Anti-H1N1 virus, cytotoxic and Nrf2 activation activities of chemical constituents from Scutellaria baicalensis

Author’s Accepted Manuscript Anti-H1N1 virus, cytotoxic and Nrf2 activation activities of chemical constituents from Scutellaria baicalensis Shuai Ji, Ru Li, Qi Wang, Wen-juan Miao, Zi-wei Li, Long-long Si, Xue Qiao, Si-wang Yu, De-min Zhou, Min Ye www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(15)30221-X http://dx.doi.org/10.1016/j.jep.2015.11.018 JEP9817

To appear in: Journal of Ethnopharmacology Received date: 17 July 2015 Revised date: 24 September 2015 Accepted date: 6 November 2015 Cite this article as: Shuai Ji, Ru Li, Qi Wang, Wen-juan Miao, Zi-wei Li, Longlong Si, Xue Qiao, Si-wang Yu, De-min Zhou and Min Ye, Anti-H1N1 virus, cytotoxic and Nrf2 activation activities of chemical constituents from Scutellaria baicalensis, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.11.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Anti-H1N1 virus, cytotoxic and Nrf2 activation activities of chemical constituents from Scutellaria baicalensis

Shuai Ji†, Ru Li†, Qi Wang, Wen-juan Miao, Zi-wei Li, Long-long Si, Xue Qiao, Si-wang Yu, De-min Zhou*, and Min Ye*

Affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, China

†

*

These two authors contributed equally to this paper.

Corresponding authors. Tel.: +86 10 82801516. Fax: +86 10 82802024. E-mail

address: [email protected] (M. Ye), or [email protected] (D.M. Zhou).

1

Abstract Ethnopharmacological relevance: Huang-Qin, derived from the roots of Scutellaria baicalensis Georgi, is a popular Chinese herbal medicine mainly used to treat influenza and cancer. This study aims to elucidate the anti-influenza, anti-cancer and anti-oxidation effective components of S. baicalensis. Materials

and

methods:

Various

column

chromatography

techniques

and

semi-preparative HPLC were used to isolate Scutellaria compounds, and their structures were identified by HRESIMS and NMR spectroscopic analysis. The pure compounds were evaluated for anti-influenza activities against A/WSN/33 (H1N1) virus in MDCK cells, cytotoxic activities against HepG2, SW480 and MCF7 human cancer cells by MTS assay, and antioxidant activities by Nrf2 luciferase reporter assay. In addition, the contents of 12 major compounds in 27 batches of S. baicalensis were simultaneously determined by a fully validated UPLC/UV method. Results: A total of thirty compounds (1-30), including four new ones (3, 7, 11 and 23), were isolated from S. baicalensis. Baicalin (15), baicalein (26), wogonin (27), chrysin (28) and oroxylin A (30) showed potent anti-H1N1 activities, with IC50 values of 7.4, 7.5, 2.1, 7.7 and 12.8 μM, respectively, which were remarkably more potent than the positive drug Osv-P (oseltamivir phosphate, IC50 45.6 μM). Most free flavones (26-28 and 30) showed significant cytotoxic activities at 10 μM (up to 61.2% inhibition rate). Furthermore, 30 could activate Nrf2 transcription by 3.8-fold of the control at 10 μM. UPLC analysis indicated the 12 major compounds (including the bioactive ones) accounted for 195.93  43.9 mg g-1 of the herbal materials.

2

Conclusion: This study demonstrated that free flavones showed potent anti-influenza, anti-cancer and anti-oxidative activities. They are important effective components of S. baicalensis, and can be used as chemical markers for quality control of this herbal medicine.

Keywords: Scutellaria baicalensis; effective components; anti-H1N1 virus; cytotoxicity; Nrf2 activation.

Main chemical compounds studied in this article: Baicalin (PubChem CID: 64982); baicalein (PubChem CID: 5281605); wogonin (PubChem CID: 5281703); chrysin (PubChem CID: 5281607); oroxylin A (PubChem CID: 5320315)

3

1. Introduction Scutellaria baicalensis Georgi (Labiatae), known as Huang-Qin in Chinese, is one of the most popular herbal medicines worldwide. It is widely used in TCM clinics to treat influenza, pneumonia, dysentery, and cancer (Chinese Pharmacopoeia Commission, 2010). It appears as a key ingredient in a number of traditional Chinese medicine prescriptions, including Huangqin Decoction, Gegen Qinlian Decoction, and Shuanghuanglian Injection (Shang et al., 2010). Thus far, more than 80 compounds have been isolated from S. baicalensis, including flavonoids (free and glycosylated forms) and phenylethanoid glycosides. Among them, baicalin and baicalein are generally considered as the major bioactive constituents. They possess anti-influenza (Miki et al., 2007; Nayak et al., 2014), anti-viral (Konoshima et al., 1992; Li et al., 2000a) and anti-inflammatory (Li et al., 2000b) activities. However, little is known on bioactivities of the other chemical constituents.

This study aims to systematically elucidate the anti-influenza, anti-cancer and anti-oxidation effective components of S. baicalensis. A total of 30 compounds (1-30) were isolated and identified, including 28 flavonoids and 2 phenylethanoid glycosides. Among them, four flavonoids (3, 7, 11 and 23) are new compounds, and their structures were established by NMR and HRESIMS data analyses. The sugar residues of 3 and 23 were established by ion chromatography coupled with pulsed amperometric detection (IC-PAD) analysis after hydrolysis. Upon cell-based assay, a number of compounds, particularly the flavone aglycones, showed significant

4

inhibitory activities against the influenza virus A/WSN/33 (H1N1) and human cancer cell lines (HepG2, SW480 and MCF7), as well as Nrf2 activation activities. In addition, the contents of 12 major compounds in 27 batches of S. baicalensis were simultaneously determined by UPLC/UV.

2. Materials and methods 2.1. General Optical rotations were measured on a Rudolph Research Autopol III automatic polarimeter. NMR spectra were recorded at 400 MHz for 1H and 100 MHz for 13C on a Bruker AVANCE III-400 spectrometer in DMSO-d6 with TMS as the reference. IR spectra were recorded as KBr disks on a Nicolet NEXUS-470 FT-IR spectrometer. UV spectra were measured on a Cary 300 Bio UV−visible spectrophotometer. HRESIMS data were obtained on a Bruker APEX IV FT-MS spectrometer. TLC was carried out on precoated silica gel GF254 plates (Qingdao Marine Chemical Inc., China), and the spots were visualized under UV light (365 nm). Open column chromatography was performed using AB-8 macroporous resin (Cangzhou Bao'en Chemical Factory, China), SBC MCI gel (75−150 μm, Sci-Bio Chem Co. Ltd., Chengdu, China), silica gel (200−300 mesh, Qingdao Marine Chemical Inc., China), ODS C18 (DAISO Company, Japan), and Sephadex LH-20 (GE Healthcare Bio-Science AB, USA). Semi-preparative HPLC was performed on an Agilent 1200 instrument equipped with a ZORBAX SB C18 column (250 × 9.4 mm, i.d. 5 μm, Agilent, USA). Reference sugar standards of D-glucose and D-glucuronic acid were from Sigma-Aldrich with

5

purity of above 97%. Scutellarin (31) was purchased from Chengdu Mansite Biological Technology Co. (Chengdu, China).

