Acylated Flavone O-Glucuronides from the Aerial Parts of Nepeta curviflora

Nepeta curviflora Boiss. (Syrian catnip) is native to the Middle East. This medicinal plant is commonly used against nervous disorders, rheumatic pains, and high blood pressure. Herbal infusions prepared from various Nepeta spp. are extensively consumed as functional food. However, limited information has been known about the phenolic constituents of Syrian catnip. In this study, two acylated flavone 7-O-glucuronides, apigenin 7-O-(2″-O-(2‴-(E-caffeoyl)-β-glucuronopyranosyl)-β-glucuronopyranoside) (1) and luteolin 7-O-(2″-O-(2‴-(E-caffeoyl)-β-glucuronopyranosyl)-β-glucuronopyranoside) (2), along with the known phenolic compounds rosmarinic acid, caffeic acid, apigenin, and apigenin 7-O-β-glucopyranoside were isolated from the aerial parts of N. curviflora. The characterizations of these compounds were based on high-resolution mass spectrometry, UV, and extensive use of multidimensional NMR spectroscopy. The new compounds (1 and 2) were identified in the unmodified state and as dimethylesters.


Introduction
The genus Nepeta (Lamiaceae) is widely distributed in Europe, North Africa, North America, India, and Asia, including the Mediterranean countries. It contains around 300 species, some of which are used in traditional medicine [1]. Intake of some Nepeta spp. has been associated with positive health effects, including antispasmodic, antiasthmatic, and anti-inflammatory activities, as well as efficiently maintaining and balancing serum lipids [2][3][4][5]. Some species (for instance Nepeta menthoides) have also been used as traditional herbal medicine against nervous disorders, rheumatic pains, and high blood pressure [6], while the aqueous extracts of N. menthoides have possible benefits in controlling the mood of patients suffering from major depression [7]. As a consequence, herbal infusions prepared from Nepeta spp. are nowadays considered as functional food [8].
Syrian catnip, N. curviflora Boiss., (syn. Glechoma curviflora (Boiss.) Kuntze), a medicinal plant native to the Middle East, has been reported to exhibit antioxidant [23], phytotoxic [24] as well as nematicidal activities [25]. While the screened antimicrobial activity of dimethylsulfoxide extract of N. curviflora apparently is low [26], however, methanolic extracts of leaves and stem of this plant have shown efficacy against more than 88.8% of the tested microorganisms [26]. Only volatile chemical constituents have previously been reported from N. curviflora [24][25][26][27]. Although phenolic compounds of some Nepeta spp. have been suggested to be responsible for a wide range of biological activities [20,28,29], the specific characterization of this group of phenolic compounds appears underinvestigated in this genus. The aim of this study was thus to isolate and elucidate individual phenolic constituents of the aerial parts of Syrian catnip.

