Identification of Sarmentosin as a Key Bioactive from Blackcurrants (Ribes nigrum) for Inhibiting Platelet Monoamine Oxidase in Humans

Abstract

Previous clinical studies indicate that monoamine oxidase-B (MAO-B) inhibition by blackcurrants must be predominantly attributed to bioactives other than anthocyanins. In this natural products discovery study, MAO-A/B inhibitory phytochemicals were isolated from blackcurrants, and a double-blind crossover study investigated the efficacy of freeze-dried whole-fruit blackcurrant powder in inhibiting MAO-B compared with blackcurrant juice in healthy adults. Platelet MAO-B inhibition was comparable between powder (89% ± 6) and juice (91% ± 4), and it was positively correlated with MAO-modulated plasma catecholamines, subjective alertness, and reduced mental fatigue, assessed using the Bond-Lader questionnaire. Sarmentosin, a nitrile glycoside, and its hydroxycinnamoyl esters were identified as novel MAO-A/B inhibitors from blackcurrant in vitro, and sarmentosin was demonstrated to inhibit platelet MAO-B activity in vivo. These findings confirm sarmentosin as the primary bioactive for MAO-A/B inhibition in blackcurrants, as well as its bioavailability and stability during freeze-drying, and suggest that consuming blackcurrant powder and juice may positively affect mood in healthy adults.

1. Introduction

Researchers have long associated both excitatory and inhibitory monoamine neurotransmitters with the modulation of mood and behavior. Stress, for example, has been correlated with norepinephrine (NE) release,¹ dopamine (DA) is associated with “hedonic tone” or feelings of enjoyment,² and serotonin (5-HT) is linked to behavioral inhibition and fear processing.³ Alterations in monoamine neurotransmitters are also associated with neurocognitive disorders such as depression, anxiety, and Alzheimer’s disease,^4,5 leading to their identification as drug targets and the subsequent development of synthetic medication that selectively inhibit monoamine oxidase (MAO), an enzyme that degrades monoamine neurotransmitters.^6,7

Monoamine oxidase exists in two isoforms, MAO-A and MAO-B, which are differentially distributed in the brain regions and have specific substrate specificity. MAO-A preferentially degrades 5-HT, whereas MAO-B has a greater affinity toward benzylamine and phenylethylamine (PEA), and the catecholamines DA, epinephrine (E), and NE are metabolized by both isoforms.⁸ Synthetic MAO inhibitors alter monoamine neurotransmitter concentrations and are associated with improvements in mood and symptoms of neurological disorders.^9,10 However, they have also been reported to induce adverse side effects such as nausea, dizziness, and insomnia, along with unfavorable drug–food interaction.¹¹ Interestingly, various medicinal plants, fruits, and vegetables produce a chemically diverse array of MAO inhibitory phytochemicals, which may be safer alternatives to synthetic drugs for the treatment of mood and neurological conditions. These include organic acids from prickly pear cacti,¹² alkaloids from Piper longum,¹³ terpenes found in Gardenia jasminoides,¹⁴ phenolics (specifically flavonoids) present in berryfruit,¹⁵ and a yet-to-be-characterized MAO inhibitor from the anthocyanin-rich berryfruit blackcurrant (Ribes nigrum).

Dietary intervention studies by our group and others have consistently reported the efficacy of blackcurrant (BC) juice in temporally inhibiting platelet MAO-B activity and in reducing the concentration of circulating 3,5-dihydroxyphenylglycine (DHPG), a marker for MAO-A activity.¹⁶⁻¹⁸ Importantly, platelet MAO-B inhibition was associated with reduced plasma prolactin,¹⁸ an indirect biomarker for increased DA release in the brain,¹⁹ providing evidence for the inhibitory effects of consuming BC on MAO-A and -B activity that consequently leads to neurotransmitter modulation. To date, the BC constituent(s) responsible for this fruit’s MAO-B inhibitory bioactivity remains unidentified, with previous studies reporting MAO-B inhibition following BC juice but not anthocyanin-enriched BC powder extract consumption.¹⁷ These findings indicate that anthocyanins are not the primary bioactive responsible for MAO-B inhibition in BC and suggest that processing procedures in the production of anthocyanin-enriched BC extract may eliminate or degrade essential bioactives responsible for MAO inhibition.

The chemical identification and nutraceutical characterization of a novel phytonutrient with MAO inhibitory bioactivity from BC will extend the scientific knowledge of bioactives from edible fruits. Moreover, understanding the bioactivity of blackcurrants consumed in different formats would allow their incorporation into a variety of functional foods offering consumers natural choices to improve their health and well-being through targeted nutrition.

In this study, we aimed to identify and isolate key bioavailable compounds in BC that inhibit MAO-B activity, which might contribute to modulations in mood and circulating neurotransmitters after BC consumption. As drying techniques can change the content of berryfruit bioactives and their antioxidant capacities,²⁰ we investigated the effect of consuming both freeze-dried BC powder and a commercially relevant dose of BC juice in healthy adults. To our knowledge, this is the first study to identify sarmentosin as a novel bioavailable MAO inhibitor in BC and characterize its efficacy in inhibiting peripheral MAO-B activity after consuming a single dose of freeze-dried BC powder and juice.

2. Materials and Methods

2.1. Study Procedures

A randomized, placebo-controlled, crossover study design was used to investigate the pharmacodynamic effect of two BC formats on platelet MAO-B activity and whether temporal changes in MAO-B are associated with modulations in mood, circulating neurotransmitters, and probable bioactive. Participants completed two treatment conditions (placebo or BC), with at least 7 days separating the trial days of each treatment. All methods and procedures were reviewed and approved by New Zealand’s Northern B Health and Disability Ethics Committee (2021 EXP 11576). This study is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12621001590853).

Thirteen adults (n = 8 males and n = 5 females) between 26 and 39 years old were recruited to this study from the wider Palmerston North community in New Zealand. Participants were screened for any contraindications to the study using a health questionnaire. Exclusion criteria included those diagnosed with a chronic disease or blood-transmissible disease, had a recent viral or bacterial illness, pregnancy, taking medication that affected blood clotting properties or mood, intolerance to BCs, or had a strong reaction to needles. All participants were familiarized with this study and provided signed consent, and their progress through the trial is illustrated in Figure 1. Enrolled participants were randomly allocated to an intervention format (BC powder (BCP) or BC juice (BCJ)) and treatment (intervention or placebo) order using Williams Latin Squares. All participants enrolled were tested synchronously as a single cohort across the study interventions from May to June 2022. Three participants withdrew from the study due to gastrointestinal response to their allocated intervention, a strong reaction to blood sampling, and loss of interest in the study. Data from two participants who completed their first treatment arm and subsequently withdrew from the study were included in the study analysis, and data from one participant were excluded due to noncompliance with study protocols. The physical characteristics of participants whose data were included for analysis are presented in Table 1.

