LC-ToF-ESI-MS Patterns of Hirsutinolide-like Sesquiterpenoids Present in the Elephantopus mollis Kunth Extract and Chemophenetic Significance of Its Chemical Constituents

A total of nine sesquiterpenoid lactones together with phenolic compounds and other terpenes were identified from the crude methanol extract of Elephantopus mollis Kunth. Compounds were isolated using different chromatographic techniques and their structures were determined by NMR and IR spectroscopy as well as mass spectrometry. The structures of some detected compounds were assigned based on LC-ToF-ESI-MS screening of main fractions/subfractions from flash chromatography and comparison with isolated analogues as standards. The findings revealed not only the in-source loss of water as the base peak in hirsutinolides but also the in-source loss of corresponding alcohol when the oxygen at position 1 is alkylated. The present study also draws up a complement of data with respect to hirsutinolide-like sesquiterpene lactones whose LC-MS characteristics are not available in the literature. The chemophenetic significance is also discussed. Some of the isolated compounds were reported for the first time to be found in the species, the genus as well as the plant family. The medium-polar fractions of the crude extract, also containing the larger amount of sesquiterpenoid lactones, exhibited activity both against a cancer cell line and bacterial strains. Isolated lactones were also active against the cancer cell line, while the chlorogenic derivatives also valuable in Elephantopus genus showed potent radical scavenging activity. This is the first report of cytotoxic and antibacterial activities of our samples against the tested strains and cell line. The present study follows the ongoing research project dealing with the characterization of taxa with antibacterial and antiparasitic activities from Cameroonian pharmacopeia.


Introduction
Elephantopus mollis Kunth is a plant used for primary healthcare since ancient times. It belongs to Asteraceae and constitutes together with roughly 31 other species the group Elephantopus [1]. E. mollis and E. scaber are the most popular species of the genus [2,3]. They have been screened for their pharmacological and phytochemical profiles [1,4,5]. Elephantopus species are sources of a class of sesquiterpenoid lactones named elephantopustype lactones with a furane ring (C-2/C-5) in germacranolides [1][2][3][4][5][6]. They also produce glaucoside-and guaianolide-type lactones even in less amount alongside phenolic compounds and other terpenes [1,4,5,7]. Sesquiterpene lactones have been proven to be the active principle of the plant against various diseases [1,4,5,7]. However, the efficacy of a plant in ethnopharmacology might be highly depending 45 on the area of collection [8]. In addition, during the chemical profiling by chromatography, 46 low abundant compounds in the extract are most of the time lost, limiting the standardi-47 zation of the plant as phytodrug. To solve such problems, efforts have been focused on 48 highly sensitive methods such as mass spectrometry (MS), which can help to identify most 49 of the constituents of an extract. Pure compounds isolated from the studied plant could 50 alternatively help to establish the MS fingerprint of this group of chemicals [9]. These 51 standards could then provide an almost exhaustive chemical profile of a taxa [9]. Accord-52 ingly, few reports are available in the literature on LC-MS characteristics of different ses-53 quiterpenoid groups found in Elephantopus genus [9]. In our attempt to elucidate the cy-54 totoxicity and antibacterial properties of E. mollis, we isolated and identified/detected re-55 ported sesquiterpenoid lactones by LC-MS. The results could be relevant in view of for-56 mulating the plant extract to relieve pains and diseases in less developing countries where 57 the use of traditional medicine still prevails. The present study follows the ongoing re-58 search project dealing with the characterization of taxa with antibacterial and antiparasitic 59 activities from Cameroonian rain forests and pharmacopeia [8,[10][11][12]. 60

