New Flavone C-Glycosides from Scleranthus perennis and Their Anti-Collagenase Activity

Three new flavone glycosides, one known flavone glycoside, and the phenolic derivative apiopaenonside were isolated and identified from the ethyl acetate fraction of the aerial parts of Scleranthus perennis. The planar structures were elucidated through extensive analysis of UV-Vis, IR, and 1H NMR and 13C NMR spectral data, including the 2D techniques COSY, HSQC, and HMBC, as well as ESI mass spectrometry. The isolated compounds were established as 5,7,3′-trihydroxy-4′-acetoxyflavone-8-C-β-d-xylopyranoside-2′′-O-glucoside (1), 5,7,3′-trihydroxy-4′-methoxyflavone-8-C-β-d-xylopyranoside-2′′-O-glucoside (2), 5,7-dihydroxy-3′-methoxy-4′-acetoxyflavone-8-C-β-d-xylopyranoside-2′′-O-glucoside (3), 5,7-dihydroxy-3′-methoxy-4′-acetoxyflavone-8-C-β-d-xylopyranoside-2′′-O-(4′′′-acetoxy)-glucoside (4), and apiopaenonside (5). Moreover, all isolated compounds were evaluated for anti-collagenase activity. All compounds exhibited moderate inhibitory activity with IC50 values ranging from 36.06 to 70.24 µM.


Introduction
The genus Scleranthus L. (Caryophyllaceae) comprises 11 named species divided into two sections, Scleranthus and Mniarum. Scleranthus includes three endemic species to Europe, Western Asia, and North Africa (S. annuus, S. perennis, S. uncinatus), as well as three Australian endemic species (S. diander, S. pungens, S. minusculus). The plants of Scleranthus are widespread perennial herbs occupying mainly dry, sandy, or disturbed habitats [1,2]. Previous phytochemical studies have revealed the presence of flavonoids in S. uncinatus [3,4]. Furthermore, phenolic acids have been isolated from S. perennis water/alcoholic extracts, while sapogenins, tannins, and sterols were identified from butanol extracts [5,6]. S. annuus water/alcoholic extracts are a source of phenolic acids and flavonoids [7]. S. perennis has not been well studied, and thus its phytochemical and pharmacological data are scarce. In folk medicine, this plant has been used for veterinary purposes as a remedy for animals that display a fluctuating temperament [8].
In our continuing phytochemical investigation of this plant, we isolated five compounds. According to high-performance liquid chromatography coupled with diode-array detection and mass spectrometry (UHPLC-DAD-MS) analysis and the UV-Vis spectra, four of the obtained structures were classified as derivatives of flavones [9,10] and one was identified as a paeonol derivative. In the present work, we performed investigations and identified C-glycosylated luteolin derivatives and apiopaenonside in an ethyl acetate fraction of S. perennis. For these compounds, 1 H NMR and 13 C NMR analyses, including the 2D techniques COSY, HMBC, and HSQC, as well as UV-Vis, IR, HR-ESI-MS, product ion scan, and acid hydrolysis, were performed. To the best of our knowledge, three of the isolated compounds are new chemical structures found in the plant kingdom. The present communication addresses their isolation and structural elucidation as well as bioactivity evaluation of these compounds.

Results and Discussion
The preliminary LC-MS screening of the ethyl acetate fraction from the dried aerial parts of S. perennis showed the presence of polyphenol derivatives [9]. Thus, the ethyl acetate fraction was separated multiple times by preparative, providing four flavone derivatives (1)(2)(3)(4) and one phenolic derivative (5) (Figure 1).

