Piperine: From Green Extraction to Clinical Translation—A Review

Abstract

Background

Piperine has diverse pharmacological and bioenhancing activities, but poor aqueous solubility, variable exposure, interaction liability, and discontinuity across production, formulation, and clinical evaluation limit translation. This review integrates these frequently separated stages.

Methods

A structured narrative PubMed search covered English-language studies published from 1 January 2021 to 27 April 2026, supplemented by ClinicalTrials.gov records checked through 13 June 2026, backward citation searching, and selected earlier seminal studies. Evidence was synthesized by experimental level, methodological limitations, and translational relevance.

Key Findings

In one solvent-circulation study, response surface methodology optimization increased the reported piperine yield from 3.87% to 5.20% of dry material, although external batch or production-scale validation was not reported. In rats, a solid piperine self-nanoemulsifying drug delivery system produced 4.92-fold higher relative oral bioavailability than pure piperine dispersion, without human pharmacokinetic validation. Preclinical studies reported anticancer, anti-inflammatory, neuroprotective, anti-infective, and metabolic effects. Head-and-neck cancer-cell IC₅₀ ranges were 102.8–176.0 µM in HEp-2 cells and 121.0–249.9 µM in SCC-25 cells, without exposure-matched in vivo confirmation. Published trials included COVID-19 (n=140), sepsis (n=66), and ischemic stroke (66 randomized; 56 completed); these trials evaluated curcumin–piperine combinations. A 2025 meta-analysis of 18 curcuminoid–piperine trials reported pooled reductions in AST and IL-6 but not ALT, ALP, CRP, or TNF-α.

Challenges

Extraction comparisons were limited by differences in botanical material, process conditions, and analytical methods. Other gaps included production-to-formulation continuity, stand-alone human exposure–response data, substrate-dependent interactions, long-term safety, and predominantly small or combination-based clinical studies.

Conclusion

Current evidence supports further development but not established stand-alone pharmaceutical efficacy. Translation requires standardized materials, scalable production, exposure-guided formulations, longer-term safety and interaction studies, and adequately powered trials with piperine-specific comparators.

Graphical Abstract

Introduction

Black pepper (Piper nigrum), known as “The King of Spices”, is an essential spice for world cuisine due to its unique flavor mainly attributed to the alkaloid piperine along with volatile and essential oils. Piperine content in Piperaceae family plants typically ranges from 2–7.4% in black and white pepper vines, with certain reports confirming up to 9% in black pepper dry fruit.1–3 Piperine (C₁₇H₁₉NO₃, MW 285.34 g/mol, mp 128–130°C), also referred to as (2E, 4E)-5-(benzo[d] [1,3]dioxol-5-yl)1-(piperidin-1-yl)penta-2,4-dien-1-one, first isolated as yellow crystals by Hans Christian Ørsted in 1819, features a piperidine ring linked via conjugated diene chains to a methylenedioxyphenyl moiety.1,3

This nitrogenous alkaloid exhibits pleiotropic bioactivity, such as antioxidant, anti-inflammatory, anticancer, neuroprotective, antimicrobial, immunomodulatory, antidepressant, and hepatoprotective.4 Present alongside minor alkaloids (piperanine, piperettine, piperylin A, piperolein B, pipericine) in black pepper, piperine’s low natural abundance necessitates efficient extraction for therapeutic use, while attracting substantial research interest for its health-promoting effects over recent decades.1,3

Piperine’s total synthesis was first achieved by Ladenburg and Scholtz in 1894 through piperic acid chloride’s reaction with piperidine, enabling scalable production beyond natural extraction.1 Over decades, research has surged from basic bioactivity profiling to advanced drug modulation mimicking enzymatic kinetics, reflecting pharmaceutical maturation. Subsequent research extended from human pharmacokinetic studies of piperine-containing combinations to mechanistic investigations of drug-metabolizing enzymes and transporters, including CYP3A4 and P-glycoprotein (P-gp), and more recent work on sustainable extraction and advanced delivery systems. Figure 1 summarizes these milestones and highlights the evidence gaps that continue to limit clinical translation.

Figure 1.

Major milestones in piperine research and pharmaceutical translation from 1819 to 2026. The timeline summarizes developments in isolation and synthetic chemistry, mechanistic and human bioenhancement research, isomer and stability characterization, sustainable processing, advanced delivery, and recent evidence synthesis. The final milestone presents the translational status identified from literature searched through 27 April 2026, including the remaining need for stand-alone human pharmacokinetic and efficacy evidence, long-term safety assessment, and scalable manufacturing.

Although piperine exhibits broad biological activity, this alone does not establish its suitability as a clinically translatable lead. Its low aqueous solubility may make absorption dissolution-limited and complicate reproducible dose delivery. Nanoparticle and Self-nanoemulsifying drug delivery systems (SNEDDS) studies have improved its solubility, dissolution, and systemic or brain exposure; however, these findings were obtained in preclinical models and therefore do not demonstrate clinical efficacy.5,6 Human pharmacokinetic information is available from short-duration studies, including piperine-capsule data used in Physiologically based pharmacokinetic (PBPK) model validation and a single-dose study of the multi-ingredient Sahastara extract, but these data do not yet define indication-specific exposure–response relationships or long-term safety.7,8 Piperine also inhibits human P-glycoprotein and CYP3A4 in vitro, while PBPK simulations predict different effects across CYP3A4 substrates; accordingly, its bioenhancing potential should be evaluated together with substrate-specific interaction risk.8,9 These linked limitations provide the scientific rationale for considering extraction quality, medicinal-chemistry optimization, formulation performance, human exposure, and safety together rather than as independent development steps.

Piperine, while possessing numerous biological activities, only constitutes a small fraction (3–9%) of dried black pepper fruit.1,4 Thus, consumption of black pepper alone is inadequate to reach the doses necessary for therapeutic uses, this emphasizes the need for efficient and large-scale extraction methods that generate highly purified piperine. Generally, simple and reproducible traditional methods, like Soxhlet extraction,10 maceration,11 and percolation are still very common approaches. Nevertheless, these processes tend to require high-solvent consumption, long extraction times, thermal degradation of heat-sensitive compounds, and relatively low extraction yields.4,12

To tackle these challenges, green chemistry principles that originated in the United States around 1990 are being integrated into piperine processing.13 Advanced extraction technologies, such as supercritical fluid extraction (SFE), ultrasound-assisted extraction (UAE), homogenizer-assisted extraction (HAE), microwave-assisted extraction (MAE), and ultrasound-microwave-assisted extraction (UMAE) can significantly reduce solvent and energy consumption, both of which shown to have high efficiency, while increasing biological activity of piperine.2,4,11

In addition to its bioactive alkaloid properties, piperine is predominantly know as a potential natural bioenhancer, able to enhance the oral bioavailability of co-administered drugs.1,14 One of the landmark studies like Bhardwaj et al demonstrated that piperine is a potent inhibitor of both major determinants of intestinal absorption and first-pass metabolism, P-glycoprotein-mediated drug transport and CYP3A4-mediated metabolism.9 In Caco-2 cell monolayers, piperine inhibited the transport of P-glycoprotein with IC₅₀ values of 15.5 μM for digoxin and 74.1 μM for cyclosporine A, while K_i values ranging from 36–77 μM were derived from plots showing mixed inhibition of CYP3A4 in human liver microsomes which is consistent with previous studies reporting elevation in plasma concentrations along of drugs such as phenytoin and rifampin following co-administration with piperine, suggesting that modulation of metabolic enzymes and efflux transporters contributes to its well-recognized bio-enhancing effect.9

Previous reviews have addressed selected components of piperine research, including isolation and purification, molecular pharmacology and clinical studies, chemistry and structure-activity relationships, and bioenhancement and delivery. Table 1 compares their principal scope, positioning relative to the present review.

Table 1.

Scope and Positioning of Selected Reviews on Piperine

Review	Principal Emphasis	Positioning Relative to the Present Review
Tiwari et al (2020)3	Isolation, purification, chemistry, and biological activities	Provides foundational coverage but predates most 2021–2026 advances in green processing, advanced delivery, and clinical translation.
Tripathi et al (2022)1	Molecular pharmacology, therapeutic applications, and clinical evidence	Provides a broad therapeutic overview; extraction standardization, scalable manufacturing, and regulatory translation are not central emphases.
Han et al (2023)15	Chemistry, piperine derivatives, SAR, biological activities, and mechanisms	Primarily addresses molecular and mechanistic developments rather than evaluating extraction-to-clinic evidence as a linked translational pathway.
Wu et al (2025)16	Total synthesis, structural modification, and biological activities	Emphasizes chemical development; integration with source-material quality, formulation, human exposure, and manufacturing or regulatory requirements remains outside its primary scope.
Tripathi et al (2025)17	Bioenhancement, drug-metabolizing enzymes, transporters, and delivery systems	Centres on bioenhancement; upstream material quality and attribution of clinical outcomes to piperine in combination studies are not central emphases.
This review	End-to-end pharmaceutical development of piperine, from sustainable extraction and purification to clinical translation (2021–2026)	Integrates evidence across sustainable extraction and purification, piperine chemistry and structure–activity relationships, formulation, pharmacokinetics, safety, and clinical studies, while identifying the remaining gaps in process scale-up, human pharmacokinetic and exposure–response characterization, and evidence of piperine-specific clinical efficacy that limit the development of a stand-alone piperine medicine.

Collectively, previous reviews provide strong domain-specific foundations, but they generally examine extraction, medicinal chemistry, bioenhancement, formulation, or therapeutic evidence as separate emphases.1,3,15–17 The present review instead evaluates the continuity of evidence across these stages, asking whether advances in extraction and formulation translate into reproducible material quality, clinically interpretable exposure, acceptable safety, scalable manufacturing, and indication-specific clinical evidence.

Taken together, the central translational gap is not a lack of reported bioactivity but the absence of a continuous evidence chain linking reproducible starting material and pharmaceutical-grade piperine with scalable manufacture, clinically interpretable exposure, long-term safety, and adequately powered indication-specific trials. Existing human evidence includes short pharmacokinetic studies and piperine-containing combination interventions, such as Risorine and curcumin–piperine trials.7,8,18,19 These studies demonstrate that piperine can be investigated in humans and used as a bioenhancer or formulation component; however, combination trials cannot isolate the efficacy of piperine itself, and short pharmacokinetic studies do not establish long-term therapeutic benefit. This discontinuity helps explain why extensive preclinical research has not yet yielded a well-established stand-alone piperine therapy supported by confirmatory clinical evidence.

Research on piperine has rapidly expanded since 2020. This growth signifies need for an updated review that brings together recent progresses spanning multiple disciplines rather than treating them separately. While previous reviews have offered valuable insights into the chemistry, pharmacology, and formulation of piperine, a cohesive pharmaceutical perspective that integrates sustainable extraction with rational drug discovery, advanced formulation technologies, clinical evidence, and translational barriers has not yet been provided. Against this background, this review addresses three critical questions: (1) Can sustainable extraction methods reproducibly produce pharmaceutical-grade piperine at scale? (2) To what extent do advanced formulations overcome solubility and exposure barriers? and (3) What evidence currently supports clinical translation? By integrating studies published from 2021 to 2026 across extraction, medicinal chemistry, formulation, pharmacokinetics, safety, and clinical development, this review identifies the remaining evidence required to advance piperine from a bioactive ingredient or bioenhancer toward an indication-specific pharmaceutical product.

Methodology

Search Strategy

A structured narrative literature search was conducted in PubMed for English-language studies published between 1 January 2021 and 27 April 2026. ClinicalTrials.gov was additionally searched to identify interventional studies involving piperine or piperine-containing products, and the relevant registry records were last checked on 13 June 2026. The core search block (“piperine” OR “Piper nigrum” OR “piperine derivative” OR “piperine derivatives”) was combined separately with terms related to: (1) extraction and purification (“extraction” OR “isolation” OR “purification” OR “green extraction”); (2) drug discovery (“drug design” OR “structure–activity relationship” OR “molecular docking” OR “molecular dynamics”); (3) biological activity (“pharmacological activity” OR “biological activity” OR “mechanism”); (4) formulation (“formulation” OR “drug delivery” OR “nanocarrier” OR “SNEDDS”); (5) pharmacokinetics (“pharmacokinetics” OR “bioavailability” OR “bioenhancer”); (6) safety (“toxicity” OR “safety” OR “drug interaction”); and (7) clinical translation (“clinical trial” OR “clinical evidence” OR “translation”).

Backward citation searching of included articles and relevant reviews was used to identify additional evidence. Studies published before 2021 were retained when required to establish historical, physicochemical, pharmacokinetic, interaction, or safety context that was not adequately represented in the date-limited literature.

Data Selection and Collection Process

Records retrieved through overlapping searches were deduplicated before titles and abstracts were assessed for relevance. Potentially eligible publications were subsequently evaluated in full text, and uncertain eligibility decisions were discussed among the authors until consensus was reached.

Studies were eligible when they investigated piperine, a clearly defined piperine derivative, or a characterized piperine-containing extract, formulation, or combination intervention relevant to the review objectives. Eligible studies were also required to provide sufficient methodological and outcome information for interpretation. Peer-reviewed original studies were prioritized, while systematic reviews and meta-analyses were used to contextualize pooled evidence and identify relevant primary studies. Trial-registry records were included to describe study status and prespecified outcomes; registration or completion status was not treated as evidence of efficacy.

Duplicate publications, editorials, conference abstracts without analyzable evidence, and studies with insufficient methodological information were excluded. Studies of uncharacterized complex Piper extracts were excluded when neither piperine-specific nor product-level interpretation was possible. Characterized extracts and multicomponent interventions were retained for relevant formulation or clinical questions, but their findings were not attributed independently to piperine. Computational studies without experimental validation were retained as hypothesis-generating evidence rather than confirmation of biological activity.

Extracted information included study design, source and form of piperine, structural modification, experimental model, extraction and purification conditions, yield and purity, formulation characteristics, biological outcomes, pharmacokinetic parameters, safety findings, and clinical-study characteristics. For clinical interventions, composition, dose, duration, comparator, sample size, endpoints, and the ability to isolate the contribution of piperine were considered.

Data Synthesis

No de novo meta-analysis was performed because the included studies differed substantially in piperine source, extraction and formulation procedures, experimental models, doses, comparators, and outcome measures. Evidence was therefore synthesized using a critical narrative approach and organized according to medicinal chemistry and SAR, computational drug discovery, biological activity, extraction and purification, formulation, pharmacokinetics and bioenhancement, safety, and clinical translation.

Numerical findings were compared only between matched compounds or interventions evaluated using equivalent assays. Biochemical, cellular, animal, and human findings were distinguished, and in vitro concentrations were not assumed to represent achievable systemic or target-tissue exposure. Outcomes from combination interventions were attributed to the complete tested formulation unless an appropriate comparator allowed the independent contribution of piperine to be determined.

Characteristics and Critical Appraisal of the Included Evidence

Methodological appraisal was conducted within the relevant thematic sections rather than by applying a single scoring system across heterogeneous study types. Computational and biological studies were evaluated according to model relevance, concentration or dose, experimental validation, target engagement, and correspondence with in vivo exposure. Extraction studies were considered in relation to botanical source, material pretreatment, solvent composition, process conditions, analytical method, reporting basis, and piperine purity. RSM studies were evaluated using the reported R², adjusted and predicted R², lack-of-fit, experimental confirmation, and external batch or scale validation where available. Formulation evidence was distinguished as physicochemical, in vitro, ex vivo, animal pharmacokinetic, animal efficacy, or human evidence. Clinical studies were considered according to sample size, power basis, comparator, analysis population, endpoint selection, multiplicity, follow-up, and whether the contribution of piperine could be isolated. Reported p-values were not interpreted independently of effect estimates, variability, study design, and clinical relevance.

Because this narrative review relied primarily on PubMed and English-language publications, relevant evidence indexed elsewhere or published in other languages may have been missed. No review protocol was registered, and no formal risk-of-bias instrument or independent certainty grading was applied. Publication bias was considered qualitatively because negative or inconclusive findings may be less likely to be published. Consequently, the predominance of positive reports was not interpreted as evidence of consistent efficacy.

Role of Piperine in Drug Discovery

Based on its already proven pharmacological and bio-enhancing properties, piperine represents a new molecular scaffold that is positioned as an attractive basis for drug development. Instead of a single predefined therapeutic position it occupies, it is now more frequently investigated as compounds that can potentially be multi-functional during all three phases of the drug development pipeline (target identification, lead optimization, and formulation strategies).20–22 The conjugated system and piperidine moiety are structural features that allow multiple molecular interactions with biological targets, which suits its application to multi-target drug design.1,23

In recent years, research has focused on the coupling of computational modeling for SAR studies with rational modification to derive piperine derivatives with higher efficacy and selectivity. Thus, recent advances (2021–2026) position piperine not merely as a bioactive compound, but as a multi-facile platform that links natural product chemistry with modern drug development. This progression is further developed as a lead compound and multi-target pharmacological potential as well as computational strategies in rational drug design are then discussed in the following section.

Chemical Structure and Properties

There are four geometric isomers of piperine found naturally in black pepper: the bioactive trans-trans isomer (piperine, A), cis-trans isomer (isopiperine, B), cis-cis isomer (chavicine, C), and trans-cis isomer (isochavicine, D) (Figure 2).1,3 Among these, trans-trans isomer, hereafter referred to as piperine, is the predominant form and the principal contributor to the characteristic pungency of black pepper.1 The light exposure initiates piperine isomerization process to form either isopiperine or chavicine or isochavicine, with chavicine spontaneously convert back during storage, resulting in gradual pungency loss.24

Figure 2.

Isomers of Piperine (C₁₇H₁₉NO₃, MW: 285.34 g/mol): (A) trans-trans, (B) cis-trans, (C) cis-cis, (D) trans-cis.

The piperine molecule consists of three key subunits: a piperidine ring amide-linked to an α,β-unsaturated carbonyl, connected via butadiene chain to a 1,3-benzodioxole (piperonal) nucleus, enabling H-bonding and π-stacking critical for its bioactivity.1

Development of Piperine and Its Derivatives

The early studies on piperine were mainly to isolate the compound and characterize its intrinsic biological activities. Nevertheless, the specificity of these compounds is hampered by poor aqueous solubility, a low bioavailability, and an intermediate potency in some applications which has led to extensive structural modification and the development of derivatives.1,17

Structurally, piperine has a methylenedioxyphenyl group within a 17‑carbon amide backbone, consisting of a conjugated system connecting the aromatic ring to a piperidine moiety by means of an amide bond (Figure 3). This specific structure plays a role in its pungency properties and biological activities, which have been more comprehensively explored over the years.17

Figure 3.

Integrated structural-feature and evidence-based SAR map of piperine derivatives. The central scaffold is color-coded to identify the methylenedioxyphenyl region (red), conjugated aliphatic linker (green), and terminal amide–piperidine region (blue). Correspondingly colored arrows connect these regions to assay-matched SAR observations across compounds E–V.

Piperine derivatives have been developed through structural modifications at three major regions of the molecule: the piperidine ring, the conjugated aliphatic linker, and the methylenedioxy-substituted aromatic ring. Representative structures illustrating these design strategies are organized in Figure 4, whereas their experimentally measured activities and within-series SAR interpretations are summarized in Table 2. The derivatives were evaluated using heterogeneous endpoints, including isolated-enzyme inhibition, cancer-cell viability, insect mortality, phenotypic cellular assays, and computational docking. Consequently, potency values obtained from different experimental systems should not be ranked as though they represent the same biological outcome; meaningful SAR comparisons should be restricted to structurally related compounds evaluated using matched assays.

Figure 4.

