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. Author manuscript; available in PMC: 2026 Mar 25.
Published in final edited form as: J Diet Suppl. 2026 Feb 5;23(2):217–239. doi: 10.1080/19390211.2026.2615924

Evaluation of in vitro cytotoxicity and induction potential of açaí (Euterpe oleracea) extracts in human hepatocytes

Zarna Raichura 1, Kelli L McDonald 1, Savanna DePriest 1, Madison Dupre 1, Meredith Almy 1, Kristen Hansen 1, Julia M Salamat 2, Satyanarayana R Pondugula 2, Angela I Calderón 1
PMCID: PMC13008539  NIHMSID: NIHMS2149547  PMID: 41641519

Abstract

Euterpe oleracea Mart. (açaí) is a widely consumed botanical supplement marketed for antioxidant, anti-inflammatory, and antiproliferative properties. Despite its popularity, limited data exist on its ability to modulate drug-metabolizing enzymes and transporters—critical determinants of pharmacokinetic botanical-drug interactions. This study evaluated the cytotoxicity and induction potential of açaí extracts on cytochrome P450 (CYP450) enzymes and drug transporters using physiologically relevant in vitro models. Test samples included aqueous, acidic methanol, methanol, and ethanol extracts of açaí berry powder (Mountain Rose) and commercial capsules (Nature’s Way, Natrol), selected to reflect consumer products and enhance translational relevance. Cytotoxicity was assessed using CellTiter-Glo® luminescent assay in sandwich-cultured human hepatocytes. Induction of CYP1A2, CYP2B6, CYP3A4, P-glycoprotein (P-gp), and organic anion transporting polypeptides (OATP1B1/B3) was evaluated at the mRNA level via RT-qPCR in the same hepatocyte model. Functional transporter activity was assessed using intracellular probe accumulation assays in LS174T human colon carcinoma cells, a complementary model for preliminary screening. A time- and dose-dependent reduction in hepatocyte viability occurred with specific extracts, notably MRAC (acidic methanol), MRME (methanol), MRET (ethanol), and F4AC (acidic methanol, Natrol). However, none significantly induced CYP450 or transporter expression. Preliminary functional assays also showed minimal impact on P-gp and OATP activity. While preliminary, this study provides a comprehensive evaluation of açaí-mediated modulation of key pharmacokinetic pathways and underscores the need for rigorous assessment of botanical extracts to better predict potential botanical-drug interactions.

Keywords: Açaí, Euterpe, cytotoxicity, cytochrome P450, transporters, P-gp, OATP, induction, plant extracts

Introduction

Botanical dietary supplements (BDS), as defined by the United States (U.S.) Department of Health and Human Services, are ingestible products made from whole plants, plant parts, or plant extracts (Botanical Dietary Supplement, n.d.). Their use has surged in recent decades, with the global market projected to reach $300 billion by 2028 (Djaoudene et al., 2023). Factors such as rising healthcare costs, fear of side effects from synthetic drugs, and concerns about privacy and misdiagnosis have fueled a growing trend of self-medication with botanical products (Ekor, 2014). According to the National Center for Complementary and Integrative Health, nearly 40% of U.S. adults and 12% of children use botanical supplements to manage chronic conditions and promote overall wellness (Complementary And Alternative Medicine Market Report, 2030). Despite their growing popularity, BDS presents significant challenges regarding safety, regulation, and quality control. Many botanical products are introduced to the market without mandatory toxicological evaluations (Djaoudene et al., 2023), and some may appear safe in the short term but cause adverse effects with prolonged use. Establishment of pharmacokinetic profiles for botanicals is essential to better predict and manage potential interactions with clinical drugs.

When a drug enters the body, it undergoes four key pharmacokinetic stages—absorption, distribution, metabolism, and excretion—that collectively determine its bioavailability, therapeutic efficacy, and potential toxicity. Throughout these stages, membrane transporters, which are specialized proteins embedded in cellular membranes, play an essential role in mediating the movement of drugs and other xenobiotics across biological barriers. Among these, P-glycoprotein (P-gp) and organic anion transporting polypeptides (OATPs) are particularly important for regulating drug absorption, distribution, and hepatic disposition. P-gp, an ATP-dependent efflux transporter, plays a key role in limiting drug absorption and promoting excretion (Giacomini et al., 2010; Bogaard et al., 2024). In the liver, P-gp is localized on the canalicular membrane facing the bile duct and contributes approximately 5–10% of hepatic drug efflux, facilitating biliary elimination of its substrates. In contrast, OATPs, especially OATP1B1 and OATP1B3, are key hepatic influx transporters, account for an estimated 30-50% hepatic drug uptake, significantly influencing metabolic clearance of many clinically used drugs (König et al., 2013; Shitara et al., 2013; Hagenbuch et al., 2025). Both P-gp and OATPs are critical for the disposition of various chemotherapy agents, including methotrexate, paclitaxel, and tyrosine kinase inhibitors (e.g., imatinib, sorafenib) (Sprowl and Sparreboom, 2014; Thakkar et al., 2015; Gurley, 2020). In parallel, cytochrome P450 enzymes (CYP450s) are a superfamily of heme-containing monooxygenases, primarily located in the endoplasmic reticulum of hepatocytes, where they catalyze Phase I oxidative metabolism. CYP450s are responsible for approximately 75–80% of oxidative drug metabolism, with the CYP1, CYP2, and CYP3 families playing dominant roles in xenobiotic biotransformation (Zhao et al., 2021). Many anticancer drugs—including paclitaxel, docetaxel, irinotecan, and cyclophosphamide—are metabolized through these pathways. Modulation of either drug transporters or CYP450 enzymes—whether by induction, inhibition, or competitive substrate interactions—can lead to altered systemic exposure and unexpected drug–drug or botanical–drug interactions (Giacomini et al., 2010; DuBuske, 2012; Tweedie et al., 2013; Gougis et al., 2022; Lee et al., 2024). These mechanisms are critical considerations in pharmacokinetic safety assessments. This study focuses on evaluating the induction potential of drug transporters and CYP450 enzymes by the botanical of interest, Euterpe oleracea Mart. (açaí), a widely consumed natural product with increasing relevance in integrative and complementary medicine.

