# Unveiling lipid and oxylipin composition in the coffee leaves throughout coffee cherry maturation

Maria Cruz Figueroa-Espinoza, Daniel Guzmán-Rosales, Lindsay Mas-Normand, Claire Vigor, Thierry Durand, Camille Oger, Jean-Marie Galano, V Gros, Guillaume Reversat, Jorge Maldonado-Veneroso, et al.

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## To cite this version:

Maria Cruz Figueroa-Espinoza, Daniel Guzmán-Rosales, Lindsay Mas-Normand, Claire Vigor, Thierry Durand, et al. Unveiling lipid and oxylipin composition in the coffee leaves throughout coffee cherry maturation. Food Chemistry, 2025, 493 (Pt 1), pp.145746. 10.1016/j.foodchem.2025.145746. hal-05236414

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# Unveiling lipid and oxylipin composition in the coffee leaves throughout coffee cherry maturation

Maria Cruz Figueroa-Espinoza<sup>a, *,</sup>, Daniel Guzmán-Rosales<sup>b</sup>, Lindsay Mas-Normand<sup>c</sup>, Claire Vigor<sup>c</sup>, Thierry Durand<sup>c</sup>, Camille Oger<sup>c</sup>, Jean-Marie Galano<sup>c</sup>, Valerie Gros<sup>c</sup>, Guillaume Reversat<sup>c</sup>, Jorge Maldonado-Veneroso<sup>b</sup>, Oscar González-Ríos<sup>b</sup>, Mirna L. Suárez-Quiroz<sup>b, *,</sup>, Erwann Durand<sup>a, d, **</sup>

## ARTICLE INFO

### Keywords:

- Lipids

- Oxylipins

- Coffee leaves

- Coffee cherry

- Drying

- Phytoprostanes

- Phytofurans

## ABSTRACT

This study investigates the evolution of lipids, oxylipins (phytoprostanes (PhytoPs) and phytofurans (PhytoFs)), and alkaloids in Mexican *Coffea arabica* leaves as coffee cherries mature. The impact of drying methods on leaf composition was also examined. Lipid extraction was performed using a binary MTBE/MeOH mixture. Fatty acid profiling was conducted by GC-FID. HPTLC was used to identify lipids (free fatty acids, acylglycerols, sterols, sterol esters, glycolipids, phospholipids, tocopherol), diterpenes (cafestol and kahweol), and alkaloids (caffeine and theobromine). A micro-HPLC-QTRAP analysis was used to identify and quantify oxylipins. The drying method influenced the leaves total lipid extraction. Freeze-dried leaves (HL) had higher lipid content than vacuum oven-dried leaves (HS). Total lipids decreased and oxylipin content varied with cherry maturity. The predominant fatty acid in coffee leaves was C18:3. This study reveals coffee leaves as a significant source of PhytoPs and PhytoFs for the first time.

## 1. Introduction

Edible leaves offer a rich source of nutrients and bioactive compounds possessing health benefits, including antioxidant, anti-inflammatory, anti-microbial, and therapeutic properties. For example, leaves of different species of berries are used to make teas, infusions, and beverages (Regolo et al., 2024), and moringa leaves are used to make soups, salads, sauces, biscuits, and cakes (Islam et al., 2021).

Coffee is one of the most traded agricultural commodities in the world (FAO, 2024). The development of sustainable strategies to address the significant quantities of residues derived from the processing of Arabica coffee (*Coffea arabica* L.) has become a challenge. Since the 1970s, several studies have been conducted to add value to residues (by- and co-products) derived from coffee processing (i.e., leaves, flowers,

coffee pulp, silver skin...) (Braham & Ressani, 1979; Hoseini et al., 2021; Klingel et al., 2020). Coffee leaves are used to prepare herbal teas and have traditional medicinal purposes in coffee-producing countries because they contain several metabolites with beneficial biological properties for health, such as phenolic compounds, alkaloids, terpenoids, carotenoids, and phytosterols (Chen, 2019; Patil et al., 2022). Infusions from coffee leaves of *C. arabica* or *C. canephora* were authorized as novel foods by the European Union (European Commission, 2020).

As far as we know, the evolution of the composition of lipids, and specifically oxylipins, in coffee leaves has not been studied during coffee cherry ripening.

In plants, oxylipins such as phytoprostanes (PhytoPs) and phytofurans (PhytoFs) are formed by non-enzymatic oxidation of α-linolenic acid (C18:3 n-3, ALA) (Imbusch & Mueller, 2000). The human body can

<footnote><sup>a</sup> Qualisud, Univ Montpellier, Avignon Université, CIRAD, Institut Agro, Université de la Réunion, Montpellier, France</footnote>

<footnote><sup>b</sup> Tecnológico Nacional de México/Instituto Tecnológico de Veracruz. UNIDA, Miguel Ángel de Quevedo N° 2779, 91860 Veracruz, Mexico</footnote>

<footnote><sup>c</sup> Institut des Biomolécules Max Mousseron, IBMM, University of Montpellier, CNRS, ENSCM, Faculty of Pharmacy, Montpellier, France</footnote>

<footnote><sup>d</sup> CIRAD, UMR QualiSud, F-34398 Montpellier, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author at: Qualisud, Univ Montpellier, Avignon Université, CIRAD, Institut Agro, Université de la Réunion, Montpellier, France.
*E-mail addresses:* maria.figueroa@supagro.fr (M.C. Figueroa-Espinoza), mirna.sq@veracruz.tecnm.mx (M.L. Suárez-Quiroz), erwann.durand@cirad.fr (E. Durand).</footnote>
uptake these molecules through food intake or pollen ingestion (Carrasco-Del Amor et al., 2017). These lipid mediators are likely to: exhibit cytoprotective responses in immature brain cells such as neuroblasts and oligodendrocytes, act as mediators of oxidative stress, possess anti-inflammatory and apoptosis-inducing activities, regulate immune function, and reach the gastrointestinal tract and interact with the intestinal microflora (Mueller, 2004, Barden et al., 2009; Durand et al., 2011; Gilles et al., 2009; Minghetti et al., 2014). Consequently, there is growing interest in studying the molecular profile and levels of these compounds in plant foods, particularly their response to cultural and post-harvest processing practices and environmental conditions. These compounds have been found in various medicinal plants and natural and processed plant foods, such as cereals, cocoa, nuts and oil-seeds, macroalgae, some fruits and legumes. For example, PhytoPs have been reported in different concentrations in olive and sunflower oils (~15.0–297.5 ng/mL) (Collado-González et al., 2015), in raw green olives flesh (5.81–10.00 ng/g w.b.) (Collado-González, Moriana, et al., 2015), wines (131.7–213.2 ng/mL) and musts (20.5–430.9 ng/mL) (Marhuenda et al., 2015), in macroalgae (0.06–13.81 ng/g d.b.) (Barbosa et al., 2015), and nuts like pecan (7.8 ng/g w.b.), macadamia (6.5 ng/g w.b.), and walnut (5.5 ng/g w.b.) (Carrasco-Del Amor et al., 2017). Cocoa contains PhytoPs (221.46–1589.83 ng/g w.b.) and PhytoFs (1.18–13.13 mg/g w.b.) (León-Perez et al., 2019), and white and brown grain flours contain 0.01–1.17 ng/g d.b. of PhytoPs and 4.22–10.3 ng/g d.b. of PhytoFs (Pinciroli et al., 2017). In our previous study, we demonstrated that coffee pulp is a promising source of PhytoPs (654.6 ng/g d.b.) and PhytoFs (543.2 ng/g d.b.) (Ruesgas-Ramón et al., 2019). This finding opens new avenues for exploring PhytoPs and PhytoFs in other residues derived from coffee processing and in different parts of the coffee plant.

