European Journal of Pharmacology 744 (2014) 147–156

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

Synthesis and biological evaluation of a novel baicalein glycoside as an anti-inflammatory agent Kyun Ha Kim a,1, Young-Don Park b,1, Heejin Park b, Keum-Ok Moon b, Ki-Tae Ha a, Nam-In Baek c, Cheon-Seok Park c, Myungsoo Joo a,n, Jaeho Cha b,nn a

School of Korean Medicine, Pusan National University, Yangsan 626-870, Republic of Korea Department of Microbiology, Pusan National University, Busan 609-735, Republic of Korea c Graduate School of Biotechnology and Institute of Life Sciences & Resources, Kyung Hee University, Yongin 446-701, Republic of Korea b

art ic l e i nf o

a b s t r a c t

Article history: Received 25 June 2014 Received in revised form 6 October 2014 Accepted 8 October 2014 Available online 29 October 2014

Baicalein-6-α-glucoside (BG), a glycosylated derivative of baicalein, was synthesized by using sucrose and the amylosucrase of Deinococcus geothermalis and tested for its solubility, chemical stability, and anti-inflammatory activity. BG was 26.3 times more soluble than baicalein and highly stable in buffered solutions and Dulbecco's modified Eagle medium containing 10% fetal bovine serum. BG treatment decreased the production of nitric oxide in RAW 264.7 cells treated with lipopolysaccharide (LPS). Luciferase reporter assays, western blots, reverse transcription-polymerase chain reaction, and flow cytometric analyses indicated that BG activated nuclear factor erythroid 2-related factor 2 (Nrf2), an antioxidant transcription factor that confers protection from various inflammatory diseases, induced Nrf2-dependent gene expression, and suppressed the production of reactive oxygen species elicited by LPS more effectively than baicalein. Cellular uptake of BG assessed by confocal microscopy and HPLC analysis of the cell-free extracts of RAW 264.7 cells demonstrated that BG was gradually converted to baicalein inside the cells. These results explain that glycosylation increased the bioavailability of baicalein by helping to protect this vital molecule from chemical or enzymatic oxidation. Therefore, BG, a glycosylated derivative of baicalein, can be an alternative to baicalein as a therapeutic drug. & 2014 Elsevier B.V. All rights reserved.

Keywords: Amylosucrase Anti-inflammation Baicalein Glycoside Nrf2 Transglycosylation Chemical compounds studied in this article: Baicalein (PubChem CID: 5281605) Baicalin (PubChem CID: 64982)

1. Introduction Scutellaria baicalensis is one of the most widely used herbal medicine against bacterial infections of the respiratory and gastrointestinal tracts and various inflammatory diseases. Its roots have been prescribed for the treatment of fever, high blood pressure, and acute pneumonia in Korean traditional medicine (Gong and Sucher, 1999; Li et al., 2004; Kumagai et al., 2007). Baicalin (baicalein 7-O-glucuronide) is a major active ingredient of S. baicalensis root; acid hydrolysis of baicalin yields glucuronic acid and a flavone aglycone named baicalein (5,6,7-trihydroxyflavone). The anti-inflammatory effects of these constituents are well documented (Sekiya and Okuda, 1982; Kubo et al., 1984). Recently, baicalein has been shown to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production in macrophages (Wakabayashi, 1999). Although flavonoids such as baicalein and baicalin have been reported to show a variety of biological activities, they have limited pharmaceutical use due to their low water solubility, fast oxidative degradation, fast metabolism, and n

Corresponding author. Tel.: þ 82 51 510 8462. Corresponding author. Tel.: þ 82 51 510 2196; fax: þ 82 51 514 1778. E-mail addresses: [email protected] (M. Joo), [email protected] (J. Cha). 1 These authors contributed equally to this study.

nn

http://dx.doi.org/10.1016/j.ejphar.2014.10.013 0014-2999/& 2014 Elsevier B.V. All rights reserved.

low absorption rate in the small intestine. Various methods have been used to overcome these issues of flavonoids. One is to use cyclodextrin, which is widely employed as an excipient to increase the solubility and stability of drugs (Oda et al., 2004; Bian et al., 2009). Zhang et al. (2011) reported that a hydroxypropyl-β-cyclodextrin-genipin complex increased water solubility 3.5-fold. Chemical modification of flavonoids with a pivaloxymethyl group is another method (Kim et al., 2010). However, these methods also have drawbacks, including compromised product purity, difficulty of preparation, and environmental pollution. Addition of a sugar moiety to the compound by enzymatic glycosylation can be an alternative to overcome these liabilities. The glycosylated flavonoids, which are synthesized by bacterial glycosidases and glycosyltransferases, have been reported to have various merits: increased water solubility, oxidative stability, bioavailability, and decreased cytotoxicity. For example, the use of maltosyltransferase from Caldicellulosiruptor bescii DSM 6725 enhanced the water solubility of piceid glucosides by 1.86
103 times compared with a piceid (Park et al., 2012). Addition of glucose to puerarin by bioconversion using Microbacterium oxydans CGMCC 1788 improved water solubility and pharmacokinetic parameters, while maintaining its bioavailability (Jiang et al., 2008). Hijiya and Miyake (1991) synthesized glucosyl hesperidin (G-hesperidin) by transglycosylation with cyclodextrin glucanotransferase from Bacillus stearothermophilus. The solubility of

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G-hesperidin was approximately 10,000 times higher than that of hesperidin, and it possessed similar antioxidant properties as hesperidin in vitro (Yamada et al., 2003). Moreover, the biological activities of G-hesperidin were greater than those of hesperidin (Ohtsuki et al., 2003). In this study, we tested whether the glycosylation of baicalein enhances the solubility and stability of baicalein. Glycosylation of baicalein was carried out by amylosucrase from Deinococcus geothermalis. The molecular structure of the transglycosylation product of baicalein was determined, and its water solubility and oxidative stability were examined. Since baicalein is used to treat inflammation, we examined whether glycosylation of baicalein affects inflammatory reaction by using LPS-treated RAW 264.7 cells.