2.2. Plant Materials In total 27 batches of Scutellaria baicalensis (S1–S27, crude drug slices) were purchased from local pharmacies of different provinces around China in 2013. The materials were authenticated by the authors, and voucher specimens were deposited at the authors’ laboratory. Scutellaria compounds (1–30) were isolated from batch S1.

2.3. Extraction and Isolation The dried slices of S. baicalensis (5 kg) were powdered and extracted with 70% (50 L × 2 h × 1) and 95% EtOH (50 L × 2 h × 2) under reflux. After concentration in vaccum, the extract (1150 g) was dispersed in H2O, and extracted with petroleum ether, ethyl acetate, and n-butanol, successively. The n-butanol extract (225 g) was separated on an AB-8 macroporous resin column eluted with EtOH-H2O (10 to 95%, v/v) to obtain fractions 1-7. Fraction 1 was subjected to MCI gel column chromatography and eluted with EtOH-H2O (10 to 95%, v/v) to afford fraction 1-1, which was then separated on a Sephadex LH-20 column eluted with 50% MeOH to obtain fraction 1-1-3. Compounds 27 (12 mg) and 29 (37 mg) were obtained from fraction 1-1-3 by ODS C18 column chromatography eluted with 50% MeOH. Compounds 28 (10 mg, tR = 33.5 min) and 30 (19 mg, tR = 42.1 min) were obtained from fraction 1-1-3-5 by semi-preparative HPLC separated on an Agilent ZORBAX

6

SB C18 column (5 μm, 250 × 9.4 mm; flow rate, 2 mL/min; MeCN-H2O-TFA, 43:57:0.03, v/v/v; 254 nm). Fractions 2-1, 2-2 and 2-3 were obtained from fraction 2 by silica gel column chromatography eluted with CH2Cl2-MeOH (1:0 to 1:1, v/v). Fraction 2-1 was purified by ODS C18 column chromatography and semi-preparative HPLC (MeCN-H2O-TFA, 15:85:0.03, v/v/v) to yield 2 (48 mg, tR = 48.5 min). Compound 5 (7 mg) was obtained from fraction 2-2 by ODS C18 column chromatography eluted with MeOH-H2O (10 to 95%, v/v). Compounds 1 (5 mg, tR = 40.5 min), 3 (2 mg, tR = 50.1 min) and 4 (4 mg, tR = 58.6 min) were isolated from fraction 2-2-5 by semi-preparative HPLC eluted with MeCN-H2O-TFA (15:85:0.03, v/v/v). Fraction 2-3 was chromatographed over an ODS C18 column eluted with 50% MeOH to produce fraction 2-3-3. Compound 8 (30 mg) was obtained from fraction 2-3-3 by Sephadex LH-20 column chromatography eluted with 50% MeOH. Compounds 6 (208 mg, tR = 25.0 min), 7 (4 mg, tR = 32.5 min), 10 (53 mg, tR = 37.8 min) and 11 (5 mg, tR = 43.2 min) were obtained from fraction 2-3-3-2 by semi-preparative HPLC eluted with MeCN-H2O-TFA (13:87:0.03, v/v/v). Fraction 3 was subjected to MCI gel column chromatography to yield fractions 3-1 to 3-7. Compounds 9 (10 mg) and 17 (13 mg) were obtained from fraction 3-1 by Sephadex LH-20 column chromatography eluted with 50% MeOH. The other subfractions from 3-1 were purified by semi-preparative HPLC eluted with MeOH-H2O-TFA (35:65:0.03, v/v/v) to obtain 12 (6 mg, tR = 35.9 min), 20 (8 mg, tR = 40.2 min), and 22 (5 mg, tR = 48.6 min). Compounds 13 (46 mg, tR = 31.8 min) and 16 (78 mg, tR = 40.5 min) were obtained from fraction 3-2 by semi-preparative HPLC eluted with

7

MeCN-H2O-TFA (21:79:0.03, v/v/v). Similarly, 21 (261 mg, tR = 45.8 min) was obtained from fraction 3-4 by semi-preparative HPLC with MeOH-H2O-TFA (65:35:0.03, v/v/v) as the eluent. Fraction 3-7 was purified over an ODS C18 column to yield fractions 3-7-1 to 3-7-8. Compounds 14 (4 mg) and 19 (79 mg) were obtained from fraction 3-7-2 by Sephadex LH-20 column chromatography. Compounds 15 (500 mg) and 24 (129 mg) were obtained from fractions 3-7-4 and 3-7-8 by recrystallization, respectively. Compounds 18 (15 mg, tR = 38.2 min), 23 (5 mg, tR = 46.7 min) and 25 (4 mg, tR = 57.2 min) were obtained from fraction 3-7-6 by semi-preparative HPLC with MeCN-H2O-TFA (22:78:0.03, v/v/v) as the eluent. Fraction 4 was chromatographed over an ODS C18 column to yield fraction 4-4. Compound 26 (20 mg) was obtained from fraction 4-4 by recrystallization. The flow chart for detailed isolation procedure is given in Fig. S1. Purities of these compounds were above 97% by HPLC/UV analysis.

(2S)-5,7,6'-trihydroxyflavanone 2'-O-β-D-glucopyranoside (3): white powder; [α]25D -52.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 287 (2.82) nm; IR (KBr) vmax 3305, 1635, 1464, 1169, 1062 cm-1; HRESIMS m/z 449.1077 [M-H]-, calcd for C21H21O11, 449.1078; for 1H and 13C NMR data, see Table 1.

Chrysin

6-C-β-L-arabinopyranoside-8-C-β-D-glucopyranoside

(7):

yellow

powder; [α]25D -15.3 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 250 (2.41) nm; IR (KBr) vmax 3427, 1751, 1629, 1224, 1100 cm-1; HRESIMS m/z 547.1464 [M-H]-, calcd for

8

C26H27O13, 547.1452; for 1H and 13C NMR data, see Table 1.