Figure 1.

Consolidated standards of reporting trials (CONSORT) flow diagram depicting the progression of participants through the study.

Table 1. Participant Physical Characteristics^a.

variable	juice (n = 5)	powder (n = 7)
males	3	5
females	2	2
age (years)	32.6 ± 3.7	31.4 ± 6.6
height (cm)	174.3 ± 8.9	172.3 ± 8.4
bodyweight (kg)	83.1 ± 19.8	77.1 ± 16.2
body mass index (BMI)	27.8 ± 8.6	24.7 ± 0.7

^aAge and anthropomorphic measurements of participants randomly allocated to the juice and powder intervention groups. Data are mean ± SD.

Enrolled participants were required to take part in two trial days where they consumed their allocated BC beverage and its corresponding placebo. Participants abstained from consuming foods and supplements high in polyphenols 24 h before their trial days. After an overnight fast (10 h), participants were given a standardized breakfast (Almond with Vanilla One Square Meal, Cookie Time Ltd., Christchurch, New Zealand) to consume on the morning of their trial day, at least 2 h before arriving at the institution’s clinical facility. After a short rest (5 min), participants completed the Bond-Lader mood questionnaire²¹ followed by a venous blood sample collection (0 min) and then given their allocated treatment beverage to be consumed as quickly as they could. At 10, 20, 120, 240, and 480 min after consuming their treatment beverage, participants completed the Bond-Lader mood questionnaire and donated venous blood. Following mood assessment and venous blood collection at 240 min post-treatment consumption, participants were provided with a low-polyphenol lunch (plain chicken or egg sandwich with mayonnaise and butter). Water was provided ad libitum after the 20 min time point. Participants were seated in the clinical trial facility and allowed Internet access for light work or entertainment between venous blood collection time points.

An a priori power analysis was conducted using Minitab 21.3.1 based on data from Lomiwes et al.¹⁶ (n = 40) to determine the appropriate sample size for this study. The power analysis indicated that a sample size of three participants per group was adequate to detect an 80% difference between blackcurrant and placebo interventions at a 5% significance level. To account for potential dropouts and ensure adequate power, six participants were recruited for the juice format and seven participants for the powder format, resulting in a total sample size of 13 participants.

2.2. Interventions

Both the BCP and BCJ interventions were provided by A̅repa IP Ltd. (Auckland, New Zealand). The BCP intervention was powdered freeze-dried New Zealand BC fruit and the BCJ intervention was prepared from New Zealand BC juice concentrate. The total amount of BC anthocyanins consumed by participants for each BC format differed approximately 7-fold: The dose of the BCP format was standardized to 7.8 mg anthocyanin/kg bodyweight, the same dose administered by Watson et al.¹⁷ was reconstituted in 300 mL water. Participants allocated to the BCJ intervention consumed 300 mL of single-strength BC juice containing 78.8 mg anthocyanins, which is equivalent to that present in a single serving of a commercial BC juice (A̅repa Performance, Auckland, New Zealand).

The placebo formulations for both formats were matched for sugar, appearance, flavor, and texture. The BCP placebo was produced by Sensient Technologies (Auckland, New Zealand) and predominantly comprised citrus fiber, maltodextrin, and caster sugar blended with BC and raspberry flavouring and artificial coloring. The BCJ placebo contained the same volume of apple juice concentrate as the BCJ beverage, BC flavouring, citric acid, and food coloring and was diluted in 300 mL water.

2.3. Platelet MAO-B Activity and Blood Glucose Concentrations

Venous blood (9 mL) was collected into an EDTA-coated vacutainer tube at each collection time point. A small subsample of blood (20 μL) was collected from the tube and immediately measured for glucose concentration using a HemoCue 201 DM System (HemoCue, Ängelholm, Sweden). The remaining blood sample was centrifuged at 600g for 3 min at room temperature, and the platelets were collected, isolated, and washed as previously described by Watson et al.¹⁷ Then, they were stored at −80 °C until analyzed.

Platelet MAO-B activity was measured using the Amplex Red Monoamine Oxidase Kit (A12214, Invitrogen), according to procedures described by Watson et al.¹⁷ The protein concentration of the platelet samples was determined using the Bicinchoninic Acid Protein Assay Kit (23255, Pierce).

2.4. Characterization of Primary Bioactive in Blackcurrant

2.4.1. Blackcurrant Juice Fractionation and Active Compound Identification

BC juice concentrate was supplied by A̅repa IP Ltd. Bioactivity-directed fractionation to identify MAO inhibitory components was performed in a series of steps. The BC juice concentrate was diluted 1:1 with water (H₂O) and adjusted to pH 5.5. The diluted concentrate was initially separated using reverse phase (RP) column chromatography by adding to an RP silica gel plug (8 cm diameter ×4 cm) and eluted sequentially with H₂O (3 fractions of 250 mL) followed by increasing proportions of ethanol in 0.1% formic acid (FA) (8 fractions, 10–90% ethanol, 250 mL each) using a low-level vacuum. Subsamples of each fraction were taken and evaporated to dryness before analysis of MAO enzyme inhibition by S9-liquid chromatography–mass spectrometry (LCMS) as described in Section 2.4.2.

The fractions showing the greatest bioactivity in inhibiting MAO activity were separated by preparative high-performance liquid chromatography (HPLC) (Gilson, Middleton, WI). Briefly, constituents were separated on a Supelco Ascentis C18 column (22 mm × 250 mm) using a gradient of acetonitrile (ACN) (1–15 and 2–60%) in 0.1% FA at a flow rate of 18 mL/min. Subsamples were taken from each fraction and screened for their efficacy in inhibiting MAO activity (see Section 2.4.2).

