Study of an inhibitory effect of plant polyphenolic compounds against digestive enzymes using bench-working experimental evidence predicted by molecular docking and dynamics.

The substantial nutritional content and diversified biological activity of plant-based nutraceuticals are due to polyphenolic chemicals. These chemicals are important and well-studied plant secondary metabolites. Their protein interactions are extensively studied. This relationship is crucial for the logical development of functional food and for enhancing the availability and usefulness of polyphenols. This study highlights the influence of protein types and polyphenols on the interaction, where the chemical bindings predominantly consist of hydrophobic interactions and hydrogen bonds. The interaction between polyphenolic compounds (PCs) and digestive enzymes concerning their inhibitory activity has not been fully studied. Therefore, we have examined the interaction of four digestive enzymes (α-amylase, pepsin, trypsin, and α-chymotrypsin) with four PCs (curcumin, diosmin, morin, and 2',3',4'-trihydroxychalcone) through in silico and in vitro approaches. In vitro plate assays, enzyme kinetics, spectroscopic assays, molecular docking, and simulations were performed. We observed all these PCs have significant docking scores and preferable interaction with the active site of the digestive enzymes, resulting in the reduction of enzyme activity. The enzyme-substrate binding mechanism was determined using the Lineweaver Burk plot, indicating that the inhibition occurred competitively. Among four PCs diosmin and morin has the highest interaction energy over digestive enzymes with IC50 value of 1.13 ± 0.0047 and 1.086 ± 0.0131 µM. Kinetic studies show that selected PCs inhibited pepsin, trypsin, and chymotrypsin competitively and inhibited amylase in a non-competitive manner, especially by 2',3',4'-trihydroxychalcone. This study offers insights into the mechanisms by which the selected PCs inhibit the enzymes and has the potential to enhance the application of curcumin, diosmin, morin, and 2',3',4'-trihydroxychalcone as natural inhibitors of digestive enzymes.

Co-encapsulation and release of apigenin and ascorbic acid in polyelectrolyte multilayer capsules for targeted polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS), a prevalent endocrine disorder in women of reproductive age, is linked to hormonal imbalances and oxidative stress. Our study investigates the regenerative potential of apigenin (AP, hydrophobic) and ascorbic acid (AC, hydrophilic) encapsulated within poly (allylamine hydrochloride) and dextran sulfate (PAH/DS) hollow microcapsules for PCOS. These microcapsules, constructed using a layer-by-layer (LbL) assembly, are found to be 4 ± 0.5 µm in size. Our research successfully demonstrates the co-encapsulation of AP and AC in a single PAH/DS system with high encapsulation efficiency followed by successful release at physiological conditions by CLSM investigations. In vitro tests with testosterone-treated CHO cells reveal that the dual-drug-loaded PAH/DS capsules effectively reduce intracellular ROS levels and apoptosis and offering protection. In an in-vivo zebrafish model, these capsules demonstrate active biodistribution to targeted ovaries and reduce testosterone levels through radical scavenging. Histopathological examinations show that the injected dual-drug-loaded PAH/DS microcapsules assist in the development of ovarian follicles in testosterone-treated zebrafish. Hence, this dual-drug-loaded system, capable of co-encapsulating two natural compounds, effectively interacts with ovarian cells, reducing cellular damage and normalizing PCOS conditions.

Skeletal and neurological risks demonstrated in zebrafish due to second-hand cigarette smoke and the neutralization of luteolin.

BACKGROUND: Cigarette smoke exposure poses significant health risks, including oxidative stress, inflammation, tissue damage, and neurodegenerative diseases. Luteolin, a natural flavonoid known for its antioxidant and anti-inflammatory properties, is of interest in countering these effects. AIM: This study aims to assess luteolin's protective potential against cigarette smoke extract (CSE) in adult zebrafish. MATERIALS AND METHODS: Adult zebrafish were exposed to CSE for 15 days, inducing smoke-related damage. Subsequent luteolin treatment assessed its impact. Evaluations included antioxidant enzymes (SOD, CAT), nitric oxide (NO), LDH activity (cellular damage), tissue integrity, fibrosis, amyloid plaque accumulation, and CSE component analysis via HPLC. KEY FINDINGS: CSE exposure heightened oxidative stress, reducing SOD and CAT activity and elevating NO levels, leading to cellular damage and tissue disruption, notably fibrosis and amyloid plaque accumulation. Inflammatory markers TNF-α and IL-1ß also increased. Luteolin treatment restored SOD and CAT activity, reduced LDH and NO activity, counteracting oxidative damage. It also mitigated fibrosis and reduced amyloid plaque deposition, preserving tissue integrity. Luteolin reduced TNF-α and IL-1ß levels and CSE components, displaying anti-inflammatory effects. SIGNIFICANCE: This study underscores luteolin's potential as a protective agent against cigarette smoke-induced harm in a zebrafish model.

