Bulbine natalensis (currently Bulbine latifolia) and select bulbine knipholones modulate the activity of AhR, CYP1A2, CYP2B6, and P-gp

• Abstract
• Introduction
• Results
• Discussion
• Materials and Methods
• Biochemicals and reagents
• Source of extracts and pure compounds
• Cell culture and treatment
• Cell viability assay
• Isolation of total RNA, cDNA synthesis, and real-time (RT) PCR analysis
• Determination of CYP2B6 and CYP1A2 enzyme activity
• AhR reporter gene assay
• Cytochrome P-450s (CYP) inhibition assay
• Statistical analysis
• Contributorsʼ Statement
• References

Abstract

Bulbine natalensis, an emerging medicinal herb on the global market with androgenic properties, is often formulated in dietary supplements that promote perceived sexual enhancement. However, to date, comprehensive safety studies of B. natalensis are lacking, particularly those related to its herb-drug interaction potential. The purpose of this study was to assess the inductive and inhibitory effects of extracts and pure compounds of B. natalensis on human cytochrome P-450 isozymes in vitro. Our findings demonstrated that both water and methanolic extracts of B. natalensis as well as knipholone, bulbine-knipholone, and 6′-O-methylknipholone dose-dependently increased mRNA expression encoded by CYP2B6, CYP1A2, and ABCB1 genes. Functional analyses showed that water (60 to 2.20 µg/mL) and methanolic (30 to 3.75 µg/mL) extracts and knipholones (10 to 0.33 µM) increased CYP2B6 and CYP1A2 activity in a dose-dependent manner. Additionally, water extract (60 µg/mL), methanolic extract (30 µg/mL), and knipholone (10 µM) caused activation of the aryl hydrocarbon receptor up to 11.1 ± 0.7, 8.9 ± 0.6, and 7.1 ± 2.0-fold, respectively. Furthermore, inhibition studies revealed that methanolic extract attenuated the activity of metabolically active CYP1A2 (IC₅₀, 22.6 ± 0.4 µg/mL) and CYP2B6 (IC₅₀, 34.2 ± 6.6 µg/mL) proteins, whereas water extracts had no inhibitory effect on either isoform. These findings suggest that chronic consumption of B. natalensis may affect normal homeostasis of select CYPs with subsequent risks for HDIs when concomitantly ingested with conventional medications that are substrates of CYP2B6 and CYP1A2. However, more in-depth translational studies are required to validate our current findings and their clinical relevance.

Introduction

Bulbine natalensis Baker (currently, Bulbine latifolia [l. f.] Spreng) is an aloe-like, succulent perennial, geophytic medicinal herb that belongs to the genus Bulbine and family Xanthorrhoeaceae. Geographically, it is widely distributed in the southern and western provinces of South Africa and Australia, and eastern African countries like Malawi and Mozambique [1]. The indigenous tribal communities of South Africa know B. natalensis by different folkloric names: ibhucu (Zulu), ingcelwane (Xhosa), and rooiwortel (Afrikaans). Traditional practitioners employ the herb to alleviate various ailments and symptoms. Herbalists and herb sellers of Eastern Cape, South Africa frequently prescribe it for the management of MSD and immunological disorders. Scientifically, the folkloric aphrodisiac activity of B. natalensis was first validated in vivo by Yakubu and Afolayan (2009) [2]. It was observed that 25 and 50 mg/kg doses of B. natalensis extract significantly augmented sexual behavior in male albino rats (Rattus norvegicus). Due to its purported aphrodisiac-enhancing properties, B. natalensis has garnered substantial popularity, resulting in both increased consumer demand for the plant and subsequent upsurges in price [2]. In addition, several unique anthraquinones have been isolated from B. natalensis [3], of which little is known about their pharmacological and toxicological attributes.

For oral ingestion, suppliers of B. natalensis are seeking its classification as a “Novel Food”. A requisition has been submitted to the European Commission (EU) under the Novel Food Regulation to obtain this status. Before May 15, 1997, B. natalensis was not used as food or a food ingredient in EU countries. Hence, before placing it as a food or food ingredient in the EU market, a safety assessment is required under the Novel Food Regulation [4]. In Canada, B. natalensis is regulated as an active ingredient of licensed natural health products (NHPs) and requires a premarketing authorization from the Natural and Non-prescription Health Products Directorate (NNHPD). As of Feb. 2020, in Canada, only three licensed multi-herb NHPs formulations are sold with B. natalensis extract as one of the active ingredients [5]. In the United States (US), several products labeled as containing B. natalensis are listed in the National Institutes of Health Dietary Supplement Label Database. These products are frequently marketed for male sexual enhancement [6]. However, they do not currently fall under the category of products designated “Generally Recognized as Safe” (GRAS) by the US-FDA. In short, these products lack systematically validated clinical efficacy or safety data. Moreover, as with many botanical supplements, people frequently consume B. natalensis-containing products along with conventional medicine without considering the risk for potential HDIs.

A botanicalʼs HDI potential often depends upon its ability to influence the activity of two key protein families involved in drug disposition: CYPs and ATP-binding cassette (ABC) transporters. CYPs are a superfamily of membrane-bound oxygenases that play a vital role in xenobiotic metabolism [7]. ABC transporters constitute a large and diverse superfamily of membrane-bound proteins whose hallmark is their ability to actively transport molecules through membranes against a concentration gradient via ATP hydrolysis. Of particular importance is the ABC isoform ABCB1, or P-gp, capable of transporting a large and diverse set of substrate molecules. While CYP and P-gp proteins are widely distributed in the body, they are highly expressed in the liver and small intestine where they function to limit xenobiotic absorption and exposure via biotransformation (i.e., CYPs) and/or efflux back into the gut lumen (i.e., P-gp) [8]. CYP and P-gp expression is regulated through the actions of various transcription factors (e.g., nuclear receptors like the PXR and constitutive androstane receptor [CAR], or basic helix-loop-helix/Per-Arnt-Sim like the AhR that are activated via specific endogenous or exogenous ligands) [9], [10], [11]. Orally ingested dietary phytochemicals may act as agonists or antagonists for specific transcription factors such as AhR, CAR, and PXR, resulting in dysregulation of drug-metabolizing enzymes and transporters, which, in turn, can affect the safety and efficacy of concomitantly ingested medications [12]. According to US-FDA guidelines [13], identifying phytochemicals capable of interacting with CYPs and transporters is of paramount importance. As mentioned earlier, the availability and consumption of B. natalensis products are on the rise. However, apart from a recent report by our group [14], where the in vitro modulatory effects of B. natalensis on PXR activation and the activity of CYP2C9 and CYP3A4, two principal CYPs involved in the metabolism of almost 70% of all medications were described, little else is known about the HDI potential of this botanical. To further our understanding of the plantʼs HDI potential, here we describe the ability of B. natalensis and select anthraquinone phytochemicals (i.e., knipholone, bulbine-knipholone, 6′-O-methylknipholone) ([Fig. 1]) to activate PXR- and AhR-mediated expression of ABCB1 and CYP1A2/2B6 mRNA, respectively, along with their influence on the functional activity of CYP1A2 and CYP2B6 isozymes.

