UHPLC-HRMS/MS Chemical Fingerprinting of the Bioactive Partition from Cultivated Piper aduncum L.

Piper aduncum L. is widely distributed in tropical regions and the ethnobotanical uses of this species encompass medicinal applications for the treatment of respiratory, antimicrobial, and gynecological diseases. Chemical studies reveal a diverse array of secondary metabolites, including terpenes, flavonoids, and prenylated compounds. Extracts from P. aduncum have shown antibacterial, antifungal, and larvicidal activities. Our study explores the activity of extracts and partitions against Mycobacterium tuberculosis H37Rv, as well as the chemical diversity of the bioactive partition. This marks the first investigation of the bioactive partition of P. aduncum from agroecological cultivation. The ethyl acetate partition from the ethanolic leaf extract (PAEPL) was found to be the most active. PAEPL was subjected to column chromatography using Sephadex LH-20 and the obtained fractions were analyzed using UHPLC-HRMS/MS. The MS/MS data from the fractions were submitted to the online GNPS platform for the generation of the molecular network, which displayed 1714 nodes and 167 clusters. Compounds were identified via manual inspection and different libraries, allowing the annotation of 83 compounds, including flavonoids, benzoic acid derivatives, glycosides, free fatty acids, and glycerol-esterified fatty acids. This study provides the first chemical fingerprint of an antimycobacterial sample from P. aduncum cultivated in an agroecological system.


Introduction
Piper aduncum L., belonging to the Piperaceae family, is a species widely distributed in tropical regions, with a relevant presence in the Americas [1,2].This plant is characterized by its leaves with secondary veins reaching the middle portion of the blade and a curved inflorescence, distinguishing it from P. mollicomum by its pubescent leaves and trichomes that feel rough to the touch.These morphological characteristics not only aid in its botanical identification but also suggest an ecological adaptation to the diverse environmental conditions found in its extensive habitat [3].
This Piper species shows a diversified ethnobotanical use, encompassing medicinal applications and traditional practices in many countries in the Pacific, Latin American, and Caribbean regions.Historically, its leaves have been used as an astringent, digestive stimulant, diuretic, antimalarial, sedative, and laxative agent [4][5][6].In Brazil, it is widely cultivated for the extraction of its essential oil and employed as an antimicrobial and antiparasitic agent [7][8][9].
The biological and pharmacological activities associated with P. aduncum extracts are extensive and noteworthy.Pharmacological studies have demonstrated the effectiveness of these extracts in antibacterial, antifungal, antiprotozoal, larvicidal, insecticidal, molluscicidal, cytotoxic, antidepressant, and anxiolytic activities [4][5][6]9,15].The wide range of biological activities described highlights the multifunctionality of this species and warrants exploration to isolate specific bioactive small molecules.This potential becomes even more evident considering that this species undergoes standardized cultivation in agroecological systems [16].In this way, exploring new compounds in Piper species may lead to the discovery of novel compounds, particularly those with antimicrobial properties.
The urgency to discover new antimicrobial agents is underscored by the global challenge posed by Mycobacterium tuberculosis H37Rv, a strain of high virulence.Tuberculosis, caused by M. tuberculosis, remains a major public health concern, with recent WHO data reporting approximately 10.6 million infections and 1.3 million deaths in 2022 alone [16,17.The disease is the second leading cause of death from infectious diseases, with the issue of multi-drug resistance exacerbating the public health crisis [17].
In this study, we investigate the chemical diversity present in the ethyl acetate partition from the ethanolic extract, which has demonstrated activity against Mycobacterium tuberculosis H37Rv.It is worth noting that this marks the first examination of the chemical composition of a bioactive partition from P. aduncum against M. tuberculosis, with leaf extract obtained from cultivation standardized by the group in an agroecological environment [16].