2. Materials and methods 2.1. Chemicals and reagents Baicalein, sucrose, baicalin, sulforaphane, fructose, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Water and methanol [high-performance liquid chromatography (HPLC)-grade] were purchased from Burdick & Jackson (USA) for purification. All other chemicals were of reagent grade and were purchased from Sigma-Aldrich. TLR4-specific Escherichia coli LPS was purchased from Alexis Biochemical (San Diego, CA, USA). Antibodies against nuclear factor erythroid 2-related factor 2 (Nrf2) and Lamin A/C were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The recombinant amylosucrase from D. geothermalis (DGAS) was prepared in E. coli as previously described (Cho et al., 2011). 2.2. Animal cell culture A murine macrophage cell line, RAW 264.7 cells, was obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA) and cultured in Dulbecco's Modified Eagle Medium supplemented with 10% (v/v) heat-inactivated FBS (cDMEM; Thermo, MA, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco; NY, USA). The cells were cultured at 37 1C under 5% CO2 in fully humidified air. Nrf2 reporter cell lines were prepared as previously described (Choi et al., 2012). 2.3. Synthesis of baicalein glycoside (BG) using DGAS To synthesize baicalein glycoside (BG), a substrate solution containing 20 mM baicalein and 40 mM sucrose in 50 mM Tris– HCl buffer (pH 8) was preincubated at 30 1C for 30 min. After preincubation, DGAS (1 mg/ml) was added to the reaction mixture and the enzymatic synthesis was carried out at 30 1C for 12 h (Cho et al., 2011). The transfer reaction was stopped by heating in boiling water for 30 min and placing the mixture tube in ice. The reaction mixture was centrifuged at 3000g for 20 min to get rid of the insoluble substances. The supernatant fraction was filtered using a 0.22-μm syringe filter (Satorius; Goettingen, Germany). 2.4. Purification of BG The transfer products were separated using a C18-T cartridge (100 mg/ml; Strata) and recycling preparative HPLC equipped with an ultraviolet (UV) detector (JAI; Tokyo, Japan). A C18-T cartridge, which was previously activated using methanol and water, was used to absorb the BG from the transglycosylation reaction mixture and to remove any sugars and salts. The transglycosylation reaction mixture was filtered using a 0.45-μm syringe filter (Sartorius) and added to the C18-T cartridge. After washing twice,

elution of the transfer products was carried out with methanol. The main transfer product in methanol was purified using a combination W-252/W-251 polymeric gel filtration column (2
50 cm2; JAI) in the recycling preparative HPLC system. The mobile phase was 100% methanol at a flow rate of 3 ml/min. The fractions corresponding to the detected peaks were collected and freeze-dried. The purity of each sample was confirmed using thin layer chromatography (TLC) analysis. 2.5. TLC analysis The purified transfer products were spotted on Whatman K5F silica gel plates (Whatman; Maidstone, UK) activated at 110 1C for 30 min. The plates were developed in developing solution composed of n-butanol:ethanol:water (5:3:2, v/v/v) for BG. The developed TLC plates were dried completely at room temperature after irrigating once or twice, and visualized using a UV lamp in combination with a UV viewing box (Camag; Muttenz, Switzerland) at 254 nm. The transfer products were also visualized by dipping in a solution containing 0.3% (w/v) N-(1-naphthyl)-ethylenediamine and 5% (v/v) H2SO4 in methanol followed by heating at 110 1C for 10 min. 2.6. HPLC analysis Cellular uptake was investigated by using HPLC. RAW 264.7 cells were seeded into 60 mm cell culture plates (2
106 cells/ml). After overnight incubation, 100 mM baicalein or BG was added, and the cells were incubated for up to 4 h at 37 1C under 5% CO2; then the medium was removed and the cells were washed with 2 ml of ice-cold PBS three times and scrapped with 2 ml of PBS. After centrifugation, the cell pellet was resuspended with 0.1 ml of distilled water by vortexing and lysed by freezing and thawing using the liquid nitrogen three times. After centrifugation, the supernatant was filtered by a 0.2 mm syringe filter and analyzed by UPLC. UPLC analysis was carried out using an Acquity UPLC H Class system (Waters, Ireland) comprised a Model bioSample ManagerFTN, a Model bioQuaternary Solvent Manager, and a PDA eλ detector. Identification of baicalein and BG was carried out by reverse-phase HPLC using a C18 (4.6
250 mm2) column (Shodex). The column temperature was 35 1C, the mobile phase was 59% methanol and 41% water containing 0.2% phosphoric acid. The flow rate was 0.8 ml/min and the injection volume was 10 μl. Baicalein and BG were observed at 276 nm. Three injections were performed for each sample and standard. 2.7. Nuclear magnetic resonance (NMR) and fast atom bombardment-mass spectrometer (FAB-MS) analysis Approximately 5.5 mg of baicalein and purified BG were dissolved in 0.5 ml of pure CD3OD and placed in 5 mm NMR tubes. The 1H and 13C NMR spectra of baicalein and purified BG were obtained with a Varian Inova AS 600 MHz NMR spectrometer (Varian; Palo Alto, CA). The sample was dissolved in CD3OD at 24 1C with tetramethylsilane (TMS) as an internal standard. Chemical shifts are reported as s (singlet), d (doublet), t (triplet), m (multiplet), or br s (broad singlet). Coupling constants are reported in Hz. The chemical shifts are reported as parts per million (δ) relative to the solvent peak. 1H NMR (600 MHz, CD3OD, δ) 7.96 (2H, br s, J ¼7.2 Hz, H-20 ,60 ), 7.55 (3H, m, H-30 ,40 ,50 ), 6.70 (1H, s, H-3), 6.53 (1H, s, H-8), 5.19 (1H, d, J ¼3.6 Hz, H-100 ), 4.29 (1H, dt, J ¼9.6, 3.0 Hz, H-500 ), 3.92 (1H, dd, J ¼9.6, 9.6 Hz, H-300 ), 3.82 (2H, d, J ¼3.0 Hz, H-600 ), 3.56 (1H, dd, J ¼9.6, 3.6 Hz, H-200 ), 3.49 (1H, dd, J ¼9.6, 9.6 Hz, H-400 ); 13C NMR (150 MHz, CD3OD, δ) 184.1 (C-4), 165.6 (C-2), 162.2 (C-8a), 155.9 (C-7), 154.6 (C-5), 133.1 (C-40 ), 132.8 (C-10 ), 131.9 (C-6), 130.4 (C-30 ,50 ), 127.6 (C-20 ,60 ), 105.7

