Green Pea (Pisum sativum L.) Hull Polyphenol Extract Alleviates NAFLD through VB6/TLR4/NF-κB and PPAR Pathways.

Green pea hull is a processing byproduct of green pea and rich in polyphenols. Nonalcoholic fatty liver disease (NAFLD) is a chronic metabolic disease characterized by accumulation of lipids in the liver for which there are no effective treatment strategies. Here, a mouse model of NAFLD induced by a DSS+high-fat diet (HFD) was established to investigate the effect of green pea hull polyphenol extract (EGPH). The results show that EGPH relief of NAFLD was a combined effect, including reducing hepatic fat accumulation, improving antioxidant activity and blood lipid metabolism, and maintaining glucose homeostasis. Increased intestinal permeability aggravated NAFLD. Combined metabolomics and transcriptomic analysis showed that vitamin B6 is the key target substance for EGPH to alleviate NAFLD, and it may be the intestinal flora metabolite. After EGPH intervention, the level of vitamin B6 in mice was significantly increased, and more than 60% in the blood enters the liver, which activated or inhibited PPAR and TLR4/NF-κB signaling pathways to relieve NAFLD. Our research could be a win-win for expanding the use of green pea hull and the search for NAFLD prophylactic drugs.

Anthocyanin-Rich Butterfly Pea Flower Extract Ameliorating Low-Grade Inflammation in a High-Fat-Diet and Lipopolysaccharide-Induced Mouse Model.

This study aimed to explore the enhancive effects of butterfly pea flower (BF) extracts on metabolic and immune homeostasis in a low-grade inflammation mouse model. The BF extract was found to contain mainly anthocyanins among other flavonoids. BF supplementation alleviated metabolic endotoxemia by lowering the plasma glucose, lipopolysaccharide (LPS), and tumor necrosis factor-α (TNF-α) levels and restored lipid metabolism and the balance between Treg and Th17 cells, thereby inhibiting the dysfunctional liver and abdominal white adipose tissues. BF extract increased the tight junction protein expression and reduced the expression of proinflammatory cytokines, therefore sustaining the colonic mucosa structure. Furthermore, BF extracts reshaped the gut microbiota structure characterized by significantly promoted SCFA-producing gut microbiota such as Akkermansia and Butyricicoccaceae. Additionally, BF extracts enhanced fecal primary bile acid (BA) levels and modulated bile acid signaling in the liver and ileum to facilitate BA synthesis for the restoration of lipid metabolism. In summary, anthocyanin-enriched BF extracts alleviated the profound negative dietary alterations and helped maintain the metabolic health by modulating the various aspects of the gut microenvironment and enhancing hepatic bile acid synthesis.

Multi-omics reveals that green pea (Pisum sativum L.) hull supplementation ameliorates non-alcoholic fatty liver disease via the SHMT2/glycine/mTOR/PPAR-γ signaling pathway.

Diets rich in various active ingredients may be an effective intervention strategy for non-alcoholic fatty liver disease (NAFLD). The green pea hull (GPH) is a processing by-product of green peas rich in dietary fiber and polyphenols. Here, a mouse model of NAFLD induced by DSS + high-fat diet (HFD) was established to explore the intervention effect of the GPH. The results showed that dietary supplements with the GPH can inhibit obesity and reduce lipid accumulation in the mouse liver to prevent liver fibrosis. GPH intervention can improve liver antioxidant capacity, reduce blood lipid deposition and maintain glucose homeostasis. DSS-induced disruption of the intestinal barrier aggravates NAFLD, which may be caused by the influx of large amounts of LPS. A multi-omics approach combining metabolomics and transcriptomic analysis indicated that glycine was the key target and its content was decreased in the liver after GPH intervention, and that dietary supplements with the GPH can relieve NAFLD via the SHMT2/glycine/mTOR/PPAR-γ signaling pathway, which was further supported by liver-associated protein expression. In conclusion, our study demonstrated that dietary GPH can significantly ameliorate NAFLD, and the future development of related food products can enhance the economic value of the GPH.

Substrate specificity of polyphenol oxidase and its selectivity towards polyphenols: Unlocking the browning mechanism of fresh lotus root (Nelumbo nucifera Gaertn.).