In addition to using coffee leaves as infusions, they could be used as functional ingredients or as a source of bioactive compounds. This could offer an economical alternative for farmers and small producers in coffee-producing countries. Extracts rich in PhytoPs and PhytoFs obtained from coffee leaves could be used to enrich foods or as nutritional supplements. Therefore, this study investigates the evolution of lipids, including oxylipins (PhytoPs and PhytoFs), in terms of total content and composition in coffee leaves at different ripening stages of the coffee cherry (C. arabica L.).

## 2. Material and methods

### 2.1. Reagents

Acetonitrile, LC-MS methanol, and HPLC grade chloroform were obtained from Fisher Scientific (Loughborough, UK). Absolute ethanol, acetic acid, ammonia, formic acid, hexane, potassium hydroxide, sodium sulfate, mix 37-component Supelco FAME (fatty acid methyl ester) mix standard, stigmasterol, theobromine (Theo), cafestol (Cafol), kahweol (Kah), trigonelline, caffeine (Caff), lysophosphatidylcholine (LPC), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and ninhydrin spray reagent (ninhydrin 0.2 % in ethanol) were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). TLC mix 34 (monocylglycerol (MAG), diacylglycerol (DAG; 1,2- and 1,3-DAG), tracylglycerol (TAG), methyl oleate, oleic acid) and the mix sterol/sterol esters were purchased from Larodan (Solna, Sweden); HPLC grade ethyl acetate and butylated hydroxytoluene (BHT) were supplied by VWR (EC) and Acros Organics (Geel, Belgium), respectively. Monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) were purchased from Avanti research (Birmingham, AL, USA). The internal standard d₄-15-F₂t-IsoPs was purchased from Cayman Chemical (Ann Arbor, MI, USA). The standards C19-16-F₁t-PhytoP, C21-15-F₂t-IsoP, d₄-10-epi-10-F₄t-NeuroP, 9-epi-9-F₁t-PhytoP, 9-F₁t-PhytoP, 9-L₁t-PhytoP, 16-epi-16-F₁t-PhytoP, 16-F₁t-PhytoP, 16-B₁-PhytoP, ent-9(RS)-12-epi-ST-Δ¹⁰⁻¹³-PhytoF, ent-16(RS)-9-epi-ST-Δ¹⁴⁻¹⁰-PhytoF, and ent-16(RS)-13-epi-ST-Δ¹⁴⁻⁹-PhytoF were synthesized at the Institut des

Biomolécules Max Mousseron. The PhytoP and PhytoF stock solutions (at 100 ng/µL) were prepared in methanol and stored at -20 °C. SPE Oasis MAX 3 cc cartridges (60 mg) were purchased from Waters (Milford, MA, USA). Water was purified on a MilliQ system (Millipore) or obtained from Fisher Scientific (Loughborough, UK).

### 2.2. Plant materials

The coffee leaves (from *Coffea arabica* var. Costa Rica) were randomly collected from September to November 2019 in Coatepec (Veracruz, Mexico) (19.47216°N, 96.97126°O), and lyophilized (HL) (FreeZone 4.5-l benchtop freeze-dryer, LABCONCO®, Kansas City, MO, USA) or dried at 60 °C in a vacuum oven (HS) (model A50980, Scorpion Scientific, Mexico City, Mexico). The harvest of coffee leaves was done as follows: September 29th (HL1), October 13th (HL2 and HS2), October 26th (HL3 and HS3), November 9th (HL4), November 23th (HL5 and HS5). The dried raw material was ground in an ultra-centrifugal mill (model ZM 200, Retsch GmbH, Haan, Germany), sieved to particle sizes between 315 and 630 µm, and finally stored under vacuum in food grade vacuum bags at -20 °C until use.

### 2.3. Lipids' extraction

Total lipids extraction was performed according to the method described by Matyash et al. (2008). Each sample (2.5 g) was added of 10 mL of a mixture of MTBE/MeOH (3:1 v/v), then shaking in a multipoint stirrer (model Cimarec i Poly 15, Thermo Fisher Scientific, Asnières-sur-Seine, France) at 200 rpm for 1 h at room temperature (22 °C). Samples were centrifuged for 5 min at 300 rpm (model CR4-12 Jouan, Fisher Scientific, Illkirch, France). Each extraction was repeated 3 times. The three supernatants of each sample were collected in a Falcon tube and added with 5 mL of distilled water. The organic phase was collected in an amber flask and dehydrated by adding 3 tablespoons of sodium sulfate. Samples were then filtered (0.45 µm, Millipore, Merck KGaA, Darmstadt, Allemagne). The filtrate was evaporated under vacuum at a temperature of 40 °C (Genevac evaporator, model EZ-2, Fisher Scientific). The content of total lipids is expressed as a percentage of the weight of the sample.

