# Detailed Metabolomic Profile of Piper borbonense Fruits, a Refined Malagasy Spice, via a Combination of Unsupervised Analytical Techniques

Elena Serino, Federica Pollastro, Paolo Luciano, David Touboul, Giovanni

Appendino, Giuseppina Chianese, Orazio Taglialatela-Scafati

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## To cite this version:

Elena Serino, Federica Pollastro, Paolo Luciano, David Touboul, Giovanni Appendino, et al. Detailed Metabolomic Profile of Piper borbonense Fruits, a Refined Malagasy Spice, via a Combination of Unsupervised Analytical Techniques. Journal of Agricultural and Food Chemistry, 2025, 73 (16), pp.9854-9866. 10.1021/acs.jafc.5c01501. hal-05367639

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Submitted on 16 Nov 2025

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Distributed under a Creative Commons Attribution 4.0 International License
Detailed Metabolomic Profile of *Piper borbonense* Fruits, a
Refined Malagasy Spice, via a Combination of Unsupervised
Analytical Techniques

Elena Serino,ª Federica Pollastro,ᶜ Paolo Luciano,ª David Touboul,ᵇ Giovanni Appendino,ᶜ
Giuseppina Chianese,*,ª Orazio Taglialatela-Scafatiª
**Abstract**

*Piper borbonense* is a wild species of the genus *Piper* native to Madagascar, particularly prized for its distinctive aroma and flavor, especially when compared to the domesticated *P. nigrum*. This prompted a comprehensive study of the secondary metabolites contained in the fruits of this plant, carried out by a combination of different analytical techniques. Headspace gas chromatography highlighted the main differences in the aroma between *P. borbonense* and *P. nigrum*, while an untargeted metabolomic analysis, based on the LC-MS²-based Feature-Based Molecular Networking tool, facilitated the annotation of several compounds belonging to different classes, including monoterpenes, sesquiterpenes, phenylpropanoids, lignans and piperamides, guiding the subsequent purification steps. In total, 40 pure metabolites were isolated including two previously undescribed ones, namely 4-hydroxyisogelehomanolide (29) and the hydroxycinnamic acid ester derivative borbonensin (38), that were fully characterized by 1D and 2D NMR. This investigation not only provided the first phytochemical profile of one of the rarest pepper species, highlighting a remarkable abundance of sesamin, but also underlined the relevance of advanced metabolomic approaches in uncovering the incredible chemical diversity contained in spices.
# 1. Introduction

The genus *Piper*, one of the largest in the Piperaceae family, includes more than 700 species that commonly grow in tropical and subtropical regions, particularly in South and Central America, as well as in Southeast Asia. *Piper* plants are well known for their economic importance, as exemplified by *P. nigrum*, the source of black pepper, one of the most widely utilized spices in the world.¹

*P. borbonense* is a rare Madagascar species, whose traditional name is *voatsipérifery*, a word derived from the Malagasy *voa*, meaning fruit, and *tsiperifery*, which literally means “which makes sores not exist”. This wild pepper, fruit of a vine that can grow up to 15 meters on the large trees of tropical forests, is native to the islands of Réunion, Mauritius and Madagascar, where it is traditionally gathered. In fact, the plant is harvested entirely by hand by Malagasy communities, whose villagers must climb high vines to collect the fresh fruits. The rarity of this species and the traditional manual labor involved in its production is reflected in its high market price, about $ 200 per kilogram, at least ten times higher than that of the domesticated *P. nigrum*.² In the last couple of decades *P. borbonense* has become very popular in the gourmet cooking in Europe, where it is especially appreciated for its distinctively delicate flavor characterized by lower pungency and more scents compared to those of the common pepper.³ This "best pepper in the world" (as it is qualified by the triple Michelin-starred chef Anne-Sophie Pic)⁴ is commonly added at the end of meat cooking to preserve its aroma, but is has been also associated with fish, fruits and other foods. Thus, since the demand has rocketed in recent years, *P. borbonense* samples can be adulterated with other genetically related Malagasy wild peppers, such as *P. malgassicum* and *P. tsarasotrae*, belonging to the same clade.⁵ Unambiguous species identification and authentication remain challenging, but the combination of botanical features along with essential oil composition and piperine content have been proposed as a combined system to make the species assignment.⁶

*P. borbonense* has also been used in folk medicine with different applications according to the plant part and to the different regions. The leaves have been used as antidiarrheic and diuretic in La Réunion, while in the Comoros the branches are used to treat dental diseases, and the leaves are used for their antidiabetic and antimalarial properties, as well as to treat intestinal worms.⁷ The traditional uses of this plant have inspired recent scientific evaluations of its medicinal properties. For example, the phenolic mixture of *P. borbonense* was evaluated for immunomodulatory activity,⁷ and the essential oils for fungicidal activity.⁸ Recently, essential oil composition has been studied, providing some
insights into its peculiar aroma composition,² while the profile of its major piperamides has
been compared with that of other species.⁹ However, a detailed analysis of the secondary
metabolites occurring in *P. borbonense* is still lacking and, to fill this gap, we report herein
an untargeted metabolomic analysis using an LC-MS²-based molecular networking that
guided the subsequent NMR-based structural characterization of its secondary
metabolites.

**2. Materials and Methods**

**2.1. Plant Material and Extraction**

*Piper borbonense* fruits (origin Madagascar) were purchased from A. Minardi & Figli,
Bagnacavallo (RA). A voucher sample is stored at xxxxxxx. Organic black piper (*Piper
nigrum*) in grain from Biojoy GmbH company (Germany). A voucher sample is stored at the
Department of Pharmacy, University of Naples Federico II (n. 2024/023).

The extraction of *P. borbonense*...

**2.2. General experimental procedures**

UV and ECD spectra were registered on a JASCO J-710 instrument. Chemicals and
solvents were from Merck Life Science S.r.l. and were used without any further purification
unless stated otherwise. Chromatographic purifications were carried out using MPLC
(Medium Pressure Liquid Chromatography) and HPLC (High Pressure Liquid
Chromatography). MPLC-DAD separations were performed on an Interschim® instrument,
puriFlash XS 520 Plus (Sepachrom s.r.l., Milan, Italy). HPLC-UV-vis separations were
performed on an Agilent instrument, using 1260 Quat Pump VL system, equipped with a
1260 VWD VL UV-vis detector and Knauer 1800 apparatus equipped with a refractive
index detector and a Rheodyne injector. ¹H (400 and 600 MHz) and ¹³C (100 and 150
MHz) NMR spectra were measured on a Bruker spectrometer. Chemical shifts are
referenced to the residual solvent signal (CDCl₃: δH 7.26, δC 77.0; CD₃OD: δH 3.31, δC
49.3). Homonuclear ¹H connectivities were determined by COSY (CORrelation
SpectroscopY) experiments. Through-space ¹H connectivities were evidenced using a
NOESY (Nuclear Overhauser Enhancement SpectroscopY) experiment with a mixing time
of 300 ms. One-bond heteronuclear ¹H–¹³C connectivities was determined by the HSQC
(Heteronuclear Single Quantum Correlation) experiment; two- and three-bond ¹H–¹³C
connectivities by gradient-HMBC (Heteronuclear Multiple Bond Correlation) experiments
optimized for a ²,³J of 8 Hz. HR-ESIMS experiments were performed on LTQ-Orbitrap
mass spectrometer controlled by Excalibur data system. Optical rotations (CHCl₃ and
MeOH) were measured at 589 nm on a JASCO P2000 polarimeter. ECD spectra were
obtained on a Jasco J-815 CD spectropolarimeter (Jasco, Mary's Court, Easton, MD,
USA) with a 0.1 mm cuvette and four accumulations. LC-MS/MS analysis were performed
on an LTQ-XL Ion Trap mass spectrometer (Thermo Fisher, Waltham, MA, USA) equipped
with an Ultimate 3000 HPLC or on a LTQ Orbitrap XL FT-MS mass spectrometer (Thermo
Fisher Scientific Spa) equipped with an Ultimate 3000 HPLC (Agilent Technology).
Chromatographic separation was achieved on a Kinetex 2.6 µm polar C18 100 Å (100 × 3
mm) column (Phenomenex).

