# Main urinary biomarkers of golden berries (Physalis peruviana) following acute and short-term nutritional intervention in healthy human volunteers

Fabrice Vaillant, Sandra Milena Llano Gil, Alberto Ángel Martín, Natalia Moreno-Castellanos

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Fabrice Vaillant, Sandra Milena Llano Gil, Alberto Ángel Martín, Natalia Moreno-Castellanos. Main urinary biomarkers of golden berries (Physalis peruviana) following acute and short-term nutritional intervention in healthy human volunteers. Food Research International, 2023, 173 (2), pp.113443. 10.1016/j.foodres.2023.113443. hal-05181769

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# Main urinary biomarkers of golden berries (Physalis peruviana) following acute and short-term nutritional intervention in healthy human volunteers

Fabrice Vaillant<sup>a,b,c,*</sup>, Sandra Llano<sup>a</sup>, Alberto Ángel Martín<sup>d</sup>, Natalia Moreno-Castellanos<sup>e</sup>

## ARTICLE INFO

**Keywords:**

Physalis peruviana

Withanolides

Sesquiterpenoids

## ABSTRACT

The metabolites entering the bloodstream and being excreted in urine as a result of consuming golden berries are currently unidentified. However, these metabolites potentially underlie the health benefits observed in various in vitro, animal, and human models. A nutritional intervention with 18 healthy human volunteers was performed, and urine was collected at baseline and after acute and short-term fruit consumption for 19 days. After UPLC-ESI/QToF-MS analysis, untargeted metabolomics was performed on the urine samples, and from the 50 most discriminant ions (VIP > 2) generated by a validated PLS-DA model (CV-ANOVA = 3.7e-35; R²Y = 0.86, Q²Y = 0.62 and no overfitting), 22 compounds were identified with relatively high confidence. The most discriminant metabolites confirmed by DHS/GC-MS² analysis of volatiles in urine were sesquiterpenes (C₁₅H₂₂): 3 stereoisomers, β-vatirenene, β-vetivenene, and β-vetispirene, and 2 isomers, eremophila-1(10), 8,11-triene and α-curcumene. Another major urinary biomarker was 4β-hydroxywithanolide E and its phase II derivatives, which were observed in urine for all individual up to 24 h after the fruit was consumed; thus, the bioavailability of this biomarker in humans was demonstrated for the first time. Additionally, the excretion of certain acylcarnitines and hypoxanthine in urine increased after golden berry consumption, which may be associated with a detoxifying effect and may occur because fats were utilized rather than carbohydrates to meet the body's energy needs. The main biomarkers of golden berry consumption are specific to this fruit, confirming its potential for the functional food market.

## 1. Introduction

Golden berries are consumed worldwide, although commercial production is concentrated in only a few countries. Colombia is currently the world's leading producer and exporter of the fruit (Gomez et al., 2022). Formerly, golden berries were used almost exclusively in importing countries as decoration for dishes and desserts; however, the fruit is increasingly being consumed for its taste and nutritional and health benefits, which are becoming known to the general public. In fact, the golden berry has recently been labeled a "superfruit", a fruit with potential health-enhancing effects (Kupska et al., 2016), and there

is increasing interest in its various unique nutritional constituents. Numerous reviews on the chemical composition and potential functional properties of golden berries have been conducted within the last decade (Puente et al., 2011; Olivares-Tenorio et al., 2016; Ramadan, 2011; Kasali et al., 2021, 2021; Puente et al., 2019; Ramadan, 2019). In summary, the fruit stands out for its content of withanolides, sucrose esters and terpenoids, three emblematic compounds of the Physalis species. The fruit is also an important source of other more common compounds, such as β-carotene (Etzbach et al., 2018); flavonols, mainly rutin and myricetin (Licodedoff et al., 2013); and phenolic acids. However, withanolides, sucrose esters and terpenoids are most often

<footnote><sup>a</sup> Corporación Colombiana de Investigación Agropecuaria – Agrosavia. Centro de Investigación La Selva, Kilómetro 7, Vía a Las Palmas, vereda Llano Grande, Rionegro Antioquia, Colombia</footnote>

<footnote><sup>b</sup> French Agricultural Research Centre for International Development (CIRAD), UMR Qualisud, Rionegro (Ant.), Colombia</footnote>

<footnote><sup>c</sup> Joint Research Unit—UMR Qualisud, Univ Montpellier, Univ. d'Avignon, CIRAD, Institut Agro, IRD, Université de La Réunion, Montpellier, France</footnote>

<footnote><sup>d</sup> Observatorio Epidemiológico de Nutrición y Enfermedades Crónicas/OENEC, School of nutrition and dietetics, Health Faculty, Universidad Industrial de Santander, Bucaramanga, Colombia</footnote>

<footnote><sup>e</sup> Centro de Investigación en Ciencia y Tecnología de Alimentos/CICTA, Department of Basic Sciences, Health Faculty, Universidad Industrial de Santander, Bucaramanga, Colombia</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail addresses: Fabrice.vaillant@cirad.fr (F. Vaillant), sllano@agrosavia.co (S. Llano), angelmar@uis.edu.co (A. Ángel Martín), nrmorcas@uis.edu.co (N. Moreno-Castellanos).</footnote>
cited as the primary compounds potentially responsible for the func-
tional health qualities attributed to golden berries.

Withanolides belong to a group of compounds characterized by a 28-
carbon (C28) steroidal backbone based on an ergostane structure bound
to a lactone. There is a large variety of withanaloid compounds, but
mainly derivatives of withanolide E and withanolide D were reported in
the fruits. Physalolactone, which is a glycosylated withanaloid, and
physagulin, another kind of withanaloid, have also been reported in
fruits (Navarro-Hoyos et al., 2022). However, to date, it has remained
unproven whether withanolide compounds are bioavailable. The other
specific constituents of Physalis fruits are sucrose esters, a group of
sugars esterified with saturated fatty acids. The main sucrose esters re-
ported in Physalis are O-isobutanoylsucrose, di- and o-tri-o-iso-
butanoylsucrose and their derivatives. Finally, terpenoids are volatile
and often determine the flavor and taste of fruits. In golden berries, all
families of terpenes with 10 and 15 carbons are present Therefore, it is
crucial to determine the bioavailability of these compounds and to
identify related biomarkers in urine that are indicative of their intake.
This information would establish their potential for bioactivity within
the body.

Biomarkers of dietary intake have emerged as a significant and contemporary subject within the realm of food science. This advancement has been facilitated through nutrimetabolomic research, which involves the combination of metabolomic tools with dietary interventions conducted directly on human volunteers. However, despite a limited number of published studies on this subject, they remain insufficient in light of the extensive diversity of food products. Specific biomarkers from golden berry remain largely unexplored, even though their understanding is crucial for comprehending the potential health-functional attributes.