### 2.4. Fatty acid composition analysis

Fatty acid methyl ester (FAME) preparation and gas chromatography (GC) analysis for fatty acid composition were made according to the NF EN ISO 12966-2 method (AFNOR, 2017). Two hundred microliters of internal standard (tridecanoic acid C13:0, Sigma Aldrich) at a concentration of 15 mg/mL, were added to each sample before saponification. Fatty acid profiling was performed using a GC Agilent 6890 (Bios Analytique, France) series and an INNOWax capillary column (length: 30 m; internal diameter: 0.32 mm; film thickness: 0.25 mm; SGE, Courtaboeuf, France). GC conditions: carried gas: helium; flow rate: 1 mL/min; split ratio: 1/80; injector temperature: 250 °C; flame ionization detector: 270 °C; oven heating: from 185 °C to 225 °C at 5 °C/min and held at 225 °C for 20 min. Each FAME was identified by comparing its retention time with those obtained from the 37-component Supelco FAME mix standard. This standard contains FAME ranging from C4:0 to C24:1, including most of the methyl esters of the major saturated, monounsaturated, and polyunsaturated fatty acids. Each FAME was quantified as a relative percentage of total fatty acids using Chromcard 2.3.32005 software (Thermo Fisher Scientific, Massachusetts, USA).

### 2.5. High Performance Thin Layer Chromatography (HPTLC)

Separation and qualitative identification of lipids and alkaloids were performed using HPTLC. The standards used were TLC mix 34, MGDG, DGDG, stigmasterol, sterol esters, cafestol, kahweol, caffeine, theobromine, and trigonelline. A 1 mg/mL MTBE/MeOH (3:1, v/v) solution of
the fat extracted from the leaves was prepared and applied to a HPTLC silica gel plate (20 × 10 cm, 60 F254, Analytical Chromatography, Merck KGaA), using an automatic TLC sampler (Model ATS4, CAMAG, Moir- ans, France). Migration was performed in an automatic developing chamber (model ADC2, CAMAG) using different types of mobile phases: for polar compounds: CHCl3:MeOH:H2O (85:20:2.5, v/v/v), for alka- loids: CHCl3:MeOH:H2O (90:10:1.5, v/v/v), or for non-polar com- pounds: hexane/diethyl ether/acetic acid (75:25:1, v/v/v/v). Development was performed in a chromatogram immersion device 3 (CAMAG). Different developing solutions were used: CuSO4:H3PO4: MeOH:H2O (50:40:25:390, v/v/v/v) or thymol (2 g thymol, 190 mL ethanol and 10 mL H2SO4) or ninhydrin spray reagent. Plates were dried with a hairdryer for a 10 s, then observed at visible light and UV light at 254 and 366 nm in a TLC visualizer (CAMAG Reprostar 3). Finally, plates were dried in an oven for 8 min at 180 °C.

## 2.6. Extraction of phytoprostanes and phytofurans

Each sample (100 mg) was weighed into a hemolysis tube and added

of 25 µL of BHT (1 % v/v in methanol) + 1 mL of methanol + 4 µL of an internal standard mix containing C19-16-F1t-PhytoP, C21-15-F2t-IsoP, d4-10-epi-10-F4t-NeuroP, and d4-15-F2t-IsoP prepared at 1 ng/µL in methanol. Only C19-16-F1t-PhytoP was used for quantification, the other standards were used to verify the repeatability between each extracted sample. Tubes were vortexed and samples were ground in a FastPrep®-24 homogenizer (1 cycle of 30 s at 6.5 m/s) (MP Bio- medicals). Samples were transferred into a Falcon tube. The lysis tube was rinsed once with 1 mL of methanol and twice with 750 µL of phosphate buffer pH 2.0 saturated in NaCl, which were added to the Falcon tube, that was then mixed during 30 min (100 rpm-45 s / 90°-15 s) at room temperature. Falcon tubes were centrifuged (4300 rpm, 5 min, room temperature), the supernatant was recovered into a new Falcon tube and 4 mL of cold chloroform was added. Tubes were vor- texed for 30 s, centrifuged (2000 rpm, 5 min, room temperature), and the chloroform phase was separated and concentrated in a Pyrex tube under a nitrogen stream at 40 °C for 1 h. KOH 1 M (950 µL) was then added and the mixture was incubated at 40 °C for 30 min (100 rpm-45 s / 90 rpm-15 s). One milliliter of formic acid (40 mM) was added,

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Precipitation rate (a) and average ambient temperature (b) in Coatepec (Veracruz, Mexico) during the harvesting season in 2019. S: September, O: October, N: November. Data provided by the National Water Commission agency (CONAGUA, Xalapa-Enríquez, Veracruz, Mexico).</figcaption></figure>
vortexed, and the mix was transferred to a hemolysis tube. Solid phase
extraction (SPE) separation was performed using SPE Oasis MAX car-
tridges in a Biotage® Extrahera automation system. After sample
loading, the cartridges were sequentially washed with 2 mL of NH₃ (2 %
v/v); 2 mL of a mixture of formic acid (20 mM):MeOH (70:30, v/v); 2 mL
of hexane, and 2 mL of a mixture of hexane:ethyl acetate (70:30, v/v).
Elution was performed using 2 × 1 mL of a mixture of hexane:ethanol:
acetic acid (70:29.4:0.6, v/v). Samples were then evaporated to dryness
under a nitrogen stream (40 °C, 1 h) and added of 100 µL of a mixture of
H₂O:ACN (83:17, v/v). After vortexing, each sample was left to stand for
10 min at room temperature, vortexed and transferred into a filtering
Eppendorf tube, then centrifuged (10,000 rpm, 1 min, room tempera-
ture), and the filtrate was injected into the micro-HPLC-QTRAP system.
Identification and quantification of Phytops and PhytoFs was performed
using a micro-HPLC-QTRAP system equipped with CTC Analytics AG
(Zwingen, Switzerland) as described by Ruesgas-Ramón et al. (2019).
Quantification of isoprostanooids was based on calibration curves ob-
tained from the ratio of the analyte to the area under the curve of
PhytoPs and PhytoFs. Linear regression of 12 concentration levels of
each standard of PhytoPs and PhytoFs (4, 8, 12, 16, 20, 40, 60, 80, 100,
200, 300, 400 pg/µL) were made, with a constant concentration of
C19-16-F₁₁t-PhytoP (50 pg/µL).

Precipitation rate and average ambient temperature data in Coatepec (Veracruz, Mexico) (Fig. 1) were provided by the National Water Commission agency (CONAGUA, Xalapa-Enríquez, Veracruz, Mexico).

## 2.7. Statistical analysis

One-way analysis of variance (ANOVA) was performed. The signifi-
cance level was set at *p* < 0.05. Results are expressed as the mean of
triplicates (means ± standard deviations). Fisher's test was used to
determine whether the means were different, using Minitab statistical
software version 22.