**2.3. Head-space gas-chromatography**

Head-space gas-chromatography analysis was carried out on a Perkin-Elmer Clarus 600 gas chromatograph equipped with a Perkin-Elmer Clarus 600 mass spectrometer and a Perkin-Elmer TurboMatrix 40 headspace sampler. 120.0 mg of the sample (*P. borbonense* fruits) were crushed and inserted in a sealed vial, which was heated in the sampler at 80°C for 10 minutes before the injection. Helium was used as gas carrier and 1 µL was injected to the transfer line at 150°C and 30 psi. The VOCs were carried by He through the transfer line maintained at 180°C for analysis in the GCMS system. The gas chromatograph was equipped with a DB-5 column (60 m x 0.25 mm × 0.25 mm inner diameter). The oven temperature was increased from 50 to 240°C at 6°C/min, to 310°C at 10°C/min, holding for 10 minutes, resulting in 46 minutes of measurement time. The column pressure was kept constant at 26 psi in the splitless mode. The mass spectra were obtained operating in electron ionization mode (EI) at 70 eV and the ion source temperature was set at 200 °C, with a scan mass range of *m/z* 40-500 and sampling rate of 0.2 seconds.

**2.4. LC-MS/MS Analysis and Molecular Networking**

The acetonic extract of *P. borbonense* was subjected to a LC-MS/MS analysis on an LTQ
Orbitrap XL FT-MS mass spectrometer (ThermoFisher Scientific Spa) equipped with an
Ultimate 3000 HPLC (Agilent Technology). Chromatographic separation was achieved on
a Kinetex 2.6 µm polar C18 100 Å (100 × 3 mm) column (Phenomenex). The LC-MS/MS
experiment was carried out using a combination of A (0.1% formic acid in H₂O, v/v) and B
(MeCN) as mobile phase; the gradient elution was optimized as follows: 50 % B for 3 min,
50 % to 95% B over 20 min, held 10 min before returning to the initial conditions. The total
run time, including the column wash and the equilibration, was 30 min, flow rate 0.5 mL/min, injection volume 5 µL. The MS and MSn spectra, in positive mode, were recorded in data-dependent acquisition mode, inducing fragmentation of the five most intense peaks for each scan. Source conditions were as follows: spray voltage, 3.5 kV (positive mode); capillary voltage, 25 V; source temperature, 320°C; normalized collision energy, 25. The acquisition range was *m/z* 150-1500. The LC-MSMS data were converted from .raw to .mzML using MSConvert part of the ProteoWizard 3.0 package before being processed on Mzmine 2.53 software using the following parameters: mass detection noise level 1000 for mass level 1; mass detection noise level was set as 50 for nose level 2; the ADAP chromatogram builder was used with a minimum of 5 consecutive scans with a minimum intensity of 1000 and a minimum absolute height of 1000, the *m/z* tolerance was set at 0.005 or 15 ppm; deconvolution was performed with the ADAP resolver with a signal to noise ratio of 30, a minimum feature height of 1000, a coefficient/area threshold set at 10, a peak duration range of 0.0 to 1.0 min and the RT wavelet range set between 0.01 and 0.10; the isotope peak grouper was used with the *m/z* tolerance set at 0.005 or 15 ppm, a RT tolerance of 0.1 min and the maximum number of charges set at 2. The molecular network was generated on MetGem, setting *m/z* tolerance 0.04, minimum matched peaks 3, keeping peak outside ±17 Th, keeping peaks above 1% and in top 6 in the 50 Th window. Then, the maximum neighbour number was set at 10, the minimal score value at 0.50, and max connected component size at 1000. The molecular networks were visualized using Cytoscape v3.7.2 software.

## 2.5. GC-MS Analysis and quantitative analysis

GC-MS analysis was performed on an Agilent 6850 Ser. II apparatus, fitted with a fused silica HP-5 capillary column (30 m x 0.25 mm), 0.33 mm film thickness, coupled to an Agilent Mass Selective Detector MSD 5973; ionization voltage 70 eV; electron multiplier energy 2000 V. Helium was used as carrier gas (1 mL/min). Gas chromatographic conditions were column temperature: 40°C, with 5 min initial hold, and then to 260°C at 2°C min-1, 260°C (20 min). Interface temperature 295°C; mass range was *m/z* 29-600, ionization energy 70 eV, multiplier energy 2000 V, scan time 1 s. Peak identification was accomplished by comparison of their mass spectra with those stored on the GC-MS data bases (NIST 02 and Wiley 275) and reported in literature. The calibration curve was obtained using piperine isomers and sesamin as standards at concentrations of 12.5, 25, 50, 100, 200 ppm. The samples (standards and extract) were dissolved in *n*-hexane.
## 2.6. Isolation of pure compounds

