Impact of ripening on the distribution of jackfruit polyphenol fractions and their inhibitory activities against metabolic enzymes

TL;DR

This study shows how ripening affects jackfruit polyphenols and their ability to inhibit enzymes linked to metabolism, suggesting ripe jackfruit has the most potential for health applications.

Contribution

The study reveals ripening-dependent changes in jackfruit polyphenol fractions and their enzyme inhibitory potential, highlighting ripe fruit's superior bioactivity.

Findings

Ripening increases conjugated phenolics' antioxidant and enzyme inhibition capabilities.

Ripe jackfruit (J3) shows the strongest conjugated phenolic bioactivities and diversity.

Molecular docking identifies cyclomorusin and cyclokievitone as strong enzyme ligands.

Abstract

This study evaluated polyphenol fractions from unripe (J1), underripe (J2), and ripe (J3) jackfruit to explore stage-dependent industrial potential. Free (FP), conjugated (CP), and bound phenolics (BP) were isolated and assessed for antioxidant and enzyme inhibitory activities. Ripening significantly altered composition and bioactivity. CP antioxidant capacity and inhibition of α-glucosidase, pancreatic lipase, and xanthine oxidase increased progressively from J1 to J3, while FP and BP showed fluctuating trends. Ripe fruit (J3) exhibited the strongest CP-related bioactivities and a more diverse conjugated phenolic profile, whereas early stages had greater FP compositional complexity. Molecular docking supported these findings, identifying cyclomorusin as a potent ligand for α-glucosidase (−9.6 kcal/mol) and xanthine oxidase (−11.8 kcal/mol), and cyclokievitone for pancreatic lipase (−6.7 kcal/mol). These results demonstrate stage-specific functional differentiation and support targeted industrial utilization for health promotion.

Unlabelled Image

•Polyphenol redistribution in jackfruit pulp is ripening-dependent.•Free phenolics increased with ripening, while conjugated and bound forms decreased.•The conjugated phenolic fraction exhibited the most diverse phenolic profile.•Conjugated phenolics represent a promising feedstock for nutraceutical development.•Jackfruit polyphenols bind metabolic enzymes via noncovalent interactions.

Polyphenol redistribution in jackfruit pulp is ripening-dependent.

Free phenolics increased with ripening, while conjugated and bound forms decreased.

The conjugated phenolic fraction exhibited the most diverse phenolic profile.

Conjugated phenolics represent a promising feedstock for nutraceutical development.

Jackfruit polyphenols bind metabolic enzymes via noncovalent interactions.

Figures

SEM micrographs of insoluble-bound phenolic residues from jackfruit at various ripening stages: (a) J1-IBPR, (b) J2-IBPR, (c) J3-IBPR, (d) J1-IBPR-NaOH, (e) J2-IBPR-NaOH, and (f) J3-IBPR-NaOH. J1, J2, and J3 represent different ripening stages. a–c depict the morphologies of residues prior to hydrolysis, whereas d–f show the corresponding residues following alkaline hydrolysis. Notable differences in fragmentation and dispersion are observed between J1-IBPR-NaOH (d) and J3-IBPR-NaOH (f), reflecting maturity-dependent responses of insoluble-bound phenolic residues to alkaline treatment.Fig. 1

Confocal laser scanning microscopy (CLSM) images of soluble conjugated phenolic residues (SCPR) and insoluble-bound phenolic residues (IBPR) from jackfruit at different ripening stages. (a) Soluble conjugated phenolic residues: (I) J1-SCPR, (II) J2-SCPR, (III) J3-SCPR, (IV) J1-SCPR-NaOH, (V) J2-SCPR-NaOH, (VI) J3-SCPR-NaOH. (b) Insoluble-bound phenolic residues: (I) J1-IBPR, (II) J2-IBPR, (III) J3-IBPR, (IV) J1-IBPR-NaOH, (V) J2-IBPR-NaOH, (VI) J3-IBPR-NaOH. Scale bars = 20 μm for all images. J1, J2, and J3 represent different ripening stages; SCPR denotes soluble conjugated phenolic residues,

FTIR spectra of soluble conjugated phenolic residues (SCPR) and insoluble-bound phenolic residues (IBPR) from jackfruit at different ripening stages. (a) Soluble conjugated phenolic residues (SCPR) and (b) insoluble-bound phenolic residues (IBPR). J1, J2, and J3 correspond to different ripening stages. SCPR-NaOH denotes SCPR residues after alkaline hydrolysis, and IBPR-NaOH denotes IBPR residues after alkaline hydrolysis.Fig. 3

Analysis of polyphenol content and antioxidant activity in jackfruit. Lowercase letters (a–c) denote significant differences (*p* < 0.05) among the groups. (a) Total phenolic content (TPC); (b) Total flavonoid content (TFC); (c) DPPH radical scavenging activity; (d) ABTS radical scavenging activity; (e) FRAP Assay for Reducing Power Assessment.Fig. 4

Inhibitory effects of jackfruit polyphenols on digestive enzymes. The reciprocal of the IC_50_ values was plotted to better visualize the trends in inhibitory activity during ripening. Different lowercase letters denote significant differences (*p* < 0.05). (a) α-Glucosidase inhibition; (b) pancreatic lipase inhibition; (c) xanthine oxidase inhibition.Fig. 5

Full text

Introduction

1

Polyphenols are widely acknowledged as natural agents capable of mitigating chronic metabolic disorders through multi-target modulation of oxidative stress, post-prandial glycaemia, lipid digestion, and serum uric acid levels (Agrawal et al., 2022). Within the plant matrix, these metabolites are not uniformly distributed but are classified into free, conjugated, and bound fractions according to their mode and strength of association with structural components of the plant tissue, which differ markedly in extractability, bioaccessibility, and functional relevance (da Silva et al., 2024). While free polyphenols are readily released by conventional solvent extraction, conjugated and bound forms are covalently or non-covalently associated with cell-wall components and require chemical or enzymatic disruption for their liberation (Zhao et al., 2025). Importantly, the relative distribution among these phenolic pools is not static but dynamically reshaped during fruit development and ripening, reflecting coordinated biosynthetic activity and cell-wall remodeling processes (Zhang et al., 2015).

Jackfruit (Artocarpus heterophyllus Lam.) represents a distinctive model for investigating such ripening-dependent phenolic dynamics. Recent studies have reported exceptionally high total phenolic contents (TPC) in jackfruit, reaching up to 1567.00 ± 33.15 mg GAE/100 g (Konsue et al., 2023), substantially exceeding those of many commonly studied fruits, such as banana (78.00 ± 0.12 mg GAE/100 g) (Mallmann et al., 2026). Beyond its high phenolic abundance, jackfruit is characterized by a complex phenolic architecture embedded within a dense polysaccharide-rich matrix (Zhu et al., 2019), suggesting a substantial proportion of phenolics may reside in conjugated or bound forms. Such structural features imply that jackfruit polyphenols are particularly sensitive to physiological transitions during ripening, yet the magnitude, directionality, and functional consequences of phenolic redistribution among free, conjugated, and bound pools remain poorly quantified (Rana et al., 2018).