Results and Discussion
The plant was identified by Dr. Munir Naser at Birzeit University, and a voucher specimen of N. curviflora has been deposited at Al-Quds University Herbarium (accession number Nc2019Lam11) and at the seeds bank of the Union of Agricultural Work Committees (UAWC) (accession number UB-435-19/s).
The HPLC chromatogram of the methanolic extract of the aerial parts (stems, leaves, and flower) of N. curviflora recorded at 360 nm showed two major and several minor compounds. This extract was purified by partition against hexane, followed by Amberlite XAD-7 absorption chromatography. The flavonoids in the purified extract were further fractionated by Sephadex LH-20 chromatography, and pure compounds (1, 2, and 4) were thereafter isolated by preparative HPLC of selected Sephadex LH-20 fractions.
Compounds 3 and 4 were identified as the methylesters of the known phenolic compounds rosmarinic acid and caffeic acid, respectively (Tables S1 and S2, supplementary materials). The esters were most probably made by the solvent (acidified methanol) during isolation. Compounds 5 and 6 were isolated from the flowers of the plant and identified as apigenin (5) and apigenin 7-O-β-glucopyranoside (6) by UV (Table S1) and NMR (Table S3) spectroscopy.
The downfield region of the 1 H-NMR spectrum of 1 ( Figure S1) showed an AA'XX' system at δ 7.94 (H-2 /6 ) and δ 6.95 (H-3 /5 ), a one proton singlet at δ 6.85 (H-3) and an AX system at δ 6.76 (d, J = 2.2 Hz; H8) and δ 6.39 (d, J = 2.2 Hz; H6), in accordance with a 7-O-substituted apigenin derivative (Tables S1 and S3). Based on HSQC ( Figure S3), HMBC ( Figure S4), and H2BC (heteronuclear 2-bond correlation) ( Figure S6) NMR spectra of 1, 15 carbon resonances belonging to the aglycone and 9 resonances corresponding to an acyl moiety, were assigned ( Table 1). The presence of two glycopyranosyl units was further suggested from both the 1 H and 13 C-NMR spectra ( Table 1). The relationships between the 1 H sugar resonances of each sugar unit were assigned by the 1 H-1 H COSY experiment ( Figure S7), and the corresponding 13 C resonances were then assigned by the HSQC experiment. The coupling constants (7.5 Hz and 8.2 Hz) for the two anomeric protons and the twelve 13 C resonances were consistent with two O-β-glucuronopyranosyl units [30][31][32][33]. The additional presence of the two singlets at δ 3.63 and δ 3.52 ppm (methoxy groups), which showed cross peaks with the carbonyl groups at δ 169. 10       The NMR resonances of 2 were very similar to those of compound 1 (Table 1). However, the main differences were shown in the aromatic region where the 1 H NMR spectrum of 2 ( Figure  S11 (Figure 1). Previously, we have reported that the free carboxyl group of glucuronyl moieties of flavonoids readily will be esterified with methanol during extraction and isolation processes involving acidified methanol as solvent [30]. This is in accord with the identification of both 1 and 2 as dimethylesters caused by methylesterfication of the two glucuronyl moieties of these flavonoids. Small amounts of parental 1 and 2 without their methylesters were indeed detected by LC-MS analysis of Sephadex LH-20 fractions of the purified extract of N. curviflora (FigureS S10 and S18).
Antibacterial activity was measured using the agar diffusion method. Sephadex LH-20 fractions containing both 1 and 2 dissolved in DMSO did not reveal antibacterial activity. However, the cruder XAD-7 purified material showed some antibacterial activity, suggesting other compounds in the extract to be considered in future antibacterial activity studies.

General
UV-Vis absorption spectra were recorded on-line during HPLC analysis using a photodiode array detector (HP 1050) (Agilent Technologies, Santa Clara, CA, USA). 1 H (600.13 MHz) and 13 C (150.90 MHz) NMR spectra were obtained on a Bruker Biospin AV-600 MHz instrument equipped with a TCI 1 H-13 C/ 15 N CryoProbe (Bruker BioSpin, Zürich, Switzerland), and on a Bruker Biospin AV-850 MHz equipped with a CryoProbe (Bruker BioSpin, Zürich, Switzerland). All experiments were recorded at 298K and the chemical shift values were set relative to the deutero-methyl 13 C signal and the residual 1 H signal of the solvent ((CD 3 ) 2 SO) at δ 39.6 and δ 2.49, respectively. Low-resolution mass spectra were recorded on a LC-MS system (Agilent Technologies, Santa Clara, CA, USA) consisting of an Agilent 1200 series LC module (binary pump, column compartment/oven, and auto sampler), equipped with an Agilent ZORBAX SB-C 18 (RRHT 2.1 × 50 mm, 1.8 µm), with an Agilent 6420A mass spectrometer equipped with a triple quadrupole (QqQ configuration) mass analyzer using electrospray ionization (ESI) as detector.