Açaí is a dark-purple berry, native to the Amazon rainforest, commonly consumed as juice or powder and known for its high antioxidant content, primarily attributed to its rich composition of polyphenols and flavonoids. It is among the top 40 selling BDS in the U.S. (Smith et al., 2021) and has gained attention for its anti-inflammatory, antioxidant, antiproliferative, and proapoptotic effects in preclinical cancer models (Silva et al., 2014; Alessandra-Perini et al., 2018). These pharmacological effects have led to claims about its potential to complement conventional chemotherapies (Jensen et al., 2008; Mertens-Talcott et al., 2008; Sadowska-Krępa et al., 2015). Despite these promising therapeutic benefits, açaí also presents notable toxicity concerns. Analysis of the 2004-2015 FDA Adverse Event Reporting System (FAERS) revealed that combining açaí with anticancer drugs metabolized by non-CYP3A4 significantly increased the odds of vascular adverse events (Fahim et al., 2019). Furthermore, data from the FDA Center for Food Safety and Applied Nutrition’s Adverse Event Reporting System (CAERS, 2007-2024) database indicated that approximately 17.5% reported adverse events linked to açaí involved cardiac-related complications. In addition, an in vivo study performed in rats by Nanjappan et al. (2022) reported that co-administration of açaí berry with atorvastatin—a substrate of both CYP3A4 and OATP1B1—resulted in a notable decrease in Cmax and AUC, suggesting possible modulation of CYP450 enzyme activity or transporter function. These findings underscore the critical need for pharmacokinetic investigations into açaí’s interaction potential, particularly its ability to induce CYP450 enzymes or hepatic transporters. As açaí is marketed and consumed as a dietary supplement, its widespread use carried significant public health implications especially for populations engaging in polypharmacy or chronic supplement use. Assessing such risks is vital for understanding and mitigating potential botanical-drug interactions in real-world settings.

In the current study, we evaluated the effects of seven distinct standardized açaí extracts—including aqueous, methanol, ethanol, and acidic methanol extracts from mountain rose açaí berry powder; an acidic methanol extract of the Nature’s Way capsule formulation; and both acidic methanol and methanol extracts of the Natrol capsule formulation—using primary human hepatocytes. We assessed their cytotoxicity as well as their potential to induce the expression and activity of major CYP450 enzymes (CYP3A4, CYP2B6, and CYP1A2) and drug transporters (P-gp and OATPs). These particular enzymes and transporters were selected in alignment with the U.S. FDA guidance for evaluating induction potential of investigational drugs (US Food and Drug Administration, 2020). Prior research on açaí-drug interactions has utilized various in vitro approaches. For instance, Zhang et al. (2019) reported that methanol-extracted açaí significantly induced CYP3A4 expression in monolayer primary human hepatocytes. Additional studies have shown that individual flavonoids and polyphenols such as quercetin, kaempferol, catechin, epicatechin, anthocyanins, and proanthocyanidins (commonly found in botanicals like açaí) can inhibit P-gp and OATP transporters in diverse cell-based models (Zhou et al., 2004; Kitagawa, 2006; Vrzal, 2016; Bajraktari-Sylejmani and Weiss, 2020; Mohos et al., 2020; Gonçalves et al., 2022). To enhance physiological relevance beyond previous models, we employed sandwich-cultured primary human hepatocytes, which more accurately recapitulate the structural and functional properties of the human liver. The utility of this model has been demonstrated in several studies (Kern et al., 1997; Yang et al., 2016; Hlengwa et al., 2023; Hagenbuch et al., 2025; McDonald et al., 2025) , noting its ability to retain transporter and enzyme expression and function over extended culture periods.

This study represents the first report of preliminary investigations utilizing sandwich-cultured primary human hepatocytes to assess the induction potential of standardized açaí extracts on CYP450 enzymes and transporters-specifically CYP2B6 and CYP1A2, which have not been previously examined in this context. While earlier transporter studies have largely focused on the inhibitory effects of individual phytochemicals, our approach uniquely investigates induction effects of whole extracts, reflecting commonly consumed consumer products (as supported by data from 2025 STRIPED database and Amazon market analysis) thus enhancing translational value. To complement transporter mRNA assessments, intracellular probe accumulation assays were employed to evaluate transporter functional activity. This study allowed for an integrated and multi-endpoint evaluation encompassing CYP450 and transporter mRNA expression analysis, transporter activity, and cytotoxicity within a physiologically relevant in vitro model. The experimental framework aligns with FDA-recommended best practice for in vitro pharmacokinetic drug-drug interactions, including appropriate concentration selection, use of positive controls, and isoform-specific evaluations. Altogether, this work reflects a methodologically broad and rigorous evaluation, contributing to a more accurate understanding of the pharmacokinetic interactions associated with widely consumed botanical supplements like açaí.

Materials and Methods

1. Chemicals and reagents

Rifampicin, phenobarbital, carbamazepine, rhodamine (R123), sulforhodamine 101 (SR101) were purchased from Sigma-Aldrich (St. Louis, MO). Omeprazole from Tocris Bio-Techne Corporation. Pravastatin (sodium) was purchased from ApexBio (Houston, TX). Vincristine (sulfate), valspodra (PSC-833) was purchased from Cayman Chemical Company (Ann Arbor, MI). Cryopreserved human hepatocytes of a 58-year-old Caucasian male (lot# RAS), which were sourced from BioIVT (Westbury, NY). Additionally, through BioIVT we obtained cell media, Torpedo antibiotics (Minneapolis, MN). BioCoat® Collagen I 48-well Clear Flat Bottom TC-treated Multiwell Plates from Corning (Corning, NY). Supplies including Trypan Blue reagent, white 96-well plates, 96-well PCR plates, and microseals were all acquired from Bio-Rad (Hercules, CA). Furthermore, we acquired RNA-to-Ct kit, Geltrex™, UltraPure™ TAE Buffer, UltraPure™ agarose, EtBr, custom 18 base pair oligos (PCR primer verification), and Triton X-100 from Thermo Fisher Scientific (Waltham, MA). The 100bp DNA ladder, Homogenizer mini columns, E.Z.N.A.® Total RNA Kit I, E.Z.N.A.® RNase-Free DNase I Set, and E.Z.N.A. ® Gel Extraction Kit was secured from Omega Bio-Tek (Norcross, GA). PerfeCTa® SYBR® Green FastMix® Reaction Mixes, Quantabio was obtained from VWR (Allentown, PA). Promega (Madison, WI) supplied CellTiter-Glo® and 6x blue/orange loading dye. PTFE syringe filters (13 mm, 0.22 μm pore size) were acquired from Cell Treat (Pepperell, MA). Luer-Lok sterile syringes (3 mL) were purchased from BD, Inc. (Franklin Lakes, NJ). Solvents like methanol, acetonitrile, water, and formic acid (optima LC/MS grade) were purchased from Fisher Scientific (Waltham, MA). PBS and DMEM were obtained from HyClone, Cytiva Lifesciences (Wilmington, DE).