Representative structural design strategies among the piperine derivatives discussed in Table 2. Compounds E–G represent carboximidamide analogues with variation in terminal aryl substitution; H–J, piperine–resveratrol hybrids; K–N, linear bisamide analogues bearing alternative terminal substituents; O–P, CF₃/piperazine-containing anilide analogues differing in conjugated-linker length; Q–S, C-2 hydroxymethyl ester derivatives; and T–V, C-2 oxime-ester derivatives. Blue highlights the retained methylenedioxyphenyl/conjugated framework, magenta indicates the principal amide or linker region, and green indicates the introduced or variable substituents; black is used for detailed R-group structures. Compound letters were assigned in the present review and correspond to Table 2.

Table 2.

Modification of Piperine and Its Derivatives

Modification	Representative Compound(s) (Figure 4)	Matched Experimental Results	Evidence-Based Structure Activity Relationship (SAR) pattern	Ref.
Piperidine ring	E (C19H16N2O4, MW = 336.35 g/mol), F (C21H20N2O6, MW = 396.40 g/mol), and G (C24H18N2O6, MW = 430.24 g/mol)	Cellular IC50 values for A549/MCF-7/Panc-1/HT-29 cells: E, 0.095/0.097/0.104/0.105 µM; F, 0.036/0.039/0.040/0.040 µM; and G, 0.032/0.035/0.036/0.038 µM. Compound G enzyme IC50: EGFR, 0.127 ± 0.010 µM; BRAFV600E, 0.040 ± 0.004 µM; and CDK2, 0.012 ± 0.002 µM.	Terminal oxygen-containing aryl substitution was associated with lower cellular IC50 values than the unsubstituted analogue, with G showing the lowest values within this series.	Al-Wahaibi et al23
Piperidine ring	H (C20H20N2O5, MW = 368.38 g/mol), I (C20H20N2O6, MW = 384.38 g/mol), and J (C20H18N2O5, MW = 366.36 g/mol)	SIRT2 inhibition at 50/5 µM: H, 72 ± 3%/19 ± 3%; I, 78 ± 3%/26 ± 3%; and J, 58 ± 3%/17 ± 3%. Compound I: SIRT2 IC50 = 21 ± 3 µM. In the single-dose NCI-60 screen at 10 µM, I was the most active, H showed moderate activity, and J was among the least active derivatives.	The 2,4-dihydroxy substitution in I was associated with greater SIRT2 inhibition and cell-growth suppression than the 4-hydroxy and 3-methoxy-4-hydroxy substitution patterns.	Tantawy et al25
Piperidine ring	K (C22H19O4N2, MW = 374.40 g/mol), L (C20H18O4N2Cl, MW = 385.82 g/mol), M (C22H23O4N2, MW = 379.44 g/mol), and N (C16H16O4N2F3, MW = 357.31 g/mol)	P. xylostella mortality at 0.2 mg/mL after 48 h: K, 0%; L, 26.7 ± 1.9%; M, 26.7 ± 1.9%; N, 43.3 ± 1.9%	Activity depended strongly on the terminal R group. The 2,2,2-trifluoroethyl derivative N was the most active, whereas K was inactive under the same conditions.	Zhang et al26
Piperidine Ring	O (C25H27N3O3F3, MW = 506.50 g/mol) and P (C23H25N3O3F3,MW = 478.47 g/mol)	IC50 values for 293T/HeLa/MDA-MB-231 cells: O, 8.06 ± 0.02/2.96 ± 0.29/7.64 ± 0.23 µM; P, 147.45 ± 6.05/11.86 ± 0.32/10.50 ± 3.74 µM	The longer conjugated diene linker in O was associated with greater potency in both cancer-cell lines but also greater cytotoxicity toward 293T cells. The shortened single-alkene linker in P reduced cancer-cell potency while producing a wider separation from the tested 293T cells.	Wang et al27
Aliphatic chain	Q (C24H24N2O5, MW = 420.46 g/mol), R (C26H27NO5, 433.50 g/mol), and S (C26H26ClNO5, MW = 467.95 g/mol)	T. cinnabarinus LC50 at 72 h: A, 14.198 mg/mL; R, 0.298 mg/mL; and S, 0.313 mg/mL. A. citricola LD50 at 48 h: A, 0.308 µg/nymph; and Q, 0.030 µg/nymph.	The preferred ester substituent was pest-dependent: R and S substituents were associated with the strongest acaricidal activity, whereas the Q produced the strongest aphicidal activity against A. citricola.	Li et al28
Aliphatic chain	T (C32H46N2O5, MW = 538.34 g/mol), U (C28H26N2O7, MW = 502.17 g/mol), and V (C30H42N2O5, MW = 510.31 g/mol)	T. cinnabarinus LC50: A, 15.02 mg/mL; T, 0.14 mg/mL; and U, 0.13 mg/mL. A. citricola LD50: A, 116.06 ng/aphid; and V, 19.12 ng/aphid.	Within the aliphatic oxime-ester series, T was optimal for acaricidal activity, whereas further chain extension reduced activity; the shorter-chain analogue V was favored in the aphicidal assay. The aromatic-conjugated analogue U independently produced the lowest acaricidal LC50.	Lv et al29

Notes: Bold capital letters A–V in the data rows denote the compound labels. These labels were sequentially assigned in the present review for consistency across Figures 2–4, Table 2, and the accompanying discussion.

Figure 4 and Table 2 summarize studies employing different biological systems, assay conditions, and endpoints. Accordingly, quantitative results should be compared only among compounds evaluated within the same study under equivalent assay conditions. Enzyme IC₅₀ values are not interchangeable with cellular antiproliferative IC₅₀ values, while insect LC₅₀ and LD₅₀ values cannot be compared directly with mammalian cellular endpoints.

Within E–G, F and G produced lower cellular IC₅₀ values than E, associating their oxygen-containing terminal aryl motifs with greater cellular potency within this series.23 Docking predicted binding poses for G within the BRAF^V600E and CDK2 active sites; however, cellular target engagement was not demonstrated. The study therefore could not determine whether the improved cellular response was mediated by these interactions or by accompanying differences in molecular shape and physicochemical properties. Moreover, because parent piperine was not evaluated in the same four-cell assay, these findings establish terminal-substituent SAR within E–G but do not demonstrate that replacement of the piperidine moiety improved activity relative to piperine.

A substituent-dependent pattern was also observed among H–J. The 2,4-dihydroxy derivative I showed greater SIRT2 inhibition and broader cell-growth suppression than the 4-hydroxy analogue H and the 3-methoxy-4-hydroxy analogue J, indicating that phenolic-group number and position influenced activity within this series.25 However, the NCI-60 evaluation was conducted at a single concentration of 10 µM. It therefore supports a qualitative activity ranking but does not provide directly comparable cellular IC₅₀ values. The two proposed binding modes of I within SIRT2 were docking-derived and require kinetic competition or direct-binding studies for confirmation.

In the linear-bisamide series K–N, mortality against Plutella xylostella ranged from 0% for K to 43.3 ± 1.9% for N at 0.2 mg/mL under identical assay conditions.26 This variation indicates that the terminal substituent influenced activity and that introduction of the linear-bisamide scaffold alone was insufficient to ensure insecticidal activity. Although N subsequently produced 90% mortality at 1 mg/mL, this higher-dose result characterizes the selected lead and cannot be used for direct SAR comparison because K–M were not reported at the same concentration. The proposed interaction of N with the GABAA receptor was based on docking to a homology model and therefore remains a mechanistic hypothesis rather than experimentally validated receptor engagement.

The matched comparison between O and P demonstrates that cellular potency and separation from the tested non-tumor cells did not improve concurrently.27 Both compounds contain the same trifluoromethylphenyl-piperazine terminal fragment, but O retains the longer piperine-like conjugated diene chain, whereas P contains a shortened single-alkene linker. Compound O produced lower IC₅₀ values in both cancer-cell lines but was also more cytotoxic toward 293T cells. Conversely, P was less potent in the cancer-cell assays but showed a wider numerical separation from the tested 293T cells, indicating a potency-cell-line selectivity trade-off. Linker shortening may alter the spacing and orientation of the terminal pharmacophore, but this explanation remains hypothetical because a direct molecular target was not established.

Compounds Q–S demonstrate that the biological consequences of C-2 ester functionalization were pest-dependent.28 The benzyl and p-chlorobenzyl derivatives R and S were favored against Tetranychus cinnabarinus, whereas the pyridin-4-yl derivative Q was favored against Aphis citricola. The C-2 formyl and hydroxymethyl intermediates alone did not improve aphicidal activity, indicating that the identity of the subsequently introduced ester substituent contributed to the observed activity.

Among the C-2 oxime esters T–V, T and U produced substantially lower LC₅₀ values than piperine in the T. cinnabarinus assay, whereas V produced a lower LD₅₀ than piperine in the A. citricola assay.29 Within the aliphatic oxime-esters, activity varied with chain length, while the aromatic-conjugated derivative U represents a separate substituent effect and should not be included in the aliphatic chain-length trend. Comparisons with ring-modified analogues in the same study additionally suggested that retention of the 3,4-methylenedioxy group was important for acaricidal activity within this series.29 Finally, SEM observations in the ester and oxime-ester studies showed treatment-associated disruption of the mite cuticle, but these morphological findings do not identify a molecular target or establish cuticular damage as the primary causal mechanism.28,29 These region-specific SAR patterns and their evidentiary limitations are integrated in Figure 3, which links the major structural regions of piperine to the assay-matched trends identified across compounds E–V.

Multi-Target Potential

Piperine shows distincts multi-target pharmacological activity mediated through modulation of key signaling pathways involved in disease progression. Piperine has been shown to modulate several oncogenic pathways in cancer models. Pressete et al showed that a piperine-chlorogenic acid hybrid molecule possesses strong multi-targeted antiproliferative effects on melanoma cells by regulating mitotic kinases and cell cycle regulation pathways.30 The lead compound (PQM-277) inhibited mitotic progression by downregulating key genes during G2/M transition (FOXM1, CCNB1, CDK1, AURKA, AURKB, and PLK1) and upregulating CDKN1A.30 Furthermore, the compound also had an apoptotic effect through increasing BAX/BCL2 ratio and binded the CUL1-RBX1 complex, suggesting that it is a multitarget agent that can modulate different signalings in tumor progression.30 Chowdhury and Kumar further demonstrated that piperine exhibits multi-target potential against Alzheimer’s disease-related pathways, showing concentration-dependent inhibition of BACE1 (29.44–51.35% at 10–100 µM), moderate inhibition of MAO-B (13.32 ± 3.91% and 37.19 ± 12.21% at 10 μM and 40 μM, respectively), and significant reduction of amyloid-β aggregation (46.37 ± 2.7%).31 These results suggest that the piperine most likely can concurrently modulate several pathological targets at once and thus reinforce piperines multi-target-directed ligand action in neurodegenerative disorders.

Moreover, Li et al pointed out that piperine functions as a multi-target agent by simultaneously modulating metabolic and inflammatory pathways, particularly through D-amino acid oxidase (DAO) metabolism and IL-17A signaling.32 Effectively targeting two targets at once highlights its ability to control both tumor metabolism and the microenvironment of inflammation in tumor, which is not only an advantage but also a strategic approach to multi-target cancer therapy.32

In dental applications, the multitarget nature of piperine has also been shown. A study by Albuquerque et al has shown antioxidant and antimicrobial properties of piperine derived from Piper nigrum. Piperine exhibited antibacterial and antifungal effects, with 0.004% concentration showing powerful bactericidal and fungicidal activity.33 Piperine also inhibited the decrease in degree of conversion of self-adhesive resin cement bonded to bleached enamel, suggesting its antioxidant protective effect on oxidative damage induced by hydrogen peroxide-based bleaching treatments.33 These data suggest that piperine has bioactive properties with both antimicrobial and anti-oxidative effects, supporting its characterization as a multifunctional bioactive compound.

Nevertheless, although polypharmacological compounds may produce additive or synergistic effects, such benefits cannot be inferred solely from activity against multiple targets and require evidence of simultaneous target engagement and synergy in appropriate experimental models.34 In designed multi-target ligands, activities must be balanced at relevant in vivo exposures while retaining adequate selectivity and suitable pharmacokinetic properties.35 Pharmacophore linking may increase molecular size and lipophilicity, potentially impairing solubility, absorption, and pharmacokinetic behaviour.35,36 These physicochemical changes may also increase safety liabilities: higher molecular weight has been associated with greater mean hERG inhibitory potency, whereas broader in vivo toxicity is more strongly associated with high lipophilicity and low polarity.37,38 When applied to piperine, these general principles indicate that its reported activity across multiple targets represents a plausible but unconfirmed opportunity rather than evidence of therapeutic synergy in vivo. Accordingly, any future piperine-derived multi-target analogue would require direct evaluation of target engagement, in vivo exposure, and safety.

Role of Computational Approaches

Computational approaches, including network pharmacology, molecular docking, quantitative structure-activity relationship analysis, ADMET prediction, and molecular dynamics (MD) simulations, can prioritize ligand-target hypotheses and guide experimental design. Nevertheless, their outputs should be interpreted according to the level of experimental validation. Docking scores are model-dependent estimates rather than experimentally measured binding affinities, and benchmark evaluations have demonstrated inconsistent performance in selecting experimentally observed binding poses and ranking ligand affinities across different protein targets.39 Ligand-protein MD simulations provide information beyond a static docking pose, but their interpretation remains dependent on methodological factors such as molecular parametrization, force fields, and conformational sampling.40 More broadly, computational models may not accurately represent the complexity of biological systems and have limitations in predicting pharmacokinetic and toxicity profiles. Therefore, computational predictions require experimental validation before they can be interpreted as evidence of target engagement or therapeutic efficacy.41

The level of validation varies substantially among piperine-related computational studies. Yu et al combined network pharmacology and molecular docking with an in vivo rat model of sciatica involving 48 animals divided into four groups, followed by inflammatory-marker measurements, histological evaluation, and analyses of PPARG and NF-κB1 expression.42 These experiments support the biological relevance of the proposed pathway but do not directly confirm the predicted piperine-protein binding poses. Zazeri et al synthesized and spectroscopically characterized piperine-inspired analogues; however, their proposed interactions with IL-1β and NF-κB were derived from molecular docking and the compounds were presented as candidates for subsequent evaluation as inhibitors.22 Similarly, the reported interactions of piperine with CDK2, CDK4, cyclin D, and cyclin T were based on docking alone, and the authors recommended subsequent in vitro and in vivo investigation. Thus, experimental confirmation of compound identity should not be regarded as validation of predicted protein binding or pharmacological efficacy.43

Al-Wahaibi et al provided a more integrated example by synthesizing piperine-carboximidamide hybrids and evaluating their antiproliferative activities and inhibition of EGFR, BRAF^V600E, and CDK2 alongside molecular docking.23 Experimental assays showed EGFR inhibition with IC₅₀ values of 96–127 nM, although the hybrids were less potent than erlotinib (80 ± 5 nM).23 Despite favourable EGFR docking scores, the predicted poses did not reproduce interactions with GLN767 and MET769 observed for the co-crystallized ligand, leading the authors to conclude that the selected EGFR binding site did not adequately explain the compounds’ antiproliferative mechanism. By contrast, the predicted interactions with key residues of BRAF^V600E and CDK2 were more consistent with the corresponding enzyme-inhibition results. This target-dependent agreement illustrates that experimental activity does not necessarily validate every predicted binding mode or cellular mechanism.

Liu et al combined fluorescence-quenching and circular-dichroism measurements with particle-size and zeta-potential analyses, molecular docking, and MD simulations to investigate piperine interactions with myofibrillar proteins.44 The experimental measurements supported molecular association and protein conformational changes, whereas docking and MD proposed non-covalent interactions involving ARG202 and PHE622. This cross-method agreement provides physicochemical support for piperine-protein association in a food-protein system; however, it does not directly establish the predicted residue-level binding mode and should not be generalized as evidence of pharmacological target engagement or therapeutic efficacy.

By contrast, Francis et al used network pharmacology, molecular docking, and 100-ns MD simulations to identify NR3C1, PPARG, FOS, CYP17A1, and H6PD as putative PCOS-related targets of piperine.45 Because the study did not include direct-binding, cellular, animal, or clinical validation, its docking energies and trajectory stability indicate computational plausibility rather than demonstrated target modulation or therapeutic efficacy in PCOS. Moreover, docking and MD alone cannot determine whether sufficient piperine reaches a proposed target after cellular uptake, metabolism, systemic exposure, and tissue distribution are considered.41 Piperine-related computational findings should therefore be regarded as hypothesis-generating and prioritized for orthogonal direct-binding, functional, cellular, and exposure-matched in vivo validation. Inconclusive or partially discordant validation results should also be reported because favourable computational predictions alone are insufficient to establish biological efficacy.

Piperine as Bioactive Compound

The broad pharmacological activities of piperine have stimulated growing interest in developing efficient extraction, formulation, and drug delivery strategies to support its therapeutic application. This section first summarizes its major biological activities before discussing advances in its production and pharmaceutical development.

Biological Activities

Numerous studies have demonstrated that piperine and its derivatives hold significant potential in drug discovery and development for various human diseases. This section provides an overview of their biological activities (Figure 5) and their impact on different pathological with recent studies (2021–2026) further advancing the understanding of their mechanisms and broadening their therapeutic scope. The diverse pharmacological activities of piperine reported in recent studies demonstrate its multi-target therapeutic potential across various disease conditions. Because these studies differ substantially in their experimental models, tested materials, doses, and outcomes, their findings are interpreted according to the level of supporting evidence. In particular, in vitro IC₅₀ and MIC values are treated as assay-specific findings and not as evidence of in vivo or clinical efficacy unless supported by pharmacokinetic and exposure-matched biological validation. A summary of the major biological activities, underlying mechanisms, and representative findings is presented in Table 3.

Figure 5.

Biological activities of piperine.

Table 3.

Summary and Evidence Appraisal of the Reported Biological Activities of Piperine and Related Test Materials (2021–2026)

Biological Activity	Experimental Model and Evidence Level	Principal Finding	Translational Limitation	References
Anticancer	HEp-2, SCC-25, and leukemia cell models; selected animal and multi-omics gastric-cancer studies	Piperine induced apoptosis and cell-cycle arrest in cellular models; selected animal studies reported reduced tumor incidence or growth	Cellular concentrations were not linked to achievable unbound tumor exposure, and human anticancer efficacy has not been established	[46–49]
Antimicrobial	In vitro bacterial, fungal, and biofilm assays using purified piperine or Piper extracts	Growth and biofilm inhibition were reported against selected microorganisms, whereas activity against P. aeruginosa was limited	Effects varied with the microorganism, assay, and test material; efficacy in animal infection models or humans was not demonstrated	[33,50–52]
Anti-inflammatory	Cellular assays and mouse models of hepatic ischemia/reperfusion injury and DSS-induced colitis; derivative and multicomponent studies	Reduced inflammatory signaling, cytokine production, and tissue injury were reported in specific experimental models	Evidence includes a piperine derivative and piperine–hydroxybenzoate complex; these effects cannot all be attributed to unmodified piperine	[46,53–56]
Immunomodulatory	Cellular, macrophage-mycobacterial, parasitic-infection, cervical-cancer, and analogue-based animal models	Modulation of Th1/Th2 responses, intracellular antibiotic activity, and NLRP3/Th17-related signaling was reported	Several studies involved an analogue, combination treatment, or cellular model; stand-alone human efficacy remains unknown	[57–59]
Neuroprotective	Cellular and rodent models of Parkinson’s disease, Alzheimer’s disease, EAE, and sepsis-associated encephalopathy	Autophagy activation, reduced neuroinflammation and oxidative stress, and improved neurological outcomes were reported in preclinical models	The studies used heterogeneous formulations and compounds; ilepcimide is chemically distinct from piperine, and human neurological efficacy has not been established	[60–64]
Antioxidant	Protein, fungal, shrimp, extract, and biomaterial models	Antioxidant-enzyme activation and reductions in oxidative damage were reported in the investigated systems	Evidence was obtained from heterogeneous nonhuman models and included piperine-containing extracts; clinical antioxidant efficacy cannot be inferred	[33,52,65–67]
Antiviral	In vitro CHIKV and LMBV assays; MERS-CoV mouse and LMBV fish models	Piperine-loaded bilosomes showed greater ex vivo permeation and oral bioavailability than piperine suspension and reduced oxidative and inflammatory markers in MERS-CoV-infected mice. Piperine also inhibited LMBV replication and improved survival in infected fish	Evidence remains preclinical and model-specific; the MERS-CoV findings apply to the optimized bilosome formulation and do not establish human antiviral efficacy	[68–70]
Hepatoprotective	Mouse models of fibrosis and chemically induced liver injury; nanopiperine and combination studies	Reduced fibrosis, liver-injury markers, inflammation, and oxidative stress were reported	Evidence is preclinical and includes nanoformulation and combination interventions; human hepatoprotection has not been established	[71–74]
Analgesic	Pediatric curcumin–piperine patch study and celecoxib–piperine cocrystal evaluation	Pain decreased after patch treatment, but addition of piperine was not superior to curcumin alone; the cocrystal improved dissolution and compressibility	The clinical intervention was a combination product, while improved formulation performance does not establish independent analgesic efficacy of piperine	[75,76]
Antidepressant	LPS-induced depression-like behaviour in mice treated with nanopiperine	Nanopiperine improved behavioural, neurotrophic, neurotransmitter, oxidative-stress, and inflammatory outcomes	Evidence is limited to a preclinical nanoformulation study; human antidepressant efficacy remains untested	[77]

Notes: Findings obtained with extracts, derivatives, formulations, or combination interventions apply to the tested material and should not be attributed independently to stand-alone piperine.

