Brain Uptake of Bioactive Flavones in Scutellariae Radix and Its

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Brain uptake of bioactive flavones in Scutellariae Radix and its relationship to anxiolytic effect in mice Sophia Yui Kau Fong, Chenrui Li, Yiu Cheong Ho, Rui Li, Qian Wang, Yin Cheong Wong, Hong Xue, and Zhong Zuo Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 20 Apr 2017 Downloaded from http://pubs.acs.org on April 22, 2017

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Molecular Pharmaceutics

Brain uptake of bioactive flavones in Scutellariae Radix and its relationship to anxiolytic effect in mice

Sophia Yui Kau Fonga, Chenrui Lia,b, Yiu Cheong Hoc, Rui Lic, Qian Wanga, Yin Cheong Wonga, Hong Xuec, and Zhong Zuoa*

.

a

School of Pharmacy, The Chinese University of Hong Kong. Shatin, N.T. Hong Kong

b

Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences,

Northwestern Polytechnical University, Xi’an, Shaanxi, P. R. China; c

Division of Life Science and Applied Genomics Center, The Hong Kong University of Science

and Technology, Hong Kong

*Corresponding author at: Dr. Zhong Zuo Professor School of Pharmacy The Chinese University of Hong Kong Shatin, N.T. Hong Kong Tel:

852 3943 6832

Fax:

852 2603 5295

Email: [email protected] 1 ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Scutellariae Radix (SR) and its bioactive flavones elicit a variety of effects in the brain. However, the brain uptake of individual SR flavones and its relationship to the elicited effects after SR administration remain unknown. Moreover, previous studies seldom measured pharmacokinetic and pharmacodynamic outcomes simultaneously. In the current study, the brain uptake of six major SR flavones and the anxiolytic behavior following oral administration of a SR extract at two clinically relevant doses (600 and 1200 mg/kg twice daily) were simultaneously investigated in mice (n=18 per group). Brain and plasma concentrations of the flavones were measured by LC-MS/MS, while the anxiolytic effect was evaluated using the elevated plus-maze. To further investigate the mechanism behind the differential brain uptake of the six SR flavones, these flavones were separately administered to mice at an equivalent molar oral dose (n=6). The brain tissue bindings of the SR flavones were also measured with the in vitro brain slice method. Our results indicated that all the six SR flavones including three aglycones (baicalein, wogonin and oroxylin A) and three glucuronides (baicalin, wogonoside and oroxyloside) could pass through the blood-brain-barrier, with brain concentrations ranging from 7.9 to 224.0 pmol/g. It provided novel evidence that oroxylin A had the highest brain uptake among the six SR flavones regardless of its limited content in SR extract, in which 3.6-3.9% of the administered oroxylin A dose was present in the brain 6 h post-dosing and with a brain-toplasma ratio of 0.42-0.46. Although SR extract contains flavones that are positive modulators of the benzodiazepine binding site of GABAA receptors (baicalein, wogonin and baicalin), our behavioral study for the first time indicated that SR extract (a mixture of six flavones) did not elicit significant anxiolytic effect at the studied doses. Oroxylin A also demonstrated the highest brain uptake when the six flavones were separately administered to mice, and the highest affinity to brain tissues in the in vitro tissue binding assay. The high brain uptake of oroxylin A, a 2 ACS Paragon Plus Environment

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GABAA antagonist which had been reported to antagonize diazepam-induced anxiolytic effect, might have suppressed the anxiolytic effects of the other flavones and account for the lack of overall anxiolytic effect of SR extract. The current study illustrates the importance of monitoring pharmacokinetics in a behavioral study, particularly for herbal medicines which consist of multiple components that might have different or even opposite pharmacological effects on the same target. Keywords Scutellariae Radix; Brain; Anxiolytic; Oroxylin A; Flavones