Chrysin

6-C-β-D-glucopyranoside-8-C-β-L-arabinopyranoside

(11):

yellow

powder; [α]25D -18.9 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 250 (2.30) nm; IR (KBr) vmax 3386, 1652, 1581, 1357, 1077 cm-1; HRESIMS m/z 547.1462 [M-H]-, calcd for C26H27O13, 547.1452; for 1H and 13C NMR data, see Table 1.

(2S)-5-hydroxy-6-methoxyflavanone 7-O-β-D-glucuronopyranoside (23): white powder; [α]25D -49.9 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 286 (2.97) nm; IR (KBr) vmax 3402, 2927, 1645, 1057 cm-1; HRESIMS m/z 461.1072 [M-H]-, calcd for C22H21O11, 461.1078; for 1H and 13C NMR data, see Table 1.

2.4. Hydrolysis and IC-PAD Analysis of 3 and 23 To identify the sugar residues of compounds 3 and 23, they were hydrolyzed and then analyzed by IC-PAD, as we had previously reported (Song et al., 2014). Compound 3 (2.0 mg) was dissolved in 5 M trifluoroacetic acid (aqueous solution, 3 mL), and heated in a water bath at 90 °C for 3 h. After extraction with CH2Cl2 (3 mL × 3), the water-soluble layer was evaporated to dryness, and reconstituted in 2 mL of water. Compound 23 (2.0 mg) was distributed in 1 mL of β-glucuronidase solution (containing 19.86 U/μL, in NaOAc buffer, pH 5.5, Sigma-Aldrich) and incubated at 37 °C for 2 h. Then 5 mL of MeOH was added into the mixture and then centrifuged at 6000 rpm for 10 min to remove protein. The supernatant was dried under a gentle

9

nitrogen flow and reconstituted in 1 mL of water. The samples were filtered through a 0.22-μm membrane before IC-PAD analysis.

IC-PAD analysis was carried out on an ICS3000 ion chromatography instrument (Thermo-Dionex Inc., USA) equipped with an ED-3000 electrochemical detector. Samples were separated on a Dionex CarboPac PA20 column (3 × 150 mm) protected with a CarboPac PA20 guard column (3 × 30 mm). The linear gradient elution program was as follows: 0−8 min, 15 mM NaOH; 8.1−15 min, 15 mM NaOH + 150 mM NaOAc; 15.1−17 min, 200 mM NaOH. The flow rate was 0.45 mL/min, and the temperature was 30 °C. The injection volume was 5 μL for each sample. Data were processed by Chromeleon 2.2 software.

2.5. Anti-H1N1 Assay Cell-based anti-H1N1 assay was performed as described in our previous report (Song et al., 2014). Briefly, MDCK cells were seeded into 96-well plates (1 105 cells per well) 24 h before infection. Compounds were mixed with H1N1, and incubated for 15 min at room temperature. The mixed compound-containing cultures were added to medium-removed MDCK cells, followed by 36 h incubation (37 °C, 5% CO2). Microscopy was performed to determine the antiviral activity, and the data were confirmed by the CellTiter-Glo luminescent cell viability assay (Promega, #G7570). Cytotoxicities of the compounds were also determined in uninfected MDCK cells after 36 h treatment (Jones et al., 2006; Evelien et al., 2010).

10

2.6. Cytotoxic Activity Assay The cytotoxicities of tested compounds against human liver carcinoma cell line (HepG2), human colorectal adenocarcinoma cell line (SW480) and human breast carcinoma cell line (MCF7) were determined using the MTS assay. Briefly, HepG2 cells were cultured at 37 °C in a humidified 5% CO2 atmosphere in DMEM medium (SW480 cells in RPMI1640 medium and MCF7 in DMEM medium) supplemented with 10% fetal bovine serum and 1×penicillin-streptomycin solution. The cells were seeded at 5 × 103 cells/well in 96-well plates and cultured overnight. Compounds at 10 μM were then added into the culture, and incubated for another 24 h. Cell viability was measured by MTS assay following the manufacturer’s protocol (Promega, Madison, WI, USA).

2.7. Nrf2 Activation Assay HepG2 cells stably transfected with Nrf2/ARE luciferase reporter (HepG2C8 cells) were used to evaluate the effects of tested compounds on Nrf2 transcriptional activity. Briefly, the cells were treated with tested compounds (10 μM) and incubated for 6 h. Then the luciferase activities were measured using the Luciferase Assay System (Promega, WI, USA) on a Centro LB 960 microplate luminometer (Berthold, Germany). Tertiary butylhydroquinone (tBHQ) was used as the positive control (Turley et al., 2015).

11

2.8. Quantitative Analysis of 12 Compounds in S. baicalensis by UPLC/UV The external standard method was used to determine the contents of 12 compounds in S. baicalensis. Since the contents of baicalin (15) and wogonoside (24) are much higher than the other compounds, their calibration curves were separately established. The detailed experimental procedures for preparation of calibration standard solutions, quality control standard solutions, quantitative analysis samples and recovery test samples were described in Supporting Information. A Waters ACQUITY H-Class UPLC system was used for quantitative analysis. The system consisted of a quaternary solvent manager, an autosampler with cooling system, a column heater, and a DAD detector (Waters, Milford, MA, USA). Samples were separated on an Agilent Eclipse Plus C18 column (1.8 µm, 2.1 mm ID  150 mm). Mobile phase A was acetonitrile and methanol (3:1, v/v), and mobile phase B was water containing formic acid (0.1%, v/v). A gradient elution program was used: 0–3 min, 20% A; 3–16 min, 20–36% A; 16–17 min, 36–60% A; 17–23 min, 60–80% A. A 7-min post-run time was set to fully equilibrate the column. The flow rate was 0.35 mL min-1 and the column temperature was maintained at 55 C. The detection wavelength was set from 210 to 400 nm. The peak areas at 330 nm were calculated for scutellarin (31) and acteoside (8), and the peak areas at 275 nm were calculated for the other 10 analytes. An aliquot of 1 μL was injected for analysis. Data were processed using the EmpowerTM 3 analytical workstation.

