Bioassay-Guided Isolation of New Flavonoid Glycosides from Platanus × acerifolia Leaves and Their Staphylococcus aureus Inhibitory Effects

Despite the rapid advances in drug R&D, there is still a huge need for antibacterial medications, specifically for the methicillin-resistant Staphylococcus aureus (MRSA). Inspired by the research where a viable class of MRSA inhibitors was found in the species Platanus occidentalis, a S. aureus inhibition screening-guided phytochemical reinvestigation on Platanus × acerifolia (London plane tree) leaves were performed with four flavonoid glycosides garnered, including two new compounds, quercetin-3-O-α-l-(2″-E-p-coumaroyl-3″-Z-p-coumaroyl)-rhamnopyranoside (E,Z-3′-hydroxyplatanoside, 1) and quercetin-3-O-α-l-(2″-Z-p-coumaroyl-3″-E-p-coumaroyl)-rhamnopyranoside (Z,E-3′-hydroxyplatanoside, 2). All of the isolates showed significant S. aureus ATCC 25904 inhibitory activity with MICs ranging from 4 to 64 μg/mL, suggesting the potential of discovering drug leads for the control of S. aureus from such a rich, urban landscaping plant in the Platanus genus.


Introduction
Owing to the long-standing misuse of antibiotics, Staphylococcus aureus has become resistant to many commonly used first-line drugs [1,2]. For instance, methicillin-resistant S. aureus (MRSA) is able to resist most of the β-lactam antibiotics and is a leading cause of high bacterial infection-related morbidity and mortality [3][4][5]. There is consequently an urgent need to develop alternatives for the control of S. aureus infections. Still, plant-derived natural products remain one of the primary sources of new drugs [6,7], as demonstrated by the isolation and characterization of a few flavonoid glycosides with promising anti-MRSA bioactivity from the American sycamore, Platanus occidentalis (family Platanaceae), as presented by Hamann et al. [4]. The genus Platanus contains seven deciduous species and twelve subspecies [8], of which three are widely distributed in China, namely P. occidentalis Linn., P. orientalis Linn. (the oriental plane) and P. × acerifolia Willd (the hybrid London plane) [9,10]. Of note, P. occidentalis and P. orientalis have long been used as traditional folk medicines for the treatment of a variety of diseases such as blepharitis, conjunctivitis, gastrointestinal disorders, diarrhea, toothache and skin disease [4,11]. Being one of the most famous urban landscaping trees in the world, P. × acerifolia is popular, and which earned the reputation of the "King of Road Trees" [12,13]. species and twelve subspecies [8], of which three are widely distributed in China, namely P. occidentalis Linn., P. orientalis Linn. (the oriental plane) and P. × acerifolia Willd (the hybrid London plane) [9,10]. Of note, P. occidentalis and P. orientalis have long been used as traditional folk medicines for the treatment of a variety of diseases such as blepharitis, conjunctivitis, gastrointestinal disorders, diarrhea, toothache and skin disease [4,11]. Being one of the most famous urban landscaping trees in the world, P. × acerifolia is popular, and which earned the reputation of the "King of Road Trees" [12,13].