LC-MS Analysis of Fractions for Structure Identification
A non-exhaustive analysis of the LC-ToF-ESI-MS profiles of main middle-polar fractions from flash chromatography of the methanol extract led to the detection of further sesquiterpene lactones (1a and 4a), chlorogenic acid derivatives (10a) and carbohydrates (20a-21a) ( Figure 2).  (6), molephantinin (7) [15], isochlorogenic acid (8) [16], 1,5-dicaffeoylquinic 70 acid (9) [17], 4,5-dicaffeoylquinic acid (10) [17], apigenin (11) [18], luteolin (12) [19], tricin 71 (13) [20], 2,6-dimethoxy-4-hydroxybenzoic acid (14) [21], 4-formylsyringol (15) [22], cryp-72 tomeridiol (16) (17) [24], β-sitos-73 terol-3-O-β-D-glucopyranoside (18) [24] and glycerine monopalmitate (19) [24]. glaucolide-type sesquiterpenoid lactones, the two sub-classes isolated in our study [9]. As 83 a result, hirsutinolide skeletons give rise to an extremely low abundance of the quasi-mo-84 lecular ion [M + H] + (2-10% RA) and sodium adduct [M + Na] + (10-20% RA) while the base 85 peak is the in-source water loss ion [M + H − H2O] + . These patterns are quite different for 86 glaucolide analogues where the base peak is the protonated molecule [M + H] + whereas 87 the in-source water loss peak is absent even when the compound bears free hydroxy 88 groups. We applied the isolated compounds 1 and 7 to ESI-MS analysis with the aim to 89 confirm these features (Figures S1-S2, supplementary data). Under the experimental con-90 ditions, the lactones 1 afforded a base peak at m/z 329.1 corresponding to the ion [M + H -91 H2O] + together with the quasi-molecular ion [M+H] + peak of low intensity at m/z 347.1 (6% 92 RA) and the sodium adduct ion [M + Na] + at m/z 369.1 (4% RA). 93 Compound 2, also belonging to hirsutinolide-like lactones, showed an in-source loss 94 of methanol [M + H -MeOH] + as the most intense peak, while the proton and sodium 95 adducts were less abundant. This variation in the fragmentation patterns of hirsutinolide-96 like lactones 1 and 2 could result from the methylation in 2 of the sole hydroxy group in 1 97 ( Figure S3, supplementary data). On the other hand, the mass spectrum of compound 7 98 presented the quasi-molecular ion [M + H] + at m/z 361.1 as the base peak, while the sodium 99 adduct [M + Na] + and the in-source water loss ion [M + H -H2O] + were absent. The quasi-100 molecular ion peaks of the lactones 1 and 2 were accompanied by the fragmentation peak 101 Da Silva et al. (2020) reported the ESI-QToF-MS characterization of hirsutinolide-and glaucolide-type sesquiterpenoid lactones, the two sub-classes isolated in our study [9]. As a result, hirsutinolide skeletons give rise to an extremely low abundance of the quasimolecular ion [M + H] + (2-10% RA) and sodium adduct [M + Na] + (10-20% RA) while the base peak is the in-source water loss ion [M + H − H 2 O] + . These patterns are quite different for glaucolide analogues where the base peak is the protonated molecule [M + H] + whereas the in-source water loss peak is absent even when the compound bears free hydroxy groups. We applied the isolated compounds 1 and 7 to ESI-MS analysis with the aim to confirm these features ( Figures S1-S2 Compound 2, also belonging to hirsutinolide-like lactones, showed an in-source loss of methanol [M + H -MeOH] + as the most intense peak, while the proton and sodium adducts were less abundant. This variation in the fragmentation patterns of hirsutinolidelike lactones 1 and 2 could result from the methylation in 2 of the sole hydroxy group in 1 ( Figure S3, supplementary data). On the other hand, the mass spectrum of compound 7 presented the quasi-molecular ion [M + H] + at m/z 361.1 as the base peak, while the sodium adduct [M + Na] + and the in-source water loss ion [M + H -H 2 O] + were absent. The quasimolecular ion peaks of the lactones 1 and 2 were accompanied by the fragmentation peak at m/z 243.1 also as the base peak. This ion might be generated from the loss of the methacrylic acid (MeacrOH, 86 Da) followed by elimination of H 2 O (18 Da) from 1 and MeOH from 2. These patterns could help numbering hydroxy and methoxy groups in hirsutinolide-like lactones. Da Silva et al. (2020) also found similar fragmentation patterns when studying hirsutinolides under ESI conditions [9]. The fragmentation pattern is compiled in Figure 3.  [9]. The fragmentation pattern is compiled in Figure 3.  The different main fractions from the crude extract were analyzed to detect potentia non-isolated lactones of the extract. The LC-chromatogram of fraction B mainly showed four peaks (I-IV) ( Figure S4, supplementary data). Peaks I-II and III-IV showed simila patterns to compound 1 for the former and to compound 2 for the latter meaning that I II could be hirsutinolide-like lactones hydroxylated at C-2 while III-IV could correspond to methoxylated ones as previously described. The ESI spectra of compounds 1-3 wer superimposable to that of peaks I, III and II, respectively. The unattributed peak IV (1a Figure 2) has been tentatively considered as the methylated analogues of compound 2 a C-2, with the molecular formula C21H26O6 [13].
In addition to the Na + adduct at m/z 397.1 (19.1%), the LC-ESI-MS of peak IV showed the most stable peak at m/z 343.1 corresponding to an in-source loss of methanol [M + H MeOH] + and the quasi-molecular ion peak [M + H] + with low intensity (17.0%) ( Figure 4) Each of the peaks I-IV gave a fragment ion at m/z 243.1 characteristic for the above men tioned losses supporting the assignment of structures to peaks as proposed ( Figure 3).  The different main fractions from the crude extract were analyzed to detect potential non-isolated lactones of the extract. The LC-chromatogram of fraction B mainly showed four peaks (I-IV) ( Figure S4, supplementary data). Peaks I-II and III-IV showed similar patterns to compound 1 for the former and to compound 2 for the latter meaning that I-II could be hirsutinolide-like lactones hydroxylated at C-2 while III-IV could correspond to methoxylated ones as previously described. The ESI spectra of compounds 1-3 were superimposable to that of peaks I, III and II, respectively. The unattributed peak IV (1a, Figure 2) has been tentatively considered as the methylated analogues of compound 2 at C-2, with the molecular formula C 21 H 26 O 6 [13].
In addition to the Na + adduct at m/z 397.1 (19.1%), the LC-ESI-MS of peak IV showed the most stable peak at m/z 343.1 corresponding to an in-source loss of methanol [M + H -MeOH] + and the quasi-molecular ion peak [M + H] + with low intensity (17.0%) ( Figure 4). Each of the peaks I-IV gave a fragment ion at m/z 243.1 characteristic for the above mentioned losses supporting the assignment of structures to peaks as proposed ( Figure 3).  [9]. The fragmentation pattern is compiled 106 in Figure 3. Figure 3. Proposed fragmentation patterns in hirsutinolide-like lactones under ESI conditions. 109 The different main fractions from the crude extract were analyzed to detect potential 110 non-isolated lactones of the extract. The LC-chromatogram of fraction B mainly showed 111 four peaks (I-IV) ( Figure S4, supplementary data). Peaks I-II and III-IV showed similar 112 patterns to compound 1 for the former and to compound 2 for the latter meaning that I-113 II could be hirsutinolide-like lactones hydroxylated at C-2 while III-IV could correspond 114 to methoxylated ones as previously described. The ESI spectra of compounds 1-3 were 115 superimposable to that of peaks I, III and II, respectively. The unattributed peak IV (1a, 116 Figure 2) has been tentatively considered as the methylated analogues of compound 2 at 117 C-2, with the molecular formula C21H26O6 [13].