Ophthalmic Intervention of Naringenin Decreases Vascular Endothelial Growth Factor by Counteracting Oxidative Stress and Cellular Damage in In Vivo Zebrafish.

Diabetes Mellitus is a metabolic disease that leads to microvascular complications like Diabetic retinopathy (DR), a major cause of blindness worldwide. Current medications for DR are expensive and report multiple side effects; therefore, an alternative medication that alleviates the disease condition is required. An interventional approach targeting the vascular endothelial growth factor (VEGF) remains a treatment strategy for DR. Anti-VEGF medicines are being investigated as the main therapy for managing vision-threatening complications of DR, such as diabetic macular oedema. Therefore, this study investigated the effect of flavonoid naringenin (NG) from citrus fruits on inhibiting early DR in zebrafish. When exposed to 130 mM glucose, the zebrafish larvae developed a hyperglycaemic condition accompanied by oxidative stress, cellular damage, and lipid peroxidation. Similarly, when adult zebrafish were exposed to 4% Glucose, high glucose levels were observed in the ocular region and massive destruction in the retinal membrane. High glucose upregulated the expression of VEGF. In comparison, the co-exposure to NG inhibited oxidative stress and cellular damage and restored the glutathione levels in the ocular region of the zebrafish larvae. NG regressed the glucose levels and cellular damage along with an inhibition of macular degeneration in the retina of adult zebrafish and normalized the overexpression of VEGF as a promising strategy for treating DR. Therefore, intervention of NG could alleviate the domestication of alternative medicine in ophthalmic research.

Morin inhibits colon cancer stem cells by inhibiting PUM1 expression in vitro.

Over the last few decades, the number of people diagnosed with cancer has increased dramatically every year, making it a major cause of mortality today. Colon cancer is the third most common cancer worldwide, and the second in mortality rate. Current cancer treatment fails to treat colon cancer completely due to the remains of Cancer Stem Cells (CSCs). Morin flavonoid present in figs (Ficus carica) and other plant sources, was found to have an anti-proliferative effect on the colon cancer model and cell line, but it is not studied for its effect on the colon CSCs. In this study, we have tested the potency of morin to inhibit CSCs. We found that morin has significantly reduced colon cancer cell proliferation, colony formation, migration, and colonospheroid formation in a dose-dependent manner. Pumilio-1 (PUM1) has been shown to play an important role in colon CSCs maintenance. We found that morin has a good binding affinity with PUM1 protein with one hydrophobic and two hydrogen bond interactions. Further, the immunofluorescence results have also shown a reduction in PUM1 expression in colon cancer cell lines after morin treatment. CD133 is overexpressed in colon CSCs and morin treatment has reduced the CD133 expression in HCT116 and CT26 colon cancer cell lines. Our research outcome has explored the anti-cancer stem cell potency of morin via targeting the PUM1 protein and further reducing the colon spheroids formation and reducing the CD133 expression in colon cancer cells.

Copper sulfate induced toxicological impact on in-vivo zebrafish larval model protected due to acacetin via anti-inflammatory and glutathione redox mechanism.