K.H. Kim et al. / European Journal of Pharmacology 744 (2014) 147–156

(C-3), 105.3 (C-4a), 104.9 (C-100 ), 96.2 (C-8), 75.3 (C-300 ), 74.9 (C-500 ), 74.0 (C-200 ), 71.1 (C-400 ), 62.4 (C-600 ). Negative FAB-MS was recorded on a JEOL JMS 700 system (JEOL; Tokyo, Japan). 2.8. Solubility determination Excess baicalein, baicalin, and BG were suspended in 1 ml of distilled water in a microfuge tube at 25 1C. A JAC-4020 ultrasonic cleaner (Kodo; Hwaseong, Korea) was used to maximize the solubility of BG. After sonication at room temperature for 1 h with intermittent pauses, the sample was centrifuged at 12,000g for 20 min. The supernatant of each sample was filtered through a 0.45-mm membrane filter and the concentration of the compound in the supernatant, which is defined as water-soluble, was estimated by measuring its absorbance at 280 nm using a Gene Quant pro UV/vis spectrophotometer (Amersham Biosciences; Buckinghamshire, UK); the absolute solubilities were calculated. 2.9. Stability against oxidative degradation Baicalein, baicalin, and BG were dissolved in various pH buffer solutions (pH 2, phosphate-buffered saline (PBS), pH 7.4, and pH 9) to reach a final concentration of 50 mM. The solutions were incubated at 37 1C and at different time points, and then an aliquot (300 ml) of the reaction mixture was taken out, filtered, and injected into the HPLC equipped with a C18 reverse phase column. Baicalein, baicalin, and BG were also dissolved in cDMEM to reach a final concentration of 50 mM. The solutions were incubated at 37 1C and 5% CO2 in a humidified incubator. The medium was harvested at different time points up to 72 h on liquid nitrogen (  196 1C), vortexed, and analyzed with HPLC under the same analysis conditions as above. 2.10. Cell viability assay Cell viability was determined by using an MTT-based colorimetric assay. RAW 264.7 cells were plated at a density of 5
105 cells/well into 24-well plates in cDMEM (37 1C, 5% CO2) for 12 h. Baicalein and BG dissolved in dimethyl sulfoxide (DMSO) were serially diluted, and the resulting solutions were added to media. After 18 h, cells were washed with PBS twice, and MTT was added to the medium for 4 h. The supernatant was removed, and the formazan crystals were dissolved using 200 ml of DMSO. The absorbance was read at 595 nm with a Wallac 1420 microplate reader. 2.11. Nitrite assay RAW 264.7 cells were plated at a density of 5
105 cells/well into 24-well plates for 12 h, followed by treatment with LPS (100 ng/ml) and different concentrations of baicalein and BG for an additional 12 h. The amount of nitrite (as an estimate of NO production) was measured by using the Griess reaction. Briefly, 100 μl of cell culture medium was mixed with 100 μl of Griess reagent (1.0% sulfanilamide in 2.5% phosphoric acid and 0.1% N-(1)-naphthyl-ethylenediamine dihydrochloride in water) in a 96-well plate, and incubated at room temperature for 5 min prior to reading at 540 nm with a microplate reader. Sodium nitrite (NaNO2) was used to generate a standard curve. 2.12. Luciferase assay Nrf2 reporter cell lines were plated at a density of 5
105 cells/ well into 24-well plates with G418 (100 mg/ml) for 12 h, and incubated with various concentrations of baicalein and BG. Nrf2 reporter cell lines were selected with G418 (Invitrogen; Carlsbad,