The acetonic extract of *P. borbonense* (4.7 g) was purified on MPLC-DAD on a Silica Cartridge 60 Å 50 µ - size 120 (180 g) - column volume (CV) 153 mL. The mobile phase was a mixture of A: 0.1% formic acid in H₂O, v/v, B: MeCN and C: methanol with the following gradient method: starting conditions: 80 % A – 20 % B for CV 0-2; 70 % A – 30 % B for CV 4-6; 60 % A – 40% B for CV 8-10; 50 % A – 50 % B for CV 13-16; 40 % A – 60 % B for CV 18-20; 10 % A – 90 % B for CV 30-32; 5 % A – 95 % C for CV 33-35; 100 % C for CV 37-42; flow rate was 40.0 mL/min. The UV detection wavelength was set at 270 nm. This separation afforded 26 fractions (A-Z). Fraction B (73.1 mg) was identified as 5-menthene-1,2-diol (23). Fraction D (18.7 mg) was purified on RI-HPLC using a Luna 5 µm C18 100 Å 250 × 4.6 mm, flow rate 1.0 mL/min, using the isocratic elution 50 % A-50 % C, affording compound 24 (0.8 mg). Fraction E (26.9 mg) was purified on RP-HPLC using a mixture of 55 % A-45 % C as mobile phase, on a Luna 5 µm C18 100 Å 250 × 4.6 mm, flow rate 1.0 mL/min, yielding to compound 30 (0.8 mg, Rt 12.4 min), 25 (0.6 mg, Rt 0.6 min), 26 (0.5 mg, Rt 33.4 min), the unprecedented 29 (1.4 mg, Rt 40.0 min) and 27 (1.0 mg, Rt 43.1 min). Fraction G (43.4 mg) was purified on a HPLC-UV, setting the UV detection wavelength at 270 nm, using a Luna 10 µm C18 100 Å 250 × 10 mm, flow rate 3.0 mL/min, to give 31 (1.5 mg, Rt 17.6 min), 32 (1.2 mg, Rt 19.2 min), 1 (2.3 mg, Rt 24.4 min) and 4 (10.3 mg, Rt 27.7 min). Fraction H (16.5 mg) was purified using an HPLC-UV, setting the UV detection wavelength at 230 nm, using Luna 5 µm C18 100 Å 250 × 4.6 mm, flowrate 1.0 mL/min, using a mixture of 50 % A-50 % C as mobile phase, affording 17 (0.4 mg, Rt 16.7 min), 33 (0.5 mg, Rt 16.7 min), 36 (0.3 mg, Rt 21.7 min), 2 (2.3 mg, Rt 23.4 min), 5 (0.6 mg, Rt 26.3 min) and 3 (1.0 mg, Rt 28.4 min). Piperine (6) was obtained from fraction I (495.6 mg) in pure form. Fraction J (29.4 mg) was purified on HPLC-UV, UV detection wavelength set at 270 nm, equipped with a Luna 5 µm C18 100 Å 250 × 4.6 mm, using a flowrate of 1.0 mL/min, using an isocratic elution of 35%A-65%C, yielding to 7 (0.5 mg, Rt 15.0 min), 35 (3.2 mg, Rt 17.1 min), 9 (0.8 mg, 21.1 min), and 8 (0.7 mg, 27.1 min). Sesamine was obtained in pure from fraction K (221.8 mg). Fraction L (92.7 mg) was purified on an MPLC using a Silica Cartridge 100Å 25 µ - size 4 (5.4 g) - column volume (CV) 5 mL, setting UV wavelength at 270 nm and at 5 mL/min flowrate, using the following gradient method: 30 % A - 70 % C for CV 0-5, 25 % A – 75 % C for CV 15-25; 20 % A – 80 % C for CV 35-45; 15 % A – 85 % C for CV 55-60; 5 % A – 95%C for CV 65-70, affording
compound **28** (14.0 mg, CV 9-11) and compound **18** (13.6 mg, CV 13-15). Fraction M (27.3 mg) was purified on an HPLC-UV, setting the UV wavelength at 270 nm, using a Luna 10 µm C18 100 Å 250 × 10 mm, flowrate 3.0 mL/min, using as mobile phase 25 % A-75 % C, obtaining compound **12** (8.8 mg, Rt 28.9 min). Fraction N (38.0 mg) was purified on HPLC-UV using a Luna 5 µm C18 100 Å 250 × 4.6 mm, flowrate 1.0 mL/min, UV wavelength set at 270nm, using 25 % A-75 % C, affording **19** (3.6 mg, 16.3 min), **10** (0.5 mg, Rt 20.3 min), **13** (1.9 mg, Rt 25.8 min). Fraction O (57.8 mg) was purified on a HPLC-UV, setting UV wavelength at 300nm, using a Luna 5 µm C18 100 Å 250 × 4.6 mm, flowrate 1.0 mL/min, with the isocratic method 20 % A-80 % C, obtaining **14** (1.8 mg, Rt 17.9 min) and the previously undescribed **38** (1.0 mg, Rt 18.8 min). Fraction P (80.6 mg) was purified on a MPLC using a Silica Cartridge 100Å 25µ - size 4 (5.4 g) – column volume (CV) 5 mL, setting UV wavelength at 270 nm, using the following gradient method: 50 % A – 50 % C for CV 0-5, 20 % A – 80 % C for CV 55-60; 5 % A – 95 % C for CV 65-67, yielding to **37** (8.9 mg, CV 16-19) and **15** (6.4 mg, CV 20-23) at 5 mL/min flowrate. Fraction Q (29.7 mg) was purified on a HPLC-UV equipped with a Luna 10 µm C18 100 Å 250 × 10 mm, flowrate 3.0 mL/min, with the isocratic method 15 % A – 85 % C, setting the UV wavelength at 270 nm, yielding to 11 (5.8 mg, 20.6 min) and **16** (10.5 mg, Rt 24.8 min). Fraction X (77.3 mg) was purified on MPLC-DAD using a Silica Cartridge 60 Å 50µ - size 4 (4.0 g) – column volume (CV) 5 mL and the following a A= Hexane, and C= Ethyl Acetate a mobile phase with the following gradient method: 90 % A – 10 % C for CV 0-5; 80 % A – 20 % C for CV 10-15, 70 % A – 30 % C for CV 20-25, setting UV wavelength at 220 and 270 nm and 10 mL/min as flowrate, yielding to compound **20** (10.5 mg, CV 3-4), **22** (3.8 mg, 5 CV). Fraction Y (98.9 mg) was purified on MPLC a Silica Cartridge 60Å 50µ - size 12 (12.0 g) – column volume (CV) 25 mL and using A= Hexane, and C= Ethyl Acetate as mobile phase with the following gradient method: 100 % A for CV 0-5; 90% A – 10 % C for CV 10-15 and 80 % A – 20 % C for CV 20-25, at 15 mL/min as flowrate, yielding to the unprecedented **21** (20.6 mg, CV 17-19).

Compound **29**: yellowish amorphous solid, [α]~D~²⁵ +99.3 (c 0.12, MeOH); HRESIMS [M+H]⁺ m/z found 249.1489 (C₁₅H₂₁O₃ requires 249.1485). ¹H NMR (CDCl₃, 600 MHz) 6.11 (1H, d, J = 17.0 Hz, H-6), 5.89 (1H, d, J = 17.0 Hz H-5), 5.11 (1H, brd, J = 11.4 Hz, H-8), 4.94 (1H, brd, J = 10.6 Hz, H-1), 2.90 (1H, brd, J = 11.9 Hz, H-10a), 2.63 (1H, m, H-2a), 2.03 (2H, overlapped, H-2b, H-3a), 2.02 (1H, overlapped, H-10b), 1.88 (3H, s, H-13), 1.81 (3H, s, H-14), 1.77 (1H, overlapped, H-3b), 1.48 (3H, s, H-15); ¹³C NMR (CDCl₃, 150 MHz) 174.9
(C-12), 159.2 (C-7), 152.9 (C-5), 139.6 (C-1), 127.1 (C-10), 121.6 (C-11), 116.3 (C-6), 77.8 (C-8), 75.5 (C-4), 49.3 (C-3), 46.1 (C-9), 27.5 (C-15), 23.9 (C-2), 16.9 (C-14), 8.8 (C-13).

Borbonensin (38): white amorphous solid, [α]D²⁵ +6.8 (c 0.12, MeOH); ESIMS [M-H]⁻ m/z found 297.1490 (C₁₉H₂₁O₃ requires 297.1485). ¹H NMR (CDCl₃, 600 MHz) 7.64 (1H, d, *J* = 15.9 Hz, H-3), 7.43 (2H, d, *J* = 8.6 Hz, H-5, H-9), 6.83 (2H, d, *J* = 8.6 Hz, H-6, H-8), 6.30 (1H, d, *J* = 15.9 Hz, H-2), 5.58 (1H, m, H-4'), 4.59 (1H, d, *J* = 12.7, 1.3 Hz H-2'a), 4.55 (1H, dd, *J* = 12.7, 1.3 Hz, H-2'b), 2.41 (1H, m, H-7'a), 2.33 (1H, m, H-5'a), 2.26 (1H, m, H-5'b), 2.17 (1H, dt, *J* = 5.5, 1.2 Hz, H-8'), 2.11 (1H, m, H-6'), 1.30 (3H, s, H-10'), 1.21 (1H, overlapped, H-7'b), 0.85 (3H, s, H-11'); ¹³C NMR (CDCl₃, 150 MHz) 167.3 (C-1), 157.4 (C-7), 144.3 (C-3), 143.1 (C-3'), 130.1 (C-5, C-9), 127.4 (C-4), 121.1 (C-4'), 115.9 (C-6, C-8), 115.8 (C-2), 66.9 (C-2'), 43.6 (C-8'), 40.6 (C-6'), 38.8 (C-7'), 31.2 (C-5'), 27.1 (C-9'), 26.0 (C-10'), 21.1 (C-11').

## 2.7. Computational Details

A preliminary conformational search for the 29 model was conducted using simulated annealing in the INSIGHT II package. The solution phase in MeOH was simulated by setting the corresponding dielectric constant. Conformational energy minimization was achieved through steepest descent, followed by the quasi-Newton-Raphson method (VA09A). Restrained simulations were run for 500 ps using the CVFF force field, as implemented in Discover software (Accelrys, San Diego, CA). The simulation began at 1000 K, and the temperature gradually decreased to 300 K. A final energy minimization step was performed to refine the obtained structures, using successive applications of steepest descent and quasi-Newton-Raphson (VA09A) algorithms. Both dynamic and mechanical calculations were conducted with flat-well distance restraints set to 1 (kcal/mol)/Å². A total of one hundred structures were generated. All optimizations were performed with the Gaussian 16W software package, using the DFT functional M06-2X and the basis set 6-311G(2d,2p). The rotatory strength values for electronic transitions from the ground state to singly excited states for the two conformers of E09-a were calculated using TDDFT at the M06-2X/6-311G(2d,2p) level with Gaussian 16W, including at least 30 excited states in each case and using the IEF-PCM model for MeOH. The resulting ECD spectra were then compared to the experimental spectra using SpecDis 1.71 software.

