Structural and Functional Characterization of New Lipid Transfer Proteins with Chitin-Binding Properties: Insights from Protein Structure Prediction, Molecular Docking, and Antifungal Activity.

Faced with the emergence of multiresistant microorganisms that affect human health, microbial agents have become a serious global threat, affecting human health and plant crops. Antimicrobial peptides have attracted significant attention in research for the development of new microbial control agents. This work's goal was the structural characterization and analysis of antifungal activity of chitin-binding peptides from Capsicum baccatum and Capsicum frutescens seeds on the growth of Candida and Fusarium species. Proteins were initially submitted to extraction in phosphate buffer pH 5.4 and subjected to chitin column chromatography. Posteriorly, two fractions were obtained for each species, Cb-F1 and Cf-F1 and Cb-F2 and Cf-F2, respectively. The Cb-F1 (C. baccatum) and Cf-F1 (C. frutescens) fractions did not bind to the chitin column. The electrophoresis results obtained after chromatography showed two major protein bands between 3.4 and 14.2 kDa for Cb-F2. For Cf-F2, three major bands were identified between 6.5 and 14.2 kDa. One band from each species was subjected to mass spectrometry, and both bands showed similarity to nonspecific lipid transfer protein. Candida albicans and Candida tropicalis had their growth inhibited by Cb-F2. Cf-F2 inhibited the development of C. albicans but did not inhibit the growth of C. tropicalis. Both fractions were unable to inhibit the growth of Fusarium species. The toxicity of the fractions was tested in vivo on Galleria mellonella larvae, and both showed a low toxicity rate at high concentrations. As a result, the fractions have enormous promise for the creation of novel antifungal compounds.

Deciphering the drug delivery potential of Type1 lipid transfer protein from Citrus sinensis for enhancing the therapeutic efficacy of drugs.

Type1 Non-specific Lipid Transfer Protein (CsLTP1) from Citrus sinensis is a small cationic protein possessing a long tunnel-like hydrophobic cavity. CsLTP1 performing membrane trafficking of lipids is a promising candidate for developing a potent drug delivery system. The present work includes in-silico studies and the evaluation of drugs binding to CsLTP1 using biophysical techniques along with the investigation of CsLTP1's ability to enhance the efficacy of drugs employing cell-based bioassays. The in-silico investigations identified Panobinostat, Vorinostat, Cetylpyridinium Chloride, and Fulvestrant with higher affinities and stability of binding to the hydrophobic pocket of CsLTP1. SPR studies revealed strong binding affinities of anticancer drugs, Panobinostat (KD = 1.40 µM) and Vorinostat (KD = 2.17 µM) to CsLTP1 along with the binding and release kinetics. CD and fluorescent spectroscopy revealed drug-induced conformational changes in CsLTP1. CsLTP1-associated drug forms showed remarkably enhanced efficacy in MCF-7 cells, representing increased cell cytotoxicity, intracellular ROS, reduced mitochondrial membrane potential, and up-regulation of proapoptotic markers than the free drugs employing qRT-PCR and western blot analysis. The findings demonstrate that CsLTP1 binds strongly to hydrophobic drugs to facilitate their transport, hence improving their therapeutic efficacy revealed by the in-vitro investigations. This study establishes an excellent foundation for developing CsLTP1-based efficient drug delivery system.

Potatoes Compared with Rice in Meals with either Animal or Plant Protein Reduce Postprandial Glycemia and Increase Satiety in Healthy Adults: A Randomized Crossover Study.

