A new flavonoid glycoside from Elsholtzia bodinieri

Abstract A new flavonoid glycoside, eriodictyol 7-O-(6″-caffeoyl)-β-D-glucopyranoside (1), along with 14 known compounds, were isolated from the whole plants of Elsholtzia bodinieri. All of the structures were determined by spectroscopic methods and chemical transformation. Compound 1 and luteolin (9) exhibited potent anti-HCV activities with a selective index of 135.85 and 20.84, respectively.


Introduction
Approximately, 170 million people worldwide are chronically infected with hepatitis C virus (HCV) (Bowen & Walker 2005). HCV infection often may lead to the development of a chronic condition that frequently progresses to liver cirrhosis and hepatocellular carcinoma (Raimondi et al. 2009). Current therapy for HCV infection includes the prolonged administration of a combination of ribavirin and pegylated interferon-α, which cures 40-50% of patients (Palumbo 2011). Recently, the FDA approved the use of two protease inhibitors, boceprevir and telaprevir in combination with ribavirin and pegylated interferon-α which have a cure rate of 60-80%, but exhibited some serious side effects in clinical trials (Poordad et al. 2011;Zeuzem et al. 2011). Consequently, there is urgent need to develop new remedies for the treatment of illness caused by HCV infection. Although natural products are considered to be an important source of antiviral leading compounds, there are few published Anti-HCV activity (SI = 135.85) reports of natural products with anti-HCV properties (Hu et al. 2007;Wohlfarth & efferth 2009).
The genus Elsholtzia (Lamiaceae) consists of at least 33 species, some of which have been used as domestic folk medicine, herbal tea, spices, beverages, perfumeries, cosmetics, aromatherapies and the source of honey manufacture (Xue et al. 2016). Elsholtzia bodinieri vaniot, belonging to the taxonomically diverse group of the Family Labiatae, is a medicinal plant growing in Yunnan and Guizhou Provinces in China. It is commonly known as 'dongzisu' and has been used as a traditional Chinese medicine for the treatment of cough, headache, pharyngitis, fever and hepatitis (Jiangshu New College of Medicine 1985). We previously found shikonin, isolated from the stem bark of Arnebia euchroma, exhibited potent anti-HCV activity with a selective index of 43.56 (Li et al. 2012). As a continuation of our search for anti-infective agents of natural origin, a phytochemical investigation of E. bodinieri was carried out, leading to the isolation of a new flavonoid glycoside, eriodictyol 7-O-(6″-caffeoyl)β-D-glucopyranoside, along with 14 known ones ( Figure 1). Here, we describe the isolation, structural determination and biological evaluation of these compounds.

Results and discussion
Compound 1 was isolated as yellow amorphous powder, whose molecular formula of C 30 H 28 o 14 was determined on the basis of HR-eSI-MS (negative-ion mode) analysis ([M-H] − , 611.1384) in combination with 1 H and 13 C NMR spectra, indicating 17° of unsaturation. The IR spectrum indicated the presence of hydroxyl (3440 cm −1 ), α,β-unsaturated carbonyl (1710 cm −1 ) and aromatic rings (1632, 1514, 1443 cm −1 ). The uV spectrum exhibited maximum absorptions at 366, 292 and 207 nm. Detailed comparison of the 1D and 2D NMR spectral data of 1 with those of miscanthoside (3) (Markham et al. 1978) revealed that they were very similar to each other, except for the presence of an extra caffeoyl group in compound 1.
The existence of eriodictyol (Wagner et al. 1992) was established by the following evidences. Signals at δ H 6.91 (s, H-2′), 6.70 (d, J = 8.0 Hz, H-5′) and 6.76 (br d, J = 8.0 Hz, H-6′) were characteristic for a 3,4-disubstituted B ring of the eriodictyol unit; two sets of doublets at δ H 6.16 (d, J = 1.8 Hz) and 6.26 (d, J = 1.8 Hz) originated from the signals of H-6 and H-8 in ring A of eriodictyol. Moreover, the signals of H-2 (δ H 5.28, dd, J = 3.0 Hz, 14.6 Hz) and H-3 (δ H 2.72, dd, J = 3.0 Hz, 17.1 Hz; δ H 3.07, dd, J = 14.6 Hz, 17.1 Hz) revealed the flavonone skeleton of the aglycon. Furthermore, acid hydrolysis of 1 followed by HPLC analysis of the hydrolysate and direct comparison with authentic sugars indicated the presence of a D-glucose unit, which was further confirmed by the signals at δ H 4.99 (d, J = 7.8 Hz, H-1″) and 4.57, 4.35 (H 2 -6″). Simultaneously, the anomeric configuration of glucosyl moiety was assigned as β on the basis of the coupling constant of its anomeric proton (J = 7.8 Hz). The glucosyl residue was located at C-7 of the aglycon eriodictyol by the appearance of HMBC cross peak of the anomeric proton H-1″ (δ H 4.99, d, J = 7.8 Hz) with C-7 (δ C 166.6).
The presence of the caffeoyl group was elucidated by a series of NMR signals. The trans-configuration at the olefinic protons (H-7″′ and H-8″′) of the caffeoyl group was established from their coupling constant (J = 15.9 Hz). Compared with the 13 C NMR data of 3, the C-5″ resonance of 1 was shifted upfield by 3.5 ppm, while the signal attributable to the adjacent C-6″ was shielded downfield by 2.4 ppm, which indicated that the caffeoyl group was attached to C-6″ of the glucosyl residue. This was further confirmed by the HMBC correlation between H 2 -6″ (δ H 4.57, 4.35) and C-9″′ (δ C 169.2). The similar 1 H and 13 C NMR spectral data for 1 and 3, together with the analysis of J values and CoSY and RoeSY experiments, showed that the relative configuration of the C2-phenyl group of 1 was the same β-configuration as that in 3.
The anti-HCV activity of compounds 1, 9, 10, 13 and 15 were evaluated, and their cytotoxicity was measured in parallel with the determination of antiviral activity, using ribavirin as positive control (CC 50 = 82.47 nM, eC 50 = 10.44 nM). Among the compounds tested, compound 1 exhibited the most potent anti-HCV activity, with an eC 50 value of 0.041 nM, which was far lower than that of rabavirin, a CC 50 value of 5.57 nM, and a SI value of 135.85. Therefore, compound 1 seemed to be an HCV replication inhibitor and might serve as a candidate for further optimisation and drug development. Moreover, compound 9 showed moderate anti-HCV activity with an SI value of 20.84 (Table 1). This study suggested that the flavonoids isolated from E. bodinieri exhibited anti-HCV activity in vitro. Till now, this was the first report on the anti-HCV constituents from E. bodinieri. This study provided a basis for further optimisation and standardisation of E. bodinieri for development of anti-HCV therapy.

