Comparison of metabolite differences and pharmacologically active constituents between Piper longum and Piper sarmentosum based on non-targeted metabolomics

TL;DR

This study compares the metabolite profiles of two Piper plants to understand their medicinal differences and uses.

Contribution

The study identifies differential metabolites and proposes biosynthesis pathways in Piper species using non-targeted metabolomics.

Findings

1,073 metabolites were identified across different tissues of Piper longum and Piper sarmentosum.

Differential accumulated metabolites were enriched in phenylalanine metabolism, including alkaloid and flavonoid pathways.

The study outlines the biosynthesis and tissue-specific accumulation of phenylpropanoid, alkaloid, and flavonoid compounds.

Abstract

Piper longum and Piper sarmentosum are plants of the Piperaceae family, rich in secondary metabolites, with various medicinal and food values. They are highly similar in morphology, but differ in their medicinal parts and pharmacological effects. To investigate the differences in the medicinal effects between P. longum and P. sarmentosum, it is of great practical significance to study and compare the metabolites of the two species. In the present work, non-targeted Liquid Chromatography-Mass Spectrometry (LC-MS) metabolomics was used to identify and measure metabolites in roots, stems, leaves, flowers, and three developmental stages of fruit from P. longum and P. sarmentosum. 1,073 metabolites were identified, including 729 metabolites in positive ion mode and 344 metabolites in negative ion mode. We identified differential accumulated metabolites (DAMs) in different tissues between the two species, and found that the DAMs were enriched in phenylalanine metabolism, including the biogenesis pathway of alkaloid and flavonoid. Based on the structural formula of identified substances, we proposed the biosynthesis pathway of phenylpropanoid, alkaloid and flavonoid and profiled the accumulation of each component in different tissues from the two Piper species. This study compares the differences in metabolites between the two species of the Piper genus. It describes the diverse accumulation of medicinal components, providing an informative scientific basis for the efficient utilization and targeted development of two important medicinal and food crops of the Piper genus.

Figures

Untargeted metabolite profiling.(A) Total ion current (TIC) chromatograms of the QC samples; (B) classification of metabolites in two Piper species; (C) principal component analysis of seven tissues between P. longum (Plo) and P. sarmentosum (Psa). RO, Root; ST, Stem; LE, Leave; ST, Stem; FL, Flower; F1, F2, F3, Three different periods of fruit.

Differential accumulated metabolite analysis.(A) Volcano plots analysis of DAMs in different tissues of P. longum and P. sarmentosum. Red dots indicate significantly increased metabolites, blue dots indicate significantly decreased metabolites, and gray dots represent metabolites with no significant differences. (B) Enrichment of DAMs in the KEGG pathways.

Proposed biosynthesis pathway and abundance of phenylpropanoids and their derivatives in two piper species.(A) Phenylpropanoids. (B) Coumarins. (C) Flavonoids.

Biosynthesis pathway of flavonoid.(A) Visualization in the metabolic pathway map and (B) heatmap of the metabolites of flavonoid glycosides.

Proposed biosynthesis pathway and abundance of alkaloid in two piper species.(A) Visualization of DAMs in tryptophan biosynthesis pathway map and heatmap of DAMs; (B) visualization of DAMs in phenylamine and tyrosine biosynthesis pathway map and heatmap of DAMs; (C) visualization of DAMs in phenylamine and lysine biosynthesis pathway map and heatmap of DAMs.

Full text

Introduction

Piper longum and Piper sarmentosum are both species of the Piper genus, commonly found in tropical and subtropical regions. These plants are not only highly valued for their medicinal properties but also widely used in culinary applications, making them prime examples of food and medicine originating from the same source. The Piper plays a significant role in various industries, including flavoring, pharmaceuticals, and insecticides, while also having a long history of traditional use (Salehi et al., 2019b). P. longum is traditionally used in the form of its fruit spikes to treat gastric disorders, arthritis, and to relieve pain. It is also commonly used as a spice (Evans, 2009). Pharmacological studies indicated that P. longum possessed different bioactivities, including anti-tumor, anti-inflammatory, anti-osteoporosis, anti-hyperlipidemia and other effects (Guo et al., 2019; Zadorozhna, Tataranni & Mangieri, 2019; Sanap et al., 2021; Li et al., 2022). The pharmacological research on P. sarmentosum primarily focuses on its leaves. Studies have shown that the leaf extracts of P. sarmentosum possess various activities, including vascular protection (Md. Salleh et al., 2021), neuroprotection (Chan et al., 2021), anti-obesity and anti-hyperlipidemic effects (Kumar et al., 2021). Its young leaves are also eaten as vegetables. In recent years, there has been a growing issue in the traditional Chinese medicine market, where immature inflorescences of P. sarmentosum are used as substitutes for P. longum. P. longum and P. sarmentosum are both species within the genus Piper of the Piperaceae family. They exhibit high morphological similarity, leading to frequent confusion during commercial circulation. However, current studies indicate that significant differences exist between them regarding their medicinal parts and pharmacologically active components. For medicinal plants,the variations observed among different plant species and their specifically employed medicinal parts are primarily attributable to differences in their profiles of pharmacologically active secondary metabolites (Kumar, Kumari & Kumar, 2023; Liu et al., 2023).

