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Investigation of Chemical Constituents of Eranthis longistipitata (Ranunculaceae): Coumarins and Furochromones

Aqueous-ethanol extracts (70%) from the leaves of Eranthis longistipitata Regel. (Ranunculaceae Juss.)—collected from natural populations of Kyrgyzstan—were studied by liquid chromatography with high-resolution mass spectrometry (LC-HRMS). There was no variation of the metabolic profiles among plants that were collected from different populations. More than 160 compounds were found in the leaves, of which 72 were identified to the class level and 58 to the individual-compound level. The class of flavonoids proved to be the most widely represented (19 compounds), including six aglycones [quercetin, kaempferol, aromadendrin, 6-methoxytaxifolin, phloretin, and (+)-catechin] and mono- and diglycosides (the other 13 compounds). In the analyzed samples of E. longistipitata, 14 fatty acid–related compounds were identified, but coumarins and furochromones that were found in E. longistipitata were the most interesting result; furochromones khelloside, khellin, visnagin, and cimifugin were found in E. longistipitata for the first time. Coumarins 5,7-dihydroxy-4-methylcoumarin, scoparone, fraxetin, and luvangetin and furochromones methoxsalen, 5-O-methylvisammioside, and visamminol-3′-O-glucoside were detected for the first time in the genus Eranthis Salisb. For all the above compounds, the structural formulas are given. Furthermore, detailed information (with structural formulas) is provided on the diversity of chromones and furochromones in other representatives of Eranthis. The presence of chromones in plants of the genus Eranthis confirms its closeness to the genus Actaea L. because chromones are synthesized by normal physiological processes only in these members of the Ranunculaceae family.


Introduction
Plant extracts are most often multicomponent mixtures and, therefore, it is difficult to analyze individual substances there. Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used as a powerful tool for the analysis of substances in plant extracts. Recently, with the development of various analytical methods, such as high-resolution mass spectrometry (HRMS), it became possible to quickly and selectively identify components in extracts. Due to its high accuracy and analytical sensitivity, LC-HRMS greatly facilitates the identification of known and unknown compounds in extracts [1,2]. Furthermore, this method does not require a large amount of plant material for the assay; this feature is undoubtedly suitable for studying plants with a small geographic range and limited occurrence in nature. Such plants include almost all members of the genus Eranthis Salisb.
The genus Eranthis belongs to Ranunculaceae Juss. Tribe Cimicifugeae Torr. & A. Grey [3]. This genus consists of 10 to 13 early flowering herbaceous perennial species that are distributed across Southern Europe and Western, Central, and temperate Asia [4][5][6][7]. Species of this genus have rarely been the subject of phytochemical research. Examination of the literature indicates that triterpene glycosides and saponins have been studied (along with their biological activity) in E. cilicica Schott & Kotschy [8,9]. The set of flavonoids has been investigated in E. sibirica DC., E. stellata Maxim., E. longistipitata Regel., and E. tanhoensis Erst [10,11]. Nonetheless, the chromones that were found in E. cilicica, E. pinnatifida Maxim., and E. hyemalis (L.) Salisb. Are the most intriguing finding [12][13][14][15][16], and the same is true for another family of benzo-α-pyrone derivatives: coumarins. In contrast to other classes of Eranthis chemical constituents, which are widespread in the plant kingdom and have been studied sufficiently, the limited occurrence of chromones and coumarins and their pharmacological activity are of much interest.
The aim of this work was to identify numerous chemical compounds in extracts from E. longistipitata leaves by LC-HRMS for subsequent chemotaxonomic, phytochemical, and pharmacological studies.

Results
Compounds belonging to such classes as amino acids, flavonoids, and fatty and organic acids-as well as their derivatives, alcohols, sugars, coumarins, furochromones, and others-were analyzed by mass spectrometry (LC-HRMS) in E. longistipitata leaves (in 70% aqueous-ethanol extracts) from natural populations growing in Kyrgyzstan (Table 1 and Figure 1). For identification of compounds, the mass spectrometry (MS) data acquired in negative and positive electrospray ionization (ESI − /ESI + ) modes were compared with information in the mzCloud database. There was a ≤99.9% match of the mass spectra between the compounds obtained by us and compounds from the mzCloud database (Table 1). A more detailed procedure for identifying substances is described in Ref. [11].  [4][5][6][7]. Species of this genus have rarely been the subject of phytochemical research. Examination of the literature indicates that triterpene glycosides and saponins have been studied (along with their biological activity) in E. cilicica Schott & Kotschy [8,9]. The set of flavonoids has been investigated in E. sibirica DC., E. stellata Maxim., E. longistipitata Regel., and E. tanhoensis Erst [10,11]. Nonetheless, the chromones that were found in E. cilicica, E. pinnatifida Maxim., and E. hyemalis (L.) Salisb. Are the most intriguing finding [12][13][14][15][16], and the same is true for another family of benzo-α-pyrone derivatives: coumarins. In contrast to other classes of Eranthis chemical constituents, which are widespread in the plant kingdom and have been studied sufficiently, the limited occurrence of chromones and coumarins and their pharmacological activity are of much interest.
The aim of this work was to identify numerous chemical compounds in extracts from E. longistipitata leaves by LC-HRMS for subsequent chemotaxonomic, phytochemical, and pharmacological studies.