This knowledge gap contributes to a paradoxical under-utilization of jackfruit bioactives. Although jackfruit is among the highest-volume tropical fruits produced globally, its commercial valorization is largely confined to the fresh consumption of fully ripe pulp, where sensory attributes such as sweetness and texture are prioritized (Omar Chavez-Santiago et al., 2022). This utilization paradigm implicitly assumes that nutritional and functional value increases monotonically with ripening. Consequently, immature (J1, unripe) and transitional (J2, underripe) fruits—despite undergoing profound metabolic reprogramming—are frequently relegated to low-value uses or discarded as agricultural by-products (Kaur et al., 2024). Rather than representing inferior raw materials, these earlier developmental stages may constitute functionally specialized biochemical resources whose optimal application lies outside the fresh-consumption pathway. Understanding how ripening governs the metabolic fate of polyphenols is therefore essential for identifying stage-specific utilization strategies and defining an optimum harvest window aligned with targeted functional outcomes.

Accordingly, the central research question of this study is how physiological transitions during jackfruit ripening drive the redistribution of polyphenols among free, conjugated, and bound states, and whether these compositional shifts selectively modulate inhibitory potency against enzymes associated with chronic metabolic disorders. To address this, polyphenol fractions from unripe (J1), underripe (J2), and ripe (J3) jackfruit were systematically profiled using HPLC-HR-TOF-MS^2^ and HPLC-DAD. Their inhibitory effects against α-glucosidase, pancreatic lipase, and xanthine oxidase were evaluated in vitro, with key ligand-enzyme interactions further explored through in silico molecular docking. By integrating ripening stage, phenolic form, and bioactivity, this framework provides a mechanistic basis for repositioning jackfruit from a single-use fresh commodity toward a developmentally informed source of high-value functional ingredients.

Materials and methods

2

Plant material

2.1

This study utilized Thai variety 5 (T5) jackfruit, harvested in June from the experimental base of the Spice and Beverage Research Institute in Wanning, Hainan Province, China (18°15′N, 110°13′E). A hybrid sampling strategy was employed to encompass both natural on-tree developmental transitions and commercial postharvest ripening processes. Two distinct stages were harvested directly from the orchard: immature fruits (J1, unripe) and commercially mature fruits (J2, underripe). J1 represents an early developmental stage characterized by physiological immaturity, where fruits lack the capacity for independent ripening off-tree. J2 was harvested at the stage of physiological maturity—the standard window for commercial logistics—identified by flattened spines and a hollow sound upon tapping (Ranasinghe et al., 2019).

To simulate the real-world supply chain, a portion of the J2 batch was subsequently subjected to controlled postharvest ripening (28 ± 1 °C, 70–75% relative humidity) to reach full edible maturity (J3, ripe) after 2–3 days. The classification of J1, J2, and J3 was validated by objective ripeness-related parameters, including color difference and soluble solids content, as detailed in Tables S1–S2. These quantitative indicators confirmed the characteristic metabolic transition from an immature state to edible ripeness. Following classification, the jackfruit pulp (after core removal) was freeze-dried, powdered, sieved (40 mesh), and stored at −20 °C until further analysis.‌.

Chemical reagents

2.2

Reagents and standards were procured from various suppliers: DPPH, ABTS, TPTZ, pancreatic lipase, pNPP, orlistat, HPLC-grade formic acid, and spectroscopic grade potassium bromide were obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Trolox, α-glucosidase, PNPG, acarbose, xanthine oxidase, xanthine, allopurinol, Congo red stain, and 18 phenolic standards (including gallic acid, chlorogenic acid, and hydroxybenzoic acid) were supplied by Shanghai YuanYe Biotechnology Co., Ltd. (Shanghai, China). HPLC-grade methanol and acetonitrile were sourced from Sigma-Aldrich (USA). Folin-Ciocalteu reagent and ethyl acetate were purchased from Beijing Solarbio Technology Co., Ltd. (Beijing, China) and Guangdong Xilong Scientific Co., Ltd. (Guangdong, China), respectively.

Extraction of phenolic fractions

2.3

Given the efficiency of acetone in preliminary solvent screening tests (Table S3), an 80% (v/v) acetone solution was employed for polyphenol extraction, following the method of Chen et al. (2015) with specific optimizations.

Free phenolics (FP)

2.3.1

Accurately weigh 1 g of jackfruit powder and mix with 80% acetone at a 1:50 (w/v) ratio. Ultrasonic extraction was performed using an ultrasonic cleaner (GT SONIC-T20, Guangdong GT Ultrasonic Co., Ltd., Meizhou, China) at 400 W for 30 min at room temperature, centrifuge at 12,000 rpm for 15 min at 4 °C. Combine extracts after double extraction. Acetone was removed by rotary evaporation at 35 °C using a vacuum controller (V-700, BÜCHI Labortechnik AG, Flawil, Switzerland), followed by liquid-liquid extraction with ethyl acetate (1:1, v/v) three times. Concentrate the organic phase under reduced pressure and dry with nitrogen blow-down to obtain FP. FP from different maturity stages were designated as J1-FP, J2-FP, and J3-FP. Each extraction was conducted in triplicate.

Conjugated phenolics (CP)

2.3.2

Mix the aqueous phase remaining after FP extraction with an equal volume of 2 mol/L NaOH and hydrolyze at room temperature for 4 h. Acidify to pH 2.0 with 6 mol/L HCl, then extract with ethyl acetate (1:1, v/v) three times. Concentrate and dry the organic phase to obtain CP, designated as J1-CP, J2-CP, and J3-CP.

Bound phenolics (BP)

2.3.3

Residues post-acetone extraction and 2 mol/L NaOH at a 1:50 (w/v) ratio and alkaline hydrolyze for 4 h at room temperature. Acidify to pH 2.0 with 6 mol/L HCl, centrifuge, and extract the supernatant with ethyl acetate (1:1, v/v) three times. Concentrate and dry the organic phase to obtain BP, designated as J1-BP, J2-BP, and J3-BP.

All extracts were stored at −40 °C prior to analysis.

Structural characterization of phenolic residues

2.4

The aqueous phase remaining after FP extraction and the residues obtained after acetone extraction were freeze-dried to yield soluble conjugated phenolic residues (SCPR) and insoluble bound phenolic residues (IBPR), respectively. Due to differences in their physicochemical properties, specific structural characterization techniques were selectively applied.

Scanning electron microscopy (SEM)

2.4.1

SEM analysis was performed only on IBPR samples. IBPR retained a relatively stable solid structure after extraction and freeze-drying, making it suitable for morphological observation under high-vacuum conditions. In contrast, SCPR originated from aqueous extracts and exhibited strong hygroscopicity and structural instability even after freeze-drying, rendering them unsuitable for SEM imaging.

Adapting the method of Kenari and Razavi (2022), a small amount of IBPR was fixed with conductive carbon glue, sputter-coated with gold for 100 s to enhance conductivity, and examined using a scanning electron microscope (Phenom ProX, Phenom-World B.V., Eindhoven, Netherlands) at 5 kV and 10 mA in high-vacuum mode.