Abbreviations ANOVA

Analysis of variance

CNS

Central nervous system

LC-MS/MS

Liquid chromatography-tandem mass spectrometry

OAT

Organic anion transporter

OATP

Organic anion transporting polypeptide

SD

Standard deviation

SR

Scutellariae Radix

Vu,brain

Unbound volume of distribution in the brain

3 ACS Paragon Plus Environment


INTRODUCTION The central nervous system (CNS) effects of flavonoids attracted research attentions over the past two decades.1 Scutellariae Radix (SR), the dried root of Scutellariae baicalensis Georgi, and its major bioactive flavones, including three aglycones (baicalein, wogonin, oroxylin A) and three glucuronides (baicalin, wogonoside, oroxyloside) (Fig. 1), demonstrated in vivo therapeutic effects against various neurological and mental disorders such as anxiety, depression, epilepsy, Parkinson’s disease, memory impairment, and other neuroinflammatory conditions.2–8 Although SR could elicit a variety of CNS effects, studies on whether the flavones can pass through the blood-brain barrier after administration of SR were limited and the findings were inconsistent. Researchers attempted but failed to detect any SR flavones in rat brain after repeated, high oral doses of SR decoction, in which the low sensitivity of the assay method might account for the failure.9 To better understand the brain uptake of SR flavones, we recently developed and fully validated a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of the six SR flavones in rat brains;10 and this method had been successfully applied in the current study. Regarding the brain uptake of SR flavones after their administration as single compounds, only limited and isolated reports are currently available. For instance, Tsai et al. reported that within 20 min after intravenous injection of baicalein to rats, baicalein appeared rapidly in brain tissues;11 whereas baicalin was not detected in brain tissue even at high doses of intravenous baicalin (up to 30 mg/kg).12 In another study, Liu et al. detected wogonin and wogonoside in the brain of tumor-bearing nude mice after intravenous administration of 20 mg/kg of wogonin.13 On the other hand, information on brain uptake after administration of oroxylin A or oroxyloside is currently lacking. In addition to the lack of knowledge of the brain uptake of SR flavones, the relationship


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between the SR flavones brain concentrations and the elicited CNS effects is largely unknown. Concurrent monitoring of the brain concentration and pharmacodynamic outcomes in behavioral studies is essential since both pharmacokinetic and pharmacodynamic factors could affect the overall CNS outcomes.14 Our previous works demonstrated that baicalein, baicalin, and wogonin are positive modulators of benzodiazepine site of GABAA receptors with Ki values ranging from 0.92 to 77.1 µM, and they elicited dose-dependent anxiolytic response in mice in the elevated plus-maze test.15,16 On the other hand, oroxylin A was found to act as an antagonist of the benzodiazepine site with a Ki of 0.89 µM. It selectively antagonized the anxiolytic effect elicited by diazepam17 while having no effect per se on anxiety-related behaviors. These observations are reproduced in recent studies by other research groups.18–20 Although studies from others and ours clearly demonstrated the roles of individual SR flavones as benzodiazepine receptor ligands, the anxiolytic effect of the herbal medicine SR itself, the common source of SR flavones, has never been reported. Furthermore, since wogonin, baicalein and baicalin represent positive modulators for the benzodiazepine site whereas oroxylin A represents an antagonist, the overall anxiolytic effect of SR (the combination of all six flavones) requires thorough assessment. In the current study, the brain and plasma samples were collected and analyzed at the end of the behavioral study in order to explore the relationship between the brain uptake of SR flavones and the overall anxiolytic effect. The objectives of the current study were to 1) investigate the brain uptake of flavones after oral administration of SR extract as well as individual SR flavones, 2) investigate the overall anxiolytic effect elicited by the oral SR extract, and 3) study the relationship between the brain uptake and the anxiolytic effect of the SR flavones.