Polyphenol oxidase (PPO) causes the browning of lotus roots (LR), negatively affecting their nutrition and shelf-life. This study aimed to explore the specific selectivity of PPO toward polyphenol substrates, thus unlocking the browning mechanism of fresh LR. Results showed that two highly homologous PPOs were identified in LR and exhibited the highest catalytic activity at 35 ℃ and pH 6.5. Furthermore, the substrate specificity study revealed (-)-epigallocatechin had the lowest Km among the polyphenols identified in LR, while (+)-catechin showed the highest Vmax. The molecular docking further clarified that (-)-epigallocatechin exhibited lower docking energy and formed more hydrogen bonds and Pi-Alkyl interactions with LR PPO than (+)-catechin, while (+)-catechin entered the active cavity of PPO more quickly due to its smaller structure, both of which enhance their affinity to PPO. Thus, (+)-catechin and (-)-epigallocatechin are the most specific substrates responsible for the browning mechanism of fresh LR.

Bioaccessibility and transport of lentil hull polyphenols in vitro, and their bioavailability and metabolism in rats.

Polyphenol-rich lentil hulls are a valuable by-product. In this study, lentil hulls were subjected to simulated in vitro digestion and caco-2 cell monolayer models to assess the bioaccessibility, transmembrane transport, and a rat model to examine the bioavailability and metabolism in vivo. Polyphenols were increasingly released during the in vitro digestion, and were found to contribute to the increased antioxidant activity. Among the bioaccessible polyphenols, catechin glucoside, kaempferol tetraglucoside, procyanidin dimer and dihydroxybenzoic acid-O-dipentoside were most efficiently transported across the caco-2 membrane, and responsible for promoting intestinal integrity as a result of enhanced expression of tight junction proteins. When ingested by rats, lentil hull polyphenols underwent extensive I and II phase metabolic reactions in vivo, including hydroxylation, methylation, glucuronidation and sulfation. Overall, results of this study showed that lentil hull polyphenols are bioaccessible and bioavailable, and lentil hulls as a by-product can be a valuable ingredient for future functional foods.

Impact of solid-state fermentation on factors and mechanisms influencing the bioactive compounds of grains and processing by-products.

Cereal and legume grains and their processing by-products are rich sources of bioactives such as phenolics with considerable health potential, but these bioactives suffer from low bioaccessibility and bioavailability, resulting in limited use. Several studies have demonstrated that solid-state fermentation (SSF) with food-grade microorganisms is effective in releasing bound phenolic compounds in cereal and legume products. In this review, we discuss the effect of SSF on cereal and legume grains and their by-products by examining the role of specific microorganisms, their hydrolytic enzymes, fermentability of agri-food substrates, and the potential health benefits of SSF-enhanced bioactive compounds. SSF with fungi (Aspergillus spp. and Rhizopus spp.), bacteria (Bacillus subtilis and lactic acid bacteria (LAB) spp.) and yeast (Saccharomyces cerevisiae) significantly increased the bioactive phenolics and antioxidant capacities in cereal and legume grains and by-products, mainly through carbohydrate-cleaving enzymes. Increased bioactive phenolic and peptide contents of SSF-bioprocessed cereal and legume grains have been implicated for improved antioxidant, anti-inflammatory, anti-carcinogenic, anti-diabetic, and angiotensin-converting-enzyme (ACE) inhibitory effects in fermented agri-food products, but these remain as preliminary results. Future research should focus on the microbial mechanisms, suitability of substrates, and the physiological health benefits of SSF-treated grains and by-products.

Free and Bound Phenolics of Laird Lentil (Lens culinaris) Hulls and the Anti-inflammatory Activity of their Digestive Products via Crosstalk between NF-κB and Keap1-Nrf2 Signaling Pathways in HT-29 Cells.

Polyphenol-rich Laird lentil hulls are a byproduct of lentil processing. In the present study, free and bound polyphenols in lentil hulls were analyzed with UHPLC-LTQ-OrbiTrap-MS2, and the anti-inflammatory mechanism of their digestive products was explored based on the NF-κB and Keap1-Nrf2 signaling pathways in the HT-29 cell model. In summary, a total of 27 polyphenols and 5 nonphenolic constituents were identified in free and bound fractions, and among them, catechin glucoside, kaempferol tetraglucoside, procyanidin dimer, and dihydroxybenzoic acid-O-dipentoside were the main polyphenols in the digestive products. These digestive products could reduce inflammatory mediators and exert anti-inflammatory activity by inhibiting NF-κB and activating Keap1-Nrf2 signaling pathways, and there was crosstalk between them, which was a mutual inhibition effect. The results show that polyphenols in lentil hulls are a good source of anti-inflammatory ingredients and have a promising development potential.