# 3. Results and Discussion
## 3.1. Analysis of the volatile compounds of *P. borbonense* fruits

Headspace gas chromatography-mass spectrometry (HS-GC-MS) has never been used to analyze the aroma composition of *P. borbonense*, previously investigated only via GC-MS.² Since it requires no sample preparation and can be carried out directly on the crushed fruit of the plant, HS-GCMS has the potential to provide a more accurate profile of the volatile components of this highly prized pepper. To highlight the main differences between *P. borbonense* and the common *P. nigrum*, we carried out a comparative analysis of the HS-GC-MS profile of the two species, identifying the compounds using the NIST library (Table 1). In agreement with GC-MS results,² the most abundant compounds were monoterpenes (98.15% in *P. borbonense*, and 97.67% in *P. nigrum*) and, among them, the most abundant compound was δ-3-carene, (40.91% in *P. borbonense* and 50.23% in *P. nigrum*). The other major peaks were assigned to D-limonene and α-pinene. The β-pinene content was 10.48% in *P. borbonense* and 12.56% in *P. nigrum*, while β-thujene reached 7.51% in *P. borbonense* and only 0.53% in *P. nigrum*. The total sesquiterpenoid composition was higher in *P. nigrum* (2.15% compared to 1.43% of *P. borbonense*), but, interestingly, *P. borbonense* showed a more diverse composition, since in *P. nigrum* the sesquiterpenoid fraction appear to be dominated by caryophyllene (1.87%, only 0.44% in *P. borbonense*). Finally, phenylpropanoids were not detected in *P. nigrum*, whereas they were found in small amounts (0.32%) in *P. borbonense*.

<table><caption>**Table 1.** Comparison of HS-GC-MS profiles of *P. borbonense* and *P. nigrum* (major compounds are highlighted in bold)</caption><thead><tr><th>Class</th><th>compound name</th><th>Rt (min)</th><th><b>P. borbonense</b><br>Area %</th><th><b>P. nigrum</b><br>Area %</th></tr></thead><tbody><tr><td>Monoterp.</td><td>α-phellandrene</td><td>12.10</td><td>0.56</td><td>0.18</td></tr><tr><td>Monoterp.</td><td><b>α-pinene</b></td><td>12.47</td><td><b>12.21</b></td><td><b>9.73</b></td></tr><tr><td>Monoterp.</td><td>bicyclo[2.2.1]heptane,7,7-dimethyl-2-methylene-</td><td>13.02</td><td>n.d.</td><td>0.02</td></tr><tr><td>Monoterp.</td><td>camphene</td><td>13.12</td><td>0.19</td><td>0.11</td></tr><tr><td>Monoterp.</td><td><b>sabinene</b></td><td>13.68</td><td><b>1.33</b></td><td><b>0.20</b></td></tr><tr><td>Monoterp.</td><td>1,3,5-cycloheptatriene, 3,7,7-trimethyl-</td><td>13.76</td><td>0.03</td><td>0.03</td></tr><tr><td>Monoterp.</td><td><b>β-myrcene</b></td><td>13.92</td><td><b>1.66</b></td><td><b>2.74</b></td></tr><tr><td>Monoterp.</td><td><b>β-pinene (PN)</b></td><td>14.00</td><td><b>10.48</b></td><td><b>12.56</b></td></tr><tr><td>Monoterp.</td><td>α-terpinolene</td><td>14.46</td><td>0.01</td><td>0.03</td></tr><tr><td>Monoterp.</td><td><b>δ-3-carene</b></td><td><b>14.80</b></td><td><b>40.91</b></td><td><b>50.23</b></td></tr><tr><td>Monoterp.</td><td>1,3-cyclohexadiene, 1-methyl-4-(1-methylethyl)-</td><td>15.02</td><td>0.07</td><td>0.09</td></tr><tr><td>Monoterp.</td><td>o-cymene</td><td>15.13</td><td>0.04</td><td>0.09</td></tr><tr><td>Monoterp.</td><td>cyclohexene, 1-methyl-5-(1-methylethenyl)-</td><td>15.24</td><td>0.01</td><td>0.08</td></tr><tr><td>Monoterp.</td><td><b>p-cymene</b></td><td>15.35</td><td><b>5.52</b></td><td><b>1.86</b></td></tr><tr><td>Monoterp.</td><td><b>D-limonene</b></td><td>15.43</td><td><b>15.91</b></td><td><b>17.81</b></td></tr><tr><td>Monoterp.</td><td><b>β-thujene</b></td><td>15.58</td><td><b>7.51</b></td><td><b>0.53</b></td></tr><tr><td>Monoterp.</td><td>eucalyptol</td><td>15.66</td><td>0.59</td><td>0.11</td></tr><tr><td>Monoterp.</td><td>trans-thujene</td><td>16.04</td><td>0.01</td><td>0.06</td></tr><tr><td>Monoterp.</td><td>γ-terpinene</td><td>16.25</td><td>0.10</td><td>0.14</td></tr><tr><td>Monoterp.</td><td>trans-4-thujanol</td><td>16.76</td><td>&lt; 0.01</td><td>&lt; 0.01</td></tr></tbody></table>
<table><tr><td>Monoterp.</td><td>isoterpinolene</td><td>16.91</td><td>0.05</td><td>0.32</td></tr><tr><td>Monoterp.</td><td>α-Terpinolene</td><td>17.08</td><td>0.64</td><td>0.70</td></tr><tr><td>Monoterp.</td><td>Linalool</td><td>17.32</td><td>0.09</td><td>0.05</td></tr><tr><td>Monoterp.</td><td>β,β-Dimethylstyrene</td><td>17.40</td><td>0.01</td><td>nd</td></tr><tr><td>Monoterp.</td><td>trans-sabinenehydrate</td><td>17.75</td><td>0.02</td><td>nd</td></tr><tr><td>Monoterp.</td><td>cosmene</td><td>18.04</td><td>nd</td><td>&lt; 0.01</td></tr><tr><td>Monoterp.</td><td>(+)-4-carene</td><td>18.43</td><td>0.01</td><td>nd</td></tr><tr><td>Monoterp.</td><td>p-mentha-1,5,8-triene</td><td>18.76</td><td>&lt; 0.01</td><td>&lt; 0.01</td></tr><tr><td>Monoterp.</td><td>(+)-2-bornanone</td><td>19.48</td><td>0.01</td><td>nd</td></tr><tr><td>Monoterp.</td><td>p-mentha-1,5-dien-8-ol</td><td>19.60</td><td>0.01</td><td>nd</td></tr><tr><td>Monoterp.</td><td>trans-sabinenehydrate</td><td>20.10</td><td>0.01</td><td>nd</td></tr><tr><td>Monoterp.</td><td>benzenemethanol, α,α,4-trimethyl-</td><td>20.29</td><td>0.01</td><td>nd</td></tr><tr><td>Monoterp.</td><td>α-terpinol</td><td>20.50</td><td>0.01</td><td>nd</td></tr><tr><td>Monoterp.</td><td>trans-sabinol</td><td>20.80</td><td>0.07</td><td>nd</td></tr><tr><td>Monoterp.</td><td>linalyl acetate</td><td>21.23</td><td>0.05</td><td>nd</td></tr><tr><td>Monoterp.</td><td>Isopiperitone</td><td>22.27</td><td>0.02</td><td>nd</td></tr><tr><td>Penylprop.</td><td>safrolene</td><td>23.22</td><td>0.15</td><td>nd</td></tr><tr><td>Sesquiterp.</td><td>δ-elemene</td><td>23.72</td><td>0.02</td><td>0.01</td></tr><tr><td>Monoterp.</td><td>camphenol</td><td>23.96</td><td>&lt; 0.01</td><td>nd</td></tr><tr><td>Sesquiterp.</td><td>α-cubebene</td><td>24.05</td><td>0.02</td><td>&lt; 0.01</td></tr><tr><td>Sesquiterp.</td><td>trans-α-copaene</td><td>24.77</td><td>0.07</td><td>nd</td></tr><tr><td>Sesquiterp.</td><td>copaene</td><td>24.95</td><td>0.11</td><td>0.13</td></tr><tr><td>Sesquiterp.</td><td>elemene</td><td>25.15</td><td>0.07</td><td>0.03</td></tr><tr><td>Penylprop.</td><td>methylugenol</td><td>25.54</td><td>0.11</td><td>nd</td></tr><tr><td>Sesquiterp.</td><td>eremophylene</td><td>25.70</td><td>nd</td><td>0.01</td></tr><tr><td>Sesquiterp.</td><td>ylangene</td><td>26.08</td><td>0.01</td><td>nd</td></tr><tr><td>Sesquiterp.</td><td><b>caryophyllene</b></td><td>26.22</td><td><b>0.44</b></td><td><b>1.87</b></td></tr><tr><td>Sesquiterp.</td><td>α-guaiene</td><td>26.30</td><td>0.01</td><td>nd</td></tr><tr><td>Sesquiterp.</td><td>copaen-4-α-ol</td><td>26.40</td><td>0.05</td><td>0.01</td></tr><tr><td>Sesquiterp.</td><td>muurola-3,5-diene</td><td>26.83</td><td>0.01</td><td>nd</td></tr><tr><td>Sesquiterp.</td><td>humulene</td><td>27.10</td><td>0.01</td><td>0.05</td></tr><tr><td>Diterp.</td><td>geranyl-α-terpinene</td><td>27.21</td><td>0.01</td><td>nd</td></tr><tr><td>Sesquiterp.</td><td>γ-muurolene</td><td>27.33</td><td>0.02</td><td>nd</td></tr><tr><td>Sesquiterp.</td><td>δ-curcumene</td><td>27.50</td><td>0.02</td><td>nd</td></tr><tr><td>Sesquiterp.</td><td>germacrene D</td><td>27.63</td><td>0.13</td><td>nd</td></tr><tr><td>Sesquiterp.</td><td>α-guaiene</td><td>27.70</td><td>0.22</td><td>nd</td></tr><tr><td>Sesquiterp.</td><td>β-bisabolene</td><td>27.78</td><td>0.07</td><td>nd</td></tr><tr><td>Sesquiterp.</td><td>isoledene</td><td>27.82</td><td>nd</td><td>&lt; 0.01</td></tr><tr><td>Sesquiterp.</td><td>epi-α-selinene</td><td>27.92</td><td>0.03</td><td>0.03</td></tr><tr><td>Sesquiterp.</td><td>δ-guaiene</td><td>27.99</td><td>0.03</td><td>nd</td></tr><tr><td>Penylprop.</td><td>myristicine</td><td>28.08</td><td>0.01</td><td>nd</td></tr><tr><td>Sesquiterp.</td><td>7-epi-α-cadinene</td><td>28.25</td><td>0.03</td><td>0.01</td></tr><tr><td>Sesquiterp.</td><td>4-epi-cubebol</td><td>28.37</td><td>0.02</td><td>nd</td></tr><tr><td>Sesquiterp.</td><td>trans-calamenene</td><td>28.51</td><td>0.01</td><td>nd</td></tr><tr><td>Penylprop.</td><td>trans-isomyristicin</td><td>28.67</td><td>0.02</td><td>nd</td></tr><tr><td>Penylprop.</td><td>elemicin</td><td>28.78</td><td>0.03</td><td>nd</td></tr></table>