Activity against Mycobacterium tuberculosis H37Rv
The results of the growth inhibition tests against M. tuberculosis H37Rv for extracts and partitions of P. aduncum are shown in Table 1 and Figure 1.According to Table 1 and Figure 1, the most active samples were the hexane partition from the ethanolic stem extract (PAHPS, 29.74 ± 1.01) and the ethyl acetate partition from the ethanolic leaf extract (PAEPL, 27.98 ± 1.01).Due to the relevant antimicrobial activity and a larger amount of material, PAEPL was purified using Sephadex-LH20 column chromatography, yielding five fractions referred to as SFR1-SFR5.SFR1 did not show the presence of any compounds in the analysis by TLC.Therefore, SFR2-SFR5 were subjected to analysis by UHPLC-HRMS/MS, as described in the experimental section.According to Table 1 and Figure 1, the most active samples were the hexane partition from the ethanolic stem extract (PAHPS, 29.74 ± 1.01) and the ethyl acetate partition from the ethanolic leaf extract (PAEPL, 27.98 ± 1.01).Due to the relevant antimicrobial activity and a larger amount of material, PAEPL was purified using Sephadex-LH20 column chromatography, yielding five fractions referred to as SFR1-SFR5.SFR1 did not show the presence of any compounds in the analysis by TLC.Therefore, SFR2-SFR5 were subjected to analysis by UHPLC-HRMS/MS, as described in the experimental section.