EXPERIMENTAL SECTION Materials. A commercially available, standardized and purified SR extract was obtained from Shanghai U-sea Biotech Co., Ltd (Shanghai, China) with batch number of 110208 and quality control standard number of WS-10001-(HD-0989)-2002. As shown in Fig. 1, The content (w/w) of the six bioactive marker compounds in this SR extract in descending order are baicalin (48.0%), oroxyloside (3.78%), baicalein (2.08%), wogonoside (1.60%), wogonin (0.40%) and oroxylin A (0.15%) as quantified by our LC-MS/MS method.10 Diazepam and phenobarbital were purchased from Sigma Chemical Co. (St. Louis, MO, USA). SR extract was prepared as a suspension (120 mg/g) in water containing 5% (w/w) propylene glycol. Authentic standards (purity >95%) of the six components of SR were obtained from Shanghai U-sea Biotech Co., Ltd (Shanghai, China) while the internal standard, 4’,5,7-trihydroxylflavone, were from Sigma Chemical Co. (WI, USA). Oasis hydrophilic-lipophilic-balanced copolymer extraction cartridges (HLB, 1 mL and 3 mL) used for solid phase extraction was supplied by Waters (Milford, USA). Acetonitrile, methanol, formic acid, and dimethyl sulfoxide, all with analytical grades, were obtained from RCI Labscan (Bangkok, Thailand), Merck (Darmstadt, Germany), BDH Laboratory Suppliers Ltd (Kampala, Ukraine), and Lab-scan Analytical Sciences (Bangkok, Thailand), respectively. All other reagents were of at least analytical grade.

Animal treatment. Male ICR mice (20-35 g, 4 weeks old) were supplied by the Animal and Plant Care Facility at The Hong Kong University of Science and Technology. The animals were housed in groups of five to ten in polypropylene cages with food and water ad libitum and kept on a 0800 h to 2000 h light cycle. All animal experiments were pre-approved by the Animal Ethics Committee of The Chinese University of Hong Kong (approval number: 13-052/GRF-3)


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and conducted in accordance with the Code of Practice for Care and Use of Animals for Experimental Purposes. The Code follows international guidelines of animal welfare and was approved by the Animal Welfare Advisory Group and the Department of Health of the Hong Kong. Mice were randomly divided into four groups (n=18 per group). The negative control group received vehicle (0.9% saline) and the positive control group received the standard drug diazepam (1 mg/kg, i.p.). Mice in the tested groups received human equivalent oral doses of SR extract that covered the clinical practice dose ranges according to Chinese Pharmacopoeia (i.e. 210 g per day in divided doses). Based on allometric scaling method according to body surface area in the FDA guideline, the conversion factor from human to mice is around 11 to 17.21 With a 5-10 g human daily dosage of SR (i.e. 83.3-166.7 mg/kg for 60 kg human) and a conversion factor of 14, the current study employed a twice daily dosage of SR at 600 mg/kg and 1200 mg/kg. The detailed experimental protocol and time schedule are presented in Fig. 2. Briefly, the mice received the first oral dose of vehicle, diazepam or SR extract (600 mg/kg or 1200 mg/kg) at 22:00 prior the day of behavioral experiment. In the following morning starting from 8:00, mice were given the secondary bolus oral dose with a 7-min interval between each treatment. The oral treatment started with 600 mg/kg SR group, followed by 1200 mg/kg SR group, diazepam and vehicle group. The mice were then put into the elevated plus maze one by one after 1 h (SR group) or 45 min (diazepam group and vehicle group). At around 14:00 (i.e. 6 h post-dosing, which is the time to reach the peak plasma concentration of SR flavones as indicated by our previous study10), the mice were anaesthetized with phenobarbital (60 mg/kg, i.p.). The sequence of mice being anesthetized followed that of the secondary oral administration with a 7-min interval. After anesthetization, blood was withdrawn by cardiac puncture and was



collected in a centrifuge tube containing heparin. The whole body blood vessels were then washed with 25 mL of 0.9% saline by perfusion at a flow rate of 3 mL/min. After perfusion, the whole brain was dissected, washed with cold 0.9% saline, wiped by tissue paper to remove excess water, and recorded the brain weight. Plasma was obtained by centrifugation of blood at 16,000 ×g for 5 min. Both the plasma and brain samples were kept frozen at -80 °C until analysis. In addition, in order to compare brain uptake among the six SR flavones, they were separately administered at equivalent molar oral doses of 52.8 µmol/kg to ICR mice (25-30 g, four weeks, n= 6/group), which corresponded to baicalein or wogonin or oroxylin A at 15 mg/kg, baicalin at 24.8 mg/kg and wogonoside or oroxyloside at the dose of 24.3 mg/kg. One hour after drug administration, mice were anaesthetized. Plasma and blood samples were collected according to the above-mentioned procedures. In order to illustrate the chosen dose was relevant to the anxiolytic effects of the flavones, the mice were subjected to elevated plus maze test after the administration of wogonin and wogonoside according to the procedures outlined below.