Results
Compounds belonging to such classes as amino acids, flavonoids, and fatty and organic acids-as well as their derivatives, alcohols, sugars, coumarins, furochromones, and others-were analyzed by mass spectrometry (LC-HRMS) in E. longistipitata leaves (in 70% aqueous-ethanol extracts) from natural populations growing in Kyrgyzstan (Table 1 and Figure 1). For identification of compounds, the mass spectrometry (MS) data acquired in negative and positive electrospray ionization (ESI − /ESI + ) modes were compared with information in the mzCloud database. There was a ≤99.9% match of the mass spectra between the compounds obtained by us and compounds from the mzCloud database (Table  1). A more detailed procedure for identifying substances is described in Ref. [11].

Discussion
In the raw data on metabolites from E. longistipitata leaves, more than 160 compounds were found, of which, 72 were identified to the class level ( Figure 1) and 58 to the individualcompound level (Table 1). After a comparison of E. longistipitata metabolomes among different populations, it turned out that these profiles of metabolites do not differ and are characterized by the presence of such classes of compounds as flavonoids, fatty acidrelated compounds, amino acid-related compounds, organic acids, sugars, alcohols, phenyl-propanoids, coumarins, furochromones, terpenoids, and quinones. Among the classes of substances that were identified in the leaves of E. longistipitata, the highest diversity was documented for flavonoids (27% of all compounds) and fatty acid-related compounds (21%). Furochromones showed lower diversity (9%), as did terpenoids, amino acid-related compounds, and sugars (8% each), coumarins (7%), and quinones (6%). Other classes of compounds were not so diverse in the extracts of this plant: organic acids (4% of all compounds), alcohols (1%), and phenylpropanoids (1%). We failed to find metabolomic data on Eranthis representatives, including E. longistipitata, in the literature.

Flavonoids
The class of flavonoids proved to be most widely represented (19 compounds), among which there were only six aglycones [quercetin, kaempferol, aromadendrin, 6-methoxytaxifolin, phloretin, and (+)-catechin] and the remaining flavonoids were mono-and diglycosides, whose sugar moiety consisted of glucose, galactose, arabinose, or other sugars. We reported about the specific features of E. longistipitata flavonoids in more detail in our previous article [11]. In addition, here we were able to identify a quercetin diglycoside: quercetin-6-O-β-D-xylopyranosyl-β-D-glucopyranoside (Table 1).

Fatty-Acid-Related Compounds
In the analyzed extracts of E. longistipitata leaves, 14 fatty acid-related compounds were identified (Table 1). Mostly, these are long-chain fatty acids, octadecanoids with hydroxyl substituents, oxylipins, and hydroxypolyunsaturated fatty acids. The identified fatty acids play an important role in the metabolism of plant organisms and have various types of biological activity [17,18]. For example, 9-oxo-ODA, by acting on peroxisome proliferator-activated receptor, inhibits cellular triglyceride accumulation in hepatocytes and can be used against disorders of lipid metabolism [19]. 12-Oxo-phytodienoic acid is affiliated with jasmonates: oxylipins that serve as key signaling compounds in immunity, germination, and development of plants [20,21]. 12-Oxo-phytodienoic acid plays an important part in plant resistance to water-deficit conditions by regulating stomatal aperture [22]; its anti-inflammatory activity is documented [23]. Previously, the fatty acid profile was characterized only in the seeds of E. hyemalis, even though the fatty acid profiles differ among individual genera of the Ranunculaceae family and may have taxonomic significance [24].

