Four flavonoids (Figure 1) were obtained from P. clypearia. Among them, compound 1 is new and its structure was elucidated by HR-ESI-MS, UV, IR, ¹H- and ¹³C-NMR, HMBC, HMQC, NOE difference and CD spectroscopy.

Compound 1 was obtained as a white amorphous powder, and its molecular formula C₂₅H₂₂O₁₁ was determined from the molecular ion peak at m/z 499.1224 [M + H]⁺ deduced by HR-ESI-MS. The IR spectrum showed hydroxyl and carbonyl groups at 3189 and 1721 cm⁻¹, respectively. IR absorption bands at 1605 and 1515 cm⁻¹ for aromatic rings suggested that 1 is likely a phenolic compound.

The ¹H-NMR spectrum of 1 exhibited two methyl proton signals at δ_H 1.51 (3H, s) and 1.45 (3H, s). The aromatic region of the ¹H-NMR spectrum showed two classic 1,3,4,5-tetrasubstitued aromatic ring patterns [6.00 (1H, s), 6.14 (1H, s); 7.02 (2H, s)], and a pentasubstituted aromatic ring [6.33 (1H, s)]. The other ¹H-NMR signals at 4.49 (1H, s), 4.17 (1H, d, J = 4.4 Hz), 2.83 (1H, dd, J = 17.2, 4.4 Hz) and 2.64 (1H, d, J = 17.2 Hz) were attributed to H-2, H-3 and H-4 of a flavan nucleus, respectively. The ¹³C-NMR spectrum of compound 1 showed the presence of 25 carbons, including two methyl carbon signals at δ_C 28.9 and 24.6; one methylene carbon signal at δ_C 25.8; one signal of carbonyl carbon at δ_C 164.9; seven methine carbon and fourteen quaternary carbon signals. Compared to that of compound 3 [13], the ¹H spectrum showed that 1 had two more methyl groups and lacked the signal of H-2' in the B ring. In addition, a relative upfield shift of H-2 (δ_H 4.49) and downfield shift of H-3 (δ_H 4.17) of 1 were also observed. The HMBC correlations (³J = 4.0 Hz) of H-3/C-7' and the HMBC correlations (³J = 8.0 Hz) of H-2/C-2' and H-8',9'/C-2' (Figure 2) suggested that the hydroxyl group at C-3 was cyclized with the substituent group at C-2'. In comparison with the ¹³C-NMR data of the known compound, (−)-epigallocatechin [14], the C-7 signal was observed to have an upfield shift of about 7.6 ppm, and downfield shifts from 4.8 to 5.4 ppm at C-6, -8 and -10, respectively, which indicated the galloyl ester group of compound 1 was at C-7.

The enhancement of H-2 observed after H-3 was pre-irradiated in a NOE difference experiment, as well as no couplings observed between H-2 and H-3, and H-2 appearing as a singlet, disclosed that the relative configuration of H-2 and H-3 was cis. To assign the absolute configuration of 1, the electronic circular dichroism (ECD) spectrum obtainable through quantum-chemical calculations were used. Along with the result of the NOE difference experiment, there were two possible candidate stereostructures 1-A (2R,3R) and 1-B (2S,3S). The ECD spectrum generated for 1-A showing the positive (211 nm) Cotton effect was in good agreement with the experimental data of 1, whereas the calculated ECD spectrum for 1-B was almost opposite to the experimental curve (Figure 3). The ECD data of 1-A was associated with the experimental Cotton effect at 211 nm. Hence, the structure of new compound 1 was elucidated unambiguously, and it was named (2R,3R)-7-O-galloylplumbocatechin A [15].

Three different subtype NAs were prepared from A/PR/8/34 (H1N1), A/Sydney/5/97 (H3N2) and B/Jiangsu/10/2003. The EtOAc extract of P. clypearia exhibited the inhibitory activity on NA from influenza H1N1 virus, with an IC₅₀ value of 26.9 ± 1.05 μg/mL. Compounds 1 and 2 possessed higher inhibitory activities on NA from influenza H1N1 virus than on those from influenza H3N2 and B viruses (Table 1).