Confocal laser scanning microscopy (CLSM)

2.4.2

Following Yu et al. (2025), both SCPR and IBPR were stained with 0.1% Congo red and analyzed using CLSM (FV10i, Olympus Corporation, Tokyo, Japan). Dietary fibers exhibited characteristic fluorescence at 488 nm excitation, while phenolic compounds were detected at 408 nm based on intrinsic fluorescence.

Fourier transform infrared spectroscopy (FTIR)

2.4.3

According to Jiang et al. (2022), SCPR and IBPR were mixed with KBr powder at a ratio of 1:100 (w/w). FTIR spectra were recorded from 4000 to 400 cm^−1^ on a Nicolet 6700 spectrometer (Thermo Fisher Scientific, Madison, WI, USA), with 64 scans at a resolution of 4 cm^−1^. Data processing was performed using OMNIC software. All measurements were conducted in triplicate.

Determination of polyphenol content

2.5

Total phenolic content (TPC)

2.5.1

TPC was assessed using the Folin-Ciocalteu method (Wekre et al., 2022). Briefly, 100 μL of jackfruit polyphenol solution was mixed with 500 μL of 50% Folin-Ciocalteu reagent, reacted for 8 min in the dark, and then 400 μL of 7.5% Na_2_CO_3_ was added for a 1 h reaction. Absorbance was measured at 765 nm. Results were expressed as mg gallic acid equivalents (GAE)/g dry weight (DW), using a gallic acid standard curve (0.02–0.24 mg/mL, R^2^ = 0.9992). Measurements were performed in triplicate.

Total flavonoid content (TFC)

2.5.2

TFC was determined using the aluminum nitrate colorimetric method (Li et al., 2022). Sequentially add 75 μL of 724.6 mM NaNO_2_, 150 μL of 266.6 mM Al(NO_3_)3, and 300 μL of 1 M NaOH to 150 μL of polyphenol solution, with 5 min dark reactions between steps. Absorbance was measured at 510 nm, and results were expressed as mg rutin equivalents (RE)/g DW based on a rutin standard curve (0.05–0.8 mg/mL, R^2^ = 0.9996). Measurements were performed in triplicate.

Antioxidant activity of phenolic compounds

2.6

DPPH radical scavenging activity

2.6.1

DPPH radical scavenging activity was assessed following Nakagawa et al. (2021). Mix 100 μL of polyphenol solution with 800 μL of 0.159 mM DPPH ethanol solution, incubate for 30 min in the dark, and measure absorbance at 517 nm. Results were expressed as mg Trolox equivalents (TE)/mL based on a Trolox standard curve (0.005–0.12 mg/mL, R^2^ = 0.9992). Measurements were performed in triplicate.

ABTS radical scavenging activity

2.6.2

ABTS radical scavenging activity was evaluated following Zhang et al. (2022). Generate ABTS radicals by mixing 10 mL of 7 mM ABTS stock solution with 176 μL of 140 mM K_2_S_2_O_8_, oxidize for 16 h at 4 °C in the dark, and dilute to an absorbance of 0.70 ± 0.02 at 734 nm. Mix 80 μL of polyphenol solution with 800 μL of working solution, react for 6 min in the dark, and measure absorbance. Results were expressed as mg TE/mL based on a Trolox standard curve (0.005–0.12 mg/mL, R^2^ = 0.9993). Measurements were performed in triplicate.

FRAP assay

2.6.3

The FRAP assay was conducted following Bouslamti et al. (2022). The FRAP working solution was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ, and 20 mM FeCl₃ in a 10:1:1 ratio and preheated at 37 °C. Mix 100 μL of polyphenol solution with 900 μL of working solution, incubate for 30 min at 37 °C in the dark, and measure absorbance at 593 nm. Results were expressed as mg TE/mL based on a Trolox standard curve (0.005–0.12 mg/mL, R^2^ = 0.9991). Measurements were performed in triplicate.

HPLC-HR TOF MS2 characterization and HPLC-PDA quantification

2.7

Jackfruit polyphenols were analyzed using a high-performance liquid chromatography (HPLC) system equipped with a photodiode array (PDA) detector. Prior to injection, the samples were centrifuged, and the supernatant was filtered through a 0.22 μm organic-phase membrane filter before chromatographic analysis. Chromatographic separation was performed on an Agilent Eclipse XDB-C_18_ column (250 mm × 4.6 mm, 5 μm particle size, 80 Å pore size) maintained at 30 °C, with an injection volume of 10 μL. The mobile phase consisted of acetonitrile (A) and 0.1% formic acid in water (B). The gradient elution program was as follows: 0–6 min, 15% A; 6–25 min, 35% A; 25–40 min, 50% A; 40–45 min, 80% A; and 45–50 min, 15% A. The flow rate was 0.8 mL/min, and detection was performed at 280 nm.

Phenolic compounds were characterized using HPLC-HR TOF MS^2^ (AB SCIEX TripleTOF™ 6600+). The system comprised an HPLC unit and a mass spectrometer with an electrospray ionization (ESI) source. Analysis was performed in positive/negative ion modes with a scan range of 50–1250 m/z for both MS and MS/MS. Ion spray voltage was set to ±5500 V/4500 V, declustering potential to ±80 V, and collision energy to ±30 V. Source gas 1 and 2 were adjusted to 50 and 60 psi, respectively, curtain gas to 35 psi, and temperature to 550 °C. Compounds were identified using PeakView 2.1 software based on exact molecular weight (error < 3 ppm), MS/MS fragments, and retention times of standards. Quantification was based on external standard curves (R^2^ > 0.999), with data processing performed using MasterView software.

Enzyme inhibition assays

2.8

α-Glucosidase inhibition assay

2.8.1

Following Gong et al. (2020), α-glucosidase solution (0.04 U/mL in 200 mM PBS, pH 6.8) was mixed with polyphenol solution in a 2:1 ratio (total volume 300 μL). After pre-incubation at 37 °C for 10 min, 200 μL of PNPG substrate (5 mM) was added to initiate the reaction. After 20 min, the reaction was terminated with 0.2 M Na_2_CO_3_, and p-nitrophenol production was monitored at 405 nm. Acarbose served as the positive control, and the half-inhibitory concentration (IC_50_) was calculated.

Pancreatic lipase inhibition assay

2.8.2

Adapting Zhou et al. (2021), the enzyme working solution (50 U/mL) was prepared in 50 mM Tris-HCl buffer (pH 7.1) containing 0.1% arabic gum and 0.2% sodium taurocholate. The substrate system comprised 1.5 mM PNPP with 0.8% isopropanol and 0.2% Triton X-100. Mix 80 μL of polyphenol solution with 100 μL of enzyme solution, pre-incubate at 37 °C for 10 min, then add 600 μL of substrate to start the reaction (37 °C, 60 min). Orlistat served as the positive control, and IC_50_ was calculated.