2. Source of açaí materials

Many studies have highlighted significant differences in the chemical composition and antioxidant properties of commercially available açaí supplements. For instance, Earling et al. (2019) found that freeze-dried powders and certain capsules contained higher levels of anthocyanins and flavonoids, whereas liquid formulations and frozen pulp contained substantially lower concentrations. Notably, lacked detectable levels of key açaí constituents, suggesting potential adulteration or substandard quality. Variations in anthocyanin content likely stem from differences in agricultural practices and post-harvest processing. While a few supplements demonstrated high phytochemical quality, the majority—particularly specific liquids and capsules—were either deficient in key bioactive compounds or included undeclared ingredients, underscoring the need for comprehensive quality testing. According to data from the 2025 STRIPED Database, capsule formulations are the most widely available form of açaí supplements, followed by powders. These trends were further validated through an independent Amazon market search, which confirmed capsules and powders as the most frequently promoted formulations. For current analysis, a certified organic açaí berry powder from Mountain Rose Herbs (MR) (Eugene, OR, USA; Catalog no. AÇAÍ4, Lot #26579) was selected. The identity of this sample was verified via high-performance thin-layer chromatography (HPTLC) conducted by Alkemist Labs (Costa Mesa, CA, USA), using authenticated reference materials for comparison. In addition, two commercially available capsule-based dietary supplements containing aqueous açaí berry extract (Euterpe oleracea Mart.) were included: one from Nature’s Way (Green Bay, WI, USA; 2022, Lot #20137327) and the other from Natrol (Chatsworth, CA, USA; 2022, Lot #2086344), hereafter referred to as F3 and F4, respectively. They were chosen based on their sustained market presence. F3 contains 1040 mg of açaí extract standardized to 20.8 mg of polyphenols, while F4 provides 1000 mg of extract per serving.

3. Preparation and quantification of açaí extracts

The açaí extracts from the above-mentioned materials were prepared using four solvents: water (AQ), methanol (ME), acidic methanol (AC; MeOH: H2O 70:30 with 0.1% HCl), and 95% ethanol (ET). These extraction types were selected to reflect traditional botanical preparations- aqueous extracts resembling decoctions or cold percolations, and alcohol-based extracts mimicking tinctures used in herbal medicines (Handa et al., 2008). Methanol and ethanol were chosen for their efficiency in extracting polyphenols and anthocyanins, key constituents in açaí, despite the inherently variable distribution of phytochemicals (Poulose et al., 2012; Zhang et al., 2019). Aqueous, hydroethanolic and ethanol extract types align with those used in previous in vivo and clinical studies assessing the therapeutic potential of açaí (Laurindo et al., 2023), supporting the physiological relevance of our findings. Solvent selection was further guided by the goal of capturing both major and minor constituents to generate chemically representative and clinically meaningful data. Acidic methanol, in particular, enhances the solubility and stability of anthocyanins and other phenolics (Friedman and Jürgens, 2000; Barnes et al., 2009; Rumalla et al., 2012; Dembczyński et al., 2015; Jakobek et al., 2015; Lu et al., 2025). A mildly acidic environment helps minimize degradation and unwanted side reactions during extraction and storage. The low acid concentration offers an effective balance-maximizing extraction efficiency while avoiding the compound degradation or equipment damage that can occur with stronger acids. Extractions were conducted at 25 °C for 24 hours with gentle shaking (75 rpm), using a powder-to-solvent ratio of 40 g/L. Organic extractions were performed twice, while aqueous extraction was performed once to minimize microbial contamination. All extracts were filtered (Whatman No. 1 or 0.45 μm PTFE syringe filters), dried using rotary evaporation and nitrogen (where applicable), lyophilized, and stored at −20 °C until analysis. Extraction yields from the açaí berry powder (MR) raw material were 12.0% (AQ), 17.0% (AC), 17.5% (ME), and 18.8% (ET). While capsule formulations yielded 18.51% (F3AC), 43.2% (F4AC), 11.2% (F4ME).

Liquid chromatography-mass spectrometry (LC-MS) method was employed for both qualitative profiling and quantification of known anthocyanins, based on previously described conditions (Heck et al., 2023). As suggested in literature, chemical standardization and quantifications of botanical extracts to known marker compounds is essential for ensuring consistency, safety, and efficacy (Van Breemen et al., 2007; Garg et al., 2012). Quantitative profiling also accounts for batch variability due to environmental influences or processing factors on phytochemical composition. Therefore, quantitative analysis of four major anthocyanins—cyanidin 3-glucoside (C3G), cyanidin 3-sambubioside, cyanidin 3-rutinoside (C3R), and peonidin 3-rutinoside—was performed, as they are predominantly found in açaí. All açaí extracts were analysed at of extract 1mg/mL. The analysis was conducted using an Agilent 6520 Q-TOF mass spectrometer coupled with a 1220 rapid resolution liquid chromatography system. Chromatographic separation was conducted on a Poroshell 120 SB-C18 column (2.1 × 100 mm, 2.7 μm) Poroshell 120 SB-C18 column. The mobile phases consisted of (A) water containing 0.1% formic acid and (B) methanol: acetonitrile (50:50, v/v) containing 0.1% formic acid. The gradient profile was as follows: 0–1 min, 5% B; 1–5 min, linear increase to 99% B; 5–6 min, return to 5% B; 6–10 min, hold at 5% B. The flow rate was set to 0.35 mL/min, with an injection volume of 10 μL, and the column temperature was maintained at 25 °C. MS was operated in positive electrospray ionization mode with a capillary voltage of 3400 V, drying gas at 350 °C and 10 L/min, nebulizer pressure at 25 psig, fragmentor voltage 175 V, and skimmer 65 V. All standards and standard solutions were injected in triplicate, with reserpine (50 ng/mL) used as an internal standard. Extracted ion chromatograms and quantitative data for four anthocyanins are presented in supplementary (Figure S1 and Table S1). Additionally, all extracts were characterized for phytochemical profiling using an untargeted LC-MS/MS metabolomics approach, as described by Heck et al. (2025), LC-MS/MS conditions for this analysis are detailed in the referenced publication. Both qualitative and quantitative data processing and analysis were performed using Agilent MassHunter software (Qualitative B.10.00, Quantitative B.08.00).