5,7,3 -Trihydroxy
Compound 1 was obtained as yellow amorphous powder. Based on the HRESIMS ion peak at m/z 623 [M + H] + , the molecular formula of C 28 H 30 O 16 was determined. The UV spectrum exhibited absorption maxima at 248 and 305 nm, which is typical of flavones. A free C7 hydroxyl group was confirmed by a bathochromic shift of 6 nm (in the presence of sodium acetate (NaOAc)), and a free C5 hydroxyl group was indicated by a bathochromic shift of 41 nm (in the presence of aluminum chloride (AlCl 3 )). Furthermore, a bathochromic shift of 4 nm (in the presence of sodium methoxide (NaOMe)) indicated that C4 is substituted [11]. The 1 H NMR spectrum showed one proton singlet at δ 6.20 characterizing a trisubstituted A-ring, while the absence of aromatic methine carbon signals in the range of 90-96 ppm suggested that C8 was substituted. Based on the HMBC data, the proton at δ 6.19 showed correlations with C5 and C8; thus, this signal was assigned to C6 [3,12]. Detailed analysis of the 13 C NMR data led to the assignment of the carbons in the B-ring. The signals at δ 7.42 (1H, d, J = 8.28 Hz) and δ 6.92 (1H, d, J = 8.28 Hz) were assigned to H-C5 and H-C6 , respectively, and these assignments were confirmed by COSY correlations. Moreover, the carbon signals at δ 150.90 (C4 ) and δ 146.92 (C3 ) display ortho coupling, as found in 3 ,4 -oxygenated flavonoids [13]. The presence of the unsaturated bond was shown by the δ 184.17 signal in the 13 C NMR spectrum, which corresponds to C4 of the C-ring [3,14]. From the HMBC analysis, the C4 carbon signal was linked with the proton signal at δ 6.51 assigned as H-C3. The presence of an acetoxy group in the structure at C4 was revealed by the chemical shift of the -CH 3 group in the 1 H NMR spectrum at δ 1.98 (s, 3H), as well as in the 13 C NMR spectrum for an acetoxyl carbonyl carbon at δ 172.98 and an acetoxyl methyl carbon at δ 20.79 [3,13]. This conclusion was further supported by HSQC and HMBC correlations. The 1 H NMR spectrum revealed the two anomeric protons at δ 5.08 (1H, d, J = 9.54 Hz) and δ 4.29 (1H, d, J = 7.78 Hz), which are characteristic of two sugars with β-configurations [15]. Based on the HMBC and HSQC correlations, the anomeric carbons appeared at δ 74.90 and δ 105.90. Extensive analysis of the 1 H NMR, 13 C NMR, DEPT, and 2D NMR spectral data, including COSY, HSQC, and HMBC, found the individual saccharide chemical shifts that are shown in Table 1 and Figure 2 [13]. According to the obtained data, one of the saccharides was β-D-glucose, and the second was β-D-xylopyranoside [4,14,16]. One sugar (terminal) was also analyzed by thin-layer chromatography (TLC) after acid hydrolysis of compound 1 and was determined to be glucose. Interference between H-C1 and H-C2 , as well as H-C1 and C8 from the HMBC data, suggests that the sugars are linked by Glc(1 →2 )Xyl bonds. Moreover, the type of bonds and substitutions were confirmed based on triplequadrupole MS fragmentation. The ion fragmentation pattern of flavonoids shows a retro-Diels-Alder reshuffling in the C-ring with the loss of neutral molecules of water, saccharides, and methyl and carbonyl groups [17].  [17]. Moreover, the IR spectrum showed typical signals for O-H (V max 3462), C-H (V max 2950), C=O (V max 1716), and C=C (V max 1616) [17]. Therefore, the new chemical structure from plants, 5,7,3 -trihydroxy-4acetoxyflavone-8-C-β-D-xylopyranoside-2 -O-glucoside ( Figure 1) named scleranthoside A, was definitively established. Table 1. 1 H and 13 C spectral data of 1, 2, and 4 (CD 3 OD, 400 Hz, δ in ppm, J in Hz).