BACKGROUND: Rice and pasta are recommended as healthier than potatoes on the basis of their glycemic index when eaten alone. OBJECTIVES: The study objective was to evaluate postprandial glycemia (PPG), appetite, and food intake (FI) at meals with potatoes or rice when consumed with either meatballs or their vegetarian substitute. METHODS: In a randomized, single-blinded, crossover design, 26 (13 males and 13 females) healthy adults (age: 18-45 y; body mass index [kg/m2]: 18.5-29.9) consumed isocaloric fixed amounts of either meatballs or vegetarian-substitute balls with ad libitum access to either baked French fries (BFF), instant mashed potatoes (IMPs), or rice (control). FI was measured at the meal and at an ad libitum pizza meal served 120 min later. Blood glucose (BG), appetite, and plasma insulin responses were measured within the meal (0-30 min), postmeal (30-120 min), within pizza meal (120-140 min), and post-pizza (140-170 min). Effects of protein source, carbohydrate (CHO) source, and sex and their interactions were analyzed using analysis of variance followed by Tukey's post hoc test. RESULTS: Participants consumed 23-25% less treatment meal energy (kcal), 32-34% less CHO energy (kcal), and 13-16% less total energy (kcal) after the BFF and IMP than rice meals (P < 0.0001). Postmeal BG was lower after IMP (6.76 ± 0.15; P < 0.0001) and rice (6.92 ± 0.15; P = 0.0012) compared with BFF (7.19 ± 0.15). Post-pizza BG was higher after rice (6.77 ± 0.09) than that after BFF (6.51 ± 0.09; P = 0.0012) and IMP (6.39 ± 0.09; P < 0.0001). Postmeal meaned insulin was higher after BFF (82.16 ± 8.58) and IMP (77.75 ± 8.60) compared with rice (56.44 ± 8.59; P < 0.002). Insulin during pizza meal was lower after BFF (17.14 ± 6.90) compared with both IMP (39.03 ± 6.90; P = 0.0060) and rice (34.21 ± 6.90; P = 0.0336). Meatballs led to lower BG (6.48 ± 0.09; P = 0.0076) and higher insulin (84.54 ± 5.87; P = 0.0406) post-pizza compared with their plant protein substitute (6.64 ± 0.09 and 73.18 ± 5.87, respectively). CONCLUSIONS: Adults consuming meatballs or plant-based substitute with ad libitum IMP had lower PPG post-treatment and at a later pizza meal compared with rice. Both IMP and BFF resulted in lower energy intake than after rice. This trial was registered at http://clinicaltrials.gov (https://register. CLINICALTRIALS: gov/prs/app/action/SelectProtocol?sid=S000CKIJ&selectaction=Edit&uid=U0000IA4&ts=2&cx=-uf51kf) as NCT05610124. Protocol ID: 43406 (Postprandial Glycemia and Satiety of Meals with Potatoes, with and without Protein).

A new trypsin inhibitor from Centrosema plumieri effective against digestive proteases from Tribolium castaneum, an eco-friendly alternative.

Tribolium castaneum, also known as the red flour beetle, is a polyphagous pest that seriously damages agricultural products, including stored and processed grains. Researchers have aimed to discover alternative pest control mechanisms that are less harmful to the ecosystem than those currently used. We conduct the purification and characterization of a protease inhibitor from C. plumieri seeds and an in vitro evaluation of its insecticidal potential against the insect pest T. castaneum. The trypsin inhibitor was isolated from C. plumieri seeds in a single-step DEAE-Sepharose column chromatography and had a molecular mass of 50 kDA. When analyzed for interaction with different proteolytic enzymes, the inhibitor exhibited specificity against trypsin and no activity against other serine proteases such as chymotrypsin and elastase-2. The isolated inhibitor was able to inhibit digestive enzymes of T. castaneum from extracts of the intestine of this insect. Therefore, we conclude that the new protease inhibitor, specific in tryptic inhibition, of protein nature from the seeds of C. plumieri was effective in inhibiting the digestive enzymes of T. castaneum and is a promising candidate in the ecological control of pests.

Purification, Structural Characterization, and Anticandidal Activity of a Chitin-Binding Peptide with High Similarity to Hevein and Endochitinase Isolated from Pepper Seeds.

With the emergence of multidrug-resistant microorganisms, microbial agents have become a serious global threat, affecting human health and various plants. Therefore, new therapeutic alternatives, such as chitin-binding proteins, are necessary. Chitin is an essential component of the fungal cell wall, and chitin-binding proteins exhibit antifungal activity. In the present study, chitin-binding peptides isolated from Capsicum chinense seeds were characterized and evaluated for their in vitro antimicrobial effect against the growth of Candida and Fusarium fungi. Proteins were extracted from the seeds and subsequently the chitin-binding proteins were separated by chitin affinity chromatography. After chromatography, two fractions, Cc-F1 (not retained on the column) and Cc-F2 (retained on the column), were obtained. Electrophoresis revealed major protein bands between 6.5 and 26.6 kDa for Cc-F1 and only a ~ 6.5 kDa protein band for Cc-F2, which was subsequently subjected to mass spectrometry. The protein showed similarity with hevein-like and endochitinase and was then named Cc-Hev. Data are available via ProteomeXchange with identifier PXD054607. Next, we predicted the three-dimensional structure of the peptides and performed a peptide docking with (NAG)3. Subsequently, growth inhibition assays were performed to evaluate the ability of the peptides to inhibit microorganism growth. Cc-Hev inhibited the growth of C. albicans (up to 75% inhibition) and C. tropicalis (100% inhibition) and induced a 65% decrease in cell viability for C. albicans and 100% for C. tropicalis. Based on these results, new techniques to combat fungal diseases could be developed through biotechnological applications; therefore, further studies are needed.