3. Results and discussion 12

3.1. Compounds Isolation from S. baicalensis The dried slices of S. baicalensis (5 kg) were extracted with 70% and 95% EtOH. The concentrated extract was suspended in water, and then partitioned successively with petroleum ether, EtOAc, and n-BuOH. The n-BuOH extract was separated by macroporous resin, MCI gel, silica gel, ODS C18, Sephadex LH-20, and semi-preparative HPLC. A total of thirty compounds (1-30) were obtained, including four new flavonoids (3, 7, 11 and 23). By comparing their spectral data with literatures, the known compounds were identified as 5,7,6'-trihydroxyflavone 2'-O-β-D-glucopyranoside

(1)

(Zhou

(2R,3R)-3,5,7,2',6'-pentahydroxyflavanone 3,5,7,2′,6′-pentahydroxyflavone 6′-O-β-D-glucopyranoside

(4)

(5)

et

(2)

(Yu (Zhang

al.,

(Yoshiyuki

et

al., et

1997) et

1984), al.,

al.,

, 1985),

viscidulin 1994),

III

chrysin

6-C-α-L-arabinopyranoside-8-C-β-D-glucopyranoside (6) (Takagi et al., 1981), acteoside

(8)

(Pan

2'-O-β-D-glucopyranoside

et

al., (9)

2011), (Yukinori

5,6'-dihydroxy-7,8-dimethoxyflavone and

Tsuyoshi,

1995),

chrysin

6-C-β-D-glucopyranoside-8-C-α-L-arabinopyranoside (10) (Takagi et al., 1981), chrysin

8-C-β-D-glucopyranoside

(12)

(Yan

et

al.,

2011),

5,2'-dihrdroxy-6-methoxyflavone 7-O-β-D-glucuronopyranoside (13) (Yukinori and Tsuyoshi, 1994), (2S)-5,7,2',6'-tetrahydroxyflavanone (14) (Michinori et al., 1981), baicalin (15) (Liu et al., 2009), baicalein 7-O-β-D-glucopyranoside (16) (Liu et al., 2009), norwogonin 7-O-β-D-glucuronopyranoside (17) (Wang et al., 1988), wogonin 5-O-β-D-glucopyranoside (18) [16], cistanoside D (19) (Li et al., 1999), chrysin

13

7-O-β-D-glucuronopyranoside 7-O-β-D-glucuronopyranoside

(20)

(21)

(Ma,

(Marques

2013), et

al.,

oroxylin

2010),

oroxylin

A A

7-O-β-D-glucopyranoside (22) (Ma, 2013), wogonoside (24) (Wang et al., 2003), 5,7,6'-trihydroxy-8,2'-dimethoxyflavone (25) (Tsuyoshi et al., 1986), baicalein (26) (Wang et al., 2003), wogonin (27) (Wang et al., 2003), chrysin (28) (Wang et al., 2003), 5,6'-dihydroxy-6,7,8,2'-tetramethoxyflavone (29) (Huen et al., 2003), and oroxylin A (30) (Marques et al., 2010). Their structures are shown in Fig. 1.

Fig. 1. Structures of compounds 1-30 from Scutellaria baicalensis.

3.2. Structural Elucidation of New Compounds 3, 7, 11 and 23 Compound 3 was obtained as yellow amorphous powder. Its molecular formula 14

was established as C21H22O11 according to the HRESIMS spectrum ([M-H]- m/z 49.1077, calcd for C21H21O11, 449.1078). The [M-H]- ion could lose 162 Da to produce m/z 287, indicating the presence of a glucosyl residue. The 1H and 13C NMR spectra showed three aliphatic proton signals at δH 5.98 (1H, dd, J = 4.0, 12.8 Hz), 4.08 (1H, dd, J = 12.8, 16.0 Hz) and 2.35 (1H, dd, J = 4.0, 16.0 Hz), and two corresponding carbon signals at δC 71.3 (CH) and 38.7 (CH2). In addition, a carbonyl resonance at δC 197.5 was observed (Table 1). These signals were consistent with the C-ring of a flavanone skeleton (Michinori et al., 1981). The other proton signals included an aromatic ABX system [δH 6.58 (1H, br d, J = 8.0 Hz), 6.65 (1H, br d, J = 8.0 Hz) and 7.13 (1H, br t, J = 8.0 Hz)], two aromatic singlets at δH 5.83 (1H, br s) and 5.85 (1H, br s), and three phenolic hydroxyl groups at δH 12.29 (1H, s, 5-OH), 10.73 (1H, br s) and 9.87 (1H, br s). The aglycone of 3 was determined to be (2S)-5,7,2',6'-tetrahydroxyflavanone by comparing its NMR spectra with those of 14 (Michinori et al., 1981). The six carbon signals at δC 101.4, 77.1, 76.6, 73.2, 69.8 and 60.8, as well as the anomeric proton signal at δH 4.74 (1H, d, J = 8.0 Hz) suggested the presence of an O-β-D-glucopyranosyl residue. In order to confirm the sugar residue, 3 was hydrolyzed by trifluoroacetic acid, and the water-soluble fraction was analyzed by IC-PAD (Song et al., 2014). As expected, the chromatogram showed one peak at the same retention time as D-glucopyranose (Fig. S2). The HMBC correlation of H-1″ (δH 4.74)/C-2′ (δC 156.7) suggested the glucosyl residue was connected to C-2′

(Fig.

2).

Therefore,

compound

3

(2S)-5,7,6'-trihydroxyflavanone 2'-O-β-D-glucopyranoside.

15

was

identified

as

Compound 23 was obtained as white amorphous powder. Its molecular formula C22H22O11 was established by the HRESIMS spectrum ([M-H]- m/z 461.1072, calcd for C22H21O11, 461.1078). The [M-H]- ion could generate a major fragment at m/z 285 ([M-176-H]-), indicating the presence of a glucuronic acid residue. The aglycone was deduced to be a flavanone skeleton according to the characteristic proton signals of C-ring at δH 5.65 (1H, dd, J = 2.8, 12.8 Hz), 3.38 (1H, dd, J = 12.8, 17.2 Hz) and 2.84 (1H, dd, J = 2.8, 17.2 Hz), as well as two corresponding carbon signals at δC 78.7 (CH) and 42.2 (CH2) (Michinori et al., 1981). The proton signals at δH 7.53 (2H, d, J = 7.2 Hz, H-2' and 6'), 7.44 (2H, m, H-3' and 5') and 7.41 (1H, m, H-4'), and the carbon signals at δC 126.7 (2×CH, C-2' and 6'), 128.5 (2×CH, C-3' and 5') and 128.6 (CH, C-4') could be assigned to the unsubstituted B-ring (Marques, et al., 2010). The other proton signals of the aglycone included an aromatic singlet at δH 6.41 (1H, s), a phenolic hydroxyl group at δH 11.94 (1H, br s, 5-OH), and a methoxyl group at δH 3.68

(3H,

s).

The

aglycone

was

determined

to

be

(2S)-5,7-dihydroxy-6-methoxyflavanone, which had been isolated from Scutellaria baicalensis (Takagi et al., 1980). The carbon signals at δC 170.0, 99.1, 75.8, 75.3, 72.8, 71.2, and the anomeric proton signal at δH 5.20 (1H, d, J = 7.6 Hz) could be assigned to an O-β-D-glucuronic acid residue (Marques, et al., 2010). This was confirmed by IC-PAD analysis with a reference standard (Fig. S2). The HMBC correlation of H-1″ (δH 5.20)/C-7 (δC 157.7) suggested the β-D-glucuronic acid residue was connected to C-7 (Fig. 2). Based on the above evidences, the structure of 23 was established as

16

(2S)-5-hydroxy-6-methoxyflavanone 7-O-β-D-glucuronopyranoside.