Untargeted metabolomics using high-throughput high-resolution mass detectors can highlight differences in metabolite composition between different biofluid states. Various biofluids can be used for this purpose, primarily plasma, serum, and urine. Urine has the advantage of being an easily accessible biological fluid that contains a considerable range of excreted metabolic by-products, including dietary compounds. It also has the enormous advantage of concentrating these exogenous metabolites and facilitating their continuous recovery over a long period, whereas blood sampling can only offer a snapshot at a given point in time.

A better understanding of the biomarkers of golden berry intake
would significantly increase in the value of this fruit and open new
markets, such as that of functional foods.. The objective of this study
focuses on discovering these main urinary biomarkers of golden berry
consumption through a nutritional intervention study with healthy
human volunteers.

## 2. Methods

2.1. Materials

The plant material utilized in this experiment was exclusively
sourced from the Dorada variety, registered by the Colombian research
institute AGROSAVIA. The plants originated from a plantation situated
in San Vicente, Antioquia (Colombia), at an elevation of 2161 m above
sea level. Typically designated for export, these fruits were cultivated
and managed in adherence to global standards. They were provided by
the company Caribbean Exotics (Rionegro, Antioquia, Colombia), and
subsequently packaged in perforated polypropylene containers of 250 g
and 150 g, which were distributed to the volunteers.

## 2.2. Clinical trial

2.2.1. Volunteers

A group of 18 volunteers participated in this single-blind nutritional
intervention, which has been described in detail elsewhere (Vaillant

et al., 2021; Sierra et al., 2022). This nutritional intervention was pre-
viously approved by the ethical committee of the Institute of Health
Sciences of CES University (Medellin, Colombia; ethical ID: 707, 20
September 2017) and registered with the International Clinical Platform
(https://rpcec.sld.cu/trials/RPCEC00000268-En; accessed on 17 April
2018). Participants were assured of anonymity and confidentiality. All
participants provided written informed consent. Briefly, the study
involved the following periods: (1) washing (one week without golden
berry consumption); (2) 12 h fasting baseline, in which placebo (250 ml
of water sweetened with 25 g of sugar) was ingested at time 0 (Day 0)
and urine was collected from 0 to 6 h, 6 h to 24 h, and at 24 h; (3) 12 h
fasting before acute consumption of 250 g of golden berries at time
0 (Day 1) and collection of urine from 0 to 6 h, 6 h to 12 h, and 12 h to 24
h; (4) short-term intervention, free living with daily consumption at any
time of the day of 150 g of golden berries for 18 days; and (5) on Day 19
at time 0, after 12 h fasting, the same placebo from Day 0 was ingested
and followed by collection of urine from 0 to 6 h, 6 h to 12 h, and 12 h to
24 h. The quantities of fruit to be consumed were established in
collaboration with the volunteers as part of a preliminary study. During
this process, a maximum acceptable quantity of 250 g for acute con-
sumption and 150 g for daily consumption over several days were
deemed appropriate. On the days of sampling (Days 0, 1 and 19), the
same standardized meals were provided to all the participants (break-
fast, lunch, and dinner). The participants, employed by Nutresa com-
pany (Medellin, Colombia) were selected based on their commitment to
exclusively utilizing the company's 24/7 catering services for their
meals during the sampling phase. They were also given the option to
bring home meals from the same restaurant. This method guaranteed a
dependable provision of standardized meals throughout the sampling
phase. Urine samples were collected in polypropylene bottles (1000 ml),
which were immediately stored at 4 °C in a cool box during the collec-
tion phase. All urine was collected and pooled between consecutive time
points 0 and 6 h, 6 h and 12 h, and 12 h and 24 h. Aliquots of pooled
urine at each time point were centrifuged and then stored at -80 °C until
analysis.

## 2.3. Metabolomic analysis

2.3.1. Sample treatment

Published guidelines for nutrimetabolomic studies (Ulaszewska
et al., 2019) were strictly followed. Briefly, the thawed urine was
centrifuged at 5000 g for 15 min, and a milliliter of supernatant was
collected, to which 1 ml of Milli-Q water was added. Subsequently, the
samples were filtered under pressure with microporous regenerated
cellulose filters with a size of 0.22 µm (Minisart-R 25). A pool of all
collected urine samples was prepared for use as quality control (QC) and
drift correction. The samples and pools were injected into the HPLC-MS
with injection of 2 quality controls (pools) for every 10 sample
injections.

2.4. UPLC-ESI/QToF-MS analysis

Ultra-performance liquid chromatography coupled to an electro-
spray ionization source and a quadrupole time-of-flight mass spec-
trometry detector (UPLC/ESI-QToF-MS) was used. Chromatographic
separation was performed using a 1.7 µm, 2.1 * 100 mm CSH 18 column
(Acquity®) coupled to a 1.7 µm Vanguard CSH 18 precolumn in a UPLC
(Waters) equipped with an automatic sampler and a quadrupole time-of-
flight detector (ESI/QToF-MS). An elution gradient was employed using
water (solvent A) and acetonitrile (solvent B), and both were adjusted
with 0.1% (w/v) formic acid. The elution started at 0% B and continued
as follows (time in min, concentration of B in % (v/v)): (initial, 100; (7,
10); (22, 95); (22,1; 0) and ended with a rebalancing stage (26. 0). The
flow rate was adjusted to 0.45 ml/min with a total running time of 26
min. The temperature of the column was maintained at 30 °C, and the
temperature in the autosampler chamber was controlled at 10 °C. The
volume of samples injected was 2 µL.

Positive and negative ESI were selected for full-scan (m/z 100–1500) data acquisition in continuous mode. The operating conditions of the ESI-QToF-MS were capillary voltage = 0.4 kV and sampling cone voltage = 40 V. Nitrogen was used as the cone gas and desolvation gas with flows of 0 L/h and 1000 L/h, respectively. Additionally, mass spectrometry analyses were performed with a constant collision energy of 6 eV. The scan time was adjusted to 0.5 s. The equipment was calibrated with leucine enkephalin (556.2771 Da) with an infusion period of 10 s and a flow of 12 µL/min. Some samples were reinjected to obtain the MS2 fragmentation pattern of selected ions. The MS2 fragmentation spectra were obtained through a complete scan from 50 to 1000 Da at the following collision energies: 10 eV, 20 eV, 30 eV, 40 eV and 50 eV in the case of ions with molecular weights >500 m/z. They were compared to the ESI-MS2 of authentic standards published in the literature or to those deposited in databases, such as Massbank of North America (MoNA, https://mona.fiehnlab.ucdavis.edu/) and HMDB (The Metabolomics innovation Center, University of Alberta, Canada).