Compounds from fractions that were most potent in inhibiting MAO activity were identified by LCMS (Information-Dependent Acquisition (IDA) Enhanced Product Ion (EPI) experiments), high-resolution LCMS (HR-LCMS), and 1H-Nuclear Magnetic Resonance (NMR) spectroscopy. For HR-LCMS, a Waters Xevo Quadrupole Time-of-Flight (QTOF) instrument (Waters, Milford, MA) was used in direct infusion mode (negative mode electrospray ionization (ESI)). The subsample from preparative HPLC was diluted with H₂O and used directly. Exact mass searching delivered molecular formulas for sarmentosin and sarmentosin esters, nigrumin caffeate, nigrumin coumarate, and nigrumin ferulate. IDA-EPI experiments utilized a 7500 QTrap triple quadrupole/linear ion trap (QqLIT) mass spectrometer (MS) equipped with a Turbo V ion source ESI probe (AB Sciex, Concord, ON, Canada) coupled to a Shimadzu Nexera LC40 UHPLC (Shimadzu, Tokyo, Japan). UHPLC conditions for separation of sarmentosin and its esters are described in Section 2.5.2. IDA-EPI experiments were triggered using the multiple reaction monitoring transitions (MRMs) listed in Table S1 as survey scans. The EPI scan was set with a CE of −16 V and a collision energy spread (CES) of 15 V to generate information for low and high-mass fragments in one spectrum. Compound identification was further supported using cochromatography with authentic standards. For NMR analysis, dried samples were dissolved in D₂O (sarmentosin) or CD₃OD (sarmentosin esters), and spectra were collected with a Bruker AVANCE NEO 500 MHz spectrometer (Bruker, Billerica, MA). Quantitative NMR (qNMR) was performed on the same instrument using samples with and without added calcium formate. Selective total correlation spectroscopy (TOCSY) was used irradiating the 6.6 ppm triplet (vinylic CH) and integrating the multiplet at 4.4 ppm (adjacent CH2).

2.4.2. MAO Inhibition: S9-LCMS Screening Assay

The bioactivity of BC fractions in inhibiting MAO activity was determined by LCMS using procedures described by Yan et al.²² with modifications. The assay employed a crude S9 microsome fraction enzyme mix comprised MAO-A and MAO-B from the porcine liver, thus measuring combined MAO-A/B activity. Briefly, each BC fraction was mixed with S9 enzyme mix, assay buffer, and kynuramine (1 mg/mL) and then incubated at 37 °C for 1 h. After cooling in ice H₂O, NaOH (2 N) and ACN were added to the mixture, and a subsample was taken for LCMS analysis. LCMS employed a Shimadzu 8040 MS with a Nexera X2 UPLC (Shimadzu, Tokyo, Japan) equipped with a Hypersil GOLD Cyano UHPLC (2.1 mm × 150 mm, 1.9 μm) column (Thermo Scientific, Waltham, MA) eluted with 0.1% FA(aq) and ACN. Chromatography was isocratic (40% ACN), and the relative levels of 4-hydroxy-quinoline (4HQ) and kynuramine were determined. MAO inhibition was calculated by comparing the amount of 4HQ with that of a buffer-only sample (no inhibition). Kynuramine is converted to 4HQ by both MAO-A and MAO-B isoforms, and 4HQ abundance was used as a measure of combined MAO inhibition.

Screening for MAO-A/B inhibition was initially conducted on crude BC fractions. The effect of purified sarmentosin (BOC Sciences; Cat. No. B2703-149954) on MAO-A/B inhibition was conducted on concentrations ranging from 0.04 to 100 μg/mL to determine the concentration of sarmentosin required to inhibit MAO activity by half of its maximum activity (IC₅₀) compared with known MAO-A (clorgyline hydrochloride; Sigma, M3778) and MAO-B (deprenyl hydrochloride; Sigma, M003) inhibitors.

2.4.3. Determination of MAO-Specific Inhibitors on MAO-A and MAO-B Activity

The effect of clorgyline hydrochloride (HCl) and deprenyl HCl on MAO-A and MAO-B, respectively, was measured using the MAO-Glo kit (Promega; Cat. No. V1402) according to the manufacturer’s instructions. The IC₅₀ of each compound in individually inhibiting MAO-A and -B was calculated from their respective dose-response curves to determine the sensitivity of the crude S9-LCMS screening assay in measuring the inhibition of both MAO isoforms.

2.5. Quantification of Blackcurrant Anthocyanins and Sarmentosin

2.5.1. Anthocyanin Quantitation of Blackcurrant Intervention Formats

Anthocyanin concentrations in BCJ and BCP were measured by UHPLC using a Dionex Ultimate 3000 Series UHPLC (ThermoFisher Scientific, San Jose, CA) with photodiode array detection at 520 and 530 nm as previously described.²³ The BCJ concentrate was diluted 1:1 with 5:95 FA/H₂O (v/v). A weighed quantity of the BCP sample was dissolved in 5:95 FA: H₂O v/v to give an aqueous solution of concentration 20 mg/mL. Anthocyanins were quantitated using a pure standard of cyanidin 3-O-glucoside (Cy-glu), and all results for individual and total anthocyanins are expressed as Cy-glu equivalents.

2.5.2. Sarmentosin Quantitation of Blackcurrant Intervention Formats

Sarmentosin concentrations in BCJ concentrate and BCP were determined by LCMS using a 5500 QqLIT MS equipped with a TurboIon-Spray interface (AB Sciex, ON, Canada) coupled to an Exion UHPLC (Shimadzu, Kyoto, Japan). The BCJ concentrate was diluted 1:1 with MeOH/H₂O (v/v). A weighed quantity of the BCP sample was dissolved in 1:1 MeOH/H₂O (v/v) to give a solution of concentration 20 mg/mL. For both samples, aliquots were taken and further diluted 100-fold with H₂O.

Sarmentosin and its esters were separated on a Poroshell 120 SB-C18 2.7 μm 2.1 mm × 150 mm ID column (Agilent Technologies, CA) maintained at 50 °C. Solvents were (A) H₂O acidified with 0.1% FA and (B) 2:98 v/v H₂O:ACN acidified with 0.1% FA with a flow rate of 600 μL min-1. The initial mobile phase, 0% B, was held for 2.5 min before ramping linearly to 60% B at 15 min and then to 100% B at 16 min and holding for 3 min before resetting to the original conditions. The injection size was 5 μL. Data acquired from MS in the negative mode using the MRM method is detailed in Table S2. Quantification of sarmentosin and its esters was performed using Sciex OS software v3.1.0 (AB Sciex), and all results are reported as sarmentosin equivalents.

2.5.3. Plasma Anthocyanin Bioavailability

Venous blood samples were collected into lithium heparin vacutainer tubes (9 mL), and the plasma was separated by centrifugation at 4000g for 10 min at 4 °C and then aliquoted to 1 mL volumes. Samples for sarmentosin and anthocyanin bioavailability were spiked with ascorbic acid (10 mmol), and samples for anthocyanin bioavailability were further acidified with FA (50%). Plasma samples for neurotransmitter analysis remained untreated. All samples were stored at −80 °C until analyzed.