# 3. Results and discussion

## 3.1. Total lipid content in coffee leaves

The total lipid content of lyophilized (HL) and oven dried (HS) coffee
leaves was analyzed at different sampling dates from September to
November 2019, coinciding with the ripening stage of the coffee cher-
ries that change from green (immature; HL1), to green-yellow (imma-
ture; HL2 and HS2), to yellow-orange (semi-mature; HL3 and HS3) and
yellow-orange-red (semi-mature; HL4) to orange-red and red (mature;
HL5 and HS5). This is the first report on the total lipid content in coffee
leaves in function of the coffee cherry ripening stage. Regarding the
drying method, the leaves dried in the vacuum oven had a lower lipid
content (HS2: 4.45 ± 0.09%; HS3: 3.90 ± 0.30 % and HS5: 3.42 ± 0.20 %)
than the same lyophilized leaves (HL1: 8.61 ± 0.31 %; HL2: 6.15 ±
0.10 % and HL3: 5.91 ± 0.22 %; HL4: 4.96 ± 0.50 % and HL5: 4.25 ±
0.26 %) (Fig. 1 a). Thus, the drying method had a significative influence
on the lipid extraction yield. Lipid extraction was more effective for
freeze-dried coffee leaves than vacuum oven-dried ones, that remained
at 60 °C for more than 10 h, which could provoke chemical and/or
enzymatical degradation of lipids. With respect to the time of sampling,
a significant decrease in fat content of up to 50.64 % was observed be-
tween HL1 and HL5 and of 30.89 % between HL2 and HL5, as well as in
the vacuum-oven-dried leaves, with a decrease of 23.14 % between HS2
and HS5. Lin and Oliver (2008) obtained similar results when quanti-
fying TAG levels on crabapple (Malus hybrid *Snowdrift*) leaves during 8
months development. TAG levels were high during the first 2 days after
bud break, then they decreased when the leaves were 90 days old and
fully matured, and finally they increased until day 240 when leaves
senesced. They observed that TAG level increased during the day and
decreased during the night, suggesting that TAG are metabolically active
and represent an energy source for the plant. *C. arabica* leaf tissue

contains cytoplasmic lipid droplets (LDs) where lipids (i.e., TAG, ter-
penoids, steryl esters...) are stored (Crisafulli & Navarini, 2023; Guzha
et al., 2023). Crisafulli and Navarini (2023) observed that LDs diameter
from C. arabica leaves (for plants grew in a greenhouse in Italy)
decreased from leaves sampled between January (cold season; minimum
temperature: 14 °C) and June (vegetative season) and that the produc-
tion of LDs was higher during the cold season, but unfortunately, they
did not analyze the lipid content of coffee leaves to conclude on the
influence of the total lipid content and lipid composition of these
changes in LDs. It would also have been interesting to analyze the LDs
characteristics during the coffee cherry development in the plant. Even
though, we can suppose that the decrease of the total lipid content in
coffee leaves during the ripening of coffee cherries (from September to
November 2019) would be relied to the LDs evolution during time.
Cheng et al. (2018) analyzed the transcriptome of the coffee bean at
three development stages (immature (green), intermediate (yellow),
mature (red)). According to these authors, lipids accumulation through
bean coffee ripening occurs predominantly in green coffee beans. It is
likely that in coffee leaves, a similar pattern is observed, as higher
contents in total lipids were present in earlier sampled coffee leaves. It
would be interesting to characterize lipids on the green coffee leaves
infusion, as it was done by Cataldo (2014) that found 4.8 % of lipids and
waxes on black tea beverage.

## 3.2. Qualitative chemical characterization of coffee leaves

From the lipid fractions extracted with MTBE/MeOH, HPTLC runs
were performed with solvents of different polarities to identify lipid
compounds such as MAG, DAG, TAG, free fatty acids (FFA), sterols (S),
sterol esters (SE), glycolipids (Gly) (galactolipids (MGDG and DGDG)),
lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), phos-
phatidylcholine (PC), as well as different compounds known to be pre-
sent in coffee, such as alkaloids (caffeine (Caff) and theobromine
(Theo)), diterpenes (cafestol (Cafol) and kahweol (Kah)), in order to
observe the presence and evolution of these compounds in the leaves.
Two sequential migrations in the same HPTLC plate were carried out,
the first from 0 cm to 4 cm for the migration of non-polar compounds
using a hexane/diethyl ether/acetic acid mixture, then a second
migration from 0 to 8 cm for polar compounds using a CHCl₃/methanol/
H₂O mixture. The results are shown in Fig. 2 b and c, where SE are
observed in all samples with no apparent change in concentration.
Almost no presence of TAG was observed, but FFA are observed,
increasing in a similar manner from HL1 to HL5 and from HS2 to HS5.
The presence of DAG is not detectable on the plate in Fig. 2 b because of
interference with S, since they migrate at almost the same height. MAG
were observed in all leaf samples (Fig. 2 b). Neither Cafol nor Kah were
present in coffee leaves (Fig. 2 b), as it was observed by Ivamoto et al.
(2017). Unfortunately, it was not possible to see neither Caff nor Theo in
HPTLC using our solvent extraction system (Fig. 2 b). C. arabica leaves'
Caff content decreases with maturation passing from 3.2 to 1.8 mg/g (d.
b.) (Ratanamarno & Surbkar, 2017) or from 15 to 2.5–6 mg/g (d.b.)
(Montis et al., 2021) from young to mature leaves. Ashihara et al. (1996)
reported a caffeine content in coffee leaves of 7.1, 2.4, and 2.4 mg/g (w.
b.) for young, mature, and aged leaves, respectively. According to these
authors Theo’s content also decreases with maturity varying from 1.8,
0.13, and 0.06 mg/g (w.b.) for young, mature, and aged leaves,
respectively. Glyc (MGDG and DGDG), LPC, PE, and PC, were present in
coffee leaves (Fig. 2 a), being more abundant in HL than in HS.