Coumarins
Natural compounds that are based on 5,6-benzo-α-pyrone are known as coumarins. Investigation into the chemistry of coumarins from the Ranunculaceae family began in the last century and continues to develop rapidly at present. The stimuli for these research advances are, on the one hand, the high variety of natural coumarins, and on the other hand, the practical value of many of these compounds as biologically-active substances. In E. longistipitata, four substances that were related to coumarins were identified here: 5,7-dihydroxy-4-methylcoumarin, scoparone, fraxetin, and luvangetin (Table 1, Figure 2). The first three compounds consist of a coumarin molecule with the following substituents: hydroxyl groups at positions C-5 and C-7 and a methyl group at C-4 in 5,7-dihydroxy-4methylcoumarin; there are methoxy groups at positions C-6 and C-7 in scoparone, whereas fraxetin also contains a methoxy group at the C-6 position and two hydroxyl groups at positions C-7 and C-8. All these simple coumarins are widespread in the plant world and have various biological activities [25,26]. Due to the high biological activity and low toxicity of coumarins, the attention of researchers in recent years was focused on the antitumor effects of these substances. Coumarins are used in the treatment of renal cell carcinoma, leukemia, and prostate cancer and have the ability to counteract the adverse effects of radiotherapy [27]. tumor effects of these substances. Coumarins are used in the treatment of renal cell carcinoma, leukemia, and prostate cancer and have the ability to counteract the adverse effects of radiotherapy [27]. Via the addition of a pyran ring, simple coumarins can be converted to pyranocoumarins, such as luvangetin which was found in E. longistipitata here. Luvangetin is a linear pyranocoumarin consisting of a six-membered ring that is linked to the benzene site of a benzopyran ring with a double bond at the C-3′(4′) position and a methoxyl group at C-8. Luvangetin possesses impressive biological properties such as antiulcer [28] and antibacterial [29] effects and antifungal activity against Fusarium graminearum Schwabe, Rhizoctonia solani J.G. Kuḧn, and Pyricularia oryzae Cavara [30]. The gastroprotective effect of luvangetin is not fully characterized because at a concentration of 1-10 μg/mL, it does not Via the addition of a pyran ring, simple coumarins can be converted to pyranocoumarins, such as luvangetin which was found in E. longistipitata here. Luvangetin is a linear pyranocoumarin consisting of a six-membered ring that is linked to the benzene site of a benzopyran ring with a double bond at the C-3 (4 ) position and a methoxyl group at C-8. Luvangetin possesses impressive biological properties such as antiulcer [28] and antibacterial [29] effects and antifungal activity against Fusarium graminearum Schwabe, Rhizoctonia solani J.G. Kuhn, and Pyricularia oryzae Cavara [30]. The gastroprotective effect of luvangetin is not fully characterized because at a concentration of 1-10 µg/mL, it does not induce additional production of prostaglandins as previously thought; luvangetin probably stimulates other protective actions of the mucous membrane [31].

Chromones
Chromones, a group of secondary metabolites that are based on 4H-1-benzopyran-4one, are considered common among a relatively limited number of plant species that are affiliated with families Ranunculaceae, Apiaceae Lindl., and Fabaceae Lindl., and some others [32]. Among the Ranunculaceae species, chromones have been found only in species of Eranthis [12][13][14][15][16]33] and of Actaea L. (sin. Cimicifuga Wermisch.) [34][35][36][37][38]. In terms of their structure, chromones are close to coumarins and flavonoids but are much less common in nature. Slightly more than 50 chromones are known, and their structural diversity has led to their tentative categorization into simple chromones and benzo-, pyrano-, and furo-chromones. Previously, in the Eranthis species that have been studied, compounds from the families of simple chromones and furochromones have been found (Table 2). Table 2. Chromones that were previously found in Eranthis species.