4. Determination of human-relevant concentration

The test concentrations of açaí extracts for CYP450 and transporter induction studies were determined based on the standardized concentration of C3G; a key anthocyanin selected due to available pharmacokinetic data. C3G serves as a reliable marker because of its well-characterized absorption profile and documented plasma Cmax and AUC values from both botanical extracts and isolated forms, as reported in in vivo performed in rats (Tsuda et al., 1999; Fornasaro et al., 2016) and clinical studies in humans (Mertens-Talcott et al., 2008; Czank et al., 2013). Reported human plasma Cmax value for C3G after açaí pulp or purée consumption range from 1.138 to 2.321 ng/mL, with the upper limit (2.321 ng/mL) chosen to represent a physiologically relevant high exposure scenario. This approach ensures that dosing reflects realistic intake from whole extract rather than isolated compounds and is consistent with C3G levels reported for other anthocyanin-rich sources like black currant (Matsumoto et al., 2001).

5. CYP450 and transporter induction evaluation using sandwich-cultured hepatocyte assay

To evaluate CYP450 and transporter induction by açaí extracts, previously optimized assay conditions using primary human hepatocytes were applied (McDonald et al., 2025). Two cell culture media were considered: BioIVT INVITROGRO™ CP medium containing bovine serum albumin (CP) and the serum-free INVITROGRO™ hepatocyte induction medium (HI). Due to minor precipitation observed in the serum-containing CP medium (as noted in the prior assay), the serum-free HI medium was used throughout this study to ensure consistency and maintain cell health. Cryopreserved human hepatocytes were thawed and plated according to BioIVT’s guidelines. Initially, hepatocytes were suspended in 5 mL of complete HI medium (INVITROGRO™ HI medium and TORPEDO™ Antibiotic Mix), and cell viability was assessed using trypan blue exclusion with a T-20 cell counter. The cell density was adjusted to 0.7 × 106 viable cells/mL, yielding a total volume suitable for plating in a 48-well Collagen I-coated plate (200 μL/well). After an incubation time of 4 hours at 37 °C, 5 % CO2, the medium was aspirated, and cells were overlaid with HI medium supplemented with 0.25 mg/mL Geltrex™ (200 μL/well) to establish the hepatocyte sandwich culture configuration. After the overlay period, the medium was replaced with fresh medium containing treatment samples for an exposure period of 24 hours. Treatment groups included a vehicle control (85:15 v/v water: acetonitrile), along with FDA-recommended positive controls (US Food and Drug Administration, 2020) for CYP450 and transporter induction—50 μM omeprazole (CYP1A2), 2 mM phenobarbital (CYP2B6), and 20 μM rifampicin (CYP3A4, P-gp). Seven standardized açaí extracts were tested: MRAQ (mountain rose aqueous extract), MRAC (mountain rose acidic methanol extract), MRME (mountain rose methanol extract), MRET (mountain rose ethanol extract), F3AC (Nature’s way acidic methanol extract), F4AC (Natrol acidic methanol extract), F4ME (Natrol methanol extract). Each extract was evaluated at two concentrations: a human-relevant concentration (2.321 ng/mL C3G equivalent), based on published pharmacokinetic data, and a higher concentration (100 ng/mL of C3G equivalent). The higher concentration reflects levels consistent with consumer use, as reported in the NIH/ODS dietary supplement label database (Dietary Supplement Label Database [DSLD], NIH Office of Dietary Supplements; https://dsld.od.nih.gov; NIH, 2023) for commonly available açaí products. Details about extract concentrations are provided in Table 1, and further elaboration on consumer intake levels is explained in the Discussion section. It is important to note that no well-established inducers are currently recognized for OATP1B1 and OATP1B3, due to the absence of validated models and reproducible data (Rodrigues et al., 2020; US Food and Drug Administration, 2020; FDA/CDER/CBER, 2024) . Therefore, changes in OATP transporter expression were assessed relative to vehicle control.

Table 1.

Extract test concentration for CYP450 & transporter induction as well as for cytotoxicity assessment.

Extract Name Extract Abbreviation ng of C3G/ mg of extract 2.321 ng/mL C3G 100 ng/mL C3G
Extract concentration (mg/mL) corresponding to C3G concentration
Mountain Rose Aqueous MRAQ 3335.559 0.0007 0.0299
Mountain Rose Acidic Methanol MRAC 8993.200 0.0003 0.0111
Mountain Rose Ethanol MRET 340.072 0.0068 0.2940
Mountain Rose Methanol MRME 1167.592 0.002 0.0856
Nature’s Way (2022) Formulation Acidic Methanol F3AC 161.286 0.0144 0.6200
Natrol’s (2022) Formulation Acidic Methanol F4AC 5983.056 0.0004 0.0167
Natrol’s (2022) Formulation Methanol F4ME 13723.480 0.0002 0.0073
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Cyanidin-3-glucoside (C3G) was used as a reference marker. Preliminary extract screening was conducted at the reported human Cmax (2.321 ng/mL) and at a higher concentration (100 ng/mL C3G equivalent).

To ensure proper solubilization, all treatment samples (including açaí extracts and controls) were first dissolved in a water: acetonitrile mixture (85:15, v/v), then sonicated for 15 minutes, incubated in a 37 °C water bath for 15 minutes, vortexed, and filtered using 0.22 μm PTFE syringe filters. A 1000x stock solution was prepared for each treatment, which was diluted 1:1000 into HI medium to maintain a final organic solvent concentration of <1%. This dilution adhered to established solvent tolerance limits for hepatocyte cultures (<0.1% for DMSO, <1% for acetonitrile/methanol), minimizing any potential impact on cell viability and metabolic phenotype (Easterbrook et al., 2001; González-Pérez et al., 2012; Verheijen et al., 2019; Wright et al., 2020).