Copper sulfate (CuSO4) as industrial effluent is intentionally or unintentionally released into water bodies and accumulates in the fish. Because of its numerous applications, CuSO4 can be hazardous to non-target creatures, producing direct alterations in fish habitats. Acacetin is a flavonoid present in all vascular plants that are extensively dispersed in plant pigments and responsible for many natural hues. However, the impact of acacetin on mitigating the toxic effect of CuSO4 in the in-vivo conditions is not known. The toxicity of acacetin was determined by measuring the survival, deformities and heart rate after treatment with various concentrations to larvae. The protective effect of acacetin was also observed in CuSO4 exposed zebrafish larvae by reducing malformation, mortality rate and oxidative stress. Meanwhile, the acacetin-protected larvae from CuSO4 effects through the molecular mechanism by suppressing pro-inflammatory genes (COX-2, TNF-α and IL-1) and upregulating antioxidant genes (GPx, GST and GR). Overall, our findings suggest that acacetin can act as a protective barrier against CuSO4-induced inflammation in an in-vivo zebrafish larval model.

Neuroprotective effect of Biochanin a against Bisphenol A-induced prenatal neurotoxicity in zebrafish by modulating oxidative stress and locomotory defects.

Exogenous toxicants cause oxidative stress and damage to brain cells, resulting in inflammation. Neuroinflammation is important in the pathobiology of various neurological illnesses, including Alzheimer's disease (AD). In this context, Bisphenol A (BPA), a common toxin, causes oxidative damage and has been linked to neurological problems. An O-methylated isoflavone known as Biochanin A (5,7-dihydroxy-4'-methoxy-isoflavone, BCA) is considered to be a phytoestrogen, which is abundant in some legume plants and soy which have preventive effects against cancer, osteoporosis, menopausal symptoms and oxidative stress. However, the mechanism by which BCA protected the prenatal neurological stress are not known. So that, in this study we investigated the BCA neuroprotective effect against BPA-induced neuroinflammation in zebrafish embryo models. For this study, fertilized zebrafish embryos are exposed to BPA (1 µM) with or without BCA. Our finding suggested that BCA co-exposure prevented the depletion of antioxidant defense enzymes by BPA and reduced the production of intracellular ROS production, superoxide anion (O2-), lipid peroxidation (LPO), lactate dehydrogenase (LDH) and nitric oxide (NO) levels in the head that aided in safeguarding neuronal development. Baseline locomotion was rendered and a total distance was calculated to assess the motor function. Exposure to BCA increased acetylcholinestrase (AChE) and improved motor neuron functions. It also reduced the pro-inflammatory response expression and prevented neuroinflammation. Our study suggests that BCA has a positive role in the attenuation or amelioration of neuronal oxidative damage and locomotory behaviour induced by BPA.

Syringol isolated from Eleusine coracana (L.) Gaertn bran suppresses inflammatory response through the down-regulation of cPLA2, COX-2, IκBα, p38 and MPO signaling in sPLA2 induced mice paw oedema.

Eleusine coracana (L.) Gaertn (E. coracana) is one of the highest consuming food crops in Asia and Africa. E. coracana is a plant with several medicinal values including anti-ulcerative, anti-diabetic, anti-viral and anti-cancer properties. However, the anti-inflammatory property of E. coracana remains to be elucidated. Therefore, the objective of present study was to investigate the potential in isolated molecule from E. coracana via a combination of in vitro, in vivo and in silico methods. In this study, we have isolated, purified and characterized an anti-inflammatory molecule from E. coracana bran extract known as syringol. Purification of syringol was accomplished by combination of GC-MS and RP-HPLC techniques. Syringol significantly inhibited the enzymes activity of sPLA2 (IC50 = 3.00 µg) and 5-LOX (IC50 = 0.325 µg) in vitro. The inhibition is independent of substrate concentration, calcium ion concentration and was irreversible. Syringol interacts with purified sPLA2 enzymes as evidenced by fluorescence and molecular docking studies. Further, the syringol molecule dose dependently inhibited the development of sPLA2 and λ-carrageenan induced edema. Furthermore, syringol decreases the expression of cPLA2, COX-2, IκBα, p38 and MPO in edematous tissues as demonstrated by western blots. These studies revealed that syringol isolated from E. coracana bran may develop as a potent anti-inflammatory molecule.

Daidzein normalized gentamicin-induced nephrotoxicity and associated pro-inflammatory cytokines in MDCK and zebrafish: Possible mechanism of nephroprotection.