Isolation of Flavones
A mixture of compounds 1, 2, and 4 was obtained in the combined fractions 25 and 26 achieved by Sephadex LH-20 chromatography (Table S4). Pure compounds 1, 2, and 4 were then isolated from fractions 25 and 26 by preparative HPLC. Pure compound 3 was eluted in Sephadex LH-20 fraction 33 (Table S4). The isolation of compounds 5 and 6 was based on the extraction of 195 g dried flowers of N. curviflora followed by the same purification and separation steps as indicated above. The dried XAD-7 purified extract (2.31 g) was fractionated by Sephadex LH-20 chromatography, and pure 5 and 6 were obtained in fractions 27 and 38, respectively (Supporting Information, Table S5).

Biological Activity
The antibacterial activities of Sephadex LH-20 fractions containing both 1 and 2, and XAD-7 purified material, were investigated against five gram-positive (Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Enterococcus faecalis, and Staphylococcus epidermidis) and four gram-negative (Escherichia coli, Klebsiella pneumonia, Proteus mirabilis, and Proteus aeruginosa) bacteria. The antibacterial test was carried out by using the agar diffusion method. The sterile saline was prepared by dissolving 0.5 g of NaCl in 500 mL of water (0.1% of NaCl) before this solution was autoclaved. The single bacterial colonies were dissolved in the sterile saline until the turbidity of the suspended cells reached the McFarland 0.5 standard. The bacterial inocula were spread on the surface of Mueller-Hinton nutrient agar using a sterile cotton swab. Then, the wells (6 mm in diameter) in the agar plate were made by using Sterile glassy borer [34,35]. The samples were dissolved in DMSO in concentrations of 6 mg/mL, and 25 µL of each were added into their respective wells before the plates were incubated at 37 • C for 12-24 h. Gentamycin (G) and Erythromycin (E) were used as positive controls, while DMSO was used as negative control. The activities of the samples were determined by measuring the inhibition zone diameter in millimeter. The results were determined by calculating the average of three trials.

Conclusions
In this investigation, individual phenolic constituents of the aerial parts of Syrian catnip have been characterized on the basis of extensive spectroscopic analyses. Two new flavonoids (1 and 2) along with two known flavonoids (5 and 6) and the methylesters of rosmarinic acid (3) and caffeic acid (4) have been identified. Flavonoids glycosylated with glucuronic acid have previously been reported from several Nepeta spp. [36][37][38]. However, the findings of 1 and 2 in N. curviflora are the first report of acylated flavone glucuronides in the genus Nepeta, which might have chemotaxonomic significance within the genus.   Figure S17: HR mass spectrum of the methyl ester form of luteolin 7-O-(2"-O-(2 -(E-caffeoyl)-β-glucuronopyranosyl)-β-glucuronopyranoside) (2). Figure S18: Mass spectrum of luteolin 7-O-(2"-O-(2 -(E-caffeoyl)-β-glucuronopyranosyl)-β-glucuronopyranoside) (2). Table S1: Chromatographic (HPLC) and spectral (UV and MS) data recorded for 1-6 from Nepeta curviflora. Table  S2: 1 H and 13 C spectral data (ppm) and coupling constants (Hz) for compounds 3 and 4 dissolved in DMSO-D6 at 25 • C. Table S3: 1 H and 13 C-NMR chemical shift values of apigenin (5) and apigenin 7-O-β-glucuronopyranoside (6) in DMSO-D 6 at 298 K. Table S4: Solvent composition and elution volumes used for separation of XAD-7 purified extract of aerial parts of Nepeta curviflora using a 100 × 5 cm Sephadex LH-20 column. The flow rate was 4 mL/min. Table S5: Composition and elution volumes used for separation of XAD-7 purified extract of flowers of Nepeta curviflora using a 100 × 5 cm Sephadex LH-20 column. The flow rate was 4 mL/min. Table S6: In-vitro anti-bacterial activity data for compound 1 and 2 and for the XAD purified materials against gram-negative bacteria. Inhibition zone diameter is in millimetre. Table S7: In-vitro anti-bacterial activity data for compounds 1 and 2 and for the XAD purified materials against gram positive bacteria. Inhibition zone diameter is in millimeter.