## 3.2. Metabolomic fingerprint of *P. borbonense* fruits through LC-UV-MS/MS and FB-MN analysis

The alkylamide composition of *P. borbonense* has been roughly compared with that of other *Piper* species,⁹ but a comprehensive phytochemical characterization of this plant has not yet been carried out. A metabolomic fingerprint of *P. borbonense* was achieved through a preliminary analysis of the crude acetone extract by LC coupled to HR-MS and feature-based molecular network (FB-MN) analysis¹⁰,¹¹ using MZmine¹² and MetGem
open-source software.¹³ The first software is used to pre-process the raw files, while MetGem compares MS/MS data, immediately visualizing the similarities among the spectra, that are then clustered according to their fragmentation pattern analogies. One of the nodes obtained in the cluster (Figure 1) was assigned to piperine (6), parent ion at m/z 286.1433, which appear to be the main component of the mixture from the FB-MN and LC-UV-MS² analysis ( **Érreur ! Source du renvoi introuvable.**).

<figure><img src="image_3.png" /><figcaption>**Figure 1.** A) Molecular networking cluster of piperamides. Nodes are labeled with the parent m/z value and sized according to the peak area; edge thickness is related to the cosine similarity score. In green: the isolated metabolites; in blue: the annotated metabolites; in pink: not annotated. B) Chemical structure of piperine and its diagnostic fragments.</figcaption></figure>

The detected fragment ions correspond to the loss of the piperidinyl moiety (m/z 201.0552), the α-cleavage to the amide group (m/z 173.0602) and the related fragment with the opposite charge retention (m/z 112.0759), and finally the loss of the benzodioxole moiety (m/z 135.0444), in agreement with the data reported in the literature.⁹ Considering the recurrence of the fragment at m/z 135.0444 in the cluster, we can assume that also the other nodes belong to benzodioxole piperamides. Consequently, they can be assigned to guineesine (16) (m/z 384.2525), clustered with m/z 396.2526, tentatively identified as 13-(1,3-benzodioxol-5-yl)-1-(1-piperidinyl) and to piperchambamide D (m/z 358.2371). This latter clustered together with two isomers with different retention time and size (nodes at m/z 274.1432), one assigned to 4,5-dihydropiperyline (1), which closely clustered with piperanine (3) (m/z 288.1586), and the other confidently assigned to piperlonguminin (5) given the different fragmentation pattern.

This preliminary information guided a detailed manual analysis of the LC-HRMS/MS, resulting in the putative annotation of 36 piperamides (Table 2), basing on their molecular formula and fragmentation pattern analysis, that matched those reported in literature.⁹
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Overlay of BPC (red) and PDA (black) chromatograms of *P. borbonense* acetic acid extract.</figcaption></figure>