Compound 7 was obtained as yellow amorphous powder. Its molecular formula C26H28O13 was established by the HRESIMS spectrum ([M-H]- m/z 547.1464, calcd for C26H27O13, 547.1452). The maximal UV absorptions at 273 and 318 nm were characteristic of a flavone skeleton (Liu et al., 2009). The [M-H]- ion at m/z 547 could fragment into m/z 487 [M-H-60]-, 457 [M-H-90]- and 427 [M-H-120]- due to cleavage of the sugar ring, indicating that 7 was a flavone C-glycoside (Vukics and Guttman, 2010). The 1H NMR spectrum showed two singlets at δH 10.24 and 13.71 for 7-OH and 5-OH, a singlet at δH 7.01 for H-3, a doublet at δH 8.20 (2H, J = 7.6 Hz) for H-2' and 6', a triplet at δH 7.55 (2H, J = 7.6 Hz) for H-3' and 5', a triplet at δH 7.63 (1H, J = 7.6 Hz) for H-4', an anomeric doublet at δH 4.73 (1H, J = 9.6 Hz), and an anomeric broad singlet at δH 5.31 (1H). These data suggested a 6,8-di-C-substituted chrysin skeleton (Takagi et al., 1981). Accordingly, the anomeric carbons resonated at δC 73.4 and 70.8 based on the HSQC spectrum. The 1D and 2D NMR spectra of 7 were very similar to those of 6, i.e. chrysin 6-C-α-L-arabinosyl-8-C-β-D-glucoside (Takagi et al., 1981). The major differences were in the arabinosyl unit. The characteristic anomeric proton (δH 5.31, br s) and the carbon resonances (δC 70.8, 72.0, 69.9, 63.2 and 66.6) suggested the presence of a β-L-arabinopyranosyl unit (Xie et al., 2003). The linkage sites for β-D-glucopyranose at C-8 and β-L-arabinopyranose at C-6 were confirmed by the HMBC correlations of H-1′′ (δH 4.73) with C-7 (δC 162.5), C-8 (δC 105.2) and C-9 (δC 157.3), as well as H-1′′′ (δH 5.31) with C-5 (δC 155.2), C-6 (δC 107.2) and C-7

17

(δC 162.5) (Fig. 2). Based on the above evidences, 7 was identified as chrysin 6-C-β-L-arabinopyranosyl-8-C-β-D-glucopyranoside.

Fig. 2. Key HMBC correlations for compounds 3, 7, 11 and 23.

Compound 11 has the same molecular formula as 7, according to its HRESIMS spectrum ([M-H]- m/z 547.1462, calcd for C26H27O13, 547.1452). Its 1D and 2D NMR spectra were almost identical to those of 7. The two anomeric protons at δH 4.61 (1H, d, J = 9.6 Hz) and δH 5.57 (1H, br s), as well as two groups of carbon resonances at δC 73.0, 71.5, 79.0, 70.0, 81.7, 61.7 and δC 70.8, 72.3, 69.8, 63.1, 67.0, indicated the presence of a C-β-D-glucopyranosyl unit and a C-β-L-arabinopyranosyl unit (Xie et al., 2003). By carefully analyzing the 2D NMR spectrum, we found that H-1′′ (δH 4.61, d, J = 9.6 Hz), instead of H-1′′′ (δH 5.31, br s) in 7, showed HMBC correlations with C-5 (δC 152.7), C-6 (δC 109.3) and C-7 (δC 162.5). Correspondingly, H-1′′′ (δH 5.57, br s) of 11 showed HMBC correlations with C-7 (δC 162.5), C-8 (δC 104.7) and C-9 (δC

159.9).

These

evidences

indicated 18

the

β-D-glucopyranose

and

β-L-arabinopyranose in 11 were attached with C-6 and C-8, respectively (Fig. 2). Thus,

the

structure

of

11

was

identified

as

chrysin

6-C-β-D-glucopyranosyl-8-C-β-L-arabinopyranoside.

3.3. Anti-H1N1 Virus Activities of Scutellaria Compounds in MDCK Cells The inhibition of Scutellaria compounds against influenza virus had been reported in several studies, though only a few compounds including baicalin, baicalein and chrysin were investigated (Miki et al., 2007; Nayak et al., 2014). In this work, we tested all the isolated compounds from S. baicalensis (except for 7 and 11 due to limited amounts) for their inhibitory activities (100 μM) against the influenza virus A/WSN/33 (H1N1) in MDCK cells using the Cell Titer-Glo luminescent cell viability assay. These compounds included 3 flavonoid C-glycosides (6, 10 and 12), 12 flavonoid O-glycosides (1, 5, 9, 13, 15-18, 20-22 and 24), 11 flavonoid aglycones (2-4, 14, 23 and 25-30), and 2 phenylethanoid glycosides (8 and 19).

According

to

the

results,

Scutellaria

compounds

showed

obvious

structure-activity relationships, and their anti-H1N1 activities followed the order of free flavonoids > O-glycosides > C-glycosides (Fig. 3). All the free flavones (26-30) except for 25 showed potent inhibitory activities against H1N1 virus, with inhibitory rates of >75% at 100 μM (Fig. S3 and Table S1). The flavanones (2 and 14) and flavonol 4 showed remarkably decreased activities. The O-glycosides of flavonoids showed moderate activities with inhibitory rates from -19% to 64%. Interestingly, the

19

glycosides of 6-OH and 6-OCH3 flavones (13, 15, 16, 21, 22) showed stronger activities than 6-H flavones (1, 5, 9, 12, 17, 18, 20, 24), with inhibitory rates of 47 ± 15% vs 0 ± 11%. The new compound 23, a 6-OCH3 flavanone glycoside, showed an inhibitory rate of 42% at 100 μM. None of the flavonoid C-glycoside (6, 10, and 12) showed observable inhibition against H1N1 virus. We further tested the IC50 values of several potent inhibitors. Baicalin (15), baicalein (26), wogonin (27), chrysin (28) and oroxylin A (30) showed IC50 values of 7.4, 7.5, 2.1, 7.7 and 12.8 μM, respectively, which were remarkably more potent than the positive drug Osv-P (oseltamivir phosphate or Tamiflu, IC50 45.6 μM).