## 2.5. DHS/GC-MS/MS analysis

Analysis by dynamic headspace solid-phase microextraction coupled to gas chromatography and mass spectrometry at different fragmentation energies (DHS/GC-MS2 analysis) was performed on urine spots. Five milliliters of urine was placed in a 10 ml glass vial. The sample was equilibrated to 30 °C, and then the headspace was swept with a nitrogen flow at 1 ml/min and stirred at 500 rpm. The volatile compounds were trapped on a Tenax TA using a Gerstel autosampler (Gerstel, Mülheim a der Ruhr, Germany) and dried with an additional purge flow at 100 ml/min at 50 °C for 2.5 min to remove residual water. The collected volatile compounds were then desorbed using an automatic thermal desorption unit (TDU) maintained at 30 °C (0.4 min) and then increased to 300 °C at 120 °C/min. The desorbed compounds were transferred to a CIS4 cooled injection system, in which compounds were cryofocused. The CIS4 temperature was raised from -10 °C to 300 °C at 12 °C/s and held for 5 min. Desorption and analysis were carried out on a GC-MS using a tandem gas chromatograph 7890B/MSD 5977 system (Agilent Technologies, Santa Clara, USA). Volatiles were analyzed on a DB-Wax column (60 m × 250 µm × 0.25 µm). Mass spectra were recorded in EI + mode at 70 eV within a range of 40 to 350 Da with a solvent delay time of 2 min and a scan speed of 4.52/s. The analyzer and source temperatures were 150 °C and 250 °C, respectively. Mass spectrometry data were analyzed using MassHunter software version B.08.00. Volatile compounds were identified by comparing their GC-MS/MS mass spectra to the NIST 08 library (Wiley, New Jersey, USA) using a match factor to assess similarity (calculated by MassHunter software). A match factor > 700 was used to decide whether a given peak was identified with relatively high confidence. A reverse match (RMatch) factor was also calculated, and it was required to be >800. RMatch is calculated by ignoring the peaks in the unknown's spectrum that are not in the library's known reference spectrum. Linear retention indices were calculated on capillary columns coated with polar stationary phase (DB-Wax) and compared with those of authentic reference standards reported in databases and literature, run on polar stationary phase under similar conditions.

## 2.6. Data processing

To center the chromatographic peaks, the measurements were taken in continuous mode; therefore, this information was retrospectively based on the *m/z* of the external reference mass. This was done with the MassLynx V3.1 SCN 639 program (Waters Inc., Milford, Massachusetts, USA). To continue with the analysis, the program Progenesis QI (Nonlinear Dynamics, Newcastle, UK) was used. In this stage, mass detection, deconvolution and alignment among samples were performed. Once the list of ions was obtained, the UPLC-ESI/QToF-MS

equipment drift was corrected using LOESS-fit signal correction (Thonnusin et al., 2017) by adjusting the intensity of the samples, accounting for the drift in the variable from quality control (pools). Then, only ions with a coefficient of variation lower than 30% in the quality control samples were selected. Additionally, only ions that were found in at least 80% of individuals from either group were examined.

## 2.7. Biological association networks

Datasets containing metabolite identifiers and their corresponding values (fold change) from the metabolomics experiment were uploaded into Ingenuity Pathway Analysis software (Ingenuity® Systems, https://www.ingenuity.com) to identify potential interactions with other biological molecules, as described previously (Vaillant et al., 2021). The *p* values were calculated using Fisher's exact test to determine the probability that the association between the metabolites in the dataset and the top pathway was due to chance alone. Data sources from Ingenuity expert findings were used with the “Core Analysis” function to interpret the data from the perspective of biological processes, pathways, and networks. For canonical pathway analysis, disease, and function, a -log (*p* value) > 2 was taken as the threshold, a Z score > 2 was defined as the threshold of significant activation, and a Z score < -2 was defined as the threshold of significant inhibition. Differentially expressed metabolite identifiers were defined as value parameters for analysis, and the relationship between metabolite alterations and related changes in biofunctions in the subcategories of molecular and cellular functions, physiological system development and function, and disease and disorders was identified. Differentially expressed metabolites with *p* < 0.05 were overlaid onto global molecular networks developed from information contained in the IPA knowledge base. Networks were then algorithmically generated based on their connectivity.

## 2.8. Statistical analysis

The ion data base obtained by UPLC-ESI/QToF-MS analysis was used to construct an experimental space that was explored by principal component analysis (PCA) using the SIMCA 15.0.2 program (Sartorius Stedim Data Analytics AB, Sweden). The space was then subdivided into groups of samples corresponding to baseline, acute consumption and short-term consumption of golden berries. These groups were subsequently subjected to a discriminant analysis of partial least squares (PLS-DA) after orthogonal signal correction (OSC). The acceptability of the model was validated by means of different proprietary parameters and ad hoc tests such as CV-ANOVA (Eriksson et al., 2008) and the test of permutations. This last test compares the goodness of fit (R²Y and Q²Y) of the original model with other models constructed by random permutations (n = 100) of the Y observations, and it is represented by a permutation plot. Variable importance of the projection (VIP) was calculated for each variable (ion) by summing the squares of the PLS loading weights, weighted by the amount of the sum of squares explained by each model component (Farrés et al., 2015). Variables (ions) were ordered according to the highest VIP among variables between baseline and acute and between baseline and short term with a *p* value <0.05 and with a *p* value inferior to their Benjamini-Hochberg critical *p* value (Glueck et al., 2008) calculated with a false discovery rate of 5%. For biological association networks, canonical pathway (CP) analysis was used to identify function-specific metabolites significantly present within the networks.

# 3. Results and discussion

All urine datasets obtained by UPLC-ESI/QToF-MS were analyzed using orthogonal signal correction-partial least square-discriminant analysis (OSC-PLS-DA). The model generated by comparing 3 groups, baseline (Day 0) after the washout period, acute consumption on Day 1,
and Day 19 after 18 days of constant consumption of golden berries, was deemed highly reliable by the usual tests (CV-ANOVA = 3.7e-35; R^2Y = 0.86, Q^2Y = 0.62, permutation test plot (n = 100) does not show overfitting (Figure S1)). Then, the OSC-PLS-DA model allowed us to establish a list of the top 50 discriminant ions among the three groups, which are presented in Table 1 and ordered by VIP scores. P values comparing baseline (B) with acute consumption (A) and baseline (B) with short-term consumption (S) are also presented as well as the average intensity in each group.

As shown in Table 1, the main discriminant ion in the PLS model after Physisalis peruviana intake is 203.18 [M + H], which appears in Table 1 at 8 different retention times (rank 1, 3, 5, 6, 19, 23, 35, and 42). The assigned formula C15H22 corresponds to sesquiterpenoid isomers with a double bond. To determine the identities among various possible C15H22 sesquiterpene isomers with a higher level of confidence, a DHS/ GC-MS2 analysis of a urine sample collected 6 h after Physisalis peruviana ingestion was performed. This analysis confirmed the presence of eight C15H22 isomers, and among them, five were identified with high confidence (match value > 700) by comparison of their GC-MS2 spectra with authentic standards reported in the NIST database (similarity factor represented by polar linear retention indices (Table 2)): α-curcumene; the 3 stereoisomers β-vatirenene, β-vetivenene, and β-vetispirene; and eremophila-1(10),8,11-triene (Fig. 1).