149

CA, USA). Nrf2 reporter cell lines were tested for responsiveness to sulforaphane, a well-documented Nrf2 activator. Luciferase activity was measured using a luciferase assay kit (Promega; Madison, WI, USA) according to the manufacturer's instructions, and normalized to the amount of total protein of the cell extract. 2.13. Western blotting RAW 264.7 cells were plated at a density of 1
106 cells/well into 60 mm cell culture dish and incubated with various concentrations of sulforaphane, baicalein, and BG for 8 h. Nuclear proteins were isolated using an NE-PER nuclear extraction kit following the manufacturer's protocol (Thermo Scientific; IL, USA). The amount of protein was measured by the Bradford assay (Bio-Rad). Equal amounts of protein were separated by NuPAGE gel electrophoresis and transferred to polyvinyldifluoride (PVDF) membranes (BioRad). Membranes were incubated with 1% bovine serum albumin and then with anti-Nrf2 and anti-Lamin A/C antibodies overnight at 4 1C. Expression of the protein was detected by using a chemiluminescence substrate (SuperSignal West Femto; Thermo Scientific). 2.14. Reverse transcription-polymerase chain reaction (RT-PCR) analysis RAW 264.7 cells were treated with the indicated compounds (0–40 mM) for 8 h and then washed with ice-cold PBS. Total RNA was isolated by Trizol reagent according to the manufacturer's instructions (Invitrogen), and the total RNA concentration was measured using a spectrophotometer. Total RNA (2 mg) was reverse-transcribed using M-MLV Reverse Transcriptase (Promega) to obtain cDNA. The cDNA was amplified by PCR using TaqPCRx DNA Polymerase (Invitrogen) with a set of specific primers as follows: the forward and reverse primers for NAD(P)H:quinine oxidoreductase-1 (NQO-1) were 50 -GCAGTGCTTTCCATCA CCAC-30 and 50 - TGGAGTGT GCCCAATGCTAT-30 , respectively; the primers for heme oxygenase-1 (HO-1) were 50 -TGAA GGAGGCCACCAAGGAGG-30 and 50 -AGAGGTCACC CAGGTAGCGG-G-30 , respectively; the primers for glutamate-cysteine ligase catalytic subunit (GCLC) were 50 -CACTGCCAGA ACACAGACCC-30 and 50 -ATGGTCTGGCTGAG AAGCCT-30 , respectively (Choi et al., 2012). PCR conditions were an initial denaturation at 95 1C for 5 min followed by 22–32 cycles of denaturation at 95 1C for 30 s, annealing at each sample's melting temperature for 30 s and extension at 72 1C for 40 s, with a final extension at 72 1C for 7 min. Amplicons were loaded on 1.2% agarose gel in 1
TBE buffer and separated at 100 V for 30 min, stained with ethidium bromide, and confirmed under UV light. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control to evaluate the relative expression of HO-1, NQO-1, and GCLC. The expression of each gene relative to GAPDH expression was determined by using the densitometric analysis software Image J (NIH; Bethesda, MD, USA). 2.15. Measurement of intracellular reactive oxygen species Production of intracellular reactive oxygen species in RAW 264.7 cells was confirmed by 5-(and-6)-carboxy-20 ,70 -dichlorodihydro-fluorescein diacetate (carboxy-H2DCFDA; Molecular Probes; Eugene, OR, USA). Briefly, after various treatments, 1
106 RAW 264.7 cells were further treated with 100 μM carboxy-H2DCFDA in cell culture medium and incubated at 37 1C for 30 min. After incubation, the cells were washed with PBS, and then fluorescence was measured using the BD FACS Canto II system (BD Biosciences; San Jose, CA, USA) at the excitation wavelength of 488 nm and the emission wavelength of 525 nm. In all cases at least 100,000 live events were collected for analysis. Data files were analyzed using FlowJo software (Tree Star, San Carlos, CA, USA).

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2.16. Confocal laser-scanning fluorescence microscopy

BG

Baicalein

80000 Response (mV)

RAW 264.7 cells were seeded onto 24-well plates and cultured on a glass coverslip. Cells were treated with 40 mM chemicals for 30 min–2 h, and then the medium was removed. Cells treated variously were washed with PBS three times, fixed with 4% paraformaldehyde for 10 min, stained with propidium iodide (PI; 100 ng/ml) for 5 min, and then directly mounted with Vecta Shield (Vector Laboratories, Burlingame, CA). Cells were imaged within 24 h of staining, which was performed by using a Zeiss LSM 700 inverted confocal microscope (Zeiss, Oberkochen, Germany). Baicalein and BG, and PI were visualized with a 405-nm and a 488-nm laser, respectively. Cells taken up baicalein or BG emit blue fluorescence, while live cells stained with PI do red fluorescence under the microscope. The percentage of cells with baicalein or BG was calculated by scoring blue cells over all the PI-stained red cells in a given microscopic field. An average percentage of three randomly picked fields under the microscope was shown.

100000

BG

60000 Baicalein

40000 20000 M 1

2

3 4

0

0

5

10

15

20

25

28

Time (min)

Fig. 1. TLC and HPLC analyses of the transglycosylation reaction by DGAS with baicalein and sucrose as the acceptor and donor, respectively. For TLC analysis, lane M contained standard markers from glucose to maltotriose; lane 1, sucrose; lane 2, fructose; lane 3, baicalein, lane 4, reaction products of the baicalein transglycosylation reaction by DGAS. In HPLC, baicalein and BG were eluted at 17.23 min and 15.61 min, respectively.