Chemical Composition Analysis of the Bioactive Partition
The UHPLC-HRMS/MS analyses in positive ionization mode afforded more comprehensive information, prompting their selection for ion investigation.The overlaid chromatograms of all fractions from the bioactive ethyl acetate partition against M. tuberculosis are depicted in Figure 2. Fraction 1, eluted from the Sephadex LH-20 chromatographic column, was excluded from this study as it did not contain any compound.
hensive information, prompting their selection for ion investigation.The overlaid chromatograms of all fractions from the bioactive ethyl acetate partition against M. tuberculosis are depicted in Figure 2. Fraction 1, eluted from the Sephadex LH-20 chromatographic column, was excluded from this study as it did not contain any compound.
The MS 2 data from the fractions of the ethyl acetate partition (PAEPL) acquired in positive mode were submitted to the online GNPS platform for the generation of the molecular network.After removing ions present in the blank (mobile phase), the resulting molecular network displayed a total of 1714 nodes and 167 clusters formed by at least two ions with similarity in the MS, using a cosine score of 0.75.
The data analysis, aided by manual inspection and/ or different libraries, as well as the specialized literature about the chemistry of the Piperaceae family, led to the annotation of 83 compounds (error up to ± 5 ppm), with 54 identified based on library suggestions and 29 after manual inspection of the data, all tentatively confirmed through fragmentation profiles.Among the annotated compound classes, non-glycosylated and glycosylated flavonoids, chromenes, cinnamic acid derivatives, amides, glycosides, glycerides, and benzoic acid derivatives (including prenylated) stand out.
Figure 3 shows the molecular network generated from GNPS, highlighting the eight annotated molecular families according to their chemical classes.The annotated substances are listed in Table 2.The MS 2 data from the fractions of the ethyl acetate partition (PAEPL) acquired in positive mode were submitted to the online GNPS platform for the generation of the molecular network.After removing ions present in the blank (mobile phase), the resulting molecular network displayed a total of 1714 nodes and 167 clusters formed by at least two ions with similarity in the MS, using a cosine score of 0.75.
The data analysis, aided by manual inspection and/ or different libraries, as well as the specialized literature about the chemistry of the Piperaceae family, led to the annotation of 83 compounds (error up to ± 5 ppm), with 54 identified based on library suggestions and 29 after manual inspection of the data, all tentatively confirmed through fragmentation profiles.Among the annotated compound classes, non-glycosylated and glycosylated flavonoids, chromenes, cinnamic acid derivatives, amides, glycosides, glycerides, and benzoic acid derivatives (including prenylated) stand out.
Figure 3 shows the molecular network generated from GNPS, highlighting the eight annotated molecular families according to their chemical classes.The annotated substances are listed in Table 2.The family of glycosylated flavonoids (Figure 3 and Supplementary Figures S1 and S2 [18].For diglycosylated flavonoids, the main fragments recorded in the fragmentation spectrum correspond to the sequential loss of glycosyl moieties, i.e., ([M+H] + )-glycosyl-1 → ([M+H] + )-glycosyl-2.It is noteworthy that glycosylated flavonoids are common in species of the genus Piper, including in P. aduncum [19][20][21][22].
The molecular family of cinnamic acid derivatives (Figures 3 and S4) presented 71 nodes where eight precursor ions represented by substances 11, 14, 18, 19, 46, 48, 49, and 52 were annotated, with higher occurrences in SFR4.The compounds in this molecular family were characterized by the loss of some neutral molecules, such as H 2 O, CH 3 OH, and C=O.Ferulic acid was annotated as the precursor ion [M+H] + (19, m/z 195.0653) and also in the form of the adduct [M + H − H 2 O]+ (18, m/z 177.0546).In both cases, the MS 2 fragmentation profile was the same, mainly presenting product ions at m/z 177 , as described by [26].Ferulic acid, like other cinnamic acid derivatives, is common in species of the Piper genus [27,28].
This GNPS analysis generated a family represented by prenylated derivatives of benzoic acid (Figures 3 and S5 The main fragment registered in the MS 2 spectra of this molecular family indicates the loss of the neutral fragment consisting of a prenyl group with 56 mass units.The class of the prenylated derivatives of benzoic acid is known in the species P. aduncum [13,29], including the annotated chromene 68 [10]. In the molecular family mostly composed of methoxybenzoic acid derivatives, as well as phenylpropanoids and C 6 -C 3 derivatives, consisting of 10 nodes (Figures 3 and S6), nine compounds (1, 3, 5, 7, 8, 10, 15, 36, and 44) were annotated in the SFR3, SFR4, and SFR5 fractions.The MS 2 spectra of these substances showed two main ions: (a) one formed from radical fragmentation and (b) another that characterizes the neutral elimination of a methanol molecule ([M+H-CH 3 OH] + ), which is generated from the fragmentation of the ester group [18].For instance, methyl vanillate ( ) as the major ion in the MS 2 spectrum.These compounds are common in Piperaceae species [20], and substance 44 has already been described in Piper species [30].However, this is the first description of methyl vanillate (3) in this genus.
In another molecular family composed of six nodes, three glycosides were annotated, namely, dihydroroseoside (12, m/z 386.2171, [M+H] + ), roseoside (13, m/z 371.2065, [M+H] + ), and ranuncoside (34, m/z 387.2016, [M+H] + ), found in the SFR3 fraction (Figures 3 and S7).Dihydroroseoside (12), for instance, was annotated using the GNPS library and exhibited the main fragment m/z 209 (C 13 H 21 O 2 + ), resulting from the loss of glucose.Substances 12 and 13 have been previously described for the Piper genus [31,32]; however, this is the first description of ranuncoside (34) for the genus.) were identified.Fatty acid derivatives were characterized by the presence of fragment ions indicating the subsequent loss of CH 2 units, as well as the ion [M+H-92] + corresponding to the elimination of the triol group [33,34].So far, there is no description of these compounds in the Piper genus.
In another molecular family consisting of 21 nodes, the GNPS library annotated seven substances, including five fatty acids (59, 71, 77, 78, and 82) and two fatty acid esters (84, 89) (Figures 3 and S9).The substance 9-hydroxy-10,12,15-octadecatrienoic acid (77, m/z 277.2160, [M + H − H 2 O] + ) is the second most abundant constituent in SFR3, and there are no previous reports of the occurrence of this compound in Piper species.These substances are known as linoleic acids and their derivatives.For example, a methyl ester of 77 isolated from the leaves of Ehretia dicksonii Hance (Boraginaceae) demonstrated interesting in vivo anti-inflammatory activity [35].
Other compounds of various classes (Supplementary Figure S10) were annotated based on the GNPS library, forming clusters of two or three nodes, or in the form of self-loops (without any spectral similarity with other ions): (a) The monoterpenic lactone loliolide (24, [44].Vanillin (6) also exhibits anticancer, antidiabetic, anti-inflammatory, and antimicrobial activities [46].
It is quite challenging to correlate the activity against M. tuberculosis with a chemically complex partition.However, some inferences can be made.For instance, the ethyl acetate partition of the ethanolic extract from the leaves of P. aduncum proved to be rich in flavonoids, with 30 substances belonging to this class of phenolics being annotated.According to [47], flavonoids have significant inhibitory potential against mycobacterial activity, acting on the inhibition of the proteasome and the inhibition of nitric oxide formation.
All the examples described here demonstrated a MIC higher than that of the ethyl acetate partition (PAEPL).This could be attributed to a synergistic effect among the compounds present in this partition.
Several biological studies have documented the antimycobacterial efficacy of essential oils derived from different species of the Piper genus, showcasing moderate to good activity against M. tuberculosis.Specifically, essential oils from the infructescences and inflorescences of Piper lhotzkyanum Kunth exhibited minimum inhibitory concentrations (MICs) of 76 µg/mL and 128 µg/mL, respectively [52].Similarly, leaf oils from Piper cernuum Vell., Piper diospyrifolium Kunth, and Piper rivinoides Kunth demonstrated MIC values of 125 µg/mL, while Piper mosenii C.DC. reported an MIC of 250 µg/mL [53].Further research indicated that oils from the roots and infructescences of Piper multinodum C.DC. showed MICs of 78.51 µg/mL and 85.91 µg/mL, respectively [54].
Moreover, supercritical fluid extracts of Piper diospyrifolium (Kunth) Kunth ex Steud.leaves and a novel benzoic acid derivative were tested against the M. tuberculosis H37Rv strain and eight clinical isolates, showing MICs of 125 µg/mL for the H37Rv strain and ≥250 µg/mL for the clinical isolates, indicating moderate activity for this species [63].Additionally, crude extracts and alkaloid fractions from Piper corcovadensis (Miq.)C.DC. roots, including isobutylamide (piperovatine), exhibited MICs of 15.6; 7.8, and 7.8 µg/mL, respectively, against the M. tuberculosis H37Rv strain, with MICs ranging from 0.98 to 3.9 µg/mL against clinical isolates, suggesting synergistic effects when combined with rifampicin [64].In 2018, the antimycobacterial activity of piperine, an alkaloid found in Piper nigrum L. and Piper longum L., was evaluated, showing MICs ranging from 31.2 to 125 µg/mL.Notably, when combined with antibiotics such as rifampicin, isoniazid, ethambutol, and streptomycin, MIC values were reduced to 0.12 to 1 µg/mL, indicating a synergistic effect against evaluated clinical isolates [65].
These findings underscore the promising antimycobacterial activity of Piper species, though further in vivo studies and explorations of their mechanisms of action are warranted.The active compounds identified within these species are likely responsible for the observed activity, making them promising candidates for the development of new anti-TB drugs.

Plant Material
Adult specimens of Piper aduncum L., in the reproductive stage, were collected in the Agroecological Cultivation System at the Socio-Environmental Responsibility Center of the Rio de Janeiro Botanical Garden Research Institute, Brazil, (S22 • 58 ′ 0 ′′ W43 • 13 ′ 43 ′′ ).Leaves (1100 g), stems (950 g), and reproductive organs (inflorescences and infructescences, 100 g) were harvested for the experiments.The material was authenticated by Dr. Elsie Franklin Guimarães and Dr. George Azevedo Queiroz, both from the Rio de Janeiro Botanical Garden Research Institute, where a voucher specimen was deposited with the number RB01426180.The studies were registered in the National System for Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under the number AE4E953.The plants were farmed in full sun, in plots arranged in 1.5 × 1.5 m spaces, with a base fertilizer application of 40 kg/ha.Irrigation was performed daily, and the soil was maintained in a field capacity condition.For more details about this Agroecological Cultivation System see [16].
As the ethyl acetate partition from leaves (PAEPL, 1.61 g) showed the highest activity in the antimycobacterial assay, for this reason, it was submitted to a chromatographic column for pre-purification.A total of 200 mg of the partition was subjected to open glass column chromatography (1000 mm × 20 mm), using Sephadex ® LH-20 (Sigma-Aldrich, São Paulo, SP, Brazil) as the stationary phase and methanol as the eluent.This procedure was repeated 4 times.The chromatographic separation resulted in 5 fractions, which were referred to as SFR1 (6.4 mg), SFR2 (61.0 mg), SFR3 (97.1 mg), SFR4 (45.5 mg), and SFR5 (84.9 mg).All solvents used were spectroscopic-grade and were obtained from Sigma-Aldrich, Brazil.
The fractions SFR1-SFR5 were analyzed via TLC and UHPLC-HRMS/MS, and the data from MS were processed using the online GNPS platform.
TLC evaluation (prepared using silica gel plates, RF254 nm, Sigma-Aldrich, Brazil, and a mobile phase composed of mixtures of hexane, ethyl acetate, and methanol in different proportions) was performed under ultraviolet light, as well as with a 5% sulfuric acid solution in ethanol (both from Sigma-Aldrich, Brazil), and subsequent heating for compound visualization.

Analysis by Ultra-High-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry in Tandem (UHPLC-HRMS/MS)
Crude extracts and partitions (10 mg/mL) were subjected to exploratory analysis by UHPLC-HRMS/MS using an UltiMate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an Orbitrap Q Exactive Plus mass spectrometer (Thermo Scientific, Waltham, MA, USA) with an electrospray ionization source.A Waters ® Acquity UPLC BEH C18 chromatographic column (100 mm × 2.1 mm I.D. × 1.7, µm particle size) (Waters, Milford, MA, USA) was employed.Mobile phases A and B were used: A-ultrapure water with 0.1% formic acid, and B-methanol with 0.1% formic acid.The gradient elution was as follows: 0.0-4.0min 15% B; 4.0 min 15% B; 10.0 min 95% B, 10.0-12.0min 95% B; 13.0 min 15% B; 13.0-17.0min 15% B. The flow rate was set at 0.35 mL/min, with a 5 µL injection volume and a column oven temperature of 40 • C. As parameters of the ionization source, sheath gas and auxiliary gas were used at 50 and 15 arbitrary units, respectively.The spray voltage was + or -3600 V, the S-lens voltage was 50 V, the capillary temperature was 320 • C, and the source temperature was 400 • C. Data acquisition was performed in Full Scan mode (total ion scan) in the m/z range of 100-1000; positive ionization mode, with a resolution of 35,000 (FWHM), AGC 1 × 10 6 , and IT 100 ms, combined with a data-dependent acquisition experiment (ddMS 2 top3) at 17,500 (FWHM), AGC 1 × 10 5 , and IT 50 ms; NCE 15-35; and an isolation window of 1.2 Da.

Processing of UHPLC-HRMS/MS Data by Molecular Network
The UHPLC-HRMS/MS data obtained in the raw format of the positive ionization mode were converted to the mzXML format using MSConvert software at version 3 (Proteowizard Software Foundation, Palo Alto, CA, USA).The data were processed using MZmine 2.53 [66], with 5.0 × 10 6 as the noise level intensity for MS 1 data and 1.5 × 10 5 for MS 2 data, 0.02 as the m/z tolerance, and 0.04 as the minimum time span.The Wavelets-ADAP algorithm was used in the chromatogram deconvolution step, and a 0.2 minimum retention time (Rt) tolerance was used in the chromatogram alignment.The processed data were then exported and submitted for analysis on the online platform GNPS (Global Natural Product Social Molecular Network, https://gnps.ucsd.edu(accessed on 04 March 2024) [67]) using the Feature-Based Molecular Networking (FBMN) workflow.For FBMN, the precursor and fragment ion mass tolerance were both set to 0.02 Da, and the edges were filtered to have a cosine score above 0.75 and more than 4 matched peaks.The molecular networks created in the GNPS were imported and visualized using Cytoscape software (Version 3.8.0).

Growth Inhibition Assay
The samples were assessed for their antimycobacterial activity using the tetrazolium salt assay in a 96-well microplate at concentrations of 32, 64, and 128 µg/mL.For this assay, the M. tuberculosis H37Rv suspension was plated (1 × 10 6 CFU/well) and incubated in the presence of samples or rifampicin.The sealed plate was incubated at 37 • C and 5% CO 2 for 5 days.After this period, the bacterial cultures were incubated for 3 h with tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) solution (5 mg/mL) in sterile phosphate-buffered saline (PBS) and then lysis buffer (20% w/v sodium dodecyl sulfate (SDS)/50% dimethylformamide (DMF) in distilled water, pH 4.7) was added overnight.The microplate was read in a spectrophotometer at 570 nm [67].Rifampicin treatment (0.008, 0.04, 0.2, and 1 µg/mL) in wells containing only bacilli was used as a positive control (C+) for antimycobacterial activity.Wells containing bacilli without treatment were used as a negative control (C−).The percentage of the inhibition of mycobacterial growth was calculated using Equation ( 1

Conclusions
This study on the chemical composition of the ethyl acetate partition (active against M. tuberculosis) from the ethanolic extract of P. aduncum leaves provides valuable insights into the chemistry of a Piperaceae species widely used in traditional medicine.This is the first investigation into the chemistry of this species under agroecological cultivation.The chemistry of this cultivated specimen is notable, particularly for free and glycosylated flavonoids, benzoic acid derivatives (including prenylated ones), glycosides, free fatty acids, and glycerol-esterified fatty acids.The chemistry of this cultivated specimen is quite similar to those previously described for specimens collected in the wild, except for some annotated substances that had not been previously reported for the species.Therefore, it can be concluded that the cultivation of the species does not substantially alter its chemistry and can be undertaken for harnessing the medicinal potential offered by P. aduncum without posing risks to the native population of this Piperaceae species.

Figure 2 .
Figure 2. Overlaid chromatograms of base peak recorded (Rt 8.0 to 16.5 min) with UHPLC-HRMS/MS in positive ionization mode of fractions 2 to 5 (SFR2-SFR5) obtained using Sephadex LH-Figure 2. Overlaid chromatograms of base peak recorded (Rt 8.0 to 16.5 min) with UHPLC-HRMS/MS in positive ionization mode of fractions 2 to 5 (SFR2-SFR5) obtained using Sephadex LH-20 column chromatography of the bioactive ethyl acetate partition from the ethanolic extract of Piper aduncum L. The bold numbers in the peaks shown correspond to the substances described in the text and Table2.
of the bioactive ethyl acetate partition from the ethanolic extract of Piper aduncum L. The bold numbers in the peaks shown correspond to the substances described in the text and Table2.

Figure 3 .
Figure 3. Molecular network of fractions 2 to 5 (SFR2-SFR5) obtained by Sephadex LH-20 column chromatography of the bioactive ethyl acetate partition from the ethanolic extract of Piper aduncum L. Only clusters containing at least two nodes are shown.Nodes present in the blank (mobile phase) were excluded.The eight annotated molecular families are highlighted, with the chemical structure of at least one example from each family.
) where O.D. = optical density.100 − (O.D.sample − O.D.C+) × 100/(O.D.C− − O.D.C+) (1) Figure S1: Molecular family monoglycosylated flavonoids; Figure S2: Molecular family diglycosylated flavonoids; Figure S3: Molecular family non-glycosylated flavonoids; Figure S4: Molecular family of cinnamic acid derivatives; Figure S5: Molecular family of prenylated acid derivatives and chromenes; Figure S6: Molecular family of derivatives of methoxibenzoic acid, phenylpropanoids, and C 6-C 3 derivative; Figure S7: Molecular family of glycosides; Figure S8: Molecular family of glyceride derivatives; Figure S9: Molecular family of fatty acids; Figure S10: Compounds annotated in clusters of two or three nodes, or in the form of self-loops in the molecular networks of the bioactive ethyl acetate partition from the ethanolic extract of Piper aduncum L.; and Figure S11: Compounds annotated in clusters of two or three nodes, or in the form of self-loops in the molecular networks.

Table 1 .
Inhibitory effect of the extracts and partitions of Piper aduncum L. against Mycobacterium tuberculosis H37RV.

Table 2 .
UHPLC-HRMS-MS analysis for the chemical composition of the bioactive partition of Piper aduncum L.