6. RNA isolation and quantitative polymerase chain reaction (qPCR) analysis

Following treatment, the hepatocytes were lysed, and total RNA was extracted using the E.Z.N.A.® Total RNA Kit I in combination with the RNase-Free DNase I Set, incorporating Homogenizer mini columns to ensure thorough sample processing. RNA concentration and purity were assessed using a NanoVuePlus spectrophotometer. Subsequent reverse transcription and quantitative PCR (qPCR) were performed on a qTOWER3 thermal cycler using the RNA-to-Ct™ kit together with PerfeCTa® SYBR® Green FastMix® Reaction Mixes. Target transcripts included housekeeping gene GAPDH (glyceraldehyde3-phosphate dehydrogenase), CYP450 genes (CYP1A2, CYP2B6, and CYP3A4), and transporter genes (P-gp, OATP1B1, and OATP1B3), amplified using gene-specific primers (Table 2). As outlined in our previous publication, 18S RNA showed substantial variability, therefore, in this study, gene expression was normalized using in-house–designed GAPDH primers (McDonald et al., 2025). Some primers were adapted from published literature (as referenced in Table 2), while others were designed in-house, using gene sequence data obtained from the National Center of Biotechnology Information database. Specificity of the PCR products from in-house primers was confirmed via 2% agarose gel electrophoresis stained with ethidium bromide. The primer sets which produced more than one PCR product were eliminated and not used further in the gene expression analysis, the details are in the supplementary (Figure S2A and S2B). To verify RNA sample integrity and the absence of genomic DNA contamination, control reactions lacking reverse transcriptase were included. The comparative Ct (ΔΔCt) method was employed for calculating relative gene expression levels (Abbott et al., 2019, 2022; McDonald et al., 2025). In alignment with FDA guidelines, a greater than 2-fold increase in mRNA expression to that of vehicle and at least 20% of the positive control induction, were considered indicative of significant gene induction (US Food and Drug Administration, 2020).

Table 2.

Forward (FWD) and Reverse (REV) primers used for quantitative RT-PCR for housekeeping, CYP450, and transporter genes.

Gene Name Primer Name Primer sequence Accession No. PCR product size Reference
GAPDH GAPDH-FWD-1AC
GAPDH-REV-1AC		 5’-ACCACAGTCCATGCCATCAC-3’
5’-GCTTCACCACCTTCTTGATG-3’		 NM_002046 265 (Abbott et al., 2019, 2022)
CYP1A2 CYP1A2-FWD-2AC
CYP1A2-REV-2AC		 5’-CCCAAAGGCCTGAAAAGTCC-3’
5’-GTCTGTGCTGAAGGTCAAGC-3’		 ENST00000343932.5 279 In-house
CYP2B6 CYP2B6-FWD-2AC
CYP2B6-REV-2AC		 5’-GTCCTCCTCTTCCTTGCACT-3’
5’-CACACCATATCCCCGGAAGA-3’		 ENST00000324071.10 327 In-house
CYP3A4 CYP3A4-FWD-1AC
CYP3A4-REV-1AC		 5’-TTGGAAGTGGACCCAGAAAC-3’
5’-CTGGTGTTCTCAGGCACAGA-3’		 NM_017460 265 (Abbott et al., 2019, 2022)
P-gp P-gp-FWD-1AC
P-gp-REV-1AC		 5’-GATCTTGAAGGGGACCGCAA-3’
5’-TCATGAAGAACCCTGTAT-3’		 NM_000927 322 (Abbott et al., 2019)
OATP1B1 OATP1B1-FWD-1AC
OATP1B1-REV-1AC
OATP1B1-FWD-3AC
OATP1B1-REV-3AC		 5’-GCTTCGTGGAATAGGGGAGA-3’
5’-CGGAGTTTGGGGCAAGAAAA-3’
5’-TGAATGCCCAAGAGATGATGC-3’
5’-GACAAGCCCAAGTAGACCCT-3’		 ENST00000256958.3 313

321		 In-house
OATP1B3 OATP1B3-FWD-2AC
OATP1B3-REV-2AC		 5’-GTGGCTTGGTTTCCTTGTGT-3’
5’-TGACCGTACTGTTGCTCCAT-3’		 ENST00000381545.8 333 In-house
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7. Determination of cell viability

Cell viability was measured by analyzing ATP levels in sandwich-cultured hepatocytes. Hepatocytes were exposed to vehicle control, positive controls, and açaí extract samples at previously described concentrations. ATP content was quantified using the CellTiter-Glo Luminescent Assay, following the manufacturer’s protocol (Promega; Madison, WI). Luminescence readings were obtained using an Infinite Microplate Reader (TECAN, Männedorf, Switzerland). Results were normalized to the vehicle control. Cytotoxicity was evaluated at 24-, 48-, and 72-hours following treatment with açaí extracts standardized to 2.321 ng/mL C3G (human-relevant concentration). For the higher test concentration (100 ng/mL C3G equivalent), cell viability was assessed after a 24-hour exposure period.

8. Transporter probe accumulation assay

To validate the observed changes in transporter gene expression, functional activity assays were conducted by quantifying the intracellular accumulation of fluorescent probe substrates in cells expressing the respective transporters (Pondugula, Ferniany, et al., 2015; Pondugula, Flannery, et al., 2015; Abbott et al., 2019). Specifically, rhodamine (R123) and sulforhodamine (SR101) were used to assess the efflux activity of P-gp and the influx activity of OATP transporters (OATP1B1 and OATP1B3), respectively.