Anticancer Activity

Piperine and its derivatives have been extensively investigated for their anticancer potential across various cancer types, demonstrating the ability to inhibit tumor progression through multiple mechanisms. Piperine suppresses cancer cell proliferation, induces apoptosis, and modulates cell cycle progression via key signaling pathways. For example, Gusson-Zanetoni et al reported piperine inhibited viability and colony formation in head and neck cancer cells, accompanied by apoptosis induction, G2/M cell cycle arrest, and downregulation of MMP2/9 and inflammatory mediators such as PTGS2 and PTGER4.78 The reported time-dependent IC₅₀ ranges of 102.8–176.0 µM in HEp-2 cells and 121.0–249.9 µM in SCC-25 cells and selected 150 µM for subsequent 24-h experiments. Under these conditions, piperine inhibited cell viability and colony formation, induced apoptosis and cell-cycle arrest, and altered the expression of MMP2/9 and inflammatory mediators, including PTGS2 and PTGER4. Similarly, Banerjee et al demonstrated that piperine induces ROS-mediated apoptosis in leukemia cells through upregulation of Bax, caspase-3, and caspase-9, along with downregulation of Bcl-2, leading to inhibition of cell proliferation.79 These studies demonstrate cellular responses under their respective experimental conditions but do not establish inhibition of tumor progression in vivo. Moreover, because the tested concentrations were not linked to achievable unbound piperine exposure in plasma or tumor tissue, their translational relevance requires exposure-matched in vivo validation.

In addition to direct cytotoxic effects, piperine also acts as a chemosensitizer. Mad-adam et al showed that piperine-containing formulations reduced tumor incidence and enhanced immune responses in vivo, while also mitigating chemotherapy-induced toxicity.46 More recent integrative studies further highlight its system-level mechanisms, where Pan et al identified ACOT1 as a key molecular target in gastric cancer through multi-omics analysis, revealing that piperine suppresses tumor growth via metabolic reprogramming and regulation of lipid metabolism pathways.47 The animal and multi-omics studies provide additional biological support beyond isolated cell assays. Nevertheless, these findings remain model-specific and do not yet establish clinically effective anticancer exposure or efficacy in humans.

Antimicrobial Activity

Piperine exhibits broad-spectrum antimicrobial activity against various pathogenic microorganisms, including Gram-positive and Gram-negative bacteria as well as fungi. For instance, Alves et al reported that piperine and Piper nigrum extracts showed effective inhibition against Staphylococcus aureus and Salmonella spp., with minimum inhibitory concentrations (MIC) below 100 μg/mL, although limited activity was observed against Pseudomonas aeruginosa.48

In addition to its antibacterial activity, piperine has demonstrated potent antibiofilm properties. Das et al showed that piperine significantly inhibits biofilm formation in methicillin-resistant Staphylococcus aureus (MRSA) at low concentrations (8–16 μg/mL), the mechanisms through which it acts include disruption of extracellular polysaccharides, reduction of cell-surface hydrophobicity, and downregulation of quorum-sensing-related genes such as icaA.49 In addition, piperine was shown to increase intracellular reactive oxygen species (ROS) production and increase membrane permeability, leading to destabilization of biofilm architecture.

Antifungal activity of piperine, which showed by Albuquerque et al, with bactericidal and fungicidal effects at low concentrations (0.004%), making it a potential for microbial infections in applied systems.33 Aligned with these findings, Al-Mamun et al investigated that Piper species extracts rich in piperine exerting notable antibacterial and antifungal activities, supporting their potential as natural antimicrobial agents.50

Taken together, the available antimicrobial evidence is predominantly in vitro and varies according to the tested microorganism, assay conditions, and test material, which included purified piperine and piperine-containing extracts. The limited activity against Pseudomonas aeruginosa reported by Alves et al also indicates that the antimicrobial effect is not uniform across microorganisms.48 Therefore, the reported MIC and antibiofilm findings support activity under their respective experimental conditions but do not establish systemically achievable antimicrobial exposure or efficacy in animal infection models or humans. Piperine should therefore be considered a candidate for further antimicrobial investigation rather than an established antimicrobial or adjunct treatment.

Anti-Inflammatory Action

Piperine has powerful anti-inflammatory actions by regulating various inflammatory mediators and signaling pathways. In addition, piperine has been shown in several studies to inhibit the release of pro-inflammatory cytokines and the expression of major inflammatory enzymes. For instance, Gusson-Zanetoni et al reported that piperine modulates inflammatory responses through the regulation of pro-inflammatory cytokine production and inhibition of MAPK signaling pathways (ERK and p38), thereby reducing inflammatory mediator expression.78 Moreover, Zhang et al showed that piperine markedly mitigates hepatic ischemia/reperfusion injury in mice by suppressing the TLR4/IRAK1/NF-κB signaling cascade, resulting in reduced production of TNF-α and IL-6, decreased inflammatory cell infiltration, and improved tissue histopathology.51

Similarly, Hu et al demonstrated that piperine alleviates dextran sulfate sodium (DSS)-induced colitis by inhibiting NF-κB activation and reducing inflammatory mediator release, as well as facilitating regenerative intestinal barrier function by increasing the level of tight junction proteins such as claudin-1, ZO-1, and occludin. In addition, piperine modulated the composition of gut microbiota, highlighting its role in the gut-immune axis.52

Structural optimization studies further support the anti-inflammatory potential of piperine derivatives. For example, Liu et al reported that a piperine-derived compound exhibited strong anti-inflammatory and analgesic activity, including potent TRPV1 antagonism (IC₅₀ = 33.06 nM), along with urate-lowering effects and reduced inflammation-associated tissue damage in hyperuricemic models.53 Moreover, formulation strategies such as multicomponent systems have been shown to enhance piperine efficacy. Sartinah et al demonstrated that a piperine-hydroxybenzoate complex exhibited improved solubility and enhanced anti-inflammatory activity compared to piperine alone.54

The level of evidence differs across these studies. The findings of Gusson-Zanetoni et al support cellular modulation of inflammatory pathways, whereas the studies by Zhang et al and Hu et al provide in vivo evidence in mouse models of hepatic ischemia/reperfusion injury and DSS-induced colitis, respectively.51,52,78 However, these findings remain model- and indication-specific. Moreover, the results reported by Liu et al concern a structurally modified derivative, while Sartinah et al evaluated a piperine-hydroxybenzoate complex; their effects therefore cannot be attributed directly to unmodified piperine.53,54 Collectively, the evidence supports further investigation but does not yet establish efficacy in human chronic inflammatory diseases. The reported findings therefore provide mechanistic and preclinical support rather than confirmatory evidence for stand-alone clinical efficacy.

Immunomodulatory Activity

Piperine exhibits significant immunomodulatory activity wherein it regulates both innate and adaptive immunity in part by modulating cytokine expression, immune cell differentiation, and host-pathogen interactions.55 Recent studies have shown that piperine can influence macrophage function and improve the potency of antimicrobial agents in immune microenvironments. For instance, Palomo et al demonstrated that piperine enhances the intracellular activity of clarithromycin in macrophages, probably due to increased drug uptake and modulation of host immunological processes enhance antimicrobial efficacy against rapidly growing mycobacteria.56

Piperine also exerts immune-polarizing properties in models of parasitic infections. Sharifi et al reported that in Leishmania major infection, piperine modulates cytokine expression by upregulating Th1-related cytokines (eg IFN-γ and IL-12) while downregulating Th2-associated responses, indicating a shift toward a protective immune phenotype.57

Furthermore, piperine has been demonstrated to regulate immune signaling pathways associated with inflammation and tumor progression. Cheng et al showed that piperine suppresses the NLRP3/IL-1β axis and reduces Th17-related cytokines (IL-17A, IL-21, IL-22), thereby inhibiting immune-mediated tumor progression in cervical cancer models. This highlights its role in modulating pro-inflammatory T cell responses.58

Neuroprotective Activity

Piperine exhibits significant neuroprotective effects across various models of neurodegenerative and neurological disorders, primarily through modulation of oxidative stress, neuroinflammation, and neuronal survival pathways.59 Recent studies have highlighted its role in regulating protein aggregation and autophagy in neurodegenerative diseases. For instance, Yu et al demonstrated that piperine promotes PI3K/AKT/mTOR-mediated autophagy in Parkinson’s disease models, leading to enhanced degradation of α-synuclein (α-Syn), reduced dopaminergic neuronal loss, and improved motor function. Mechanistically, piperine increased the LC3II/LC3I ratio and reduced p62 expression, indicating activation of autophagic flux.60

In Alzheimer’s disease models, piperine has also shown protective effects against neurotoxicity. Mishra et al reported that piperine-encapsulated casein micelles significantly reduced Aβ(1-42)-induced oxidative stress, restored mitochondrial homeostasis, and improved neuronal viability compared to free piperine. These effects were associated with reduced intracellular ROS, improved mitochondrial membrane potential, and modulation of autophagy and mitophagy pathways.61

In neuroinflammatory and autoimmune conditions, piperine has demonstrated the ability to protect neuronal integrity and modulate immune-mediated damage. Nasrnezhad et al showed that piperine alleviates experimental autoimmune encephalomyelitis (EAE) by reducing demyelination, glial activation, and inflammatory cytokines (TNF-α, IL-1β), while enhancing antioxidant defenses (Nrf2, HO-1) and neurotrophic factors such as BDNF.62

Additionally, piperine has shown protective effects in acute brain injury models. Ferreira et al reported that piperine reduces oxidative stress, inflammatory mediators (IL-6, TNF), and neuronal degeneration in sepsis-associated encephalopathy, while improving cognitive function and increasing antioxidant enzyme activity.63 Similarly, Hsieh et al demonstrated that piperine protects against excitotoxic neuronal damage by modulating the NGF/TrkA/Akt/GSK3β signaling pathway, restoring neurotransmission balance and reducing hippocampal injury.64

Beyond the neuroprotective evidence for piperine itself, antiepilepsirine (ilepcimide) represents a clinically used antiseizure drug in China that was synthetically developed from the piperine scaffold.65,66 More recently, ilepcimide was investigated for neuroprotective repurposing in a mouse model of experimental autoimmune encephalomyelitis, where it reduced disease severity, demyelination, blood–brain barrier disruption, and CNS infiltration by pathogenic T cells.67 These effects were associated primarily with inhibition of dihydroorotate dehydrogenase and reduced inflammatory responses, while NRF2 activation was proposed to contribute to its protection against neuronal oxidative stress.67 Nevertheless, these findings remain preclinical, and ilepcimide is chemically distinct from piperine and its geometric isomers; therefore, its clinical use and neuroprotective activity should not be interpreted as evidence of established neurological efficacy or regulatory approval for piperine itself.

In conclusion, these findings indicate that piperine exerts neuroprotective effects through multiple mechanisms, including activation of autophagy, suppression of neuroinflammation, reduction of oxidative stress, modulation of neurotrophic signaling, and prevention of protein aggregation. These multifaceted actions highlight its potential as a therapeutic agent in neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, and neuroinflammatory disorders.

Antioxidant Activity

Piperine exhibits strong antioxidant activity across various systems. At the molecular level, piperine can interact directly with proteins to influence oxidative stability.68 Wu et al demonstrated that piperine binds non-covalently to myoglobin via hydrophobic interactions and static quenching, preventing free iron release and thereby limiting oxidative reactions. This interaction also induced structural modification of the protein, including a 5% reduction in α-helix content, contributing to enhanced antioxidant capacity in a concentration-dependent manner.69

In microbial systems, piperine has also been reported to regulate antioxidant defense pathways. Buitimea-Cantúa et al showed that piperine upregulated antioxidant-related genes (sod1, catA, cat2) and significantly increased catalase (CAT) activity by up to 65.50%, highlighting its role in activating enzymatic antioxidant responses.70 Furthermore, dietary supplementation studies highlight systemic antioxidant benefits of piperine. Albaqami reported that piperine significantly enhanced antioxidant enzyme activities and reduced lipid peroxidation (MDA levels) in Litopenaeus vannamei, alongside improvements in immune response and metabolic profiles.71

Additionally, piperine-containing extracts have demonstrated notable free radical scavenging activity. Al-Mamun et al reported that Piper chaba extracts exhibited strong DPPH scavenging activity with IC₅₀ values of 39.62 ± 0.95 μg/mL and 43.85 ± 1.50 μg/mL, indicating significant antioxidant potential.50 Moreover, Albuquerque et al demonstrated that piperine exhibits antioxidant activity in biomaterial systems, maintaining structural integrity and preventing oxidative degradation while simultaneously showing antimicrobial properties.33

Antiviral Activity

Piperine has emerged as a promising antiviral agent with activity against various viral pathogens through interference with viral entry, replication, and host-virus interactions. In the context of arboviral infections, Rocha et al demonstrated that piperine exhibits significant antiviral activity against Chikungunya virus (CHIKV) in vitro, with time-dependent inhibition observed particularly during post-entry stages of infection. Molecular docking and dynamics simulations revealed that piperine binds stably to the E1-E2 glycoprotein fusion pocket, interacting with key residues such as MET88, LEU16, and TYR15, thereby disrupting viral fusion and entry processes.72

In addition to direct antiviral mechanisms, formulation strategies have been shown to enhance piperine’s efficacy. Zakaria et al developed piperine-loaded bilosomes with an entrapment efficiency of 97.2 ± 0.8%, a particle size of 220.2 ± 20.5 nm, and 88.2 ± 5.6% drug release after 8 h.73 Compared with a piperine suspension, the optimized formulation showed greater ex vivo permeation and increased oral bioavailability in a pharmacokinetic study. It also produced greater antiviral activity and reduced oxidative markers and inflammatory cytokines in MERS-CoV-infected mice.73

Piperine also demonstrates antiviral activity against aquatic viral pathogens. Wang et al showed that piperine effectively inhibited largemouth bass virus (LMBV) replication in vitro, achieving >95% inhibition of viral gene expression and an IC₅₀ of 34.61 μM. In vivo, piperine administration (25 mg/kg) improved survival rates of infected fish by approximately 20% and significantly reduced viral loads across multiple organs. Mechanistically, piperine was found to interfere with viral adsorption and exert inhibitory effects during later stages of viral replication.74

Hepatoprotective Activity

Piperine exhibits significant hepatoprotective activity against various models of liver injury through modulation of oxidative stress, inflammation, and fibrogenic signaling pathways. In liver fibrosis models, Abdelhamid et al demonstrated that piperine suppresses hepatic stellate cell (HSC) activation by inhibiting the TGF-β/Smads signaling pathway. This effect was associated with downregulation of miR-17 expression and restoration of Smad-7 levels, leading to reduced collagen deposition and attenuation of liver fibrosis progression.75

In drug-induced hepatotoxicity, Coelho et al reported that piperine significantly alleviates paracetamol (APAP)-induced liver injury. Piperine treatment reduced necrotic areas, decreased liver enzymes (ALT, AST), and suppressed inflammatory mediators such as IL-1β, IL-6, and TNF-α. Additionally, piperine modulated inflammasome-related pathways by reducing NLRP3 and caspase-1 expression, while enhancing antioxidant status, indicating its protective role against oxidative and inflammatory liver damage.76

Advanced formulations further enhance the hepatoprotective potential of piperine. Hussain et al demonstrated that nano-piperine effectively mitigates cypermethrin-induced hepatotoxicity by reducing oxidative stress markers (LPO), restoring antioxidant enzymes (GSH, SOD, catalase), and suppressing pro-inflammatory cytokines and caspase expression. Histopathological analysis confirmed significant improvement in liver tissue architecture following treatment.77

Moreover, piperine has been shown to act as a bioenhancer in combination therapies. Biswas et al reported that co-administration of piperine with ursolic acid significantly improved hepatoprotective efficacy in CCl₄-induced liver injury, reducing hepatic enzyme levels and enhancing pharmacokinetic properties. Notably, piperine increased the oral bioavailability of ursolic acid by approximately tenfold, highlighting its role in improving therapeutic outcomes.80

Analgesic Activity

Piperine has demonstrated analgesic potential through modulation of inflammatory mediators and enhancement of drug efficacy, particularly via its bioavailability-enhancing properties. In a clinical setting, Maulina et al evaluated the analgesic effect of a piperine-combined curcumin patch in pediatric patients following orofacial surgery. The study showed a significant reduction in pain intensity (FLACC scores, p <0.01) across treatment groups, alongside modulation of salivary prostaglandin E₂ (PGE₂), a key mediator of inflammation and pain. Although pain scores decreased following the curcumin-piperine patch intervention, the addition of piperine was not statistically superior to curcumin alone. Consequently, this study does not establish an independent analgesic contribution of piperine and should be interpreted as inconclusive evidence for piperine-specific clinical efficacy.81

At the formulation level, Fitriani et al developed a celecoxib-piperine cocrystal system, demonstrating that piperine can enhance the physicochemical performance of analgesic drugs. The cocrystal exhibited improved dissolution rate and compressibility compared to pure celecoxib, suggesting improved drug release and potential enhancement of analgesic efficacy. Given that celecoxib is a selective COX-2 inhibitor, this formulation strategy indicates that piperine may indirectly potentiate analgesic effects through improved drug delivery and bioavailability.82

Antidepressant Activity

Piperine has also demonstrated promising antidepressant-like effects, particularly through modulation of neuroinflammatory, oxidative stress, and neurotrophic pathways. Basavaraju et al developed a nanoemulsion-based piperine formulation (nanopiperine) to enhance its bioavailability and evaluated its efficacy in a lipopolysaccharide (LPS)-induced depression model in mice. Treatment with nanopiperine (5 and 10 mg/kg for 14 days) significantly reversed depression-like behaviors, as evidenced by reduced immobility time in the tail suspension test (TST) and forced swim test (FST).83

Mechanistically, nanopiperine increased hippocampal brain-derived neurotrophic factor (BDNF) levels and promoted progenitor cell proliferation, indicating its role in neurogenesis and synaptic plasticity. In addition, neurotransmitter analysis revealed elevated levels of key monoamines, including serotonin and dopamine, alongside a reduction in oxidative stress and inflammatory markers. Notably, the nanoformulation exhibited improved oral bioavailability and stability compared to free piperine, contributing to its enhanced therapeutic efficacy.83

These findings suggest that piperine exerts antidepressant effects via a multi-target mechanism involving neuroprotection, neurotransmitter regulation, and anti-inflammatory activity, with nanodelivery systems further amplifying its pharmacological potential.