All of these isomers, except *m/z* 203.1807 at RT 10.53, exhibit the same LC-MS2 fragmentation pattern (Table 1), corresponding to the bicyclic sesquiterpenoid structure with an eremophilane backbone (Fig. 1). Alpha-curcumene, detected in urine by DHS/CG-MS, was tentatively attributed to ion *m/z* 203.1807 at RT 10.53 because of the absence of the *m/z* 175.15 and 161.13 fragments typical of the bicyclic structure of the eremophilane backbone (Fig. 2).

All C15H22 sesquiterpenoids present a very similar pattern of excretion (Fig. 3), and they appeared in all individuals in urine collected between 0 and 12 h after fruit intake. This means that this compound is readily bioavailable with probable absorption in the upper GI tract, and it is cleared quite quickly almost 12 h after ingestion, even after almost 3 weeks of daily consumption.

Other ions among the top discriminant ions correspond to phase I and II reactions that occur in vivo to increase the polarity and the excretion of C15H22 sesquiterpenes. This includes ion *m/z* 413.21 [M + H] (rank 4) that shows a main fragment at *m/z* 219.17 consistent with glucuronide loss (-194.04 amu) and corresponds to the formula C15H22O, which can be attributed to a hydroxy or epoxy form of C15H22 sesquiterpenoids after hydroxylation or epoxidation of the alkene by human cytochrome P450. Experimental MS2 spectra with main fragments at *m/z* 201.16 and 135.11 confirm this assignment when compared to MS2 spectra generated in silico (Wang et al., 2021; Figure S2). Consequently, *m/z* 413.21 [M + H] can be tentatively attributed to the glucuronide form of the hydroxy or epoxy-β-vatirenene stereoisomers. Another ion corresponding to two phase I reactions is *m/z* 237.18 [M + H] (rank 9; C15H24O2), which has an MS2 spectrum similar to those of the preceding compound and shows a fragment at *m/z* 219.17 following the loss of a water molecule. This ion can be attributed to dihydroxy or hydroxy-epoxy-sesquiterpene.

Therefore, C15H22 sesquiterpenoid isomers and derivatives are among the most important biomarkers of golden berry intake, and their concentration peaks in urine soon after fruit ingestion. Sesquiterpenes are secondary metabolites of plants that, in addition to giving the fruit its specific aroma and slightly bitter taste, also have interesting functions for health, including anti-inflammatory, cardiovascular, gastro-protective and anticancer properties (Durán et al., 2021). Among the C15H22 sesquiterpenes, curcumene has been the most studied compound for its health effect, and it was found that curcumene has the potential ability to modulate cancer, neurodegeneration, diabetes, and inflammation (Obrenovich et al., 2010). Some authors have shown that curcumene (a derivative of curcumin) inhibits the activation of NF-κB and downstream signaling, reducing inflammatory stress and

concomitant cytokine expression from adipose tissue, such as visceral fat (Gonzales and Orlando, 2008). Additionally, some data suggest that curcumene may improve cardiovascular function and insulin sensitivity (Obrenovich et al., 2010), which could also provide some clues to confirm the impact of golden berry consumption on insulin-associated signaling pathways observed previously in this same study (Vaillant et al., 2021).

The next discriminant ion is *m/z* 260.18 [M + H] (rank 7), which was assigned to L-hexanoyl carnitine with the formula C13H25NO4 according to the fragmentation pattern, which is in agreement with MS2 spectra published for the authentic compound by Mass bank (MoNA). L-hexanoyl-carnitine belongs to the acylcarnitine group, with the prominent fragment ion at *m/z* 85.03 reported in common for all acylcarnitines (Giesbertz et al., 2015). In fact, other acylcarnitines, such as undecanoyl-carnitine (*m/z* 330.26 [M + H], rank 14) and 3-hydroxynonanoyl carnitine (*m/z* 318.22 [M + H], rank 47), are among the top 50 most discriminant ions, and they were tentatively identified against the MS2 spectra of authentic compounds published by the Mass Bank (MoNA; Table 1). For all these acylcarnitines, urinary excretion appeared to be impaired immediately after fruit consumption, with an increase of >10-fold, in the case of L-hexanoyl-carnitine, in urine fractions collected up to 12 h. After 12 and 24 h, the excretion of acylcarnitines decreased and returned to normal in the urine collected 24 h after short-term consumption of fruit (Fig. 4).

Notably, healthy volunteers who participated in the study still consumed their normal diet with an estimated daily intake above 100 g of animal protein. The study was supervised by a nutritionist who assessed this quantity by reviewing food records and administering a questionnaire to volunteers. The concomitant consumption of golden berries appeared to increase the excretion of acylcarnitines, which is generally associated with the use of fat over carbohydrates to produce body energy (Stephens and Galloway, 2014). Increased urinary excretion of acylcarnitines is related to the enhancement of fatty acid oxidation and CoA recycling by removing short-chain acyl groups that accumulate in mitochondria. This increases free CoA levels, which are then available to continue lipid β-oxidation and the Krebs cycle, thereby releasing more ATP (Tonazzi et al., 2021). The esterification of L-carnitine to form acylcarnitine derivatives is part of the metabolism of fatty acids and the synthesis of urea under negative energy balance. Interestingly, the increased urinary excretion of acylcarnitine metabolites suggests a possible detoxifying effect in the elimination of ammonia, hypoxanthine and uric acid; therefore, it plays an important role in the catabolism of purines, avoiding the toxic effect of nitrogenous products (Tonazzi et al., 2021). The presence of purine bases, such as hypoxanthine, among the top 50 biomarkers after golden berry intake tends to confirm this statement. Hypoxanthine sulfate (C5H4N4O + SO3) was identified in negative mode (ion *m/z* 214.99 [M-H + SO3] (rank 38)) by comparison of experimental MS2 spectra with MS2 spectra of the authentic compound (MoNA). When biomarkers of golden berry intake and their fold change were uploaded to IPA software for bioinformatic computation analysis, a significant connection (p < 0.05) and effects on the life cycle related to canonical pathways, such as the mitochondrial L-carnitine pathway (Fig. S3A), L-carnitine biosynthesis, adenine and adenosine salvage III, and adenosine nucleotide degradation II (Fig. S3B), were predicted.