2.17. Statistical analysis Statistical analysis of results (n ¼ 3 independent experiments) was performed using SPSS version 14.0. Values are expressed as the mean 7S.E.M. The significance of the differences between the group means was evaluated by analysis of variance (ANOVA).

3. Results 3.1. Synthesis of BG by DGAS DGAS transfers the glucosyl unit of sucrose to synthesize an amylose-type polymer as well as glycoconjugates (Emond et al., 2008). DGAS reacted with baicalein in the presence of sucrose as the donor molecule, and the reaction products were detected by TLC and HPLC analyses. On the TLC plate, the spot corresponding to baicalein was invisible, whereas another spot, possibly newly produced BG, appeared after dipping the TLC plate into the sulfuric acid solution (Fig. 1 inset). A spot in the upper position was also observed under UV light (data not shown), implying that it originated from baicalein, since baicalein was the only chromophore in the reaction. An additional spot located below sucrose in the reaction products was detected in the reaction without baicalein, which was likely a transglycosylated product transferred to sucrose. HPLC analysis of the reaction products showed that baicalein and BG were eluted at 17.23 and 15.61 min, respectively (Fig. 1). The conversion yield of BG was 59.1% estimated by HPLC. The results suggested that DGAS was successfully used to produce the BG. Other polyphenols, such as aesculin, arbutin, genistin, and salicin, were also good acceptors for transglycosylation by DGAS (data not shown). When DGAS was used to transglycosylate salicin, the major saliclin transfer product was determined to be glucosyl salicin (Jung et al., 2009). Likewise, newly produced BG observed in the DGAS transglycosylation reaction here was believed to be a glucosyl unit attached to baicalein. 3.2. Separation and identification of BG The sugars included in the transglycosylation reaction mixture were first removed by a reverse phase C18-T column, and then the mixture of baicalein and BG was separated by using W-251 and W252 polymeric gel filtration columns through preparative recycling HPLC. Two separate compounds were detected by using UV and refractive index (RI) detectors. Each peak fraction showed one single spot on the TLC plate. The purified fractions were concentrated by using a rotary vacuum evaporator and confirmed as a

single peak in the HPLC analysis. The molecular weight of the newly synthesized BG was determined to be 432 Da from the pseudomolecular ion peak m/z 431 [M-H]  in the negative FAB-MS spectrum, and the molecular formula was determined to be C21H19O10 based on the high-resolved molecular ion peak, 431.09. The 1H NMR spectrum exhibited one doublet hemiacetal proton signal at δH 5.19 with a coupling constant of 3.6 Hz, four oxygenated proton signals in the region from δ 4.29 to δ 3.49, and one oxygenated methylene proton signal at δH 3.82 (2H, d, J¼ 3.0 Hz), indicating the existence of an aldohexose. The 13C NMR spectrum showed 21 carbon signals, indicating that the compound was a flavonoid monoglycoside. One ketone carbon signal at δC 184.1 (C-4), five oxygenated olefin quaternary carbon signals at δC 165.6 (C-2), δC 162.2 (C-8a), δC 155.9 (C-7), δC 154.6 (C-5), and δC 131.9 (C-6), two olefin quaternary carbon signals at δC 132.8 (C-10 ) and δC 105.3 (C-4a), and seven olefin ethane carbon signals at δC 133.1 (C-40 ), δC 130.4 (C-30 ,50 ), δC 127.6 (C-20 ,60 ), δC 105.7 (C-3), and δC 96.2 (C-8) indicated that the compound was a baicalein monoglycoside (Fig. 2A). The carbon resonance due to the sugar moiety, such as a hemiacetal carbon signal at δC 105.3 (C-1″), four oxygenated ethane carbon signals at δC 75.3 (C-3″), δC 74.9 (C-5″), δC 74.0 (C-2″), and δC 71.1 (C-4″), and one oxygenated methylene carbon signal at δC 62.4 (C-6″), indicated that the sugar was D-glucopyranose. The configuration of the anomer carbon was determined to be α based on the coupling constant of the anomer proton signal (J¼3.6 Hz) in the 1H NMR spectrum. In the heteronuclear multiple-bond correlation (HMBC) spectrum, the anomer proton signal (δH 5.19, H-1″) showed a correlation with the oxygenated olefin quaternary carbon signal (δC 131.9, C-6) (Fig. 2B), suggesting the glycosidic bond to be at C-6. From the combination of the above-described data, the BG was identified as a baicalein 6-O-α-D-glucopyranoside. 3.3. Effects of glucosylation on water solubility and stability against oxidative degradation The solubility of BG in water was evaluated by comparing it with that of baicalein and baicalin (baicalein 7-O-glucuronide), which were determined to be 5.4 mg/l and 50 mg/l, respectively. The water solubility of BG was 141.9 mg/l, which was 26.2 and 2.8 times greater, respectively, than those of natural baicalein and baicalin. This implies that the attachment of a glucosyl residue to baicalein enhanced the water solubility of the original compound. In general, flavonoids have short half-lives of less than 10 h in buffered aqueous solutions because they undergo fast autooxidative degradation (Walle et al., 2007; Meng et al., 2009). Transient

K.H. Kim et al. / European Journal of Pharmacology 744 (2014) 147–156

Fig. 2.