158
In contrast with ESI patterns of compounds 1-2, peaks VI, IX and XIV showed the 159 double molecule-Na + complex ion [2M + Na] + at m/z 815.2, 751.2 (32% RA) and 779.2 (26% 160 RA), respectively. All these similarities, when combining ions occurring for hirsutinolides 161 under ESI conditions and the double molecule sodium adduct ions, are indicative of sim-162 ilar core skeleton shared by peaks VI, XIV and compound 4 (peak IX). One could also 163 notice a difference in relative abundances of protonated, sodium adduct and water loss 164 In contrast with ESI patterns of compounds 1-2, peaks VI, IX and XIV showed the double molecule-Na + complex ion [2M + Na] + at m/z 815.2, 751.2 (32% RA) and 779.2 (26% RA), respectively. All these similarities, when combining ions occurring for hirsutinolides under ESI conditions and the double molecule sodium adduct ions, are indicative of similar core skeleton shared by peaks VI, XIV and compound 4 (peak IX). One could also notice a difference in relative abundances of protonated, sodium adduct and water loss ions when the hydroxy groups in elephantane-type skeleton, such as in 4 or 4a, are free or substituted. This is the first report of elephantane-type lactone ESI-patterns that could serve as standards for the detection and characterization of hirsutinolide-like sesquiterpenoids in complex mixtures such as plant extracts using ESI-MS facilities.
The main fraction P1E was constituted in large extent by carbohydrates as proven by the MS profile of peaks ranging from t R 0.847-1.202 min ( Figures S9-10, supplementary data) with quasi-molecular ions [M + Na] + and [M + K] + at m/z 203.1 and 219.1; at m/z 365.1 and 381.1, and at m/z 527.1 and 543.1, respectively. They correspond to a mono-, di-and trisaccharide tentatively attributed to glucose (20a) and sucrose (21a) for the first two, while the third one was not assigned. Compounds 20a and 21a were also identified by exploiting the NMR data of the mixture of these compounds. However, it was not possible to elucidate the structure of the trisaccharide (Figure 2).

