Phenolic Compounds from the Flowers of Bombax malabaricum and Their Antioxidant and Antiviral Activities

Three new phenolic compounds 1–3 and twenty known ones 4–23 were isolated from the flowers of Bombax malabaricum. Their chemical structures were elucidated by spectroscopic analyses (IR, ESI-MS, HR-ESI-MS, 1D- and 2D-NMR) and chemical reactions. The antioxidant capacities of the isolated compounds were tested using FRAP and DPPH radical-scavenging assays, and compounds 4, 6, 8, 12, as well as the new compound 2, exhibited stronger antioxidant activities than ascorbic acid. Furthermore, all of compounds were tested for their antiviral activities against RSV by the CPE reduction assay and plaque reduction assay. Compounds 4, 10, 12 possess in vitro antiviral activities, and compound 10 exhibits potent anti-RSV effects, comparable to the positive control ribavirin.


Introduction
Bombax malabaricum DC. (Bombacaceae) is a very common native tree in Guangdong Province of China. In the folklore of southern China its flowers are often used as a healthy food material, which is stewed in soup with meat and cooked congee with rice [1]. This flower and four other flowers from medicinal herbs are mixed as a health care tea called "five flowers tea" [2]. According to the traditional Chinese medicine theory, the flower of B. malabaricum is sweet and cool-natured [1], and traditionally used for the treatment of diarrhea, chronic inflammation, fever, hepatitis, and contused wounds [3]. Modern pharmacology research has shown that the Bombax plant possesses many biological activities, such as antioxidant [4], anti-inflammatory [5], anticancer [6], and protection of the hepatic and cardiovascular systems [7]. Phytochemical investigations have shown that flavonoids, sesquiterpenes and phenylpropanoids are the major constituents of B. malabaricum [8][9][10].
In the present work, we found that the ethanol extract of B. malabaricum flower possessed in vitro antiviral activity against respiratory syncytial virus (RSV). Furthermore, a previous study had showed that its ethanol extract displayed good antioxidant activity [4]. As a part of our continuing study on the isolation of interesting and biologically active compounds from this plant, three new phenolic compounds and 20 known ones ( Figure 1) were isolated from the ethanol extract of this plant. In this paper, the isolation and structural elucidation of the new compounds are reported. In addition, the anti-RSV activities of the isolates were evaluated by cytopathic effect (CPE) and plaque reduction assays, and their antioxidant activities were tested by using ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picryhydrazyl (DPPH) radical-scavenging assays.
Molecules 2015, 20 3 correlations between H-2 and H-3/H-5 suggested that H-2/H-3 were on the same side and H-4 was on the other side. Thus the structure of 1 was elucidated and it was named 4-epi-bombalin. 2' 4'  6, and two benzene rings at δC 103-159 were exhibited by the 13 C-NMR spectrum. The 1 H-and 13 C-NMR data (Table 1) showed a number of similarities to those of 2-O-(3,4dihydroxybenzoyl)-2,4,6-trihydroxyphenylacetic acid [11], except that 2 had an extra glucose and the chemical shifts of H-3 and H-5 at δH 6.27 and 6.18 shifted downfield by about 0.3 ppm to δH 6.56 and 6.47, suggesting the glucose was connected to C-4. And it was confirmed by the HMBC correlation between δH 4.89 (H-1′′) and δC 158.6 (C-4) ( Figure 2). Acid hydrolysis of 2 afforded D-glucose (Figure S17, see Supplementary Materials), which was identified by HPLC analysis [12].

Identification of Compounds 1-23
Compound 1 was isolated as a colorless oil. Its molecular formula was determined as C 16 [9] showed that they were very similar, except for some differences of the chemical shifts on C-4, C-5 and C-6. These differences were about 2.0-4.7 ppm, indicating the configuration of C-4 was different from that of 14.