Extraction, Purification, and Green Processing

Following the identification of piperine as a bioactive scaffold and the evaluation of its derivatives, reproducible production of chemically characterized piperine represents an important upstream stage of development. Extraction and purification supply the material used in subsequent formulation, pharmacokinetic, and biological studies; however, extraction recovery alone does not demonstrate suitability for dosage-form development because recovery, piperine content or purity, and batch reproducibility are distinct considerations. This section provides an updated overview (2021–2026) of conventional extraction methods, advanced technologies, green-based approaches, and optimization strategies for scaling-up and industrial translation.2,4,11,33,84–95 Table 4 summarizes the reported extraction methods for piperine isolation from varieties of Piper species, highlighting the extraction media and corresponding piperine yields.

Table 4.

Extraction Methods Employed for Piperine Isolation from Piper Species

Methods	Piperine Source	Solvent/System	Piperine Yield/Content (%)	Ref.
Maceration	White pepper	Ethyl acetate	0.094	[84]
Percolation (solvent-circulation)	Vietnamese white pepper	85% Ethanol	5.199 dry material	[85]
Reflux Extraction	Black pepper	Ethanol	92.86	[33]
Soxhlet Extraction	Vietnamese black pepper	Methanol	4.551	[86]
	Kampot black pepper	Methanol	4.381	[86]
	Dapo black pepper	Methanol	4.356	[86]
	White pepper	Ethyl acetate	0.96	[84]
	Black pepper	Ethanol	10.2 ± 0.5	[87]
	Black pepper	Ethanol (96%)	32.77 (oleoresin)	[88]
	Black pepper	Ethanol-acetone mixture	25.66 (oleoresin)	[88]
	Black pepper	Acetone (98%)	23.56 (oleoresin)	[88]
Ultrasound-assisted Extraction (UAE)	Vietnamese black pepper	Methanol	4.204	[86]
	Kampot black pepper	Methanol	4.027	[86]
	Dapo black pepper	Methanol	3.843	[86]
	Green pepper	Ethanol	19.25	[11]
	Black pepper (BPMH sample)	Methanol	31.85	[89]
	Black pepper	ChCl:Citric acid:Propylene glycol (1:2:2)	3.876	[2]
	White pepper	Lidocaine:Valeric acid (1:1)	3.375	[90]
Microwave-asssisted Extraction (MAE)	Black Trang peppercorns	Water	3.57	[91]
	Red Trang peppercorns	Water	2.64	[91]
	White Trang peppercorns	Water	2.82	[91]
	Green pepper	Ethanol	19.81	[11]
Ultrasound-Microwave-assisted Extraction (UMAE)	Green pepper	Ethanol	28.32	[11]
Supercritical Fluid Extraction (SFE)	Black pepper	Supercritical CO2	2.13	[92]
	Black pepper	Supercritical CO2	25.31	[93]
Multistage Extraction	Black pepper	Absolute ethanol (reflux)	79	[94]
Ultrasonic bath extraction (UBE)	Black pepper	ChCl:Glycerol:Urea (1:1:1)	2.567	[4]
Ultrasonic probe extraction (UPE)	Black pepper	ChCl:Glycerol:Urea (1:1:1)	4.997	[4]
Steam Explosion	White pepper	High-pressure steam	8.80	[95]
Conventional soaking	White pepper	Water soaking	6.47	[95]

The reported values should be interpreted within their respective experimental settings rather than used to rank the extraction methods directly. Piperine recovery is influenced by differences in Piper species and origin, plant-part preparation, solvent composition, solid-to-liquid ratio, extraction time and temperature, reporting basis, and analytical quantification. Conventional Soxhlet, reflux, and maceration methods provide useful reference points, whereas ultrasound-, microwave-, deep-eutectic-solvent-, and supercritical-fluid-based approaches have been reported to reduce extraction time, solvent use, or other process requirements under specific experimental conditions. Nevertheless, no method can be regarded as universally superior or inherently greener without matched information on piperine purity, energy consumption, solvent preparation and recovery, waste generation, batch reproducibility, and scale-up performance. The available studies therefore identify promising laboratory-scale process options but provide limited evidence connecting optimized extraction directly with reproducible downstream formulation and pharmaceutical development.

Conventional Extraction Methods

Maceration and Percolation

Conventional extraction techniques such as maceration and percolation (Figure 6a) remain widely used for isolating piperine due to their simplicity, low operational cost, and minimal equipment requirements.4,96 Among these, maceration is one of the most traditional approaches, involving prolonged soaking of plant material in organic solvents to facilitate the diffusion of target compounds.97

Figure 6.

Illustration of (a) conventional extraction methods and (b) advanced extraction methods.

Zhang et al evaluated the effect of different extraction techniques on pepper oleoresin, including maceration, ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and ultrasound-microwave-assisted extraction (UMAE). The results showed that, although maceration yielded lower extraction efficiency compared to advanced techniques, it produced extracts with the highest total phenolic content (4.99 ± 0.12 mg GAE/g DW) and superior antioxidant activity.11 This indicates that the mild extraction conditions of maceration are advantageous for preserving thermolabile bioactive compounds.

Moreover, a comparative study by Harimurti et al investigated the extraction of piperine from white pepper (Piper nigrum) using ethyl acetate through both maceration and Soxhlet extraction. In the maceration process, white pepper powder was extracted with ethyl acetate (1:3 w/v) for five days through two extraction stages. The resulting piperine crystals yielded only 0.094%, indicating relatively low extraction efficiency. The result attributed to the absence of heating and the reliance on passive diffusion at room temperature, which limited the release of piperine from the plant matrix. Nevertheless, the isolated piperine exhibited physicochemical characteristics consistent with reference standards, as confirmed by Thin layer chromatography (TLC), Fourier Transform Infrared (FTIR), and UV-Vis analyses.84

However, maceration suffers from several limitations, including long extraction time, high solvent consumption, and relatively low mass transfer efficiency.4,97 In the same study, UAE and MAE significantly reduced extraction time while improving yield, whereas UMAE demonstrated the highest extraction efficiency for both oleoresin and piperine content. These findings highlight that, although maceration remains relevant as a baseline conventional method, it is increasingly being replaced by more efficient and sustainable extraction technologies.11

Percolation is a conventional extraction technique in which solvent continuously passes through a packed bed of plant material, allowing repeated solvent-matrix contact and facilitating mass transfer of target compounds.98 Compared with maceration, percolation generally offers higher extraction efficiency and shorter extraction times because fresh solvent is continuously supplied throughout the extraction process.97,98 Historically, piperine extraction from Piper nigrum has been performed using ethanol, methanol, or acetone as extraction solvents. However, conventional percolation still requires relatively large solvent volumes and prolonged extraction times, limiting its sustainability and industrial efficiency.3,98

The newest advancement in piperine extraction have introduced solvent-circulation percolation systems that used to enhance mass transfer and solvent utilization. Le et al employed an industrial-scale solvent circulation percolator for the extraction of piperine from Vietnamese white pepper.85 In this system, ethanol was pumped through the extraction chamber and recirculated in a continuous manner, passing through the packed pepper bed multiple times while being recirculated through the extraction chamber, allowing repeated contact between the solvent and plant matrix. The extractor utilizes temperature control and solvent recovery units to essentially maximize the efficiency of extraction, as well as minimizing any losses that may occur with the solvents. Under operating conditions of 85% ethanol, a solvent-to-solid ratio of 3.4:1 mL/g, and 78 minutes extraction time, the process produced approximately 5.199% dry material.85 Because the reported endpoint represents recovered dry material rather than the purity of isolated piperine, this result supports process-level extraction efficiency but does not by itself establish the production of pharmaceutical-grade piperine. Nevertheless, these findings demonstrate that solvent-circulation percolation can provide efficient piperine recovery while maintaining operational simplicity and industrial applicability.

Although advanced extraction technologies such as UAE, MAE, UMAE, and supercritical fluid extraction (SFE) have gained increasing attention due to their shorter extraction times and higher extraction efficiencies, solvent-circulation percolation is still an attractive method for industrial scale applications since it has a less complex equipment design, easy scalability and compatible with available extraction infrastructure.

Reflux Extraction

Reflux extraction (Figure 6a) is a traditional hot extraction method, which the solvent continues to boil, condensed, and returned to the vessel in order to ensure multiple contact of new solvent with plant matrix with very little loss of solvent.99 In contrast with maceration and percolation, the reflux extraction often has a more efficient extraction in a shorter time period since this process has an enhanced mass transfer through higher temperature and continuous solvent circulation.3,99,100 However, prolonged exposure to heat may limit its application for thermolabile compounds.100

Recent studies reveal that using reflux extraction continue to be an effective method for the isolation of piperine from Piper nigrum. Sulman used reflux extraction with diethyl ether as the extraction solvent, followed by repeated recrystallization to obtain purified piperine crystals. The extraction was conducted for 2 h under reflux conditions, after which the extract was concentrated and recrystallized 7–8 times until pale-yellow crystals were obtained. Purity evaluation using TLC, UV-Vis spectroscopy, FTIR, and gas chromatography-mass spectrometry (GC-MS) confirmed the identity of piperine, with GC-MS analysis showing approximately 85% purity. The study revealed that reflux-assisted extraction improved the degradation of plant cell walls and enhanced the recovery of piperine suitable for use as a reference standard in analytical and research applications.101

In a more recent work by Albuquerque et al also utilized a reflux system to extract piperine from black pepper and reported a high extraction yield of 92.86% and purity of 99.9% from 78 mg of piperine. The isolated piperine has activity as an antibacterial and antifungal agent, while also retaining antioxidant activity. These results indicate that reflux extraction can still be employed for piperine-rich extract production with the desired purity and bioactivity.33

Despite reflux extraction yields a higher extraction efficiencies than static methods like maceration and conventional percolation, it still requires increased temperatures and associated high energy consumption. Consequently, recent research trends increasingly focus on advanced extraction technologies, including UAE, MAE, UMAE, and SFE, which offer shorter processing times, reduced solvent consumption, and improved sustainability.

Soxhlet Extraction

Soxhlet extraction (Figure 6a) is the traditional solid-liquid extraction method, which was derived from classical decoction methods and works based on a continuous recycling of the solvent in order to enhance extraction efficiency. The method includes reflux extraction and percolation features, where fresh solvent repeatedly passes through the plant matrix via a siphoning mechanism.102 Compared to maceration and percolation, soxhlet extraction usually has greater extraction efficiency with less solvent usage since the same solvent is continuously recirculated throughout the process. Nonetheless, extended extraction at high temperatures could put thermally-sensitive compounds at risk of thermal degradation, and is also much slower compared with modern extraction technologies.100

Despite these limitations, soxhlet extraction is still widely used as a standard method for obtaining piperine from Piper nigrum owing to its robustness and high efficiency in extracting compounds. In a new study by Zhang et al, soxhlet extraction, UAE, and steam distillation (SD) were compared for several black pepper varieties. The outcomes revealed that soxhlet extraction is the most effective method among others regarding piperine content. In Vietnamese pepper samples, soxhlet extraction yielded 4.551%, while Kampot and Dapo pepper yielded 4.381% and 4.356%, respectively. Conversely, UAE yielded lower piperine contents ranging from 3.843–4.204%. These results show that, despite the emergence of newer extraction technologies, soxhlet extractions are still very effective at maximizing piperine recovery from black pepper matrices.86

Harimurti et al, who extracted white pepper using ethyl acetate through both maceration and soxhlet extraction, further support the effectiveness of soxhlet extraction. Soxhlet extraction produced 0.96% piperine crystals, which was approximately ten times higher than that obtained by maceration, which was only 0.094%. It has been proved that continuous solvent circulation and heating improve solvation and mass transfer from the plant matrix, making it more likely for these conditions to occur. These results show that soxhlet extraction is still a more effective conventional method for piperine isolation than static maceration.84

Reinforcing these findings, more recent work by Chang et al emphasized the efficiency of soxhlet extraction for piperine recovery from black pepper. Under optimized conditions consisting of an ethanol-to-solid ratio of 10 mL g⁻¹, extraction temperature of 85 °C, and extraction time of 215 min, soxhlet extraction produced a crude extract yield of 34.6 ± 3.6 wt% containing 10.2 ± 0.5 wt% piperine. The crude extract was then processed via antisolvent crystallization, which successfully yielded piperine with a final purity of 98.9 ± 0.3 wt%. Overall, these results demonstrate that soxhlet extraction is still a valid upstream extraction technique and managed to produce enough piperine yield for further purification operations.87

The excellent recovery of piperine by soxhlet extraction is explained on the fact that, during this process, the plant matrix is in contact with freshly condensed hot solvent, which favors solute diffusion and mass transfer.100 However, soxhlet extraction is becoming increasingly supplemented or overturned due to its high energy demand and long extraction duration by advanced techniques, such as UAE, MAE, UMAE, and SFE, which provide reduced extraction times and better process sustainability.11 Thus, soxhlet extraction is still used as pivotal benchmark method of determining the efficacy of new technologies for piperine extraction.

As a result, in the last years several studies have increasingly focused on developing intensified extraction technologies capable of maintaining or improving piperine yield and simultaneously reduce extraction time, solvent consumption, and energy requirements, leading to the emergence of ultrasound-assisted, microwave-assisted, and other green extraction approaches.

Advanced Extraction Technologies

Ultrasound-Assisted Extraction (UAE)

Ultrasound-assisted extraction (UAE) (Figure 6b) is one of the most investigated non-conventional techniques for recovering piperine from Piper nigrum. The method works based on acoustic cavitation produced by ultrasonic waves that generates microbubbles that collapse close to the plant tissues, giving rise of the cell wall disruption, thereby improves solvent diffusion and mass transfer.4,103 Compared with conventional extraction methods such as maceration, percolation, and soxhlet extraction, UAE generally offers shorter extraction times, less solvent consumption, and improved extraction efficiency while operating under relatively mild conditions.4,100,104

The utility of UAE has been established previously for the extraction of piperine from black pepper. Zhang et al compared UAE, soxhlet extraction, and steam distillation for different P. nigrum varieties using methanol as the extraction solvent. UAE was performed at a solid-to-liquid ratio of 1:10 (w/v), 40 kHz frequency, and 840 W power for 3 h. Among the tested solvents, methanol produced the highest extraction yield under UAE, reaching 8.24%, exceeding the yields obtained using ethyl acetate (6.68%), petroleum ether, and n-hexane. Analysis by ultra-performance liquid chromatography (UPLC) revealed variations in piperine content by pepper variety, with Vietnamese pepper exhibiting the highest concentration (4.204%), followed by Kampot pepper (4.027%) and Dapo pepper (3.843%). Although Soxhlet extraction yielded slightly higher piperine levels (Vietnamese pepper: 4.551%, Kampot pepper: 4.381%, Dapo pepper: 4.356%), UAE better preserved volatile organic compounds and aroma complexity, which is an especially attractive option in case both bioactive components and flavour potential or characteristics play an important role.86

Additional evidence supporting the effectiveness of UAE was reported by Zhang et al, who compared maceration, UAE, MAE, and UMAE for the extraction of green pepper oleoresin. UAE was performed using ethanol as the extraction solvent and achieved an extraction yield of 11.40%, which was higher than MAE (10.22%) and slightly higher than conventional maceration. Furthermore, UAE required only 90 min to reach extraction equilibrium, considerably shorter than the 600 min required for maceration. The piperine content in the oleoresin increased with extraction time and reached a maximum value of 19.25%. The enhanced extraction performance was attributed to ultrasonic cavitation, which promotes solvent penetration and facilitates the release of intracellular compounds from plant tissues. Because UAE operates at relatively low temperatures, it can also reduce the risk of thermal degradation of heat-sensitive compounds compared with microwave-based extraction methods.11

The superiority of UAE over traditional extraction methods has also been demonstrated in quantitative studies. Alqarni et al compared traditional extraction and ultrasound-assisted extraction of piperine from several commercial black pepper samples. Quantification using high-performance thin-layer chromatography (HPTLC) showed consistently higher piperine concentrations in UAE extracts. For example, the piperine content of the BPMH sample increased from 30.95% in the traditional extract to 31.85% after UAE. Similar improvements were observed for other pepper samples, leading the authors to conclude that UAE is a superior extraction procedure for piperine recovery while maintaining excellent green analytical performance.89

More recently, UAE has been integrated with environmentally friendly solvent systems to further improve extraction sustainability. You et al developed temperature-responsive deep eutectic solvents (TRDESs) for UAE of alkaloids from white pepper. Under optimized conditions consisting of a lidocaine/valeric acid (1:1) TRDES, solid-to-liquid ratio of 1:40 mg/mL, extraction temperature of 75°C, and ultrasonic treatment for 30 min, the piperine yield reached 3.375%. The TRDES-UAE system showed great selectivity toward alkaloids, allowing the identification of 274 alkaloid components in comparison to conventional ethanol extract which only yielded 159 compounds. Furthermore, more than 85% extraction efficiency was retained even after five reuses of the solvent system while a higher Green Analytical Procedure Index (GAPI) value compared with ethanol-based extraction suggested its great potential for environmentally friendly industrial piperine production.90

Overall, UAE is a very efficient extraction technique for piperine, providing large savings on time and solvent while achieving relatively high yields. Recently, research focuses on a shift from conventional UAE toward hybrid systems based on green solvents and process optimization strategies, which may increase the extraction efficiency, selectivity, and environmental sustainability for large-scale piperine production.

Microwave-Assisted Extraction (MAE)

Microwave-assisted extraction (MAE) (Figure 6b) has gained significant interest as an effective substitute to conventional extraction methods for recovering piperine. MAE can enhance cell disruption, mass transfer and release of intracellular compounds through rapid extraction mediated by microwave irradiation.4,103 Compared with conventional techniques such as maceration and percolation, MAE is a green extraction method markedly shortens extraction time while consumption of solvent decreased significantly without sacrificing the efficiency of extraction. Owing to the rapid heating volumetric, mass transfer is also accelerated providing effective recovery of piperine and other bioactives from pepper.4,97,100,103

The potential of MAE for pepper extraction was further evidenced by recent studies showing rapid and selective recovery of piperine from various sources. Pothipongsa et al studied the extraction of black, red, and white Trang peppercorns using water as a green solvent under various microwave conditions. The optimal extraction condition was found to be 500 W for 30s, which produced higher extraction yields, total phenolic content, total flavonoid content, and piperine content than extraction at 300 W for 50s. Under these conditions, extraction yields reached 11.58%, 17.42%, and 7.54% for black, red, and white pepper, respectively. The corresponding piperine contents were 3.57%, 2.64%, and 2.82%. In addition to enhanced phytochemical recovery, the extracts exhibited α-glucosidase inhibitory activity and immunostimulatory effects, indicating that MAE can efficiently produce biologically active pepper extracts using water as an environmentally friendly extraction solvent.91

The extraction performance of MAE has also been compared with other extraction technologies. Zhang et al evaluated maceration, UAE, MAE, and UMAE for the extraction of green pepper oleoresin. MAE was conducted using ethanol as the solvent at 300 W and 60°C. The extraction yield obtained by MAE reached 10.22%, which was comparable to maceration but required only 18 min compared with 600 min for conventional maceration. The highest piperine content obtained by MAE was 19.81%, demonstrating that microwave irradiation effectively promotes the release of piperine from pepper tissues. The improved extraction efficiency was attributed to the rapid heating effect of microwaves, which enhances solvent penetration and accelerates the rupture of cellular structures.11

However, MAE has also some shortcomings. Piperine is highly susceptible to heat, light and oxygen, so microwaving for long periods may cause thermal degradation or change composition of volatile compounds. Zhang et al observed that while extraction yield generally increased with extraction temperature, piperine content decreased as the temperature increased which indicates a need for process optimization to improve extraction efficiency while maintaining compound stability. Thus, recent trends focus more on the synergistic role of MAE and innovative technologies, such as UAE, to improve its extraction performance while minimizing thermal damage.11

In general, MAE could serve as a fast and green alternative to traditional extraction methods for piperine recovery. MAE promises both (1) efficient extraction yields within seconds to minutes, and (2) reduced solvent use, making this technology a good candidate for laboratory- and industrial-scale applications compatible with green solvents.