Protein fraction from Sesbania virgata (Cav.) Pers. seeds exhibit antioxidant and antifungal activities.

Sesbania virgata (Cav.) Pers. seeds are protein sources with health and environmental benefits. In this research, proteins with lectin activity were identified in a protein fraction from S. virgata seeds (PFLA), as well its antioxidant and antimicrobial potentials, in addition to cytotoxic effects. To obtain PFLA, seed flour was homogenized in Glycine-NaOH (100 mM; pH 9.0; NaCl 150 mM) and precipitated in ammonium sulfate. PFLA concentrates bioactive lectins (32 HU/mL, 480 HU/gFa, 18.862 HU/mgP) and essential amino acids (13.36 g/100g protein). PFLA exerts antioxidant activity, acting as a promising metal chelating agent (~77% of activity). Analyzes of cell culture assay results suggest that antioxidant activity of PFLA may be associated with the recruitment of essential molecules to prevent the metabolic impairment of cells exposed to oxidative stress. PFLA (256 - 512 µg/mL) also exhibits antifungal activity, inhibiting the growth of Aspergillus flavus, Candida albicans, Candida tropicalis and Penicillium citrinum. Cytotoxic analysis indicates a tendency of low interference in the proliferation of 3T3 and HepG2 cells in the range of PFLA concentrations with biological activity. These findings support the notion that PFLA is a promising adjuvant to be applied in current policies on the management of metal ion chelation and fungal infections.

Potato protein hydrolysate inhibits RANKL-induced osteoclast development by inhibiting osteoclastogenic genes via the NF-κB/MAPKs signaling pathways.

In recent times, there has been growing attention towards exploring the nutritional and functional aspects of potato protein, along with its diverse applications. In the present study, we examined the anti-osteoclast properties of potato protein hydrolysate (PP902) in vitro. Murine macrophages (RAW264.7) were differentiated into osteoclasts by receptor activator of nuclear factor-κB ligand (RANKL), and PP902 was examined for its inhibitory effect. Initially, treatment with PP902 was found to significantly prevent RANKL-induced morphological changes in macrophage cells, as determined by tartrate-resistant acid phosphatase (TRAP) staining analysis. This notion was further supported by F-actin analysis using a confocal microscope. Furthermore, PP902 treatment effectively and dose-dependently down-regulated the expression of RANKL-induced osteoclastogenic marker genes, including TRAP, CTR, RANK, NFATc1, OC-STAMP, and c-Fos. These inhibitory effects were associated with suppressing NF-κB transcriptional activation and subsequent reduced nuclear translocation. The decrease in NF-κB activity resulted from reduced activation of its upstream kinases, including I-κBα and IKKα. Moreover, PP902 significantly inhibited RANKL-induced p38MAPK and ERK1/2 activities. Nevertheless, PP902 treatment prevents RANKL-induced intracellular reactive oxygen species generation via increased HO-1 activity. The combined antioxidant and anti-inflammatory effects of PP902 resulted in significant suppression of osteoclastogenesis, suggesting its potential as an adjuvant therapy for osteoclast-related diseases.

Fermented Wheat Germ Protein with Histone Deacetylase Inhibitor AR42 Demonstrates Enhanced Cytotoxicity against Lymphoma Cells In Vitro and In Vivo.

Current treatments for lymphoma are plagued by substantial toxicity and the inability to overcome drug resistance, leading to eventual relapse and rationalizing the development of novel, less toxic therapeutics and drug combinations. Histone deacetylase inhibitors (HDACis) are a broad class of epigenetic modulators that have been studied in multiple tumor types, including lymphoma. Currently, HDACis are FDA-approved for treating relapsed T-cell lymphomas and multiple myeloma, with ongoing trials in other lymphomas and solid tumors. As single agents, HDACis frequently elicit toxic side effects and have limited efficacy; therefore, many current treatment strategies focus on combinations to boost efficacy while attempting to minimize toxicity. Fermented wheat germ extract (FWGE) is a complementary agent that has shown efficacy in several malignancies, including lymphoma. Here, we utilize a more potent FWGE derivative, known as fermented wheat germ protein (FWGP), in combination with the HDACi AR42, to assess for enhanced activity. We report increased in vitro killing, cell cycle arrest, and in vivo efficacy for this combination compared to each agent alone with minimal toxicity, suggesting a potentially new, minimally toxic treatment modality for lymphoma.

Angiotensin-Converting Enzyme and Renin-Inhibitory Activities of Protein Hydrolysates Produced by Alcalase Hydrolysis of Peanut Protein.