Results

HepG2 cells were incubated with consecutively increasing concentrations of extracts and pure compounds of B. natalensis for 24 and 48 h to determine the concentration-related effect on cell viability. The cell viability remained > 80% upon exposure to extract and purified knipholones up to 48 h (data not shown). In contrast, upon exposure to 10 µM doxorubicin, cell viability was decreased to 56%. These results suggest that B. natalensis and knipholones have no adverse effects on the viability of HepG2 cells up to the highest concentrations tested in various experiments.

The effect of extracts and pure compounds of B. natalensis (stems) on CYP2B6 and CYP1A2 mRNA expression was examined by RT-PCR analysis. As shown in [Fig. 2 a], both concentrations of the water extract (60 and 20 µg/mL) had insignificant inductive effects. In contrast, the methanolic extract at 30 µg/mL significantly increased CYP2B6 mRNA levels (8.3 ± 1.2-fold) but was inactive at 10 µg/mL. Among pure compounds, knipholone markedly increased CYP2B6 mRNA expression by 97 ± 3.9-fold and 5.8 ± 2.2-fold at 10 and 3.3 µM, respectively. Both concentrations of bulbine-knipholone (10 and 3.33 µM) also had significant inductive effects and increased CYP2B6 mRNA by 15.2 ± 1.4-fold and 4.2 ± 0.7-fold, respectively. However, the highest concentration of 6′-O-methylknipholone (10 µM) elevated CYP2B6 mRNA by only 2.2 ± 1.6-fold, while the lower concentration (3.3 µM) was inactive. The results presented in [Fig. 2 b] indicate that methanolic and water extracts of B. natalensis stems increased mRNA expression of CYP1A2 up to 4.8 ± 1.0- and 2.7 ± 0.1-fold at 30 µg/mL and 60 µg/mL, respectively. In contrast, the lower concentrations of both extracts did not show significant inductive effects. Among pure compounds, knipholone and bulbine-knipholone increased the mRNA expression of CYP1A2 by 5.7 ± 0.6-fold and 6.8 ± 0.6-fold, respectively, at 10 µM. However, less significant induction was observed for 6′-O-methylknipholone (10 and 3.3 µM).

As presented in [Fig. 3], incubation of cells with 60 and 20 µg/mL of the water extract increased P-gp mRNA expression up to 10.9 ± 0.2- and 3.5 ± 0.2-fold, respectively. Similarly, the methanolic extract also showed significant inductive effects and increased P-gp mRNA by 3.7 ± 0.4- and 2.4 ± 0.1-fold at 30 and 10 µg/mL, respectively. Among the pure compounds, 6′-O-methylknipholone was most active in increasing transcription of P-gp mRNA 5.2 ± 0.1- and 5.8 ± 0.9-fold at 10 and 3.3 µM, respectively. Bulbine-knipholone exhibited modest transcriptional inductive effects (2.0 ± 0.2-fold increase) at 10 µM; however, lower concentrations (3.3 µM) had no effect. In contrast, both concentrations of knipholone (10 and 3.3 µM) were found to be inactive. Overall, RT-PCR analysis suggested that both extracts and pure compounds of B. natalensis can activate the transcriptional machinery of CYP2B6, CYP1A2, and P-gp (ABCB1) genes. Whether these findings translate to increased expression at the functional protein level remains to be determined.

Enzyme activity results presented in [Fig. 4 a] indicated that the water extract at a concentration of 60 µg/mL increased CYP2B6 activity 3-fold, whereas the methanolic extract at 30 and 10 µg/mL increased CYP2B6 activity up to 4.7 ± 0.1- and 3.0 ± 0.01-fold, respectively. Among purified phytoconstituents, the 3 concentrations of knipholone (10, 3.3, and 1.1 µM) had significant translational effects and increased CYP2B6 enzyme activity 4.7 ± 0.5-, 4.3 ± 0.5-, and 2.6 ± 0.5-fold, respectively. Similarly, bulbine-knipholone augmented CYP2B6 activity by 7.4 ± 1.90, 3.9 ± 1.2, and 2.9 ± 0.5-fold at 10, 3.33, and 1.11 µM, respectively. In contrast, 6′-O-methylknipholone was not as strong as other knipholones and imparted only moderate effects at all concentrations. The results presented in [Fig. 4 b] depict that incubation with water extract at 60 and 20 µg/mL enhanced CYP1A2 activity by 3.9 ± 0.1- and 2.7 ± 0.1-fold, respectively. In contrast, lower concentrations (6.6 and 2.2 µg/mL) produced no substantial changes. Subsequently, 30 µg/mL of the methanolic extract produced significant increases in CYP1A2 activity (5.7 ± 1.3-fold), while lower concentrations did not. The effect of knipholone was more pronounced than bulbine-knipholone and 6′-O-methylknipholone at 10 µM. These results collectively suggest that both extracts and purified anthraquinone phytoconstituents of B. natalensis increase CYP2B6 and CYP1A2 enzyme activities in a concentration-dependent manner, thus mirroring the mRNA expression data.