Ultrasound-Microwave-Assisted Extraction (UMAE)

Ultrasound-microwave assisted extraction (UMAE) which combines the cavitation effect from ultrasound with rapid volumetric heating, is an emerging hybrid extraction method as shown in Figure 6b.11,105 The combined application of both mechanisms maximizes cell wall disruption, increases solvent penetration, and boosts mass transfer, which contributes to more efficient recovery of bioactive compounds than conventional extraction methods.11,105

In a comparative study conducted by Zhang et al, the extraction performance of maceration, UAE, MAE, and UMAE was evaluated for green pepper oleoresin. UMAE exhibited the highest extraction efficiency amongst all the tested techniques, with an oleoresin yield of 12.14%, and extracting at extraction equilibrium in only 18 minutes. This yield surpassed that of UAE (11.40%) and MAE (10.22%), revealing the synergistic influence of ultrasound and microwave energy addition on extraction efficiency.11

The superiority of UMAE was also evident in piperine recovery. For pure piperine, the highest content (28.32%) was obtained from UMAE and thus exceeded those of maceration (25.66%), UAE (19.25%) and MAE (19.81%). The improved extraction performance was mainly ascribed to the synergistic effect of ultrasonic cavitation and microwave heating on rapid breaking of plant tissues, accelerated release of intracellular materials. Microwave heating enhanced permeability of cell membranes and facilitated diffusion of piperine into the solvent, while ultrasound promoted penetration of solvents and mass transfer.11

Furthermore, the best conditions for UMAE were reported as a particle size of 80 mesh and solid-to-liquid ratio (1:10) (g/mL). Under these conditions, UMAE showed the best combination of extraction yield and piperine recovery. Thus, UMAE could easily be classified as a rapid, green and economical extraction technique for piperine-containing oleoresin from pepper. However, regulatory of its extraction temperature is still significant owing to instability of piperine under heat, light and oxygen for a protracted time.11

Supercritical Fluid Extraction (SFE)

Supercritical fluid extraction (SFE) (Figure 6b) is a green extraction technique, employing carbon dioxide (CO2) above the critical temperature and pressure to extract bioactive compounds from plant materials.106,107 Supercritical CO2 has gas-like diffusivity and liquid-like solvating power, allowing it to penetrate plant matrices efficiently while leaving no toxic solvent residues in the final extract. SFE has advantages over conventional solvent extraction like lower solvent utilization, high selectivity, mild operating conditions and easier removal of the solvent.92,107

Luca et al evaluated supercritical CO2 extraction (SFE) of piperamides from Piper nigrum at pressures of 100–300 bar and temperatures of 40–60 °C. Piperine was identified as the major piperamide (1.71%) in black pepper. Piperine recovery increased markedly with pressure, reaching its highest extraction efficiency at 300 bar due to the enhanced solvating power of supercritical CO2. Conversely, increasing the temperature from 40 to 60 °C generally reduced piperine recovery because of the decrease in CO2 density and solvent strength. A two-step pressure-gradient process (100 bar followed by 300 bar at 60 °C) successfully produced a piperamide-rich fraction with higher piperine content (2.13%) than conventional solvent extraction (1.71%), demonstrating the high selectivity of SFE for piperine enrichment.92

Additional evidence supporting the effectiveness of SFE for black pepper extraction was reported by Sutil et al where black pepper extract produced by supercritical CO2 extraction at 20 MPa (200 bar) and 40 °C for 240 min contained approximately 25.31% piperine, confirming that piperine is one of the major compounds efficiently recovered using supercritical fluid technology. The study further added that SFE is able to give bioactive-rich extracts without the use of large volumes of organic solvents.93

Overall, SFE yields an eco-friendly and very selective methods for isolation of piperine from Piper nigrum. The extraction efficiency can be optimized by adjusting pressure and temperature, with higher pressures generally favoring piperine recovery. In addition, the ability of SFE to give piperine-fractionated extracts with negligible solvent residues has made this technique a good choice as a substitute for conventional extraction methods in pharmaceutical, nutraceutical and food applications.107

Molecularly Imprinted Polymer (MIP)

Molecularly imprinted polymer (MIP) (Figure 6b) technology has been widely explored for the selective recognition and extraction of piperine. MIPs are polymeric materials with recognition sites produced during polymerization through the presence of a target analyte.108,109 Following template removal, complementary cavities remain within the polymer matrix, preserving the spatial arrangement and functional characteristics of the template and thereby facilitating highly selective molecular recognition.110,111 An early study by Roland and Bhawani in 2016 synthesized piperine-imprinted polymer microspheres using precipitation polymerization with acrylic acid as the functional monomer and Ethylene glycol dimethacrylate (EGDMA) as the cross-linker. The optimized MIP exhibited a binding capacity of 84.94%, significantly higher than the corresponding non-imprinted polymer, and successfully extracted 81.18% of piperine from spiked urine samples. These results confirmed the initial hypothesis that MIPs exhibit a very high specificity toward piperine, therefore being potentially excellent sorbents for extracting this compound from food matrices or as clean-up step during sample preparation.112

While early MIP studies established a foundation for selectively extracting piperine, recent works propelled the application into the analytical sensing applications. Banerjee et al developed a molecularly imprinted graphite electrode (PDMAM-MIP@G) for selective electrochemical detection of piperine, employing piperine as the template, PDMAM as the functional monomer, and EGDMA as the cross-linker. The cavities were able to imprint and even sense piperine recognition with high sensitivity and selectivity using cyclic voltammetry.113 Subsequently, Banerjee et al integrated this MIP sensor with convolutional neural network (CNN)-assisted regression models for piperine quantification in black pepper, achieving remarkable agreement with reverse phase-high performance liquid chromatography (RP-HPLC) results (R² = 0.9999; MAPE = 0.034%). The success in gradually transitioning from extraction-based MIPs to electrochemical sensors shows how the same molecular recognition principle can also be used for analyte isolation and rapid quantification of piperine.114

Multistage Extraction

Li et al developed a multistage extraction process for black pepper (Piper nigrum) to sequentially recover piperine and related compounds while maximizing biomass utilization. Following reflux extraction with absolute ethanol at 80 °C for 2 h, the extract underwent concentration, acid-base treatment, and recrystallization to obtain yellow piperine crystals. The method achieved a 79% recovery with a purity exceeding 99%, as verified by nuclear magnetic resonance (NMR), UV-Vis, fluorescence spectroscopy, and HPLC, demonstrating the potential of multistage processing to produce highly purified piperine.94

In contrast to conventional extraction methods for piperine solely, the multistage method approach also used piperine as a precursor to synthesize downstream bioactive compounds. In the second step, a 72% yield of piperic acid was achieved through hydrolysis of isolated piperine in ethanolic KOH under reflux. Piperic acid was further esterified under methanol with a solid acid to produce methyl piperate in 61% yield. The stepwise extraction-conversion strategy allowed for multiple high-value compounds to be extracted from the same biomass source.94

This study also proved that the multistage extraction is a sustainable and energy efficient alternative to traditional single-step extraction, which allows improving а resource-saving innovativeness of processing technology with the product diversification. Using sequential extraction and transformation processes, the black pepper biomass gave rise to piperine, piperic acid, and methyl piperate that showed remarkable antibacterial and anticancer properties. This makes multistage extraction a promising method for the simultaneous utilization of black pepper and piperine-derived functional ingredients.

Green-Based Approaches

Ethanol, Acetone, and Green Solvent Systems

The role of solvent selection in piperine extraction from black pepper was identified as an important factor based on recent studies. Malinda Prabhath Madhusankha et al analyzed the extraction effectiveness of black pepper oleoresin by soxhlet extraction using ethanol (96%), acetone (98%), and an ethanol-acetone mixture.88 Among the tested solvents, ethanol gave the highest piperine yield (32.77% oleoresin), followed by the solvent mixture (25.66%) and acetone (23.56%). Although piperine is generally considered soluble in acetone, the authors observed that extraction at elevated temperatures favored ethanol because its higher boiling point (78 °C) enabled more efficient recovery of piperine than acetone, which boils at 56 °C. Additionally, the study showed that it is not the solvent polarity alone that governs extraction efficiency but, rather, a sum of influences including polarity, solvent-solute interactions and extraction temperature determines piperine recovery rate.88 In addition, at the same time that an ethanol-acetone mixture gave the greatest overall oleoresin yield, ethanol was also shown to have more successful selective extraction potency against piperine from black pepper. The results prove that food-grade ethanol can be efficiently used as a green solvent for the extraction of piperine, especially in the approaches when high recoveries of pungent bioactive compounds are needed.88

Deep Eutectic Solvents (DES)

Interest in deep eutectic solvents (DESs) and natural deep eutectic solvents (NADESs) as greener alternatives to traditional organic solvents for piperine extraction from black pepper is rising because of the increasing demand for sustainable extraction processes. NADES are usually created by combination of hydrogen bond acceptor (HBA) with one or more hydrogen bond donors (HBDs), yielding solvent systems which exhibit tunable polarity, high potential to make H-bonds and low volatility with lower environmental impact.4,115,116 These features enhance the solubilization of plant-based bioactive agents, while at the same time decreasing the use of hazardous organic solvents.115,117

In this study, Lwamba et al focused on the extraction of piperine from Piper nigrum using UAE coupled with various NADES formulations.2 Sixteen NADES systems composed of choline chloride or L-proline as HBAs and different HBDs, including citric acid, malic acid, urea, glycerol, glucose, and 1,2-propylene glycol, were evaluated. High viscosity of NADES may also be a limitation to mass transfer, therefore 25% of water was incorporated in the solvent systems with the intention of reducing viscosity and decreasing diffusion barriers for piperine extraction from the plant matrix. Out of all tested formulations, choline chloride-citric acid-1,2-propylene glycol (1:2:2) provided the most efficient extraction with an overall yield of 3.876% piperine. The improved extraction of piperine was related to the synergistic hydrogen-bonding interactions between NADES components and piperine, as well as the improved cell disruption provided by ultrasound-assisted extraction. Compared with conventional solvents such as ethanol and aqueous methanol, the optimized NADES system demonstrated superior extraction capability while maintaining the advantages of a biodegradable and environmentally friendly solvent.2

More recently, Al Adhreai et al expanded the application of NADES-based extraction by comparing ultrasonic bath extraction (UBE) with ultrasonic probe extraction (UPE) for piperine isolation.4 Among the six NADES formulations evaluated, the ternary mixture of choline chloride, glycerol, and urea (1:1:1) provided the highest extraction efficiency. Consistent with previous studies, the addition of water reduced solvent viscosity, facilitating mass transfer between the solvent and the plant matrix. UPE outperformed UBE under the same solvent conditions, yielding 4.997% piperine compared with 2.567%. The superior performance of UPE was attributed to its stronger cavitation effect, which enhanced cell wall disruption and promoted more efficient release of piperine. The results underscore the role of solvent composition and extraction technique for determining extraction efficiency and further support the use of NADES as green solvents.4

You et al developed temperature-responsive deep eutectic solvents (TRDESs) as an integrated platform for extracting and separating piperine from white pepper.90 Unlike conventional DESs, TRDESs exhibit temperature-dependent phase behavior, allowing piperine extraction and recovery to be achieved in the same solvent system with minimal use of organic solvents. The authors prepared eighteen TRDES formulations using pharmaceutical amines, including lidocaine, procaine, and tetracaine, as hydrogen bond acceptors combined with fatty acids as hydrogen bond donors. Among them, the lidocaine-valeric acid (1:1) system produced the highest piperine yield (3.375%) under UAE. In addition to improving extraction efficiency, the piperine separation was achieved by regulating temperature and water content using these temperature-responsive solvent, which can be easily recycled for it simplified downstream purification. This work confirms the feasibility of using TRDES not only as extraction solvents, but also as integrated extraction-separation systems for more sustainable processing of natural products.90

Taken together, recent studies show that DES- and NADES-based solvent systems are environmentally friendly alternatives to conventional organic solvents for piperine extraction. They exhibit tunable physicochemical properties, strong hydrogen-bonding interactions with target compounds promote efficient piperine recovery.115,117 However, current research remains largely limited to laboratory-scale optimization. Further work is needed to evaluate large-scale implementation, long-term solvent recyclability, extraction selectivity, and techno-economic feasibility before these solvent systems can be adopted for industrial piperine production.

Steam Explosion

Recently steam explosion has emerged as sustainable pretreatment method for the processing and extraction of pepper.95,118 Unlike conventional soaking methods, which require prolonged processing times and generate considerable wastewater, steam explosion utilizes high-pressure steam followed by rapid pressure release to disrupt plant tissue structures in a short period. It is an environmentally friendly culture method as it reduces processing time, water consumption and does not require the use of organic solvents.95,118,119

Jiang et al explored the use of steam explosion for processing white pepper and found that this method markedly increased retention of piperine in comparison to conventional soaking. White pepper produced through conventional soaking contained 6.47% piperine, whereas steam-exploded samples reached up to 8.80%. The increased piperine content associated with shorter soaking duration was largely due to significantly lower degradation and leaching losses, both common during prolonged soaking. Furthermore, steam explosion improved peeling efficiency by inducing rapid cell wall destruction through thermal treatment and instantaneous decompression.95

The mechanism underlying steam explosion involves high-pressure steam penetrating plant tissues and a subsequent release of that pressure. As a result, this mechanism causes mechanical disruption of the intracellular structure, degradation of pectin and cellulose in the pepper skin, accompanied by easy separation of tissues. These structural modifications can enhance the release and stability of bioactive compounds while minimizing processing.95,118,120

However, the effectiveness of steam explosion is highly dependent on processing conditions. Jiang et al observed that excessive steam pressure (>0.55 MPa) or prolonged treatment times (120 s) resulted in reduced piperine content, with values decreasing to approximately 8.05%. The decrease was due to the thermal degradation and chemical instability of piperine under progressive high-temperature and high-pressure conditions. Hence, there is a need for optimizing the steam pressure and residence time to attain balance between efficient tissue disintegration with minimum loss of valuable thermolabile bioactive compounds.95

In conclusion, steam explosion is a potentially viable green pretreatment technology for promoting pepper processing with high piperine retention and reduced processing time compared with traditional methods. Nevertheless, its application in direct piperine extraction remains relatively underexplored, and further studies are needed to evaluate its integration with downstream extraction techniques and its influence on extraction yield, purity, and bioactivity of piperine-rich extracts.

Optimization Strategies

Response Surface Methodology (RSM)

Since Response Surface Methodology (RSM) allows to investigate the influence of multiple extraction variables as well as their interactions while minimizing experimental runs, it has found great application for optimization of piperine extraction processes from Piper nigrum.2,4,121 Among the available RSM approaches, Central Composite Design (CCD) and Box-Behnken Design (BBD) are the preferred designs for developing predictive models and determining optimum extraction conditions.2,122

Nguyen et al applied CCD-based RSM to maximize the piperine extraction from Vietnamese black pepper with ethanol as the extraction solvent. Screening experiments suggested that extraction temperature, extraction time, and material-to-solvent ratio were the primary factors determining piperine recovery.122 Among the solvents screened, ethanol 96% proved to be the most effective extraction solvent relative to water, as well as 50% and 70% ethanol for yield of piperine at 0.79% dry matter content. The CCD model demonstrated excellent predictive performance with an R² value of 0.975 and an adjusted R² of 0.955. The optimum extraction conditions were determined as a material-to-solvent ratio of 1:3.9 (g/mL), extraction time of 104 min, and extraction temperature of 37°C. Under these optimized conditions, the piperine content reached approximately 0.808% of the dry material. Subsequent crystallization and recrystallization yielded 0.4954 g of purified piperine per gram of extract, demonstrating that the optimized extraction process also facilitated efficient downstream purification.122

A different optimization strategy was reported by Lwamba et al, who combined BBD, UAE, and NADES to maximize piperine recovery from black pepper.2 Before RSM optimization, sixteen NADES formulations were screened to identify the most suitable extraction medium. Among them, the system composed of choline chloride, citric acid, and propylene glycol (1:2:2) containing 25% water achieved the highest piperine yield (3.876%). The BBD model was then used to evaluate the effects of extraction time, extraction temperature, water content in the NADES, and the solid-to-liquid ratio. Among these variables, extraction time and the solid-to-liquid ratio had the greatest influence on piperine recovery. The optimized conditions predicted by RSM consisted of 50 min extraction time, 30°C extraction temperature, 14.5% water content, and a solid-to-liquid ratio of 30 mL/g, yielding 3.907% piperine. In addition, the recovered piperine-rich extract achieved approximately 90% purity, demonstrating the potential of combining green solvents and ultrasound-assisted extraction with RSM optimization.2

Table 5 summarizes the RSM designs, investigated factors, model-derived optimum conditions, piperine yields, and principal model diagnostics reported in these studies.

Table 5.

Application of Response Surface Methodology (RSM) for Optimization of Piperine Extraction Processes

Study	RSM Design	Factors Optimized	Optimum Conditions	Piperine Yield	Model Appraisal
Reflux extraction122	CCD	Material-to-solvent ratio, extraction time, extraction temperature	1:3.9 g/mL, 104 min, 37°C	0.808% dry matter	R2 = 0.975; adjusted R2 = 0.955; transferability across independent pepper batches or production scales was not evaluated
Ultrasound-assisted extraction (UAE)2	BBD	Extraction time, extraction temperature, water content in NADES, solid-to-liquid ratio	50 min, 30°C, 14.5% water, 30 mL/g	3.907%	R2 = 0.824; non-significant lack-of-fit; only extraction time and liquid-to-solid ratio were significant

Although both RSM models identified study-specific optimum conditions, model adequacy cannot be inferred from R² alone. Nguyen et al reported high R² and adjusted R² values, whereas Lwamba et al reported a lower R² with a non-significant lack-of-fit, and only extraction time and liquid-to-solid ratio significantly affected piperine yield.2,122 Because the studies differed in starting material, solvent system, extraction mode, and quantification and reporting basis, their yields do not establish the intrinsic superiority of BBD-NADES-UAE over CCD-based ethanol extraction. The reported optima therefore remain specific to the investigated experimental domains.