Elevated plus maze test. Since our previous studies demonstrated that the elevated plus maze provided an effective model for evaluation of the anxiolytic effect of SR flavones, it was adopted for the present study.17 Briefly, the maze was built and kept in a dimly lit room and elevated 40 cm above the ground. Following the hole board test, mice were then placed individually in the center of the maze facing an enclosed arm. The number of entries and time spent in the open arms and closed arms were recorded over a 5-min period. At the end of the test, the number of entries into and the time spent in the open/closed arms were expressed as percentage of total number of entries into the arms and the total time spent in each arm,


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respectively. It is well established that the proportion of entries into the open arms in the elevated plus maze is a good indicator of anti-anxiety activity while the number of entries into the closed arms is related to drug effects on locomotor activities.

Evaluation of brain tissue binding of six SR flavones via brain slice method.22 Preparation of brain slices: ICR mice were anesthetized and blood was collected by cardiac puncture. The isolated brain was immediately placed into blank ice-cold artificial extracellular fluid (aECF). A 3 mm piece was cut from the rostral area on a coronal plane, leaving a piece of about 10 mm glued to the slicing platform for subsequent slicing. The razor blade (Gillette, super-stainless) was then mounted and the clearance angle was fixed at 21°. A motorized blade holder sectioning speed of 0.8 mm/s with 1 mm amplitude was used, in 0.05 mm steps. After discarding the first one or two brain slices, ten consecutive 300 µm brain slices were cut on a coronal plane, starting approximately 1.7 mm anterior to the bregma (rostral striatum). Preparation of cassettes and incubation: The HEPES-buffered aECF contained 129 mM NaCl, 3 mM KCl, 1.4 mM CaCl2, 1.2 mM MgSO4, 0.4 mM K2HPO4, 25 mM HEPES, 10 mM glucose and 0.4 mM ascorbic acid (pH 7.6). In a beaker with 10 brain slices, 10 ml of aECF was added with each flavone at the concentration of 100 nM (total DMSO of 0.6%). The beaker was then filled with humidified 100% oxygen over the aECF and covered with a custom-fabricated lid. The beakers were kept in a shaking water bath at 37 °C with the speed of 45 rpm and incubated for 5 h. Sample treatment: A 200 µl sample of aECF was taken directly from the beaker at the beginning of the study and after 5 h incubation for analysis of initial and final concentrations of



the SR flavones in aECF. After sampling the aECF, the brain slices in the aECF were individually removed, dried on filter paper, weighed (~13 mg per mouse brain slice) and homogenized separately in 3 volumes (w/v) of aECF with an ultrasonic processor. The brain slice homogenates were treated by the solid-phase extraction method described below and the concentration of each flavone was quantified by LC-MS/MS. Calculation of Vu,brain: Vu,brain (ml/g brain) is equivalent to the ratio of the amount of drug in the brain slice (Abrain, nmol/g brain) to the measured final aECF concentration (Cbuffer, µmol/L). It was calculated by the equation of Vu,brain = Abrain/Cbuffer.

Measurement of brain uptake and plasma concentration of SR flavones. The collected brain and plasma samples were subjected to solid-phase extraction followed by LCMS/MS analysis per our previously validated method. Such analytical method had been validated according to the guidelines for Bioanalytical Method Validation published by the U.S. Food and Drug Administration.10 Briefly, 450 mg of each brain sample was spiked with 50 µL of 4’,5,7-triflavone as internal standard (5 µg/mL) followed by homogenization for three times in acidified water (1st time) and acetonitrile (2nd and 3rd time). After centrifugation of the mixture at 16,000 ×g at 4 °C for 10 min, the supernatant was subject to solid-phase extraction using 3-mL HLB cartridge. For the plasma samples, 100 µL of each plasma sample was spiked with 50 µL of internal standard and diluted by 1 mL of 35% methanol in water, followed by solid phase extraction using 1 mL HLB cartridge. The extracted samples were then subjected to LC-MS/MS analysis as previously described.10 The LC-MS/MS system consisted of Agilent 1200 series LC pumps and autosampler (Agilent, CA, USA), coupled with an ABI 2000 Q-Trap triple quadrupole mass spectrometer with an electro-spray ionization source (AB Sciex Instruments,