The virus-induced “cytokine storm” appears to contribute to the severe pathogenesis of the reconstructed 1918 H1N1 and H5N1influenza viruses pandemics [16,17]. Therefore, the inhibition of virus-induced cytokine release is also important for the treatment of influenza. Supernatants of human A549 lung carcinoma cells treated or untreated with flavonoids were compared by ELISA for the respective expression levels of IL-6 and MCP-1. Mock-infected cells maintained a level of IL-6 or MCP-1 as low as 73.01 ± 90.31 (Figure 4) or 221.36 pg/mL (Figure 5), while H1N1 infection dramatically increased the level of IL-6 or MCP-1 by 9-fold (657.73 ± 267.43 pg/mL) (Figure 4) or 5-fold (1116.06 ± 67.89 pg/mL) (Figure 5). All four flavonoids screened were found to significantly inhibit the expressions of IL-6 or MCP-1 induced by influenza H1N1 virus at various doses. However, their behaviors were very different. Flavonoids 1–4 all showed dose responses in which only higher doses displayed any inhibitory effects against IL-6. Among them, flavonoid 2 showed the best inhibitory effect against IL-6 and flavonoid 3 showed the weakest inhibition (Figure 4). Flavonoids 1–2 were both shown to inhibit MCP-1 expression best at the lowest concentrated dilution of 3 μg/mL (Figure 5). One apparent reason for these different results is the difference in chemical structure. Among these four compounds compound 3, for example, is the only one that bears a hydroxyl group at C-3, and it displayed the worst inhibition of both IL-6 and MCP-1. However, exactly how the chemical structure affects IL-6 or MCP-1 expression and the potent anti-inflammatory effects seen at the lowest dose tested for these compounds still need further investigation.

The leaves and twigs of P. clypearia were collected from Shankou, Beihai City, Guangxi Province in May 2012 and were identified by Professor Lin Ma, Department of Natural Products Chemistry, Institute of Materia Medica (IMM), Chinese Academy of Medical Sciences and Peking Union Medical College (CAMS & PUMC), Beijing, China. A voucher specimen (2012032) was deposited in IMM, CAMS & PUMC.

Optical rotations were measured with a JASCO P-2000 polarimeter (Tokyo, Japan). UV spectra were collected in MeOH on a JASCO V-650 spectrophotometer (Tokyo, Japan). IR spectra were recorded on a Nicolet 5700 spectrometer (Madison, WI, USA) by the FT-IR transmission electron microscopy method. ¹H- and ¹³C-NMR spectra were recorded on a MP-400 spectrometer (Palo Alto, CA, USA). HRESIMS were recorded on an Agilent 1200 SL series LC / 6520 QTOF spectrometer (Boleblingen, Germany). Purification was performed using silica gel (160–200 mesh, Qingdao Marine Chemical Factory, Qingdao, China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), and ODS (50 μm, YMC, Kyoto, Japan). Fractions obtained from column chromatography were monitored by TLC (silica gel GF254, Qingdao Marine Chemical Factory, Qingdao, China).

A549 cells (an epithelial cell line through explant culture of lung carcinomatous tissue) were maintained in RPMI1640 medium containing 10% FBS. Before infection, cells were washed with PBS (pH 7.2–7.4) and cultured in media supplemented with 2 μg/mL trypsin TPCK treated from bovine pancreas (Sigma–Aldrich, Saint Lousis, MO, USA, Lot# 031M7358V).

The human influenza virus strains A/PR/8/34 (A/H1N1), A/Sydney/5/97 (H3N2) and B/Jiangsu/10/2003 were kindly donated by the Institute for Viral Disease Control and Prevention, China Centers for Disease Control and Prevention (Beijing, China). Viral stocks of these laboratory-adapted strains were prepared by passaging them in 9-day-old embryonated chicken eggs for 48 or 72 h.