20

100

8 9 10 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Osv-P DMSO

Inhibitory rates (%)

1 2 3 4 5 6 120

Anti-H1N1 activities (100 μM)

80 60 40 20 0 -20

Inhibitory rates (%)

-40 80 60

HepG2 cytotoxic activities (10 μM)

40 20 0

×

Inhibitory rates (%)

-20 70 50

SW480 cytotoxic activities (10 μM)

30 10

×

-10

Inhibitory rates (%)

-30 70 50

MCF7 cytotoxic activities (10 μM)

30 10

×

-10

4

Nrf2 activation activities (10 μM)

3 2 1 0

1 2 3 4 5 6

8 9 10 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Flavonoid aglycone Flavonoid C-glycoside

tBHQ DMSO

Folds of control

-30 5

Flavonoid O-glucoside and O-glucuronoside Phenylethanoid glycoside

Fig. 3. Anti-H1N1 (100 μM), cytotoxic (10 μM) and Nrf2 activation (10 μM) activities of Scutellaria compounds. ×, not tested.

21

3.4. Cytotoxic Activities of Scutellaria Compounds against HepG2, SW480 and MCF7 Cells Scutellaria baicalensis and its chemical constituents had been reported to possess remarkable anti-tumor activities (Li, 2009; Shieh et al., 2000). In this study, the cytotoxicities of Scutellaria compounds (10 μM) against three human cancer cell lines, namely human hepatocellular carcinoma cell line (HepG2), human colorectal adenocarcinoma cell line (SW480), and human breast carcinoma cell line (MCF7) were evaluated. The free flavones 26–28 and 30 showed obvious cytotoxic activities against all the three cell lines (Fig. 3 and Table S1). For example, wogonin (27) could inhibit HepG2, SW480 and MCF7 cell viability by 61.2%, 28.9% and 39.1% at 10 μM, respectively. Nevertheless, none of flavonoid C-glycosides, flavonoid O-glycosides or phenylethanoid glycosides showed significant cytotoxic activity.

3.5. Nrf2 Activation Activities of Scutellaria Compounds in HepG2C8 Cells The antioxidant activities of Scutellaria compounds (10 μM) were evaluated by measuring their activation effects on Nrf2 (nuclear factor erythroid 2-related factor 2) transcriptional activity using a luciferase reporter assay (Beyotime Biotech Inst, Jiangsu, China) in HepG2 cells stably transfected with Nrf2 luciferase reporter (HepG2C8 cells) (Lee and Johnson, 2004; Xi et al., 2013). Seven compounds (5, 6, 10, 17, 23, 26 and 30) could activate Nrf2 transcription by >1.4-fold of the control (the positive control tBHQ, 4.3-fold of the control at 100 μM or 1.5-fold at 10 μM). The most active compound, oroxylin A (30), could activate Nrf2 transcription by 3.8-fold

22

(Fig. 3 and Table S1) at 10 μM.

3.6. Quantitative Analysis of 12 Compounds in S. baicalensis by UPLC/UV The contents of 12 compounds (2, 6, 8, 10, 15, 17, 21, 24, 26, 27, 30, 31) in S. baicalensis were simultaneously determined by UPLC/UV. They represented the major chemical constituents of S. baicalensis, and several compounds showed significant bioactivities (like 15, 21, 26, 27 and 30). Although scutellarin (31) was not isolated in our phytochemical experiments, it has been considered as a representative bioactive compound of Scutellaria species, and was also determined in this study.

In order to extract the compounds completely and efficiently, extraction methods (ultrasonic bath, reflux, soaking), extraction solvents (40%, 70%, 100% methanol or ethanol, v/v), solvent volume (50, 100, 200, 300, 400 and 500 folds), and extraction time (15, 30, 45 and 60 min) were optimized (Figs. S4–S7). In addition, different UPLC columns (Eclipse Plus C18, XTerra, Atlantis dC18, Zorbax SB-C18 and Extend-C18), the organic mobile phase (methanol, acetonitrile, and their mixtures), aqueous mobile phase (different concentrations of formic acid), column temperatures (50C and 55C), flow rates (0.30 mL min-1 and 0.35 mL min-1) were also optimized to obtain desirable chromatographic resolutions (Figs. S8–S11). The detailed experimental results were described in Supporting Information. With the optimized conditions, the system pressure was 8000 psi, and all the 12 analytes could be well separated within 20 min. According to maximal UV absorptions of the analytes, 330

23

nm was selected for the detection of scutellarin (31) and acteoside (8), and 275 nm was used for the other ten compounds. A typical chromatogram is shown in Fig. 4. 15

Reference standards

0.20

27

AU

21 2

6 31 8

17

24

10

26 30

0.00

Scutellaria baicalensis (S1)

15

AU

1.00

24

0.00 0.10

Scutellaria baicalensis (enlarged)

24

15 17

AU

6 2

31 8

27

21

26

10

30

0.00 0.00

5.00

10.00

15.00

20.00

Time (min)

Fig. 4. UPLC/UV chromatograms (275 nm) for quantitative analysis of 12 compounds in Scutellaria baicalensis. Compounds

identification:

(2R,3R)-3,5,7,2',6'-pentahydroxyflavanone

6-C-α-L-arabinoside-8-C-β-D-glucoside

(6),

acteoside

(2), (8),

chrysin chrysin

6-C-β-D-glucoside-8-C-α-L-arabinoside (10), baicalin (15), norwogonin 7-O-β-D-glucuronoside (17), oroxylin A 7-O-β-D-glucuronoside (21), wogonoside (24), baicalein (26), wogonin (27), oroxylin A (30), and scutellarin (31).

We used the external standard method to establish the calibration curves, as shown in Table S2. All the 12 compounds showed good linearity (r2 > 0.999) in a dynamic range of 48–100 folds (0.47–10.20 to 35.51–1020.00 μg mL-1). Limit of detection (LOD) and limit of quantitation (LOQ) were determined at a signal-to-noise (S/N) ratio of 3 and 10, respectively. LOD of the 12 compounds varied from

24

0.005–0.05 μg mL-1, and the LOQ ranged from 0.02–0.32 μg mL-1. The method validation details (accuracy, precision, repeatability and stability) were described in Supporting Information.

In total 27 batches of S. baicalensis samples were analyzed using the validated method. Representative chromatograms are shown in Fig. S12. Contents of the 12 analytes were summarized in Table 2. The 12 analytes accounted for 123.6–267.2 mg g-1 of the herbal materials, except for sample S21 (68.7 mg g-1). Content variation between batches were moderate for most analytes (RSD 30%-50%), except for 8 (acteoside, RSD 145%). The contents of baicalin, wogonoside and their aglycones in S. baicalensis had been extensively studied, and our results were consistent with literature data (Makino et al., 2008; Islam et al., 2012). All the 27 batches except for S3, S6, S14 and S21 fulfilled the requirement of Chinese Pharmacopeia, which states the content of baicalin (15) should be no lower than 8.0% (Chinese Pharmacopoeia Commission,

2010).