5,7,3 -Trihydroxy-4 -methoxyflavone-8-C-β-D-xylopyranoside-2 -O-glucoside (2)
Compound 2 was also isolated as a yellow amorphous powder. Its molecular formula of C 27 H 30 O 15 was established based on the positive HRESIMS ion peak at m/z 595 [M + H] + . Detailed analysis of the 1 H NMR and 13 C NMR data (Table 1), including the 2D techniques COSY, HMBC, and HSQC, of compound 2 showed that its planar structure and sugar side chain were identical to those of compound 1, but some slight differences in chemical shifts were observed, mainly concerning the C4 moiety. The 1 H NMR spectrum showed a signal at δ 4.01 (s, 3H) corresponding to the -CH 3 instead of the acetoxy group observed in compound 1. Furthermore, the 13 C NMR data confirmed the methoxyl group at δ 56.84 [13], which was assigned to the 4 carbon in the C-ring. The site of methylation was further supported by the HMBC data observed for C4 (δ 151.88) with a methyl group (δ 4.01) (  [18]. Thus, the structure of 2, which is a new natural product, was established as 5,7,3 -trihydroxy-4 -methoxyflavone-8-C-β-D-xylopyranoside-2 -O-glucoside and named scleranthoside B (Figure 1).

5,7-Dihydroxy
Compound 3 showed an [M + H] + ion at m/z 637 in its HRESIMS spectrum, corresponding to the molecular formula C 29 H 32 O 16 . This structure exhibited flavone and sugar skeletons similar to those of compounds 1 and 2 except for the signals at the 3 and 4 carbons of the C-ring. The presence of a methoxyl group in the molecule was indicated by a peak in the 1 H NMR spectrum at δ 4.01, which appeared as a singlet and integrated to 3H, and in the 13 C NMR spectrum it appeared as one signal at δ 56.71. This methoxyl group was placed on carbon 3 (δ 149.50) based on the HMBC correlations of this group with C3 . In addition, from long-range COSY connectivities, the position of the methoxyl group on the B-ring was confirmed due to the cross-peaks from H2 (δ 7.65). Furthermore, in the 1 H NMR spectrum, we observed a chemical shift for the -CH 3 of the acetoxyl group (δ 1.94, s, 3H), and in the 13 C NMR spectrum, signals for an acetoxyl carbonyl carbon at δ 172.93 and an acetoxyl methyl carbon at δ 20.73 were observed, which, according to the HMBC and COSY data, were assigned to the C4 position.  [18]. Hence, the structure of 4 was identified as 5,7-dihydroxy-3 -methoxy-4acetoxyflavone-8-C-β-D-xylopyranoside-2 -O-(4 -acetoxy)-glucoside and this compound was given the trivial name scleranthoside C (Figure 1).

Plant Material
The aerial parts of Scleranthus perennis were collected between August and September 2018 in the Bialystok area (53 • 06 39.0 N 23 • 07 13.4 E) in Poland. The plant was authenticated based on its morphological characteristics by one of the authors (MT) according to Rutkowski [22]. A plant voucher specimen (No. SP-18041) was deposited in the Herbarium of the Department of Pharmacognosy at the Medical University of Białystok, Poland.

Extraction and Isolation
The dried and powdered aerial parts of S. perennis (1100 g) were partitioned successively with petrol, chloroform, and methanol. The MeOH extract was concentrated to dryness under vacuum at a controlled temperature (30 ± 2 • C) and subjected to lyophilization until a constant weight was obtained (108 g). The extract was dissolved in MeOH (110 g) and subjected to CC (85 cm × 5 cm) on a Sephadex LH-20 column. The column was eluted with MeOH to give 33 fractions (~50 mL each). Based on TLC silica gel plate developed with EtOAc:H 2 O:FA at a ratio of 18:1:1 and derivatized with 1% NA and LC-MS analyses (UPW:ACN 5→95), all fractions were pooled into five main fractions (F1-F5). The aqueous residue of F3 was fractionated by liquid−liquid extraction with Et 2 O, EtOAc, and finally n-BuOH. The combined layers were evaporated and purified. LC-MS analysis of the EtOAc fraction showed compounds that could be classified as derivatives of flavonoids. The EtOAc fraction (2 g) was dissolved in DMSO, and part of this fraction (2.5 g) was separated by preparative HPLC (0-35 min, 0%-7% UPW-ACN, 20 mL/min) to obtain compound 1 (11.75 mg), compound 2 (13.8 mg), compound 3 (385.56 mg), compound 4 (31.3 mg), and compound 5 (8.69 mg). The purified compounds were identified based on chromatographical products of acid hydrolysis (TLC; Rf: 0.55 corresponds to glucose standard) and the recorded 1 H, 13 C, COSY, HSQC, and HMBC spectra in CD 3 OD, as well as MS, IR, and UV spectra, and the product ion scan.