Paradigm Shift in Phytochemicals Research: Evolution from Antioxidant Capacity to Anti-Inflammatory Effect and to Roles in Gut Health and Metabolic Syndrome.

Food bioactive components, particularly phytochemicals with antioxidant capacity, have been extensively studied over the past two decades. However, as new analytical and molecular biological tools advance, antioxidants related research has undergone significant paradigm shifts. This review is a high-level overview of the evolution of phytochemical antioxidants research. Early research used chemical models to assess the antioxidant capacity of different phytochemicals, which provided important information about the health potential, but the results were overused and misinterpreted despite the lack of biological relevance (Antioxidants v1.0). This led to findings in the anti-inflammatory properties and modulatory effects of cell signaling of phytochemicals (Antioxidants v2.0). Recent advances in the role of diet in modulating gut microbiota have suggested a new phase of food bioactives research along the phytochemicals-gut microbiota-intestinal metabolites-low-grade inflammation-metabolic syndrome axis (Antioxidants v3.0). Polyphenols and carotenoids were discussed in-depth, and future research directions were also provided.

Polyphenol Content of Green Pea (Pisum sativum L.) Hull under In Vitro Digestion and Effects of Digestive Products on Anti-Inflammatory Activity and Intestinal Barrier in the Caco-2/Raw264.7 Coculture Model.

Green pea hulls are a byproduct of the processing of green pea and are rich in phenolic substances. In the present study, in vitro digestion, human colonic adenocarcinoma cell line (Caco-2) monolayer, and the Caco-2/macrophage cell lines of the murine origin (Raw264.7) coculture model were established to investigate the release of polyphenols, absorption, and transport of digestive products and their effects on inflammation and intestinal barrier. During the digestive process, polyphenols were constantly released from the pea hulls, reaching the maximum amount in the small intestine (total phenolic content (TPC): 5.41 ± 0.04 mg gallic acid (GAE)/g dry weight (DW)), and the digestive products (800 µg/mL) could reduce the secretion of NO (50.9%), IL-6 (50.6%), and TNF-α (24.6%) and inhibit the mRNA expression of cyclooxygenase-2 (COX-2) (37.2%) and inducible nitric oxide synthase (iNOS) (91.1%) compared with the lipopolysaccharide (LPS) group. A total of 12 phenolic components were quantified by ultraperformance liquid chromatography-linear ion trap orbitrap tandem mass spectrometry (UHPLC-LTQ-OrbiTrap-MS) technology. Kaempferol trihexoside in digestive products could be absorbed and transported (1.25 ± 0.13 ng quercetin/mL). The digestive products could promote the expression of claudin-1 (210.8%), occludin (64.9%), and zonulin occludin-1 (ZO-1) (52.0%) compared with the LPS group and exert anti-inflammatory effects after being absorbed. The results indicated that pea hull polyphenols could be continuously released and absorbed to play a positive role in protecting the intestinal barrier and anti-inflammatory activity.

Green Pea (Pisum sativum L.) Hull Polyphenol Extracts Ameliorate DSS-Induced Colitis through Keap1/Nrf2 Pathway and Gut Microbiota Modulation.

As a processing by-product, green pea hull (GPH) was found to be rich in phenolic components in our previous studies. In this study, UHPLC-LTQ-OrbiTrap-MS (Ultra performance liquid chromatography-linear ion trap orbitrap tandem mass spectrometry) technique was used to quantify polyphenols, and DSS (sodium dextran sulfate)-induced colitis mouse model was established to explore the effect of GPH extracts on colitis. The results showed that quercetin and its derivatives, kaempferol trihexanside and catechin and its derivatives were the main phenolic substances in the extract, reaching 2836.57, 1482.00 and 1339.91 µg quercetin/g GPH extract, respectively; GPH extracts can improved inflammatory status, repaired colonic function, regulated inflammatory factors, and restored oxidative balance in mice. Further, GPH extracts can activate Keap1-Nrf2-ARE signaling pathway, regulate downstream antioxidant protease and gut microbiota by increasing F/B value and promoting the growth of Lactobacillaceae and Lachnospiraceae, and improve the level of SCFAs (short-chain fatty acids) to relieve DSS-induced colitis in mice. Therefore, GPH may be a promising dietary resource for the treatment of ulcerative colitis.

Phenolics of Yellow Pea (Pisum sativum L.) Hulls, Their Plasma and Urinary Metabolites, Organ Distribution, and In Vivo Antioxidant Activities.