The

contents

of

oroxylin

A

(30),

oroxylin

A

7-O-β-D-glucuronoside (21), scutellarin (31) and acteoside (8) had also been reported, and our results were comparable to the literature data (Xie et al., 2002). The contents of 2, 6, 10 and 17 in S. baicalensis were reported for the first time in this study.

Among the 12 compounds, baicalin (15), oroxylin A 7-O-β-D-glucuronoside (21), baicalein (26), wogonin (27) and oroxylin A (30) showed potent anti-H1N1 virus activities. They had average contents of 116.8, 11.6, 12.0, 4.4 and 1.7 mg g-1 of the

25

herbal materials in the 27 batches of samples, respectively. Moreover, 26, 27 and 30 showed significant cytotoxic activities against HepG2, SW480 and MCF7 cells, and 30 also showed potent Nrf2 activation activity. These compounds are important anti-influenza, anti-cancer and anti-oxidation effective components of S. baicalensis, and could be used as chemical markers for its quality control.

4. Conclusions A total of thirty compounds (1-30) were isolated and identified from Scutellaria baicalensis, including four new flavonoids (3, 7, 11 and 23). Their biological activities were tested by cell-based assay. The results indicated that a number of free flavones, including baicalein, wogonin and oroxylin A, showed potent anti-H1N1, cytotoxic, and Nrf2 activation activities, which are closely related with the therapeutic effects of S. baicalensis. They should be important effective components of S. baicalensis. The contents of 12 major compounds (including the bioactive ones) were determined by a fully validated UPLC method. This study systematically elucidated the anti-influenza, anti-cancer, and antioxidant effective components of S. baicalensis. The established UPLC method could be used for rapid and efficient quality control of this popular herbal medicine.

Acknowledgments This work was supported by National Natural Science Foundation of China (No. 81222054, No. 81470172), the Program for New Century Excellent Talents in

26

University from Chinese Ministry of Education (No. NCET-11-0019), and National Science and Technology Mega Project for Primary Drug Innovation of China (No. 2014ZX09304307-001-011). We wish to thank Drs. Li-na Liang and Ren-yong Li (ThermoFisher Scientific Ltd.) for their kind help in IC-PAD analysis.

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viscidula Bunge, Acta Pharm. Sin. 19, 397–398. Yukinori, M., Tsuyoshi, T., 1994. Constituents of Scutellaria species XVI. On the phenol glycosides of the root of Scutellaria baicalensis Georgi, J. Nat. Med. 48, 215–218. Yukinori, M., Tsuyoshi, T., 1995. Studies on the constituents of Scutellaria species XVII. Phenol glycosides of the root of Scutellaria baicalensis Georgi (2), J. Nat. Med. 49, 350–353. Zhang, Y.Y., Guo, Y.Z., Hashimoto, K., Hashimoto, K., Ikeya, Y., Okada, M., Maruno. M., 1994. Four flavonoids from Scutellaria baicalensis, Phytochemistry 35, 511–514. Zhou, Y., Hirotani, M., Yoshikawa, T., Furuya, T., 1997. Flavonoids and phenylethanoids

from

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root

Phytochemistry 44, 83–87.

32

cultures

of

Scutellaria

baicalensis,

DMSO-d6)

for 3, 7, 11, and 23. δH

162.9, C

δC,

-

δH (J in Hz)

11 -

7 δC,

23 163.4, C

3 δH

type

5.65, dd (2.8, 12.8)

(J in Hz)

δC,

type

78.7, CH

(J in Hz)

δH type

5.98, dd (4.0, 12.8)

(J in Hz)

δC, type

Table 1. 1H and 13C NMR spectral data (400 MHz, Position 71.3, CH 42.2,

3.38, dd (12.8, 17.2)

103.5, CH

7.01, s

102.4, CH

-

7.06, s

2 4.08, dd (12.8, 16.0)

182.4, C

-

38.7,

-

152.7, C

-

3

182.4, C

-

109.3, C

5

4

105.2, C

-

-

-

130.5, C

103.4, C

159.9, C

104.7, C

162.5, C

-

-

-

-

-

2.84, dd (2.8, 17.2) 197.4, C

155.2, C

-

157.3, C

-

CH2

154.6, C

107.2, C

95.4, CH

104.6, C

5.85, s

6 166.3, C 157.7, C

131.0, C

2.35, dd (4.0, 16.0) 197.5, C 130.0, C

162.5, C

94.7, CH 103.4, C

CH2

163.9, C 158.1, C

7 163.6, C 138.5, C 128.5, CH

126.7, CH

7.41, m

7.44, m

7.53, d (7.2)

132.1, CH

129.1, CH

126.9, CH

7.55, t (7.6)

7.63, t (7.6)

7.55, t (7.6)

8.20, d (7.6)

129.2, CH

132.1, CH

129.2, CH

126.4, CH

7.56, dd (6.4, 6.8)

7.63, t (6.4)

7.56, dd (6.4, 6.8)

8.16, d (6.8)

6.41, s

8 101.7, C 156.7, C 6.65, d (8.0)

128.6, CH

129.1, CH

8.16, d (6.8)

94.4, CH

9 112.9, C

2' 106.6, CH 7.13, t (8.0)

7.44, m

126.4, CH

5.83, s

1'

10

3' 130.2, CH

128.5, CH

8.20, d (7.6)

4.61, d (9.6)

4' 6.58, d (8.0)

126.9, CH

73.0, CH

110.0, CH

7.53, d (7.2)

4.73, d (9.6)

5'

126.7, CH

73.4, CH

3.83, m

157.4, C

5.20, d (7.2)

71.5, CH

6'

99.1, CH

3.73, m

4.74, d (8.0)

70.8, CH

101.4, CH

3.39, m

1''

72.8, CH

3.10, m

3.18, m

73.2, CH

79.0, CH

2''

3.25, m

75.3, CH

78.7, CH

3.25, m

3.31, m

76.6, CH

3.10, m

3''

3.15, m

3.40, m

70.0, CH 3.98, d (9.6)

81.7, CH

71.2, CH

3.40, m 75.8, CH

3.22, m

3.13, m

5.57, br s

3.39, m; 3.69, m

70.6, CH 3.31, m

70.8, CH

CH2

81.9, CH

69.8, CH

4''

77.1, CH

5''

61.4,

70.8, CH

5.31, br s

61.7,

170.0, C

60.8,

CH2

3.47, m; 3.70, m

6'' 1'''

CH2

3.53, m; 3.75, m

33

3'''

2''' 69.9, CH

72.0, CH

3.82, m

3.72, m

69.8, CH

72.3, CH

4.01, m

3.90, m

3.84, m

13.47, s

63.1, CH

9.95, s

3.95, m 13.71, s

63.2, CH

10.24, s

4''' 11.94, s

3.63, m; 3.77, m

12.29, s

67.0, CH2

10.73, s

3.55, m; 3.69, m

5-OH

3.68, s

66.6, CH2

7-OH 9.87, s 60.3, CH3

5'''

6'-OH 8-OCH3

34

S21

S20

S19

S18

S17

S16

S15

S14

S13

S12

S11

S10

S9

S8

S7

S6

S5

S4

S3

S2

S1

Batch No.

