Rhinacanthin-C Mediated Herb-Drug Interactions with Drug Transporters and Phase I Drug-Metabolizing Enzymes.

Rhinacanthin-C is a major active constituent in Rhinacanthus nasutus (L.) Kurz, a plant widely used in herbal remedies. Its potential for pharmacokinetic herb-drug interaction may exist with drug transporters and drug metabolizing enzymes. This study assessed the possibility for rhinacanthin-C-mediated drug interaction by determining its inhibitory effects against major human efflux and influx drug transporters as well as various human cytochrome P450(CYP) isoforms. Rhinacanthin-C demonstrated a moderate permeability through the Caco-2 monolayers [Papp (AP-to-BL) = 1.26 × 10-6 cm/s]. It significantly inhibited transport mediated by both P-glycoprotein (P-gp) (IC50 = 5.20 µM) and breast cancer resistance protein (BCRP) (IC50 = 0.83 µM) across Caco-2 and BCRP-overexpressing Madin-Darby canine kidney II cells (MDCKII) cells. This compound also strongly inhibited uptake mediated by organic anion-transporting polypeptide 1B1 (OATP1B1) (IC50 = 0.70 µM) and OATP1B3 (IC50 = 3.95 µM) in OATP1B-overexpressing HEK cells. In addition to its inhibitory effect on these drug transporters, rhinacanthin-C significantly inhibited multiple human CYP isoforms including CYP2C8 (IC50 = 4.56 µM), 2C9 (IC50 = 1.52 µM), 2C19 (IC50 = 28.40 µM), and 3A4/5 (IC50 = 53 µM for midazolam and IC50 = 81.20 µM for testosterone), but not CYP1A2, 2A6, 2B6, 2D6, and 2E1. These results strongly support a high propensity for rhinacanthin-C as a perpetrator of clinical herb-drug interaction via inhibiting various influx and efflux drug transporters (i.e., P-gp, BCRP, OATP1B1, and OATP1B3) and CYP isoforms (i.e., CYP2C8, CYP2C9, and CYP2C19). Thus, the potential for significant pharmacokinetic herb-drug interaction should be addressed when herbal products containing rhinacanthin-C are to be used in conjunction with other prescription drugs.

Flavonoids kaempferide and 4,2'-dihydroxy-4',5',6'-trimethoxychalcone inhibit mitotic clonal expansion and induce apoptosis during the early phase of adipogenesis in 3T3-L1 cells.

OBJECTIVE: Kaempferide and 4,2'-dihydroxy-4',5',6'-trimethoxychalcone (DTMC) are two major flavonoids found in Chromolaena odorata Linn. leaf extract. The aim of this study was to elucidate the mechanism by which these two flavonoids exerted their effect on adipogenesis. The inhibitory effect of kaempferide and DTMC on adipocyte differentiation and their mechanisms involving mitotic clonal expansion (MCE) and apoptosis during the early stage of adipogenesis were investigated. METHODS: Confluent 3T3-L1 preadipocytes were induced to differentiate and exposed to the flavonoids during various phases of differentiation. Intracellular lipid accumulation, cell density and expression of the transcription factors peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding proteins α were assessed using AdipoRed, Oil red O and Western blot assays. Effects of both flavonoids on cell proliferation and apoptosis were also determined by carboxyfluorescein diacetate succinimidyl ester and annexin V-fluorescein isothiocyanate/propidium iodide-staining assays, respectively. RESULTS: Kaempferide and DTMC showed significant, concentration-dependent anti-adipogenic activity and effect on cell density in the early phase of adipogenesis. The expression of the transcription factors seemed to be reduced when the treatment was prolonged or in the early phase of adipogenesis. These flavonoids interrupted MCE via inhibition of preadipocyte proliferation and induction of apoptosis. DTMC was nearly three times more potent than kaempferide in inducing apoptosis. CONCLUSION: Kaempferide and DTMC exerted their anti-adipogenic activity through inhibition of MCE, either by suppressing cell proliferation or by inducing apoptosis during the early phase of differentiation.

Potential P-glycoprotein-mediated herb-drug interaction of phyllanthin at the intestinal absorptive barrier.

OBJECTIVES: This study investigated the absorptive potential of phyllanthin across the polarized Caco-2 monolayers and the potential role of phyllanthin in P-glycoprotein (P-gp)-mediated drug interaction. METHODS: The absorptive potential of phyllanthin was predicted from its apparent permeability (Papp ) across the Caco-2 monolayers under the pH gradient condition (pH 6.5AP -7.4BL ) at 37°C. Integrity of paracellular transport was assessed by monitoring transepithelial electrical resistance (TEER) and lucifer yellow (LY) leakage. P-gp-mediated interaction was evaluated by transport studies of phyllanthin and rhodamine-123. KEY FINDINGS: The absorptive Papp of phyllanthin (34.90 ± 1.18 × 10-6 cm/s) was in the same rank order as the high permeable theophylline and antipyrine. Phyllanthin transport in the absorptive and secretive directions was comparable (the efflux ratio (ER) of 1.19 ± 0.01). Phyllanthin caused no changes in TEER nor LY leakage in the monolayers. However, phyllanthin increased rhodamine-123 ER in a concentration-dependent manner, suggesting its inhibition on P-gp function. In addition, phyllanthin aqueous solubility was <5 µg/ml at 37°C. CONCLUSIONS: Phyllanthin is a highly permeable compound that could passively diffuse through the absorptive barrier via transcellular pathway with little hindrance from P-gp. Phyllanthin could interfere with transport of P-gp drug substrates, when concomitantly administered. In addition, aqueous solubility could be a limiting factor in phyllanthin absorption.