To investigate the AhR agonistic activity of B. natalensis extracts and selected anthraquinones, we utilized a standard AhR reporter assay system. The results presented in [Fig. 5] illustrate that the methanolic extract dramatically induced transcriptional activity of AhR by 9.0 ± 0.6-fold and 5.1 ± 0.4-fold at 30 and 10 µg/mL, respectively. Likewise, the water extract showed marked AhR activation. Among the anthraquinones, knipholone activated AhR transcription at all concentrations dose-dependently, while bulbine-knipholone and 6′-O-methylknipholone showed modest effects. Collectively, AhR assay results suggest that extracts and knipholones of B. natalensis dramatically upregulated transcriptional activity of AhR, which likely accounts for the observed changes in enzyme activity in vitro but may also translate into in vivo regulation of CYP1A2.

Fluorescence-based high-throughput screening (HTS) assays have been successfully employed for in vitro assessment of herb- or drug-drug interactions. Hence, to determine the inhibitory effects of extracts and knipholones of B. natalensis on CYP2B6 and CYP1A2 catalytic activity, a fluorescence-based HTS assay was used. This methodʼs principle is based upon the metabolic conversion of a nonfluorescent substrate into a highly fluorescent metabolite by a specific CYP isozyme. Enzyme inhibition results are presented in [Table 1]. The methanolic extract weakly inhibited the catalytic activities of CYP2B6 and CYP1A2 with IC₅₀ values of 34.2 ± 6.6 and 22.6 ± 0.4 µg/mL, respectively. In contrast, the water extract and knipholones showed no inhibitory effect toward CYP2B6 and CYP1A2, even at the maximum tested concentrations (25 µg/mL and 25 µM, respectively). On the other hand, the inhibition potentials of control drugs, miconazole for CYP2B6 and α-naphthoflavone for CYP1A2, were very strong with IC₅₀ values of 0.16 ± 0.03 µM and 0.04 ± 0.01 µM, respectively. Hence, B. natalensis extracts and individual knipholones appear to pose no inhibitory potential for CYP2B6 and CYP1A2 catalytic activity.

Discussion

For millennia, plants have been staples in the human diet while also serving as sources of essential medicines. Over the last 2 decades, however, BDS, many of which utilize plant extracts rarely found in diets worldwide, have gained a significant foothold in the global market. BDS demand and consumption are validated by the fact that > 75 000 products, many of which incorporate multi-ingredient formulations, have been marketed in the US [12]. In addition, several surveys note that both patients and healthy individuals willingly consume BDS along with conventional medicines, frequently without informing their physician [15]. Such actions may increase the risk for HDIs [16], [17]. At present, the BDS recognized as having the greatest HDI risk is Hypericum perforatum (St. Johnʼs wort), a popular botanical marketed for mild to moderate depression that renders many medications ineffective due to its ability to upregulate CYP3A4 expression via PXR activation [18]. This activation is the result of hyperforin, a phytochemical unique to H. perforatum that also is a potent ligand for PXR [19].

Other phytochemicals and xenobiotics have been found to act as agonists or antagonists for different xenoreceptors (e.g., AhR, CAR, and PXR) that, in turn, modulate the expression of drug-metabolizing enzymes and transporters, especially CYPs and efflux pumps (e.g., P-gp) [20]. B. natalensis (currently B. latifolia), a medicinal herb and emerging component of several mainstream herbal supplement formulations targeted at MSD may pose risks for HDI similar to that of St. Johnʼs wort [1], [21]. In line with this notion, previous examinations from our group found that extracts and pure compounds (knipholone, bulbine-knipholone, and 6′-O-methyl knipholone) of B. natalensis impart a strong inductive effect on PXR-mediated upregulation of CYP3A4, and CYP2C9 [14]. To expand upon these findings, we describe herein the inductive potential of B. natalensis extract and several of its purified anthraquinones on AhR activation, P-gp mRNA expression, and both expression and activity of CYP1A2 and CYP2B6.

The AhR belongs to a family of basic helix-loop-helix transcription factors encoded by the AhR gene located on chromosome 7 [11]. Several naturally occurring exogenous molecules, including various dietary compounds (e.g., carotenoids, curcumin, flavonoids, polyphenolics, indoles, and tryptophan) as well as synthetic polycyclic aromatic hydrocarbons (e.g., 3-methylcholanthrene, benzo[a]pyrene, benzanthracenes, and benzoflavones), have been identified as ligands for AhR [10], [22]. Upon binding with the ligand, the cytosolic complex (chaperon HSP90) of AhR is dissociated, translocated into the nucleus, and dimerized with AhR-nuclear translocator (ARNT). The dimerized protein binds at the promoter region and initiates transcription of targeted genes such as CYP1A2, CYP1B1, and TCDD-inducible poly-ADP-ribose polymerase (TIPARP) [9].

Our findings indicate that B. natalensis anthraquinones can be added to the growing list of natural compounds capable of acting as ligands for AhR [10], [22]. Water and methanolic extracts, as well as purified knipholones, upregulated AhR in a concentration-dependent manner. Interestingly, the AhR induction potency of both extracts was dramatically higher than individual knipholones. As a result, this may reflect the collective influence of multiple anthraquinones present in the extracts, especially in methanolic extract. As previously mentioned, the CYP1A2 gene is a key target of the AhR receptor, and we noticed that extracts and pure compounds of B. natalensis substantially upregulated AhR expression, CYP1A2 mRNA, and subsequently CYP1A2 activity. This sequence of effects would appear to have its origin in the several knipholones that serve as ligands for AhR.