S and SE were observed in all samples, TAG were not, but there was a
strong presence of FFA. In coffee leaves, TAG are a minor component of
the overall lipid content (Guzha et al., 2023), that would explain why
TAG were not visible at our experimental conditions. FFA content in
coffee leaves increased with maturity from HL1 to HL5 and from HS2 to
HS5. MAG and DAG (1,3-DAG, 1,2-DAG) were present in all the samples,
with a slight increase in the concentration of 1,3-DAG and 1,2-DAG with
respect to the sampling time from HL1 to HL5 and from HS2 to HS5. No
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** **a.** Total lipids content (% w/w of dry matter). **b.** HPTLC profile using double migration (hexane/diethyl ether/acetic acid (75:25:1, v/v/v/v) and CHCl₃: MeOH:H₂O (85:20:2.5, v/v/v)) to qualitatively reveal polar and non-polar lipids. **c.** TLC profile of non-polar lipids. Mix34 standard, S/SE (sterol/sterol ester), Glyc (glycolipids: monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG)), LPC (lysophosphatidylcholine), PE (phosphatidylethanolamine), PC (phosphatidylcholine), Caferol (cafestol), Kah (kahweol), Toco (tocopherol), Caff (caffeine), The (theobromine), HL (lyophilized coffee leaves), HS (oven dried coffee leaves). Results are the mean of triplicates (mean values ± standard deviation values).</figcaption></figure>

reports on the evolution of S, SE, Gly (MGDG, DGDG), TAG, DAG, MAG, or FFA with the degree of maturity of coffee leaves was found in literature.

### 3.3. Fatty acid profile

The results in Table 1 show that the main FA found in HL and HS were C18:3Δ⁹,¹²,¹⁵ (ALA), C16:0, C18:2Δ⁹,¹², cis and trans, C18:0, C24:1, and C18:1. The other FA were present at concentrations inferior to 0.27 mg/g of sample (d.b.). These results are in agreement with Mongrand et al. (2005) who reported that the principal FA in C. arabica leaves are C18:3Δ⁹,¹²,¹⁵, C16:0, and C18:0. The highest contents in ALA were observed at intermediate degrees of coffee cherry maturity in HL2 (1.96 ± 0.01 mg/g of sample (d.b.)) and HL3 (1.99 ± 0.18 mg/g of sample (d. b.)). No significant effect of maturity on the ALA content was observed in the HS series, and their contents were significantly inferior to those observed in the HL series. The drying mode had thus an influence on the ALA content in coffee leaves, that was significantly higher in HL than in HS. Similar results were observed with C18:2Δ⁹,¹², cis and trans, C16:1, and C22:2. Thus, freeze-drying was more efficient than the oven drying process in preventing ALA and other unsaturated FA oxidation.

In the HL series, two tendencies can be observed when comparing FA individually: (i) an increase of FA content from HL1 to HL2 then a decrease to HL5, with FA content in HL5 inferior to HL1 (C16:0, C20:0),

not significantly different between HL5 and HL1 (C16:1, C22:2, C18:1, C18:3Δ⁹,¹²,¹⁵) or FA content in HL5 superior to HL1 (C18:2Δ⁹,¹², cis and trans); (ii) a decrease of FA content from HL1 to HS2, with FA HL5 content inferior to HL1 (C12:0, C14:0, C14:1, C15:0, C17:0, C18:3Δ⁶,⁹,¹², C22:1, C18:0, C24:1) or not significantly different between HL5 and HL1 (C17:1). In the HS series, the evolution of the individual FA content showed two tendencies: (i) no significant effect of maturity from HS2 to HS5 (C14:1, C15:0, C16:1, C17:0, C17:1, C18:0, C18:1, C18:2Δ⁹,¹², cis and trans, C18:3Δ⁹,¹²,¹⁵, C20:0, C16:0, C22:1, C22:2), (ii) a decrease of FA content from HS2 to HS5 (C12:0, C14:0, C18:3Δ⁶,⁹,¹², C24:1).

When comparing total FA content in leaves, in the HL series total FA increases from HL1 (12.8 mg/g d.b.) to HL2 (15.0 mg/g d.b.), then decreases from HL3 (14.1 mg/g d.b.) to HL5 (9.9 mg/g d.b.). For the HS series total FA variation is less pronounced than in the HL series: HS2 (8.1 mg/g d.b.), HS3 (9.0 mg/g d.b.), and HS5 (8.2 mg/g d.b.). Thus, more FA were extracted from the HL samples than from the HS.