Xanthine oxidase inhibition assay

2.8.3

Following Dolati et al. (2018), xanthine oxidase was dissolved in 50 mM Tris buffer (pH 7.5) to prepare a 0.1 U/mL working solution. Mix 100 μL of polyphenol solution with an equal volume of enzyme solution, pre-incubate at 37 °C for 10 min, then add 400 μL of 0.5 mM xanthine to start the reaction (total volume 600 μL). After 30 min at 37 °C, terminate the reaction with 300 μL of 1 M HCl and measure absorbance at 290 nm. Allopurinol served as the positive control, and IC_50_ was calculated.

Molecular docking simulations

2.9

The crystal structures of α-glucosidase (PDB ID: 2QMJ), pancreatic lipase (PDB ID: 1GPL), and xanthine oxidase (PDB ID: 1N5X) were retrieved from the RCSB Protein Data Bank. Receptor preprocessing was performed using PyMOL, including the removal of crystallographic water molecules and non-covalently bound ligands.

Ligand screening was conducted based on phenolic compounds identified by HPLC–HR TOF MS^2^. Compounds were ranked according to their VIP values, and the top 50 candidates were selected for further analysis. Z-score normalization was applied across nine sample groups (J1-FP/CP/BP, J2-FP/CP/BP, and J3-FP/CP/BP), and compounds with Z-scores greater than 0 were retained, resulting in the selection of 46 ligands (Fig. S2). Ligand structures were retrieved from PubChem and converted from SDF to PDB format using Open Babel.

Receptor preparation, including the addition of polar hydrogen atoms, optimization of protonation states, and assignment of Gasteiger charges, was performed using AutoDock Tools. Molecular docking was subsequently carried out using AutoDock Vina version 1.5.7, following the protocol described by (Sui et al., 2016).

The grid box parameters were individually defined for each target protein and centered on the respective active sites to ensure complete coverage of the binding pockets. For α-glucosidase (2QMJ), the grid box center was set at x = −29.211, y = 4.011, z = −18.494, with dimensions of 97.65 × 97.65 × 86.8 Å. For pancreatic lipase (1GPL), the grid box center was set at x = −29.211, y = 4.011, z = −18.494, with dimensions of 62.72 × 66.27 × 66.27 Å. For xanthine oxidase (1N5X), the grid box center was set at x = 116.428, y = 79.353, z = 43.095, with dimensions of 126.0 × 126.0 × 124.0 Å. For each docking run, 15 binding modes were generated.

The top nine high-affinity complexes for each target enzyme were selected based on binding energy. Hydrogen bonding, hydrophobic interactions, and π–π stacking interactions were analyzed using PyMOL and Discovery Studio 4.5.

Statistical analysis

2.10

Statistical analysis was performed using IBM SPSS Statistics 26. Duncan's multiple range test was employed to assess significant differences at p < 0.05, with results expressed as mean ± standard deviation. All experiments were conducted in triplicate.

Results and discussion

3

Structural characterization of phenolic compounds

3.1

Scanning electron microscopy

3.1.1

SEM analysis highlighted distinct morphological changes in IBPR residues across different jackfruit maturity stages. As depicted in Fig. 1a–b, residues from the early stages (J1 and J2) displayed irregular ellipsoidal shapes with pronounced surface wrinkling, likely attributable to structural collapse or shrinkage during freeze-drying or ultrasonic extraction. In contrast, residues from the fully mature stage (J3) exhibited curled lamellar morphologies (Fig. 1c), akin to those observed in insoluble-bound phenolic residues of lychee pulp (Xu et al., 2020). These differences may stem from the high starch content in early ripening stages, which diminishes as dietary fiber accumulates with advancing maturity (Ranasinghe et al., 2019).Fig. 1SEM micrographs of insoluble-bound phenolic residues from jackfruit at various ripening stages: (a) J1-IBPR, (b) J2-IBPR, (c) J3-IBPR, (d) J1-IBPR-NaOH, (e) J2-IBPR-NaOH, and (f) J3-IBPR-NaOH. J1, J2, and J3 represent different ripening stages. a–c depict the morphologies of residues prior to hydrolysis, whereas d–f show the corresponding residues following alkaline hydrolysis. Notable differences in fragmentation and dispersion are observed between J1-IBPR-NaOH (d) and J3-IBPR-NaOH (f), reflecting maturity-dependent responses of insoluble-bound phenolic residues to alkaline treatment.Fig. 1

Post-alkaline hydrolysis, the residue surfaces exhibited fragmentation and severe disruption of the cell wall network (Fig. 1d–f). Notably, distinct differences were observed between J1-IBPR-NaOH (Fig. 1d) and J3-IBPR-NaOH (Fig. 1f). Residues from the early ripening stage (J1) remained as relatively large and compact aggregates after alkaline treatment, whereas residues from the fully mature stage (J3) displayed extensive fragmentation and a highly dispersed morphology composed of fine particles. This enhanced disintegration in J3 may be attributed to maturity-dependent loosening of cell wall structures and alterations in the binding modes of insoluble-bound phenolics, which facilitate the cleavage of ester or weak covalent linkages under alkaline conditions.

This structural breakdown likely results from the hydrolysis of dietary fiber components under strongly alkaline conditions, facilitating the release of bound phenolics. Consistent with these observations, Jiang et al. (2022) reported that phenolic-bound dietary fibers exhibit denser and more compact structures, whereas alkaline treatment induces surface fragmentation, spicule formation, and increased porosity. Overall, SEM imaging confirms that both maturity and alkaline treatment significantly influence the morphological characteristics of jackfruit residues.

Confocal laser scanning microscopy

3.1.2

CLSM was used to examine the morphological features of jackfruit residues and their interaction with polyphenols before and after alkaline hydrolysis. Dietary fibers were stained with Congo red to visualize their microstructure, producing green fluorescence (Fig. 2). The red signal, linked to the intrinsic fluorescence of polyphenols, particularly ferulic acid bound to dietary fibers (Guo & Beta, 2013), was also observed. SCPR from different maturity stages showed elongated structures (Fig. 2a I-III), suggesting strong and complex interactions between polyphenols and dietary fibers. IBPR residues from the early stages (J1 and J2) exhibited similar morphologies (Fig. 2b I, II), but significant differences were observed in the fully mature stage (J3) (Fig. 2b III), aligning with SEM findings. These variations are likely due to changes in carbohydrate composition during ripening. After alkaline hydrolysis, the surface of dietary fibers became more porous and loose, with visible pores, cracks, and fragmented debris (Fig. 2a IV-VI, b IV-VI). Similar observations were reported by Xu et al. (2020). CLSM fluorescence analysis confirmed the binding of polyphenols to dietary fibers and provided evidence that alkaline hydrolysis disrupts dietary fiber structures, promoting the release of phenolic compounds.Fig. 2. Confocal laser scanning microscopy (CLSM) images of soluble conjugated phenolic residues (SCPR) and insoluble-bound phenolic residues (IBPR) from jackfruit at different ripening stages. (a) Soluble conjugated phenolic residues: (I) J1-SCPR, (II) J2-SCPR, (III) J3-SCPR, (IV) J1-SCPR-NaOH, (V) J2-SCPR-NaOH, (VI) J3-SCPR-NaOH. (b) Insoluble-bound phenolic residues: (I) J1-IBPR, (II) J2-IBPR, (III) J3-IBPR, (IV) J1-IBPR-NaOH, (V) J2-IBPR-NaOH, (VI) J3-IBPR-NaOH. Scale bars = 20 μm for all images. J1, J2, and J3 represent different ripening stages; SCPR denotes soluble conjugated phenolic residues, and SCPR-NaOH represents SCPR residues after alkaline hydrolysis; IBPR denotes insoluble-bound phenolic residues, and IBPR-NaOH represents IBPR residues after alkaline hydrolysis.Fig. 2