CHROMONES
hibits allergic inflammatory reactions [48]. Some chromones that were isolated from Eranthis exert antioxidant action on the superoxide anion [16]. According to comprehensive molecular and morphological analyses, the genus Eranthis is affiliated with Ranunculaceae tribe Cimicifugeae, which includes genera Actaea (incl. Cimicifuga and Souliea Franch.), Anemonopsis Siebold & Zucc., Beesia Balf.f. & W.W.Sm., and Eranthis [3]. On the basis of morphological similarity with the genus Helleborus L., some investigators have assigned Eranthis to the tribe Helleboreae DC., subtribe Helleborinae Jensen [49][50][51]. On the other hand, ongoing research in the field of Helleborus phytochemistry indicates that a distinctive feature of this genus is the accumulation of substances from the class of cardioactive glycosides: cardenolides and bufadienolides [52], which have not been found in Eranthis species. In phytochemical characteristics, Eranthis is closer to the genus Actaea because chromones are synthesized in these genera via normal physiological processes [53]. Our detection of furochromones in E. longistipitata is another proof of the close relationship between Eranthis and the genus Actaea.
Most often, the presence of simple chromones has been registered in the underground part of Eranthis species, whereas furochromones have been identified in both the underground and aboveground parts ( Table 2).
The furochromones that have been registered in Eranthis species are represented by cimifugin (15) and two of its derivatives: cimicifugin β-D-glucopyranoside (16) and norcimifugin (17). Khellin (19) is a furanochromone derivative in which there are methoxy groups at positions C-4 and C-9 and a methyl group at the C-7 position (Table 2). Visnagin (18) is based on the furanochromone framework too but has substituents at positions C-4 and C-7 (methoxy and methyl groups, respectively), but there are no substituents at the C-9 position. Khellol (20) is an aglycone of khellol glucoside (21), whose sugar moiety is attached at the C-7 position. The next two compounds that were previously found in the leaves and stems of E. pinnatifida differ from each other by the presence of a hydroxymethyl group at the C-9 position [in norammiol (23)] or its absence [in norkhellol (22)] [13]. The furochromone that was identified in tubers of E. cilicica (24) is a diglucoside [16].
In our extracts of E. longistipitata, the class of chromones is represented by seven compounds belonging to the family of furochromones (Table 1, Figure 2). The latter are formed via condensation of simple chromones with a furan ring at positions C-6 and C-7. Cimifugin (15) has been detected previously in the leaves and stems of E. pinnatifida and in the tubers of E. cilicica; visnagin (18), khellin (19), and khellol glucoside (21) have been found in E. hyemalis, whereas khellol (20) in the leaves and stems of E. pinnatifida (Table 2). In our study, methoxsalen, 5-O-methylvisammioside, and visamminol-3 -O-glucoside were found in Eranthis for the first time. Methoxsalen is a naturally occurring analog of psoralen and is found in various species of Rutaceae Juss, Fabaceae, and Apiaceae [39][40][41]. Earlier, 5-O-methylvisammioside was isolated from the underground part (radix) of Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk. (Apiaceae) [42].
The pharmacological activity of furochromones and their limited occurrence are of interest. Chromone derivatives possess anti-inflammatory, antiviral, and antitumor activities and are employed as antioxidants. In addition, due to their photochemical properties, they can serve as fluorescent labels in biochemical experiments and clinical practice. Chromones, with electron-withdrawing substituents at the C-3 position, form the basis for the synthesis of various heterocyclic compounds [43][44][45]. The first chromone to be used in clinical practice was khellin, which is isolated from the seeds of Ammi visnaga (L.) Lam. [45]. Khellin and visnagin can serve as bioherbicides [46] and an anti-inflammatory activity of 5-O-methylvisammioside has been reported [47], whereas cimifugin effectively inhibits allergic inflammatory reactions [48]. Some chromones that were isolated from Eranthis exert antioxidant action on the superoxide anion [16].
According to comprehensive molecular and morphological analyses, the genus Eranthis is affiliated with Ranunculaceae tribe Cimicifugeae, which includes genera Actaea (incl.  [49][50][51]. On the other hand, ongoing research in the field of Helleborus phytochemistry indicates that a distinctive feature of this genus is the accumulation of substances from the class of cardioactive glycosides: cardenolides and bufadienolides [52], which have not been found in Eranthis species. In phytochemical characteristics, Eranthis is closer to the genus Actaea because chromones are synthesized in these genera via normal physiological processes [53]. Our detection of furochromones in E. longistipitata is another proof of the close relationship between Eranthis and the genus Actaea.

Plant Material and Preparation of the Extract
The leaves of E. longistipitata were collected during the flowering-fruiting period in 2019 (Table 3). The plant material was dried by means of silica gel. The leaves were ground up to obtain a homogeneous powder. The set of compounds was studied in 70% aqueousethanol extracts of the leaves; these extracts were prepared at 72 C in a WB-4MS water bath (Biosan, Riga, Latvia). A certain portion (0.200 g) of the crushed air-dried material was extracted twice: first, with 30 mL for 30 min, and then, with 20 mL for 20 min. "Yellow ribbon" filter paper (LLC Melior XXI, Reutov, Russia) was used to filter extracts. After filtration, the residue in the flask and on the filter was washed with 5 mL of 70% ethyl alcohol. The mixed extract was then concentrated in porcelain cups down to 5 mL. Before the analysis, the solutions were filtered and stored at 4 • C.