**Fig. 3** shows the profile of SFA, MUFA, PUFA, and TUFA (total unsaturated FA) in HL and HS. SFA increased from HL1 to HL2, then decreased at the same level as HL1 in HL3, and decreased again in HL4 and stayed stable until HL5. MUFA increased from HL1 to HL2, stayed stable until HL3, then decreased in HL4 and stayed stable until HL5. PUFA decreased from HL1 to HL2 and stayed stable until HL3, then decreased in HL4 and stayed stable until HL5. TUFA decreased from HL1 to HL4 and stayed stable until HL5. No significant differences were
<table><caption>**Table 1**
Fatty acid profile of lipid content in coffee leaves (mg/g of sample, d.b.).</caption><thead><tr><th rowspan="2">Fatty acid</th><th colspan="10">Fatty acid content (mg/g of sample, d.b.)</th></tr><tr><th>HL1</th><th>HL2</th><th>HL3</th><th>HL4</th><th>HL5</th><th>HS2</th><th>HS3</th><th>HS5</th></tr></thead><tbody><tr><td>C12:0</td><td>0.01</td><td>± 0.00</td><td>a</td><td>0.00</td><td>0.00</td><td>b,<sup>#</sup></td><td>0.00</td><td>0.00</td><td>b,<sup>#</sup></td></tr><tr><td>C14:0</td><td>0.16</td><td>± 0.01</td><td>a</td><td>0.11</td><td>± 0.00</td><td>c,<sup>#</sup></td><td>0.05</td><td>± 0.00</td><td>B,<sup>#</sup></td></tr><tr><td>C14:1</td><td>0.01</td><td>± 0.00</td><td>a</td><td>0.00</td><td>± 0.00</td><td>b,<sup>#</sup></td><td>0.00</td><td>± 0.00</td><td>A,<sup>#</sup></td></tr><tr><td>C15:0</td><td>0.02</td><td>± 0.00</td><td>a,b</td><td>0.02</td><td>± 0.00</td><td>b,<sup>#</sup></td><td>0.01</td><td>± 0.00</td><td>A,<sup>#</sup></td></tr><tr><td>C16:0</td><td>3.36</td><td>± 0.07</td><td>b</td><td>4.19</td><td>± 0.03</td><td>a,<sup>#</sup></td><td>3.76</td><td>± 0.00</td><td>A,<sup>#</sup></td></tr><tr><td>C16:1</td><td>0.02</td><td>± 0.00</td><td>a</td><td>0.03</td><td>± 0.00</td><td>a,<sup>#</sup></td><td>0.03</td><td>± 0.00</td><td>c,<sup>#</sup></td></tr><tr><td>C17:0</td><td>0.11</td><td>± 0.00</td><td>a</td><td>0.10</td><td>± 0.00</td><td>b,<sup>#</sup></td><td>0.07</td><td>± 0.00</td><td>b,<sup>#</sup></td></tr><tr><td>C17:1</td><td>0.01</td><td>± 0.00</td><td>a</td><td>0.01</td><td>± 0.00</td><td>a,<sup>#</sup></td><td>0.01</td><td>± 0.00</td><td>A,<sup>#</sup></td></tr><tr><td>C18:0</td><td>1.51</td><td>± 0.03</td><td>a</td><td>1.56</td><td>± 0.01</td><td>a,<sup>#</sup></td><td>1.29</td><td>± 0.14</td><td>A,<sup>#</sup></td></tr><tr><td>C18:1</td><td>0.39</td><td>± 0.02</td><td>c</td><td>0.56</td><td>± 0.00</td><td>a,<sup>#</sup></td><td>0.53</td><td>± 0.06</td><td>b,<sup>#</sup></td></tr><tr><td>C18:2<sup>#</sup>,12, cts and trans</td><td>1.01</td><td>± 0.02</td><td>c</td><td>1.96</td><td>± 0.01</td><td>a,<sup>#</sup></td><td>1.99</td><td>± 0.18</td><td>a,<sup>#</sup></td></tr><tr><td>C18:3<sup>#</sup>,6<sup>#</sup>,9,12</td><td>0.02</td><td>± 0.00</td><td>a</td><td>0.02</td><td>± 0.00</td><td>a,b,<sup>#</sup></td><td>0.01</td><td>± 0.00</td><td>c,<sup>#</sup></td></tr><tr><td>C18:3<sup>#</sup>,9<sup>#</sup>,12,15</td><td>3.88</td><td>± 0.11</td><td>b</td><td>5.08</td><td>± 0.02</td><td>a,<sup>#</sup></td><td>5.59</td><td>± 0.47</td><td>a,<sup>#</sup></td></tr><tr><td>C20:0</td><td>0.19</td><td>± 0.00</td><td>a</td><td>0.27</td><td>± 0.00</td><td>a,<sup>#</sup></td><td>0.21</td><td>± 0.00</td><td>b,<sup>#</sup></td></tr><tr><td>C22:1</td><td>0.01</td><td>± 0.00</td><td>a</td><td>0.01</td><td>± 0.00</td><td>a,b,<sup>#</sup></td><td>0.01</td><td>± 0.00</td><td>a,<sup>#</sup></td></tr><tr><td>C22:2</td><td>0.03</td><td>± 0.00</td><td>b</td><td>0.06</td><td>± 0.00</td><td>a,<sup>#</sup></td><td>0.05</td><td>± 0.01</td><td>a,<sup>#</sup></td></tr><tr><td>C24:1</td><td>2.04</td><td>± 0.15</td><td>a</td><td>1.04</td><td>± 0.03</td><td>b,<sup>#</sup></td><td>0.44</td><td>± 0.03</td><td>c,<sup>#</sup></td></tr></tbody></table>

HL: lyophilized leaves, HS: oven dried leaves. Results are the mean of triplicates (mean values ± standard deviation values). Values in the same row with the same superscript letter (HL1 to HL5 (lowercase letters), HS2 to HS5 (capital letters) or symbol (HL2 vs HS2 (\*), HS3 vs HS3 (\#), HL5 vs HS5 (\#)) are not significantly different at *p* < 0.05 (ANOVA; *n* = 3). In bold: the major fatty acids.

observed between HS2, HS3 and HS5 neither in SFA, nor MUFA, nor PUFA, nor TUFA contents. When comparing HL2 to HS2, all SFA, MUFA, PUFA and TUFA contents were higher in HL. The same was observed for HL3 vs HS3 and HS5 vs HS5, excepting for PUFA contents which were lower in HS3 and HS5, maybe due to a chemical or enzymatic oxidation in oven dried leaves. Thus, drying method had a significant effect (*p* < 0.05) on the FA family content. Lyophilization made it easier to see the evolution of FA contents by FA family. It is likely that oven drying masked the differences in FA contents by family in the coffee leaves. This work made it possible, for the first time, to observe the effect of maturation and of the drying method on the FA composition (individual FA and by FA family) in randomly obtained coffee leaves.

C. arabica leaf infusions possess a range of health benefits, including antioxidant, anti-inflammatory, and antihyperlipidemic activities (Chen et al., 2018; Sansri et al., 2024). These benefits are attributed to the chemical composition of leaves, which contain, among others, phenolic compounds (~868 mg gallic acid equivalents/g d.b.), alkaloids such as caffeine (~7.9 g/kg d.b.), and macrominerals such as K, Ca, and Mg (17,323.2 µg/g), along with microminerals like Mn, Cu, Zn, Co, and Ni (100.0 µg/g). However, no toxic metals such as Cr, Cd, or Pb were detected (Dubale et al., 2017; Jeon et al., 2024). Patil et al. (2024) reported the presence of oxalic acid (1.48 ± 0.02 %) in a C. robusta coffee leaf powder, which could potentially bind to minerals, thereby decreasing their absorption. No reports were found concerning the FA composition of coffee leaf infusions. For instance, a 250 mL cup of infusion prepared with 5 g of HL3/day, with 1.99 ± 0.18 mg ALA/g of HL3 (d.b.), would meet a maximum of 0.6 % of the recommended minimal physiological requirement for ALA (1.8 g/day for an energy intake of 2000 Kcal/day) (AFSSA (Agence Française de Sécurité Sanitaire des Aliments), 2006). Therefore, further research is needed to investigate the FA composition of coffee leaf infusion to understand its full health benefits and nutritional composition.