One-Factor-at-a-Time (OFAT) and RSM Coupling

To improve the efficiency of piperine extraction, several recent studies have adopted a combination of One-Factor-at-a-Time (OFAT) and Response Surface Methodology (RSM). In this sequential approach, OFAT is first used to screen the most influential extraction variables and define appropriate experimental ranges, after which RSM is applied to evaluate interactions among these variables and identify the optimal extraction conditions.85,121 Compared with OFAT alone, which evaluates only one variable at a time, the combined OFAT-RSM strategy provides a more comprehensive optimization by accounting for factor interactions that would otherwise be overlooked.4,85

Le et al employed this optimization strategy to improve piperine extraction from Vietnamese white pepper using a solvent-circulation extraction system.85 The initial OFAT experiments examined the effects of ethanol concentration, solvent-to-solid ratio, extraction temperature, extraction time, extractor loading capacity, and extraction cycles before the most influential variables were selected for subsequent RSM optimization. The optimum conditions obtained through OFAT consisted of 90% ethanol, a solvent-to-solid ratio of 3:1 mL/g, extraction temperature of 70°C, extraction time of 60 min, and three extraction cycles, yielding 3.87% dry material. Subsequently, ethanol concentration, extraction time, and solvent-to-solid ratio were selected for further optimization using CCD-RSM. The developed model reported an R² value of 0.9734, and the optimum conditions were determined as 85% ethanol, a solvent-to-solid ratio of 3.4 mL/g, and an extraction time of 78 min. Under these conditions, the piperine yield increased to 5.2% dry material, representing an improvement of approximately 34% compared with the OFAT-derived conditions.85

A similar optimization strategy was reported by Al Adhreai et al, who employed NADES combined with ultrasound-assisted extraction for piperine recovery.4 OFAT experiments were first conducted to evaluate the effects of liquid-to-solid ratio, extraction time, extraction temperature, and water content on extraction performance using UBE and UPE. For UBE, the optimum OFAT conditions were 25 mL/g, 45 min, 45°C, and 20% water content, producing 2.68% piperine. Meanwhile, UPE achieved a substantially higher OFAT yield of 4.97% under conditions of 30 mL/g, 50 min, 50°C, and 30% water content. The selected variables were subsequently optimized using BBD-RSM. The optimized UBE conditions generated a piperine yield of 2.71%, whereas the optimized UPE system achieved 5.37%. Although the improvement observed for UBE was relatively small, the UPE process showed an increase of approximately 7.9% following RSM optimization. Furthermore, the UPE model demonstrated excellent statistical performance with an R² value of 0.9817, indicating a high level of agreement between predicted and experimental results.4

Overall, both studies demonstrated that integrating OFAT with RSM significantly enhances process optimization by enabling the identification of variable interactions that cannot be captured through single-factor experimentation. The improvement was noticeable in the study by Le et al, where piperine yield increased from 3.87 to 5.2% dry material after RSM optimization.85 Similarly, Al Adhreai et al reported enhanced extraction performance after applying BBD-RSM, especially in the UPE system.4 Overall, combining OFAT with RSM provides a practical strategy for optimizing piperine extraction by utilizing interactive effects among some extraction variables which cannot be determined by OFAT alone. As summarized in Table 6, RSM consistently identified more favorable operating conditions and generally produced higher piperine yields, highlighting its value as a multivariate optimization approach.

Table 6.

Optimization of Piperine Extraction Processes Using Response Surface Methodology (RSM) Compared with One-Factor-at-a-Time (OFAT) Approaches

Study	OFAT Conditions	OFAT Yield	RSM Design	Optimized Conditions	RSM Yield	Improvement
Percolation (solvent cyclic extraction)85	90% ethanol, 3:1 mL/g, 70°C, 60 min, 3 extraction cycles	3.87% dry material	CCD	85% ethanol, 3.4:1 mL/g, 78 min	5.2% dry material	Predicted increase of 34.3%
Ultrasonic bath extraction (UBE)4	25 mL/g, 45 min, 45°C, 20% water	2.68%	BBD	26.82 mL/g, 47.44 min, 44.81°C, 22.41% water	2.71%	Predicted increase of 1.3%
Ultrasonic probe extraction (UPE)4	30 mL/g, 50 min, 50°C, 30% water	4.97%	BBD	28.12 mL/g, 47.26 min, 44.58°C, 29.85% water	5.37%	Predicted increase of 7.9%

Notes: Relative changes were calculated by the review authors as [(RSM yield − OFAT yield)/OFAT yield] × 100. For UBE and UPE, these percentages represent model-predicted rather than independently confirmed improvements.

OFAT-RSM coupling enabled the evaluation of factor interactions after preliminary univariate screening, but the reported improvements should be interpreted according to their validation status. Le et al reported a higher yield under the RSM-optimized conditions, whereas the UBE and UPE yields reported by Al Adhreai et al were model-predicted optima rather than independent experimental confirmations.4,85 Although the Al Adhreai models showed favourable internal diagnostics, these statistics support model adequacy only within the investigated design spaces. Neither study reported validation across independent pepper batches or at production scale; consequently, the improvements summarized in Table 6 remain study-specific and, for UBE and UPE, represent predicted rather than experimentally verified gains.

Green Analytical Assessment Tools

In light of this growing concern over sustainable extraction and analytical methodologies, novel quantitative greenness assessment tools like the Analytical GREEnness Metric (AGREE) and Green Analytical Procedure Index (GAPI) have emerged over time.123,124 These tools focus on a systematic evaluation of the environmental performance of extraction and analytical procedures by considering factors such as reagent toxicity, solvent consumption, energy requirements, waste generation, and occupational safety. Table 7 summarizes representative studies that applied these tools to evaluate the greenness of piperine extraction methods.

Table 7.

Green Assessment of Recent Piperine Extraction Methods Using Analytical Sustainability Metrics

Method	Piperine Source	Green Assessment Tool	Main Findings	Greenness Score	Reference
Traditional extraction (TE) and ultrasound-based extraction (UBE) coupled with HPTLC analysis	Piper nigrum (black pepper)	AGREE	UBE produced higher piperine content than TE; highest piperine content reached 31.85%	0.90 (excellent greenness)	[89]
TRDES-based UAE	White pepper	GAPI	TRDES enabled piperine extraction and solvent recycling; piperine yield reached 3.375%	82 (TRDES) vs 68 (ethanol extraction)	[90]

One of the earliest piperine-related studies incorporating a formal greenness assessment was reported by Alqarni et al The authors developed a sustainable normal- and reversed-phase HPTLC method for the quantification of piperine in traditional extracts (TE) and UAE of Piper nigrum.89 Using the AGREE metric, both analytical methods achieved a greenness score of 0.90, indicating excellent compliance with the principles of Green Analytical Chemistry.125 In addition, UAE produced higher piperine contents than conventional extraction across all black pepper samples, with the highest piperine content reaching 31.85% in UAE compared with 30.95% obtained by TE. The results showed that ultrasound-assisted extraction coupled with an eco-friendly chromatography could enhance the extraction efficiency while displaying a high level of environmental sustainability.89

More recently, You et al assessed the green aspect of a TRDES-based ultrasound-assisted extraction system for white pepper piperine recovery.90 GAPI was used in the study to assess the environmental performance of the TRDES extraction system versus conventional ethanol-based extraction. The optimized TRDES composed of lidocaine and valeric acid (1:1) produced a piperine yield of 3.375% while simultaneously enabling solvent recycling and facile piperine recovery through temperature-induced phase separation. The TRDES method showed a substantially higher greenness profile (82) against the simplest and most widely used conventional ethanol extraction process (68), extending the GAPI assessment. The increases in score was mainly due to lower solvent consumption, better recyclability, and easier purification. These results indicate the potential of enabling advanced green solvents using formal greenness assessment tools to promote more sustainable piperine extraction processes.90

Noticeably, the interest in exploring green extraction technologies for piperine is growing, but application of AGREE and GAPI is limited. The majority of studies focus on extraction efficiency and yield of piperine while quantitative assessment of environmental performance is seldomly reported. More generalized greenness assessment tools are essential for comparison of extraction methods with an objective approach that can help to establish real green sustainable piperine production processes; however, the resulting scores should be interpreted together with extraction recovery, piperine content or purity, solvent and energy requirements, waste generation, and process reproducibility. A favourable greenness score alone does not establish the chemical quality or suitability of the recovered material for subsequent formulation and pharmaceutical development. Future studies should therefore integrate environmental assessment with process performance and material characterization rather than treating greenness as an isolated endpoint.

Formulation and Dosage Forms

Building on the extraction and purification approaches discussed in Extraction, Purification, and Green Processing, formulation development represents the next stage in determining whether chemically characterized piperine can be delivered reproducibly. Although several studies have carried extracted, isolated, or standardized piperine-containing materials into formulation testing, controlled evidence directly relating upstream purity, composition, or batch attributes to dosage-form performance remains limited. Piperine has poor aqueous solubility, which may limit its dissolution and systemic exposure following oral administration. Accordingly, solubility-enhancing systems, lipid-based formulations, nanoparticles, mesoporous carriers, and multifunctional delivery platforms have been investigated to improve dissolution, stability, permeation, or preclinical exposure. This section first discusses the key physicochemical and pharmacokinetic barriers that limit piperine delivery, followed by recent advances in formulation strategies aimed at improving its therapeutic performance and their remaining translational limitations.

Physicochemical and Pharmacokinetic Limitations

Despite its broad pharmacological activities, the pharmaceutical development of piperine remains challenging due to its unfavorable physicochemical and pharmacokinetic properties. Piperine is a highly lipophilic alkaloid containing a hydrophobic piperidine ring and a conjugated diene chain, structural features that contribute to its poor aqueous solubility and limited oral bioavailability.1,17 The compound exhibits a reported log P value of 3.5 and very low water solubility, which limits its dissolution in gastrointestinal fluids, thereby reducing the amount of drug available for absorption. As a result, piperine often exhibits dissolution-limited bioavailability despite possessing favorable membrane permeability characteristics.7,14 These physicochemical and metabolic characteristics contribute to the variable oral bioavailability reported for piperine.1 Based on its low aqueous solubility and relatively high membrane permeability, piperine is generally classified as a Biopharmaceutics Classification System (BCS) Class II compound, although its poor dissolution behavior may substantially restrict oral bioavailability in vivo.5,126

The combination of poor aqueous solubility and dissolution-rate-limited absorption contributes to the low oral bioavailability of piperine.1,14 These biopharmaceutical limitations have driven the development of a wide range of formulation strategies aimed at improving piperine solubility, dissolution rate, oral absorption, and overall systemic exposure.14,127

Formulation-based approaches should therefore be evaluated according to the specific endpoint demonstrated, including solubility, dissolution, in vitro permeability, or measured systemic exposure. Improved solubility, dissolution, or in vitro release does not by itself establish enhanced in vivo pharmacokinetics, while increased preclinical exposure does not necessarily demonstrate therapeutic efficacy or clinical benefit. Long-term safety, manufacturing reproducibility, and scalability also remain separate requirements for pharmaceutical translation. This evidence hierarchy provides the basis for evaluating the advanced delivery systems discussed in the following subsection.

Advanced Drug Delivery Systems (DDS)

Conventional oral dosage forms do not adequately address the major biopharmaceutical challenges associated with piperine, including its poor aqueous solubility and dissolution-limited absorption.1,14 As a result, recent research has shifted toward advanced drug delivery systems (DDS) designed to enhance piperine solubility, bioavailability, stability, and overall therapeutic performance.16,128 Table 8 summarizes recent advances in piperine formulation technologies, ranging from solubility-enhancing systems to nanotechnology-based carriers and bio-enhancing platforms. These strategies collectively aim to address the intrinsic biopharmaceutical limitations of piperine and facilitate its therapeutic translation.

Table 8.

Summary of Recent Formulation Strategies Developed to Improve the Biopharmaceutical Performance of Piperine (2021–2026)

Formulation Strategy	Formulation/Carrier	Key Findings	Main Improvement	Ref.
Deep Eutectic System (DES)	Piperine-based DES	Increased piperine solubility by up to 294% compared with native piperine	Solubility enhancement	[129]
Solid Dispersion (QbD-optimized)	Fourth-generation ternary solid dispersion of Piper longum extract	Relative bioavailability increased to 188.8%; improved dissolution and melanoma therapeutic efficacy	Solubility, dissolution, oral bioavailability	[130]
SNEDDS	Glyceryl monolinoleate–Poloxamer 188–Transcutol HP	Improved intestinal permeation and ~4.9-fold higher oral bioavailability; enhanced antihypertensive activity	Dissolution, permeability, oral bioavailability	[6]
Solid SNEDDS (S-SNEDDS)	Mesoporous mannitol-based SNEDDS	Maintained nanoemulsion properties after solidification and improved dissolution performance	Stability and dissolution	[131]
Albumin Nanoparticles	Piperine-loaded albumin nanoparticles	Reduced IL-17 and TNF-α levels and improved anti-inflammatory activity	Targeted delivery, therapeutic efficacy	[132]
Lipid-Polymer Hybrid Nanoparticles (LPHNPs)	Piperine-loaded LPHNPs	6.02-fold higher intestinal permeation and 4.55-fold higher bioavailability; improved anticancer activity	Permeability, bioavailability, anticancer efficacy	[133]
Nanoemulsion	Piperine nanoemulsion	Improved drug release, permeability, stability, and biological activity	Solubility and permeability	[134]
Liposome	Piperine-loaded liposomes and chitosan-coated liposomes	Particle size 166–243 nm; encapsulation efficiency 60–80%; chitosan coating improved cellular uptake and cytotoxicity against MCF-7 cells compared with free piperine	Solubility, cellular uptake, anticancer efficacy	[135]
Polymeric Nanoparticles	Polycaprolactone nanoparticles	Encapsulation efficiency 84.8 ± 3.5%; sustained release profile	Controlled release and stability	[136]
Antileishmanial Nanoparticles	Piperine–amphotericin B nanoparticles	IC50 reduced to 21.9 ng/mL (promastigotes) and 4.87 ng/mL (amastigotes); up to 96% parasite inhibition	Therapeutic efficacy and toxicity reduction	[137]
Mesoporous Nanocarriers	Mesoporous silica nanoparticles (MSNs)	Drug loading ~60%; sustained release ~75% within 24 h; efficacy up to 94.67%	Controlled release and loading capacity	[138]
Ionic Liquid Bioenhancer System	Ketoprofen–piperine ionic liquid	Solubility increased by 71–83%; skin permeation increased by >218%; improved anti-inflammatory effect	Bioenhancement and transdermal delivery	[139]

Viewed together with the extraction and purification studies discussed in Piperine as Bioactive Compound, the investigations summarized in Table 8 demonstrate partial rather than complete continuity between upstream material production and downstream formulation development. Kusumorini et al isolated piperine with a purity of at least 95% from white pepper and subsequently incorporated it into liquid and solid SNEDDS; mechanistically, the self-emulsifying system maintained piperine in a solubilized formulation and increased its in vitro dissolution.129 Coco et al directly encapsulated a characterized ethanolic black-pepper extract into polycaprolactone nanoparticles, achieving piperine encapsulation and sustained release together with storage stability, but without evaluating systemic exposure.130 Mohapatra et al formulated a standardized Piper longum extract into a ternary solid dispersion that improved wettability and dissolution and extended the evaluation to preclinical pharmacokinetics and efficacy.131 These examples show that different formulations address distinct stages of delivery, including solubilization, dissolution, encapsulation, release, and, in the case of the solid dispersion, preclinical systemic exposure.

Nevertheless, improvements in solubility, dissolution, or controlled release do not by themselves demonstrate enhanced systemic exposure, which requires comparative in vivo pharmacokinetic measurements such as AUC and Cmax. Moreover, these studies did not compare materials with different piperine purity, composition, or batch attributes under matched formulation and pharmacokinetic conditions. The remaining gap is therefore not the absence of extraction-to-formulation studies, but the limited systematic evidence showing how upstream material attributes influence downstream drug loading, release, stability, reproducibility, and systemic exposure.

Solubility Enhancement Systems

Improving aqueous solubility represents one of the primary strategies for overcoming the biopharmaceutical limitations of piperine. Among the emerging approaches, deep eutectic systems (DESs) have attracted increasing attention as environmentally friendly solubilization platforms capable of enhancing the dissolution behavior of poorly water-soluble compounds. Hassan et al reported that piperine-based DES formulations increased piperine solubility by up to 294% compared with native piperine. This improvement was attributed to favorable intermolecular interactions between piperine and the DES components, which facilitated drug solubilization and highlighted the potential of DES technology as a green alternative to conventional solubilizing agents.132

Moreover, the solid dispersion technology has also become an important way to enhance dissolution and oral absorption of piperine in addition to DESs. Mohapatra et al designed a quality-by-design (QbD)-optimized fourth-generation ternary solid dispersion containing standardized Piper longum fruit ethanolic extract.131 The physicochemical characterization confirmed that piperine transformed into an amorphous form to some extent, exhibiting better wettability and dissolution behaviour. The optimized formulation had a relative oral bioavailability of 188.8% for the plain extract and improved therapeutic efficacy in a melanoma model.131 Together, these datasets suggest that solubility-enhancing systems can significantly enhance the pharmaceutical performance of piperine and serve as a basis for formulating higher-order delivery platforms.

Nanotechnology-Based Delivery Systems

Nanoformulations are being investigated as one strategy to improve piperine solubilization, release, permeation, and preclinical exposure; however, their effects remain carrier- and model-dependent.128 Recent studies have demonstrated that nanotechnology-based delivery systems significantly enhance the therapeutic potential of piperine as seen in Figure 7. For example, Golijani et al piperine-loaded albumin nanoparticles exhibited significant anti-inflammatory effects in vivo by reducing cytokines such as IL-17 and TNF-α, supporting their potential in disease management.133

Figure 7.

Nanotechnology-based delivery systems. The large downward arrow indicates the movement of nanocarriers into the systemic circulation. The curved arrow indicates the application of external ultrasound at the target site, while the blue ultrasound waves represent ultrasound-induced disruption or lysis of the responsive nanocarrier, followed by intracellular drug release.

Notably, Kazmi et al developed lipid-polymer hybrid nanoparticles (LPHNPs) with particle sizes below 160 nm and positive surface charge (>+20 mV), which demonstrated enhanced cytotoxicity against MCF-7 and MDA-MB-231 breast cancer cells, along with significantly improved intestinal permeation (6.02-fold) and oral bioavailability (4.55-fold) compared to free piperine.134 In addition to nanoparticles, nanoemulsion-based systems have also been explored by Alshehri et al, showing improved drug release, permeability, and biological activity with droplet sizes ranging from 105 to 250 nm and enhanced stability.135 Liposomal delivery systems have also been investigated as versatile carriers for piperine. Imam et al developed piperine-loaded liposomes and chitosan-coated liposomes with particle sizes ranging from approximately 166 to 243 nm and encapsulation efficiencies of 60–80%. The chitosan coating enhanced mucoadhesive properties and cellular uptake, resulting in significantly greater cytotoxic activity against MCF-7 breast cancer cells compared with free piperine.136 These findings suggest that liposomal formulations can improve piperine solubilization, retention, and therapeutic efficacy while maintaining favorable carrier stability.

Moreover, polymeric nanoparticle systems, such as polycaprolactone-based formulations, further demonstrated high encapsulation efficiency (84.8 ± 3.5%) and sustained drug release, improving stability and delivery performance.130 Additionally, nanotechnology-based delivery systems have also demonstrated potential in the treatment of parasitic diseases. Ray et al developed a nanoparticle formulation combining piperine with amphotericin B for leishmaniasis therapy.137 This nanoformulation displayed significantly improved antileishmanial activity with promastigote and amastigote IC₅₀ values of 21.9 ng/mL and 4.87 ng/mL, respectively, while the corresponding values for amphotericin B alone were 33.5 ng/mL and 40.8 ng/mL, respectively. Additionally, in vivo studies in hamsters revealed up to 96% inhibition of parasites following oral administration of the nanoformulation, highlighting the ability of nanoparticle carriers to improve therapeutic efficacy while potentially providing a lower toxicity that may accompany amphotericin B.137

Collectively, these nanocarrier systems have been shown to enhance the solubility, bioavailability, and targeted delivery of piperine while reducing systemic toxicity, thereby positioning them as promising platforms for next-generation therapeutic applications.

Mesoporous Nanocarriers

Aside from lipid- and polymer-based nanocarriers, mesoporous and inorganic delivery platforms have also been highlighted as promising candidates for enhancing piperine encapsulation capabilities and achieving controlled-release drug effects. In this context, mesoporous silica nanoparticles (MSNs) (Figure 7) are of high interest due to their considerable surface area, their adjustable pores architecture and a great drug-loading capacity.138,139 Sahebi et al developed piperine-loaded MSNs with a drug-loading efficiency close to 60% and a sustained-release nature reaching nearly 75% over 24 h.139 The formulation also showed promising biological performance, with efficacy values of up to 94.67% while maintaining cell viability above 90%, indicating low cytotoxicity. These enhancements were attributed to the ability of mesoporous silica matrix to keep piperine dispersed with high efficiency and gradually release drug over time.139

While these findings are promising, research on mesoporous silica-based piperine delivery remains at an early stage. More studies are needed to ascertain their long-term biocompatibility, biodegradation, manufacturability at scale and regulatory issues prior to clinical translation of these systems.