The next most discriminant ion corresponded to 606.27 *m/z* [M + H] (rank 10) at RT 11.4 min, but it also had another discriminant isomer at RT 12.08 min (rank 17). Additionally, two isomers of ion *m/z* 624.28 [M + H + H2O] (rank 16 and 18) were observed. The MS2 fragmentation patterns of all these ions were similar, and they exhibited a main fragment at *m/z* 169.08 [M + ] corresponding to γ-lactone ring loss followed by fragments typical of the fragmentation patterns of withanolides (Musharraf et al., 2011). These fragments were *m/z* 449.24 [M + H-3H2O] and 431.23 [M + H-4H2O] due to loss of water molecules, *m/z* 299.16 [M + H-Lact] due to the loss of lactone moieties and *m/z* 281.15 [M + H-Lac-H2O] and *m/z* 263.15 [M + H-Lac-2H2O] due to the loss of
<table><caption>**Table 1**

List of the most discriminant Ion (VIP > 2) in human urine considering an OSC-PLS model considering base-line, acute and short term consumption of golden berries.</caption><thead><tr><th rowspan="2">Rank</th><th rowspan="2">Obs m/z<sup>1</sup><br/>[M + H]/<br/>[M-H]</th><th rowspan="2">RT<br/>(min)</th><th rowspan="2">Formula</th><th rowspan="2">VIP score</th><th rowspan="2">Tentative identification</th><th rowspan="2">M</th><th rowspan="2">Error<br/>(ppm)</th><th rowspan="2">Main MSMS fragments (% mas peak)</th><th colspan="2">P-Value</th><th colspan="3">Average relative intensity</th></tr><tr><th>B/A</th><th>B/C</th><th>B</th><th>A</th><th>S</th></tr></thead><tbody><tr><td>1</td><td>203,1807</td><td>11,54</td><td>[C15H22 + H]</td><td>4,00</td><td>Sesquiterpene isomer</td><td>202.1730</td><td>4.5</td><td>203.18 (20); 161.13 (30) 147.11 (100); 119.10 (60); 105.07 (60); 133.107 (40); 91.05 (20)</td><td>2,4E-07</td><td>4,0E-04</td><td>&lt;50</td><td>12.490</td><td>490</td></tr><tr><td>2</td><td>552,3488</td><td>13,42</td><td></td><td>3,90</td><td>Unknown</td><td></td><td></td><td>130.05 (100); 495.27 (60); 439.28 (60); 375.17 (60); 331.18 (90)</td><td>1,6E-09</td><td>5,0E-05</td><td>&lt;50</td><td>535</td><td>165</td></tr><tr><td>3</td><td>203,1807</td><td>10,53</td><td>[C15H22 + H]</td><td>3,80</td><td>α-curcumene</td><td>202.1730</td><td>4.5</td><td>146.11 (100); 119.10 (60); 105.07 (60); 133.107 (40); 91.05 (20)</td><td>1,2E-07</td><td>1,6E-02</td><td>&lt;50</td><td>327</td><td>&lt;50</td></tr><tr><td>4</td><td>413,2168</td><td>11,88</td><td>[Glu-C15H22 + H + H2O]</td><td>3,80</td><td>hydroxy or epoxy-sesquiterpene glucuronide</td><td>412.2096</td><td>1.4</td><td>219.17 (20); 201.16 (20); 135.11 (100); 107.08 (90); 93.07 (90)</td><td>2,9E-10</td><td>3,2E-03</td><td>&lt;50</td><td>5 257</td><td>1 665</td></tr><tr><td>5</td><td>203,1807</td><td>9,62</td><td>[C15H22 + H]</td><td>3,80</td><td>Sesquiterpene isomer</td><td>221.19</td><td>4</td><td>203.0180 (30); 147.11 (100); 119.10 (90); 105.07 (80); 133.107 (50); 91.05 (20)</td><td>1,2E-06</td><td>4,9E-03</td><td>&lt;50</td><td>488</td><td>&lt;50</td></tr><tr><td>6</td><td>203,1807</td><td>11,12</td><td>[C15H22 + H]</td><td>3,70</td><td>Sesquiterpene isomer</td><td>202.1730</td><td>4,5</td><td>147.11 (100); 119.10 (90); 105.07 (80); 133.107 (50); 91.05 (20)</td><td>2,5E-06</td><td>8,0E-05</td><td>&lt;50</td><td>417</td><td>&lt;50</td></tr><tr><td>7</td><td>260,1872</td><td>7,51</td><td>[C13H26NO4 + H]</td><td>3,70</td><td>L-hexanoyl carnitine</td><td>259.1784</td><td>6</td><td>85.03 (100); 201.11 (20); 144.10 (10)</td><td>1,1E-10</td><td>1,8E-03</td><td>9 979</td><td>74 579</td><td>28 925</td></tr><tr><td>8</td><td>552,3459</td><td>12,8</td><td></td><td>3,60</td><td>Unknown</td><td></td><td></td><td>130.05 (100); 495.27 (60); 439.28 (60); 375.17 (60); 331.18 (90)</td><td>3,9E-06</td><td>3,1E-01</td><td>&lt;50</td><td>168</td><td>&lt;50</td></tr><tr><td>9</td><td>237,1848</td><td>13,02</td><td>[C15H2402 + H]</td><td>3,40</td><td>Dihydroxysesquiterpene or hydroxy-epoxy-sesquiterpene</td><td>237.1849</td><td>0</td><td>219.1767 (30); 191.1150 (30); 147.1158 (100); 95.1027 (50)</td><td>5,1E-06</td><td>3,1E-01</td><td>207</td><td>825</td><td>259</td></tr><tr><td>10</td><td>606,2719</td><td>11,41</td><td>[C28H38O8-Cystein + H]</td><td>3,40</td><td>4β-hydroxywithanolide E-cystein</td><td>605.2657</td><td>1.3</td><td>30 eV: 169.08 (100); 281.15 (80); 299.16 (60); 449.23 (10); 123.08 (50); 125.1 (30)</td><td>3,9E-06</td><td>5,5E-04</td><td>&lt;50</td><td>502</td><td>65</td></tr><tr><td>11</td><td>276,0823</td><td>10,44</td><td></td><td>3,40</td><td>Unknown</td><td></td><td></td><td>201.0469 (100); 183.0359 (50)</td><td>4,9E-01</td><td>3,4E-05</td><td>885</td><td>1009</td><td>2220</td></tr><tr><td>12</td><td>778,4536</td><td>12,84</td><td></td><td>3,30</td><td>Unknown</td><td></td><td></td><td></td><td>5,0E-08</td><td>4,1E-02</td><td>&lt;50</td><td>388</td><td>&lt;50</td></tr><tr><td>13</td><td>163,0398</td><td>10,44</td><td>[C9H6O3 + H]</td><td>3,30</td><td>Hydroxycoumarine</td><td>162.0317</td><td>1.8</td><td>77.046 (100); 107.04 (40); 91.05 (30); 119.05 (10)</td><td>&gt;0.05</td><td>4,3E-06</td><td>&lt;50</td><td>15</td><td>74</td></tr><tr><td>14</td><td>330,2635</td><td>10,2</td><td>[C18H35NO4 + H]</td><td>3,20</td><td>Undecanoyl carnitine</td><td>329,2566</td><td>-2,7</td><td>130.06 (100); 85.03 (20)</td><td>&gt;0.05</td><td>3,7E-04</td><td>374</td><td>550</td><td>1 157</td></tr><tr><td>15</td><td>568,3397</td><td>12,43</td><td></td><td>3,10</td><td>Unknown</td><td></td><td></td><td></td><td>4,9E-07</td><td>1,5E-02</td><td>&lt;50</td><td>105</td><td>&lt;50</td></tr><tr><td>16</td><td>624,2806</td><td>11,41</td><td>[C28H38O8 + Cystein + H + H2O]</td><td>3,09</td><td>4β-dihydroxywithanolide E cystein</td><td>623.2762</td><td>5.4</td><td>606.27 (50); 169.08 (100); 281.15 (80); 299.16 (50)</td><td>2,0E-04</td><td>3,7E-01</td><td>&lt;50</td><td>1565</td><td>319</td></tr><tr><td>17</td><td>606,2732</td><td>12,09</td><td>[C28H38O8 + Cystein + H]</td><td>3,08</td><td>4β-dihydroxywithanolide E cystein</td><td>605.2657</td><td>1.3</td><td>30 eV: 169.08 (100); 281.15 (80); 299.16 (60); 449.23 (10); 123.08 (50); 125.1 (30)</td><td>5,8E-06</td><td>8,9E-02</td><td>&lt;50</td><td>664</td><td>185</td></tr><tr><td>18</td><td>624,2806</td><td>11,64</td><td>[C28H38O8 + Cystein + H + H2O]</td><td>3,07</td><td>4β-dihydroxywithanolide E cystein</td><td>623.2762</td><td>5.4</td><td>606.27 (50); 169.08 (100); 281.15 (80); 299.16 (50)</td><td>8,1E-04</td><td>8,7E-03</td><td>&lt;50</td><td>1 393</td><td>180</td></tr><tr><td>19</td><td>203.1809</td><td>13,3</td><td>[C15H22 + H]</td><td>3,07</td><td>Sesquiterpene isomer</td><td>202.1730</td><td>4.5</td><td>147.11 (100); 105.07 (60); 119.10 (50); 133.107 (40); 161.13 (30)</td><td>8,5E-07</td><td>1,9E-01</td><td>&lt;50</td><td>723</td><td>&lt;50</td></tr><tr><td>20</td><td>320.0198</td><td>3,85</td><td>[C10H9NO6 + SO3 + H]</td><td>3,06</td><td>Unknown</td><td></td><td></td><td>240.0507 (30); 212.05 (100); 194.04 (70); 176.03 (50); 166.05 (70)</td><td>6,5E-06</td><td>&gt;0.05</td><td>&lt;50</td><td>324</td><td>&lt;50</td></tr><tr><td>21</td><td>486,3047</td><td>11,14</td><td></td><td>3,05</td><td>Unknown</td><td></td><td></td><td></td><td>2,4E-03</td><td>1,0E-02</td><td>&lt;50</td><td>296</td><td>&lt;50</td></tr><tr><td>22</td><td>519,1871</td><td>10,72</td><td></td><td>3,02</td><td>Unknown</td><td></td><td></td><td></td><td>1,3E-08</td><td>1,9E-02</td><td>348</td><td>2 133</td><td>542</td></tr><tr><td>23</td><td>543,257</td><td>12,82</td><td>[C28H38O8 + Na + H2O]</td><td>3,01</td><td>4β-dihydroxywithanolide E</td><td>542,2491</td><td>0,2</td><td>503.26 (40); 449.23 (20); 229.16 (30); 281.15 (20); 169.08 (20)</td><td>2,4E-04</td><td>7,5E-04</td><td>&lt;50</td><td>2321</td><td>1054</td></tr><tr><td>24</td><td>203,1809</td><td>12,76</td><td>[C15H22 + H]</td><td>3,00</td><td>Sesquiterpene isomer</td><td>202.1730</td><td>4.5</td><td>203.18 (40); 147.11 (100); 119.10 (60); 105.07 (60); 133.107 (40); 161.13 (30), 186.05 (30)</td><td>1,1E-06</td><td>3,3E-02</td><td>&lt;50</td><td>2 477</td><td>125</td></tr></tbody></table>