Plasma samples were spiked with an internal standard (IS) (malvidin 3-O-galactoside,1 ng) and acidified with 4% phosphoric acid(aq) prior to cleanup on a SOLAμ solid phase extraction (SPE) 96-well plate (reverse phase polymeric (HRP) 2 mg/1 mL) (Thermo Scientific, Waltham, MA). Plates were conditioned prior to sample loading with MeOH and acetic acid(aq) (0.2%). The loaded plates were washed with H₂O and acetic acid(aq) (0.2%), and the retained anthocyanins were eluted with 95:5 (v/v) MeOH:FA. The eluted samples were evaporated to dryness in a CentriVap concentrator (Labconco, Kansas City, MO) and reconstituted in 5:3:92 (v/v/v) ACN:FA: H₂O prior to analysis by LCMS. LCMS experiments were carried out on a 7500 QqLIT as described in Section 2.4.1.

Anthocyanin separation was achieved on a Poroshell 120 SB-C18 2.7 μm 2.1 × 150 mm ID column maintained at 70 °C. Solvents were (A) 5:3:92 (v/v/v) ACN/FA/H₂O and (B) 99.9:0.1 (v/v) ACN/FA, and the flow rate was 600 μL/min. The initial mobile phase, 100% A, was held isocratically for 0.5 min, then ramped linearly to 15% B at 5 min, followed by another linear ramp to 90% B at 5.1 min, and held for 1.9 min before resetting to the original conditions. The sample injection volume was 10 μL. MS data were acquired in the positive mode using an MRM method with details listed in Table S3.

Quantification of individual and total anthocyanins were performed using the IS ratio method and Sciex OS software v3.1.0. All results are reported as Cy-glu equivalents.

2.5.4. Plasma Sarmentosin Bioavailability

Plasma samples were spiked with a d₄-salicyclic acid glycoside as IS (0.5 ng) and applied to a Phree phospholipid removal 96-well plate (Phenomenex, Torrance, CA) for cleanup to remove proteins and phospholipids and enable concentration. In brief, acidified ACN was added to each plate well, and the mixture vortexed before the Phree plate was placed on top of a 96 Multi-Tier Micro Plate System (TOPAS) for collection of the filtrate under positive pressure. The filtrate was evaporated to dryness under nitrogen (Microvap triple microplate evaporator; Organomation, Berlin, MA) at 35 °C and then reconstituted in H₂O prior to analysis by LCMS with a 7500 QqLIT as described in Section 2.4.1. MS data were acquired in the negative mode using an MRM method with details listed in Table S1. Quantification of sarmentosin was performed using the IS ratio method and SCIEX OS software.

2.6. Quantitation of Neurotransmitters in Plasma

Plasma neurotransmitters, precursors, and catabolites from the tyrosine (TYR) and tryptophan (TRP) metabolic pathways were measured using two separate analytical workflows (Method A and Method B). Metabolites measured in the TYR metabolic pathway were phenylalanine (PHE), phenylethylamine (PEA), tyrosine (TYR), 3,4-dihydroxyphenylalanine (L-DOPA), DA, 3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), NE, 3,4-dihydroxyphenylethylene glycol (DHPG), 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), NM, E, metanephrine (MN), and vanillylmandelic acid (VMA). Metabolites measured in the TRP metabolic pathway were TRP, kynurenine (KYN), kynurenic acid (KA), xanthurenic acid (XA), quinolinic acid (QA), 5-hydroxytryptophan (5-HTP), 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), and melatonin (MT). Method A utilizes an MS-probe and stable isotope coding LCMS method.^18,24 This method uses derivatization to quantitatively convert the neurotransmitters and related compounds to their corresponding acetate or ester to increase their analysis sensitivity. Method B is used for the quantitation of KA, QA, and XA, which derivatize poorly using method A, and for MT, which does not have a functional group compatible with derivatization by method A. These analytes and their labeled internal standards are measured directly by LCMS and are underivatized. Experimental details underlying both methods are outlined in Appendix ‘Neurotransmitter Analysis Methodology’.

2.7. Mood Measures

The Bond-Lader mood questionnaire allows self-evaluation of mood.²¹ The participants are required to rate 16 mood items on a 100 mm line to what extent each item was appropriate to them at that moment in time, with each item relating to “alert”, “calm,” or “content” mood dimensions. For individual Visual Analogue Scale (VAS) assessments of stress, anxiety, and mental fatigue, scales were anchored “not at all” and “extremely” at opposite ends of the 100 mm line.

2.8. Sarmentosin Intervention Study

To determine the efficacy of sarmentosin in temporally inhibiting platelet MAO-B activity, we conducted a randomized, placebo-controlled crossover study. Participants completed three treatment conditions (placebo, 42 mg sarmentosin, and 84 mg sarmentosin), with each trial day separated by at least 48 h. All methods and procedures were reviewed and approved by New Zealand’s Northern B Health and Disability Ethics Committee (2023 EXP 19223), and this study is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12624000021572).

Five healthy male adults aged between 25 and 36 years old were enrolled in this study. All participants met the same study criteria as described in Section 2.1 and provided signed consent before attending their first trial day. Participants were randomly allocated one of the three trial treatment beverages. All participants completed all three treatment arms of the study, and data from all participants were included in the study analysis.

Enrolled participants were required to take part in three trial days where they consumed their allocated sarmentosin beverages or the placebo. Participants adhered to the same dietary restrictions and consumed the same standardized breakfast as previously described in the BC intervention study (Section 2.1). A venous blood sample was collected at the beginning of each trial day, and then, participants were given their treatment beverage to consume immediately. A venous blood sample was collected 120 and 480 min after the treatment beverage. Water was provided ad libitum during the trial day, and participants were seated in the clinical trial facility between venous blood collection time points.

2.8.1. Sarmentosin Intervention

BC juice concentrate sourced from A̅repa IP Ltd. was diluted 1:1 with RO-purified H₂O and slowly applied to a column (10 cm diameter × 125 cm height) filled with Diaion HP20 resin (Mitsubishi Chemical Industries, Japan). The eluant was collected until it was determined that most of sarmentosin had eluted and was quantified as described in Section 2.4. Sarmentosin fractions were combined and concentrated using a rotary evaporator, and the resulting sarmentosin-rich fraction was then sterile filtered (Millipore Stericup (0.22 μM), Sigma-Aldrich). Participants consumed a 250 mL beverage containing 42 and 84 mg total sarmentosin, similar to that present in one and two servings, respectively, of a commercial BC juice (A̅repa Performance). Sucrose, BC flavouring, and coloring were added to the beverages to blind for taste and appearance.

The placebo beverage was matched for sucrose, contained the same volume of added BC flavouring and coloring produced by Sensient Technologies (Auckland, New Zealand) as the sarmentosin beverages, and diluted to 250 mL to match for sweetness, appearance, and flavor.