Acid Hydrolysis
Approximately 3 mg of compounds 1-4 was refluxed in 2N HCl (5 mL) for 2 h. The aglycones from the post-hydrolyzed solution (PHS) were extracted with Et 2 O and identified by TLC with standard. The TLC plate was developed with 30:3:10 solvent system (HCl:AcOH:H 2 O). TLC analysis of monosaccharides residues was conducted by spotting standards, the PHS water layer, and developing with a solvent system of 20:1:4 (EtOH:NH 4 OH:H 2 O). TLC chromatograms were derivatized using freshly prepared aniline phthalate, heating, and comparing with Rf values of standards.  Table 1 and Figures S1-S9 Table 1 and Figures S10-S18 Table 1 and Figures S24-S32 [19], NMR spectral data, see [20].

In Vitro Collagenase Inhibition Assay
The previous spectrophotometric procedure was modified and subsequently employed to determine the anti-collagenase activity of the isolated compounds [23]. This assay was performed in 50 mM Tricine buffer (pH = 7.5; 400 mM NaCl, 10 mM CaCl 2 ). The mixed solution included 25 µL of 0.1 U/mL collagenase from Clostridium histolyticum, 25 µL Tricine buffer, and 25 µL of various levels of the sample were incubated at 37 • C for 20 min. After incubation, 75 µL of 0.8 mM FALGPA substrate was present. Then, absorbance was measured at 335 nm wavelength. Negative control was performed using Tricine buffer instead of sample and positive control was conducted with EGCG.
The percentage inhibition for assay was calculated by: where C is the negative control and S is the sample.

Statistical Analysis
All results are expressed as the mean ± standard deviation (SD) and analyses were performed in triplicate. Significant statistical analysis was performed using GraphPad Prisma 9 software (GraphPad Software, San Diego, CA, USA). Statistical differences were assessed using one-way ANOVA.

Conclusions
The occurrence of 1-4 constitutes this as the first report of flavone C-glycosides from the Scleranthus perennis. Compound 5, a derivative of paeonol, was also newly found in the Caryophyllaceae family. Furthermore, to the best of our knowledge, compounds 1, 2, and 4 are new chemical structures occurring in the plant kingdom. Their discovery not only extends the structural and chemical diversity of phenolic compound, but also underlines the potential source for bioactive natural products. Further investigations on their biological activities are in progress.

Supplementary Materials:
The following are available online, Figure S1: Product ion scan in positive mode of 1; Figure S2: Product ion scan in negative mode of 1; Figure S3: UV spectrum of 1; Figure S4: IR spectrum of 1 in KBr; Figure S5: 1 H NMR spectrum (400 MHz) of 1 in CD 3 OD; Figure S6: 13 C NMR spectrum (400 MHz) of 1 in CD 3 OD; Figure S7: 1 H-1 H COSY spectrum of 1 in CD 3 OD; Figure S8: HSQC spectrum of 1 in CD 3 OD; Figure S9: HMBC spectrum of 1 in CD 3 OD; Figure S10: Product ion scan in positive mode of 2; Figure S11: Product ion scan in negative mode of 2; Figure S12: UV spectrum of 2; Figure S13: IR spectrum of 2 in KBr; Figure S14 Acknowledgments: Special thanks to A. Bajguz and A. Piotrowska-Niczyporuk from the University of Białystok for the accepting authors as visiting scientists and providing a laboratory to conduct the study with a triple-quadrupole LC-MS system. The authors express thanks to Wojciech Miltyk from Medical University of Bialystok for providing the laboratory to conduct IR spectroscopic measurements and Piotr Olejnik for technical assistance in obtaining IR spectra. We also thank to Urszula Sierniewska from the Medical University of Warsaw for her participation in the process of isolation and identification of the compound.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 1-5 are available from the authors.