(continued on next page)
<table><caption>Table 1 (continued)</caption><thead><tr><th rowspan="2">Rank</th><th rowspan="2">Obs m/z<sup>1</sup><br/>[M + H]/<br/>[M-H]</th><th rowspan="2">RT<br/>(min)</th><th rowspan="2">Formula</th><th rowspan="2">VIP<br/>score</th><th rowspan="2">Tentative identification</th><th rowspan="2">M</th><th rowspan="2">Error<br/>(ppm)</th><th rowspan="2">Main MSMS fragments (% mas peak)</th><th colspan="2">P-Value</th><th colspan="3">Average relative intensity</th></tr><tr><th>B/A</th><th>B/C</th><th>B</th><th>A</th><th>S</th></tr></thead><tbody><tr><td>25</td><td>573,2831</td><td>13,42</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>1,6E-05</td><td>1,0E-01</td><td>472</td><td>1069</td><td>608</td></tr><tr><td>26</td><td>325,1314</td><td>10,72</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>8,0E-04</td><td>&gt;0.05</td><td>3 094</td><td>5 780</td><td>3 411</td></tr><tr><td>27</td><td>303.0120</td><td>11.89</td><td>[C11H12O5 + SO3-H]</td><td>3,00</td><td>Sinapic acid sulphate</td><td>304,0252</td><td>17,7</td><td>223.05 (90); 208.03(100);164.03(50);<br/>149.02(20; NOMA)</td><td>1,8E-07</td><td>4,1E-01</td><td>172</td><td>839</td><td>185</td></tr><tr><td>28</td><td>851,277</td><td>13,24</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>3,9E-05</td><td>&gt;0.05</td><td>&lt;50</td><td>354</td><td>045</td><td>000</td></tr><tr><td>29</td><td>566,1879</td><td>11,4</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>8,9E-05</td><td>&gt;0.05</td><td>597</td><td>3 156</td><td>655</td></tr><tr><td>30</td><td>525,2446</td><td>13,09</td><td>[C28H38O8 + Na]</td><td>2,93</td><td>4β-hydroxywithanolide E</td><td>524,2385</td><td>-3,2</td><td>503.2612(20); 299.16(50); 281.15<br/>(20);169.08(100);125.09(10)</td><td>8,4E-04</td><td>2,5E-04</td><td>&lt;50</td><td>14 304</td><td>4 003</td></tr><tr><td>31</td><td>559,2324</td><td>12,64</td><td>[C28H38O8 + K +<br/>H2O]</td><td>2,93</td><td>4β-dihydroxywithanolide E</td><td>558,223</td><td>2,8</td><td>299.16(50);281.15(20);169.08<br/>(100);125.09(10)</td><td>3,7E-05</td><td>1,2E-05</td><td>&lt;50</td><td>322</td><td>162</td></tr><tr><td>32</td><td>219,1204</td><td>7.20</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>1,0E-12</td><td>2,4E-02</td><td>338</td><td>3467</td><td>608</td></tr><tr><td>33</td><td>261.1594</td><td>13.70</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>9,0E-04</td><td>3,3E-07</td><td>&lt;50</td><td>66</td><td>147</td></tr><tr><td>34</td><td>411.1814</td><td>12.17</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>1,0E-13</td><td>3,5E-03</td><td>2533</td><td>8660</td><td>3402</td></tr><tr><td>35</td><td>307.0023</td><td>12.64</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>9,6E-06</td><td>3,9E-07</td><td>64</td><td>370</td><td>745</td></tr><tr><td>36</td><td>203,1808</td><td>12,43</td><td>[C15H22 + H]</td><td>2,80</td><td>Sesquiterpene isomer</td><td>202.1730</td><td>0</td><td>147.11(100); 105.07(60); 119.10(50);<br/>133.107(40); 161.13 (30)</td><td>1,9E-06</td><td>3,1E-01</td><td>&lt;50</td><td>2091</td><td>176</td></tr><tr><td>37</td><td>412.1709</td><td>13.47</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>6,81E-11</td><td>4,24E-01</td><td>6285</td><td>12,808</td><td>6360</td></tr><tr><td>38</td><td>214.989</td><td>12.26</td><td>[C5H4N4O + SO3-H]</td><td>2,63</td><td>Hypoxanthine sulphate</td><td>215.9953</td><td>7</td><td>92.01(100);65.02 (10)</td><td>3,01E-12</td><td>8,54E-03</td><td>1993</td><td>9602</td><td>2857</td></tr><tr><td>39</td><td>412.1866</td><td>11.95</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>1,60E-09</td><td>3,03E-01</td><td>1388</td><td>3757</td><td>1282</td></tr><tr><td>40</td><td>411.1793</td><td>12.65</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>5,00E-11</td><td>2,28E-01</td><td>13,178</td><td>32,195</td><td>14,579</td></tr><tr><td>41</td><td>327.1010</td><td>10.55</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>2,87E-10</td><td>5,18E-05</td><td>2155</td><td>6525</td><td>4456</td></tr><tr><td>42</td><td>535.1607</td><td>10.44</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>6,78E-11</td><td>6,06E-03</td><td>1668</td><td>9409</td><td>2619</td></tr><tr><td>43</td><td>203,1808</td><td>15,06</td><td>[C15H22 + H]</td><td>2,40</td><td>Sesquiterpene isomer</td><td>202.1730</td><td>0</td><td>147.11(100); 105.07(60); 119.10(50);<br/>133.107(40); 161.13 (30)</td><td>2,00E-05</td><td>1,70E-04</td><td>&lt;50</td><td>287</td><td>121</td></tr><tr><td>44</td><td>519.1822</td><td>13.02</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>5,14E-07</td><td>8,39E-02</td><td>1196</td><td>4800</td><td>1367</td></tr><tr><td>45</td><td>287.0889</td><td>10.22</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>7,18E-02</td><td>2,55E-04</td><td>6805</td><td>5389</td><td>14,655</td></tr><tr><td>46</td><td>399.1340</td><td>11.10</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>5,62E-08</td><td>1,01E-02</td><td>3249</td><td>5679</td><td>3687</td></tr><tr><td>47</td><td>135.0397</td><td>12.26</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>7,49E-10</td><td>5,50E-02</td><td>579</td><td>1365</td><td>733</td></tr><tr><td>48</td><td>318.2282</td><td>8.94</td><td>[C16H31NO5 + H]</td><td>2,04</td><td>3-hydroxynonanoyl carnitine</td><td>317,2202</td><td>0,6</td><td>176.07 (40); 130.0669(100); 85.02(60)</td><td>3,66E-04</td><td>8,83E-01</td><td>2 666</td><td>5 354</td><td>2 603</td></tr><tr><td>49</td><td>461.2234</td><td>14.63</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>5,14E-02</td><td>6,04E-06</td><td>4207</td><td>3257</td><td>2170</td></tr><tr><td>50</td><td>537.1756</td><td>11.08</td><td></td><td></td><td>Unknown</td><td></td><td></td><td></td><td>1,48E-06</td><td>6,86E-02</td><td>1756</td><td>12,258</td><td>2160</td></tr></tbody></table>
<table><caption>**Table 2**