Due to limitations, one restriction in funding and second, high cost of the primary hepatocyte-based assays, we utilized the LS174T human colon carcinoma cell line (obtained from ATCC) as an alternative model. This cell line has been well characterized as a robust model for pregnane X receptor (PXR) mediated induction of P-gp (Kota et al., 2010; Pondugula, Flannery, et al., 2015). Given the absence of FDA-recommended (US Food and Drug Administration, 2020; FDA/CDER/CBER, 2024) in vitro models for assessing OATP induction, LS174T cells were employed as a preliminary screening platform in this exploratory study. LS174T cells are not conventionally used for evaluating OATP transporter function due to limited endogenous activity, however, previous studies have reported detectable transcript levels and functional activity of OATPs in carcinoma-derived cell lines, such as those originating from colon tissue (Pressler et al., 2011; Obaidat et al., 2012; Thakkar et al., 2013; Sun et al., 2014; Alam et al., 2018). In this context, the use of LS174T cells was intended to provide supportive functional insights rather than serve as a quantitative surrogate for hepatic OATP1B1/1B3 activity. Furthermore, using the same in vitro model for both P-gp and OATP assessments allowed for consistency in cellular background and assay conditions, minimizing inter-system variability in this proof-of-concept evaluation. Accordingly, this approach was utilized as a complementary system to hepatocyte-based assays to investigate potential transporter modulation by açaí extracts.

The cells were cultured in DMEM with phenol red supplemented with 10% FBS and incubated at 37°C with 5% CO2. For the transporter probe accumulation assay, cells were treated with vehicle (85:15 v/v, water: acetonitrile) and 20 μM rifampicin as a positive control for P-gp induction in Phenol red-free DMEM supplemented with 5% charcoal-dextran treated FBS and incubated at 37°C with 5% CO2. Açaí extracts standardized to 100 ng/mL C3G were also included as test treatments. All treatments were exposed for a 48-hour time period. After treatment, cells were washed with PBS and incubated for 15 minutes at 37°C in phenol red-free DMEM, either in presence or absence of transporter- specific inhibitors valspodar (PSC-833, 10 μM) for P-gp, pravastatin (20 μM) for OATP1B1, and vincristine (20 μM) for OATP1B3. These inhibitors have been validated in previous studies (Karlgren et al., 2012) and Pondugula, Flannery, et al, 2015). Subsequently, probe substrates R123 (5 μM) and SR101 (10 μM) were added to the cells, and incubation continued for 45 minutes under the same conditions. Afterward, the cells were washed with ice-cold PBS and lysed using 0.1 % triton-PBS. Fluorescence intensities were measured using Infinite microplate reader (TECAN), R123 was detected at excitation/emission wavelengths of 485/538 nm, and SR101 at 586/605 nm. The functional activity of each transporter was evaluated by comparing intracellular fluorescence levels in the absence and presence of specific inhibitors.

9. Statistical analysis

Identical treatments were applied to triplicate wells of primary human hepatocytes, with qPCR conducted in triplicate and repeated three times for robustness and reliability. Statistical significance of mRNA fold induction was assessed using ANOVA and Dunnett’s test, with p < 0.05 deemed significant. Data analysis was performed in Excel 365, and graphs were created using GraphPad Prism version 9.00 (GraphPad Software, San Diego, California, USA, www.graphpad.com).

Results

1. Assessment of hepatocyte viability following exposure to açaí extracts

Cell viability of human hepatocytes was assessed using the CellTiter-Glo Luminescent Assay, which quantifies ATP as an indicator of metabolically active, viable cells via firefly luciferase activity. Results were expressed as Relative Luminescence Units (RLU), normalized to vehicle control. A time-dependent decline in cell viability was observed at the human-relevant concentration (2.321 ng/mL C3G equivalent) for several açaí extract treatments. Notably, MRAC (acidic methanol extract of mountain rose powder), MRME (methanol extract of mountain rose powder), and MRET (ethanol extract of mountain rose powder) showed a progressive reduction in viability over 24, 48, and 72 hours, suggesting potential cytotoxicity upon prolonged exposure [Figure 1(A)]. Furthermore, a decrease in hepatocyte viability was evident with the F4AC (acidic methanol extract of Natrol capsule) extract, with the higher concentration (100 ng/mL C3G equivalent), causing a pronounced decrease in luminescence compared to vehicle control [Figure 1(B)]. Together, these preliminary findings highlight both exposure period and concentration influence hepatocyte viability, emphasizing the need to define safe exposure thresholds for these extracts in potential therapeutic use.

Figure 1.

Figure 1.

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(A) Cell viability after 24-hour (dark blue), 48-hour (purple), and 72-hour (pink) treatment period with açaí extracts at a human-relevant concentration (2.321 ng/mL C3G equivalent) (B) Cell viability following 24-hour treatment at a higher concentration (100 ng/mL C3G equivalent). Treatment groups include positive controls: omep = omeprazole, pheno = phenobarbital, rif = rifampicin; test samples: MRAQ = mountain rose aqueous extract, MRAC = mountain rose acidic methanol extract, MRET = mountain rose ethanol extract, MRME = mountain rose methanol extract, F3AC = nature’s way acidic methanol extract, F4AC = natrol acidic methanol extract, F4ME = natrol methanol extract.

2. Impact of extracts on mRNA expression of CYP450 enzymes and transporters

In the sandwich culture assays, positive controls like omeprazole, phenobarbital, and rifampicin demonstrated significant induction of CYP1A2, CYP2B6, and CYP3A4, validating assay performance. Rifampicin also demonstrated an upward trend in P-gp mRNA expression, consistent with published literature (Kota et al., 2010; Pondugula, Flannery, et al., 2015). As previously noted, no established inducers are available for OATP transporters; therefore, their expression levels were compared against vehicle control. According to FDA guidelines (2020), treatment is considered a significant inducer if it causes more than a 2-fold increase in mRNA expression relative to the vehicle and exceeds 20% of the induction observed with the positive control. Açaí extracts were tested at two concentrations: a human-relevant concentration (2.321 ng/mL C3G equivalent) and a high concentration (100 ng/mL C3G equivalent). None of the extracts significantly induced the mRNA expression of CYP1A2, CYP2B6, or CYP3A4 at either concentration (Figure 2). However, a significant reduction in mRNA levels was observed for CYP1A2 and CYP3A4 following treatment with F3AC and MRAQ at higher concentration [Figure 2(B)]. Additionally, a moderate decrease in CYP1A2 and CYP2B6 mRNA expression was noted for MRME at human-relevant concentration [Figure 2(A)]. For P-gp, OATP1B1, and OATP1B3, no significant induction could be inferred (Figure 3); however, a modest trend toward induction was noted at higher concentration, particularly for F4AC [Figure 3(B)].

Figure 2.

Figure 2.