Antioxidant Activities
The ethanol extract of B. malabaricum flower was reported to have good antioxidant activity [4]. Among the compounds isolated from the ethanol extract of B. malabaricum flower, compounds 2, 4, 6, 8 and 12 showed potent antioxidant activities under both DPPH and FRAP assays (Table 2) [34,35]. Compound 2, a new compound from the flower of B. malabaricum, showed a strong radical scavenging activity with SC 50 value of 11.3˘1.6 µM, while the SC 50 value of ascorbic acid of 16.3˘0.7 µM. In addition, FRAP assay also gave the same result that this new compound possessed potent antioxidant activity. Regarding the structure of compound 2, the 3,4-dihydroxyphenyl moiety and phenolic hydroxyls were considered as the antioxidant functional groups [36]. Compounds 4, 6, 8 and 12 also exhibited potent DPPH radical-scavenging activities with SC 50 values of 6.0˘0.3, 10.8˘1.2, 9.6˘0.7 and 14.5˘2.3 µM, respectively. According to the structures of these four compounds, the ring B of catechol groups may play an important role in their antioxidant activities [7], while the number of the free hydroxyl also has a positive effect on the antioxidant activities [7].

Anti-RSV Activities
In this work, we found that the ethanol extract of B. malabaricum flower possessed in vitro anti-RSV activity with an IC 50 value of 50.0 µg/mL. Therefore, we investigated the in vitro anti-RSV activities of the compounds isolated from this extract. First of all, we evaluated the anti-RSV effects of the compounds with the CPE reduction assay, and found that compounds 4 [37], 10, 12 possessed this effect to a different extent. The anti-RSV activities of these three compounds were further confirmed by the plaque reduction assay which is a quantitative method. As shown in Table 3, compounds 4, 10, 12 possessed in vitro antiviral activities against RSV with IC 50 values of 20.0˘0.6, 6.3˘0.2 and 40.0˘0.7 µM, and SI values of 12.9, >79.3, >12.5, respectively. Among the active compounds, kaempferol-3-O-(6"-O-E-p-coumaroyl)-β-D-glucopyranoside (10) showed potent anti-RSV activity comparable to the positive drug ribavirin. In our previous studies, we have found that caffeoyl acid derivatives from natural medicines had potent anti-RSV activities [36,[38][39][40]. Compound 10 is a flavonoid glycoside with a cis-coumaroyl connection. These results suggest (di)hydrocinnamoyl might be the active functional groups providing potent antiviral activity against RSV. Besides, mangiferin (12), a main constituent of the flower of B. malabaricum, also demonstrated anti-RSV activity. The anti-RSV activities of compounds 10 and 12 are reported for the first time.

General Procedures
Melting points were determined on an X-5 micro-melting point detector (Tech, Beijing, China). Optical rotations were measured using a JASCO P-1020 polarimeter (JASCO, Hachioji-shi, Tokyo, Japan). UV spectra were recorded on a JASCO V-550 UV/VIS spectrophotometer (JASCO). IR spectra were determined using a JASCO FT/IR-480 plus spectrophotometer with KBr pellets (JASCO). NMR spectra were recorded on a Bruker AV 300 or 400 MHz spectrometer (Bruker, Faellanden, Switzerland) with TMS as internal standard. ESI-MS data were determined by a Finnigan LCQ Advantage Max mass spectrometer (Thermo Electron, Billerica, MA, USA). HR-ESI-MS data were obtained by an Agilent 6210 LC/MSD TOF mass spectrometer (Agilent, Santa Clara, CA, USA). A Dionex chromatograph was used for analytical HPLC with a P680 pump, a PDA-100 photodiode array detector, and a 5C 18

Acid Hydrolysis and Sugar Analysis of 2
Compound 2 (3.0 mg) was dissolved in 10 mL 2N HCl and heated at 80˝C for 2 h. The mixture was evaporated to dryness, and the residue was suspended in water and partitioned with dichloromethane. The aqueous phase was concentrated in vacuum, anhydrous pyridine (1.0 mL) and L-cysteine methyl ester hydrochloride (4.0 mg) were added, and the mixture was heated at 60˝C for 1 h. After the reaction mixture was evaporated to dryness, o-tolyl isothiocyanate (10 µL) was then added, and the mixture was heated at 60˝C for 1 h. The reaction mixture was directly analyzed by an Agilent 1260 HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a photodiode array detector and a Capcell pak C 18 column (4.6ˆ250 mm, 5 µm, Cosmosil, Nacalai Tesque, Kyoto, Japan) at 25˝C with isocratic elution of 25% CH 3 CN in 0.1% formic acid solution for 40 min at a flow rate of 0.8 mL/min. The injection volume was 10 µL and peaks were detected at 250 nm. The standards D-glucose and L-glucose were treated by the same reaction and chromatographic conditions. As a result, D-glucose from the hydrolyzate of 2 was detected by the same retention time of standard sugar derivatives.