Fourier transform infrared spectroscopy

3.1.3

FTIR analysis was performed on residues of soluble conjugated phenolics and insoluble bound phenolics from jackfruit at different maturity stages, both before and after alkaline hydrolysis, to identify functional group vibrations characteristic of phenolic compounds. As shown in Fig. 3, peak shapes for both SCPR and IBPR residues remained consistent across maturity stages, but peak intensities varied significantly. Conjugated phenolics exhibited more peaks than bound phenolics, indicating a more diverse phenolic composition, a result corroborated by HPLC analysis. Alkaline hydrolysis reduced peak intensities compared to unhydrolysis spectra, suggesting effective extraction of phenolic compounds bound to dietary fibers. The peak at 1666 cm^−1^ corresponds to carboxyl group bending vibrations, while the peak at 1016 cm^−1^ likely arises from ether bonds (C–O–C) in lignin or hemicellulose (Zheng et al., 2025). The marked reduction in peak intensity post-hydrolysis indicates cleavage of these ether bonds and release of bound phenolics. The FTIR fingerprint region (1750–500 cm^−1^) reflects structural features linked to antioxidant potential, as phenolic and flavonoid compounds with antioxidant activity typically exhibit vibrations in this range (Indrianingsih et al., 2024). Peaks observed between 3669 and 3289 cm^−1^ correspond to –OH stretching vibrations in carbohydrates, water, and organic acids, confirming the presence of phenolic compounds (Kaur et al., 2025). In summary, FTIR analysis confirmed the presence of substantial bound phenolics in both SCPR and IBPR residues and demonstrated that alkaline hydrolysis effectively liberates these compounds from dietary fiber matrices.Fig. 3FTIR spectra of soluble conjugated phenolic residues (SCPR) and insoluble-bound phenolic residues (IBPR) from jackfruit at different ripening stages. (a) Soluble conjugated phenolic residues (SCPR) and (b) insoluble-bound phenolic residues (IBPR). J1, J2, and J3 correspond to different ripening stages. SCPR-NaOH denotes SCPR residues after alkaline hydrolysis, and IBPR-NaOH denotes IBPR residues after alkaline hydrolysis.Fig. 3

Polyphenol content and antioxidant activity in jackfruit

3.2

As Fig. 4a in, the TPC of FP exhibited a progressive increase with ripening, whereas the TPC of CP and BP demonstrated a decreasing trend. During the unripe stage, fruits accumulate substantial amounts of bound phenolic compounds as a defense mechanism against biotic and abiotic stressors. As ripening advances, endogenous hydrolytic enzymes, such as β-glucosidase and esterase, catalyze the hydrolysis of bound phenolics into their free forms. The TFC of jackfruit polyphenols was found to increase significantly during ripening (Fig. 4b). This phenomenon is likely attributable to the activation of key enzymes in the flavonoid biosynthesis pathway (phenylpropanoid metabolism), including phenylalanine ammonia-lyase, chalcone synthase, and chalcone isomerase. Additionally, the hydrolysis of starch into soluble sugars (e.g., glucose and fructose) during ripening provides carbon skeletons (e.g., malonyl-CoA), further promoting the accumulation of flavonoid compounds (Kaur et al., 2025).Fig. 4. Analysis of polyphenol content and antioxidant activity in jackfruit. Lowercase letters (a–c) denote significant differences (p < 0.05) among the groups. (a) Total phenolic content (TPC); (b) Total flavonoid content (TFC); (c) DPPH radical scavenging activity; (d) ABTS radical scavenging activity; (e) FRAP Assay for Reducing Power Assessment.Fig. 4

Polyphenols are recognized for their robust redox properties, functioning as reducing agents, hydrogen or oxygen radical scavengers, and participating in processes such as excited-state reactions, energy transfer, complex formation, and collisional quenching. These characteristics render polyphenols critical contributors to antioxidant activity. In this study, three complementary antioxidant assays were employed to evaluate the antioxidant capacity of jackfruit polyphenols: the DPPH and ABTS radical scavenging assays, which rely on the decolorization of radicals by antioxidants, and the FRAP assay, which measures the ability of antioxidants to reduce Fe^3+^ to Fe^2+^. The antioxidant activity of FP exhibited a biphasic trend, initially decreasing slightly and subsequently increasing significantly with ripening (Fig. 4c–e). This pattern may be attributed to the transient upregulation of polyphenol oxidase (PPO) activity during early ripening, which catalyzes the oxidation of free phenolics to quinones, thereby reducing the availability of hydroxyl groups and temporarily diminishing antioxidant capacity. In later ripening stages, the enhanced activity of antioxidant enzymes, such as superoxide dismutase and catalase, likely protects free phenolics from excessive oxidation, thereby preserving their reducing capacity (Fecka et al., 2021). Although the TPC of CP decreased with ripening, its antioxidant activity was significantly enhanced. This enhancement is likely due to enzymatic hydrolysis of bound phenolics, which exposes reactive functional groups, such as hydroxyl groups, thereby amplifying antioxidant potential (Minnaar et al., 2022). Conversely, the antioxidant activity of BP exhibited an inverse trend to FP. During early ripening, enzymes such as pectinase and cellulase partially hydrolyze cell wall polysaccharides (e.g., pectin and hemicellulose), releasing tightly bound phenolic compounds (e.g., ferulic acid and lignin-bound phenolics), which enhances antioxidant activity. However, in the mid-to-late stages of ripening, elevated PPO activity promotes the irreversible covalent binding of free phenolics to proteins and polysaccharides, forming large molecular complexes that mask antioxidant groups and reduce activity (Qi et al., 2022).

Our study indicates that the total phenolic content (TPC) does not directly correlate with antioxidant activity in polyphenols. Polyphenols from different fractions vary in antioxidant capacity, potentially attributed to differences in their structural functional groups and substituents. Moreover, other components present in plant extracts, such as vitamins and carotenoids, can interact synergistically with polyphenols, thereby influencing overall antioxidant activity (Rudrapal et al., 2024). This interplay highlights the importance of considering both quantitative and qualitative aspects of phenolic compounds when evaluating their functional properties.