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Fold induction of açaí extracts [mountain rose aqueous extract (MRAQ), mountain rose acidic methanol extract (MRAC), mountain rose ethanol extract (MRET), mountain rose methanol extract (MRME), nature’s way acidic methanol extract (F3AC), natrol acidic methanol extract (F4AC), natrol methanol extract (F4ME)] at (A) human-relevant concentration (2.321 ng/mL C3G equivalent) and (B) higher concentration (100 ng/mL C3G equivalent) against CYP1A2, CYP2B6, and CYP3A4 mRNA expression, relative to vehicle (solvent mixture; 85:15 v/v water: acetonitrile); omeprazole (OMEP), phenobarbital (PHENO) and rifampicin (RIF) as positive controls. Data was calculated as mean ± SD with n = 3. Statistical analysis performed using one-way ANOVA: Dunnett comparison of all treatments v/s positive control. *Indicates significance (*= p-value <0.05; **= p-value <0.005; ***= p-value <0.001; ****= p-value <0.0001).

Figure 3.

Figure 3.

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Fold induction of açaí extracts [mountain rose aqueous extract (MRAQ), mountain rose acidic methanol extract (MRAC), mountain rose ethanol extract (MRET), mountain rose methanol extract (MRME), nature’s way acidic methanol extract (F3AC), natrol acidic methanol extract (F4AC), natrol methanol extract (F4ME)] at (A) human-relevant concentration (HED) (2.321 ng/mL C3G equivalent) and (B) higher concentration (100 ng/mL C3G equivalent) against P-gp, OATP1B1 and OATP1B3 mRNA expression, relative to vehicle (solvent mixture; 85:15 v/v water: acetonitrile); and rifampicin (RIF) as positive controls. Data was calculated as mean ± SD with n = 3. Statistical analysis performed using one-way ANOVA: Dunnett comparison of all treatments v/s positive control. *Indicates significance (*= p-value <0.05; **= p-value <0.005; ***= p-value <0.001; ****= p-value <0.0001).

3. Intracellular transporter probe accumulation

As observed in the mRNA expression data, treatment with açaí extracts at a concentration corresponding to 100 ng/mL C3G, particularly F4AC, indicated a potential trend toward transporter gene induction. To assess the functional relevance of these changes, preliminary intracellular probe accumulation assay was performed using F4AC extract. Functional activity of transporters was evaluated by measuring the intracellular accumulation of fluorescent probe substrates in LS174T cell lines following 48-hour treatments. Transporter-specific inhibitors were employed to assess inhibition-mediated accumulation, with the ratio of intracellular substrate in the absence versus presence of inhibitor serving as a representation for transporter activity. As shown in Figure 4 and Figure S3, F4AC treatment did not significantly alter substrate accumulation, suggesting a lack of substantial modulation of transporter function under the tested conditions.

Figure 4.

Figure 4.

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Functional transporter activity following 48- hour treatment with vehicle (solvent mixture; 85:15 v/v water: acetonitrile), RIF (rifampicin), or F4AC (natrol acidic methanol extract) in LS174T cells. P-gp activity was assessed via intracellular accumulation of R123. Fluorescence values were normalized to vehicle. Statistical analysis performed using ANOVA: Dunnett comparison of all treatments v/s control. No significant change in P-gp activity was observed; p-values are indicated where applicable.

Discussion

1. Relevance of açaí test concentration and induction results

Assuming 100% oral bioavailability of the extracts, the estimated systemic dose was calculated using the formula: dose (calculated, mg dose) = mg/mL extract concentration × 1000 mL/L × 7 L (represents the average volume of blood and plasma in the human). Based on this model, the calculated dose corresponding to the test concentrations used for induction studies ranged from approximately 0.7 mg to 4500 mg (see Supplementary Data for extract-specific details, Table S2). These estimated doses align well with the range reported in the NIH/ODS dietary supplement label database (Dietary Supplement Label Database [DSLD], NIH Office of Dietary Supplements; https://dsld.od.nih.gov; NIH, 2023), where commercially available açaí-only products (açaí berry/açaí juice/ açaí fruit extracts) span a daily intake range of 25.5 mg to 6000 mg. The dataset included ~143 product formulations—capsules, powders, liquids, and tea bags—underscoring the diverse forms of açaí in the consumer market and reinforcing the physiological relevance of the tested concentrations and extract types. Importantly, given that açaí is marketed and consumed as a dietary supplement, not a regulated pharmaceutical, changes in CYP450 or transporter expression- even at supplement-relevant concentrations-carry significant public health implications. The lack of regulatory oversight or standardized dosing may lead consumers to unknowingly ingest excessive or subtherapeutic amounts, potentially resulting in clinically relevant drug interactions. It is worth noting, however, that this dose calculation does not account for pharmacokinetic parameters such as the absorption rate constant (Ka), volume of distribution (Vd), or the free/unbound fraction of active constituents. Incorporating these parameters would likely result in higher dose estimates necessary to achieve comparable systemic exposure.

As noted in the results, reduced mRNA expression of CYP450 enzymes was observed following treatment with specific açaí extracts. Such downregulation may arise from various biological mechanisms, including transcriptional repression, epigenetic modulations, post-transcriptional regulation, cellular stress responses, and/or cytotoxicity. There have been reports (Ciolino et al., 1999; Dong et al., 2010; Smutny and Pavek, 2014; Küblbeck et al., 2020; Tang et al., 2021) suggesting that polyphenolic constituents may suppress gene expression by interfering with nuclear receptors such as PXR (Pregnane X Receptor), CAR (Constitutive Androstane Receptor) or AhR (Aryl Hydrocarbon Receptor), which are key regulators of CYP450 expression. However, further efforts will be required to confirm those findings. While a prior study reported significant CYP3A4 induction by methanol açaí extract at 1.5 μg/μL (equivalent to 1.5 mg/mL) (Zhang et al., 2019), our results showed no such effect. This discrepancy could be attributed to differences in extract lot numbers, which may reflect environmental or processing variability. Additionally, Zhang et al. (2019) used a monolayer hepatocyte culture model, whereas our study employed the more physiologically relevant sandwich-culture system, and used different concentration ranges. Although in vitro CYP450 induction often translates to clinical outcomes (Sprouse and Van Breemen, 2015), product-to-product variability among botanical preparations can lead to divergent results. A limitation to acknowledge is that the current findings are based on hepatocytes from a single donor, which may not capture inter-individual variability. Future studies using hepatocytes from multiple donors are essential to capture genetic and metabolic differences that influence CYP450 and transporter induction. Additionally, in vivo studies are warranted to assess long-term exposure effects and systemic interactions across organs, providing a more detailed understanding of the potential metabolic impact of açaí extracts.