As a byproduct, large amounts of yellow pea hull (YPH) are used as low-value or worthless feed worldwide each year, which is a major waste of these polyphenol-rich hulls. The metabolism, bioavailability, and in vivo activities of these polyphenols have not been reported. In the present study, the chemical profiles of YPH extract, their metabolites, and organ distribution were analyzed with UHPLC-LTQ-OrbiTrap-MS, and their in vivo antioxidant activities were studied using the d-gal model in rats. In summary, a total of 42 ingredients were identified in YPH extracts, and 54 metabolites were found in plasma or urine samples. The distribution of metabolites in plasma and organs may have a positive effect on SOD, GSH-Px, MDA, and T-AOC, and the liver and kidneys were the main distribution organs of these metabolites. Our results are of great significance for the development and utilization of the polyphenol-rich hull of yellow pea.

Role of dietary polyphenols on gut microbiota, their metabolites and health benefits.

The beneficial health roles of dietary polyphenols in preventing oxidative stress related chronic diseases have been subjected to intense investigation over the last two decades. As our understanding of the role of gut microbiota advances our knowledge of the antioxidant and anti-inflammatory functions of polyphenols accumulates, there emerges a need to examine the prebiotic role of dietary polyphenols. This review focused onthe role of different types and sources of dietary polyphenols on the modulation of the gut microbiota, their metabolites and how they impact on host health benefits. Inter-dependence between the gut microbiota and polyphenol metabolites and the vital balance between the two in maintaining the host gut homeostasis were discussed with reference to different types and sources of dietary polyphenols. Similarly, the mechanisms behind the health benefits by various polyphenolic metabolites bio-transformed by gut microbiota were also explained. However, further research should focus on the importance of human trials and profound links of polyphenols-gut microbiota-nerve-brain as they provide the key to unlock the mechanisms behind the observed benefits of dietary polyphenols found in vitro and in vivo studies.

Do short chain fatty acids and phenolic metabolites of the gut have synergistic anti-inflammatory effects? - New insights from a TNF-α-induced Caco-2 cell model.

Fermentation of dietary fiber and metabolism of polyphenols by the gut microbiota produce short chain fatty acids (SCFAs) and some simple phenolic acids, both of which have been independently shown to have anti-inflammatory effects and to improve gut health. While synergistic interactions between the two colonic metabolite groups have been speculated, direct evidence on whether and how these compounds work together towards collective anti-inflammatory effect and gut health remain unclear. In this study, the most important SCFA butyric acid (BtA) and three phenolic metabolites benzoic acid (BzA), phenylacetic acid (PAA) and phenylpropionic acid (PPA) were selected and assessed for the anti-inflammatory effects and the underlying mechanisms in a TNF-α-induced Caco-2 cell model. Significantly stronger inhibition of TNF-α-induced IL-8 production was found in cells pre-treated with different concentrations of mixtures of BtA and the phenolic metabolites than individual compounds. Further study showed that the synergistic effect was via significant down-regulation of the gene expressions of IL-8, TNF-α and VCAM-1, and of phosphorylation of JNK, p38 and IĸBα, cellular signaling mediators of the MAPK and NF-κB pathways. Although these are results of a cell model, they do provide significant insights into the synergistic anti-inflammatory effects between SCFAs and phenolic metabolites. As both metabolite groups are simultaneously present in the gut, our findings therefore also suggest a potential synergistic effect between the two colonic metabolite groups in maintaining gut health.

Anti-Inflammatory Effect and Cellular Transport Mechanism of Phenolics from Common Bean (Phaseolus vulga L.) Milk and Yogurts in Caco-2 Mono- and Caco-2/EA.hy926 Co-Culture Models.

The bioavailability and anti-inflammatory activity of the phenolic compounds derived from gastrointestinal digestates of navy bean and light red kidney bean milks and yogurts were investigated in both Caco-2 mono- and Caco-2/EA.hy926 co-culture cell models. Instead of being transported directly, the ferulic acid ester derivatives in common bean milks and yogurts were found to be metabolized into ferulic acid and then be transported through the Caco-2 cell monolayer with an average basolateral ferulic acid concentration of 56 ± 3 ng/mL after 2 h. Strong anti-inflammatory effects were observed in the basolateral EA.hy926 cells of the co-culture model, and modulations of oxLDL-induced inflammatory mediators by the transported phenolics were verified to be through the p38 MAPK pathway. The present results suggest that the common bean-derived phenolics can be metabolized and absorbed by the intestinal epithelial cells and have antioxidant and anti-inflammatory effects against oxidative stress injury in vascular endothelial cells, hence contributing to the amelioration of vascular diseases.