CYP1A2 is an important drug-metabolizing enzyme that accounts for 4 to 16% of the total hepatic CYP pool. It is constitutively expressed in hepatic tissues with an average abundance of ~ 18 to 25 pmol/mg of microsomal protein but weakly expressed in the intestine [23], [24]. In contrast, some researchers claim that in vitro cellular models of the intestine, such as LS174T and Caco-2 cells, sufficiently express CYP1A1 and 1A2 isoforms [25]. Because CYP1A2 is highly expressed in the human liver, it plays a significant role in the biotransformation of several drug categories like analgesics (e.g., acetaminophen, phenacetin, and lidocaine), antipsychotics (e.g., olanzapine, clozapine), antidepressants (e.g., duloxetine, agomelatine), anti-inflammatory agents (i.e., nabumetone), cardiovascular drugs (e.g., propranolol, guanabenz, and triamterene), anti-Alzheimerʼs (i.e., tacrine), muscle relaxants (i.e., tizanidine), and others [23], [26], [27]. CYP1A2 is also involved in the detoxification of carcinogens like polyaromatic hydrocarbons (i.e., benzo[a]pyrene) present in cigarette smoke and cooked meat and heterocyclic amines (e.g., nitroarenes and arylamines) formed in fried or grilled meat and fish [25]. CYP1A2 is also involved in the bioactivation of drugs like the antiandrogen flutamide [28]. RT-PCR analysis indicated that B. natalensis extracts, especially the methanolic extract, increased mRNA expression of the CYP1A2 gene by several fold. To our knowledge, this is the first report of B. natalensis-mediated expression of CYP1A2, placing it among a growing number of plant extracts, including Camellia sinensis [25], Ginkgo biloba [29], H. perforatum [30], Eschscholzia californica [31], and Mitragyna speciosa [20], [32] with strong inductive effects on CYP1A2 mRNA expression.

In this study, we also found that 10 µM of the B. natalensis anthraquinones (knipholone and bulbine-knipholone) increased CYP1A2 mRNA expression by almost 6-fold, an effect equal to the positive control, omeprazole. Thus, contributions from these more lipophilic anthraquinones are likely responsible for the greater effect on CYP1A2 mRNA expression and enzyme activity observed for the methanolic extract.

CYP2B6, another important monooxygenase, makes up 2 to 10% of total hepatic CYPs and plays a vital role in the metabolism and detoxification of select xenobiotics [33]. Several clinically important life-saving therapeutics such as anticancer agents (e.g., cyclophosphamide, ifosfamide, and tamoxifen) [34], [35], [36], antiretrovirals (e.g., efavirenz and nevirapine) [37], [38], and anesthetics (e.g., ketamine and propofol) [39] are metabolized by CYP2B6. The xenoreceptors such as CAR and PXR are predominantly involved in regulating the expression of CYP2B6 [18], [40]. To analyze the inductive effect on transcription of CYP2B6, transfected HepG2 cells were treated with extracts or pure compounds of B. natalensis. The water extracts slightly increased mRNA expression of CYP2B6 (1.4-fold), suggesting a weak inductive effect. In contrast, methanolic extracts markedly increased transcription of CYP2B6 (~ 8-fold). This discrepancy is likely due to differences in the water versus methanol extraction efficiency of various phytochemicals. Among the purified anthraquinones tested, knipholone and bulbine knipholone dramatically increased CYP2B6 mRNA expression, while 6′-O-methylknipholone had only marginal effects. This disparity may be the result of topological alterations imparted to small changes in molecular architecture.

Apart from examining the effects of B. natalensis on xenoreceptor activation, cell-based functional assays were employed to test the effect of B. natalensis extracts and phytochemicals on CYP2B6 enzyme activity. Both extract types and pure compounds increased CYP2B6 activity in a concentration-dependent manner, indicating that observed xenoreceptor activation translated into increased enzyme activity. These findings suggest that chronic consumption of B. natalensis supplements may result in CYP2B6-mediated HDIs.

The membrane efflux pump P-gp is an important mediator of xenobiotic exposure whose inhibition/induction is considered a key player in many HDIs [41]. RT-PCR analyses showed that both extracts and pure compounds of B. natalensis increased P-gp mRNA expression in a concentration-dependent fashion. Although the effect of B. natalensis on P-gp functionality was not investigated here, our findings concerning P-gp mRNA expression lend further credence to the HDI potential of this up-and-coming botanical supplement.

Finally, earlier studies from our group [14], [42] and others [32], [43], [44] have noted that dietary phytochemicals can exhibit dual-functional behaviors concerning the modulation of xenobiotic metabolism and transport. In other words, phytochemicals may act as agonists for nuclear receptors like CAR, PXR, and AhR while also functioning as catalytic inhibitors for post-translationally matured proteins like CYPs and P-gp. Such duality may lead to fluctuations in plasma concentrations of co-administered drugs that could, in turn, produce adverse drug reactions, or, on the contrary, this simultaneous induction and inhibition may neutralize such untoward effects. Thus, to measure precisely the magnitude and mechanism of a potential HDI, both induction and inhibition analyses are equally important. Hence, we examined the CYP inhibitory effects of B. natalensis and its constituents using recombinant CYP baculosomes and noticed that the methanolic extract and knipholones moderately inhibited CYP2B6 activity but not CYP1A2. The results of this study suggest that the gross cumulative inductive potential of extracts and knipholones of B. natalensis is appreciably higher than that for CYP inhibition. Consequently, elevated levels of CYP2B6 and 1A2 may shift the interaction balance in favor of induction, much like that seen for St. Johnʼs wort. Nevertheless, to determine the translational relevance of these in vitro investigations, clinical HDI studies with B. natalensis products are warranted.

In conclusion, crude extracts and purified anthraquinones of B. natalensis were examined for possible CYP- and P-gp-mediated HDIs. We found that both extracts and knipholones significantly increased the mRNA expression of CYP2B6, CYP1A2, and P-gp, as well as the functional activities of CYP2B6 and CYP1A2 isozymes. However, they did not considerably inhibit the catalytic activity of either isoform. Collectively, our findings indicate that B. natalensis induces the expression of key drug-metabolizing enzymes and efflux transporters. Hence, dietary supplements containing B. natalensis may lead to possible CYP-mediated HDIs when concomitantly ingested with conventional medicines primarily metabolized by CYP2B6 and 1A2 isozymes; however, further investigations into the clinical relevancy of these findings are warranted.