The leaves and twigs of P. clypearia (15 kg) were extracted three times with 95% EtOH (4 L) under reflux. The 95% EtOH extract (2 kg) was subjected to column chromatography over silica gel, eluted with petroleum ether, CHCl₃, EtOAc, acetone and CH₃OH. The EtOAc fraction (100 g) was then chromatographed on a silica gel column, with gradient elution (CHCl₃–MeOH = 9:1 to 1:1). The fraction eluted with CHCl₃–MeOH = 9:1 was repeatedly chromatographed over a Sephadex LH-20 column eluted with MeOH, then over a C18 column eluted with a linear gradient elution system (30% to 90% MeOH in H₂O) to yield ten fractions. Repeated chromatography of fraction 2 over Sephadex LH-20 (MeOH) yielded compound 4 (10 mg). Fraction 4 was rechromatographed over Sephadex LH-20, eluted with MeOH to furnish 1 (33 mg) and 2 (28 mg). Rechromatography of fraction 3 on C₁₈ using MeOH–H₂O (from 30% to 90% MeOH) as eluent furnished 3 (20 mg).

Compound 1: white amorphous solid; HR-ESI-MS: m/z 499.1224 [M + H]⁺, (calcd. for C₂₅H₂₃O₁₁, 499.1235), [α]²⁰_D +90.9 (c 0.11, MeOH), UV (MeOH) λ_max: 211, 289 nm, CD (MeOH): 211.0 (∆ε +37.74) nm; IR (KBr) ν_max: 3189, 1721, 1605, 1515 cm⁻¹. ¹H- and ¹³C-NMR data, see Table 2. According to the data and reported literature values [13,14,15], compound 1 was identified as (2R,3R)-7-O-galloylplumbocatechin A.

Compound 2: white amorphous solid; ESI-MS: m/z 465 [M + Na]⁺, [α]²⁰_D −5.2 (c 0.18, MeOH), ¹H- and ¹³C-NMR data, see Table 2. According to the data and reported literature values [18], compound 2 was determined to be (−)-5,3',4',5'-tetrahydroxyflavan-7-gallate.

Compound 3: brown amorphous solid; ESI-MS: m/z 457 [M − H]⁻, [α]²⁰_D +38.4 (c 0.17, MeOH), ¹H- and ¹³C-NMR data, see Table 2. According to the data and reported literature values [13], compound 3 was determined to be (+)-3,5,3',4',5'-pentahydroxyflavan-7-gallate.

Compound 4: brown amorphous solid; ESI-MS: m/z 593 [M − H]⁻, [α]²⁰_D −17.9 (c 0.12, MeOH), ¹H- and ¹³C-NMR data, see Table 2. According to the data and reported literature values [19], compound 4 was determined to be (−)-7,4'-di-O-galloyltricetiflavan.

Because there had only one chiral center (C-2) in the structures of 2 and 4, and the optical rotation values of them were both negative, their absolute configuration at C-2 should both be S [9,18].

For compound 3, due to the value of coupling constant (J_2,3 = 6.4 Hz), the relative configuration of H-2 and H-3 should be trans [13]. In addition, the optical rotation values of 3 was positive, therefore, the absolute configurations of C-2 and C-3 should be R and S, respectively [13].

A standard fluorimetric assay was used to measure influenza virus NA activity [19]. The substrate MUNANA is cleaved by NA to yield a fluorescent product which can be quantified. The reaction mixture containing tested extract of P. clypearia or compounds 1–4, and NA enzyme or virus suspension in 32.5 mM MES buffer and 4 mM calcium chloride (pH 6.5) was incubated at 37 °C for 40 min. After incubation, the reaction was terminated by adding 34 mM NaOH. Fluorescence was quantified with excitation wavelength at 360 nm and emission wavelength at 450 nm. The 50% inhibitory concentration (IC₅₀) was defined as the concentration of NA inhibitor necessary to reduce NA activity by 50% relative to a reaction mixture containing virus but no inhibitor.

Confluent monolayers of A549 cells in 96-well plates at 37 °C in 5% CO₂ infected with influenza virus (H1N1) at MOI 0.1 were treated with flavonoids at the serially diluted, non-cytotoxic concentrations of 3, 10 and 30 μg/mL. Supernatants from mock or H1N1-infected cells at 24 h post-infection were compared for the expression of IL-6 and MCP-1 by ELISA (4A Biotech Co. Ltd., Beijing, China) respectively according to the manufacturer’s protocol [20]. The experiments were repeated three times with similar results in three parallel measurements.

All data are given as the mean ± SD. Statistical analysis of the results was performed with Students’ t-test. p values of <0.05 were considered to be statistically significant.