### 3.4. Quantification and identification of PhytoPs and PhytoFs

The nonenzymatic peroxidation of ALA in plants leads to the formation of oxylipins (PhytoPs and PhytoFs) (Cuyamendous et al., 2015; Imbusch & Mueller, 2000). The total contents of PhytoPs and PhytoFs in leaves showed the same evolution with the advancement of the coffee cherries maturity. They increased from stage 1 to stage 4 for HL and from stage 2 to 3 for HS, and then decreased. In the HL group, an increase in total PhytoPs was observed from HL1 (3777.2 ng/g d.b.) to HL4 (6103.0 ng/g d.b.), followed by a decrease in HL5 (5309.1 ng/g). The variation in PhytoFs content was less marked but followed the same pattern: it increased from HL1 (2545.9 ng/g d.b.) to HL4 (2970.2 ng/g d.b.), then decreased in HL5 (2373.7 ng/g d.b.) at a level even lower than in HL1. In the HS group, an increase in total PhytoPs was observed from HS2 (3735.6 ng/g d.b.) to HS3 (5932.8 ng/g d.b.), followed by a decrease in HS5 (5108.6 ng/g). The variation in PhytoFs content was less marked but followed the same pattern: it increased from HL1 (2545.9 ng/g d.b.) to HL4 (2970.2 ng/g d.b.), and from HS2 (1863.8 ng/g d.b.) to HS3 (3304.5 ng/g d.b.), then decreased in HL5 (2373.7 ng/g d.b.) and HS5 (2416.7 ng/g d.b.). These results could be explained by the environmental and climatic conditions that prevailed during sampling (Fig. 1). The temperature and relative humidity of the farm environment were monitored and rainfall data were collected from the nearest CONAGUA agency. After stage 1, temperatures below 18 °C and precipitation values below 10 mm/d were observed, which could cause temperature and water stress in the plant. In other crops, such as olive trees, it has been shown that water stress increases the content of certain F1t-PhytoP and B1t-PhytoP in extra virgin olive oil (Collado-González et al., 2016; Collado-González, Pérez-López, et al., 2015). Consequently, these authors proposed that 9-F1t-PhytoP, 9-epi-9-F1t-PhytoP, and a racemic mixture of 16-B1-PhytoP and ent-16-B1-PhytoP could be biomarkers of water stress in olive trees. Yonny et al. (2016) showed that melon leaves' PhytoPs and PhytoFs can be considered as oxidative stress
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Total saturated (SFA), monounsaturated (MUFA), polyunsaturated (PUFA) and unsaturated (TUFA) fatty acids in coffee leaves. HL: lyophilized leaves, HS: oven dried leaves. Results are the mean of triplicates (mean values ± standard deviation values). Values by family of FA with the same superscript letter (HL1 to HL5 (lowercase letters), HS2 to HS5 (capital letters) or symbol (HL2 vs HS2 (*), HS3 vs HS3 (#), HL5 vs HS5 (α)) are not significantly different at *p* < 0.05 (ANOVA; *n* = 3).</figcaption></figure>

biomarkers in thermally stressed melon plants. Therefore, PhytoPs and PhytoFs could be used as biomarkers of water or heat stress in coffee plants.

The PhytoPs identified in all samples by micro-HPLC-QTRAP were: ent-16-F₁t-PhytoP, ent-16-epi-16-F₁t-PhytoP, 9-F₁t-PhytoP, 9-epi-9-F₁t-PhytoP, ent-16-B₁t-PhytoP, ent-9-L₁t-PhytoP, 16(RS)-16-A₁t-PhytoP. Concerning PhytoFs, two molecules were found: ent-16(RS)-9-epi-ST-Δ¹⁴⁻¹⁰-PhytoF and ent-16(RS)-13-epi-ST-Δ¹⁴⁻⁹-PhytoF. These molecules, excepting the 16(RS)-16-A₁t-PhytoP, were also found in coffee pulp, cocoa husk hull, and cocoa pod husk in a previous work (Ruesgas-Ramón et al., 2019). Other works have also reported molecules similar to those found in the present work, such as the 9-L₁t-PhytoP series

reported in melon leaves (Yonny et al., 2016), extra virgin olive oil (Collado-González, Pérez-López, et al., 2015), and in walnut, macadamia and pecan nuts as minor constituents (Carrasco-Del Amor et al., 2017). The 9-F₁t-PhytoP series was predominant in raw pecan nuts, up to 4.72 ng/g w.b. (Carrasco-Del Amor et al., 2017). On the other hand, the B₁t-PhytoP series was identified at high levels in flaxseed oil (Karg et al., 2007) and is likely involved in the regulation of gene expression in *Arabidopsis thaliana* and tobacco (Loeffler et al., 2007). In coffee leaves the most abundant oxylipins were the 9-F₁t-PhytoP (1606.4 ng/g d.b. in HL4 and 1751.9 ng/g d.b. in HS3) and ent-16(RS)-13-epi-ST-Δ¹⁴⁻⁹-PhytoF (1568.4 ng/g d.b. in HL1 and 1899.4 ng/g d.b. in HS3).

Total oxylipins content in the HL group were different from those of

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Qualitative and quantitative evaluation of the phytoprostanes (PhytoPs) and phytofurans (PhytoFs) present in the lipid fractions for each sample. HL: lyophilized leaves, HS: oven dried leaves.</figcaption></figure>
the HS group (Fig. 4). Total oxylipins in HS3 were slightly higher than those in HL4, and HS3 was the sample with the highest total amounts of PhytoPs (5932.8 ng/g d.b.) and PhytoFs (3304.5 ng/g d.b.), which could be due to a thermal stress provoked by the oven drying.

In our previous work on coffee pulp (Ruesgas-Ramón et al., 2019), we reported the F₁t series as the major quantified PhytoPs (with a total content of 527.2 ng/g d.b.; 9-epi-9-F₁t-PhytoP (171.4 ng/g d.b.) > 9-F₁t-PhytoP > ent-16-F₁t-PhytoP > 16-epi-16-F₁t-PhytoP), and total contents of PhytoPs and total PhytoFs of, respectively, 654.6 ng/g d.b. and 543.2 ng/g d.b. As in coffee pulp, in coffee leaves, the major PhytoPs series was also the F₁t, being 9-F₁t-PhytoP the major one (1113.8–161.4 ng/g d.b. for HL; 1065.5–1751.9 ng/g d.b. for HS) > ent-16-F₁t-PhytoP or 9-epi-9-F₁t-PhytoP > ent-16-epi-16-F₁t-PhytoP, for both HL and HS samples. Total PhyoPs' concentration ranged from 2567.8 to 3133.1 ng/g d.b. from HL1 to HL5, with a maximum at 3474.0 ng/g for HL4, and of 2138.6, 3400.7, and 2761.0 ng/g d.b. for HS2, HS3, and HS5, respectively. HL4 and HS3 contained, respectively, 11- and 12-fold more 9-F₁t-PhytoP, 9.3- and 9.1-fold more total PhytoPs, 5.5- and 6.1-fold more total PhytoFs than coffee pulp. In coffee pulp (Ruesgas-Ramón et al., 2019) and coffee leaves, the series B₁t, L₁t, and A₁t were detected at lower levels: ent-16B₁t-PhytoP (60.7 ng/g d.b. for coffee pulp; 393.9–795.2 ng/g d.b. for HL; 428.9–727.7 ng/g d.b. for HS), ent-9L₁t-PhytoP (66.7 ng/g d.b. for coffee pulp; from 455.8 to 1131.7 ng/g d.b. for HL; 789.4–1225.5 ng/g d.b. for HS), and 16(RS)-16-A₁t-PhytoP (not detected on coffee pulp; 359.7–701.7 ng/g d.b. for HL; 378.7–578.8 ng/g d.b. for HS). In cocoa beans, the series F₁t (209.46 to 1558.44 ng/g) was the major PhytoPs, and the series B₁t (3.04 to 46.35 ng/g), L₁t (2.11 to 26.34 ng/g), and D₁t (1.82 to 21.79 ng/g) were detected at lower levels (León-Perez et al., 2019). These results show for the first time, that PhytoPs are present in higher concentrations in coffee leaves than in coffee pulp or cocoa beans.