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CA, USA). The MS/MS system was operated under positive mode. Chromatographic separation was achieved by an Alltima C18 column (150 mm × 4.6 mm i.d., 5 µm particle size, Alltech) equipped with a guard filter (Unifilter 0.5 µm, Thermo). The two mobile phases were acetonitrile and 0.1% formic acid in water. Data acquisition and integration of the LC-MS/MS chromatograms were performed with Analyst software 1.4.1 (AB Sciex Instruments, CA, USA). The concentration of the six SR flavones was calculated from the peak area ratio compared to internal standard.

Statistical analysis. Data are presented as mean ± standard deviation (SD) for the individual group. Pharmacokinetic and behavioral data obtained was subjected to one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison post-hoc test by SPSS® Statistics 16.0 (SPSS Inc.). A p oroxyloside (2.0-5.7 nmol/mL) > oroxylin A (0.28-0.71 nmol/mL) > wogonoside (0.10-0.28 nmol/mL) ~ baicalein (0.10-0.17 nmol/mL) > wogonin (0.06-0.11 nmol/mL). It should be noted that the sequence of plasma abundance followed that of the original SR flavone content, except for oroxylin A which was the least abundant flavone in SR extract but was the third most abundant flavone detected in plasma 6 h post-dose. Comparing the brain and plasma concentrations of the flavones in mice receiving 600 mg/kg of SR extract with that receiving 1200 mg/kg of SR extract revealed that doubled dose resulted in a proportionally doubled brain and plasma concentrations. After obtaining the brain and plasma concentrations of SR flavones, the percentage of dose in brain as well as the brain-to-plasma ratio of each flavone were calculated and the results are shown in Table 1. Doubling the oral dose of SR extract (600 mg/kg vs. 1200 mg/kg) resulted in no significant different in both the percentage of dose in brain and brain-to-plasma ratio of all the SR flavones (p>0.05). Among the six SR flavones, oroxylin A had the highest percentage of dose in brain (3.6-3.9%) and brain-to-plasma ratio (0.42-0.46), both of which were statistically different (p2-fold higher than that of the glucuronides including oroxyloside, baicalin and wogonoside (0.014-0.082). Except for oroxylin A, the percentages of administered dose detected in brain for baicalein, baicalin, wogonoside and oroxyloside were considerably low, ranging from 0.01% to 0.06%. Wogonin had a slightly higher percentage of administered dose in brain that was approximately 0.1%.


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In order to compare the brain uptake of the six SR flavones, their plasma and brain concentrations after single oral dose of each flavone at the same molar dose at 52.8 µmol/kg were obtained and compared in Table 2. This dose was selected based on our previous in vivo studies on wogonin.

16

The anxiolytic effect observed after wogonoside administration was a

novel finding which had not been reported before (Fig. 3). The sampling time was optimized conducted to ensure the sensitive detection of all six SR flavones in both plasma and brain. 1 h post-administration was eventually chosen as the optimized sampling point. It was noticed that the plasma concentrations of these flavones were much higher than their corresponding brain concentrations. In plasma, baicalin was identified after the administration of not only baicalein but also oroxylin A, which indicated that oroxylin A was firstly demethylated into baicalein and further metabolized by glucuronidation. The aglycone form of each flavone could not be detected in plasma. For brain uptake, all the aglycone forms (oroxylin A, wogonin and baicalein) together with wogonoside were detected, whereas baicalin and oroxyloside were not detectable in brain under the current experimental conditions.