This study investigates the therapeutic activity of daidzein, an isoflavone that occurs naturally in plants and herbs, against gentamicin-induced nephrotoxicity in Madin-Darby canine kidney (MDCK) cells in-vitro and zebrafish model in-vivo. The in-vitro studies revealed that daidzein protected MDCK cells from gentamicin-induced inflammation by suppressing oxidative stress and apoptosis. The zebrafish were divided into groups and injected with gentamicin (140 mg/mL) to induce nephrotoxic conditions. After injection, renal dysfunction, nitric oxide production, antioxidant consumption, exaggerated apoptosis, and inflammation were all observed in the zebrafish model. We also observed that during kidney inflammation in zebrafish, pro-inflammatory cytokines such as cyclooxygenase (COX-2), tumor necrosis factor (TNF-α), and interleukin-1ß (IL-1ß) are upregulated. Furthermore, daidzein treatment after gentamicin injection showed a strong protective anti-inflammatory effect. Daidzein activity was associated with an increase in antioxidant biomarkers such as superoxide dismutase (SOD) and glutathione reductase (GSH), whereas lipid peroxidation (LPO) and nitric oxide (NO) production were decreased in a dose-dependent factor. Moreover, histopathological alteration caused by gentamicin in zebrafish kidneys was normalized due to daidzein treatment. Daidzein also downregulated the pro-inflammatory cytokines gene expression in gentamicin-induced kidney inflammation in zebrafish. These results revealed that daidzein could potentially prevent nephrotoxic conditions through pro-inflammatory cytokines inhibition and its antioxidant property.

Separation and identification of bioactive peptides from stem of Tinospora cordifolia (Willd.) Miers.

Enzyme hydrolysates (trypsin, papain, pepsin, α-chymotrypsin, and pepsin-pancreatin) of Tinospora cordifolia stem proteins were analyzed for antioxidant efficacy by measuring (1) 1,1-diphenyl-2-picrylhydrazyl (DPPH•) radical scavenging activity, (2) 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) radical scavenging capacity, and (3) Fe2+ chelation. Trypsin hydrolysate showed the strongest DPPH• scavenging, while α-chymotrypsin hydrolysate exhibited the highest ABTS+ scavenging and Fe2+ chelation. Undigested protein strongly inhibited the gastrointestinal enzymes, trypsin (50% inhibition at enzyme/substrate ratio = 1:6.9) and α-chymotrypsin (50% inhibition at enzyme/substrate ratio = 1:1.82), indicating the prolonged antioxidant effect after ingestion. Furthermore, gel filtration purified peptide fractions of papain hydrolysates exhibited a significantly higher ABTS+ and superoxide radical scavenging as compared to non-purified digests. Active fraction 9 showing the highest radical scavenging ability was further purified and confirmed by MALDI-TOF MS followed by MS/MS with probable dominant peptide sequences identified are VLYSTPVKMWEPGR, VITVVATAGSETMR, and HIGININSR. The obtained results revealed that free radical scavenging capacity of papain hydrolysates might be related to its consistently low molecular weight hydrophobic peptides.

Constitutive cyclic GMP accumulation in Arabidopsis thaliana compromises systemic acquired resistance induced by an avirulent pathogen by modulating local signals.

The infection of Arabidopsis thaliana plants with avirulent pathogens causes the accumulation of cGMP with a biphasic profile downstream of nitric oxide signalling. However, plant enzymes that modulate cGMP levels have yet to be identified, so we generated transgenic A. thaliana plants expressing the rat soluble guanylate cyclase (GC) to increase genetically the level of cGMP and to study the function of cGMP in plant defence responses. Once confirmed that cGMP levels were higher in the GC transgenic lines than in wild-type controls, the GC transgenic plants were then challenged with bacterial pathogens and their defence responses were characterized. Although local resistance was similar in the GC transgenic and wild-type lines, differences in the redox state suggested potential cross-talk between cGMP and the glutathione redox system. Furthermore, large-scale transcriptomic and proteomic analysis highlighted the significant modulation of both gene expression and protein abundance at the infection site, inhibiting the establishment of systemic acquired resistance. Our data indicate that cGMP plays a key role in local responses controlling the induction of systemic acquired resistance in plants challenged with avirulent pathogens.