<table><caption>**Table 2.** Annotated compounds from LC-HRMS/MS; compounds are listed in order of elution; t.id. stands for tentative identification.</caption><thead><tr><th>Compound name</th><th>Molecular formula</th><th>t<sub>R</sub> (min)</th><th>Precursor ion (m/z)</th><th>Δ (ppm)</th><th>Fragments (m/z)</th></tr></thead><tbody><tr><td>4,5-Dihydropiperyline (1)</td><td>C<sub>16</sub>H<sub>19</sub>NO<sub>3</sub></td><td>2.03</td><td>274.1430</td><td>- 2.663</td><td>203.0707;<br>175.0758;<br>161.0601; 135.0443</td></tr><tr><td>Piperyline (4)</td><td>C<sub>16</sub>H<sub>17</sub>NO<sub>3</sub></td><td>2.14</td><td>272.1276</td><td>- 2.058</td><td>201.0551; 173.0602;<br>143.0494; 135.0443;<br>115.0503</td></tr><tr><td>Dihydropiperlonguminine (2)</td><td>C<sub>16</sub>H<sub>21</sub>NO<sub>3</sub></td><td>2.58</td><td>276.1590</td><td>- 1.630</td><td>203.0704; 175.0757;<br>161.0599; 135.0441</td></tr><tr><td>Piperlonguminin (5)</td><td>C<sub>16</sub>H<sub>19</sub>NO<sub>3</sub></td><td>2.69</td><td>274.1432</td><td>- 1.897</td><td>201.0553;<br>175.0760; 145.0651;<br>135.0444; 115.0541</td></tr><tr><td>Piperanine (3)</td><td>C<sub>17</sub>H<sub>21</sub>NO<sub>3</sub></td><td>2.81</td><td>288.1586</td><td>- 2.950</td><td>203.0709; 175.0761;<br>161.0602; 135.0444</td></tr><tr><td>Piperine (6)</td><td>C<sub>17</sub>H<sub>19</sub>NO<sub>3</sub></td><td>3.03</td><td>286.1427</td><td>- 3.739</td><td>201.0552; 173.0602;<br>135.0444; 112.0759</td></tr><tr><td>7-(1,3-Benzodioxol-5-yl)-1-(1-pyrrolidinyl)-2,4-heptadien-1-one</td><td>C<sub>18</sub>H<sub>21</sub>O<sub>3</sub>N</td><td>3.20</td><td>300.1590</td><td>- 1.499</td><td>229.0863, 201.0914,<br>135.04442</td></tr><tr><td>Piperdardine (8)</td><td>C<sub>19</sub>H<sub>23</sub>NO<sub>3</sub></td><td>4.75</td><td>314.1742</td><td>- 2.706</td><td>229.0867; 201.0915;<br>135.0443</td></tr></tbody></table>
<table>
  <tr>
    <td>Sarmentine</td>
    <td>C<sub>14</sub>H<sub>23</sub>NO</td>
    <td>4.98</td>
    <td>222.1849</td>
    <td>- 1.624</td>
    <td>151.1120; 133.1014;<br/>124.0759; 95.0492</td>
  </tr>
  <tr>
    <td>Marginatine</td>
    <td>C<sub>15</sub>H<sub>20</sub>O<sub>2</sub></td>
    <td>5.10</td>
    <td>233.1533</td>
    <td>- 1.357</td>
    <td>215.1438; 187.1487;<br/>177.0916; 133.1015</td>
  </tr>
  <tr>
    <td>Pellitorine (18)</td>
    <td>C<sub>14</sub>H<sub>25</sub>NO</td>
    <td>6.00</td>
    <td>224.2004</td>
    <td>- 2.235</td>
    <td>168.1358; 151.1119;<br/>133.1013; 109.0648</td>
  </tr>
  <tr>
    <td>Neopellitorine B (19)</td>
    <td>C<sub>15</sub>H<sub>25</sub>NO</td>
    <td>7.23</td>
    <td>236.2004</td>
    <td>- 2.121</td>
    <td>151.1120; 133.1013;<br/>123.1171; 112.0757</td>
  </tr>
  <tr>
    <td>Dehydropipernonaline (12)</td>
    <td>C<sub>21</sub>H<sub>25</sub>NO<sub>3</sub></td>
    <td>7.46</td>
    <td>340.1905</td>
    <td>- 0.588</td>
    <td>255.1027; 227.1076;<br/>179.1311; 161.0602;<br/>112.0759</td>
  </tr>
  <tr>
    <td>Pipernonaline (10)</td>
    <td>C<sub>21</sub>H<sub>27</sub>NO<sub>3</sub></td>
    <td>8.21</td>
    <td>342.2056</td>
    <td>- 2.251</td>
    <td>255.1390; 229.1226;<br/>220.1698; 201.0354</td>
  </tr>
  <tr>
    <td>1-(Pyrrolidinyl)-11-(3',4'-<br/>methylenedioxyphenyl)-2,4,10-<br/>undecatrien-1-one<br/>(13)</td>
    <td>C<sub>22</sub>H<sub>27</sub>NO<sub>3</sub></td>
    <td>9.02</td>
    <td>354.2059</td>
    <td>- 1.328</td>
    <td>283.1345; 255.1391;<br/>232.1705; 215.1073;<br/>135.0443</td>
  </tr>
  <tr>
    <td>Piperacide (14)</td>
    <td>C<sub>22</sub>H<sub>29</sub>NO<sub>3</sub></td>
    <td>9.40</td>
    <td>356.2218</td>
    <td>- 0.703</td>
    <td>283.1339; 255.1389;<br/>234.1859; 215.1074;<br/>135.1171</td>
  </tr>
  <tr>
    <td>Piperchabamide D</td>
    <td>C<sub>22</sub>H<sub>31</sub>O<sub>3</sub><br/>N</td>
    <td>10.50</td>
    <td>358.2370</td>
    <td>- 1.955</td>
    <td>285.1393; 235.1862;<br/>135.1170</td>
  </tr>
  <tr>
    <td>Piperundecalidine (15)</td>
    <td>C<sub>23</sub>H<sub>29</sub>NO<sub>3</sub></td>
    <td>10.85</td>
    <td>368.2213</td>
    <td>- 1.848</td>
    <td>283.1332; 255.1383;<br/>135.0441</td>
  </tr>
  <tr>
    <td>Piperchabamide B (11)</td>
    <td>C<sub>23</sub>H<sub>31</sub>NO<sub>3</sub></td>
    <td>11.74</td>
    <td>370.2372</td>
    <td>- 1.405</td>
    <td>285.1492; 257.1545;<br/>161.0601; 135.0442</td>
  </tr>
  <tr>
    <td>Brachyamide A</td>
    <td>C<sub>24</sub>H<sub>31</sub>NO<sub>3</sub></td>
    <td>12.24</td>
    <td>382.2372</td>
    <td>- 1.361</td>
    <td>313.1805; 283.1699;<br/>135.0442</td>
  </tr>
  <tr>
    <td>Guineensine (16)</td>
    <td>C<sub>24</sub>H<sub>33</sub>NO<sub>3</sub></td>
    <td>12.73</td>
    <td>384.2525</td>
    <td>- 2.057</td>
    <td>311.1646; 283.1698;<br/>161.0598; 135.0442</td>
  </tr>
  <tr>
    <td>13-(1,3-Benzodioxol-5-yl)-1-(1-<br/>piperidinyl)-2,4,12-tridecatrien-<br/>1-one<br/>N-Isobutyl-2,4,10-<br/>hexadecatrienamide</td>
    <td>C<sub>25</sub>H<sub>33</sub>NO<sub>3</sub></td>
    <td>13.98</td>
    <td>396.2526</td>
    <td>- 1.768</td>
    <td>311.1646; 283.1697;<br/>135.0442</td>
  </tr>
  <tr>
    <td>1-(1-Piperidinyl)-2,4,10-<br/>hexadecatrien-1-one</td>
    <td>C<sub>20</sub>H<sub>35</sub>NO</td>
    <td>14.09</td>
    <td>306.2786</td>
    <td>- 1.800</td>
    <td>233.1911; 205.1961</td>
  </tr>
  <tr>
    <td>Piperolein B</td>
    <td>C<sub>21</sub>H<sub>35</sub>ON</td>
    <td>15.79</td>
    <td>318.2784</td>
    <td>- 2.203</td>
    <td>233.1910; 112.0758</td>
  </tr>
  <tr>
    <td>N-Isobutyl-hexadeca-2,4-<br/>dienamide</td>
    <td>C<sub>20</sub>H<sub>37</sub>ON</td>
    <td>16.78</td>
    <td>308.2943</td>
    <td>- 1.756</td>
    <td>265.4167; 235.2061;<br/>182.1539</td>
  </tr>
  <tr>
    <td>N-Isobutyl-2,4,12-<br/>octadecatrienamide</td>
    <td>C<sub>22</sub>H<sub>39</sub>NO</td>
    <td>17.42</td>
    <td>334.3100</td>
    <td>- 1.231</td>
    <td>261.2218; 233.2266</td>
  </tr>
  <tr>
    <td>N-Isobutyl-2,4,10,12-<br/>Octadecatetraenamide</td>
    <td>C<sub>22</sub>H<sub>37</sub>NO</td>
    <td>18.10</td>
    <td>332.2942</td>
    <td>- 1.810</td>
    <td>261.2218; 233.2277</td>
  </tr>
  <tr>
    <td>1-(1-Piperidinyl)-2,4,12-<br/>octadecatrien-1-one</td>
    <td>C<sub>23</sub>H<sub>39</sub>NO</td>
    <td>18.94</td>
    <td>346.3097</td>
    <td>- 2.083</td>
    <td>261.2221; 233.2267;<br/>112.0759</td>
  </tr>
  <tr>
    <td>Pipericine</td>
    <td>C<sub>22</sub>H<sub>41</sub>NO</td>
    <td>19.68</td>
    <td>336.3256</td>
    <td>- 1.580</td>
    <td>263.2369; 234.0333;<br/>157.7024</td>
  </tr>
</table>
<table><tr><td>1-(Piperidinyl)-2,4-octadecadien-1-one</td><td>C<sub>23</sub>H<sub>41</sub>NO</td><td>19.85</td><td>348.3257</td><td>- 1.095</td><td>264.2330; 236.2014; 112.0758</td></tr><tr><td>N-Isobutyl-2,4,14-eicosatrienamide</td><td>C<sub>24</sub>H<sub>43</sub>NO</td><td>20.09</td><td>362.3407</td><td>- 2.957</td><td>289.2535; 261.2585</td></tr><tr><td>1-(1-Pyrrolidinyl)-2,4,15-eicosatrien-1-one (22)</td><td>C<sub>24</sub>H<sub>41</sub>NO</td><td>20.94</td><td>360.3257</td><td>- 1.142</td><td>289.2533; 262.2183</td></tr><tr><td>Pipercitine</td><td>C<sub>23</sub>H<sub>43</sub>NO</td><td>21.41</td><td>350.3412</td><td>- 1.403</td><td>264.2398; 212.1963</td></tr><tr><td>1-(Piperidinyl)-2,4,14-eicosatrien-1-one (21)</td><td>C<sub>25</sub>H<sub>43</sub>NO</td><td>21.50</td><td>374.3412</td><td>- 1.473</td><td>318.28; 289.2535; 261.2588</td></tr><tr><td>1-(Piperidinyl)-2,4-eicosadien-1-one</td><td>C<sub>25</sub>H<sub>45</sub>NO</td><td>22.50</td><td>376.3568</td><td>- 1.545</td><td>291.2700; 112.0760</td></tr></table>