2.9. Data Analysis and Visualization

R version 4.2.1 was used for data analysis and visualization. For MAO-B activity and glucose data, comparisons of means between time points, treatments, and formats were made using analysis of variance (ANOVA) from a linear mixed effects model, with fixed effects for the format, study day, treatment, time, sex, and their interactions, and random effects for the participant, participant × study day, participant × treatment and participant × time point. The models were fitted with R package lmerTest,²⁵ and the least significant differences (LSDs) for comparing means were calculated post hoc using the R package predictmeans.²⁶ For comparative analysis of neurotransmitter concentrations, mood descriptors, and their relationship with MAO-B inhibition, observations were normalized to reflect the proportional change from baseline (first measurement of the time course at 0 min), and MAO-B inhibition was expressed as the inverse of MAO-B activity. The distribution of means was assessed using Shapiro–Wilk testing as implemented in the “MVN” package²⁷ as normality was rejected for most variables. Nonparametric rank-sum testing was used for pairwise comparisons (Wilcoxon-signed rank test) via “rstatix”²⁸ and for correlation analysis (Spearman’s rank-sum correlation) via “corrr”.²⁹ Unless stated otherwise, the level of significance was set at α < 0.05. Graphical summaries were prepared using the R-packages “ggpubr” (boxplots),³⁰ “gplots” (heatmaps),³¹ and the R base function.³²⁻³⁴

3. Results and Discussion

3.1. Platelet MAO-B Activity and Blood Glucose

Consuming a single dose of either the BCJ or BCP intervention similarly reduced platelet MAO-B activity (format effect; p = 0.170), with >80% inhibition observed for each participant 120 min post-intervention consumption (Figure 2). A significant treatment × time interaction was measured for both BCJ (p = 0.026) and BCP (p = 0.004) interventions so that MAO-B activity at 10 min after consuming these interventions was significantly lower compared with baseline (0 min) activity. This effect dissipated at 240 and 480 min after BCJ and BCP consumption, respectively, returning to baseline levels for both treatment formats at the latter time point. Further analysis of combined MAO-B inhibition results from both formats revealed no significant difference between males and females in this study (sex effect; p = 0.223). Additionally, no significant sex × treatment or sex × time × treatment interactions were observed (p = 0.416 and 0.700, respectively).

Figure 2.

Platelet monoamine oxidase (MAO)-B activity before (0 min) and after consuming blackcurrant (BC) juice or powder interventions and their corresponding placebo (PL). Values are mean ± SEM. Data points labeled “a” and “c” indicate significant difference (p < 0.05) from 0 min for juice and powder interventions, respectively. Data points labeled “b” and “d” indicate significant difference (p < 0.05) from PL at the corresponding time point for juice and powder interventions, respectively.

Consuming the corresponding BCJ and BCP placebo beverages had no significant effect on platelet MAO-B activity. Small and nonsignificant decreases in MAO-B activity (<8–20% MAO inhibition) were observed after placebo consumption and suggest that trial conditions such as food restriction and prolonged sedentary sitting, or a combination of these factors, may contribute to some MAO-B inhibition.

Postprandial changes in blood glucose concentrations temporarily affect mood and cognitive performance.³⁵ Therefore, blood glucose was measured post-intervention consumption. Our results showed no significant change in blood glucose following the consumption of the BCJ and BCP interventions and their corresponding placebos (Figure S1A and S1B; format × treatment × time interaction; p = 0.827).

This is the first study to demonstrate the efficacy of a BC powder format in inhibiting platelet MAO-B activity, confirming that freeze-dried BC powder comparably inhibits platelet MAO-B activity as BC juice. Functional foods in the format of powders offer several advantages, including ease of storage, stability, and versatility, making them appealing to consumers and food manufacturers. In contrast to the ineffective anthocyanin-enriched BC powder used by Watson et al.,¹⁷ the freeze-dried BC powder in this study had not undergone phytochemical extraction, which is speculated to remove or degrade MAO-B inhibitory phytonutrients. This highlights the importance of processing procedures in retaining platelet MAO-B inhibitory bioactivity of BCs.

Most clinical studies investigating the MAO-B inhibitory bioactivity of BCs utilized a range of doses normalized to participant’s bodyweight to mitigate variations in treatment effects arising from differences in bodyweight.¹⁶⁻¹⁸ However, commercial ready-to-consume BC-based functional foods are usually marketed at fixed serving sizes where consumers ingest the same absolute dose of BC bioactive, which is lower than those tested in previous studies. Our results confirm the efficacy of consuming an absolute dose of BC juice found in a commercial BC nootropic drink (A̅repa Performance), in inhibiting platelet MAO-B to a similar degree as reported in studies utilizing significantly higher doses of BC juice.

3.2. Sarmentosin is a Novel MAO Inhibitor from Blackcurrants

Bioactivity-directed RP fractionation of BCJ concentrate was used to isolate compounds that inhibited MAO-A/B activity (Figure 3). Of the 11 fractions retrieved from the initial RP fractionation, RP2 and RP10 inhibited MAO-A/B by over 80%, with RP2 having greater bioactivity (Figure 3A).

Figure 3.

Bioactivity of the initial 11 blackcurrant fractions (prepared from blackcurrant juice concentrate) in inhibiting monoamine oxidase (MAO)-A/B activity (MAOI) using the S9-liquid chromatography–mass spectrometry (LCMS) assay (A). The reduction in 4-hydroxy-quinoline (4HQ) produced from gradient elution of reverse phase fraction RP2 (B) and RP10 (C) with acetonitrile. Information-dependent acquisition (IDA) enhanced product ion (EPI) spectra and chemical structures for each of the identified compounds proposed to contribute to the MAOI bioactivity in the most bioactive bands collected from RP2, sarmentosin (SAR) B₁, and from RP10, SAR-caffeoyl ester (nigrumin caffeate) C₁, SAR-coumaroyl ester (nigrumin coumarate) C₂, and SAR-feruloyl ester (nigrumin ferulate) C₃ (D). The characteristic product ion (m/z) for each hydroxycinammic acid generated in negative mode by cleavage of the sarmentosin-ester bond is illustrated.