Application of accelerated solvent extraction coupled with online two-dimensional countercurrent chromatography for continuous extraction and separation of bioactive compounds from Citrus limon peel.

Drug discovery from complex mixtures, like Chinese herbs, is challenging and extensive false positives make it difficult to obtain compounds with anti-Alzheimer's activity. In this study, a continuous method comprised of accelerated solvent extraction coupled with online two-dimensional countercurrent chromatography was developed for the efficient, scaled-up extraction and separation of six bioactive compounds from Citrus limon peels: neoeriocitrin, isonaringin, naringin, hesperidin, neohesperidin, and limonin. These active compounds were isolated and purified from the raw plant materials by two-dimensional countercurrent chromatography separation via two sets of an n-hexane/n-butanol/methanol/water solvent system: 0.23:1.00:0.25:1.13 and 0.47:1.00:0.38:1.46, v/v/v/v. The compounds were collected in yields of 0.22, 0.25, 0.10, 0.31, 0.29, and 0.28 mg/g, respectively, with purities of 95.79, 96.47, 97.69, 97.22, 98.11, and 98.82%, respectively. Subsequently, a simple and efficient in vitro method was developed for rapidly evaluating the acetylcholinesterase inhibitory activities of six bioactive components. Furthermore, the PC12 cell model and the in vitro metabolism of cytochromes P450 were employed to verify the monomers obtained from the continuous method. The results demonstrated that these six bioactive extracts from the C. limon peels were strong acetylcholinesterase inhibitors.

Synergistic antioxidant effects of petunidin and lycopene in H9c2 cells submitted to hydrogen peroxide: Role of Akt/Nrf2 pathway.

Phenolics and carotenoids coexist in fruits and vegetables and could possess interaction effects after consumption. The present study aims to elucidate the possible mechanisms of the antioxidant interactions between anthocyanins and carotenoids using petunidin and lycopene as examples in hydrogen peroxide (H2 O2 )-induced heart myofibroblast cell (H9c2) line model. The results revealed that petunidin and lycopene showed antioxidant effects and petunidin in a larger proportion mixed with lycopene, for example, petunidin: lycopene = 9:1 significantly protected against the loss of the cell viability (8.98 ± 1.03%) and intracellular antioxidant enzyme activities of superoxide dismutase (SOD, 27.07 ± 3.51%), catalase (CAT, 29.51 ± 6.12%), and glutathione peroxidase (GSH-Px, 20.33 ± 2.65%). Moreover, the messenger RNA (mRNA) and protein expressions of NAD(P)H quinone reductase (NQO1) and heme oxygenase (HO-1) of the nuclear factor erythrocyte 2-related factor 2 (Nrf2) signaling pathway were significantly induced in petunidin, lycopene, and synergistic combinations, suggesting that the antioxidant action was through activating the Nrf2 antioxidant response pathway. This was further validated by Nrf2 siRNA, and the results that petunidin significantly induced more of NQO1 expression and lycopene more of HO-1 suggested that the synergism may be a result of concerted actions by the two compounds on these two different target genes of the Nrf2 pathway. The two compounds also significantly increased the phosphorylation of Akt in synergistic combinations. Findings of the present study demonstrated that petunidin and lycopene exerted synergistic antioxidant effects when petunidin in a larger proportion in the combinations and contribute to the prevention of cellular redox homeostasis, which might provide a theoretical basis for phenolics and carotenoids playing beneficial effects on the cardiovascular risk. PRACTICAL APPLICATION: In this study, we revealed that the combined treatments of petunidin and lycopen inhibited H2 O2 -induced oxidative damage in myocardial cells. Moreover, the treatments contributed to the Nrf2 pathway and the restoration of cellular redox homeostasis might provide a theoretical basis for phenolics and carotenoids playing beneficial effects on the cardiovascular risk.

A comprehensive profiling of free, conjugated and bound phenolics and lipophilic antioxidants in red and green lentil processing by-products.