SR extract did not demonstrate anxiolytic effect. The effects of oral administration of vehicle, diazepam (1 mg/kg), and SR extract at two studied doses (600 mg/kg and 1200 mg/kg) on the anxiolytic behavior of mice in elevated plus-maze are presented in Fig. 4. As a positive control, diazepam significantly increased the percentage of open arm entries and open arm time spent [F(3,71)=5.954, p baicalein (111.1±27.1) > oroxyloside (40.3±9.1) > wogonoside (10.3±2.8) > baicalin (9.7±2.0), which was consistent with our findings in the in vivo brain concentrations of SR flavone in ICR mice shown in Table 2.

DISCUSSION While the CNS effects of SR and its bioactive flavones had been reported, the question on whether the flavones can pass through the blood-brain-barrier. The current study provides novel evidence that all the six flavones could be taken up into the brain after oral administration of a commercially available and standardized SR extract at human equivalent doses. According to the Chinese Pharmacopoeia, the clinical dosage of SR is 2-10 g daily. In the current study, the employed dosages of SR (600-1200 mg/kg, twice daily) were calculated based on a 5-10 g human daily dosage of SR divided into two doses. At these clinically relevant oral doses, both the aglycones (baicalein, wogonin and oroxylin A) and the glucuronides (baicalin, wogonoside


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and oroxyloside) were detected in mice brain, and the percentages of dose in brain and the brainto-plasma ratios of the aglycones were much higher than that of the glycosides. Moreover, for the first time, the potential anxiolytic effect of SR in an extract form was studies. Although all the SR flavones were detected in brain and some of which possess anxiolytic properties, it is surprising that at both studied clinical relevant doses, oral treatment of SR extract to mice did not elicit significant anxiolytic effect in the elevated plus-maze test. The discrepancy between the pharmacokinetic and pharmacodynamic observations thus caught our attention for further exploration. Such discrepancy could be explained by the differential brain uptake of SR flavones. The current study provides novel evidence that oroxylin A, among the six flavones, had the highest brain concentration after oral administration of SR extract. This finding is very interesting since oroxylin A was the least abundant flavone in the SR extract (only 0.15% in original content) yet it was the most abundant flavone quantified in mice brain. Our study on single compound administration at equivalent molar dose also confirmed the observation that oroxylin A displayed the highest brain concentration. The calculated brain-to-plasma ratio of oroxylin A (0.4) was comparable to that of the other CNS GABAergic drugs in mice, including the anxiolytic midazolam (0.23) and the hypnotics zolpidem (0.29),23 indicating that oroxylin A had an appreciable brain uptake. As discussed in the Introduction section, oroxylin A acts as an antagonist of the benzodiazepine site, whereas other SR flavones such as wogonin, baicalein and baicalin are positive modulators of the benzodiazepine binding site of GABAA receptor. Since oroxylin A represented the major flavone being taken up into the brain in vivo, its antagonistic effect could, to a certain extent, balance off the previously reported in vivo anxiolytic effects of baicalin, baicalein, wogonin and the newly identified wogonoside, which were less abundantly



present in the brain. Cancellation of the positive modulatory effects of baicalein, wogonin and their glucuronides by benzodiazepine site antagonists had also been previously demonstrated. Our in vitro electrophysiological studies showed that wogonin16 and baicalin24 enhanced the GABAactivated current in cells expressing GABAA receptors, which was reversed by the co-application of flumazenil, a selective benzodiazepine receptor antagonist. In addition, while baicalein could significantly reduce the production of β-amyloid in CHO cells expressing GABAA receptors, such effect was blocked by the GABAA antagonist bicuculline.5 Consistent findings were also shown in in vivo studies, in which the anxiolytic effects induced by the administration of wogonin,16 baicalein and baicalin25 were reduced by co-administration of flumazenil. The follow-up question is why oroxylin A has the highest brain uptake compared to the other SR flavones? To compare the CNS distribution of the flavones, the brain slice method was applied, which is a robust technique for estimating the overall uptake of drugs into brain tissue through determination of the unbound volume of distribution in the brain (Vu,brain).22 The Vu,brain value of oroxylin A was 2-8 folds higher than the other aglycones and was 20-100 folds higher than the studied glucuronides. Recent physiologically-based pharmacokinetic models suggested that a high brain tissue binding could enhance the overall brain uptake of lipophilic compounds having a high passive clearance across the blood-brain barrier, since the brain tissue would act as a “sink” and more compound molecules are retained within the bulk of the brain (boundunbound cycling).26 Additional in vitro assays could help to identify the exact mechanisms (e.g. nonspecific binding to brain tissue, lysosomal trapping, and active uptake into the cells) that differentiates oroxylin A from the other flavones. Differential involvement of active transporters at the blood-brain barrier could also