Materials and Methods

Biochemicals and reagents

DMEM-F12, HEPES, trypsin EDTA, penicillin-streptomycin solution, and sodium pyruvate were purchased from GIBCO BRL, Invitrogen Corp. FBS was purchased from Hyclone Lab Inc. Omeprazole and rifampicin were obtained from Sigma Aldrich Chem. Co. AhR reporter assay kits were procured from Indigo Biosciences. RNA isolation kits were from Takara Bio Inc. PCR reaction mixture iTaq universal SYBR Green supermix and standard primers were acquired from Bio-Rad Laboratories. CYP assay kits (P450-Glo) were from Promega Corp. CYP inhibition kits (CYP2B6 and CY1A2) were from Invitrogen. All other reagents and solvents were of analytical grade and procured from authentic sources.

Source of extracts and pure compounds

Methanolic extract, water extract, and purified B. natalensis anthraquinones (knipholone bulbine-knipholone, and 6′-O-methylknipholone, purity > 95%) were available in-house from the repository of the National Center for Natural Products Research (NCNPR), School of Pharmacy, University of Mississippi, MS, USA. Both extracts were prepared from the stems of B. natalensis (NCNPR# 17 131), and pure compounds were isolated from the methanolic extract [3]. Both extracts and pure compounds were stored at 4 °C. The working stock solutions of extracts (20 mg/mL) and pure compounds (10 mM) were prepared in DMSO.

Cell culture and treatment

Human hepatocellular carcinoma (HepG2) cells were obtained from ATCC. The cells were cultured in DMEM/F12 supplemented with 10% FBS, 2.4 g/L sodium bicarbonate, 100 µg/mL streptomycin, and 100 units/mL penicillin at 37 °C in an environment of 5% CO₂ and 95% relative humidity. Stock solutions of tested compounds and controls were diluted in serum-free media to desired concentrations, and the DMSO concentration did not exceed 0.3% (v/v) during treatments.

Cell viability assay

The effects of the extracts and pure compounds on the viability of HepG2 cells were investigated as described earlier [45]. In brief, HepG2 cells were grown exponentially and 1.2 × 10⁴ cells/200 µL were seeded in 96-well plates in DMEM/F12 medium and allowed to grow overnight. Cells were then treated with different concentrations of extract (water extract: 120, 60, 30, 15, 7.5, and 3.8 µg/mL; methanolic extract: 60, 30, 15, 7.5, 3.8, and 1.9 µg/mL) or purified knipholones (20, 10, 5, 2.5, 1.3, and 0.6 µM) and doxorubicin (10, 5, 2.5, 1.3, 0.6, and 0.3 µM) for 24 and 48 h, respectively. At the end of incubation, 10 µL of MTT dye from a 5 mg/mL stock solution was added to each well, and plates were incubated for 4 additional hours. Subsequently, media was aspirated, and cells were washed with PBS. Formazan blue crystals formed by viable cells were dissolved in 200 µL of DMSO, plates were shaken for 5 min, and color intensity was measured at 580 nm. The absorbance of the control sample was considered 100%, and the % decrease in cell viability was calculated.

Isolation of total RNA, cDNA synthesis, and real-time (RT) PCR analysis

Validated PCR^™ SYBR Green Assay forward and reverse primers for CYP2B6 (qHsaCED0038976), CYP1A2 (qHsaCID0015160), and P-gp (qHsaCED0002291) were purchased from Bio-Rad Laboratories. HepG2 cells during their logarithmic growth phase were transfected with pSG5-hPXR (25 µg) and PCR-5 plasmid DNA (25 µg) and seeded at a density of 2 × 10⁶ per mL in 6-well plates as described previously [14], [20]. After 24 h of incubation, cells were treated with different concentrations of extracts (aqueous extract: 60 and 20 µg/mL; methanolic extract: 30 and 10 µg/mL) and select knipholones (10 and 3.3 µM) for 24 h. Rifampicin and omeprazole (10 µM) were used as positive controls for CYP2B6 and CYP1A2, respectively. Subsequently, media was removed, cells were washed with ice-cold PBS twice, and total RNA was isolated using the Quick-Start protocol (Qiagen kit), according to manufacturer directions. The quantity of isolated RNA was measured using the Bio-Tek, Synergy HT Multi-Mode Microplate reader. The purity of RNA was confirmed by the ratio of absorbance of the isolated nucleic acid at 260 and 280 nm (A260/A280 ≥ 1.8).

The complementary DNA (cDNA) sequence was synthesized using 1 µg of RNA as a template in Bio-Rad iScript Reverse Transcription Supermix. Two microliters of cDNA were used as the template with Bio-Rad, iTaq Universal SYBR Green Supermix. RT-PCR was performed in a 96-well plate by CFX connect real-time PCR detector system (Bio-Rad) with the following program: 1 step at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 10 s, annealing at 65 °C for 15 s, and elongation at 72 °C for 15 s. In addition, the relative quantification of CYP2B6 and CYP1A2 gene was normalized to the housekeeping gene GAPDH (qHsaCED0038674), and fold increase of mRNA in treated cells was calculated by comparing with vehicle-treated cells.

Determination of CYP2B6 and CYP1A2 enzyme activity

Exponentially growing HepG2 cells were transfected with pSG5-hPXR (25 µg) and PCR-5 (25 µg) plasmid DNA by electroporation at 180 V (1 pulse) for 80 msec as described previously [14], [20]. Cells were seeded in 96-well, clear bottom, white plates at a density of 0.7 × 10⁵ cells per well per 200 µL. Plates were incubated at 37 °C in a CO₂ incubator for 24 h. After attaining > 90% confluency, cells were treated with various concentrations of water extract (60, 20, 6.66, and 2.22 µg/mL), methanolic extract (30, 10, 3.3, and 1.1 µg/mL), knipholones (10, 3.3, 1.1, and 0.4 µM), and positive controls (10, 3.3, 1.1, and 0.4 µM) for 48 h, with test sample-containing media changes occurring at 24-h intervals. After incubation, culture media was aspirated from each well, and cells were washed twice with 100 µL sterile PBS (pH 7.4). Cells were incubated with luminogenic specific substrates of CYP2B6 (Luciferin-2B6) and CYP1A2 (Luciferin-A2) according to kit instructions (Promega, P450-Glo). After adding 50 µL of luciferin detection reagent and vortex mixing for 2 min, luminescence was measured using the Spectramax M5 plate reader. Vehicle-treated (DMSO) cells were used as controls, and fold change in enzyme activity was calculated in treated cells compared to controls.