Gradient separation of RP2 revealed that fractions eluted at 10–11% ACN exhibited the highest MAO-A/B inhibition (Figure 3B). Subsequent HR-LCMS suggested that the bioactivity of this fraction was associated with a single compound with the formula C₁₁H₁₇O₇N. The main ion observed in negative mode was the formic adduct m/z 320, and a representative NMR spectrum of this compound (Figure S2) indicated the presence of a sugar (likely glucose), leading to the identification of sarmentosin, a γ-hydroxy nitrile glycoside. This identification was confirmed through NMR spectral comparison using authenticated sarmentosin data.³⁶

Separation for fraction RP10 resulted in two bioactive bands with <2% 4HQ production, respectively, eluting at 26% and 31–40% ACN (Figure 3C). Fractions eluting at 55% ACN also reduced 4HQ (<5%) but not to the same degree as the bands with higher polarity. LCMS analysis utilizing IDA-EPI experiments determined the presence of three sarmentosin esters and conjugated with three different hydroxycinnamic acids (Figure 3D; C₁–C₃). The coumaroyl ester (nigrumin coumarate) coeluted with the feruloyl ester (nigrumin ferulate), with the latter being most enriched between 35 and 38% ACN. The identity and bioactivity of nigrumin ferulate and -coumarate were verified using the purified compound. The chemical identification of the third least polar bioactive fraction (55% ACN) was inconclusive. It should be noted that RP10 is a mix of polyphenolic compounds, including flavanol-3-O-glycosides and anthocyanins, which are particularly enriched in fractions eluting at 19–35% ACN. It is, therefore, possible that these compounds contribute to the MAO-A/B inhibition observed in this fraction.

Following the identification of sarmentosin as the primary bioactive in RP2, the IC₅₀ of this compound on MAO-A/B activity was determined to be 2.67 μM using the S9-LCMS screening assay (Figure 4). Clorgyline HCl and deprenyl HCl were also tested to determine the sensitivity of this assay in measuring individual MAO-A and MAO-B activity, respectively. The IC₅₀ for clorgyline HCl on MAO-A/B activity was 18.8 μM, magnitudes greater than the IC₅₀ of this compound in inhibiting MAO-A enzyme activity using a commercial kit (Figure S3A) and reported values for this selective MAO-A inhibitor.³⁷ In contrast, the IC₅₀ of deprenyl HCl on MAO-A/B activity (1.49 μM), as measured by the S9-LCMS assay, closely matched the IC₅₀ for inhibiting MAO-B using a commercial kit (1.0 μM) (Figure S3B). Collectively, these findings suggest that the S9-LCMS assay exhibits greater sensitivity to MAO-B inhibition. Therefore, measured MAO-A/B inhibition of sarmentosin from this assay likely reflects its MAO-B inhibitory properties, with caution advised when inferring sarmentosin’s MAO-A inhibitory bioactivity from the S9-LCMS assay. Further research is required to elucidate whether sarmentosin is equally bioactive in inhibiting both MAO isoforms.

Figure 4.

Dose effect of sarmentosin extract, clorgyline hydrochloride, and deprenyl hydrochloride in inhibiting MAO-A/B enzyme activity using the S9-liquid chromatography–mass spectrometry assay. The concentration of each compound required to inhibit MAO-A/B activity by 50% of its maximal activity (IC₅₀) is indicated. Data are mean ± SD.

To our knowledge, this is the first report on the MAO-A/B inhibitory bioactivity of sarmentosin and its caffeoyl-, coumaroyl-, and feruloyl esters as well as the first published report of sarmentosin as a constituent in BCs. Sarmentosin has been identified in Crassulaceae species, Ribes fasciculatum var. chinense, Rhodiola species, and Ribes uva-crispa(38) and is known to be sequestered by lepidopteran species of Parnassius butterflies.³⁹ Additionally, the sarmentosin esters nigrumin-5-coumarate and nigrumin-5-ferulate have been identified in BC seeds and pomace,^40,41 and closely related β-hydroxynitriles were extracted from BC flowers.⁴² The lack of studies identifying sarmentosin in BC may be because of its high polarity and poor ultraviolet absorption sensitivity (λmax° = 210–212 nm), requiring specific solvent extraction protocols and analytical detection methodologies to detect. Indeed, our initial analysis of BC interventions failed to detect sarmentosin because of highly polar eluting compounds, including sugars, were diverted to waste to protect the integrity of the mass spectrometer, as is typical for LCMS analysis.

3.3. Bioavailability of Blackcurrant Anthocyanins and Sarmentosin

The anthocyanin and sarmentosin compositions of the placebo and BC interventions in both formats, along with relative and absolute amounts of these constituents consumed by participants in the BCJ and BCP doses, are detailed in Table S4. The peak bioavailability of total anthocyanins in plasma was measured at 120 min (T_max) post BCJ and BCP consumption, indicating a similar rate of anthocyanin absorption for both formats (Figure 5A). However, the peak concentration (C_max) of total anthocyanins varied between the two formats (BCJ = 3.2 ± 0.3 nm; BCP = 21.3 ± 4.2 nM). This reflects the total anthocyanin in a dose of BCP being approximately seven times greater than a dose of BCJ intervention (Table S4). No anthocyanins were detected in plasma samples from participants after consuming the placebos of the BCJ and BCP formats, as these did not contain measurable amounts of anthocyanins (Table S4).

Figure 5.

Concentration of total anthocyanins (A) and sarmentosin (B) in plasma over time after consuming a single dose of the blackcurrant juice and powder interventions. Values are mean ± SEM and total anthocyanins are calculated as cyanidin 3-O-glucoside (Cy-glu) equivalents.

The T_max of sarmentosin in plasma was observed at 120 min post BCJ and BCP consumption, with both interventions exhibiting similar C_max concentrations at this time point (BCJ = 603 ± 62 nM; BCP = 575 ± 134 nM) (Figure 5B). Plasma sarmentosin concentrations declined at comparable rates from T_max in both BCJ and BCP groups, reaching concentrations of 40.6 ± 3.7 and 41.6 ± 4.8 nM, respectively, at 480 min post-intervention consumption. Spiking experiments demonstrated the successful recovery and measurement of sarmentosin esters in plasma (data not shown); however, no endogenous levels of these compounds or their predicted phase II metabolites were detected in plasma for either BC format. Sarmentosin was not detected in plasma following the consumption of the placebo interventions, which did not contain any sarmentosin.

Characterizing sarmentosin bioavailability after BC consumption is important for linking its bioactivity with the inhibition of platelet MAO-B activity observed in this study and others.¹⁷ Despite the dose of sarmentosin in the BCJ being 2-fold higher than the BCP intervention (Table S4), the bioavailability of sarmentosin in both BC formats did not significantly differ. Phenolic compounds such as sarmentosin esters, likely undergo hydrolysis after ingestion by carboxylesterases, leading to the biotransformation of these esters to sarmentosin that then enter circulation.⁴³ Therefore, the comparable relative dose of combined sarmentosin (sarmentosin and sarmentosin esters) in BCJ and BCP, when normalized to bodyweight, may explain the equivalent sarmentosin bioavailability after consuming the BCJ and BCP beverages. If this is confirmed, then future clinical trials investigating the nootropic benefits of BCs should consider both sarmentosin and its esters when determining a suitable dose for participants.