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contribute to the higher brain uptake of oroxylin A. Regarding efflux transporters, baicalein is a P-glycoprotein substrate11 while baicalin could be effluxed by multiple ABC transporters including breast cancer resistance protein and various multidrug resistant proteins.27 Involvement of efflux transporters has not been reported for oroxylin A and oroxyloside. As a further effort to assess the relative importance of brain efflux transporters on different flavones, the organic anion transporter (OAT) OAT3, which is involved in the transport of several flavonoid glucuronides,28 was studied. We performed a preliminary study in HEK293 cell line with and without the overexpression of OAT3, which acted as cell uptake transporter. It was found that the presence of OAT3 would significantly increase the cell uptake of baicalein but not oroxylin A and wogonin (unpublished data). Since OAT3 is an efflux transporter in rodents at both the blood-brain barrier29 and the blood-cerebrospinal fluid barrier,30 this further supports that efflux transporters might limit the brain uptake of baicalein but not oroxylin A. On the other hand, the brain uptake of the relatively bulky and hydrophilic glucuronides might require influx transporters. The most likely involved transporters are the organic anion transporting polypeptides (OATPs) OATP1A2 and OATP2B1 since they are identified as uptake transporters located in the apical side of the brain capillary endothelial cells.31 We previously found that OATP2B1 contributed to the hepatic uptake of baicalin in OATP-transfected cell lines.32 Whether OATP1A2 and OATP2B1 also involve in the brain uptake of these glucuronides is currently unknown and requires further investigation. Moreover, it is proposed that during their passage across blood-brain-barrier, flavonoid conjugates may be metabolized back to their parent aglycones before entering the CNS.1 For example, the glucuronides of narigenin and hesperetin were found to enter the brain in their aglycone forms.1 The conversion of baicalin, wogonoside and oroxyloside back to their corresponding aglycone forms during passage through blood-brain-barrier might thus be another



reason accounting for the presence of the aglycones in brain and the low brain concentrations (or even absence) of the glucuronides. The higher brain concentration of oroxylin A could also be in part due to the pharmacokinetic interactions between SR flavones at systemic and brain levels when they were administered as a mixture in SR extract. Oroxylin A, wogonin and baicalein are very similar in chemical structures, and they share common metabolic pathways (e.g. glucuronosyltransferasemediated glucuronidation33) and active transporters. The only difference between oroxylin A and wogonin is the location of the methoxy (-OCH3) group, with the -OCH3 group of oroxylin A at the 6-C position while that of wogonin is at the 8-C position (Fig. 1). The difference between oroxylin A and baicalein is at the 6-C position, in which the hydroxyl group at 6-C of baicalein was methylated in oroxylin A (Fig. 1). Based on others’ and our previous works, two possible mechanisms could have enhanced the plasma concentration and subsequently the brain concentration of oroxylin A. The first possible reason is the higher systemic bioavailability of oroxylin A resulted from metabolic competition between the three aglycones in vivo. We previously compared the plasma pharmacokinetics of baicalein, wogonin and oroxylin A when they were administered separately with that when co-administered as a mixture to rats. After oral co-administration of the three flavones, only the absorption of oroxylin A was significantly increased in comparison to that when they were administered alone.34 Similar results were observed in the current single time point study, in which the plasma concentration of oroxylin A was significantly higher (p

Brain Uptake of Bioactive Flavones in Scutellariae Radix and Its

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