AhR reporter gene assay

AhR reporter assays were performed according to AhR kit manufacturer instructions (Indigo Biosciences). In brief, a 200 µL suspension of reporter cells was dispensed in round-bottom 96-well plates. Plates were incubated in a CO₂ chamber for 5 h to facilitate cell attachment. After incubation, media was removed and 200 µL of serially diluted test samples in screening media (water extract: 60, 20, 6.7, and 2.2 µg/mL; methanolic extract: 30, 10, 3.3, and 1.1 µg/mL; knipholones: 10, 3.3, 1.1, and 0.4 µM) and positive control (Me-Bio: 1000, 333.3, 111.1, and 37.0 nM) were added and plates were incubated for 24 h. Following incubation, the culture medium was aspirated from the plates, and 100 µL of luciferase detection reagent was added. Plates were allowed to rest for 5 min at room temperature, and luminescence was measured using a Spectramax M-5 plate reader. The increase in luciferase activity in treated cells was calculated compared to vehicle-treated cells.

Cytochrome P-450s (CYP) inhibition assay

CYP inhibition assays were performed according to CYP2B6 and CY1A2 Vivid kits (Invitrogen). Briefly, stock solutions of extracts, purified knipholones, and positive controls were serially diluted in methanol (extracts: 50, 16.7, 5.6, 1.9, 0.6, and 0.2 µg/mL; knipholones: 25, 8.3, 2.8, 0.9, 0.3, and 0.1 µM; positive control: 1, 0.3, 0.1, 0.03, 0.01, and 0.004 µM) and incubated with human recombinant cytochrome P-450 (BACULOSOMES), NADP⁺, and kit-provided regeneration reagents in 96-well black, round bottom plates at room temperature for 10 min. The reaction was started by adding 10 µL of 10X specific fluorescent substrate of CYP2B6 (Vivid BOMCC) or CYP1A2 (Vivid EOMCC). After incubation, 50 µL of stop reagent (0.5 M Tris base) was added to halt the reaction, and fluorescence was measured on a Spectramax M-5 plate reader at excitation and emission wavelengths of 530 nm and 590 nm, respectively, for 90 min. The concentration of the test sample responsible for causing 50% inhibition (IC₅₀) was calculated using XLfit 4.2 software (IDBS) with fit model 201.

Statistical analysis

All experiments were performed in triplicate and results were expressed as mean ± SD. Data were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroniʼs multi-comparison test to determine the statistical significance using GraphPad Prism Software Version 7. The value of p < 0.05 was considered to be statistically significant.

Contributorsʼ Statement

Conceptualization: S. I. K., A. G. C., B. J. G., I. A. K.; methodology: I. H., O. R. D., V. M., Z. A.; formal analysis: I. H., O. R. D., V. M., S. I. K.; resources: S. I. K., A. G. C., I. A. K.; original draft preparation: I. H., S. I. K., A. G. C., B. J. G.; project administration: S. I. K., I. A. K.; funding acquisition: I. A.K and A. G. C. All authors have read and agreed to the published version of the manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

This research is supported in part by “Science-Based Authentication of Botanical Ingredients” funded by the Center for Food Safety and Applied Nutrition, US Food and Drug Administration grant number 5U01FD004246 and “Discovery & Development of Natural Products for Pharmaceutical & Agricultural Applications” funded by the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No. 58-6060-6-015.

Acknowledgements

The authors are thankful to Dr. Alvaro M. Viljoen, Tshwane University of Technology, Pretoria, South Africa, for providing plant material, Dr. Bharathi Avula for her valuable insights into the overall chemical composition of aqueous and methanolic extracts, and Dr. Siddharth Tripathi for assisting in IC₅₀ calculations.

^# Currently at Covance Inc., Salt Lake City, Utah, United States

• References

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• 32 Lim EL, Seah TC, Koe XF, Wahab HA, Adenan MI, Jamil MFA, Majid MIA, Tan ML. In vitro evaluation of cytochrome P450 induction and the inhibition potential of mitragynine, a stimulant alkaloid. Food Chem Toxicol 2013; 68: 117-127
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• 42 Fantoukh OI, Dale OR, Parveen A, Hawwal MF, Ali Z, Manda VK, Khan SI, Chittiboyina AG, Viljoen A, Khan IA. Safety assessment of phytochemicals derived from the globalized south African rooibos tea (Aspalathus linearis) through interaction with CYP, PXR, and P-gp. J Agric Food Chem 2019; 67: 4967-4975
  CrossrefPubMedSearch in Google Scholar
• 43 Liu C, Lim Y, Hu M. Fucoxanthin attenuates rifampin induced cytochrome P450 3A4 (CYP3A4) and multiple drug resistance 1 (MDR1) gene expression through pregnane X receptor (PXR)-mediated pathways in human hepatoma HepG2 and colon adenocarcinoma LS174T cells. Mar Drugs 2012; 10: 242-257
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Correspondence