Results and Discussion
The preliminary bioactivity evaluation of subfractions (F1-F6) from the 75% ethanolic extract of P. × acerifolia leaves revealed that F4 (60% EtOH) showed a mild inhibitory effect (MIC: 256 μg/mL) on S. aureus ATCC 25904. This subfraction was then subjected to repeated column chromatography over silica gel, macroporous resin D101, MCI gel and Sephadex LH-20 followed by semi-preparative HPLC to afford two new (1 and 2) and two known (3 and 4) flavonoid glycosides as shown in Figure 1. The new ones were elucidated to be quercetin-3-O-α-(2″-E-p-coumaroyl-3″-Z-p-coumaroyl)-rhamnopyranoside (1) and quercetin-3-O-α-(2″-Z-p-coumaroyl-3″-E-p-coumaroyl)-rhamnopyranoside (2) through the 1D-/2D-NMR data analysis in conjunction with the HRMS experiments. By comparing the observed and reported spectroscopic data and physicochemical properties, the known structures were identified as kaempferol- [4]. The HRMS and NMR spectra of compounds 1 and 2 are available in Supporting Information. Compound 1 was obtained as a yellowish-brown powder, and its positive-mode HRESIMS data showed a sodium adduct ion at m/z 763.1621 [M + Na] + , which established a molecular formula of C39H32O15 with the aid of the 13 C NMR (Table 1) data. The UV spectrum showed characteristic absorption bands at 272 and 314 nm, suggesting the existence of a 3-OH substituted flavanol skeleton [27,28]. The 1 H NMR spectrum (Table 1)  Compound 1 was obtained as a yellowish-brown powder, and its positive-mode HRESIMS data showed a sodium adduct ion at m/z 763.1621 [M + Na] + , which established a molecular formula of C 39 H 32 O 15 with the aid of the 13 C NMR (Table 1) data. The UV spectrum showed characteristic absorption bands at 272 and 314 nm, suggesting the existence of a 3-OH substituted flavanol skeleton [27,28]. The 1 H NMR spectrum (Table 1)  . Undoubtedly, the data above delineated a quercetin backbone [29][30][31]. In addition, the 1 H-1 H COSY motif ( Figure 2) of H-1"/H-2"/H-3"/H-4"/H-5"/H 3 -6" in conjunction with the 13 C (Table 1) and HSQC NMR experiments ( Figure S3) revealed the presence of an α-rhamnose moiety [29,32], which was found to locate at C-3 due to the key HMBC correlation from H-1" to C-3 (δ C 135.56) (  Figure 2) from H-2 to C-9 (δ C 167.77) and H-3" to C-9 (δ C 167.58) allowed the assembly of the (E)-and (Z)-p-coumaryl units that connected to C-2" and C-3" via the ester bonds, respectively. Thus, compound 1 was determined as quercetin-3-O-α-(2"-E-p-coumaroyl-3"-Z-p-coumaroyl)-rhamnopyranside. in conjunction with the 13 C (Table 1) and HSQC NMR experiments ( Figure S3) revealed the presence of an α-rhamnose moiety [29,32], which was found to locate at C-3 due to the key HMBC correlation from H-1″ to C-3 (δC 135.56) (    Compound 2 exhibited the same molecular formula (C 39 H 32 O 15 ) as 1 based on the HRESIMS data ( Figure S13). A comparison of the NMR data of 2 ( Table 2) with those of 1 showed the very much close structural similarity between the two compounds. Differing from 1, the bonding positions of the (E)-and (Z)-p-coumaryl units with the rhamnose were found to be swapped upon closer inspection of the HMBC spectrum of 2 ( Figure 2), with the (E)-p-coumaryl unit connecting to C-3", while (Z)-p-coumaryl unit connecting to C-2". Thus, compound 2 was identified as quercetin-3-O-α-(2"-Z-p-coumaroyl-3"-E-pcoumaroyl)-rhamnopyranoside. The ability of compounds 1-4 to inhibit the growth of S. aureus ATCC 25904 was then tested by in vitro antibacterial susceptibility assays. Methicillin and chloramphenicol were used as the positive controls. Notably, all the compounds were found to possess antibacterial activity against S. aureus with MICs ranging from 4 to 64 µg/mL (Table 3). Among them, compounds 3 and 4 showed considerable S. aureus inhibition activities with MICs at the level of 4 and 16 µg/mL, respectively. Along with the relatively weaker inhibition effects of 1 and 2, both MIC values are 64 µg/mL-this study is in good agreement with the reported data in terms of the structure-activity relationship [4]. Briefly, all the compounds in this study showed antibacterial activity, confirming that a flavonoid moiety connected to the p-coumaroyl groups via a sugar unit (e.g., rhamnose) and hydroxy groups at positions 5, 7, and 4 are essential for the antibacterial activity against S. aureus. Meanwhile, the bioactivity results of compounds 1 and 2 implied that the presence of a hydroxy group at position 3 presumably has a negative impact on such antimicrobial activity while the Eor Z-configuration of the p-coumaroyl units does not exert effects. In general, the data presented herein suggest that the genus Platanus (especially the abundant P. × acerifolia leaves) could serve as a promising source for the development of drugs to treat S. aureus infections, more specifically, glycosides from this genus with flavonoids and p-coumarinoids being the aglycone parts.