The differences in the medicinal efficacy of these plants are primarily due to the distinct secondary metabolites they produce. Secondary metabolites evolve on top of primary metabolism and serve as an effective arsenal to help plants cope with the biotic and abiotic stresses encountered during their life cycle (D’Auria & Gershenzon, 2005; Wang et al., 2019). Additionally, the accumulation of secondary metabolites plays an important role in the quality assessment of fruits and medicinal ingredients (Robertson, Reily & Baker, 2007; Wu et al., 2022). Research on the Piper has revealed the presence of various bioactive chemical constituents, including unsaturated amides, flavonoids, phenylpropanoids, and alkaloids (Parmar et al., 1997). The most abundant phytochemical of the P. longum plant is alkaloids. Piperlongumine is a key alkaloid bioactive compound in P. longum, and studies have reported its pharmacological activities, including anti-cancer, anti-inflammatory, and anti-oxidant effects (Conde et al., 2021; Zhu et al., 2021; Girol et al., 2021). Some flavonoids, such as quercetin, myricetin and apigenin, were identified from the whole plant (Biswas et al., 2022). Quercetin exhibited anti-inflammatory and immune-enhancing effects in vitro and in vivo (Li et al., 2016). Quercetin generally exists in the form of glycosides. Quercetrin is a glycoside compound formed by the connection of quercetin. Quercetrin may activate carcinogenic factors in liver cancer, posing certain carcinogenic risks. Its glycosylated products, isoquercitrin and rutin, can reduce the activation risk of liver cancer carcinogens (Vrba et al., 2012). Myricetin has a variety of pharmacological activities, including anticancer, anti-inflammatory, and antidiabetic effects (Badr et al., 2020). Apigenin has anticancer, antioxidant, antidiabetic, and anti-inflammatory effects (Salehi et al., 2019a). In the study of metabolic constituents in P. sarmentosum. Its metabolites are primarily represented by alkaloids and flavonoids (Ware et al., 2024). To date, 40 alkaloids have been identified from P. sarmentosum (Adib et al., 2024). Alkaloids and phenolic compounds in the leaves account for 70% of the total alkaloid and phenolic content in this species (Zhou, Wang & Zhou, 2021). Alkaloids and phenolic compounds can prevent pathogen invasion in plants by scavenging free radicals or binding with catalysts involved in oxidation reactions. This supports a strong correlation between the structure of these compounds and their functional activity (Roy, 2017; Lin et al., 2013). The alkaloids piperlotine C and piperlotine J, identified in P. sarmentosum, have been shown to antiplatelet activity (Li et al., 2007). The antioxidant activity of P. sarmentosum is largely attributed to its flavonoid content. Due to the structural features of flavonoids, particularly the phenolic groups, they protect against oxidative stress by scavenging reactive oxygen species through their multiple hydroxyl groups. Additionally, glycosylation enhances solubility and bioavailability (Agati et al., 2012). Research on P. longum and P. sarmentosum mainly focuses on the composition and bioactivity of their extracts, particularly the bioactive properties of the fruit spikes of P. longum and the leaves of P. sarmentosum. The biosynthesis and distribution of secondary metabolites in plants vary depending on the species, tissue, organ, and growth stage (Wang et al., 2016; Chen et al., 2022). Significant differences also exist in the types and quantities of metabolites between different species and within different tissues of the same species (Dong et al., 2014; Wu et al., 2023). However, little is known about the metabolite composition and medicinal value of other organs. Moreover, the metabolite profiles and inter-organ chemical differences have not been systematically elucidated between P. longum and P. sarmentosum.

With the continuous development of metabolomics, high-throughput techniques such as chromatography, mass spectrometry, and nuclear magnetic resonance can be used to detect metabolic products in organisms, providing a foundation for the systematic study of plant physiological metabolism (Song et al., 2017). Metabolites are the final products of replication, transcription, and protein expression in organisms and are the material basis of the plant phenotype. The content and variation of metabolites can directly reflect the transcriptional and protein expression levels of related genes. Therefore, the changes in metabolite types and quantities are key to understanding the causes of plant variations (Alseekh & Fernie, 2018; Guo et al., 2022; Antignac et al., 2005). Utilizing complex metabolomics data to identify metabolites related to specific phenomena and potential biomarkers has become a hot and challenging topic in the biomedical field.

In this study, we detected and indentified metabolites in different tissues of P. longum and P. sarmentosum, comparing the metabolites present in both species and the differentially accumulated metabolites (DAMs) between them. We also explored the possible biosynthetic pathways of important secondary metabolites in both plants, further elucidating the bioactive secondary metabolites present in these two species. We believe that a systematic comparative analysis and understanding of the metabolic components in different tissues of P. longum and P. sarmentosum will provide theoretical support for utilizing these resources and scientific guidance for the targeted development and application of products derived from these plants.

Methods

Experimental materials

The Piper longum and Piper sarmentosum samples were collected from the Xishuangbanna Tropical Botanical Garden in China (21°55′N, 101°16′E). Free from pests and diseases, healthy plant tissues were selected from P. longum and P. sarmentosum. The plant materials were laboratory-authenticated as P. longum and P. sarmentosum using DNA barcoding. Samples of roots, stems, young leaves, flowers, small fruits, medium fruits, and large fruits were collected. Immediately immerse them in liquid nitrogen for brief storage, then preserve them in a laboratory freezer at −80 °C for subsequent metabolite extraction. All plant materials were subjected to at least three biological replicates. Each repetition comes from a separate plant.

Metabolite extraction

All samples were lyophilized and ground into a fine powder, which was transferred to two ml centrifuge tubes and mixed with the extraction solution. The extraction solution was prepared with 70% high-performance liquid chromatography (HPLC) grade methanol, and internal standards acyclovir (final concentration 1 ppm) and roxithromycin (final concentration 0.5 ppm) were added (Two internal standards are used to test the stability of the instrument). Approximately 50 ± 1 mg of the powdered sample was placed in the two ml centrifuge tube. The extraction solution was added at a ratio of 10 µl per one mg of sample powder. The mixture was thoroughly vortexed to ensure complete mixing of the powder and extraction solution. The samples were then subjected to ultrasonic extraction for 30 min using the KQ52000DE model ultrasonic cleaner from Kunshan Shumei Ultrasonic Instrument Co., Ltd. (Kunshan, Jiangsu, China) (power set to 100%). Finally, the samples were centrifuged at 5,000 g for 10 min at 4 °C, and the supernatants were filtered using a 0.22 µm organic filter membrane (Tian et al., 2024).

All chemicals and solvents used for the sample treatment and analysis were of analytical or  HPLC grade. Water and methanol were purchased from Sinopharm Group Co. Ltd (Shanghai, China). Acyclovir and roxithromycin were purchased from Selleck chemicals (Houston, TX, USA).

Non-targeted metabolic analysis by ultra-high-performance liquid chromatography-triple/time-of-flight mass spectrometry (UHPLC-Q-TOF/MS)

Analysis was performed by using an Ultra High Performance Liquid Chromatography (UHPLC) (1290 Infinity II LC; Agilent Technologies) equipped with Waters ACQUITY UPLC T3 Column (2.1  × 100 mm, 1.8 µm, Phenomenex) and coupled to a quadrupole time-of-flight (Q-TOF) (Agilent Technologies 6545 Q-TOF). The mobile phase comprises water (A) with 0.04% acetic acid added and acetonitrile (B) with 0.04% acetic acid added. The gradient procedure was as follows: 0 min, 2% B; 0.5 min, 2% B; 15 min, 98% B; 17 min, 98% B; 17.1 min, 2% B; 20 min, 2% mobile phase B; The flow rate was set at 0.35 mL/min. Injection volume: two µl, column temperature: 40 °C. Full mass spectrometry (MS) and ddMS2 scans were acquired in positive and negative ion mode, respectively. In the positive ion modes, full MS scan parameters were set as follows: spray voltage 4 kV, gas temperature 300 °C, sheath gas temperature 350 °C, sheath gas flow 11 L/min, collision energy (CE) 45 V. In MS-only acquisition, the mass range of the instrument was from m/z 100 to 1,200 Da, and the accumulation time for triple/time-of-flight mass spectrometry (TOF MS) scan was set at 1.667 ms/spectra. Additionally, in auto MS/MS mode, the mass range was set to m/z 50–1,200 Da, with an accumulation time of 1.667 ms per spectrum for the product ion scan. The MS/ MS secondary scanning was a data-dependent scan. In the negative ion mode, MS parameters were the same as the positive ion mode.

Processing of metabolomics data

The raw mass spectrometry data were converted using the msconvert (v2.0; ProteoWizard, Richland, WA, USA). This step allowed for basic filtering of retention time, mass-to-charge ratio, and other parameters. Additionally, peaks were extracted based on a set of criteria, including a mass deviation of 5 ppm, a signal intensity deviation of 30%, a signal-to-noise ratio of 3, a minimum signal intensity of 100,000, and the presence of additive ions. software, and then analyzed using the R package xcms (v3.22.0) (Smith et al., 2006; Tautenhahn, Böttcher & Neumann, 2008; Domingo-Almenara & Siuzdak, 2020; Du, Kibbe & Lin, 2006). Peak areas were then quantified, and target ions were integrated. The molecular formula was predicted based on the molecular ion peak and fragment ions. Background ions were removed using blank samples, and quantitative results were normalized. The metabolites were identified by comparing them to a standard library and characteristic ion peaks, assisted by databases such as Plant Metabolome Hub (PMhub, https://pmhub.org.cn/), PubChem (https://pubchem.ncbi.nlm.nih.gov/), and Human Metabolome Database (https://www.hmdb.ca/). Further, the NP Classifier was used to classify substance. Finally, the identification and relative quantification results of the metabolites were obtained.

Principal component analysis (PCA) was performed using R. Differential metabolites across tissues were identified by applying the following criteria: Variable Importance in the Projection (VIP) > 1, P-value < 0.01 from Student’s t-test, and fold change (FC) > 2. Subsequently, the identified differential metabolites were uploaded to MetaboAnalyst (https://www.metaboanalyst.ca/) for metabolic pathway enrichment analysis

Results

Metabolite profile in P. longum and P. sarmentosum

To investigate the metabolic divergency between P. longum and P. sarmentosum, we performed untargeted metabolite profiling on seven different tissues of both plants, including leaves, stems, roots, flowers, and fruits from three different developmental stages. To ensure the reliability of the analytical results, quality control (QC) samples were injected at regular intervals throughout the analytical sequence to monitor instrument stability. The reproducibility of metabolite detection was evaluated by calculating the relative standard deviation (RSD) of the peak intensities for each metabolite across the QC samples (Table S1 and Figs. S1, S2). The results reveals distinct differences in metabolite compositions among mixed tissues, as evidenced by the total ion chromatograms (TIC), which also served as a quality control sample. Furthermore, each tissue of P. longum and P. sarmentosum exhibited unique characteristics (Fig. 1A and S3). In total, we identified 1,073 metabolites across all samples, including 729 in ESI+ mode and 344 in ESI- mode (Table S2). These metabolites were classified into eight categories, as shown in Fig. 1B. Among these categories, shikimates and phenylpropanoids (255 compounds) represented the largest group, accounting for 23.76% of the identified metabolites. The second largest group was alkaloids (233 compounds), comprising 21.71%. The third largest group was unclassified compounds (195 compounds), accounting for 18.17%. Other categories include fatty acids (171 compounds, 15.94%), terpenoids (128 compounds, 11.93%), amino acids and peptides (47 compounds, 4.38%), polyphenols (28 compounds, 2.61%), and carbohydrates (16 compounds, 1.49%). We then conducted principal component analysis (PCA) on all the identified metabolites. The PCA results demonstrate that samples from different tissues of both species can be clearly distinguished, with intra-group variations smaller than inter-group variations (Fig. 1C). Except for the root, the PCA showed notable separation in leaf, stem, flower and early stages of fruit between the two Piper species (Fig. 1C), suggesting their metabolite divergence. The metabolite differences among tissues and species imply their potential different pharmacological effects.

Untargeted metabolite profiling.(A) Total ion current (TIC) chromatograms of the QC samples; (B) classification of metabolites in two Piper species; (C) principal component analysis of seven tissues between P. longum (Plo) and P. sarmentosum (Psa). RO, Root; ST, Stem; LE, Leave; ST, Stem; FL, Flower; F1, F2, F3, Three different periods of fruit.

Differential accumulated metabolites analysis between P. longum and P. sarmentosum

To further explore DAMs between P. longum and P. sarmentosum in different tissues, we compared the metabolites across various tissues and identified the DAMs between each tissue of the two Piper species. Volcano plots were used to visualize representative DAMs. The significant DAMs were defined with a relative abundance fold change ≥2 and a p-value < 0.01. Consequently, in the vegetative tissues and the late stage of fruit (Fruit_2 and Fruit_3), we observed that there were many more up-regulated metabolites in P. longum than in P. sarmentosum (Fig. 2A). Only in the flower and the first stage of fruit, more up-regulated metabolites were found in P. sarmentosum. To elucidate the divergent metabolic strategies between the two Piper species, we conducted a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the pooled differential accumulated metabolites (DAMs) identified across all examined tissues. This comprehensive approach revealed that the metabolic differences were predominantly enriched in pathways related to aromatic amino acid metabolism. Most notably, the phenylalanine metabolism and biosynthesis of phenylalanine, tyrosine, and tryptophan pathways were significantly enriched (p-value < 0.01, FDR < 0.05; Table S3), suggesting a fundamental divergence in the synthesis and utilization of aromatic amino acids and their derivatives. The phenylalanine metabolism pathway is a major metabolic route, serving as a precursor for various compounds such as coumarins, alkaloids, and flavonoids (Vogt, 2010). Tryptophan and tyrosine are involved in the synthesis of alkaloids (Lichman, 2021). The products in these pathways also play critical physiological roles in plants, influencing growth, development, reproduction, defense, and environmental responses. We further classified the metabolites enriched in these two pathways and found that they mainly concentrated in tryptophan alkaloids, tyrosine alkaloids, lysine alkaloids, C6-C3 structural compounds, coumarins, and flavonoids. These DAMs may play an important role in the efficacy divergency between P. longum and P. sarmentosum.

Differential accumulated metabolite analysis.(A) Volcano plots analysis of DAMs in different tissues of P. longum and P. sarmentosum. Red dots indicate significantly increased metabolites, blue dots indicate significantly decreased metabolites, and gray dots represent metabolites with no significant differences. (B) Enrichment of DAMs in the KEGG pathways.

DAMs in the phenylpropanoid biosynthesis pathway

As aforementioned, we found DAMs between both species are mainly involved in phenylalanine metabolism and the biosynthesis of phenylalanine. The phenylpropanoid pathway is a major source of various compounds derived from phenylalanine and serves as a precursor for producing valuable compounds such as coumarins, flavonoids, and lignans (Jaini et al., 2017). To explore the divergency of metabolite accumulation in the phenylalanine pathway between P. longum and P. sarmentosum, we analyzed the relative content of identified metabolites in different tissues (Fig. 3). The main metabolites identified in this pathway include phenolic acid derivatives, coumarin derivatives, and flavonoids. In total, 22 phenylpropanoids (including 2-hydroxycinamic acid in Fig. 3B) were identified in our analysis (Fig. 3A). All of these phenylpropanoids were categorized into four groups based on the hydroxylation and methylation of benzene ring (Fig. 3A). Interestingly, phenylpropanoids without hydroxylation and methylation modification of benzene ring showed higher accumulation in P. sarmentosum (highlighted in blue in Fig. 3A), meanwhile, phenylpropanoids with multiple hydroxylation or methylation modifications were accumulated in a relatively higher level in P. longum (highlighted in yellow and red in Fig. 3A), suggesting enzymatic hydroxylation and methylation of cinnamic acid might be stronger in P. longum. Myristicin, known for its antibacterial, antioxidant, and cancer chemopreventive effects (Salehi et al., 2019a), was accumulated at the highest level in the fruit of P. longum, which may contribute to its medicinal properties. Methylisoeugenol, which has anxiolytic and antidepressant effects (Fajemiroye et al., 2014), was present in significantly higher accumulation in the reproductive organs of P. longum compared to P. sarmentosum, suggesting its potential contribution to the antidepressant activity of P. longum. Besides phenylpropanoids, we also checked the abundance of coumarin and flavonoid derivatives. Among the coumarin derivatives identified, dephnetin, a bioactive compound extracted from Daphne species, showed significantly higher abundance in P. sarmentosum than in P. longum (Fig. 3B), which possesses many biological activities, including anti-inflammatory, anti-oxidant, neuroprotective, analgesic, anti-pyretic, anti-malarial, anti-microbial, anti-arthritis, hepatoprotective, nephroprotective, and anti-cancer properties (Javed et al., 2022). We found that the identified flavonoid metabolites exhibited a consistent expression pattern in both P. longum and P. sarmentosum, with the highest abundance observed in flowers. The flavonoid myricetin was notably higher in the flowers of P. longum than in other tissues or P. sarmentosum (Fig. 3C), which has a variety of pharmacological activities, including antitumor, anti-inflammatory, and antidiabetic effects (Song et al., 2021).

Proposed biosynthesis pathway and abundance of phenylpropanoids and their derivatives in two piper species.(A) Phenylpropanoids. (B) Coumarins. (C) Flavonoids.

DAMs in the flavonoid glycosylation pathway

Methylation, glycosylation, and pentanoylation are common substitution modifications that confer structural complexity and diversity to flavonoid compounds. Glycosylation effectively regulates their biological activities by altering the water solubility and stability of flavonoids. Flavonoid glycosides exhibit various biological activities, including antioxidant, immune-regulating, and anticancer effects (Yang et al., 2018). In the enriched phenylalanine metabolic pathway, we identified 22 flavonoid glycosides. We proposed the metabolic pathways of flavonoid glycosides and their potential precursors based on the structure similarity (Fig. 4A). The expression patterns of flavonoid glycosides in P. longum and P. sarmentosum were generally consistent, with both species showing lower flavonoid glycoside content in the roots and the highest content in the leaves and flowers (Fig. 4B), indicating the glycosylation modification possesses similar tissue preference in both Piper species. In total, there are 14 identified flavonoid glycosides derived from apigenin. Among them, two are mono-glycosylated, ten are di-glycosylated, and the remining two are tri-glycosylated (Figs. 4A and 4B). Most mono-glycosylateds flavonoid and di-glycosylated flavonoids showed similar expression patterns as their substrate, apigenin (Fig. 4B). Icariin, a bi-glycosylated flavonoid derived from apigenin which is known for promoting osteoblast differentiation and improving osteoporosis (Yin et al., 2007), showed high accumulation in fruit rather than in leaves and flowers where apigenin was highly accumulated (Fig. 4B). Robinin, a tri-glycosylated flavonoid derived from apigenin with antitumor, cardioprotective, and anti-inflammatory effects (Zhang, Liu & Hu, 2023; Janeesh & Abraham, 2013; Janeesh et al., 2014) also showed different expression pattern compared to its substrate apigenin (Fig. 4B). These alterations of expression patterns of robinin and icariin imply the variation in glucuronosyltransferase (UGT) expression levels might influence the accumulation of different flavonoid glycosides, particularly relevant for products with multiple glycosylation. In addition, both isoquercitrin and rutin are significantly higher in the early developmental stages of the fruit spikes of P. sarmentosum, which may be associated with the pharmacological activities of the fruit spikes of this plant.

Biosynthesis pathway of flavonoid.(A) Visualization in the metabolic pathway map and (B) heatmap of the metabolites of flavonoid glycosides.

DAMs in the alkaloid biosynthesis pathway

The primary metabolic components of most alkaloids include common amino acids, such as ornithine, lysine, phenylalanine, tyrosine, and tryptophan. In previous studies of P. longum and P. sarmentosum, many alkaloids have been identified, most of which were derived from tryptophan, phenylalanine, tyrosine, and lysine. KEGG enrichment analysis between these two species revealed that the metabolic pathways of phenylalanine, tyrosine, and tryptophan are the main pathways enriched for DAMs. Further analysis of these pathways showed that in the tryptophan metabolic pathway, about 80% of tryptophan-derived metabolites had significantly higher levels in P. sarmentosum, which is consistent with the metabolic expression pattern of tryptophan (Fig. 5A). These metabolites are mainly concentrated in the reproductive organs, suggesting that tryptophan-derived alkaloids may be a key factor contributing to the medicinal activity of P. sarmentosum’s reproductive organs. Serotonin, widely recognized for its role in various active drugs, exhibits antidepressant, antipsychotic, and anxiolytic effects (Ligneul & Mainen, 2023), was notably higher accumulated in the reproductive organs of P. sarmentosum (Fig. 5A).

Proposed biosynthesis pathway and abundance of alkaloid in two piper species.(A) Visualization of DAMs in tryptophan biosynthesis pathway map and heatmap of DAMs; (B) visualization of DAMs in phenylamine and tyrosine biosynthesis pathway map and heatmap of DAMs; (C) visualization of DAMs in phenylamine and lysine biosynthesis pathway map and heatmap of DAMs.

In the metabolic pathways of phenylalanine and tyrosine, high levels of DL-noradrenaline are detected in the flowers and fruit of P. sarmentosum. In contrast, DL-adrenaline is detected at higher accumulation in the flowers and fruit of P. longum (Fig. 5B). The tyrosine pathway gives rise to various isoquinoline alkaloids, which possess excellent medicinal properties. Boldine inhibits osteoclastogenesis and improves bone damage by downregulating signaling pathways, making it a potential therapeutic agent for rheumatoid arthritis (Zhao et al., 2017). Boldine shows similar tissue expression patterns in both P. longum and P. sarmentosum, with the highest abundance in the flowers of P. sarmentosum (Fig. 5B), possibly linked to this ‘species’ ability to improve bone and joint diseases. Nuciferine and Roemerine are accumulated at the highest level in the mature fruit of P. sarmentosum (Fig. 5B). Numerous reports suggest nuciferine possesses bioactivities such as improving osteoarthritis, managing hyperlipidemia, and exhibiting antitumor effects (Qi et al., 2017; Yu et al., 2021; Peng et al., 2024).

Additionally, lysine-derived alkaloids are the most abundant types of alkaloids identified, synthesized via the lysine metabolic pathway into compounds such as piperidine and pyrrolidine, which are further incorporated into various bioactive alkaloids (Fig. 5C). Both P. longum and P. sarmentosum contain various bioactive piperidine and pyrrolidine alkaloids, such as Piperlotine C. More than two-thirds of the lysine-derived alkaloids are more highly accumulated in P. longum than in P. sarmentosum (Fig. 5C). The most widely studied bioactive component in P. longum is piperlongumine, which has shown significant anticancer, anti-inflammatory, antidiabetic, and antidepressant activities. Piperlongumine is more abundant in P. longum than in P. sarmentosum, with higher abundance in the early stages of flower and fruit development. Moreover, Perhexiline, which has been extensively studied for its efficacy in treating angina (Cole et al., 1990), is found in significantly higher concentrations in the flowers of P. longum.

Alkaloids generally exhibit strong biological activities, and both species contain various alkaloids, which may be the reason for their respective medicinal effects. However, the differences in their distribution and concentration across tissues may account for the observed differences in their therapeutic effects.

Discussion

Plants are an important source for discovering new products with medicinal value in drug development, and plant secondary metabolites serve as unique resources for pharmaceuticals, food additives, and fragrances, among other industrial applications. In recent years, due to the significant commercial value of these secondary metabolites, there has been considerable interest in their production (Tiwari & Rana, 2015). Secondary plant metabolites are the active ingredients in herbal medicines and many modern pharmaceuticals. Therefore, exploring new secondary metabolites in plants not only helps discover new metabolites but may also lead to better therapeutic approaches for treating diseases (Pagare et al., 2015). A substantial body of research has reported the key bioactive compounds found in medicinal plants. Among them, paclitaxel, which can be extracted from Taxus species, has been extensively studied and is widely applied in the clinical treatment of various cancers, including breast cancer, ovarian cancer, and lung cancer (Wani et al., 1971; Swain et al., 1995; Ahmed Khalil et al., 2022). The primary bioactive compound in Coptis chinensis is berberine, which has been shown to possess significant lipid-lowering effects, anti-diabetic properties, and antimicrobial activity (Xu et al., 2020; Reyes et al., 2024). Artemisinin, an active compound extracted from Artemisia annua, has been identified as one of the most effective drugs for treating drug-resistant malaria. In addition, artemisinin exhibits a wide range of pharmacological activities, including antitumor and antidiabetic effects (White, Hien & Nosten, 2015; Efferth, 2017; Zhang, Lin & Li, 2023). With the continuous advancement of mass spectrometry technologies, the development of key bioactive compounds from plants has progressed significantly. The acquisition of metabolic information specific to medicinal plants has become increasingly convenient, providing a solid foundation for the identification and confirmation of key bioactive constituents. In this study, metabolomics was applied to perform qualitative and relative quantitative analyses of metabolites in different tissues of P. longum and P. sarmentosum. A large number of alkaloids were identified, consistent with previous reports on the alkaloid-rich nature of the Piper genus. These findings confirm the presence of known bioactive compounds in various tissues of both P. longum and P. sarmentosum, offering valuable metabolic insights for their further development and efficient utilization.

Research on P. longum and P. sarmentosum has traditionally focused on their pharmacological activities, with studies demonstrating their significant medicinal value. P. longum shows remarkable effects in the treatment of tumors, diabetes, inflammation, depression, as well as in improving arthritis and modulating the immune system (Zhu et al., 2021; Gou et al., 2023; Campo-Grande et al., 2023; Kim et al., 2024), while P. sarmentosum exhibits notable anti-inflammatory, anti-bacterial, anti-depressant, anti-oxidant, and rheumatic pain-relieving activities (Hussain et al., 2010; Shi et al., 2017; Wang et al., 2021; Adib et al., 2024). Additionally, both plants hold considerable culinary value. Current studies suggest that the main bioactive compounds in P. longum are piperlongumine and piperine, although its pharmacological metabolites require further investigation, with limited reports on comprehensive metabolite research. The pharmacological studies of P. sarmentosum mainly focus on the bioactivity of its extracts, with its active compounds still needing further exploration. The adaptation of plants to their environment is intimately associated with, and manifested through, their fascinating ability to synthesize a wide variety of metabolites with diverse structures (Xu & Gaquerel, 2025). Studies also indicate considerable differences in the types and amounts of metabolites between different species and even between different tissues of the same species (Dong et al., 2014; Kumar, Kumari & Kumar, 2023; Liu et al., 2023). This study compared the metabolite differences between P. longum and P. sarmentosum within the same tissues and found that P. longum had more differential metabolites in most tissues. The only exception was in the early stages of flowers and fruits, where P. sarmentosum exhibited higher upregulated metabolites than P. longum. However, in mature or nearly mature fruit spikes, P. longum showed a greater number of upregulated metabolites, which could be related to the fruit spike being the primary medicinal part of the plant. Moreover, after integrating the differential metabolites from various tissues and performing KEGG pathway enrichment analysis, it was found that these metabolites were predominantly enriched in amino acid metabolism-related pathways, which are crucial for the biosynthesis of various alkaloids. This result aligns with the identification of a large number of alkaloid compounds in these species.

The rapid depletion of wild medicinal plant resources has made the development and utilization of these plants more urgent. Secondary metabolites, as key pharmacological components in medicinal plants, are critical to understanding their pharmacological effects and to the development of heterologous synthesis pathways. Nett, Lau & Sattely (2020) successfully achieved the heterologous synthesis of colchicine, a compound with high pharmacological value, in tobacco, offering a sustainable and efficient method for its utilization. Jiang et al. (2024) reconstructed the biosynthetic pathway of key precursors for the anticancer compound paclitaxel and applied synthetic biology techniques to achieve efficient and sustainable production of paclitaxel. In this study, we enriched the differential metabolites into relevant metabolic pathways and visualized these results. Based on the identified compound structures, we hypothesized potential precursor substances, thereby providing insights into the biosynthetic pathways of possible pharmacologically active compounds. Furthermore, we compared the pharmacological components of different species and tissues of P. longum and P. sarmentosum, focusing on the tissue-specificity of these bioactive compounds. This provides data support for the future development of similar pharmacological constituents in these two plants. By comparing the pharmacological components in different tissues of P. longum and P. sarmentosum, we offer new perspectives for discovering alternative biosynthetic pathways for these active compounds. In conclusion, comparing the metabolic differences between these two plants and their various tissues at the metabolite level lays a theoretical foundation for further exploring the medicinal potential of these species. However, the present study only revealed metabolic-level differences between P. longum and P. sarmentosum, without further targeted verification of the metabolite identification results. Future studies should validate these findings through targeted approaches. Moreover, the putative biosynthetic pathways inferred from KEGG enrichment and metabolite structural analysis require further investigation at the key gene level to fully elucidate the underlying regulatory mechanisms.

Conclusions

This study utilized non-targeted Liquid Chromatography-Mass Spectrometry (LC-MS) metabolomics analysis to elucidate the characteristics of metabolites in different tissues of P. longum and P. sarmentosum, providing valuable information for developing their medicinal properties. A total of 729 and 344 metabolites were annotated in positive and negative ion modes. These identified and known metabolites offer solid reference points for the further development of P. longum and P. sarmentosum. The accumulation of metabolites in both species shows strong tissue specificity, with similar metabolite profiles within the same tissue. KEGG pathway analysis revealed that the biosynthesis of phenylpropanoids and alkaloids derived from phenylalanine, tryptophan, and lysine is the main pathway responsible for the metabolic differences between P. longum and P. sarmentosum tissues*.* Finally, by examining the content of differentially accumulated metabolites in these tissues, we identified the distribution of known bioactive compounds in various tissue parts of P. longum and P. sarmentosum. This study provides theoretical support for the efficient separation of bioactive components and lays the foundation for a clearer understanding of the biosynthetic pathways of differentially accumulated metabolites in these tissues.

Supplemental Information

10.7717/peerj.20719/supp-1Supplemental Information 1RSD report

10.7717/peerj.20719/supp-2Supplemental Information 2Identification metabolites

10.7717/peerj.20719/supp-3Supplemental Information 3Identification DAM

10.7717/peerj.20719/supp-4Supplemental Information 4Total ion current (TIC) chromatograms of different tissues