Piperamides, the most abundant class of metabolites present in the extract, include two main subclasses, according to the terminal part of the fatty acid chain, one bearing a benzodioxole moiety, as deduced from the FBMN analysis, and another simply ending with a methyl group. The positive ion mode used for the LC-MS² experiment was well suited for the analysis of piperamides, but the LC-UV analysis showed an interesting pattern (Figure 2). In particular, as shown in Figure 2, the Photo Diode Array (PDA) chromatogram included several additional peaks compared to the LC-MS base peak chromatogram (BPC), suggesting the presence of additional metabolites. When LC-UV-MS² analysis was performed in negative mode, the BPC resulted flat, suggesting that these metabolites are not ionizing and cannot be detected neither in positive nor in negative mode.

### 3.3. Chromatographic purification and GC-MS quantification of sesamin

In order to carry out a comprehensive and detailed analysis of the secondary metabolites present in the acetonic extract of *P. borbonense* fruits, chromatographic purification was needed. To this aim, the acetone extract (about 5 g) was purified by MPLC as described in detail in the Experimental section. Piperamides were confirmed to be the most abundant class of metabolites present in the extract, but also terpenes and lignans were largely represented. In fact, this first purification step yielded, as major compounds, 5-*p*-menthene-1,2-diol (23, 73 mg),¹⁴ a deoxygenated monoterpene never reported in the *Piper* genus, piperine (6, 495.6 mg)¹⁵ and sesamin (34, 221.8 mg),¹⁶ while the other fractions needed further purification.

The unexpected relative abundance of sesamin, Rt 3.79 in the PDA chromatogram (see Fig. 2), prompted us to carry out a quantitative analysis. The measurement of sesamin and
of the main piperine isomers in the extract of *P. borbonense* was carried out via GC-MS analysis (Figure 3).

<figure><img src="image_5.png" /><figcaption>**Figure 3.** GC-MS chromatograms of *P. borbonense* (upper) and *P. nigrum* (lower) acetonic extracts</figcaption></figure>

Sesamin and a mixture of piperin isomers were used as external standards, each at concentrations of 12.5, 25, 50, 100, and 200 ppm. The calibration curve demonstrated a high coefficient of determination (R² = 0.9986-0.9993), indicating the reliability of the method. Due to their structural similarity, piperine was quantified as the sum of all stereoisomers, which eluted at retention times of 24.89, 25.04, and 26.88 minutes (Figure 3). The results confirmed a very high content of sesamin (152 mg/g) in the extract of *P. borbonense*, comparable with the content of piperine isomers (233 mg/g). Despite it is one of the major components of the acetonic extract, this is the first report of sesamin in *P. borbonense*. Interestingly, this lignan was not detectable in our samples of *P. nigrum* (Figure 3) and only a couple of papers have reported the presence of small amounts of sesamin in common black pepper.¹⁷,¹⁸ The relatively high concentration of sesamin had been probably missed in earlier studies of *P. borbonense* due to the almost exclusive use of LC-ESI-MS/MS for the quantitative determination of its metabolites.

According to recent studies conducted on sesame oil, where this lignan is found in high concentrations, sesamin can affect aroma¹⁹ directly and also indirectly, because it can
affect flavor formation and improve sensory perception by interfering with lipid oxidation.²⁰

Moreover, sesamin content can influence the processing steps of the biomass, but it has never been taken into account in the previous studies on the processing of *P. borbonense*.²¹

**3.4. Isolation and characterization of *P. borbonense* secondary metabolites**

Several piperamides, previously annotated via LC-MS/MS were also isolated and unambiguously identified (Figure 4), generally comparing their ¹H NMR profile with that reported in the literature. Along with the main compound piperine (6), the benzodioxole piperamides 4,5-dihydropiperyline (1),²² dehydropiperlonguminin (2),²³ piperanine (3),¹⁵ piperiline (4),²⁴ piperlonguminin (5),¹⁵ isochavine (7),²⁵ piperdardine (8),²⁶ chingchengenamide A (9),²⁷ pipernonaline (10),¹⁵ piperchabamide B (11),²⁸ dehydropipernonaline (12),²⁹ (2E,4E,10E)-11-(1,3-benzodioxol-5-yl)-1-(1-pyrrolidinyl)-2,4,10-undecatrien-1-one (13),³⁰ piperacide (14),³¹ piperundecalide (15),³² guineesine (16),¹⁵ and piperodione (17)³³ were isolated. In addition, the following members of the N-linked long-chain fatty acids class were obtained (Fig. 3): pellitorine (18),³⁴ neopellitorine B (19),²⁷ N-isobutyl-2E,4E,14Z-eicosatrienamide (20),¹⁵ 1-(piperidinyl)-2,4,14-eicosatrien-1-one (21) and 1-(1-pyrrolidinyl)-2,4,15-eicosatrien-1-one (22).³⁵
<figure><img src="image_6.png" /><figcaption>**Figure 4.** Piperamides isolated from *P. borbonense*</figcaption></figure>

Among the isolated piperamides, chingchengenamide A (9), piperacide (14) and neopellitorine B (19) have never been reported not only in *P. borbonense* but also in the common *P. nigrum*. The 14Z configuration of 20 and 21 (and 15Z of 22) was supported through the study of fragment peaks, supporting their location, and of the chemical shifts of two adjacent methylene carbons (δ<sub>C</sub> 27.2), in agreement with data reported in the literature.<sup>36</sup> Interestingly, this configuration has been associated with a prolonged tingling sensation<sup>35</sup> and, given their significant presence in *P. borbonense*, they may contribute to the peculiar taste of this spice.

As previously stated, metabolites of the terpene class constituted a rich part of the extract. Besides the above mentioned **23**, the related monoterpene 5β-hydroxy-2-oxo-p-menth-6(1)-ene (24)<sup>37</sup> was also isolated. The sesquiterpenoids multistalactones E (25) and D (26)<sup>38</sup> and neolitacumone B (27)<sup>39</sup>, all never reported before in genus *Piper*, were also isolated, along with the previously undescribed 4-hydroxyisogelehumanolide (29), an analogue of the germacrenolide gelehumanloide (28)<sup>40</sup> co-occurring in the plant (Figure 5).
<figure><img src="image_7.png" /><figcaption>**Figure 5.** Terpenoids isolated from *P. borbonense*</figcaption></figure>

Finally, among the lignans, beside the abundant sesamin (34), other isolated compounds were 5-methoxysesamin (35),<sup>41</sup> (+)-acuminatolide (30),<sup>42</sup> never reported before in genus *Piper*, compound 31,<sup>43</sup> piperitol (32),<sup>44</sup> pluviatiol (33),<sup>45</sup> and verrucosin (36),<sup>45</sup> also unreported before in the genus *Piper*. Furthermore, two monoterpenyl hydroxycinnamic esters derivatives were isolated, namely the known pressafonin A (37)<sup>46</sup> and the unprecedented myrtenoyl ester 38, named borbonensin (Figure 6**Figure** ).