151

13

C NMR (A) and HMBC (B) spectra of baicalein 6-O-α-D-glucopyranoside.

protection of the oxidative group with a promoiety would stabilize the flavonoids against oxidative degradation. The stability of baicalein, baicalin, and BG was determined by their half-lives, t1/2 (h), in various buffer solutions (PBS, pH 7.4, pH 2.0, or pH 9.0) as well as in a cell culture medium (cDMEM) (Table 1). Baicalein underwent fast degradation at low pH and in cell culture medium, whereas it showed a moderate stability profile under alkaline and neutral conditions with half-lives of 8.1 and 5.5 h, respectively. Compared to baicalein, BG showed increased stability in buffered aqueous solutions at different pHs. This tendency is consistent with the results obtained from baicalin. As anticipated, glucosylation of the oxidative group of baicalein resulted in a stable baicalein derivative without oxidative decomposition in buffered solutions even after 72 h of incubation at neutral and alkaline pHs (Table 1). The increased stability of BG in cDMEM was also observed. At low pH, baicalin was very stable unlike other two compounds. The glucuronic acid moiety attached to baicalein may increase the stability of baicalin at acidic pH. The high stability of baicalin at pH 3.0–4.0 has been reported (Xing et al., 2005). It was explained due to that acidic environment can prevent reactions intermediated by radicals.

3.4. Effects of BG on NO production and cell viability in LPS-treated RAW 264.7 cells The effect of BG on NO production was evaluated with RAW 264.7 cells that were treated with LPS. NO levels in the culture supernatant were measured as an index of inflammatory mediators. NO production was significantly inhibited by approximately 15–60% in a dose-dependent manner upon administration of BG, although the inhibitory effect of BG seemed slightly less compared to baicalein (Fig. 3A). Reduction of NO by BG was not due to the cytotoxicity of BG, since an MTT assay showed that when

Table 1 Stability of baicalein, baicalin, and its glucoside in buffer solutions and cell culture medium. Half-life, t1/2 (h)

Buffer

cDMEM

pH 2.0 PBS pH 9.0

Baicalein

Baicalin

BG

4.9 5.5 8.1 1.1

51.1 472 38.2 4.8

5.0 472 472 8.4

incubated with BG for 18 h, RAW 264.7 cells were viable within the range of 40 mM of BG. (Fig. 3B). 3.5. Effects of BG on Nrf2 activation and the production of reactive oxygen species in LPS-treated RAW 264.7 cells It has been reported that baicalein activates Nrf2, a key transcription factor that regulates inflammation, and enhances the expression of HO-1 in Hct116 human colon carcinoma cells (Havermann et al., 2013). To examine the effect of BG on Nrf2 activation, we took RAW 264.7-derived reporter cells that harbors a 1-kb long NQO-1 proximal promoter fused with the firefly luciferase gene, and treated them with BG (5–40 mM) for 8 h. Total cell lysate prepared for a luciferase reporter assay showed increased luciferase activity in a dose-dependent manner, suggesting the possibility that BG activates Nrf2 (data not shown). To examine this possibility further, we determined whether BG induces the nuclear localization of Nrf2, indicative of activated Nrf2. RAW 264.7 cells were treated with indicated concentrations of BG, along with baicalein. At 16 h after treatment, nuclear fractions were isolated and subjected to western blot analysis. As shown in Fig. 4A, BG induced the nuclear localization of Nrf2 in the range

K.H. Kim et al. / European Journal of Pharmacology 744 (2014) 147–156

Nitrite ( μM)

Cell viability (%)

152

-

-

5

10

20

40

BG (μM)

-

5

10

20

40

LPS (100 nM) Fig. 3. Effect of BG on the nitrite production and cell viability of RAW 264.7 cells. (A) Cells were treated with each compound at the indicated amounts and incubated further for 12 h in the presence or absence of LPS. The amounts of nitrite were measured as described in Section 2. (B) MTT assay was performed to determine a cytotoxicity of BG. RAW 264.7 cells were treated with indicated amounts of BG for 12 h. Data were derived from three independent experiments and expressed as mean 7S.E.M. nPo 0.05, nn P o0.01, and nnnPo 0.005 indicate significant differences from the LPS-treated group.

of 20–40 mM. It is notable that nuclear localization of Nrf2 by BG was more robust than baicalein. Thus, we examined whether BG was more effective than baicalein in expressing Nrf2-dependent genes. After treatment of RAW 264.7 cells with BG or baicalein, total RNA was extracted from the cells and analyzed by semiquantitative RT-PCR for the expression of Nrf2-dependent genes including NQO-1, GCLC, and HO-1. Similar to Fig. 4A, BG treatment strongly induced the expression of Nrf2-dependent genes compared to baicalein (Fig. 4B). Given that reactive oxygen species activates Nrf2 (Johnson et al., 2008), we sought to exclude the possibility that BG induces reactive oxygen species production, resulting in Nrf2 activation. To this end, RAW 264.7 cells were treated with BG (40 mM) or baicalein (40 mM) for 16 h and stained with carboxy-H2DCFDA prior to flow cytometric analysis for intracellular reactive oxygen species. As shown in Fig. 4C, neither BG nor baicalein elicited reactive oxygen species production, whereas LPS (100 ng/ml) treatment induced reactive oxygen species production. Together, these results show that BG, the derivative of baicalein, activated Nrf2 more effectively than baicalein, without mediation of reactive oxygen species. Given that Nrf2-dependent genes play a key role in scavenging reactive oxygen species produced during inflammation, we examined the effect of BG and baicalein on the production of reactive oxygen species in LPS-treated macrophages. RAW 264.7 cells were treated with BG (40 mM) or baicalein (40 mM) 2 h prior to LPS (100 ng/ml). At 16 h after LPS treatment, cells were stained for intracellular reactive oxygen species and analyzed by FACS. As shown in Fig. 5A, baicalein treatment reduced the level of intracellular reactive oxygen species (pink line) elicited by LPS treatment (dark line). Similarly, as shown in Fig. 5B, BG treatment reduced the level of reactive oxygen species (red line) produced by LPS treatment (dark line). However, BG was more potent in suppressing reactive oxygen species production than baicalein. In parallel experiment where cells were treated with LPS for 8 h along with BG or baicalein, we obtained similar results (data not shown). Therefore, these results show that BG was more effective than baicalein in activating Nrf2 and thereby suppressing reactive oxygen species produced in an inflammatory environment. 3.6. Uptake of baicalein and BG by RAW 264.7 cells Baicalein and BG have intrinsic blue fluorescence, which allowed a direct visualization of their intracellular uptake. When RAW 264.7