Self-Nanoemulsifying Drug Delivery Systems (SNEDDS)

The self-nanoemulsifying drug delivery systems (SNEDDS) is a typical lipid-based and advanced nanotechnology-based delivery system, which unlike the other conventional nanocarriers, they do not rely on preformed nanoparticles. Instead, SNEDDS, as shown in Figure 8, consists of isotropic mixtures of oils, surfactant and co-surfactants which spontaneously forms fine oil-in-water nanoemulsions upon contact with gastrointestinal fluids under gentle mechanical agitation.140 This in situ nanoemulsification can improve the dissolution, solubilization, and intestinal absorption of poorly soluble compounds compared with conventional nanocarriers but has simpler formulation and superior physical stability than many conventional nanocarriers.140,141 A piperine-loaded SNEDDS based on glyceryl monolinoleate, poloxamer 188, and Transcutol Highly Purified was developed by Zafar et al, which produced nanosized droplets and significantly improved piperine release and intestinal permeation.6 The prepared formulation resulted in about 4.9-fold higher oral bioavailability compared with piperine dispersion and also demonstrated enhanced antihypertensive activity, suggesting that the potential of SNEDDS can improve both pharmacokinetic and pharmacological behavior.6

Figure 8.

Proposed mechanism after ingestion of self-nanoemulsifying drug delivery systems (SNEDDS). The left-to-right arrows indicate the proposed sequence of oral administration, spontaneous nanoemulsion formation in gastrointestinal fluids, passage across enterocytes, and entry into the systemic circulation.

Kusumorini et al reported similar results with piperine-loaded liquid SNEDDS (L-SNEDDS) using Miglyol 812N, Cremophor RH40, and Polyethylene Glycol (PEG) 400.129 The optimized formulation rapidly formed a transparent nanoemulsion with nano-sized droplets immediately upon dilution, confirming rapid self-emulsification in aqueous media. Then liquid formulation converted into solid SNEDDS (S-SNEDDS) by adsorbing on mesoporous mannitol to improve storage stability and handling. This indicates that S-SNEDDS is a potential platform for developing stable oral formulations of piperine and upon reconstitution the solid formulation exhibited properties characteristic of nanoemulsions, as well as an improved dissolution rate which was up to 10 times higher compared to pure piperine.129

Available studies indicate that SNEDDS can improve piperine solubilization and dissolution and, in rats, increase oral exposure relative to piperine dispersion. However, the in vivo evidence remains limited, and comparative human pharmacokinetics, long-term stability, excipient safety, and manufacturing scale-up have not yet been established.

Ionic Liquids and Multifunctional Delivery Platforms

Among the various applications of piperine as a bioenhancer, recent studies have demonstrated its utility as a functional component in advanced pharmaceutical delivery systems. Hassan et al developed a ketoprofen-piperine ionic liquid (KP-PI IL) by pairing ketoprofen with piperine through hydrogen-bond-mediated ionic liquid formation.142 The resulting formulation transformed crystalline ketoprofen into a stable amorphous form, leading to substantial improvements in its pharmaceutical properties. Specifically, ketoprofen solubility increased by approximately 71–83%, while transdermal permeation through rat skin was enhanced by more than two-fold compared with the corresponding ketoprofen-piperine physical mixture. Furthermore, the ionic liquid exhibited superior anti-inflammatory activity in vivo, producing significantly lower paw edema in carrageenan-induced rats than the non-ionic formulation.142

Importantly, the enhanced performance of the ketoprofen-piperine ionic liquid was not solely attributable to improved solubility. Ionic liquid formation simultaneously increased ketoprofen lipophilicity, skin permeability, and anti-inflammatory efficacy, demonstrating the multifunctional role of piperine within the delivery platform as can be seen in Figure 9. In addition to its well-established ability to inhibit P-glycoprotein-mediated efflux and CYP3A4-mediated metabolism, piperine contributed structurally to the formation of a stable pharmaceutical ionic liquid, thereby facilitating drug transport across biological barriers and improving therapeutic outcomes.142

Figure 9.

Proposed formulation effects of an ionic liquids (ILs) on transdermal delivery. The downward arrow represents the conceptual transport of the formulation across the skin. Upward arrows indicate increases in physical or formulation stability, drug solubility, and skin permeation. Bold labels are used only for visual emphasis.

These findings illustrate the expanding role of piperine beyond its traditional use as a pharmacokinetic enhancer. Rather than functioning solely as a co-administered absorption promoter, piperine can be incorporated directly into advanced drug delivery systems, including ionic liquids, nanocarriers, and hybrid formulations, where it contributes both bio-enhancing and formulation-stabilizing functions. Such multifunctional applications may provide new opportunities for improving the delivery and therapeutic performance of poorly soluble drugs.

Clinical Evidence and Translational Status of Piperine

Improvements in formulation performance address only one component of pharmaceutical translation. Whether enhanced dissolution or preclinical systemic exposure translates into reproducible human pharmacokinetics, acceptable safety, and indication-specific efficacy must be established independently. Accordingly, this section examines the limited human evidence on piperine exposure and the pharmacokinetic bioenhancement of co-administered compounds, together with clinical studies of piperine-containing interventions. Evidence directly attributable to piperine is distinguished from findings obtained with multicomponent herbal remedies, standardized extracts, commercial combination products, and co-supplementation interventions.

The development of piperine as a bioenhancer represents one of the most successful examples of translational research involving natural products. Black pepper (Piper nigrum) and long pepper (Piper longum), have been widely used for centuries in traditional medical systems, including Ayurveda, Unani, and Siddha, laying the foundation for modern pharmacological research.143 Black pepper is one of the major ingredients in Ayurveda preparations, Trikatu, which is a classical polyherbal formula for various gastrointestinal and respiratory ailments, proved for its bio-enhancing action at scientific level.144,145 Similarly, black pepper has been used in Unani and Siddha medicine in different formulations for digestive, inflammatory and rheumatic conditions.143

Based on its centuries-old usage in traditional medicine followed by subsequent pharmacological and pharmacokinetic studies, piperine showed as an excellent bioenhancer of drug availability and potency in many therapeutic classes without significant independent pharmacologic activity.16,143 These studies further showed that piperine enhances systemic exposure to a variety of other pharmacologically active compounds, including antibiotics, antituberculosis drugs, anticonvulsants, cardiovascular drugs, and phytochemicals.1,143 Mechanistic studies then showed that the bio-enhancing effects are mediated through mechanistically distinct pathways, including effects on intestinal permeability, inhibition of drug-metabolizing enzymes, and repression of efflux transporters such as P-glycoprotein.9,14,16 The available evidence therefore supports substrate-dependent pharmacokinetic modulation rather than universal bioenhancement or independent therapeutic efficacy.

One of the most important translational milestones was that demonstrated piperine dramatically increased curcumin oral bioavailability in humans.146 This discovery subsequently led the incorporation of piperine into numerous nutraceutical and pharmaceutical formulations, including commercially available products such as BioPerine^® (Sabinsa Corporation, East Windsor, New Jersey, USA) and Risorine^® (Cadila Pharmaceuticals Ltd., Ahmedabad, Gujarat, India).18,147,148 Nevertheless, evidence from these products or from curcumin-piperine interventions should not be interpreted as confirmatory evidence for piperine as a stand-alone medicine.

Building off these fundamental findings, more recent studies have moved from proof-of-concept bioenhancement studies all the way through clinical validation and therapeutic potential. Between 2021 and 2026, additional clinical studies have extended the therapeutic applications of piperine formulations into other areas, such as infectious diseases, inflammatory disorders, cardiovascular diseases, autoimmune conditions, and cancer-related indications. Because most used multicomponent interventions and several remained exploratory or preclinical, their findings are evaluated according to study design, statistical strength, and the extent to which the independent contribution of piperine can be determined.

Clinical Studies

Despite most piperine research has remained at the preclinical stage, clinical interest has grown steadily over the past decade, driven by its therapeutic potential and well-recognized bio-enhancing properties. Recent clinical studies have mainly investigated piperine in combination with curcumin or other bioactive compounds for the management of infectious, inflammatory, autoimmune, cardiovascular, metabolic, and oncological disorders. These studies have provided preliminary evidence supporting the clinical potential of piperine while also highlighting the need for larger and more rigorous trials. Table 9 provides an overview of representative clinical studies published or registered between 2021 and 2026, summarizing their study design, intervention regimen, sample size, and key clinical outcomes.19,149–151

Table 9.

Clinical Studies and Registered Trials Involving Piperine or Piperine-Containing Interventions

Condition	Country	Phase/Status	Intervention/Dose	Duration	Sample Size	Findings	Trial Registration/Ref.
COVID-19	India	Double-blind RCT/ Completed	Curcumin 525 mg co-administered with piperine 2.5 mg twice daily as an adjunct to conventional COVID-19 treatment	During hospitalization; treatment given for 14 days from admission	140 total; 70 intervention, 70 control	Faster symptom recovery, improved oxygen saturation, reduced hospitalization duration in moderate-severe cases, fewer deaths observed	CTRI/2020/05/025482149
Critically ill COVID-19 ICU patients	Iran	Randomized, double-blind, placebo-controlled trial protocol / Registered	Curcumin 1500 mg/day co-administered with piperine 15 mg/day, divided into three daily doses	7 days intervention + 28-day mortality follow-up	60 (30 intervention; 30 placebo)	Clinical status, fever resolution, oxygen saturation, ICU stay, ventilator dependency, oxidative stress, inflammatory markers (CRP, ESR), mortality	IRCT20121216011763N52150
Sepsis ICU patients	Iran	Randomized double-blind placebo-controlled trial/ Completed	Curcumin-piperine combination, two tablets daily; each tablet contained 500 mg curcuminoids and 5 mg piperine (total daily dose: 1000 mg curcuminoids and 10 mg piperine)	7 days	66	CRP, ESR, bilirubin, hematological parameters, clinical outcomes, 28-day mortality	IRCT20150613022681N419
Ischemic stroke (rehabilitation phase)	Iran	Double-blind placebo-controlled RCT/ Completed	Curcumin 500 mg co-administered with piperine 5 mg once daily	12 weeks	66 randomized (56 completed)	Significant reductions in hs-CRP, CIMT, total cholesterol, triglycerides, systolic and diastolic blood pressure, and increased TAC; intervention was well tolerated with no major adverse effects	IRCT20121216011763N48151
STEMI after PPCI	Indonesia	Not applicable/ Completed	Curcumin 390 mg co-administered with piperine 20 mg once daily	28 days	50	hsCRP, MDA, liver and renal safety markers	* NCT07149961152
SLE with hypovitaminosis D	Indonesia	Phase II/Completed	Three groups: vitamin D3 with placebo; curcumin-piperine with placebo; and vitamin D3 co-administered with curcumin-piperine.	3 months	45	SLE disease activity, fatigue, IL-6, TGF-β	* NCT05430087153
Prostate cancer, MGUS, low-risk SMM	United States	Phase II/Completed	Curcumin 4 g co-administered with piperine 5 mg orally twice daily	12 months	30	Disease-specific response, progression-free status, safety	* NCT04731844154
Curcuminoid pharmacokinetics	Jordan	Not applicable/ Completed	Turmeric extract with piperine 5 mg/capsule, single dose	8 h PK sampling	24	Plasma curcuminoid and piperine-curcuminoid levels by LC-MS/MS	* NCT05542394155
Postmenopausal women with obesity	United States	Early Phase I/ Completed	GLY-LOW supplement containing piperine 15 mg/capsule; 2 capsules/day	6 months	13	Insulin resistance, body composition, inflammation, biological aging, safety	* NCT06242535156

Notes: * Referenced from the US National Library of Medicine: https://www.clinicaltrials.gov/ct2/results?cond=&term=piperine&cntry=&state=&city=&dist=.

Table 9 includes published clinical studies, trial registrations without publicly available results, and one preliminary non-peer-reviewed report; these evidence categories should not be assigned equivalent weight. Registration or completion indicates that a study was planned or conducted but does not itself demonstrate efficacy. Moreover, most interventions combined piperine with curcumin, herbal extracts, or other supplement components and did not include a formulation-matched comparator that differed only in the presence or absence of piperine. Their outcomes therefore apply to the tested combinations rather than to stand-alone piperine.

The published randomized trials reported mixed rather than uniformly beneficial outcomes. Pawar et al powered their study specifically for the oxygen-support outcome, but equivalent power was not established for the remaining outcomes or severity-subgroup analyses.149 In the ICU COVID-19 trial registered as IRCT20121216011763N52, the published study enrolled 40 participants rather than the 60 planned in the protocol. Curcumin-piperine supplementation reduced CRP and AST and increased hemoglobin relative to placebo, whereas other biochemical, hematological, and arterial-blood-gas outcomes were not significantly different, and 28-day mortality was identical between groups.150 Alikiaii et al reported changes in selected inflammatory and laboratory indices, but no significant between-group differences in APACHE II, NUTRIC, SOFA, or GCS scores.19 Boshagh et al reported improvements in selected cardiovascular and inflammatory outcomes, although several lipid, coagulation, and quality-of-life outcomes were not significantly different between groups.151 In the SLE trial, the combined vitamin D–curcumin–piperine group showed greater changes in selected outcomes than either intervention alone, whereas vitamin D alone and curcumin–piperine alone produced similar changes.152 None of these trials isolated the independent clinical contribution of piperine

The remaining registry-linked studies require different interpretation. NCT07149961 and NCT04731844 are listed as completed, but publicly available outcome results were not identified by the literature-search cutoff.153,154 NCT05542394 is methodologically relevant because it prespecified turmeric formulations with and without piperine, potentially allowing piperine-mediated bioenhancement to be evaluated more directly; however, no publicly available results were identified, and its completion status cannot be interpreted as evidence of bioenhancement.155 A preliminary report associated with NCT06242535 involved a single-arm, multicomponent supplement study in which 13 participants were enrolled and seven completed follow-up. Because the report was non-peer-reviewed, lacked a control group, and could not isolate piperine, it was regarded as exploratory evidence only.156

A 2025 GRADE-assessed systematic review and meta-analysis of 18 randomized controlled trials reported that curcuminoid-piperine co-supplementation significantly reduced AST and IL-6, whereas the pooled effects on ALT, ALP, CRP, and TNF-α were not statistically significant.157 This endpoint-specific pattern indicates that the pooled evidence was not uniformly positive. Moreover, these outcomes were laboratory biomarkers rather than direct measures of disease-specific clinical efficacy. Because all included interventions combined curcuminoids with piperine, the pooled estimates cannot establish the independent clinical effect or stand-alone efficacy of piperine.

Overall, the clinical literature supports further investigation of piperine-containing interventions but does not establish broad therapeutic applicability or confirm the efficacy of piperine as a stand-alone pharmaceutical agent. Future trials require adequately powered primary clinical endpoints, transparent control of multiple comparisons, longer follow-up, and formulation-matched comparator groups capable of isolating the contribution of piperine.

Clinical Evidence Supporting Piperine-Mediated Bioenhancement

Along with its direct pharmacological effects, piperine is also extensively studied as a natural bioenhancer for augmenting the absorption and systemic exposure of co-administered compounds.16 Piperine’s bio-enhancing effect is largely ascribed to inhibition of cytochrome P450 enzymes including CYP3A4, down-regulation of P-glycoprotein-mediated efflux, alteration in intestinal membrane dynamics, and an overall attenuation of first-pass metabolism. Piperine may increase drug bioavailability, systemic circulation time, and therapeutic efficacy through these mechanisms.16,127,158

Piperine has been shown to enhance the bioavailability of many conventional drugs and phytochemicals in various pharmacokinetic studies.16,159 Co-administration of piperine concurrently has also been shown to enhance the systemic exposures of numerous compounds, including but not limited to propranolol, theophylline, phenytoin, carbamazepine, sodium valproate, rifampicin, cyclosporine, and curcumin through increases in peak plasma concentration (C_max), area under the concentration-time curve (AUC), and elimination half-life.3,14,143,158 Of all these examples, the bioavailability improvement of curcumin is the most explored topic and human studies showed that piperine can significantly increase absorption of curcumin via inhibition in intestinal and hepatic metabolism.146 The study supports a pharmacokinetic bioenhancing effect of piperine for curcumin under those dosing conditions, but it does not characterize the plasma pharmacokinetics or independent therapeutic efficacy of piperine itself.

A 2023 systematic review and meta-analysis of five randomized studies in healthy participants also found that piperine co-administration increased the pooled C_max, AUC, and elimination half-life of selected CYP-metabolized drugs.158 However, the small number of included trials and their restriction to selected substrates and dosing regimens prevent these findings from being generalized to all co-administered drugs. The available human evidence therefore supports substrate-specific bioenhancement under the investigated conditions rather than a universal increase in drug absorption.

Bioenhancement using piperine has translational relevance to its inclusion in commercially available products. The anti-tuberculosis formulation Risorine^® (Cadila Pharmaceuticals Ltd., Ahmedabad, Gujarat, India) consists of rifampicin, isoniazid, and piperine to improve rifampicin bioavailability, while standardized piperine preparations like BioPerine^® (Sabinsa Corporation, East Windsor, New Jersey, USA) has been used extensively in nutraceutical formulations containing curcuminoids and other poorly bioavailable phytochemicals.18,147,148 More recently, human studies specifically evaluating pharmacokinetics have continued to characterize piperine-containing formulations and assess systemic absorption of curcuminoids and their metabolites (Table 9), effectively further supporting its use as a bioavailability-enhancing agent.155

Overall, these findings support piperine as a clinically relevant pharmacokinetic enhancer, marking it as more than just a traditional phytochemical. Nonetheless, the specific dosage fraction of piperine that exerts such additive effects towards therapeutic outcomes remains unknown as it is typically administered within combination formulations along with other compounds. Thus, further pharmacokinetic and exposure-response studies are required to obtain a definitive assessment of the size and clinical significance of piperine-mediated bioenhancement in humans.

Current Challenges and Future Perspectives

Despite some advances in excipient functionality and formulation strategies are tailored to improved piperine permeability, therapeutic translation of this compound has not progressed as much compared to preclinical development in pharmacokinetics. A series of scientific, clinical, regulatory and manufacturing barriers still need addressing prior to translation into approved therapeutic products, in spite of promising biological performance and the potential for enhanced pharmaceutical delivery with more sophisticated systems. Overcoming these limitations will be critical for piperine to evolve from a potential phytochemically active agent to being a clinically validated and commercially viable pharmaceutical agent.

Challenges

Lack of Robust Clinical Data

The limited availability of high quality clinical evidence for piperine represents one major obstacle precluding its clinical translation. Despite the numerous randomized controlled trials evaluating piperine-containing interventions, such as knee osteoarthritis, COVID-19, sepsis, ischemic stroke, and autoimmune disorders, most studies are limited by small sample sizes, short treatment durations, and between-study variability which limits the generalizability of their findings.149–151 Table 9 shows that, in many cases, participant numbers are less than 100 subjects, and follow-up periods cannot sufficiently assess long-term efficacy and safety outcomes.

Additionally, piperine is often given in combination with other bioactive compounds, particularly curcumin, complicating assessment of its independent therapeutic contribution versus that of the co-administered agent.149,150 Therefore, although there are many studies indicating favourable clinical results, exact attributable benefit from piperine remains uncertain. Dataset heterogeneity is also compounded by differences in formulation design, dosing regimens, and treatment protocols among studies.