Biological Endpoints of Compounds and Fractions
The crude extract, the flashed main fractions (P1A-P1E) and isolated compounds in high yields (1-4, 8-10) were evaluated against the cervix carcinoma cell line KB-3-1. The crude extract showed low activity with IC 50 > 100 mg/mL. The middle-polar fractions P1B and P1C were, however, more potent to inhibit the growth of the cell line with IC 50 = 87 µM for P1B and IC 50 = 21 µM for P1C compared to the reference drug griseofulvin (IC 50 = 17-21 µM). Among the isolated compounds, only 2 and 3 were weakly active against KB-3-1 cells with IC 50 = 22.6 µM for 2 and 45.2 µM for 3.
Likewise, the samples were also screened against a panel of Gram-negative (Escherichia coli DSMZ 1058 and Pseudomonas agarici DSMZ 11810) and Gram-positive microorganisms (Micrococcus luteus DMSZ 1605, Staphylococcus warneri DSMZ 20036 and Bacillus subtilis DSMZ 704). The crude extract showed relatively low potential to inhibit the growth of the bacterial strains (DZI < 7 mm) at concentrations of 0.5 mg/mL. Nevertheless, and for the same concentrations, the middle-polar fractions P1B and P1C showed potent antibacterial properties against four of five strains tested including P. agarici (DZI = 15-16 mm), M. luteus (DZI = 13-15 mm), S. warneri (DZI = 10-11 mm) and B. subtilis (DZI = 14-15 mm). According to the MIC endpoints, P1B and P1C were moderately active against P. agarici and B. subtilis with MIC ranging from 25-54 µg/mL while the activity was low against S. warneri (MIC > 200 µg/mL) with 0.8-0.0015 mg/mL for each one. Conversely, fractions P1A and P1D showed moderate activity with DZI of 8 mm against E. coli and B. subtilis for P1A and DZI of 8-9 mm towards B. subtilis and P. agarici for P1D. The other strains were not sensitive to the tested samples including P1E. Our results were compared to those of gentamycin (reference drug), DZI = 17-21 mm at 0.5 mg/mL and MIC = 1.6-2.1 µg/mL. Likewise, only compound 2 showed a low activity against P. agarici DSMZ 11810 (DZI = 9 mm), M. luteus DMSZ 1605 (DZI = 7 mm) and B. subtilis DSMZ 704 (DZI = 8 mm). The isolated compounds were also screened for their scavenging effects against the radical DPPH. Amongst the tested samples, only compounds 8 and 10 displayed moderate activity with IC 50 of 118 and 34 µg/mL, respectively, compared to the reference drug trolox (IC 50 = 20 µg/mL). The absence of activity of compound 9 could be related to its insolubility in DMSO used for these assays as solvent.

Discussion
To date, more than 20 sesquiterpenoid lactones have been reported from Elephantopus mollis worldwide [1,4,5,13,15,26]. Chemophenetic uses chemical ingredients to define new clades that were previously not recognized or cannot be defined based on morphology [27]. In general, germacranolides and guaianolides are the chemophenetic markers of the studied plant genus [1,4,5,13,15,26,28]. Nine of them were identified or detected in the frame of our study, while compound 3 is being reported for the first time in the species and even in the genus Elephantopus. When comparing Cameroonian and Chinese species, one could highlight that seven of the lactones encountered so far were unique in the Chinese species while three were present only in the Cameroonian variant [5,13,15]. The Chinese species highlighted derivatives where substituents such as ethoxy at C-4 in compound 4 or angelate or tiglate at C-8 in compound 6 instead of hydroxy and acrylate, respectively, and a second lactone moiety due to the oxidized C-15/C-2 positions or the methoxylation of the αmethylene butyrolactone in 1 and 2 [5]. Some sesquiterpenoid skeletons already relayed in the literature such as guaianolide-type sesquiterpene lactones were not encountered in the present study. Our study is also highlighting the occurrence of an elanane-type sesquiterpene, compound 16, in the genus Elephantopus. This class of sesquiterpenes has been reported in some species of the Asteraceae plant family including Vernonia.
Our study also points out the occurrences of phenolic acids (14-15), flavonoids (11-13) and quinic acid derivatives (8)(9)(10) in Elephantopus species. Apart from compound 10 reported in E. mollis and compounds 8, 11 and 12 reported in E. scaber, each of these phytoconstituents are herein reported for the first time in the genus Elephantopus [28,29]. Methoxylated flavonoids are abundant in Asteraceae species while dicaffeoylquinic acids are scarce [24]. The occurrences of phenolic acids in E. mollis could be related to the presence of quinic acid since the latter is an intermediate in the biosynthesis of the former. Some chemophenetic markers of Asteraceae plant species namely the steroids 17, the carbohydrates 21a and the monoglyceride 19, were also elucidated [24]. The monoglyceride was isolated for the first time in the studied genus.
Overall, the samples were moderately active against both bacterial strains and cancer cell line used. The studied plant and sesquiterpene lactones are reputed for their anticancer and anti-inflammatory activities because of the α-methylene-γ-butyrolactone moiety in their structures [5,6,8,13,15]. The MeOH extract of E. mollis has been reported to exhibit significant activity against a panel of colorectal, liver and breast carcinoma [30]. We report herein for the first time the cytotoxicity of the plant towards the cervix carcinoma cell line KB-3-1. The results obtained from the middle-polar main fractions P1B and P1C were similar to those reported for the EtOAc extract of E. mollis against the liver carcinoma HepG2 [3]. Compounds 2, 4a, 3 and 7 have been reported to be active against leukemia K562 and HL−60 and neuroblastoma B104 cell lines with IC 50 less than 5 µM [13,15,31]. As for the extract, this is the first report on the cytotoxicity of both main fractions and sesquiterpenoid lactones against the cancer cell lines KB-3-1. Likewise, we highlight here for the first time the antibacterial activity of the samples against the tested strains.
Nonetheless, only disubstituted quinic acid shows antiradical scavenging effect toward DPPH probably due to caffeoyl moieties as indicated in the literature. The radical scavenging activity in the chlorogenic series increased with the number of caffeoyl substituents. Compound 10 was the most active antioxidant constituent as reported in the literature [28]. The other sesquiterpenes and compounds were not active. Our results confirm readily data available in the literature on the study plant E. mollis [13,15,26,32]. The active constituents of this plant have been identified to sesquiterpenoid lactone which is known to bind sulfhydryl-containing enzymes and proteins in pathogens resulting in a programmed death of organisms [13,15,26,32]. Apart from their antioxidant capacities, caffeoyl derivatives of quinic acids are well-known for their antiviral potency and extracts of E. scaber have been already patented to alleviate virus form of symptoms of diseases [33].