Characterization by HPLC-HR TOF MS2 and quantification by HPLC-PDA

3.3

HPLC-HR TOF MS^2^ analysis revealed 263 differential features (VIP > 1, p < 0.05) in jackfruit pulp extracts. Among these, 55.51% were putatively annotated as phenolic acids and 17.49% as flavonoids (Fig. S1). However, only 18 phenolic compounds (nine phenolic acids and nine flavonoids) met stringent validation criteria (S/N ≥ 10, CV ≤ 15%, authentic standard available, matrix-matched r^2^ ≥ 0.995, bias ≤ 20%); the remaining 245 features lacked sufficient confidence for quantification.

As illustrated in Table 1, the compositional complexity of FP decreased with advancing ripeness, a trend consistent with the findings of Belwal et al. (2019), who observed a reduction in free phenolic species in wild berries as ripeness increased. Conversely, the phenolic profiles of CP and BP became more diverse. Notably, gallic acid content in FP, CP, and BP followed a bimodal trend, initially increasing and then decreasing, while sinapic acid and (+)-catechin exhibited continuous upward trajectories. In CP and BP, chlorogenic acid, homovanillic acid, 3-hydroxyphenylacetic acid, ferulic acid, and hyperoside also demonstrated sustained increases. Ferulic acid and glycitein as unique compounds during the J3 stage, whereas 2-methoxycinnamaldehyde and mulberrin were specific to CP. Gupta et al. (2023) highlighted the antioxidant and protective effects of 2-methoxycinnamaldehyde against H_2_O_2_-induced muscle atrophy, while also noting that not all antioxidants possess anti-atrophic properties. Furthermore, research by Zhang et al. (2024) demonstrated that morin enhances antioxidant enzyme activity, mitigates oxidative damage in ESMCs via Nrf2 pathway activation, and improves mitochondrial respiration and cellular energy metabolism. Potentially accounting for the elevated antioxidant activity observed in CP. In FP, compounds such as chlorogenic acid, p-hydroxybenzoic acid, vitexin-2″-o-rhamnoside, taxifolin, and quercetin were exclusively detected in the early stages of ripeness, whereas caffeic acid, sinapic acid, (+)-catechin, and naringenin were only detectable in the later stages. In CP, 3-hydroxyphenylacetic acid, sinapic acid, ferulic acid, hyperoside, taxifolin, and glycitein were predominantly observed in the later stages of ripeness. In contrast, chlorogenic acid, p-hydroxybenzoic acid, caffeic acid, vanillic acid, 3-hydroxyphenylacetic acid, and quercetin were undetectable in J1 and J2 but became abundant in J3.Table 1. Detailed characteristics of HPLC-determined compounds.Table 1. ClassificationTarget compoundsMolecular formulaMolecular weightRT(min)Adduct(±)Mass errorppmObservedm/zJ1-FPJ1-CPJ1-BPJ2-FPJ2-CPJ2-BPJ3-FPJ3-CPJ3-BPPhenolic acidGallic acidC_7_H_6_O_5_170.11.77[M-H]^−^1.85169.0123.34 ± 0.11^Eh^32.54 ± 0.17^Cd^17.9 ± 0.41^Bi^27.45 ± 0.08^Ce^41.53 ± 0.34^Da^36.3 ± 0.85^Bb^24.77 ± 0.34^Dg^34.69 ± 0.45^Gc^25.29 ± 1.05^Gf^Chlorogenic AcidC_16_H_18_O_9_354.33.09[M-H]^−^0.34353.0919.74 ± 0.07^Gd^19.17 ± 0.57^Ge^ND20.61 ± 0.09^Fc^19.77 ± 0.18^Kde^NDND23.5 ± 0.14^Kb^34.47 ± 0.26^Ea^p-Hydroxybenzoic acidC_7_H_6_O_3_138.12.27[M + HCOO]^−^2.93183.0323.77 ± 0.17^Dd^44.41 ± 0.46^Bb^ND19.26 ± 0.17^Ge^39.7 ± 0.13^Ec^NDND76.6 ± 0.27^Ca^15.84 ± 0.15^Kf^Caffeic acidC_9_H_8_O_4_180.23.66[M-H]^−^0.94179.04ND26.64 ± 0.29^Eb^ND24.58 ± 0.24^Dd^24.61 ± 0.35^Id^ND25.64 ± 3.63^Cc^63.94 ± 0.5^Da^25.82 ± 0.09^Gc^Homovanillic acidC_9_H_10_O_4_182.23.98[M-H]^−^0.66181.0527.33 ± 0.16^Ce^29.87 ± 0.16^Dd^NDND30.53 ± 0.45^Gc^NDND44.28 ± 0.27^Fb^60.3 ± 0.12^Ca^3-Hydroxyphenylacetic acidC_8_H_8_O_3_152.24.71[M-H]^−^1.92151.04NDNDND31.67 ± 0.02^Bc^NDNDND33.69 ± 0.21^Ha^31.94 ± 0.01^Fb^Sinapic acidC_11_H_12_O_5_224.24.59[M-H]^−^0.47223.06NDND26.66 ± 0.31^Ae^ND48.6 ± 0.28^Cc^28.63 ± 0.29^Dd^26.54 ± 0.42^Be^164.85 ± 0.61^Aa^74.32 ± 0.28^Ab^Ferulic acidC_10_H_10_O_4_194.24.61[M-H]^−^0.28193.05NDNDNDNDNDNDND1.46 ± 0.33^Pa^0.77 ± 0.09^Lb^2-MethoxycinnamaldehydeC_10_H_10_O_2_162.25.51[M + NH_4_]^+^2.39325.25ND15.45 ± 0.09^Ha^NDND15.27 ± 0.1^Ma^NDND15.25 ± 0.11^Na^ND

Flavone(+)-catechinC_15_H_14_O_6_290.33.29[M-H]^−^2.29289.07ND24.57 ± 0.18^Fe^NDND25.46 ± 0.47^Hd^33.18 ± 0.21^Cb^24.68 ± 0.31^De^25.78 ± 0.14^Jc^41.68 ± 0.55^Da^vitexin-2″-o-rhamnosideC_27_H_30_O_14_578.54.22[M-H]^−^4.05577.1616.45 ± 0.08^Hc^19.57 ± 0.27^Ga^NDND18.48 ± 0.17^Lb^15.29 ± 0.21^Fd^ND19.72 ± 0.33^Ma^19.59 ± 0.5^Ja^HyperosideC_21_H_20_O_12_464.44.47[M-H]^−^5.93463.09NDNDNDND20.59 ± 0.3^Jd^21.4 ± 0.23^Ec^ND22.37 ± 0.12^Lb^24.4 ± 0.69^Ha^(−)-Epicatechin gallateC_22_H_18_O_10_442.44.88[M-H]^−^0.15441.0842.46 ± 0.24^Ae^59.9 ± 0.29^Ab^ND42.48 ± 0.16^Ae^50.28 ± 0.18^Bd^39.23 ± 0.46^Af^35.76 ± 0.72^Ag^52.39 ± 0.41^Ec^60.72 ± 0.06^Ba^TaxifolinC_15_H_12_O_7_304.34.67[M-H]^−^0.73303.0531.46 ± 0.12^Bb^NDND31.47 ± 0.08^Bb^31.36 ± 0.06^Fb^NDND32.48 ± 0.13^Ia^NDGlyciteinC_16_H_12_O_5_284.35.31[M-H]^−^1.53283.06NDNDNDNDNDNDND10.59 ± 0.03^O^NDQuercetinC_15_H_10_O_7_302.25.41[M + CH_3_COO]^−^3.25361.0520.36 ± 0.01^Fb^NDNDNDNDNDNDND22.64 ± 0.3I^a^NaringeninC_15_H_12_O_5_272.34.31[M + HCOO]^−^0.28317.07NDNDND22.29 ± 0.21^Eb^NDND23.6 ± 0.08^Ea^NDNDMulberrinC_25_H_26_O_6_422.53.68[M + NH_4_]^+^4.59440.20ND59.7 ± 0.01^Ac^NDND122.7 ± 0.33^Aa^NDND110.27 ± 0.14^Bb^NDND indicates not detected. Values within each cell represent the mean ± standard deviation. Different lowercase letters within the same row and different uppercase letters within the same column denote significant differences (p < 0.05).

