Studies on the Bioactive Flavonoids Isolated from Pithecellobium clypearia Benth

One new flavonoid named (2R,3R)-7-O-galloylplumbocatechin A (1) and three known flavonoids, (−)-5,3',4',5'-tetrahydroxyflavan-7-gallate (2), (+)-3,5,3',4',5'-penta-hydroxyflavan-7-gallate (3), and (−)-7,4'-di-O-galloyltricetiflavan (4), were isolated from Pithecellobium clypearia Benth. Their structures were elucidated based on spectroscopic analysis, including homonuclear and heteronuclear correlation NMR (HSQC and HMBC) experiments. In vitro assays, compounds 1 and 2 showed moderate inhibitory effects against influenza H1N1 virus neuraminidase (NA). Compounds 1–4 were all found to inhibit the expression of proinflammatory cytokines IL-6 or MCP-1 induced by influenza H1N1 virus in human A549 lung carcinoma cells.


Introduction
The influenza virus is a highly infective agent that causes acute pulmonary diseases [1][2][3]. The influenza virus contains eight pieces of segmented RNA, with neuraminidases (NAs) as surface antigens. NAs are involved in the release of progeny virus from infected cells, by cleaving sugars that OPEN ACCESS bind the mature viral particles. Specifically, NAs cleave the α-ketosidic bond that links a terminal neuraminic-acid residue to the adjacent oligosaccharide moiety, therefore NAs are essential for the movement of the virus to and from sites of infection in the respiratory tract [4,5].
Recent studies have shown that certain strains of influenza virus, such as H5N1, are more likely to induce excessive cytokine release and immune cell exudation [6,7]. This so-called "cytokine storm" scenario, features elevated levels of cytokines and chemokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and monocyte chemotactic protein (MCP)-1, which causes tissue damage, impairs normal mucosal membrane and may induce airway blockage, making it a risk factor for the higher mortality associated with these virulent strains. Therefore, alleviating inflammation during influenza virus infection could potentially be beneficial [8].
Pithecellobium clypearia Benth, a member of Mimosaceae family, is widely distributed in the South of China, such as Sichuan, Yunnan, and Guangdong provinces. P. clypearia has been found to contain various flavonoids. It has been used as a herbal medicine in the treatment of respiratory tract diseases in China for many years [9][10][11][12]. One Chinese patent medicine manufactured from the aqueous extract of the leaves and twigs of P. clypearia has been recorded in the Pharmacopeia of China, and used for the treatment of upper respiratory tract infections, pharyngitis, laryngitis, and acute tonsillitis, [9][10][11][12]. Though some of flavonoids from P. clypearia were reported to have anti-viral activity against H1N1 and herpes simplex [9], the mechanisms underlying the effects of P. clypearia have not been identified clearly.
Our previous screening showed that the EtOAc extract of P. clypearia exhibited in vitro influenza virus neuraminidase (NA)-inhibitory activity, with IC 50 values of 26.9 ± 1.05 μg/mL. In the present study, the chemical constituents of the EtOAc extract were separated, and their NA inhibitory and anti-inflammatory activities were both evaluated to partially reveal the responsible compounds and the mechanism of action against influenza virus.

Characterisation of Compound 1
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, 1 H-and 13 C-NMR, HMBC, HMQC, NOE difference and CD spectroscopy.
Compound 1 was obtained as a white amorphous powder, and its molecular formula C 25 13 C-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 1 H 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 ( 3 J = 4.0 Hz) of H-3/C-7' and the HMBC correlations ( 3 J = 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 13 C-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].

NA Inhibitory Effects
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 IC 50 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).

Inhibition of Cytokine Production in H1N1-Infected Cells
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.

Characterization of Compounds
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].

Neuraminidase Inhibition Assay
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 (IC 50 ) 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.

Cytokine Secretion Assay
Confluent monolayers of A549 cells in 96-well plates at 37 °C in 5% CO 2 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.

Statistical Analysis
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.
The virus-induced cytokine response contributes to the activation of the immune system and the damage to the host [27,28]. Suppression of these cytokines can potentially control the severity of the virus-induced inflammatory complications and ultimately lower the mortality [27,28]. These findings provide a possibility that an agent with antiviral and anti-inflammatory activities can be a drug of choice for the treatment of patients with severe influenza-associated complications. A certain types of flavonoids may possess the activity [29].
As far as we know, a lot of sub-groups of flavonoids have been studied for their H1N1 NA-inhibitory activity, including flavones, like apigenin and luteolin; isoflavones, like daidzein and genistein; bioflavonoids, like ginkgetin and hinokiflavone; chalcones; aurones and stilenes, etc., with a range of IC 50 values from 2.2 to 97.1 μM [23][24][25][26]. The relationship between structures and in vitro anti-viral activities can be summarized as follows: hydroxyl groups in position 4' and 7, an oxo group in position 4, and a double bond between position 2 and 3 are necessary for good NA inhibition [23,25]. In the present study, flavonoids 1 and 2 showed moderate inhibitory effects against neuraminidase (NA) of influenza virus, with IC 50 values of 29.77 ± 6.12 and 36.91 ± 3.80 μg/mL, respectively. The four flavonoids all significantly inhibited the expression of IL-6 and MCP-1 induced by influenza H1N1 virus at various doses from 3 to 30 μg/mL. This study showed that the skeleton of flavonoids 1-4 is favorable to NA inhibition. Among them, the new compound 1, having an unique E ring between rings B and C, showed the best results both in terms of inhibition of NA and anti-inflammatory effects, which indicated that this extra ring is more favorable for activity than the other functional groups substituted at the same positions.
Thus, two possible pathways might be deduced for their antiviral effects: (1) inhibition of virus release from host cells; (2) reduction of the serious inflammatory cytokine storm induced by the influenza virus infection. Nonetheless, our data did not rule out the possibility that other mechanisms may be involved. Although a complete pharmacokinetic study of flavonoids in P. clypearia is needed to further precisely address its bioavailability, the present research partly revealed the mechanisms of P. clypearia used as a herbal medicine in the treatment of respiratory tract diseases in China for many years.