cells were incubated with baicalein or BG, blue fluorescence was detected inside the cells, which was markedly higher than basal fluorescence in untreated cells (data not shown). The degree of cellular uptake of each compound was measured by counting blue cells among PI-stained, live cells in three randomly picked microscopic fields. As shown in Fig. 6, blue fluorescent cells were apparent after 30 min incubation of baicalein (open column) or BG (closed column); cells taken up baicalein or BG constituted less than 80% and 60% of live cells, respectively. However, statistics analysis showed no significance between the uptakes of each compound. When cells were incubated up to 2 h, the percentage of blue fluorescent cells among live cells was not significantly changed either, although fluorescence appeared to be more intense in general (data not shown). These results suggest that cellular uptake of BG occurs similar to baicalein. The cellular uptake of each compound was also investigated in cell lysates by HPLC. Baicalein or BG was added to RAW 264.7 cells, and the concentrations of the compound present in the cDMEM medium and taken up by the RAW 264.7 cells were determined at different time points, up to 4 h (Fig. 7). The amount of BG remaining in the extracellular medium during incubation decreased to less than 50% of the initial amount of BG after 4 h (data not shown). During 4 h exposure of RAW 264.7 cells to BG, an increase of the intracellular baicalein content was observed instead of BG, indicating that BG was converted to baicalein within cell. In the case of baicalein treatment, intact baicalein was observed in culture medium and cell lysates.

4. Discussion Baicalein and baicalin, major flavonoids derived from the roots of S. baicalensis, are widely used in traditional Asian remedies for the treatment of fever, viral and bacterial infections, and inflammation (Ishimaru et al., 1995; Makino et al., 2008). Although many flavonoids have several beneficial properties, low water solubility, short half-life in serum, and high cytotoxicity limit pharmaceutical application. In particular, drugs with poor aqueous solubility exhibit dilution rate-limited absorption in the membrane of the GI tract; therefore, enhancing the solubility of poorly watersoluble drugs is an important issue in pharmaceutical research. These limitations of flavonoids can be overcome by forming an inclusion complex with soluble cyclodextrin or by changing the chemical modification of the structure (Bian et al., 2009).

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Fluorescent intensity Fig. 4. Effect of BG on Nrf2 and Nrf2-dependent gene expression. (A) Western blot analysis of nuclear Nrf2 expression. RAW 264.7 cells were treated with various amounts of baicalein or BG along with 5 mM sulforaphane (SFN) for 8 h. Nuclear proteins were analyzed by western blotting for Nrf2 and for Lamin A/C as internal controls. (B) RT-PCR analysis of Nrf2-dependent genes. RAW 264.7 cells were treated with the indicated concentrations of compounds for 8 h, and total RNA was extracted for semi-quantitative RT-PCR analysis of Nrf2-dependent genes including HO-1, NQO-1, and GCLC. GAPDH was used as an internal control. The intensity of each band was analyzed by ImageJ, and relative expressions of HO-1, NQO-1 and GCLC over GAPDH were shown in graphs. nPo 0.0001 and nnPo 0.005, compared with baicalein-treated group. (C) FACS analysis was performed to measure intracellular reactive oxygen species from the cells treated with LPS (100 ng/ml), an inducer of reactive oxygen species, baicalein (40 μM), or BG (40 μM). All experiments were repeated at least three times.

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Fig. 5. Effect of BG on reactive oxygen species production in LPS-treated RAW 264.7 cells. RAW 264.7 cells were treated with baicalien (A) or BG (B) 2 h prior to LPS treatment. Production of reactive oxygen species in RAW 264.7 cells was measured using carboxy-H2DCFDA and FACS analysis. Gray lines in (A) and (B) represent basal levels of reactive oxygen species in mock-treated cells, and dark lines indicate reactive oxygen species producing cells among LPS-treated ones. BG treatment strongly suppressed reactive oxygen species production (red line), whereas baicalein did it modestly (pink line). This experiment was repeated three times independently, and representatives are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Observation of baicalein and BG uptake in RAW 264.7 cells by fluorescence microscopy. RAW 264.7 cells were incubated with baicalein or BG (50 μM each) for indicated periods. The percentage of cells taken up baicalein or BG was calculated over all the PI-stained, live cells scored in a given microscopic field. The percentage represented an average of three, randomly picked microscopic fields. Cells taken up baicalein were not significantly different from those with BG (denoted as NS).