Moreover, most clinical investigations that have been registered remain exploratory pilot or early-phase studies while the adequately powered multicenter Phase III RCTs to evaluate piperine containing interventions are still lacking.19,149–156 In the absence of large-scale confirmatory studies, piperine-independent evidence-based dose recommendations may not be established and regulatory authorities may reject piperine-based products. Hence, standardised formulations, larger patient numbers, longer time follow-ups, and well-designed multicenter trials should be the ultimate clinical future to elucidate clear efficacy and safety profiles.

Pharmacokinetic Challenges

The pharmacokinetic properties of piperine are less favourable for therapeutic translation, creating challenges for appropriate clinical dose selection. As a highly lipophilic compound that has low aqueous solubility and thus shows dissolution-rate-limited oral absorption leading to relatively poor oral bioavailability.1,160 Enterohepatic recirculation and negligible urinary excretion of unchanged piperine have also been reported in clinical pharmacokinetic studies, indicating a complex disposition that may additionally hinder the prediction of plasma concentrations for orally administered drugs.7

A further major challenge is that piperine also functions as a natural bioavailability enhancer.16 Piperine inhibits CYP3A4 and P-glycoprotein, reducing presystemic metabolism and intestinal efflux of many co-administered drugs.1,9 While these mechanisms enhance the oral bioavailability some poorly bioavailable substrates, they may simultaneously increase systemic drug exposure and affect pharmacokinetic profiles to generate clinically relevant drug-drug interactions (DDIs), especially for narrow therapeutic index medications.8 The magnitude of this effect should not be generalized across drug classes because it depends on the substrate, piperine dose and formulation, treatment duration, and relative contribution of the affected metabolic or transport pathway. Therefore, although the bio-enhancing effects of piperine provide substantial clinical benefits in improving therapeutics, consideration should be given to drug disposition changes together with dose adjustments, and therapeutic monitoring during combination therapy.

Substrate-specific findings illustrate this variability. In rats, co-administration of piperine altered warfarin metabolism and reduced anticoagulation at 24 h, but this preclinical finding has not been confirmed in patients.161 A PBPK study additionally predicted an approximately 31%, 34%, 35%, 36%, 39%, and 59% increase in ritonavir, nifedipine, cyclosporine, triazolam, alfentanil, and simvastatin AUC, respectively, following simulated administration of piperine at 20 mg/day for seven days.8 These results indicate that patients taking CYP3A4 substrate medications should be careful when consuming black pepper in quantities that provide 20 mg of piperine daily, as this combination could drastically modify the drugs’ pharmacokinetic profiles. Because this result was model-derived rather than observed in a clinical interaction study, it should be treated as a signal requiring prospective validation. These findings demonstrate that piperine-drug interactions are substrate-dependent and should not be described simply as universal enhancement of drug exposure.

To address these challenges, improved pharmacokinetic characterization will be required through carefully designed human pharmacokinetic studies, population pharmacokinetic analyses, physiologically based pharmacokinetic (PBPK) modeling and pharmacogenomic investigations. These strategies would enhance the prediction of systemic exposure, allow for individualised dose optimisation, improve an occurrence-based and mechanistic DDI risk assessment, and provide a broader framework to facilitate the safer clinical development of piperine-containing formulations.

Safety and Toxicological Considerations

Despite piperine’s exhibiting a good safety profile in most preclinical and short-term clinical studies, certain toxicological implications still need to be deemed critical for long-term sustainable medicinal use. Piperine has a largely dose-dependent safety profile and route-dependent bioavailability. Experimental animals have shown LD₅₀ values between approximately 15–514 mg/kg reflecting large discrepancies in toxicity with respect to the experimental model and route of exposure.162 Adverse effects such as respiratory depression, gastrointestinal irritation, and histopathological alterations in hepatic and adrenal tissues have been reported for piperine at sufficiently high doses.162 Nevertheless, human intervention studies generally employ substantially lower doses, typically ranging from 4–40 mg/day, and available clinical evidence suggests that piperine is generally well tolerated, with only occasional reports of mild gastrointestinal discomfort, diarrhea, or skin irritation.1,163 However, the limited number of long-term safety studies warrants continued caution regarding the use of isolated piperine at pharmacological doses.1

Published genotoxicity findings have not been entirely uniform. Thiel et al noted conflicting results among earlier studies but reported negative in vitro and in vivo micronucleus tests under their experimental conditions.164 These findings support the absence of a genotoxic response under the tested conditions rather than proving that genotoxic risk is absent under all exposure scenarios. Reproductive evidence is also limited. In a 30-day study involving six male rats per group, piperine at 5 and 10 mg/kg/day altered testicular steroidogenic activity and inhibited spermatogenesis.165 The relevance of these findings to long-term human exposure has not been established.

Evidence concerning liver injury is similarly context-dependent. A 21-day study involving six male mice per group reported increased liver histological-damage scores at oral doses of 35–140 mg/kg/day.166 However, the small sample size, short exposure period, and reliance principally on histological scoring limit its relevance to human hepatotoxicity. In a separate study, piperine potentiated carbon-tetrachloride-induced liver injury in rats, representing a conditional co-exposure effect rather than evidence that piperine alone produced the injury.167 By contrast, subchronic toxicity studies have generally supported the favorable safety profile of piperine under controlled experimental conditions. Repeated oral administration for up to 90 days at doses as high as 35 mg/kg/day did not produce significant hematological, biochemical, or histopathological abnormalities in rats.168 Nevertheless, safety thresholds remain a matter of debate. The European Food Safety Authority (EFSA) proposed a conservative no-observed-adverse-effect level (NOAEL) of 5 mg/kg/day based on alterations in serum cholesterol concentrations, whereas other investigators argued that these biochemical changes were not toxicologically relevant and suggested that substantially higher doses may remain safe.168 These differences illustrate why the safety profile should not be characterized as uniformly toxic or uniformly safe.

Drug-interaction liability also represents an important safety consideration. As discussed in Pharmacokinetic Challenges, piperine altered warfarin metabolism and anticoagulation in rats, while PBPK modelling predicted increased exposure to several CYP3A4 substrates, including ritonavir, nifedipine, cyclosporine, triazolam, alfentanil, and simvastatin.8,161 Because the warfarin evidence is preclinical and the cyclosporine finding is model-derived, neither should be presented as a confirmed clinical interaction. Nevertheless, these signals support substrate-specific evaluation, particularly for medications with narrow therapeutic indices, before recommendations concerning dose adjustment or therapeutic monitoring can be established.

Overall, short-term clinical evidence suggests that piperine is generally tolerated under the investigated conditions, but uncertainties remain concerning chronic exposure, reproductive effects, high-dose organ toxicity, and clinically relevant interactions in susceptible populations. Long-duration controlled studies, exposure-based toxicological assessment, substrate-specific interaction studies, and post-marketing surveillance are therefore required before the safety of pharmacological-dose piperine can be established.168

Translational and Commercialization Challenges

Despite the impressive amount of therapeutic claims and formulation advancements that piperine has achieved, translating piperine-product innovation from laboratory discovery to commercial application in humans is still a major hurdle. As discussed in Advanced Extraction Technologies, many advanced delivery systems, including nanoparticles, nanoemulsions, self-emulsifying systems, mesoporous carriers, deep eutectic systems, and ionic liquid-based formulations, have shown great improvements over solubility, bioavailability and therapeutic performance. However, most of these technologies are still at the proof-of-concept or laboratory scale with little advancement toward industrial production and clinical transformation.

Commercialization still is impeded by a few technical barriers, including process scalability, batch-to-batch reproducibility, long-term physicochemical stability, raw material variability, and cost-effective large-scale production. Furthermore, the complexity of nano-enabled and hybrid delivery systems could pose further quality control, manufacture repeatability, and storage stability challenges before successfully making its way through to the marketplace.169–171

Regulatory considerations represent another major obstacle. New and upcoming regulatory frameworks for both phytopharmaceuticals, botanicals drugs, as well as nanomedicine-based products are continuing to develop across various jurisdictions creating uncertainties around product classification, quality requirements, and approval pathways. The lack of uniform standards for formulations containing piperine may also impede the timeline to market authorization and industrial acceptance. Hence, a greater integration of QbD inputs, process analytical technologies (PAT) measurements and methodologies, life-cycle management strategies, and techno-economic assessments into future translational research should assist in the regulatory acceptance of these approaches, enable scale-up for manufacture, minimise investment risk and enhance commercialisation success after bioprocess development phases are completed.172–174

Future Perspectives

Integrating Advanced Formulation Strategies

The future pharmaceutical development of piperine is likely to be driven by new formulation technologies that can overcome multiple biopharmaceutical barriers at the same time. Present delivery systems have made remarkable advancements in terms of its efficacy on solubility and oral bioavailability, but the upcoming DDS is expected to be more multi-functional integrating targeted delivery, controlled release, enhanced metabolic stability and stimulus-responsive characteristics. Among various types of nanocarriers, the theranostic capabilities of multifunctional nanocarrier offers great potential to support more precise treatment strategies for cancer and chronic inflammatory diseases. Also, intelligent nanocarriers, hybrid lipid-polymer systems and biomimetic delivery platforms may offer even greater therapeutic impact with reduced off-target cytotoxicity. Progresses in computational modelling, artificial intelligence-assisted formulation design, and QbD principles are also anticipated to support the rational design, optimization, and scale-up of piperine-based drug products; thus facilitating their clinical translational.

Sustainable Production and Extraction Methods

Similar to formulation development, sustainable production has emerged as an important component of the future commercialization of piperine-based products. The adoption of green extraction technologies that reduce solvent use, energy consumption and waste generation without loss of efficiency has been accelerated by increasing environmental awareness and less stringent regulatory expectations. UAE, MAE, UMAE and NADES-based extraction are presented as methodologies that could replace conventional solvent extraction with much more sustainable alternatives. On the other hand, high piperine recovery by itself will not suffice for industrial application. Future studies will also need to focus on process scalability, solvent recyclability, long-term operational stability as well as life-cycle sustainability and economic viability. Techno-economic analysis and life-cycle assessment should be included in the process-development phase so that a more holistic view of these technologies can be obtained, which would ultimately facilitate the transition from laboratory-scale research to commercial-scale production.

Clinical Validation and Regulatory Pathways

Any future progress in reaching the therapeutic application of piperine will be critically dependent on comprehensive confirmation in the clinic and elaboration of explicit steps for regulatory approval. Despite a lot of preclinical and early-phase clinical studies showing potential pharmacological and bio-enhancing properties, large-scale multicenter RCTs data still have limited supporting evidence. Future clinical investigations should prioritize standardized formulations, adequately powered study populations, longer follow-up periods, and clinically relevant endpoints to establish definitive efficacy and safety profiles across different disease indications.

In addition, a deeper understanding of the pharmacokinetic-pharmacodynamic (PK-PD) relationships of piperine is required to optimize therapeutic outcomes and minimize variability in clinical response. The integration of population pharmacokinetic analyses, PBPK modelling, and exposure-response assessments may facilitate rational dose selection, prediction of DDI, and individualized treatment strategies. Such approaches are particularly important given the dual role of piperine as both a therapeutic agent and a bioavailability enhancer.

From a regulatory perspective, harmonized evaluation frameworks for phytopharmaceuticals, bioenhancers, and nano-enabled formulations remain underdeveloped in many jurisdictions. Differences in product classification, quality requirements, and approval pathways may hinder the global development and commercialization of piperine-containing products. Therefore, advances in clinical validation, PK-PD integration, PBPK-guided dose optimization, and regulatory standardization will be critical for accelerating the successful translation of piperine from promising research candidate to clinically accepted therapeutic product. As summarized in Figure 10, these interdependent priorities form an evidence-gated development sequence from standardized material and scalable manufacturing through nonclinical validation, phased clinical evaluation, regulatory review, market authorization, and post-marketing surveillance.

Figure 10.

Evidence-gated clinical development roadmap for translating piperine into a pharmaceutical product. The roadmap links the current evidence base—which remains predominantly preclinical and largely based on combination interventions—to six consecutive development stages: (1) current evidence, defining the principal limitations that remain to be addressed; (2) material quality, requiring qualified reference materials, validated analytical methods, and defined purity and batch specifications; (3) scalable product development, integrating GMP-compatible processing, batch consistency, formulation development, and CMC requirements; (4) nonclinical validation, evaluating comparative PK/PD, toxicology, target-organ safety, and substrate-specific drug-interaction risks before human testing; (5) phased clinical development, progressing from Phase I safety and human PK evaluation through Phase II proof-of-concept studies to adequately supported Phase III trials and longer-term safety assessment; and (6) the regulatory and market pathway, comprising regulatory submission, benefit–risk review, market authorization, and post-marketing surveillance. The arrows indicate the proposed sequence of standardization, scale-up, validation, translation, and authorization. Progression between stages is conditional on predefined quality, safety, pharmacokinetic, and efficacy criteria.

Conclusion

Piperine has been widely investigated as a natural-product scaffold across multiple biological pathways, alongside advances in sustainable extraction, purification, medicinal chemistry, and formulation development. However, most pharmacological evidence remains preclinical, computationally proposed targets frequently lack direct experimental validation, and improvements in extraction recovery, solubility, dissolution, or animal systemic exposure do not establish pharmaceutical readiness. Translation remains constrained by variability in starting-material quality, insufficient analytical standardization and scale-up evidence, limited pharmacokinetic characterization of stand-alone piperine in humans, substrate-dependent drug interactions, uncertain long-term safety, and clinical evidence derived predominantly from small studies of combination interventions.

Five priorities should therefore guide further development: (1) establish qualified reference materials and validated analytical methods for piperine quantification and impurity control; (2) optimize scalable green extraction and purification under GMP-compatible conditions, considering batch consistency, solvent recovery, energy demand, and techno-economic feasibility; (3) develop standardized formulations supported by integrated pharmacokinetic, pharmacodynamic, and drug-interaction assessments; (4) conduct prospective human safety studies with longer follow-up, including periods of 12 months or more where appropriate; and (5) advance indication-specific candidates through appropriately sequenced dose-finding, pharmacokinetic, safety, and proof-of-concept studies, followed, when supported by preceding evidence, by adequately powered multicenter Phase III trials. These priorities should be accompanied by harmonized quality and regulatory requirements for piperine-containing products. Without resolving the clinical-evidence and manufacturing-scale-up gaps, piperine is likely to remain primarily a nutraceutical or bioenhancing ingredient rather than become a well-established pharmaceutical agent.

Funding Statement

This publication charge is funded by Universitas Padjadjaran through the Indonesian Endowment Fund for Education (LPDP) on behalf of the Indonesian Ministry of Higher Education, Science and Technology and managed under the EQUITY Program (Contract No. 4303/ B3/DT.03.08/2025 and 3927/UN6. RKT/HK.07.00/2025); and Universitas Padjadjaran through Beasiswa Unggulan Pascasarjana Padjadjaran (BUPP) Grant.

Abbreviations

α-Syn, α-synuclein; ACOT1, acyl-CoA thioesterase 1; ADMET, Absorption, distribution, metabolism, excretion, and toxicity; AGREE, Analytical GREEnness Metric; ALP, alkaline phosphatase; ALT, alanine aminotransferase; APAP, acetaminophen (paracetamol); AST, aspartarte aminotransferase; AUC, area under the plasma concentration-time curve; BBD, Box-Behnken design; BCS, Biopharmaceutics classification system; BDNF, Brain-derived neurotrophic factor; CAT, Catalase; CCD, Central composite design; CHIKV, Chikungunya virus; CIMT, Carotid intima-media thickness; CMC, chemistry, manufacturing, and controls; Cmax, maximum plasma concentration; CNN, Convolutional neural network; COX-2, cyclooxygenase-2; CRP, C-Reactive Protein; CYP3A4, cytochrome P450 3A4; DAO, D-amino acid oxidase; DDI, Drug-drug interactions; DDS, Drug delivery system; DES, Deep eutectic solvent; DSS, Dextran sulfate sodium; EAE, Experimental autoimmune encepalomyelitis; EE, encapsulation efficiency; EFSA, European Food Safety Authority; EGDMA, Ethylene glycol dimethacrylate; ESR, Erythrocyte Sedimentation Rate; FST, Forced swim test; FTIR, Fourier Transform Infrared; GABA, γ-aminobutyric acid; GAPI, Green analytical procedure index; GC-MS, Gas chromatography-mass spectrometry; GMP, Good Manufacturing Practice; GSH, reduced glutathione; HAE, Homogenizer-assisted extraction; HBA, Hydrogen bond acceptor; HBD, Hydrogen bond donor; HO-1, heme oxygenase-1; HPTLC, High-performance thin-layer chromatography; HSC, Hepatic-stellate cell; IC₅₀, Inhibitory Concentration 50%; ICU, Intensive care unit; IL, interleukin; ILs, Ionic liquids; IKP-PI IL, Ketoprofen-piperine ionic liquid; IRAK1, interleukin-1 receptor-associated kinase 1; LC₅₀, Lethal Concentration 50%; LC-MS/MS, Liquid chromatography-tandem mass spectrometry; LD₅₀, Lethal Dose 50%; LMBV, Largemouth bass virus; LPHNPs, Lipid-polymer hybrid nanoparticles; LPO, lipid peroxidation; LPS, Lipopolysaccharide; MAE, Microwave-assisted extraction; MAPE, Mean absolute percentage error; MAPK, mitogen-activated protein kinase; MD, Molecular dynamics; MDA, Malondialdehyde; MERS-CoV, Middle East respiratory syndrome coronavirus; MGUS, Monoclonal gammopathy of undetermined significance; MIC, Minimum inhibitory concentration; MIP, Molecularly imprinted polymer; MRSA, Methicillin-resistant Staphylococcus aureus; MSNs, Mesoporous silica nanoparticles; MW, Molecular weight; NADES, Natural deep eutectic solvent; NF-κB, nuclear factor kappa B; NLRP3, NOD-like receptor family pyrin domain containing 3; NMR, Nuclear magnetic resonance; NOAEL, No-observed-adverse-effect level; Nrf2, nuclear factor erythroid 2-related factor 2; OFAT, One-factor-at-a-time; PAT, Process analytical technologies; PBPK, Physiologically based pharmacokinetic; PCOS, Polycystic Ovarian Syndrome; PDMAM, Poly (N,N-dimethylacrylamide); PEG, Polyethylene Glycol; PGE2, Prostaglandin E2; P-gp, P-glycoprotein; PK-PD, Pharmacokinetic-pharmacodynamic; PPCI, Primary percutaneous coronary intervention; QbD, Quality by design; QSAR, Quantitative structure-activity relationship; RCT, Randomized controlled trial; RMSD, Root mean square deviation; ROS, Reactive oxygen species; RP-HPLC, Reverse phase-high performance liquid chromatography; RSM, Response surface methodology; SAR, Structure-activity relationship; SASA, Solvent accessible surface area; SD, Steam distillation; SFE, Supercritical fluid extraction; SLE, Systemic lupus erythematosus; SMM, Smoldering multiple myeloma; SNEDDS, Self-nanoemulsifying drug delivery systems; SOD, superoxide dismutase; STEMI, ST-elevation myocardial infarction; TAC, Tacrolimus; TE, Traditional extracts; TGF- β, transforming growth factor-beta; Th, T-helper; TLC, Thin layer chromatography; TLR4, Toll-like receptor 4; TNF- α, tumor necrosis factor-alpha; TRDES, Temperature-responsive deep eutectic solvent; TRPV1, transient receptor potential vanilloid 1; TST, Tail suspension test; UAE, Ultrasound-assisted extraction; UBE, Ultrasonic bath extraction; UMAE, Ultrasound-microwave-assisted extraction; UPE, Ultrasonic probe extraction; UPLC, Ultra-performance liquid chromatography.

Data Sharing Statement

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

Disclosure

The authors declare no conflicts of interest in this work.