Subsequent analysis revealed that both plasma sarmentosin and anthocyanins (nonmetabolized and metabolized cyanidin methyl ester glucuronide) concentrations were significantly positively correlated with platelet MAO-B inhibition (Figure 6A–C). The correlation of plasma sarmentosin with MAO-B inhibition was consistent for both BC formats. In comparison, the higher anthocyanin dose contained in the BCP intervention, which was reflected in the higher bioavailability of nonmetabolized and metabolized anthocyanins, did not translate to higher MAO-B inhibition than the BCJ intervention. These findings provide further support for sarmentosin and not anthocyanins as key constituents in BCs responsible for their MAO inhibitory bioactivity.

Figure 6.

Correlation of platelet monoamine oxidase (MAO)-B inhibition with the concentration of plasma sarmentosin (A), anthocyanins (B), and the anthocyanin phase II metabolite cyanidin methyl ester glucuronide (C) after consuming blackcurrant juice and powder formats. Shading represents to the 95% confidence interval for the line of best fit for the corresponding blackcurrant format.

Previous studies have investigated the MAO inhibitory bioactivity of plant phenolics (see review by Chaurasiya et al.⁴⁴). Among the examined compounds, berryfruit anthocyanins and their aglycones were found to exhibit moderate to weak MAO-A and MAO-B inhibitory bioactivity (IC₅₀: > 15 μM).¹⁵ Interestingly, naringenin, which is an important precursor for flavonoid biosynthesis, was found to be more selective in inhibiting MAO-B than MAO-A activity (IC₅₀: 0.27 and 8.64 μM, respectively).⁴⁵ Thus, it is likely that other BC phytochemicals contribute to the efficacy of BCs in inhibiting MAO-B activity, and the bioactivity of these compounds, relative to sarmentosin, requires further investigation.

3.4. Circulating Tyrosine- and Tryptophan-Derived Neurotransmitters

Given that MAO inhibition is a likely mechanism through which plant bioactives offer neurocognitive benefits, we investigated the effect of BC consumption on circulating neurotransmitters associated with the TYR and TRP metabolic pathways. These pathways are prominently regulated by both MAO isoforms and other metabolic enzymes that modulate the concentrations of key catecholamines in the brain, blood, and other organs. As both BCJ and BCP interventions comparably inhibited platelet MAO-B activity and their corresponding placebos had no inhibitory effect, data from both formats were combined to compare the effects of BC and placebo on circulating neurotransmitters and mood. Twenty-two neurotransmitters associated with TYR and TRP metabolic pathways were quantified in plasma collected at allocated time points following BC and placebo consumption (Table S5).

Overall, plasma TYR and TRP metabolites decreased from baseline in both BC and placebo groups following intervention consumption (Figure 7A,B). For TYR metabolites, a 40% reduction in plasma DA was observed 10 min after BC consumption, while a negligible change in this neurotransmitter was observed in the placebo group (Figure 7C). This trend was consistent with 3-MT, a methylated DA intermediate, and its major catabolite HVA, via MAO-A/B metabolism (Figure 7C,7E). Plasma DA remained significantly lower in the BC group compared with the placebo group at 20 min post-intervention consumption, with no further treatment differences observed at subsequent time points.

Figure 7.

Proportional changes in plasma neurotransmitter-associated metabolites from baseline (0 min). Heatmaps indicate relative changes in tyrosine (TYR)- (A) and tryptophan (TRP)-derived (B) metabolites during the time course of the study. Red and blue hues indicate higher and lower concentrations, respectively, when compared with baseline and placebo intervention (scaled by row). * and ** denote significant responses to the blackcurrant treatment at the corresponding time point (p < 0.1 and 0.05, respectively). Boxplots of proportional change in plasma metabolites that were significantly different when participants received the blackcurrant (BC) compared with the placebo (PLA) intervention at 10 and 20 min (C), and 120 and 240 min (D). Schematic representation of TYR (E) and TRP (F) pathways-derived neurotransmitters significantly changed following blackcurrant consumption. The up arrow represents an increase and down arrow decrease in proportional concentration from the baseline when compared with the placebo at the time points indicated. The monoamine oxidase (MAO)-A/B substrate preference where known is indicated by the increased size of the letter depicting the preferred enzyme. An asterisk indicates that the substrate is metabolized by MAO-B; albeit only slowly.⁵⁰

A reduction was also measured in plasma DHPG and MHPG 120 min following BC intervention but not placebo consumption. These compounds are metabolic products of NE and NM, respectively, via MAO-A/B metabolism (Figure 7D,E). Plasma VMA also decreased in the BC group at 240 min post-consumption. In comparison, plasma MHPG increased by 10 and 40% in both BC and placebo groups, respectively, at 240 min post-intervention consumption.

For TRP metabolites, contrasting trends in the proportional changes of plasma KY and 5-HT concentrations were observed after consuming the dietary interventions (Figure 7B). Plasma concentrations of KYN-associated metabolites (KA, XA, and QA) were at their lowest at 120 and 240 min post-BC and placebo consumption (Figure 7B). Conversely, an increasing trend in plasma 5-HT and its precursor 5-HTP was observed at 120 min in the BC group, but not the placebo group. A reduction in plasma 5-HIAA, a catabolite of 5-HT via MAO-A/B, was measured at 120 and 240 min post-BC consumption (Figure 7D,F).

Although preclinical studies have demonstrated the modulatory effects of polyphenols on 5-HT and DA in specific brain regions of rodents,^46,47 research on their influence on circulating neurotransmitters in humans is limited. Among these studies, consuming a single dose of BC juice corresponded with reduced DHPG and increased NM concentrations in plasma.^17,18 The decrease in plasma DHPG, which is largely dependent on the preferential metabolism of NE by MAO-A, suggests the MAO-A inhibitory bioactivity of BCs.⁴⁸ Conversely, the increase in plasma NM concentrations is attributed to a potential increase in NE metabolism by catechol-O-methyl transferase due to MAO-A inhibition (Figure 7E). Similar to previous studies, our findings show that significant changes in TYR- and TRP-related metabolites following BC consumption were primarily end products of MAO-metabolized neurotransmitters. Those that were modulated shortly after BC ingestion were mainly associated with DA metabolism and preferentially metabolized by MAO-B. Conversely, neurotransmitters modulated at latter time points were those preferentially metabolized by MAO-A or slowly by MAO-B (Figure 7E,7F). These findings largely align with those by Watson et al.,¹⁷ particularly regarding results for DHPG. While reduced concentrations of peripheral HVA, DHPG, MHPG, and 5-HIAA suggest MAO-A/B inhibition post-BC consumption, significant changes in their precursors DA, NE, or 5-HT were not observed, although positive trends were noted for 5-HT and 5-HTP.