Anti-adipogenic effect of flavonoids from Chromolaena odorata leaves in 3T3-L1 adipocytes.

OBJECTIVE: The leaves of Chromolaena odorata, a highly invasive shrub found growing wild worldwide, are traditionally used for wound healing. Due to its high flavonoid contents, we aimed to find a new application for this plant. Preliminary tests using its ethanolic leaf extract showed that it could suppress the accumulation of lipids in adipocytes. We therefore studied the anti-adipogenic effect of several C. odorata leaf extracts and the relationship between molecular structure and bio-activity of its isolated flavonoid constituents using 3T3-L1 preadipocytes/adipocytes as a model. METHODS: Three leaf extracts and thirteen flavonoids isolated from C. odorata were tested for their effect on lipid accumulation in 3T3-L1 adipocytes using AdipoRed reagent, with quercetin as the positive control. The effects of active flavonoids on the adipocytes were confirmed by oil red O staining and visualized under a light microscope. RESULTS: n-Hexane and ethyl acetate extracts of C. odorata leaves displayed anti-adipogenic activity. The latter extract was the more potent one, especially at 40 µg/mL. Four flavonoids, pectolinarigenin, kaempferide, 4,2'-dihydroxy-4',5',6'-trimethoxychalcone and dillenetin, exhibited significant, concentration-dependent inhibitory effects on lipid accumulation in 3T3-L1 adipocytes. The most potent flavonoid obtained in this study was 4,2'-dihydroxy-4',5',6'-trimethoxychalcone, which caused 75% and 90% inhibition of cellular lipid accumulation at 30 and 50 µmol/L, respectively. Both kaempferide and 4,2'-dihydroxy-4',5',6'-trimethoxychalcone were major constituents in the ethyl acetate extract of this plant. CONCLUSION: C. odorata leaves contained several flavonoids with anti-adipogenic effects against lipid accumulation in 3T3-L1 adipocytes. The plant, normally considered a useless weed, may actually provide an abundant source of biologically active flavonoids.

Effects of rhinacanthin-C on function and expression of drug efflux transporters in Caco-2 cells.

Rhinacanthin-C is a bioactive naphthoquinone ester found in Rhinacanthus nasutus Kurz (Acanthaceae). This compound has potential therapeutic value as an anticancer and antiviral agent. The purposes of this study were to determine the effects of this compound on the function and the expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2), using the in vitro model of Caco-2 cells. The activities of P-gp and MRP2 were determined by following the intracellular accumulation of calcein and 5(6)-carboxy-2',7'-dichlorofluorescein in the uptake assays with fluorescence spectroscopy. The expression of P-gp after prolonged exposure was evaluated by flow cytometry with the use of a fluorescein isothiocyanate-conjugated anti-human P-gp antibody. Our results showed that the inhibitory effect of rhinacanthin-C was more potent toward P-gp than MRP2, and was reversible. Short-term exposure of Caco-2 cells with rhinacanthin-C (100 µM) resulted in increase in P-gp expression without any significant change in its function. Extended exposure of Caco-2 cells to the naphthoquinone at the highest non-cytotoxic concentration (0.625 µM) for 7 days had no effect on the expression and the function of P-gp. These findings suggested that rhinacanthin-C might raise the problem of herb-drug interaction when co-administered with other P-gp substrates.

Influence of incorporation methods on partitioning behavior of lipophilic drugs into various phases of a parenteral lipid emulsion.

The purpose of this study was to investigate the effect of drug incorporation methods on the partitioning behavior of lipophilic drugs in parenteral lipid emulsions. Four lipophilic benzodiazepines, alprazolam, clonazepam, diazepam, and lorazepam, were used as model drugs. Two methods were used to incorporate drugs into an emulsion: dissolving the compound in the oil phase prior to emulsification (de novo emulsification), and directly adding a concentrated solution of drug in a solubilizer to the emulsion base (extemporaneous addition). Based on the molecular structures and determination of the oil and aqueous solubilities and the partition coefficients of the drugs, the lipophilicity was ranked as diazepam > clonazepam > lorazepam > alprazolam. Ultracentrifugation was used to separate the emulsion into four phases, the oil phase, the phospholipid-rich phase, the aqueous phase and the mesophase, and the drug content in each phase was determined. Partitioning of diazepam, which has the highest lipophilicity and oil solubility among the four drugs, was unaffected by the drug incorporation method, with both methods giving a high proportion of drug in the inner oil phase and the phospholipid-rich phase, compared to the aqueous phase and mesophase. Partitioning of the less lipophilic drugs (alprazolam, clonazepam, and lorazepam) in the phases of the emulsion system was dependent on the method of incorporation and the drug solubility properties. Emulsions of the three drugs prepared by de novo emulsification exhibited higher drug localization in the phospholipid-rich phase compared to those made by extemporaneous addition. With the latter method, the drugs tended to localize in the outer aqueous phase and mesophase, with less deposition in the phospholipid-rich phase and no partitioning into the inner oil phase.