MS Settings and the Spectral Library
An aqueous-ethanol extract (1 mL) was diluted with double-distilled water to 5 mL and passed through a Diapak C16 concentrating cartridge (ZAO BioKhimMak, Moscow, Russia). The substances were washed off the cartridge with a small amount (3 mL) of 70% ethanol and then with 2 mL of 96% ethanol. The combined eluate was passed through a membrane filter with a pore diameter of 0.45 µm (Restek, Shanghai, China).
LC-HRMS was carried out at the Core Facility of Mass-Spectrometric Analysis at the Institute of Chemical Biology and Fundamental Medicine SB RAS (Novosibirsk, Russia). An Ultimate 3000 liquid chromatograph (Thermo Fisher Scientific, San Jose, CA, USA) that was coupled with a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) was utilized to determine metabolomic profiles of E. longistipitata leaves. The chromatographic separation was attained at a 0.4 mL/min flow rate on a Zorbax Eclipse XDB-C18 reversed-phase column (150 × 3.0 mm, 5 µm, Agilent Technologies, Santa Clara, CA, USA) thermostatted at 40 • C. The mobile phase was composed of 0.1% aqueous formic acid (eluent A) and acetonitrile (eluent B). The elution gradient was implemented as follows: from 5% to 70% B for 40 min, followed by an increase to 90% B for 8 min, a decrease to 5% B for 5 min, and re-equilibration under the initial conditions for 7 min.
The settings of the ESI source were as follows: electrospray voltage: 3.2 kV in the negative mode and 4.2 kV in the positive mode; capillary temperature: 320 • C; and the S lens RF level: 50. The data were obtained by full-scan data-dependent acquisition (FSdd-MS2) in the positive and negative modes at a resolving power of 45,000 full-width at half maximum (FWHM) at m/z 200. The following settings of the mass spectrometer were employed: the scan range, m/z 80-1200; automatic gain control (AGC), 3e6; injection time, 100 ms; and the isolation window, m/z 2.0. The normalized collision energy for the fragmentation of molecular ions was set to 40 eV. A targeted MS/MS (dd-MS2) analysis was performed in both the positive and negative modes at 15,000 FWHM (at m/z 200). AGC for dd-MS2 was set to 1 × 10 5 , with an injection time of 50 ms and a loop count of 5. In the section of dd settings, the AGC target was programmed at 8 × 10 3 , and the maximum injection time was set to 100 ms. The data were analyzed using Xcalibur 4.0 and Compound Discoverer 3.1 software (Thermo Fisher Scientific). All the samples, including the blank samples, were assayed in triplicate.
All the samples were processed in Compound Discoverer 3.1 via a common workflow called "Environmental Unknown ID w Online and Local Database Searches" (Figure S1 The chemical constituents were identified on the basis of both accurate mass and fragment mass "fingerprint" spectra via searches against the spectra of compounds that were available in the mzCloud database (https://www.mzcloud.org; last accessed on 10 March 2021). If compounds were absent in mzCloud, they were tentatively identified using a ChemSpider search. According to the workflow, the masses that were extracted from the chromatograms were aligned and filtered to remove (i) the background compounds that were present in the blank sample, (ii) substances that failed to become fragmented, (iii) compounds' masses that were absent in the databases, and (iv) signals with low intensity.
All the samples, including the blank samples, which consisted of the pure solvent, were analyzed as two biological replicates with three technical replicates per treatment group.

Chemicals
All chemicals were of MS or analytical grade. The chemical reference standards of quercetin and kaempferol were purchased from Sigma-Aldrich (Taufkirchen, Germany). Rutin and chemical reference standards of hyperoside were purchased from Fluka Chemie AG (Buchs, Switzerland).

Conclusions
Data on the overall metabolite profile of E. longistipitata leaves were obtained by LC-HRMS for the first time. This method requires only a small amount of plant material and, therefore, is certainly suitable for the research on endemic plants of the genus Eranthis. A comparison of samples of E. longistipitata among different natural populations revealed no differences in the metabolome among the studied samples. Next, we more closely examined flavonoids in the metabolite profile (19 compounds). Additionally, coumarins (four compounds), amino acids (six), fatty (14) and organic acids (three), sugars (three), furochromones (seven), and other compounds were identified in the leaves. Flavonoids and fatty acids manifested the highest diversity; however, coumarins and furochromones that were found in E. longistipitata are more intriguing and are relatively rare in the plant kingdom. The proposed fine-tuned technique for the identification of substances in E. longistipitata leaves will help investigators to conduct further phytochemical and chemotaxonomic research on plant species from the genus Eranthis.