Sesquiterpenoids C15H22 identified by DHS-SPME/GC-MS<fnref n="2" /> analysis in urine of volunteer 6 h after consumption of physalis peruviana.</caption><thead><tr><th>#</th><th>Compound</th><th>RT</th><th>LRI<sup>1</sup>calc</th><th>LRI<sup>2</sup>teorico</th><th>Match</th><th>R-match</th><th>IUPAC name</th></tr></thead><tbody><tr><td>1</td><td>Eremophila-1(10),8,11-triene</td><td>48,9</td><td>1833</td><td>1507</td><td>759</td><td>811</td><td>(1S,7S,8aR)-1,8a-Dimethyl-7-(prop-1-en-2-yl)-1,2,3,7,8,8a-hexahydronaphthalene</td></tr><tr><td>2</td><td><strong>Unknown</strong></td><td>49,2</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>3</td><td>α-Curcumene</td><td>50,4</td><td>1862</td><td>1777</td><td>894</td><td>943</td><td>Benzene, 1-(1,5-dimethyl-4-hexenyl)-4-methyl-</td></tr><tr><td>4</td><td>β-Vetispirene</td><td>50,6</td><td>1866</td><td>1737</td><td>819</td><td>850</td><td>(5R,10R)-10-Methyl-6-methylene-2-(propan-2-ylidene)spiro[4.5]dec-7-ene</td></tr><tr><td>5</td><td>β-Vatirenene</td><td>53,7</td><td>1927</td><td>1852</td><td>711</td><td>814</td><td>8-Isopropyl-2,5-dimethyl-1,2,3,4-tetrahydronaphthalene</td></tr><tr><td>6</td><td><strong>Unknown</strong></td><td>56,1</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>7</td><td><strong>Unknown</strong></td><td>56,8</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>8</td><td>β-Vetivenene</td><td>59,5</td><td>2042</td><td>1868</td><td>819</td><td>850</td><td>(8R,8aS)-8,8a-Dimethyl-2-(propan-2-ylidene)-1,2,3,7,8,8a-hexahydronaphthalene</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** C₁₅H₂₂ sesquiterpenoids isomers identified in urine spots collected 6 h after ingestion of goldenberries by DHS /CG-MS.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** MS<fnref n="2" /> fragmentation pattern of bicyclic sesquiterpenoids (C15H22) based on eremophilane structure (CFM-ID) and possible structure of fragments.</figcaption></figure>

both the lactone moiety and water. This fragmentation pattern was specific to 4β-hydroxywithanolide E (4βHWE; C28H38O8 m/z 503.26 [M + H]), as published previously (Ballesteros-Vivas et al., 2019; Musharraf et al., 2011).