Fig. 5 shows the total contents of PhytoPs and PhytoFs in various plant sources. It can be seen that HL5 and HS5 coffee leaves are among the richest in both PhytoPs and PhytoFs, making coffee leaves an excellent source of oxylipins.

With our results, it is difficult to establish a correlation between the contents of ALA (Table 1) and total oxylipins (Fig. 4) in coffee leaves during maturity of coffee cherries and in function of the drying method. Since fresh leaves oxylipin contents were not analyzed, what it's seeing from these results is both the effect of the stage of maturity and of the drying method on the ALA and oxylipin content. In general, it could be observed that: (i) more ALA and oxylipins were extracted in lyophilized leaves, (ii) HL2 contained more ALA and more oxylipins than HS2, (iii) HL3 contained more ALA but less oxylipins than HS3, (iv) HL5 contained more ALA and slightly more oxylipins than HS5, and (v) ALA increases

with maturity for HS and in HL it increases until HL3 then it decreases. Finally, it can only be concluded that in general more ALA is extracted from freeze-dried leaves, maybe because the oven drying treatment promotes oxidation. Following the analysis of these results, it is difficult to highlight a trend in the ALA and/or oxylipin content depending on the maturity of the leaves and of the drying method. It would have been interesting to quantify the ALA and oxylipins of fresh coffee leaves, to better see the effect of both maturity and drying method. In our case, it was decided to dry coffee leaves in order to analyze them under the same conditions, as it was difficult to ensure the correct freezing of the coffee leaves during transport from Mexico to France. In addition, not all oxidized derivatives of ALA are quantified, but only certain non-enzymatic ones such as PhytoPs and PhytoFs. ALA in coffee leaves can also be oxidized by lipoxygenase (Rojas et al., 1993). It should therefore be interesting to study the lipoxygenase activity and the presence of linotrins (dihydroxylated ALA derivatives resulting from the oxidation of ALA by lipoxygenase), possessing anti-inflammatory activity (Balas et al., 2022) of coffee leaves during the different stages of maturity of coffee cherries.

Finally, since coffee leaves can be used to produce infusions, it would be interesting to characterize the composition of ALA, PhytoPs, and PhytoFs in the resulting beverage and to study their physiological effects in *in vivo* studies and on human health. As far as we know, only three studies related to the metabolism of oxylipins in humans have been published (Barden et al., 2009; Karg et al., 2007; Lara-Guzmán et al., 2020). However, none of these studies address the dose-response relationship of oxylipin concentrations after taking PhytoPs and/or PhytoFs. It would be of particular interest to examine the contribution of oxylipins from coffee leaf infusions in regulating anti-inflammatory activity, possibly in interaction with other bioactive molecules such as polyphenols.

## 4. Conclusion

Coffee leaves have been used for a long time in various cultures as tea-like beverages due to their potential health benefits and unique flavors. They are currently authorized as novel food by the European Union. Coffee leaves are also employed for traditional medicinal purposes, as they contain several bioactive molecules, including phenolic compounds, diterpenes, and alkaloids. This study, for the first time, (i) describes the evolution of several classes of lipids and alkaloids in coffee leaves at different stages of maturity of the *C. arabica* cherry, (ii) highlights the importance of the drying method used for coffee leaves, as it influences the chemical characterization results of the aforementioned mentioned bioactive molecules, and (iii) enriches the portfolio of

<figure><img src="image_8.png" /><figcaption>Fig. 5. Average of total phytoprostanes (left) and phytopurans (right) (ng/g d.b.; * ng/g w.b.) of some plant products reported in the literature (Carrasco-Del Amor et al., 2016, Carrasco-Del Amor et al., 2017; León-Perez et al., 2019; Medina et al., 2017; Pinciroli et al., 2017; Ruesgas-Ramón et al., 2019; Vigor et al., 2018; Yonny et al., 2016). HL: lyophilized leaves, HS: oven dried leaves.</figcaption></figure>
interesting bioactive molecules present in coffee leaves by demonstra-
tion that they are rich in oxylipins (PhytoPs and PhytoFs). Coffee leaves
can be valorized to produce infusions, as functional ingredients or as a
source of bioactive compounds. Adding value to this agricultural re-
sidual resource would contribute to sustainability, to promote a bio-
economy approach, and to offer an economical alternative for farmers
and small producers in coffee-producing countries.

CRediT authorship contribution statement

**Maria Cruz Figueroa-Espinoza:** Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.
**Daniel Guzmán-Rosales:** Writing – review & editing, Investigation, Formal analysis, Data curation.
**Lindsay Mas-Normand:** Writing – review & editing, Investigation, Formal analysis, Data curation.
**Claire Vigor:** Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation.
**Thierry Durand:** Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation.
**Camille Oger:** Writing – review & editing, Investigation, Formal analysis, Data curation.
**Jean-Marie Galano:** Writing – review & editing, Investigation, Formal analysis, Data curation.
**Valerie Gros:** Writing – review & editing, Investigation, Formal analysis, Data curation.
**Guillaume Reversat:** Writing – review & editing, Investigation, Formal analysis, Data curation.
**Jorge Maldonado-Veneroso:** Writing – review & editing, Investigation, Formal analysis, Data curation.
**Oscar González-Ríos:** Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.
**Mirna L. Suárez-Quiroz:** Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.
**Erwann Durand:** Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Funding sources

This project was supported by Agropolis Fondation [Labex AGRO
2011-LABX-002, project number 2002-012, I-Site Muse framework];
and the National Council of Science and Technology (CONACyT) in
Mexico.

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.

Acknowledgments

Daniel Guzmán-Rosales thanks the National Council of Science and Technology (CONACyT) in Mexico for the Master fellowship grant.

Data availability

Data will be made available on request.

References

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