General experimental procedure
optical rotations were obtained using a Jasco DIP-370 digital polarimeter. uV spectra were run on a uV-210A spectrophotometer. IR spectra were recorded on a Bio-Rad FtS-135 spectrophotometer in a KBr disk. NMR spectra were recorded over Bruker AM-400 and DRX-500 instruments, using TMS as internal standard. eIMS were measured on a VG Auto-Spec-3000 spectrometer; eSIMS and HReSIMS were obtained with an API-Qstar-ToF instrument. Column chromatography (CC) was performed with silica gel (200-300 mesh, Qingdao Marine Chemical Factory, Qingdao, China), Lichroprep RP-18 (43-63 mm, Merck, Darmstadt, Germany) and Sephadex LH-20 (Amersham Biosciences AB, uppsala, Sweden); thin-layer chromatography (TLC) was performed on TLC plates (Si gel GF 254 ), and spots were detected by spraying with 5% H 2 So 4 -etoH, followed by heating on a hot plate.

Material
The sample of E. bodinieri was collected from Honghe, Yunnan Province, PR China, in May 2010, and was identified by Prof Hai-Zhou Li. A voucher specimen (KMuST 20101201) was deposited at the Laboratory of Phytochemistry, Faculty of Life Science and Technology, Kunming university of Science and Technology. Ribavirin (99%) and gentamycin (607 Iu/ mg, anhydrous) were purchased from Shandong Phoenix Pharmaceutical Co Ltd and Apollo Scientific Ltd, respectively. The purity (> 95%) of compounds 1, 9, 10, 13 and 15 used for biological assay was determined by HPLC.

Acid hydrolysis of compound 1
Compound 1 was spotted on a HPLC precoated plate and hydrolysed with concentrated HCl vapour (80 °C water bath for 20 min) followed by co-TLC with authentic sugar developing with CHCl 3 -MeoH-H 2 o-HoAc (7:3:0.5:1). The HPLC plate was sprayed with 10% H 2 So 4 for detection. The D-glucose was detected with an R f value of 0.40.

Anti-HCV activity
Inhibitor preparation. For the inhibitory activity assays, compounds 1, 9, 10, 13 and 15 were dissolved and then serially diluted with DMSo, using DMSo and ribavirin as blank and positive control, respectively. Cell line and cell culture. Stocks of infectious HCV J6/JHH-1 viral particles were generated as previously described (Li et al. 2012) and aliquoted for storage at -80 °C. The HCV load (RNA copies) in stocks was measured with real-time quantitative RT-PCR.
Anti-HCV assay in vitro. Anti-HCV assay was carried out in clear-bottom 96-well plates. Huh7.5.1 cells were seeded at a density of 1.5 × 10 4 cells/well in 100 μL of DMeM culture medium and incubated overnight, and viral stocks were added into the wells and incubated for 8 h. Before the samples were added, the supernatants were removed and cells were washed with completed medium for five times. The samples or controls were serially diluted in DMeM and then added to the appropriate wells. After incubation for three days, the supernatants were collected to determine their viral load with real-time quantitative RT-PCR.
Cytotoxicity assay. The toxicities of the compounds were assayed by a modified MTT method (Li et al. 2012). In brief, the test samples were prepared at different concentrations. After Huh7.5.1 cells had been seeded in a 96-well microplate for 4 h, the samples (20 μL) were placed in each well and incubated for three days at 37 °C; then, 0.1 mL MTT was added for 4 h. After removal of the MTT medium, DMSo (100 μL/well) was added onto the microplate for 10 min. The formazan crystals were dissolved, and the absorbance was measured on a microplate reader at 490 nm.

Conclusion
our present work on the plants of E. bodinieri yielded one new flavonoid glycoside, along with 14 known compounds. The new compound and luteolin (9) exhibited potent anti-HCV activities with a selective index of 135.85 and 20.84, respectively. This investigation should provide valuable information for further understanding of E. bodinieri.