000723 in the Main Botanical Garden of the Institute of Botany and Phyto-introduction, Almaty, Kazakhstan. The same sample was assigned with the NCNPR number 25173 and stored in the National Center for Natural Product Research Botanical Repository, University of Mississippi, USA.

Extraction and Isolation
The collected aerial parts of A. heptapotamica were first dried in the shade and then crushed into small pieces. The dried plant material (1.65 kg) was extracted by maceration with 95% methanol three times at room temperature and was concentrated under reduced pressure to yield 253.35 g of the total extract. The total extract was mixed with a small amount of distilled water and successively fractionated with n-hexane and ethyl acetate. The fractions were concentrated under reduced pressure to produce n-hexane (29.1 g) and ethyl acetate fractions (58.74 g).

Evaluation of Antimicrobial Activity
The antimicrobial activity of the total extract and different fractions was evaluated using the method reported by Samy et al. [32].

Liquid Chromatography-Diode Array Detector-Quadrupole Time-of-Flight Mass Spectrometry (LC-DAD-QToF)
About 25 mg of extract was sonicated in 1.0 mL of methanol for 5 min, followed by centrifugation for 15 min at 7000 rpm. The clear filtered supernatant solution was used for analysis. The liquid chromatographic system was an Agilent Series 1290, and separation was achieved on an Acquity UPLC TM HSS C18 column (100 mm × 2.1 mm I.D., 1.8 µm). The mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) at a flow rate of 0.23 mL/min. Analysis was performed using the following gradient elution: 15% B to 40% B in 30 min, then to 100% B in the next 15 min. Each run was followed by a 5 min wash with 100% B and an equilibration period of 15 min with 85% A/15% B. Two microliters of the sample were injected. The column temperature was 40 • C.
The mass spectrometric analysis was performed with a QToF-MS-MS (Model #G6545B, Agilent Technologies, Santa Clara, CA, USA) equipped with an ESI source with Jet Stream technology, using the following parameters: drying gas (N 2 ) flow rate, 13 L/min; drying gas temperature, 325 • C; nebulizer pressure, 30 psi; sheath gas temperature, 300 • C; sheath gas flow, 11 L/min; capillary voltage, 3500 V; nozzle voltage, 0 V; skimmer, 65 V; Oct RF V, 750 V; and fragmentor voltage, 125 V. All the operations, acquisition of data, and analysis of data were controlled using Agilent MassHunter Acquisition Software ver. A.10.1 and processed with MassHunter Qualitative Analysis software ver. B.07.00. Each sample was analyzed in positive and negative modes over the range of m/z 50-1700 and an extended dynamic range. Accurate mass measurements were obtained by employing ion correction techniques using reference masses at m/z 121.0509 (protonated purine) and 922.0098 (protonated hexakis [1H, 1H, 3H-tetrafluoropropoxy] phosphazine or HP-921) in positive ion mode, while m/z 112.9856 (deprotonated trifluoroacetic acid-TFA) and 1033.9881 (TFA adducted HP-921) were used in negative ion mode. Samples were analyzed in all-ion MS-MS mode, where experiment 1 was carried out with a collision energy of zero and experiment 2 with a fixed collision energy of 45 eV.

Conclusions
Ten compounds were isolated from the ethyl acetate fraction of A. heptapotamica, including four flavonoids, four alkyl coumarate, one sesquiterpene lactone, and one phenolic acid. Additionally, chemical characterization of the methanolic extract of A. heptapotamica using LC-QToF analysis led to the identification of 43 compounds, of which sesquiterpene lactones were the major secondary metabolites, followed by flavonoids. The chemical characterization of the methanolic extract of A. heptapotamica also showed the presence of the isolated compounds (1-10), which matched with the authentic samples of A. heptapotamica.