Plant Material
The whole plant of Elephantopus mollis was collected on the savanna hills of Bamendjing (Mbouda) in the western Region of Cameroon in December 2019. The identification was performed by Mr. Victor Nana, an experienced botanist, at the Cameroon National Herbarium (Yaoundé) by comparison with the voucher specimen kept under the voucher number 35121/HCN.

Extraction and Purification
Dried materials of Elephantopus mollis (1.0 kg) were ground and macerated with methanol (3 × 5 L, 72 h each) at room temperature to yield a semi-solid crude extract

Bioactivity
The antibacterial activity of some isolated compounds was carried out by using the agar disk diffusion method to determine the diameter zone of inhibition (DZI) against five bacteria including two Gram-negative (Escherichia coli DSMZ 1058 and Pseudomonas agarici DSMZ 11810) and three Gram-positive (Micrococcus luteus DMSZ 1605, Staphylococcus warneri DSMZ 20036 and Bacillus subtilis DSMZ 704) strains [10,11]. The microorganisms were provided by DSMZ (German Collection of Microorganisms and Cell Cultures).
The cytotoxicity assay was performed with the human cervix carcinoma cell line KB-3-1 as previously reported [10,11]. The KB-3-1 cells were provided by DSMZ (number ACC 158) and cultivated as a monolayer in DMEM (Dulbecco's modified Eagle medium) with glucose (4.5 g/L), L-glutamine, sodium pyruvate and phenol red, supplemented with 10% (KB-3-1) foetal bovine serum (FBS). On the day before the test, the cells (70% confluence) were detached with trypsin-ethylenediamine tetraacetic acid (EDTA) solution (0.05%; 0.02% in DPBS) and placed in sterile 96-well plates in a density of 10,000 cells in 100 µL medium per well. The dilution series of the compounds were prepared from stock solutions in DMSO of concentrations of 1 mM or 10 mM. The stock solutions were diluted with culture medium (10% FBS) at least 50 times. Some culture medium was added to the wells to adjust the volume of the wells to the wanted dilution factor. The dilution prepared from stock solution was added to the wells and each concentration was tested in six replicates. The control contained the same concentration of DMSO as the first dilution. After incubation at 37 ºC and 5.3% CO 2 -humidified air for 72 h, 30 µL of an aqueous resazurin solution (175 µM) was added to each well. The cells were incubated for 6 h at the same conditions. Thereafter, the fluorescence was measured. The excitation was affected at a wavelength of 530 nm, whereas the emission was recorded at 588 nm. The IC 50 that is the values equal the drug concentrations, at which vitality is 50%, were calculated as a sigmoidal dose response curve using GRAPHPAD PRISM 4.03. The antioxidant activity of compounds using DDPH was determined according to the methods described by Nguemo et al. 2020 [34].

Data Availability Statement:
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher