Flavonoids are promising bioactive agents against cancer, inflammatory disease, cardiovascular disease, neurological disorders, metabolic syndrome, obesity, and osteoporosis [1
], and epidemiological studies show that consumption of flavonoid-rich foods is associated with reduced risks of cancer and cardiovascular disease [5
]. Moreover, clinical and experimental studies of individual flavonoids such as baicalin, genistein, and tea catechin have reported such efficacies [7
]. Flavonoids are converted to inactive metabolites like glucuronides in the gut, and circulate mainly as glucuronides in the blood stream, resulting in low concentrations of circulating active aglycones [12
]. It is therefore unclear how oral flavonoids exert bioactivity in tissues, an issue termed “the flavonoid paradox”.
We and others have demonstrated multiple biological actions of flavonoid glucuronides, and have shown that β-glucuronidase-expressing macrophages convert flavonoid glucuronides into active aglycones, especially at local lesions with inflammation [15
]. Our recent study revealed that genistein 7-O
-glucuronide activates macrophages and promotes microbe phagocytosis both in vitro and in vivo through deconjugation of the glucuronide and ensuing activation of estrogen signaling [15
]. Collectively, these studies have revealed intriguing biological properties of glucuronides as carrier and precursor forms of active flavonoids (aglycones) rather than as simply products of detoxification for excretion. However, detailed studies on the pharmacokinetics and metabolism of flavonoids are needed to understand how glucuronides involve in pathophysiological responses in peripheral tissues.
Keigairengyoto (KRT) is a pharmaceutical-grade traditional Japanese (kampo) medicine comprised of seventeen crude drugs, and Scutellariae radix, Aurantii fructus immaturus, Schizonepetae spica, and Glycyrrhizae radix especially contains abundant flavonoids. Keigairengyoto is used for several disorders but mainly to treat microbial infection, and is also prescribed for purulent inflammation such as dermatoses, empyema, and rhinitis. The three-dimensional high-performance liquid chromatograph (3D-HPLC) profile of KRT provided by Tsumura & Co., shows that KRT includes various compounds, especially flavonoids, triterpenoids, and isoquinoline alkaloids (Figure 1
). We previously reported that oral administration of KRT suppressed microbe-induced dermatosis in mice and enhanced bacterial clearance through modulation of the immune system [18
]. It is likely that bioactive flavonoids derived from KRT mediate the adjuvanticity against microbes. However, it is unclear which specific flavonoids are absorbed in plasma after oral administration of KRT and contribute to these antibacterial effects.
The flavonoid glucuronide is one possible mediator of KRT efficacy. Our aims in this study are to examine the plasma pharmacokinetics of KRT flavanoid-derived aglycones and glucuronides and to identify active flavonoids contributing to the antibacterial efficacy of KRT.
4. Materials and Methods
4.1. Test Drugs
Keigairengyoto was supplied by Tsumura & Co. (lot number 2140050010, Tokyo, Japan) as a powdered extract obtained by spray-drying a hot water extract mixture of the following seventeen crude drugs: Scutellariae radix, Phellodendri cortex, Coptidis rhizoma, Platycodi radix, Aurantii fructus immaturus, Schizonepetae spica, Bupleuri radix, Gardeniae fructus, Rehmanniae radix, Paeoniae radix, Cnidii rhizoma, Angelicae radix, Menthae herba, Angelicae dahuricae radix, Saposhnikoviae radix, Forsythiae fructus, and Glycyrrhizae radix.
Amoxicillin and clavulanic acid used as reference antimicrobial drugs were purchased from Toronto Research Chemicals (Toronto, ON, Canada). Pure samples of apigenin, baicalein, baicalin, genistein, hesperidin, hesperetin, liquiritin, liquiritigenin, naringenin, glycyrrhizic acid, and glycyrrhetinic acid were purchased from Wako Pure Chemical Industries (Osaka, Japan). Apigenin 7-O-glucoside was purchased from Sigma-Aldrich (St. Louis, MO, USA), narirutin from Extrasynthese (Genay, France), and isoliquiritin from Chemfaces (Hubei, China). Baicalein, baicalin (baicalein 7-O-glucuronide), luteolin, genistin, wogonin, and wogonoside (wogonin 7-O-glucuronide), and berberine chloride were obtained from Tsumura & Co., with high purities for pharmacokinetic study.
Male Sprague–Dawley rats were purchased from Charles River Laboratories (Yokohama, Japan) and used at 8 week of age for pharmacokinetic study. Male BALB/c mice were purchased from Japan SLC (Shizuoka, Japan) and used at 7 weeks old. All studies were approved by and conducted according to the guidelines of the experimental animal ethics committees of Tsumura & Co. (permit no.: 16-072 (19 December 2016) and 17-002 (5 April 2017)). All surgeries were performed under ketamine or isoflurane anesthesia, and all efforts were made to minimize suffering. In the present study, KRT was administrated orally to the experimental animals at 2 g/kg in distilled water. Although the dose of KRT was higher than the clinical dose used in humans, this dose was based on previous publications of experimental KRT studies [18
] reporting the beneficial effects of KRT related to clinical efficacy. No adverse effects were observed in animals given 2 g/kg KRT in the present experiment.
4.3. Superficial Skin Infection Model
Cutaneous infection by S. aureus was induced in BALB/c mice weighting 17.9 to 21.8 g according to the procedure described by Kugelberg et al. with a minor modification [47
]. Forty mice were randomly divided into seven groups as follows: normal (n = 4) non-infection group, vehicle control (n = 6 × 2), KRT-treated (n = 6 × 2), and antibiotics-treated (n = 6 × 2) infection groups. The hair on the back was shaved with an electric razor and depilatory cream applied under ketamine anesthesia. An area of skin approximately 4 cm2
was stripped with clothmade cohesive tape once a day for two days. After stripping on the second day, S. aureus MW2 (strain: BAA-1707, ATCC, Manassas, VA, USA) was inoculated on the skin at 1 × 107
CFU in 100 μL saline. Keigairengyoto was administrated orally at 2 g/kg in distilled water 1 h before and 1, 2, and 3 days after the S. aureus inoculation (preventive administration) or 1, 2, and 3 days after inoculation (therapeutic administration). As reference drugs, a mixture of amoxicillin (100 mg/kg) and clavulanic acid (50 mg/kg) was prepared in water and administrated orally following the same schedules as for KRT. The macroscopic evaluation of skin lesion was examined at days 2 and 4 after the inoculation. The histological evaluation and the count of living S. aureus on the skin were performed at day 4 after the inoculation.
Living S. aureus
on the skin were counted 4 days after inoculation with S. aureus
, according to a previous paper [19
]. Immediately after the mice were killed, the wounds, approximately 2 cm2
, were excised and homogenized together with 1 mL of phosphate-buffered saline in Stomacher lab system bags using a Stomacher 80 machine (Seward Ltd., Thetford, UK) set at 260 strokes per min for 120 s. Suitable dilutions of the homogenates were plated on S. aureus
agar plates to determine the number of living bacteria (CFU). The number of living bacteria is reported as CFU per weight of the skin tissues (CFU/g).
The macroscopic evaluation was performed 2 and 4 days after inoculation with S. aureus
. The severities of erosion and the papule were evaluated according to a previously published report with a minor modification [48
]: 0 (none), 1 (mild), 2 (moderate), and 3 (severe), and are reported as the sum of each score. The observer was blinded to the treatments for all animals.
To characterize the histopathology, biopsy specimens were taken 4 days after inoculation with S. aureus. Immediately after the animals were killed, 5-mm punch biopsy specimens of excised skin were fixed in phosphate-buffered (pH 7.4) formalin (4%). The formalin-fixed biopsy specimens were embedded in paraffin and stained with hematoxylin and eosin. The histological findings of the crust, acanthosis, pastule and spongiosis in the epidermis, and edema and inflammation in the dermis were scored according to the following criteria, respectively: 0 (none), 1 (slight, <25% in full field), 2 (mild, 25–50%), 3 (moderate, 50–75%), and 4 (marked, >75%). The observer was blinded to the treatments for all biopsy specimens.
4.4. Pharmacokinetic Analysis of KRT-Derived Flavonoids and Glucuronide Metabolites by Liquid Chromatography–Mass Spectrometry with Tandem Mass Spectrometry
Keigairengyoto prepared in water was given orally to 16-h fasted rats (weighing 253 to 273 g) at a dose of 2 g/10 mL/kg. Blood was withdrawn from the abdominal inferior vena cava with a heparinized syringe at 0.25, 0.5, 1, 2, 4, 6, 10, or 24 h after administration (n
= 3). Plasma was obtained by centrifugation at 1700× g
for 15 min at 4 °C and stored at −75 °C or colder until analysis. To specifically deconjugate glucuronides, 80 μL of plasma sample was incubated with 50 μL of β-glucuronidase solution containing 200 units/mL β-glucuronidase from Escherichia coli
(Sigma-Aldrich) for 2 h at 37 °C. The deconjugation reaction was stopped by adding ethyl acetate or by refrigerating the mixture considering the following procedures. This pretreated solution was injected into the two liquid chromatography–mass spectrometry with tandem mass spectrometry (LC-MS/MS) systems after pooling with an internal standard solution of niflumic acid (Sigma-Aldrich) or atropine (Wako Pure Chemical Industries, Osaka, Japan). Analytical conditions are summarized in Tables S1 and S2
For quantification of apigenin, wogonin, wogonoside, hesperetin, naringenin, liquiritigenin, genistein, and luteolin in β-glucuronidase-naive (untreated) plasma, 200 µL plasma samples were pretreated by liquid-liquid extraction using ethyl acetate. The total supernatant was collected, dried, and concentrated using a centrifugal evaporator. The dried residue was then dissolved in 60 μL of the specific HPLC mobile phase used for each analytical method, and a 10 or 20 μL volume injected into the LC-MS/MS system.
For quantification of baicalin, baicalein, and berberine in untreated plasma, 80 µL of plasma was pretreated by solid-phase extraction using Oasis HLB 96-well µElution plate (Waters, Milford, MA, USA). The methanol eluates were collected, dried, and dissolved in 60 μL of the specific HPLC mobile phase. A 10 or 20 μL volume was then injected into the LC-MS/MS system.
For quantification of glycyrrhizic acid and glycyrrhetinic acid in untreated plasma, 100 µL plasma samples were pretreated by deproteinization using methanol. The supernatant was collected, dried, and concentrated using a centrifugal evaporator. The dried residue was then dissolved in 60 μL of the specific HPLC mobile phase used for the analytical method, and a 10 μL volume injected into the LC-MS/MS system.
For quantification of wogonin, wogonoside, hesperetin, naringenin, liquiritigenin, and genistein in treated plasma, the reaction solutions after deconjugation reactions were pretreated by liquid-liquid extraction. The supernatant was dissolved in 120 μL of the specific HPLC mobile phase, and a 10 or 15 μL volume injected into the LC-MS/MS system.
For quantification of apigenin, luteolin, baicalin, baicalein, and berberine in treated plasma, the reaction solutions after deconjugation were pretreated by solid-phase extraction by the method described above, and 10 or 15 μL of the solution dissolved in 50 μL of the specific HPLC mobile phase was injected into the LC-MS/MS system.
Pharmacokinetic parameters of flavonoids and glucuronide metabolites in KRT-administrated plasma with or without β-glucuronidase treatment were analyzed by noncompartmental modeling using Phoenix WinNonlin (version 6.3, Certara L.P., St. Louis, MO, USA) to determine various pharmacokinetic constants, including Cmax, tmax, t1/2, and AUC0–last. The t1/2 was divided by loge2/ke, where ke is the terminal elimination rate constant determined from at least three data points on the descending linear limb.
4.5. Macrophage Culture Assay
Cells of the mouse macrophage cell line RAW264.7 (ATCC, Manassas, VA, USA) were grown in Dulbecco’s modified Eagles medium supplemented with 10% fetal bovine serum (FBS), 4.5 g/L glucose, 2 mmol/L l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10 mmol/L HEPES. Cells were seeded in 96-well culture plates at 5 × 103 cells/well and cultured with the test compound (30 μmol/L) in the presence or absence of a suboptimal dose (0.5 ng/mL) of mouse interferon-γ (IFN-γ) (PeproTech, Rocky Hill, NJ, USA). After 3 days’ incubation at 37 °C, culture fluids were removed and replaced by warm medium containing 30 μg/mL FITC-conjugated S. aureus. After 30 min incubation in a 5% CO2 incubator, cells were harvested using cold PBS containing 2 mmol/L EDTA, washed, and fixed for 15 min at 4 °C with phosphate buffer containing 4% paraformaldehyde (pH 7.4). FITC-positive cells were determined using a FACSaria II flow cytometer and DIVA 8.0.1 software (Becton Dickinson, San Jose, CA, USA). Phagocytic activity is expressed as the mean fluorescence intensity (MFI) from individual cells.
4.6. Conversion Assay of Baicalin to Baicalein
Naïve mice were sacrificed by exsanguination to obtain skin samples from the back. After shaving with an electric razor, the skin was excised and homogenized in cold PBS at 20 mg/mL. After centrifugation at 10,000× g for 20 min at 4 °C, supernatants were harvested and stored at −80 °C until use.
To examine β-glucuronidase activity in skin tissue, baicalin (final 10 μmol/L) was added as a substrate to skin homogenate in 24-well plates. Alternatively, to examine β-glucuronidase activity in macrophages, baicalin (final 10 μmol/L) was added to live RAW264.7 cells seeded at 1 × 105 cells/0.5 mL/well or to cell lysates in 24-well plates. After 24 h incubation in a 5% CO2 incubator, supernatants were harvested and stored at −80 °C until measurement of baicalin and baicalein by LC-MS/MS as described above.
4.7. Statistical Analysis
Paired group means were compared by Student’s t-test and multiple group means by Steel, Steel-Dwass, or Dunnett test. For all tests, a probability of less than 0.05 (2-tailed) was considered significant.