Shandong

Hunan

Henan

Henan

Gansu

Zhejiang

Shandong

Hebei

Hubei

Zhejiang

Anhui

Liaoning

Zhejiang

Shanxi

Shanghai

Liaoning

Liaoning

Liaoning

Shandong

Guangxi

Inner Mongolia

Beijing

Purchased from

Yunnan

Shandong

unknown

unknown

Henan

Gansu

Gansu

Hebei

Hebei

Anhui

Anhui

Anhui

Shanxi

Shanxi

Shanxi

Shanxi

Inner Mongolia

Inner Mongolia

Inner Mongolia

Inner Mongolia

Inner Mongolia

Inner Mongolia

Inner Mongolia

Cultivated in

2.46

2.62

0.52

3.01

3.05

3.31

2.09

3.21

2.93

1.15

2.34

2.64

2.16

2.12

3.15

1.67

2.71

1.98

2.38

2.66

1.96

2.73

1.46

2

0.96

0.79

0.28

0.85

0.97

0.98

0.54

0.82

0.82

1.08

1.01

1.05

0.92

0.87

0.89

0.74

0.82

0.55

1.76

0.66

0.58

1.35

2.12

31

3.13

3.92

1.08

3.74

4.13

3.57

3.14

3.64

4.38

2.53

3.56

3.78

4.26

4.21

3.95

4.66

3.65

3.24

4.5

3.55

2.5

3.06

4.84

6

0.83

0.85

0.87

0.90

1.05

2.91

0.59

0.92

1.10

6.46

4.05

2.34

0.91

0.87

1.14

1.43

0.74

1.04

11.42

1.16

0.57

6.20

15.83

8

3.26

3.80

0.95

3.60

4.02

3.11

3.25

3.91

4.28

2.40

3.36

3.35

4.16

4.13

3.77

4.28

3.45

3.47

4.38

3.72

2.75

2.91

4.61

10

122.12

138.84

36.71

122.54

143.21

110.08

107.51

128.12

142.99

77.23

111.92

92.94

152.45

146.99

133.56

157.85

101.26

53.81

133.34

112.23

79.48

85.77

163.88

15

6.80

7.02

2.06

6.08

7.55

2.49

5.55

6.63

7.26

5.36

6.53

5.15

7.81

7.52

7.81

9.24

7.35

3.22

9.62

6.16

4.38

4.61

9.97

17

9.24

13.08

3.16

12.27

13.92

26.13

8.29

8.39

14.64

6.87

11.88

10.67

17.47

17.14

14.84

16.02

8.88

7.42

11.62

8.35

7.03

7.56

13.42

21

37.57

36.43

8.83

29.92

34.35

31.07

29.67

34.69

35.53

21.20

30.49

26.29

38.06

37.08

35.66

37.38

24.29

16.81

30.68

29.47

22.35

22.56

38.69

24

18.82

9.12

8.94

10.78

11.18

2.84

16.64

14.79

9.70

4.66

7.81

14.21

4.64

4.73

10.01

7.36

15.00

19.57

16.79

24.81

17.01

8.08

7.67

26

5.20

2.74

3.74

3.35

4.19

1.65

5.32

6.10

2.97

2.27

3.67

6.44

1.72

1.81

5.19

3.27

5.10

9.19

6.61

6.61

5.24

3.88

3.62

27

1.60

1.07

1.56

1.37

1.52

1.12

1.64

1.76

1.21

0.86

1.48

2.94

0.76

0.79

2.32

1.43

2.05

4.29

2.54

2.10

1.72

1.54

1.17

30

211.98

220.28

68.69

198.42

229.13

189.26

184.22

212.98

227.80

132.06

188.09

171.79

235.31

228.23

222.30

245.32

175.31

123.55

235.63

201.49

145.58

150.26

267.27

total

Table 2. Contents of 12 compounds in 27 batches of Scutellaria baicalensis.

S22

Yunnan

Contents (mgg-1)

S23

35

S26

S25

S24 Beijing

Fujian

Jiangxi unknown

unknown

unknown 3.41

2.73

2.41 0.96

0.78

0.79 4.05

3.54

3.31 0.92

0.73

0.67 3.13

3.55

3.45

104.48

102.52

134.37

6.06

5.31

6.97

12.62

7.54

9.17

26.27

26.89

37.91

9.21

25.76

15.97

3.37

7.35

4.63

1.10

2.14

2.49

1.41

237.66

176.61

189.77

221.04

195.93

3.02

1.70

22.4

7.84

4.36

44.4

35.27

12.00

41.4

14.22

30.20

50.0

7.17

11.55

24.5

156.51

6.36

39.9

4.02

116.77

30.4

0.86

3.52

27.2

3.97 2.49

20.9

0.81 3.62 145.2

2.87 0.92 21.2

unknown 2.43 39.6

Tianjin Average 27.8

S27 RSD (%)

36

Figure legends Fig. 1. Structures of compounds 1–30 from Scutellaria baicalensis. Fig. 2. Key HMBC correlations for compounds 3, 7, 11 and 23. Fig. 3. Anti-H1N1 (100 μM), cytotoxic (10 μM) and Nrf2 activation (10 μM) activities of Scutellaria compounds. ×, not tested. Fig. 4. UPLC/UV chromatograms (275 nm) for quantitative analysis of 12 compounds in

Scutellaria

baicalensis.

Compounds

identification:

(2R,3R)-3,5,7,2',6'-pentahydroxyflavanone (2), chrysin 6-C-α-L-arabinoside-8-C-β-D-glucoside (6), acteoside (8), chrysin 6-C-β-D-glucoside-8-C-α-L-arabinoside (10), baicalin (15), norwogonin 7-O-β-D-glucuronoside (17), oroxylin A 7-O-β-D-glucuronoside (21), wogonoside (24), baicalein (26), wogonin (27), oroxylin A (30), and scutellarin (31).

37