Flavonoid Glycosides and Their Derivatives from the Herbs of Scorzonera austriaca Wild

Five flavonoid glycosides and two derivatives were isolated from the herbs of Scorzonera austriaca Wild by silica gel column chromatography and preparative HPLC. Their structures were identified, using chemical and spectroscopic methods, as 5,7,4′-trihydroxyflavone 6-C-(2''-O-β-d-glucopyranosyl β-d-glucopyranoside) (1), 5,7,3′,4′-tetrahydroxyflavone 6-C-(2''-O-β-d-glucopyranosyl β-d-glucopyranoside) (2), quercetin 3-O-rutinoside (3), 5,7,4′-trihydroxyflavone 6-C-β-d-glucopyranoside (4), 3′-methoxy-5,7,4′-trihydroxyflavone 6-C-β-d-glucopyranoside (5), 5,7,4′-trihydroxyflavone 8-C-(6''-O-trans-caffeoyl β-d-glucopyranoside) (6), and 5,7,3′,4′-tetrahydroxyflavone 8-C-(6''-O-trans-caffeoyl β-d-glucopyranoside) (7). Compounds 6 and 7 are new flavonoid glycoside derivatives, and compounds 1–5 were isolated from the herbs of Scorzonera austriaca for the first time. Compounds 6 and 7 were also assayed for their hepatoprotective activities with rat hepatocytes in vitro.


Introduction
Scorzonera austriaca Wild, a perennial herb of Compositae, is widely distributed in the northeast and northwest regions in China, especially abundant in Jilin Province, and has been widely used for curing fever, carbuncle, and mastitis as a traditional herbal medicine [1]. Since S. austriaca is used to treat hepatitis B as a folk medicine in our district, we have investigated its bioactivities and found that the total flavonoids from S. austriaca have hepatoprotective effects and inhibitory effects on hepatitis B virus [2][3][4]. Chronic infection with hepatitis B virus (HBV) often leads to the development of liver cancer and cirrhosis, creating immense sociological, clinical, and economic burdens worldwide [5]. Globally, 240 million people are infected with the HBV [6], and 650,000 people die every year from HBV-related cirrhosis or hepatocellular carcinoma [7]. Currently, seven drugs have been approved by FDA for the treatment of HBV infection: two interferons (standard and pegylated) and five nucleoside/nucleotide analogues (lamivudine, adefovir, telbivudine, entecavir, and tenofovir) [8]. Although interferons can restitute the host immune system, and have finite duration and no risk of drug resistance, the need for parenteral administration, the poor long-term response, and the high frequency of adverse side effects make them not ideal [8]. Nucleoside/nucleotide analogues are less expensive and orally available, have minimal side-effects comparing to interferons and can be used for decompensated cirrhosis and after liver transplantation [9]. However, because they have to be taken on a long-term basis, in general, drug resistance may evolve, and there is also a significant risk of HBV reactivation and sometimes HBV flare up after withdrawal of the antiviral agents [10]. Moreover, all of these drugs have low percentage of HBV e antigen seroconversion rate and HBV surface antigen loss, and none of them are able to clear chronic HBV infection [9,11,12]. Thus, new efforts are being directed to develop new and are able to clear chronic HBV infection [9,11,12]. Thus, new efforts are being directed to develop new and more effective anti-HBV therapeutics. In order to screen the new drug candidate for treating hepatitis B, the isolation and structure identification of flavonoid glycosides and their derivatives from the herbs of S. austriaca have been carried out, and we report the isolation and identification of two new flavonoid glycoside derivatives (6 and 7), together with five flavonoid glycosides 1-5 in the present study ( Figure 1

Results and Discussion
The air-dried S. austriaca herbs were extracted with 70% aqueous ethanol solution (v/v), the extract was subjected to D101 polyporous resin column chromatography eluated with water and 60% aqueous ethanol solution (v/v), and the crude flavonoid extracts were obtained from 60% aqueous ethanol eluate. The crude flavonoid extracts were loaded on D4020 polyporous resin column eluting successively with water and different aqueous ethanol solutions, and four fractions were obtained. The four fractions were further separated and purified by silica gel column chromatography and semi-preparative RP-HPLC to afford compounds 1-7, including two new flavonoid glycoside derivatives (6 and 7).