The phenolic composition of different fractions undergoes distinct transformations during ripening, which may contribute to variations in their bioactivities. Elucidating the relationship between ripeness and phenolic composition is essential for the targeted isolation of phenolic compounds with specific bioactive properties.

Enzyme inhibition activity

3.4

α-Glucosidase, a key player in carbohydrate metabolism, is critical for managing blood glucose levels in type 2 diabetes. Similarly, pancreatic lipase is indispensable for lipid digestion, breaking down lipids into free fatty acids and monoglycerides (Subramaniyan & Hanim, 2025). Xanthine oxidase drives the conversion of hypoxanthine to xanthine and subsequently to uric acid. When serum uric acid levels escalate due to overproduction or underexcretion, hyperuricemia (HUA) ensues, laying the biochemical groundwork for gout. Inhibiting xanthine oxidase can lower serum uric acid levels, disrupt the gout development cascade (crystal deposition-inflammatory activation-tissue damage), and forestall HUA (Xue et al., 2023). This study sets out to evaluate the enzyme inhibitory potential of jackfruit polyphenols by focusing on α-glucosidase, pancreatic lipase, and xanthine oxidase.

The results of the α-glucosidase inhibition assay indicated that the inhibitory activity of CP increased significantly with ripening, whereas FP exhibited only minor variations without statistically significant differences among different ripening stages; in contrast, the inhibitory activity of BP initially decreased and then increased (Fig. 5a). All three phenolic fractions exhibited maximum inhibitory activity at J3, with CP showing the lowest IC_50_ value of 0.0378 mg/mL at this stage. Although this value did not match that of acarbose (0.824 × 10^−6^ mg/mL), it was significantly more effective than those of kiwifruit (0.18 mg/mL) (Qiao et al., 2025) and scoparium (181.7 μg/mL) (Lachkar et al., 2021). These findings suggest that jackfruit polyphenols could serve as partial alternatives to acarbose, potentially reducing the dosage required for managing symptoms of type 2 diabetes.Fig. 5. Inhibitory effects of jackfruit polyphenols on digestive enzymes. The reciprocal of the IC_50_ values was plotted to better visualize the trends in inhibitory activity during ripening. Different lowercase letters denote significant differences (p < 0.05). (a) α-Glucosidase inhibition; (b) pancreatic lipase inhibition; (c) xanthine oxidase inhibition.Fig. 5

Polyphenols, as natural bioactive compounds, can influence lipid metabolism and absorption by inhibiting pancreatic lipase, thereby aiding in weight management. The inhibitory activity of FP against pancreatic lipase initially increased slightly and then decreased significantly with ripening, while CP exhibited a gradual increase with ripening, whereas BP increased from J1 to J2 and then slightly decreased at J3 (Fig. 5b). The highest inhibitory activity for FP and BP was observed at J2, whereas CP exhibited maximum activity at J3. The inhibitory activity CP (0.422 mg/mL) > BP (0.458 mg/mL) > FP (1.711 mg/mL). The pancreatic lipase inhibitory activity of jackfruit polyphenols was significantly higher than that of passion fruit (1.58 mg/mL) (Sukketsiri et al., 2023) and broad bean (2.97 mg/mL) (Lu et al., 2018), indicating substantial potential for reducing blood lipid levels.

Allopurinol was used as a positive control for xanthine oxidase inhibition assays. The inhibitory activity of FP and BP initially decreased and then slightly increased with ripening, with maximum activity observed at J1 (Fig. 5c). In contrast, the inhibitory activity of CP increased consistently and reached a maximum at J3, with CP exhibiting the strongest activity (0.358 mg/mL). Jackfruit polyphenols demonstrated remarkable xanthine oxidase inhibitory capacity, surpassing that of pear (10.75 mg/mL) (Baltas, 2017) and cherry (2.619 mg/mL) (Kamaraj et al., 2025). This suggests potential applications of jackfruit polyphenols in the treatment and daily management of hyperuricemia. Jackfruit polyphenols exhibit potent digestive enzyme inhibitory activities, show promise as digestive enzyme inhibitors.

Molecular docking results analysis

3.5

Molecular docking, a computational technique that visualizes the interactions between small-molecule ligands and macromolecular receptors, serves as an effective tool for elucidating binding characteristics (Jin & Wei, 2024). In this study, molecular docking was employed to investigate the interactions between representative jackfruit polyphenols from different ripening stages and three key enzymes, namely α-glucosidase, pancreatic lipase, and xanthine oxidase. Based on HPLC–HR TOF MS^2^ analysis, 263 differential metabolites were ranked by VIP values, and the top 50 compounds were further screened using Z-score normalization. Compounds with Z-scores greater than zero across nine sample groups (J1-FP/CP/BP, J2-FP/CP/BP, and J3-FP/CP/BP) were retained, yielding a final set of 46 representative polyphenols for molecular docking analysis. The corresponding binding energies are summarized in Table 2.Table 2. Molecular docking binding energy values (kcal/mol).Table 2. No.CompoundBinding energy (kcal/mol)α-GlucosidasePancreatic lipaseXanthine oxidase12’-Hydroxydaidzein−8.4−6.0−7.621-Caffeoylquinic acid−6.9−5.8−8.032-Naphthol−6.7−4.9−8.34Ginkgolic acid−5.7−5.6−7.45Kanzonol J−8.0−6.1−9.86Butylparaben−6.0−4.6−7.27Cyanidin 3-(6″-succinyl-glucoside)−8.5−5.9−10.482-Methylbenzoic acid−5.9−4.1−6.19Cyclomorusin−9.6−7.4−11.810Sinapic acid−7.1−5.0−7.411CID 153946−7.6−6.1−10.012Acetyleugenol−6.2−4.5−6.813Calabaxanthone−7.8−6.1−9.314Artemetin−7.0−5.3−8.915Diosmetin−8.4−6.2−8.9163-Hydroxyphenylacetic acid−6.2−4.1−6.8172-Hydroxybenzyl alcohol−6.3−4.0−5.718moracin I−7.7−5.5−9.5194-Ethylbenzoic acid−6.1−4.0−6.420Vanilloloside−6.8−5.6−7.721Hydrocinnamic acid−6.8−4.4−7.0224-Nonylphenol−5.8−3.8−6.823(+)-Medicarpin−7.2−5.5−8.4246-Demethoxytangeretin−7.3−5.7−8.725Cyclokievitone−9.0−6.7−9.5263,4-Dihydroxyphenylglycol−6.2−4.4−6.427Nateglinide−7.6−4.8−8.628Pinostrobin chalcone−7.1−5.6−8.029(R)-Hispaglabridin B−8.0−6.2−9.8305-Hydroxyauranetin−6.6−5.4−7.831Isosinensetin−7.3−5.2−8.932[8]-Paradol−6.5−4.2−6.333Xanthohumol−7.7−5.3−8.7343,4-Dihydroxybenzyl alcohol−5.9−4.3−7.035Apigenin−8.6−6.1−9.2362-Aminobenzoic acid−6.1−4.7−6.637Trimethoprim−5.8−5.1−6.438Terephthalic acid−6.1−4.7−6.939Curculigoside−7.3−5.7−8.140Rhamnazin−7.6−6.2−9.941Cianidanol−8.3−6.3−9.1424-Hydroxy-3-methoxyphenylacetone−5.6−4.5−6.043Ethyl gallate−6.1−5.0−6.544Vitexin 2″-O-rhamnoside−8.9−5.8−9.945(−)-Epicatechin gallate−9.4−6.2−9.946Asebogenin−7.9−5.2−8.3