3.5. Mood Modulation Post-Intervention Consumption

To investigate the effect of BC consumption on participant’s moods in relation to MAO-B inhibition, correlation clustering was performed on normalized mood descriptor scores (proportional change from 0 min) (Figure 8). The trend of normalized platelet MAO-B inhibition increased within 120 min, irrespective of treatment with the degree of MAO-B inhibition greater in the BC group peaking at 20 min after BC consumption and declining thereafter.

Figure 8.

Correlation cluster heatmaps visualizing the relationship between monoamine oxidase (MAO)-B inhibition with mood descriptors at allocated time points after consuming the blackcurrant (A) and placebo (B) interventions. Red and blue hues denote positive and negative correlations, respectively, and data were scaled on columns (time).

Mood descriptors clustered in five and seven groups following BC (Figure 8A) and placebo (Figure 8B) consumption, respectively. MAO-B inhibition clustered positively with mood descriptors “Proficient”, “Quick-witted”, “Relaxed”, and “Sociable” at 120 min post-treatment consumption in both treatment groups. These descriptors were also positively associated with MAO-B inhibition at 10 and 20 min after BC, but not placebo, consumption. This trend corresponded with similar associations with positive mood descriptors at 10 min (“Tranquil”, “Strong”, “Calm”, “Alert”, and “Contented”) and 20 min (“Clear-headed”, “Energetic”, and “Friendly”) after BC consumption. Negative mood descriptors (“Anxious”, “Stressed”, and “Mentally fatigued”) clustered in both treatment groups and were most strongly perceived at 240 min, coinciding with decreased MAO-B inhibition.

Overall, platelet MAO-B inhibition was negatively correlated with perceived “Mental fatigue” and positively correlated with mood descriptors related to “Alertness” (“Proficient”, “Strong”, “Quick-witted”, “Attentive”, and “Energy”) (Figure S4). Our findings indicate that BCs attenuate mental fatigue and support a more positive mood profile over 120 min after consumption, with correlations in positive mood most strongly perceived by participants within the first 20 min after BC consumption.

The small sample size of participants enrolled in each BC format is an important limitation to consider when interpreting mood and neurotransmitter data and associations made with MAO-B inhibition. This study was designed and powered to determine the effect of the BCP intervention on temporally inhibiting platelet MAO-B activity as the primary outcome. Nevertheless, these findings align with previous BC intervention studies that have reported similar associations between MAO-B inhibition and positive cognitive and mood outcomes in humans. BC-induced MAO-B inhibition corresponded with enhanced cognitive performance and reduced anxiety in healthy adults completing a prolonged cognitive task.^17,49 Additionally, BC consumption attenuated perceived exertion and supported a positive affective response during a mild walking exercise, which was inversely related to platelet MAO-B activity.¹⁶

3.6. Consuming Blackcurrant Sarmentosin Inhibits Platelet MAO-B Activity

To confirm our in vitro discovery indicating that sarmentosin is a key bioactive in BCs in inhibiting MAO-B activity, we conducted a pilot clinical study where platelet MAO-B activity was measured after participants consumed sarmentosin doses approximately equivalent to one and two doses of the BCJ intervention (Table S4). Blood samples were collected 2 and 4 h after treatment consumption, corresponding to maximal MAO-B inhibition and subsequent dissipation following BC consumption, respectively.

Our results confirm that consuming BC-derived sarmentosin significantly inhibited platelet MAO-B activity (Figure 9) with significant treatment (p < 0.001) and time (p < 0.001) effects measured for this outcome, and a significant treatment × time interaction (p < 0.001) also detected. Consuming both 42 and 84 mg sarmentosin similarly inhibited platelet MAO-B activity with significant MAO-B activity measured 2 h after sarmentosin consumption compared with baseline (0 h) activity.

Figure 9.

Platelet monoamine oxidase (MAO)-B activity before (0 min) and 2 and 4 h after consuming sarmentosin (42 mg and 84 mg) beverages and the corresponding placebo. Values are mean ± SEM. Data points labeled with * indicate significantly lower (p < 0.05) MAO-B activity from 0 h for the corresponding treatment group. Data points labeled with ∧ indicate significant difference (p < 0.05) in MAO-B activity between time points within the corresponding treatment group.

The effect of sarmentosin in inhibiting platelet MAO-B activity diminished in both sarmentosin treatment groups by 4 h after consumption, resulting in MAO-B activity comparable to baseline in both groups at this time point. However, the magnitude of this change varied between treatment groups, such that the difference in MAO-B activity between 2 and 4 h after sarmentosin ingestion was significant (p < 0.05) only when participants consumed 42 mg of sarmentosin. This suggests a dose-dependent effect in the duration of temporal platelet MAO-B inhibition by sarmentosin.

Taken together, this study is the first to report the efficacy of sarmentosin derived from BCs in inhibiting platelet MAO-B activity, mirroring the MAO-B inhibition following BC consumption presented in this study and by others [18]. These findings also confirm the significance of sarmentosin as a key constituent within BCs, contributing to the fruit’s MAO-B inhibitory bioactivity.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.4c03802.

• Blood glucose concentrations after consuming the blackcurrant interventions and their corresponding placebo (Figure S1), representative nuclear magnetic resonance spectra of sarmentosin purified from blackcurrant juice concentrate (Figure S2), dose effects of clorgyline hydrochloride and deprenyl hydrochloride in inhibiting monoamine oxidase (MAO)-A and MAO-B activity (Figure S3), and correlations of platelet MAO-B inhibition with neurotransmitter-associated metabolites and mood descriptors (Figure S4). Supplementary tables present multiple reaction monitoring transitions used for sarmentosin (Table S1 and S2) and anthocyanin analysis (Table S3), sarmentosin and anthocyanin composition in blackcurrant juice and powder interventions (Table S4), and neurotransmitter concentrations in plasma following consumption of dietary interventions (Table S5). Finally, the methodology for neurotransmitter analysis in plasma samples is described (PDF)

This research was cofunded by New Zealand’s National Science Challenge, High-Value Nutrition (HVN) “Boosting immune responses and cognitive support with A̅repa Nootropic Powder (HVN1921)” and AlphaGen Ltd., as a collaboration between The New Zealand Institute for Plant and Food Research Ltd. and AlphaGen Ltd. HVN was underwritten by New Zealand’s Ministry of Business, Innovation, and Employment (MBIE). The funders were not involved in the collection, analyses, or interpretation of data; the drafting of the manuscript; or in the decision to publish the results.

The authors declare no competing financial interest.