Results and Discussion
The air-dried S. austriaca herbs were extracted with 70% aqueous ethanol solution (v/v), the extract was subjected to D101 polyporous resin column chromatography eluated with water and 60% aqueous ethanol solution (v/v), and the crude flavonoid extracts were obtained from 60% aqueous ethanol eluate. The crude flavonoid extracts were loaded on D4020 polyporous resin column eluting successively with water and different aqueous ethanol solutions, and four fractions were obtained. The four fractions were further separated and purified by silica gel column chromatography and semi-preparative RP-HPLC to afford compounds 1-7, including two new flavonoid glycoside derivatives (6 and 7).
The hepatoprotective activities of compounds 6 and 7 were assessed by measuring the content of alanine aminotransferase (ALT) of the cultures of rat hepatocytes injured by CCl 4 . Compounds 6 and 7 exhibited hepatoprotective activities with values of 71.2% and 81.2%, respectively, at a concentration of 100 µM, comparable to that of silibinin [22], which was used as a positive control (68.3% at 50 µM) (Table 2). Therefore, the flavonoid glycoside derivatives 6 and 7 are considered to be two of the hepatoprotective principles in this plant. All data were analyzed using SPSS version 20.0 (International Business Machines Corporation, Armonk, NY, USA); the each value represents the mean˘SD (n = 3); the % of protection is calculated as 100ˆ(values of CCl 4´v alue of sample)/(value of CCl 4´v alue of control); * p < 0.01, compared with control group; # p < 0.05, ## p < 0.01, compared with

Extraction and Isolation
Two kilograms of air-dried whole S. austriaca herbs were extracted twice with 20 L of 70% aqueous ethanol solution (v/v) at room temperature. The extraction solution was concentrated under reduced pressure to remove ethanol, and the water concentrate was filtered and then passed through a D101 polyporous resin column eluting successively with water and 60% aqueous ethanol solution (v/v). The crude flavonoid extracts were obtained from 60% aqueous ethanol eluate by vacuum distillation recovery and used for the next experiments. The crude flavonoid extracts were loaded on D4020 polyporous resin column eluting successively with water, 15%, 20%, 25%, 30%, and 40% aqueous ethanol solutions (v/v), and six fractions (Fractions 1-6 , v/v, lower layer) to afford Fraction 6a. They were further isolated by semi-preparative RP-HPLC using acetonitrile and 0.1% formic acid solution in water as the mobile phase and the eluate was monitored at 337 nm. Compound 1 (30 mg), 2 (30 mg), and 3 (20 mg) were obtained from Fraction 2a with gradient elution (10%-11% acetonitrile from 0.00-20.00 min, 11%-18% acetonitrile from 20.00-40.00 min), compound 4 (30 mg) and 5 (20 mg) from Fraction 4a by using 15% acetonitrile, and compound 6 (60 mg) and 7 (200 mg) from Fraction 6a with gradient elution (20%-22% acetonitrile from 0.00-45.00 min).

In Vitro Hepatoprotective Activity
Isolated rat hepatocytes from female Wistar rats were prepared by the collagenase perfusion technique with minor modifications [23]. Culture medium was composed of RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% Pen/Strep (100 IU¨mL´1 penicillin and 100 mg¨mL´1 streptomycin). The isolated cells were diluted to 8ˆ10 6 / mL using the culture medium, and every 8ˆ10 5 cells (0.1 mL) were seeded into a 48-well plate and incubated at 37˝C in a humidified atmosphere containing 5% CO 2 . After preincubation for four hours, the medium was replaced with fresh medium containing CCl 4 (15 mM) and test specimens at various concentrations, and four hours later, the medium was taken to measure ALT. Enzyme-linked immunosorbent assay kits and DNM-9602 enzyme immunoassay spectrophotometer were used to measure ALT. For the results, see the Table 2.

Conclusions
Compounds 6 and 7 are new flavonoid glycoside derivatives. Compounds 1-5 were isolated from S. austriaca Wild for the first time. Compounds 6 and 7 were also assayed for hepatoprotective activities with rat hepatocytes, and the data proved that compounds 6 and 7 exhibited hepatoprotective activities. Therefore, the new flavonoid glycoside derivatives 6 and 7 are considered to be two of the hepatoprotective properties in this plant.