Publication History

Received: 23 April 2021

Accepted after revision: 18 July 2021

Article published online:
06 August 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 Musara C, Aladejana EB. Review of studies on Bulbine natalensis Baker (Asphodelaceae): ethnobotanical uses, biological and chemical properties. J Appl Pharm 2020; 10: 150-155
  PubMedSearch in Google Scholar
• 2 Yakubu MT, Afolayan AJ. Effect of aqueous extract of Bulbine natalensis (Baker) stem on the sexual behaviour of male rats. Int J Androl 2009; 32: 629-636
  CrossrefPubMedSearch in Google Scholar
• 3 Bae JY, Ali Z, Wang YH, Chittiboyina AG, Zaki AA, Viljoen AM, Khan IA. Anthraquinone-based specialized metabolites from rhizomes of Bulbine natalensis . J Nat Prod 2019; 82: 1893-1901
  CrossrefPubMedSearch in Google Scholar
• 4 European Commission. Novel Food Catalogue. Brussels, Belgium: European Commission; 2020
  Search in Google Scholar
• 5 (LNHPD) Licensed Natural Health Products Database. Natural and Non-prescription Health Products Directorate. Ottawa, ON, Canada: Health Canada; 2020
  Search in Google Scholar
• 6 Dietary Supplement Label Database (DSLD). Office of Dietary Supplements and the National Library of Medicine. Bethesda, MD: National Institutes of Health; 2020
  Search in Google Scholar
• 7 Cederbaum AI. Molecular mechanisms of the microsomal mixed-function oxidases and biological and pathological implications. Redox Biol 2015; 4: 60-73
  CrossrefPubMedSearch in Google Scholar
• 8 Thelen K, Dressman JB. Cytochrome P450-mediated metabolism in the human gut wall. J Pharm Pharmacol 2009; 61: 541-558
  CrossrefPubMedSearch in Google Scholar
• 9 Hankinson O. The aryl hydrocarbon receptor complex. Annu Rev Pharmacol Toxicol 1995; 35: 307-340
  CrossrefPubMedSearch in Google Scholar
• 10 Busbee PB, Rouse M, Nagarkatti M, Nagarkatti PS. Use of natural AhR ligands as potential therapeutic modalities against inflammatory disorders. Nutr Rev 2013; 71: 353-369
  CrossrefPubMedSearch in Google Scholar
• 11 Nebert DW. Aryl hydrocarbon receptor (AHR): “pioneer member” of the basic-helix/loop/helix per-Arnt-sim (bHLH/PAS) family of “sensors” of foreign and endogenous signals. Prog Lipid Res 2017; 67: 38-57
  CrossrefPubMedSearch in Google Scholar
• 12 Gurley BJ, Yates CR, Markowitz JS. “…Not intended to diagnose, treat, cure, or prevent any disease.” 25 years of botanical dietary supplement research and the lessons learned. Clin Pharmacol Ther 2018; 104: 470-483
  CrossrefPubMedSearch in Google Scholar
• 13 US-FDA guideline 2020. In vitro drug interaction studies – cytochrome P450 enzyme- and transporter-mediated drug interactions: Guidance for industry. Accessed July 28, 2021 at: https://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm
  PubMed
• 14 Husain I, Manda V, Alhusban M, Dale OR, Bae JY, Avula B, Gurley BJ, Chittiboyina AG, Khan IA, Khan SI. Modulation of CYP3A4 and CYP2C9 activity by Bulbine natalensis and its constituents: An assessment of HDI risk of B. natalensis-containing supplements. Phytomedicine 2021; 81: 153416
  CrossrefPubMedSearch in Google Scholar
• 15 Eichhorn T, Greten HJ, Efferth T. Self-medication with nutritional supplements and herbal over-the-counter products. Nat Prod Bioprospect 2011; 1: 62-70
  CrossrefPubMedSearch in Google Scholar
• 16 Koe XF, Muhammad TST, Chong AS, Wahab HA, Tan ML. Cytochrome P450 induction properties of food and herbal-derived compounds using a novel multiplex RT-qPCR in vitro assay, a drug-food interaction prediction tool. Food Sci Nutr 2014; 2: 500-520
  CrossrefPubMedSearch in Google Scholar
• 17 Gouws C, Hamman JH. What are the dangers of drug interactions with herbal medicines?. Expert Opin Drug Metab Toxicol 2020; 16: 165-167
  CrossrefPubMedSearch in Google Scholar
• 18 Xie W, Evans RM. Orphan nuclear receptors: the exotics of xenobiotics. J Biol Chem 2001; 276: 37739-37742
  CrossrefPubMedSearch in Google Scholar
• 19 Moore LB, Goodwin B, Jones SA, Wisely GB, Singh CJS, Willson TM, Collins JL, Kliewer SA. St. Johnʼs wort induces hepatic drug metabolism through activation of the pregnane X receptor. Proc Natl Acad Sci 2000; 97: 7500-7502
  CrossrefPubMedSearch in Google Scholar
• 20 Manda VK, Avula B, Olivia RD, Ali Z, Khan IA, Walker LA, Khan SI. PXR mediated induction of CYP3A4, CYP1A2, and P-gp by Mitragyna speciosa and its alkaloids. Phytother Res 2017; 31: 1935-1945
  CrossrefPubMedSearch in Google Scholar
• 21 Yakubu MT, Afolayan AJ. Anabolic and androgenic activities of Bulbine natalensis stem in male Wistar rats. Pharm Biol 2010; 48: 568-576
  CrossrefPubMedSearch in Google Scholar
• 22 Denison MS, Nagy SR. Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu Rev Pharmacol Toxicol 2003; 43: 309-334
  CrossrefPubMedSearch in Google Scholar
• 23 Zanger UM, Schwab M. Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol Ther 2013; 138: 103-141
  CrossrefPubMedSearch in Google Scholar
• 24 Kapelyukh Y, Henderson CJ, Scheer N, Rode A, Wolf RC. Defining the contribution of CYP1A1 and CYP1A2 to drug metabolism using humanized CYP1A1/1A2 and Cyp1a1/Cyp1a2 knockout mice. Drug Metab Dispos 2019; 47: 907-918
  CrossrefPubMedSearch in Google Scholar
• 25 Netsch MI, Gutmann H, Schmidlin CB, Aydogan C, Drewe J. Induction of CYP1A by green tea extract in human intestinal cell lines. Planta Med 2006; 72: 514-520
  Thieme ConnectPubMedSearch in Google Scholar