A systemic approach was taken in profiling the hydrophilic and lipophilic antioxidants in lentil hulls using a combination of HPLC, LC-ESI-MS2 and GC techniques. A total of 37 phenolics were tentatively identified in the hydrophilic fractions, while four carotenoids and three tocopherols were found in the lipophilic fraction. Results showed that in addition to the high free extractable phenolics, phenolic compounds in conjugated and bound forms also exist in similar amounts. Information on conjugated and bound phenolics are particularly important as these forms of phenolics often go unnoticed by chromatographic profiling of extractables. All phenolic, carotenoid and tocopherol fractions contributed to antioxidant activities. Information about bioactives from lentil hulls, specifically conjugated and bound phenolics are reported here for the first time. The comprehensive profiling of these bioactives lays a good foundation for further assessment of the value-added uses of lentil hulls which are by-products of pulse processing.

Lactobacillus pentosus S-PT84 Prevents Low-Grade Chronic Inflammation-Associated Metabolic Disorders in a Lipopolysaccharide and High-Fat Diet C57/BL6J Mouse Model.

A long-term exposure to lipopolysaccharides results in the gut inflammation and its impaired barrier function, leading to the development of metabolic disorders. In this study, the role of dietary heat killed Lactobacillus pentosus S-PT84 on preventing endotoxemia to maintain metabolic homeostasis was studied. We demonstrated that the treatment of L. pentosus S-PT84 improved the gut integrity by maintaining tight-junction protein expression, in order to suppress the infiltration of endotoxin into plasma. The systemic inflammatory responses were inhibited via reducing the secretion of TNF-α and MCP-1. Furthermore, the blood lipid profile and glucose level as well as adiponectin in both plasma and white adipose tissues (WAT) were preserved by L. pentosus S-PT84 through upregulation of PPAR-γ and IRS-1 expression in WAT. The above findings suggest that the metabolic homeostasis in mice treated with HFD and LPS was sustained by L. pentosus S-PT84, leading to reducing the early risk for progression into metabolic disorders.

LC-MS/MS for simultaneous detection and quantification of Amadori compounds in tomato products and dry foods and factors affecting the formation and antioxidant activities.

In order to establish an efficient detection method to evaluate the formation of Amadori compounds (ACs) in food products and study the potential health effects, an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method using caffeine as internal standard was developed to determine eight ACs. The detection limits ranged from 0.0179 to 0.0887 mg/L for the ACs. The accuracy of the method was tested through measuring recovery of the spiked samples that varied from 81.90 ± 2.98% to 108.74 ± 2.34%. This method was further applied to detect ACs in 10 food products. Results showed that dry fruits and vegetables were rich in ACs, the total content of ACs varied from 1.36 ± 0.26 to 3415.91 ± 147.96 mg/100 g. The total amount of ACs in tomato juice heated under vacuum condition showed significant increment (P < 0.05) in 25 min at 80 °C comparing with that under atmospheric pressure due to the rapid loss of water. Besides, the amino acid content shows positive correlation with the corresponding AC formation in Maillard reaction during food drying. After heated at fixed water activity (Aw) for 4 hr by sous-vide process, the ACs content in tomato powder increased significantly and the antioxidant activity improved as well. PRACTICAL APPLICATION: Results of this study provided a valuable tool to evaluate the formation of ACs in complex dry food products, facilitated the quality control of food products. The knowledge obtained will offer useful information to food processors. The synthesized ACs would facilitate further study into the antioxidant activities and potential health effects of specified AC.

Extraction and isolation of acetylcholinesterase inhibitors from Citrus limon peel using an in vitro method.

A simple and efficient ultrafiltration-liquid chromatography-mass spectrometry-based method was developed for the rapid screening and identification of ligands from Citrus limon peel, which are suitable acetylcholinesterase inhibitors. Subsequently, the anti-Alzheimer's activity of these compounds was assessed using a PC12 cell model. Six major compounds, viz. neoeriocitrin, isonaringin, naringin, hesperidin, neohesperidin, and limonin, were identified as potent acetylcholinesterase inhibitors. A continuous and efficient online method, which involved the use of a microwave-assisted extraction device, solvent concentration tank, and centrifugal partition chromatography column, was developed for the scale-up of these compounds, and the obtained compounds presented high purity. Next, their bioactivity was evaluated using a PC12 cell model. This novel approach, which was based on ultrafiltration-liquid chromatography-mass spectrometry, microwave-assisted extraction online coupled with solvent concentration tank, and centrifugal partition chromatography along with in vitro evaluation, could represent a powerful tool for the screening and extraction of acetylcholinesterase inhibitors from complex matrices, and could be a useful platform for the large-scale production of bioactive and nutraceutical ingredients.