Plant Material
The green leaves of P. × acerifolia were collected from dozens of trees in January 2018 along the roadside at Zhangjiang campus, Fudan University in Shanghai, China, and identified by Prof. Ze-Xin Jin (Taizhou University). A voucher specimen (No. 20180508) has been deposited at the Herbarium of the School of Pharmaceutical Sciences, Taizhou University, China.

In Vitro Antibacterial Susceptibility Assays
The minimum inhibitory concentration (MIC) was evaluated on the basis of Clinical Laboratory Standards Institute (CLSI) guidelines by the conventional two-fold microbroth gradient dilution assay [33,34]. S. aureus ATCC 25904 was inoculated to the Brain Heart Infusion (BHI) agar plates and cultured for 18~24 h at 35 • C for activation. The diluted bacterial suspension in Cation-adjusted Mueller-Hinton II broth (CAMHB) with a turbidity of (1~2) × 10 6 CFU/mL was ready for detection according to the direct bacterial suspension method. The subfractions and isolates were firstly dissolved in DMSO to produce solutions at the concentrations of 10.0 mg/mL and 2.0 mg/mL, respectively. The resulting solutions were then diluted with fresh CAMHB medium to produce working concentrations of 1024 (from 512 µg/mL to 1 µg/mL in 96-well plates) and 128 µg/mL (from 64 µg/mL to 0.125 µg/mL in 96-well plates), respectively. Then, 100 µL of the working isolates solution was distributed in each well, while the growth controls contained equal amounts of DMSO. Finally, the bacteria-containing suspension (100 µL) was added to each well. The 96-well plates were incubated at 35 • C for 16~20 h, and MIC was determined as the lowest concentration of the drugs that completely inhibited the growth of bacteria. Chloramphenicol and Methicillin were used as positive controls, which were active against S. aureus. All the tests were performed in triplicate.

Statistical Analysis
The MIC results were analyzed for their variances by ANOVA using the SAS program (version 9.2). Differences among means of different compounds were assessed by Tukey's multiple comparison statistical analysis at a p = 0.05 level of significance.

Conclusions
Previous phytochemical studies on the buds [8,[14][15][16][17][18][19][20] and the bark [21][22][23][24][25] of P. occidentalis and P. orientalis led to the isolation and characterization of a large number of secondary metabolites, including flavonoids, glycosides, furan coumarins, terpenoids, sterols and organic acids. However, as a hybrid of these two medicinally used plants [4,11], P.× acerifolia is still lacking phytochemical and biological investigations to a large extent. In the present work, we hence focused on the flavonoid glycosides from the leaves of P.× acerifolia, which gleaned two previously undescribed flavonoid glycosides (compounds 1 and 2) as well as two known ones (compounds 3 and 4). Regarding the bioactivity evaluations, all of the isolates showed considerable S. aureus ATCC 25904 inhibition effects with MICs ranging from 4 to 64 µg/mL. While the mechanism of action requires further study, which is currently in progress in our laboratory and would trigger the next stage of research, including animal testing and the survey of plant resources available for providing bulk drug substances, the above findings expanded the structural diversity of P.× acerifolia and could provide useful clues for discovery and development of new therapeutic or preventive agents for the treatment of S. aureus infection-related diseases.