• 26 Fasinu PS, Bouic PJ, Rosenkranz B. The inhibitory activity of the extracts of popular medicinal herbs on CYP1A2, 2C9, 2C19, and 3A4 and the implications for herb-drug interaction. Afr J Tradit Complement Altern Med 2014; 11: 54-61
  CrossrefPubMedSearch in Google Scholar
• 27 Guengerich FP. Cytochrome P450 and chemical toxicology. Chem Res Toxicol 2008; 21: 70-83
  CrossrefPubMedSearch in Google Scholar
• 28 Kang P, Dalvie D, Smith E, Zhou S, Deese A, Nieman JA. Bioactivation of flutamide metabolites by human liver microsomes. Drug Metab Dispos 2008; 36: 1425-1437
  CrossrefPubMedSearch in Google Scholar
• 29 Hellum BH, Hu Z, Nilsen OG. The induction of CYP1A2, CYP2D6, and CYP3A4 by six trade herbal products in cultured primary human hepatocytes. Basic Clin Pharmacol Toxicol 2007; 100: 23-30
  CrossrefPubMedSearch in Google Scholar
• 30 Silva SM, Martinho A, Moreno I, Silvestre S, Granadeiro LB, Alves GA, Duarte AP, Domingues F, Gallardo E. Effects of Hypericum perforatum extract and its main bioactive compounds on the cytotoxicity and expression of CYP1A2 and CYP2D6 in hepatic cells. Life Sci 2016; 144: 30-46
  CrossrefPubMedSearch in Google Scholar
• 31 Manda VK, Ibrahim MA, Dale OR, Kumarihamy M, Cutler S, Khan IA, Walker LA, Muhammad I, Khan SI. Modulation of CYPs, P-gp, and PXR by Eschscholzia californica (California Poppy) and its alkaloids. Planta Med 2016; 82: 551-558
  Thieme ConnectPubMedSearch in Google Scholar
• 32 Lim EL, Seah TC, Koe XF, Wahab HA, Adenan MI, Jamil MFA, Majid MIA, Tan ML. In vitro evaluation of cytochrome P450 induction and the inhibition potential of mitragynine, a stimulant alkaloid. Food Chem Toxicol 2013; 68: 117-127
  PubMedSearch in Google Scholar
• 33 Zanger UM, Klein K. Pharmacogenetics of cytochrome P450 2B6 (CYP2B6): advances on polymorphisms, mechanisms, and clinical relevance. Front Genet 2013; 4: 24
  CrossrefPubMedSearch in Google Scholar
• 34 Roy P, Yu LJ, Crespi CL, Waxman DJ. Development of a substrate-activity-based approach to identify the major human liver P-450 catalysts of cyclophosphamide and ifosfamide activation based on cDNA expressed activities and liver microsomal P-450 profiles. Drug Metab Dispos 1999; 27: 655-666
  PubMedSearch in Google Scholar
• 35 Sridar C, Kent UM, Notley LM, Gillam EM, Hollenberg PF. Effect of tamoxifen on the enzymatic activity of human cytochrome CYP2B6. J Pharmacol Exp Ther 2002; 301: 945-952
  CrossrefPubMedSearch in Google Scholar
• 36 Wang D, Li L, Yang H, Ferguson SS, Baer MR, Gartenhaus RB, Wang H. The constitutive androstane receptor is a novel therapeutic target facilitating cyclophosphamide-based treatment of hematopoietic malignancies. Blood 2013; 121: 329-338
  CrossrefPubMedSearch in Google Scholar
• 37 Erickson DA, Mather G, Trager WF, Levy RH, Keirns JJ. Characterization of the in vitro biotransformation of the HIV-1 reverse transcriptase inhibitor nevirapine by human hepatic cytochromes P-450. Drug Metab Dispos 1999; 27: 1488-1495
  PubMedSearch in Google Scholar
• 38 Ward BA, Gorski JC, Jones DR, Hall SD, Flockhart DA, Desta Z. The cytochrome P450 2B6 (CYP2B6) is the main catalyst of efavirenz primary and secondary metabolism: implication for HIV/AIDS therapy and utility of efavirenz as a substrate marker of CYP2B6 catalytic activity. J Pharmacol Exp Ther 2003; 306: 287-300
  CrossrefPubMedSearch in Google Scholar
• 39 Yanagihara Y, Kariya S, Ohtani M, Uchino K, Aoyama T, Yamamura Y, Iga T. Involvement of CYP2B6 in N-Demethylation of ketamine in human liver microsomes. Drug Metab Dispos 2001; 29: 887-890
  PubMedSearch in Google Scholar
• 40 Tolson AH, Wang H. Regulation of drug-metabolizing enzymes by xenobiotic receptors: PXR and CAR. Adv Drug Deliv Rev 2010; 62: 1238-1249
  CrossrefPubMedSearch in Google Scholar
• 41 Fasinu PS, Manda VK, Dale OR, Egiebor NO, Walker LA, Khan SI. Modulation of cytochrome P450, P-glycoprotein and pregnane X receptor by selected antimalarial herbs-implication for herb-drug interaction. Molecules 2017; 22: 2049
  CrossrefPubMedSearch in Google Scholar
• 42 Fantoukh OI, Dale OR, Parveen A, Hawwal MF, Ali Z, Manda VK, Khan SI, Chittiboyina AG, Viljoen A, Khan IA. Safety assessment of phytochemicals derived from the globalized south African rooibos tea (Aspalathus linearis) through interaction with CYP, PXR, and P-gp. J Agric Food Chem 2019; 67: 4967-4975
  CrossrefPubMedSearch in Google Scholar
• 43 Liu C, Lim Y, Hu M. Fucoxanthin attenuates rifampin induced cytochrome P450 3A4 (CYP3A4) and multiple drug resistance 1 (MDR1) gene expression through pregnane X receptor (PXR)-mediated pathways in human hepatoma HepG2 and colon adenocarcinoma LS174T cells. Mar Drugs 2012; 10: 242-257
  CrossrefPubMedSearch in Google Scholar
• 44 Zheng YF, Bae SH, Choi EJ, Park JB, Kim SO, Jang MJ, Park GH, Shin WG, Oh E. Bae SK. Evaluation of the in vitro/in vivo drug interaction potential of BST204, a purified dry extract of ginseng, and its 4 bioactive ginsenosides through cytochrome P450 inhibition/induction and UDP-glucuronosyltransferase inhibition. Food Chem Toxicol 2014; 68: 117-127
  CrossrefPubMedSearch in Google Scholar
• 45 Husain I, Sharma A, Kumar S, Malik F. Purification and characterization of glutaminase free asparaginase from Pseudomonas otitidis: Induce apoptosis in human leukemia MOLT-4 cells. Biochimie 2016; 121: 38-51
  CrossrefPubMedSearch in Google Scholar