2. Implications of reduced hepatic cell viability

The observed decline in hepatocyte viability upon treatment with açaí extracts indicates potential cytotoxic effects at higher concentrations or with prolonged exposure. To further dive deep into the mechanistic insight of the observed cytotoxicity, cytokine profiling could serve as a valuable next step and an important avenue for future work. Additionally, it is important to note that a reduction in cell viability does not directly or proportionally equate to hepatotoxicity. Therefore, in vitro hepatocyte cell models must be complemented with hepatotoxicity-specific assays—such as assessment of mitochondrial integrity, caspase activation, and liver-specific enzyme release (e.g., ALT, AST)—to establish a definitive link to hepatotoxicity.

However, these initial findings serve as a preliminary indication warranting further toxicological evaluation. While açaí has been predominantly reported to exert hepatoprotective effects in in vivo studies due to its antioxidant and anti-inflammatory properties (Carvalho et al., 2018; de Bem et al., 2018; Zhou et al., 2018; Romão et al., 2020), the possibility of adverse outcomes cannot be completely ruled out. An analysis of the FDA CAERS (2007–2024) revealed sporadic reports of adverse events involving gastrointestinal (50.5 %), cardiovascular (17.5 %), and hepatic (7.8 %) systems following consumption of açaí-containing products. Although hepatic-related events constitute a smaller proportion of these reports, their occurrence signals a need for further safety investigation. Based on our literature searches, a single peer-reviewed clinical case has reported cholestatic jaundice (drug-induced liver injury/ potential toxicity) linked to daily consumption of an açaí berry supplement (Stratton, 2014). This suggests that hepatotoxicity is rare, it may occur in certain individuals or contexts (e.g. high-dose supplements). Additionally, given the increasing consumer use of botanical supplements and variability in extract quality and composition, these findings underscore the importance of further conducting in vitro and in vivo toxicity assessments. Future studies should aim to confirm whether the reduction in hepatocyte viability reflects a genuine hepatotoxic risk or is a transient, concentration-dependent cellular response.

3. Preliminary assessment of transporter activity in response to açaí extracts

Although mRNA expression results indicated a modest trend toward potential induction by the F4AC açaí extract, this was not accompanied by a corresponding functional increase in the transporter activity studies. In the preliminary accumulation assays, rifampicin showed expected modulation of P-gp activity, indicating assay responsiveness. However, the positive controls did not demonstrate substantial activity, suggesting limited endogenous OATP in LS174T cell model. This highlights a potential limitation of the system and points to the need of for future studies to incorporate overexpression models or alternative cell lines with more robust OATP activity.

While F4AC açaí extract treatment resulted in marginal changes in substrate uptake, this effect did not meet the expected criteria for definitive transporter induction. These subtle shifts in transporter activity highlight the need for further optimization of experimental conditions, particularly with respect to preincubation time, total exposure duration, and choice of cell model (such as LS180, transfected HEK293 or CHO cells), to better capture subtle or delayed effects on transporter activity. Recognizing the complex nature of botanical mixtures and their potential for subtle pharmacokinetic interactions, these findings underscore the importance of applying a rigorous, stepwise validation strategy when assessing botanical–drug interactions at the transporter level.

Conclusion

This study provides preliminary yet methodologically rigorous evaluation of the induction potential of açaí extracts on FDA-recommended cytochrome P450 enzymes (CYP1A2, CYP2B6, CYP3A4) and hepatic drug transporters (P-gp, OATP1B1, and OATP1B3) using physiologically relevant sandwich-cultured primary human hepatocytes. Cytotoxicity profiling revealed a time-dependent reduction in cell viability with MRAC, MRME, and MRET extracts, while F4AC exhibited a dose-dependent effect. Across seven distinct extracts, no significant induction of CYP450 enzymes was observed at either human-relevant (2.321 ng/mL C3G equivalent) or higher (100 ng/mL C3G equivalent) concentrations. Notably, F3AC and MRAQ extracts showed significant reduction of CYP1A2 and CYP3A4 mRNA expression at higher concentration, while MRME moderately suppressed CYP1A2 and CYP2B6 expression at human-relevant concentration. Although preliminary functional transporter assays did not confirm induction, modest trends observed for F4AC suggest potential for weak modulation that warrants further study. Overall, these findings emphasize the need for rigorous pharmacokinetic and toxicological evaluation of botanical dietary supplements. Given the increasing popularity of açaí and its marketing for antiproliferative and health-promoting effects, such assessments are especially critical when used alongside medications with narrow therapeutic indices, such as chemotherapeutics. Future studies should incorporate donor variability, in vivo validation, and clinical correlation to better define the risk of botanical-drug interactions and guide safe use in polypharmacy contexts.

Supplementary Material

Supplementary material

Acknowledgements

We are grateful to the National Institutes of Health National Center for Complementary and Integrative Health and Office of Dietary Supplements (NIH NCCIH/ODS) for funding. We are thankful to Drs. Chen-Che Huang, Ya-Xiong Tao, and Emily Graff for sharing their research facilities as well as Xu Chuanling for her assistance in maintaining the lab facilities.

Funding

This work was supported by the National Institutes of Health National Center for Complementary and Integrative Health and Office of Dietary Supplements (NIH NCCIH/ODS) [Grant R15AT011047] and Auburn University Animal Health and Disease Research Grant.

“This manuscript is the result of funding in whole or in part by the National Institutes of Health (NIH). It is subject to the NIH Public Access Policy. Through acceptance of this federal funding, NIH has been given a right to make this manuscript publicly available in PubMed Central upon the Official Date of Publication, as defined by NIH”

Footnotes

Disclosure Statement

No potential conflict of interest was reported by the author.

Data Availability Statement

The authors declare that all the data supporting the findings of this study are available within the paper and its supplemental data.

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Supplementary Materials

Supplementary material
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Data Availability Statement

The authors declare that all the data supporting the findings of this study are available within the paper and its supplemental data.

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