The interpretation of docking results primarily relies on binding energy and non-covalent interactions, including hydrogen bonding, electrostatic interactions, and hydrophobic contacts (Morris et al., 2009). Negative binding energy values indicate stable enzyme–ligand complexes, with lower values generally reflecting stronger binding affinity. The docking results revealed that polyphenols from different fractions exhibited favorable binding affinities toward the three target enzymes. Notably, the CP fraction showed particularly strong binding to α-glucosidase, which is consistent with the IC_50_ results and may be attributed to the formation of multiple hydrogen bonds between CP polyphenols and key amino acid residues. These interactions were visualized in Fig. 6, and the detailed residue-level interactions are summarized in Tables S4–S6.Fig. 6(a) Optimal docking conformations and interaction modes of jackfruit polyphenols with α-glucosidase. (I) Cyclomorusin, (II) 2′-Hydroxydaidzein, (III) Diosmetin, (IV) (−)-Epicatechin gallate, (V) 2″-O-alpha-L-Rhamnopyranosyl-isovitexin, (VI) Pinostrobin chalcone, (VII) Apigenin, (VIII) Isorangeatin, (IX) 4′,5,7,8-Tetramethoxyflavone. (b) Optimal docking conformations and interaction modes of jackfruit polyphenols with pancreatic lipase. (I) Cyclomorusin, (II) (+)-Catechin, (III) Diosmetin, (IV) (−)-Epicatechin gallate, (V) 2″-O-alpha-L-Rhamnopyranosyl-isovitexin, (VI) Vanilloloside, (VII) Apigenin, (VIII) 4′,5,7,8-Tetramethoxyflavone, (IX) Ginkgolic acid. (c) Optimal docking conformations and interaction modes of jackfruit polyphenols with xanthine oxidase. (I) Cyclosakuranetin, (II) Isochlorogenic acid B, (III) (+)-Catechin, (IV) 2″-O-alpha-L-Rhamnopyranosyl-isovitexin, (V) (−)-Epicatechin gallate, (VI) Pinostrobin chalcone, (VII) Apigenin, (VIII) Isorangeatin, (IX) 4′,5,7,8-Tetramethoxyflavone.Fig. 6

A residue-level analysis further demonstrated that polyphenol binding to α-glucosidase predominantly involved residues such as Arg647, Asp649, Glu767, Lys776, and Pro676, which frequently participated in hydrogen bonding and electrostatic interactions, while hydrophobic contacts with Tyr636, Leu286, Phe535, and Ile734 contributed to ligand stabilization within the catalytic pocket. For pancreatic lipase, key interacting residues including Tyr71, Lys74, Arg77, Asp79, and Glu146 were repeatedly involved in ligand binding, along with hydrophobic interactions with Pro149, Tyr93, Ile147, and Leu129, suggesting that polyphenols occupied regions close to the catalytic site and may interfere with substrate access (Visvanathan et al., 2024). In the case of xanthine oxidase, ligand interactions mainly involved residues such as Arg60, Lys249, Lys256, Leu257, Val259, Ile353, and Leu404, with additional contributions from aromatic and hydrophobic residues including Trp283, Leu398, and Pro400, facilitating π–π stacking and hydrophobic stabilization within the active site.

Although molecular docking suggested that FP exhibited relatively strong binding affinities toward pancreatic lipase and xanthine oxidase, these predictions were not fully consistent with the in vitro inhibition results, highlighting the limitations of docking simulations in fully capturing enzyme inhibition behavior. Overall, the superior inhibitory effects observed for the CP fraction may be attributed to its favorable binding energies and stable interactions with key catalytic or substrate-recognition residues, as supported by the residue-level docking analysis.

Conclusion

4

This study systematically elucidated the stage-dependent redistribution of polyphenol fractions and their functional implications during jackfruit ripening. Ripening significantly altered both the composition and relative abundance of free (FP), conjugated (CP), and bound phenolics (BP). A progressive transformation among phenolic fractions was observed, accompanied by a marked increase in CP-mediated antioxidant capacity and bioactivity from J1 to J3. Notably, fully ripe jackfruit (J3) exhibited the strongest antioxidant and inhibitory activities against α-glucosidase, pancreatic lipase, and xanthine oxidase. This enhanced bioactivity in J3 was closely associated with the enrichment and diversification of conjugated (CP) and bound (BP) phenolic compounds, highlighting that qualitative structural changes, rather than merely total phenolic content, drive these functional properties, whereas the compositional complexity of free phenolics (FP) decreased with ripening. These findings highlight the functional differentiation of jackfruit at different maturity stages and underscore the superior antioxidant and enzyme inhibitory potential of the conjugated phenolic fraction in ripe fruit. The results provide a scientific basis for stage-specific processing strategies and high-value utilization of jackfruit resources. Nevertheless, further in vivo and clinical validation is required to fully establish the health-promoting effects of these phenolic fractions.

CRediT authorship contribution statement

Liru Ma: Writing – original draft, Visualization, Methodology, Formal analysis, Conceptualization. Zhen Feng: Methodology, Investigation, Conceptualization. Chao Zhang: Methodology, Investigation, Conceptualization. Haode Chang: Methodology, Formal analysis. Wenjing Zhang: Methodology, Investigation. Quanmiao Zhang: Visualization, Software. Yihong Bao: Writing – review & editing, Validation, Supervision, Resources. Chunhe Gu: Writing – review & editing, Supervision, Formal analysis.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.