In the present study, we examined the effect of glycosylation on the properties and functions of baicalein. We glycosylated baicalein by using amylosucrase from D. geothermalis and analyzed the water solubility, oxidative stability, and the effects of glycosylated baicalein on the modulation of inflammatory activity via NO production and the Nrf2 signaling pathway. Our results suggest the possibility that BG can be an effective anti-inflammatory compound compared to baicalein. The enhanced solubility observed in our study is consistent with the result that the solubility of ampelopsin, a flavonol found in Cedrus deodara, was increased 89-fold with the addition of one unit of glucose (Woo et al., 2012). It has also been reported that glycosylation of

epigallocatechin gallate (Moon et al., 2006), naringin (Lee et al., 1999), and puerarin (Ko et al., 2012) with amylases or dextransucrases increased the water solubility of the unglycosylated flavonoids by 14–200 fold. The stability of flavonoids in buffered aqueous solutions at different pHs and in biological fluids such as plasma is important for absorption in the gut because there is a sharp increase in pH from the acidic stomach to the slightly alkaline intestine. The stability of modified polyphenols in plasma has been examined. The quercetin–amino acid conjugates were synthesized and their stability estimated against PBS (Kim et al., 2009b). Several amino acid conjugates were stable in PBS buffer (t1/2 417 h), and there was strong resistance against hydrolases compared to quercetin. It has been suggested that the addition of conjugate compounds prevents the decomposition of quercetin by blocking its susceptible positions. The substitution of phenolic hydroxyl groups with fluoride or the amino group of quercetin also increased the oxidative stability of quercetin in various buffer solutions as well as in cDMEM (Cho et al., 2012). In our study, glycosylation significantly extended the half-life of baicalein in PBS and cDMEM. Therefore, we hypothesize that the glycosylation helps to protect baicalein from chemical and enzymatic oxidation. This result was also supported by the results of the experiment evaluating the effect of the glycosylation of resveratrol on its enzymatic oxidation (Regev-Shoshani et al., 2003). Glycosylation of the p-hydroxy group of resveratrol abolished the enzymatic oxidation induced by mushroom tyrosinase. Generally, glycosides would not be easily absorbed systemically due to great hydrophilicity of them. On the other hand, it is also known that the glycosides linked with various sugars in plant foods enhance the absorption of dietary flavonoids in the gut (Hollman et al., 1999). The results of the pharmacokinetic parameters, such as the maximum concentration that a drug achieves after dosing (Cmax) and the values for the area under the plasma concentration–time curve (AUC) in serum, revealed that glycosylated purarin or hesperidin were absorbed more rapidly and efficiently than their aglycones (Yamada et al., 2006; Jiang et al., 2008). This result

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Retention time (min) Fig. 7. HPLC chromatograms of the cell lysate samples after uptake of baicalein (C, D) or BG (E, F). (A) the standard peak of 10 mM baicalein and BG, (B) a control cell lysate without treatment of baicalein or BG, (C, E) cell lysate 2 h after treatment of baicalein or BG, (D, F) cell lysate 4 h after treatment of baicalein and BG. A contaminant peak appeared at the retention time close to BG in the chromatogram of cell lysate without treatment of baicalein or BG.

indicates that flavonoid absorption in the gut can be occurred through active transport in the intestine. Baicalein and baicalin have been shown to exhibit antiinflammatory activity in vivo and in vitro (Lin and Shieh, 1996; Kim et al., 2009a). We compared the anti-inflammatory activity of BG with that of its aglycone, baicalein. During the inflammatory process, large amounts of the proinflammatory mediator NO were generated by inducible nitric oxide synthase (iNOS) (Marletta, 1993). BG inhibited LPS-induced NO production, albeit slightly less than baicalein. To further examine the mechanism of the baicalein-mediated inhibition of NO, we investigated its effects on the Nrf2 signaling pathway. Nrf2 is normally sequestered in the cytosol by binding with Kelch-like ECH-associated protein 1 (Keap1). When stimulated by inflammatory mediators such as LPS, Keap1 is ubiquitinated and rapidly degraded via proteosomal degradation, thus releasing Nrf2 to travel to the nucleus and induce the expression of genes involved in phase II detoxification and antioxidation (Itoh et al., 1997; Jaiswal, 2000; Tong et al., 2006). In vitro studies using macrophage cell lines showed that

although baicalein and BG both activated the anti-inflammatory factor Nrf2 and expressed Nrf2-regulated genes, including GCLC, NQO-1, and HO-1, BG appeared to be more potent than baicalein. Consistent with these results, BG was more effective in suppressing reactive oxygen species production in RAW 264.7 cells induced by LPS. Since many of Nrf2 dependent genes are involved in scavenging reactive oxygen species and reactive oxygen species are key mediators for exacerbating inflammation, it is possible that BG is more effective in suppressing inflammation. The uptake experiment of BG in RAW 264.7 cells by confocal microscopy and HPLC analysis of the cell-free extracts demonstrated that BG was uptaken as an intact form (not aglycone) and then intracellular BG was gradually converted to baicalein inside the cells. In conclusion, this study provides evidence for the enhancement of water solubility and stability of baicalein by glycosylation. We propose that glycosylated baicalein increases the bioavailability of baicalein by helping to protect this vital molecule from chemical or enzymatic oxidation, thereby extending its half-life in the cell and maintaining its beneficial anti-inflammatory property.

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