Therefore, ions *m/z* 606.27 [M + H] and *m/z* 624.28 [M + H + H₂O] were assigned with relatively high confidence to 4β-hydroxywithanolide E (4βHWE) and 4β-dihydroxywithanolide E conjugated to a cysteine residue (loss of 103.00 amu), as this conjugation probably occurred during the phase II reaction. This identity of 4βHWE was also confirmed by the presence of unconjugated ions among the top VIPs in urine with the same typical fragmentation pattern, such as ions 543.25 [M + Na +

H₂O] (rank 23), *m/z* 525.24 [M + Na] (rank 30), and *m/z* 559.24 [M + K + H₂O] (rank 31). In the fruit extract, ions at *m/z* 503.26 [M + H] also appeared with a similar fragmentation pattern at a similar retention time of 12.7 min, confirming the presence of 4βHWE in the edible fruit (Figure S3). 4βHWE is reported as the main withanolide in the fruit of Physalis peruviana (Yang et al., 2020; Navarro-Hoyos et al., 2022). In addition, the ion *m/z* 606.27 [M + H] was also observed in the fruit at only one retention time (11.4 min), showing that cysteine conjugation may also occur in plants, as the amino acid composition of golden berry fruits is known for its high level of sulfur amino acids (cystine/methionine; Mokhtar et al., 2018). According to various authors (White et al.,

<footnote n="1">¹ Calculated for a DB-Wax column.</footnote>

<footnote n="2">² Retention indices reported in literature for polar column.</footnote>
<figure><img src="image_5.png" /><figcaption>Fig. 3. Urinary excretion of the main isomer of sesquiterpenoid C15H22 (Tr = 11.54 min) by the 18 individuals after consuming golden berry fruit.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 4. Urinary excretion of L-hexanoyl carnitine by the 18 individuals after consuming golden berry fruit.</figcaption></figure>

2016; Yang et al., 2020), cysteine residues are most often implicated to react with key electrophilic sites on the withanolide molecule during phase II reactions but also in the plant. The reaction of cysteine residue with 4βHWE could probably also explain why cysteine, L-cysteine, cystine and L-cystine were significantly impaired after acute consumption of golden berries with a significant reduction in plasma

concentration as observed in this same clinical study (Vaillant et al., 2021).

As shown in Fig. 5, the excretion of 4βHWE derivatives was highest in urine collected between 0 and 6 h after ingestion, meaning that this withanolide is readily bioavailable almost immediately after ingestion. Similar to sesquiterpenoids, withanolide is quickly removed from the

<figure><img src="image_7.png" /><figcaption>Fig. 5. Urinary excretion of 4β-hydroxywithanolide E (4βHWE; C₂₈H₃₈O₈) cysteine conjugated (Ion 606.27 m/z [M + H] at tr 11.4 min) in the 18 individuals after golden berry consumption.</figcaption></figure>
body 12 h after acute consumption and 24 h after a period of constant
consumption of the fruit for almost 3 weeks. Given its relatively high
molecular weight, this compound is probably absorbed at the level of the
upper GI tract.

This is the first time that the bioavailability of 4βHWE and its de-
rivatives has been reported in humans, and this is a relevant result that
could confirm the potential functional interest of golden berry fruit. The
observation of urinary excretion of 4βHWE and its derivatives proves
their systemic presence in the body with the capacity to potentially exert
all the biological activity observed in vitro and in vivo. The withanolide
4βHWE has been linked repeatedly to a positive impact on chronic
diseases, anti-inflammatory and anticancer activities in vitro and in cell
and animal models (White et al., 2016; xxxx; Yen et al., 2010; Chiu et al.,
2013; Wang et al., 2019; Park et al., 2016; Ye et al., 2019; Xu et al.,
2021). This significant result could explain some of the results observed
in different studies after golden berry consumption.

Next, tentatively identified ion *m/z* 163.039 [M + H] (rank 13) was
discriminant only after short-term intervention. The exact mass and
comparison of experimental MS2 published (Nguyen et al., 2020) was
apparently consistent with assignation to hydroxycoumarin (C9H6O3),
a phase I product of the metabolism of coumarin in the human body.
Coumarin has recently been detected in considerable quantities in the
calyxes of Physalis peruviana (Puente et al., 2019), but although some
authors have repeatedly reported its presence in the fruits, it has not
been possible to find an original article with the scientific basis for
confirmation.

Finally, *m/z* 303.01 [M–H] observed in negative mode was tenta-
tively assigned to sinapic acid sulfate [C11H12O + SO3] in accordance
with MS2 spectra published by Mass Bank (NoMA). Sinapic acid is
among the major biomarkers of hydroxycinnamate consumption. In
golden berry fruits, p-coumaric acid and *trans*-ferulic acid have been
detected (Vega-Gálvez et al., 2014), and both can eventually be trans-
formed into sinapic acid after phase I hydroxylation.

Finally, it is worth discussing what was expected but was not
observed in urine. Sucrose esters, which are known to be present in
physalis fruits, did not generate any identifiable ions among all ions
observed (significant and not significant in the PLS model). Sucrose
esters are probably metabolized like sugars due to the nonspecific
endogenous esterase present in the human body. These enzymes may
cleave the link between short fatty acids and the sucrose moiety, liber-
ating these compounds, which are subsequently metabolized. Therefore,
the possible benefits of these compounds should be limited to the GI
tract.

4. Conclusion

This research highlights the distinct nutritional value of golden
berries, revealing that their consumption introduces uncommon com-
pounds into the circulatory system. These compounds potentially ac-
count for the observed health benefits associated with their
consumption. Importantly, the identification of key biomarkers for di-
etary intake opens a pathway to infer potential health impacts of a given
food. This inference is supported by studies employing the same purified
compounds in laboratory and animal settings, demonstrating discernible
bioactivity. This analytical approach can be further strengthened by
integrating endogenous biomarkers influenced by food consumption
into established biological networks, elucidating plausible mechanisms
of action. However, a primary limitation of this nutrimetabolomic
methodology is the absence of established physiological concentrations
for these biomarkers. Thus, subsequent investigations are essential to
determine exposure doses for meaningful comparisons with studies
involving pure compounds.

Finally, recognizing that biomarkers of dietary intake cannot be
directly deduced from untargeted conventional food analyses is crucial.
The case of golden berries exemplifies this: specific compounds, such as
sesquiterpenes and withanolides, despite being present in low

concentrations in the food matrix, emerge as the primary indicators of
golden berry consumption. This underscores the critical need to enhance
the recognition of biomarkers of dietary intake, enabling their incor-
poration in the assessment of food quality, impact of processing tech-
niques, and even plant breeding considerations.

Funding

This research was funded by Corporacion Colombiana de Investigacion Agropecuaria—Agrosavia through recurring transfer from the government of Colombia, Ministry of Agriculture and Rural Development through the macroproject “Comprehensive development of sustainable production models for the cultivation of cape gooseberry in Colombia” and Vidarium -Nutrition, Health and Wellness Research Center, Nutresa in charge of the organization of the clinical study.

CRediT authorship contribution statement

**Fabrice Vaillant:** Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data curation, Visualization, Supervision, Project administration. **Sandra llano:** Investigation, Writing – review & editing. **Alberto Ángel Martín:** Formal analysis, Writing – review & editing. **Natalia Moreno-Castellanos:** Methodology, Validation, Formal analysis, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

Data availability

Data will be made available on request.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodres.2023.113443.

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