Antioxidant Assay
FRAP assays of compounds were estimated in triplicate according to our previous report [36]. TPTZ (10 mM) was dissolved into 40 mM HCl. FRAP reagent was prepared as required by mixing 25 mL of 0.3 M acetate buffer, 2.5 mL of 10 mM TPTZ solution and 2.5 mL of 20 mM FeCl 3 solution. 20 µL of sample (100 µM) and 180 µL of FRAP reagent were added to a 96-well microplate. The mixtures were vortexed for 1 min and incubated for 5 min in the dark at room temperature. And then, the absorbance was detected at 593 nm with a multi-mode detection microplate reader. FeSO 4¨7 H 2 O solution at different concentrations (0.15-1.5 mM) were used to establish a calibration curve. Ascorbic acid was used as the positive group. The FRAP assay results were expressed as the concentration of sample (µM) giving an absorbance increase equivalent to 1 mM Fe 2+ solution.
The antioxidant activities were also determined by the scavenging activity of stable DPPH free radicals [36]. In a 96-well microplate, 100 µL of DPPH solution (200 µM in ethanol) was added to 100 µL of the tested compound at final concentrations (0-500 µM) in ethanol. The mixtures were shaken adequately and considered to stand for 30 min in the dark. The absorbance of the mixture was detected at 517 nm with a multi-mode detection microplate reader, and ascorbic acid was used as the positive control. The scavenging capacity of DPPH was calculated in the following way: scavenging activity (%) = 100ˆ(A control -A sample )/A control , A control is absorbance of control, A sample is absorbance of sample. The concentration of sample scavenged 50% of DPPH radical was defined as the SC 50 value.

Cell and Virus
Human larynx epidermoid carcinoma (HEp-2, ATCC No.: CCL-23) cell and human respiratory syncytial virus Long (ATCC No.: VR-26) strain, which were purchased from Medicinal Virology Institute, Wuhan University, China. The RSV Long strain was grown and titered in HEp-2 cells. The HEp-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 U/mL penicillin & streptomycin solution (Sigma, St. Louis, MO, USA). The antiviral and cytotoxic assays were tested in the medium only contained 2% FBS. Ribavirin (Sigma) was used as the positive control in the anti-RSV tests. All the cells were cultured at 37˝C in a humidified atmosphere of 5% CO 2 (v/v).

Anti-RSV Activities
CPE reduction assay was adopted to screen the anti-RSV activities of the isolated compounds as described in the previous reports [36,38]. First of all, the cytotoxic activities of the ethanol extract and isolates on host cells were observed under a light microscope (DP70 Olympus, Melville, NY, USA). The maximal non-cytotoxic concentration (MNCC) of the sample was defined as the maximal concentration of the sample that did not exert toxic effect (0% CPE) under microscopic monitoring. Then, the antiviral activities of the samples were tested in the beginning concentrations of their MNCCs. Briefly, 100 µL of 100 TCID 50 virus suspension and a serial two-fold diluted samples were added into a 96-well microplate containing confluent cell monolayer. The medium and virus suspension without sample were added as cell and virus controls, respectively. The 96-well microplate was incubated for 3-4 days. The virus-induced CPE were observed under light microscopy in comparison with the virus control and cell control.
The samples showing anti-RSV activities in CPE assay were further determined by plaque reduction assay [38]. HEp-2 cells were inoculated in 24-well plate for 24 h. The virus suspension with 60-80 plaque forming unit (PFU) and two-fold diluted samples were added a 24-well microplate containing confluent cell monolayer. The medium and virus suspension without sample were added as the cell and virus controls, respectively. The medium was inspirited by intermittent shaking at 15 min intervals for 2 h. The cell monolayers were washed twice with PBS, and then covered with agarose overlay medium. After the agarose solidified, a serial two-fold diluted samples and controls were added to the corresponding wells, respectively. And then, the plates were incubated for 4-5 days to form RSV plaques. The cells were fixed with 10% formalin and stained by 1% crystal violet. The number of plaques was counted. In the assay, half of the maximal concentration inhibiting the RSV-induced plaque formation by the sample was defined as the IC 50 value.