<figure><img src="image_8.png" /><figcaption>**Figure 6.** Phenols isolated from *P. borbonense*. Chart A: lignans; Chart B: hydroxycinnamic esters.</figcaption></figure>
4-Hydroxyisogelehumanolide (29) was isolated as colorless amorphous solid with HR-ESIMS *m/z* 249.1485, in agreement with the molecular formula of C₁₅H₂₀O₃, implying six degrees of unsaturation. The ¹H NMR spectrum of 29 compound was very similar to that of 28, although it included one additional oxygen atom. A close comparison of the ¹H NMR spectra of the two compounds revealed that the most remarkable difference was a pair of mutually coupled olefinic protons resonating at δH 6.10 (1H, d, *J* = 17.0 Hz) and 5.90 (1H, d, *J* = 17.0 Hz) in place of the doublet at δH 4.85 (1H, d, *J* = 12.0 Hz, H-5). This assignment was further supported by the inspection of ¹³C NMR and 2D HSQC spectra allowing the identification of 15 carbon signals including two allylic methyls (δC 16.9, C-14 and 23.9, C-2), one aliphatic methyl (δC 8.8, C-13), three methylenes (δC 23.9, C-2; 46.3, C-9 and 49.3, C-3), three olefinic methines (δC 116.3, C-5; 139.6, C-1; 152.9, C-6), three olefinic quaternary carbons (δC 121.6, C-11; 127.1, C-10; 159.2, C-7), three oxygenated carbons, including an oxygentated methine resonanting at δC 77.8 (C-8), one quaternary sp³ carbon (δC 75.5, C-4) and one quaternary sp² carbon (δC 174.9, C-12). Analysis of the 2D NMR COSY experiments led to the definition of two spin systems, spanning from C-1 to C-3 and C-5 to C-6, and the assembly, guided by the 2D NMR HMBC cross-peaks, of the ten-membered ring of germacrene skeleton. In particular, the diagnostic HMBC correlations between H₃-15 with C-3, C-4, C-5 and of H-6 with C-4, C-7, C8, C-11 unambiguously assigned the position of the Δ⁵ double bond, while the cross peak between H-8 and C-12 confirmed the ring closure of the lactone moiety (Figure 7). While the E geometry of Δ⁵ double bond could be easily deduced by the large *J*<sub>H-H</sub> coupling constant, the configuration at the two stereogenic centers C-4 and C-8 remained to be determined. Given the biogenetic relationship with the known 28, we postulated that C-8 share the same absolute configuration. But the configuration of C-4 still remained to be determined.

<figure><img src="image_9.png" /><figcaption>**Figure 7.** COSY (red bold) and HMBC (blue arrows) of 29.</figcaption></figure>

Borbonensin (38) exhibited HR-ESIMS peak at *m/z* 297.1490 [M-H]⁻, suggesting C₁₉H₂₂O₃ as molecular formula. The ¹H NMR spectrum of 38 showed typical signals of a *p*-hydroxycinnamyl moiety, namely the two coupled doublets at δH 7.43 (2H, d, *J* = 8.6 Hz,
H-5, H-9) and 6.83 (2H, d, J = 8.6 Hz, H-6, H-8), belonging to the *p*-disubstituted phenyl ring, and δ<sub>H</sub> 7.64 (1H, d, J = 15.9 Hz, H-2) and 6.30 (1H, d, J = 15.9 Hz, H-2), belonging to the conjugated *trans* olefinic group in the conjugated system. In the mid-field region, a signal resonating at δ<sub>H</sub> 5.58 and two mutually coupled doublets at δ<sub>H</sub> 4.59 and 4.56 (both 1H, d, J = 12.7, 1.3 Hz) were present, while the high-field region appeared crowded with partially overlapping multiplets, along with two methyl singlets at δ<sub>H</sub> 1.30 and 0.85. Once each proton has been associated to its directly bonded carbon atom by HSQC, a combined analysis of COSY and HMBC 2D NMR spectra allowed the structural elucidation of **38** (Figure 10). In particular, the COSY correlations grouped the high-field signals from H-7 to H<sub>2</sub>-4 to the olefinic proton H-3 (1H, m, H-3'). The HMBC cross-peaks of H<sub>3</sub>-10' and H<sub>3</sub>-11' with C-5', C-7' and C-8' located the unprotonated carbon at δ<sub>C</sub> 37.7, bearing the two methyl groups, on a four membered-ring. The HMBC correlations of the isolated methylene (δ<sub>H</sub> 4.55, 4.59) with C-2', C-3' and C-7', placed the allylic oxymethylene at C-1', thus fully defining a myrtenol unit. Finally, the HMBC cross peaks H<sub>2</sub>-1'/C-1 allowed us to join the hydroxycinnamic acid unit and the myrtenol moieties, confirming that **38** is an unprecedented hydroxycinnamic acid ester derivative, which we named borbonensin. Due to the positive [α]<sub>D</sub> value and the absence of other chiral centers in the hydroxycinnamic acid residue, the myrtenol moiety can be tentatively assigned as (+)-1*S*-myrtenol. Myrtenol has been detected in the essential oils of various plants, such as *Myrtus communis*, *Aegle marmelos*, and *Rhodiola rosea*.<sup>47</sup> The natural compound is commonly reported as (-)-(1R)-myrtenol but, since the presence of myrtenol in essential oils is commonly investigated via GC-MS, this aspect has not been sufficiently explored. Notably, in our analyses of volatile components of *P. borbonense*, both through GC-MS and HS-GC-MS, we did not detect the presence of free myrtenol. Thus, apparently, all the myrtenol production in the plant is devoted to the elaboration of borbonensin.

<figure><img src="image_10.png" /><figcaption>**Figure 10.** COSY (red bold) and key HMBC correlations (blue arrow) of compound **38**</figcaption></figure>

**4. Conclusions**
A combination of different experimental approaches has contributed to define the first detailed characterization of *P. borbonense* fruits, highlighting the main differences between this priced gourmet specialty and the common *P. nigrum*. In particular, the LC-MS/MS-based Feature-Based Molecular Networking analysis provided the piperamides fingerprint, supported by the subsequent purification steps, while the HS-GC-MS analysis revealed a rich and diverse terpene composition of *P. borbonense*, as well as the presence of phenylpropanoids, absent in *P. nigrum*. Indeed, the mono- and sesquiterpenes proved to be an important class of compounds in the extract, enriched by the presence of several compounds previously unreported in the *Piper* genus. In addition, a rich variety of lignans was identified, including the very abundant sesamin, quantified by GC-MS. Finally, the structure of two previously undescribed metabolites, a sesquiterpenoid and a hydroxycinnamic ester (borbonensin) was characterized in detail.

In conclusion, the peculiar aroma of this spice can be related to the variety of monoterpenes and sesquiterpenes detected, while its peculiar taste can be certainly attributed to the rich abundance of fatty acid amides, including those bearing a Z-olefinic group, but it is certainly influenced by the surprisingly high content of sesamin. Further studies are needed to evaluate in detail the presences of lignans within the different *Piper* species and how they could affect the organoleptic properties of these spices.

Overall, our investigation confirms that *P. borbonense* is endowed with a peculiar profile when compared to *P. nigrum*. However, analytical chemistry can only give an idea of the richness of an aroma: as in a musical symphony, reading the score of the individual instruments cannot convey the magic that is created when they are all played together.
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