Hypertension is a major controllable risk factor associated with cardiovascular disease (CVD) and overall mortality worldwide. Most people with hypertension must take medications that are effective in blood pressure management but cause many side effects. Thus, it is important to explore safer antihypertensive alternatives to regulate blood pressure. In this study, peanut protein concentrate (PPC) was hydrolyzed with 3-5% Alcalase for 3-10 h. The in vitro angiotensin-converting enzyme (ACE) and renin-inhibitory activities of the resulting peanut protein hydrolysate (PPH) samples and their fractions of different molecular weight ranges were determined as two measures of their antihypertensive potentials. The results show that the crude PPH produced at 4% Alcalase for 6 h of hydrolysis had the highest ACE-inhibitory activity with IC50 being 5.45 mg/mL. The PPH samples produced with 3-5% Alcalase hydrolysis for 6-8 h also displayed substantial renin-inhibitory activities, which is a great advantage over the animal protein-derived bioactive peptides or hydrolysate. Remarkably higher ACE- and renin-inhibitory activities were observed in fractions smaller than 5 kDa with IC50 being 0.85 and 1.78 mg/mL. Hence, the PPH and its small molecular fraction produced under proper Alcalase hydrolysis conditions have great potential to serve as a cost-effective anti-hypertensive ingredient for blood pressure management.

In silico screening of potential plant peptides against the non-structural proteins of dengue virus.

BACKGROUND OBJECTIVES: Peptides isolated from different sources of plants have the advantages of specificity, lower toxicity, and increased therapeutic effects; hence, it is necessary to search for newer antivirals from plant sources for the treatment of dengue viral infections. METHODS: In silico screening of selected plant peptides against the non-structural protein 1, NS3 protease domain (NS2B-NS3Pro) with the cofactor and ATPase/helicase domain (NS3 helicase domain/NS3hel) of dengue virus was performed. The physicochemical characteristics of the peptides were calculated using Protparam tools, and the allergenicity and toxicity profiles were assessed using allergenFP and ToxinPred, respectively. RESULTS: Among the tested compounds, Ginkbilobin demonstrated higher binding energy against three tested nonstructural protein targets. Kalata B8 demonstrated maximum binding energy against NSP-1 and NSP-2, whereas Circulin A acted against the NSP3 protein of dengue virus. INTERPRETATION CONCLUSION: The three compounds identified by in silico screening can be tested in vitro, which could act as potential leads as they are involved in hampering the replication of the dengue virus by interacting with the three prime non-structural proteins.

A Comparison of Phenolic, Flavonoid, and Amino Acid Compositions and In Vitro Antioxidant and Neuroprotective Activities in Thai Plant Protein Extracts.

The leaves of mulberry, Azolla spp., sunflower sprouts, cashew nut, and mung bean are considered rich sources of plant protein with high levels of branched-chain amino acids. Furthermore, they contain beneficial phytochemicals such as antioxidants and anti-inflammatory agents. Additionally, there are reports suggesting that an adequate consumption of amino acids can reduce nerve cell damage, delay the onset of memory impairment, and improve sleep quality. In this study, protein isolates were prepared from the leaves of mulberry, Azolla spp., sunflower sprouts, cashew nut, and mung bean. The amino acid profile, dietary fiber content, phenolic content, and flavonoid content were evaluated. Pharmacological properties, such as antioxidant, anticholinesterase, monoamine oxidase, and γ-aminobutyric acid transaminase (GABA-T) activities, were also assessed. This study found that concentrated protein from mung beans has a higher quantity of essential amino acids (52,161 mg/100 g protein) compared to concentrated protein from sunflower sprouts (47,386 mg/100 g protein), Azolla spp. (42,097 mg/100 g protein), cashew nut (26,710 mg/100 g protein), and mulberry leaves (8931 mg/100 g protein). The dietary fiber content ranged from 0.90% to 3.24%, while the phenolic content and flavonoid content ranged from 0.25 to 2.29 mg/g and 0.01 to 2.01 mg/g of sample, respectively. Sunflower sprout protein isolates exhibited the highest levels of dietary fiber (3.24%), phenolic content (2.292 ± 0.082 mg of GAE/g), and flavonoids (2.014 mg quercetin/g of sample). The biological efficacy evaluation found that concentrated protein extract from sunflower sprouts has the highest antioxidant activity; the percentages of inhibition of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical were 20.503 ± 0.288% and 18.496 ± 0.105%, respectively. Five plant-based proteins exhibited a potent inhibition of acetylcholinesterase (AChE) enzyme activity, monoamine oxidase (MAO) inhibition, and GABA-T ranging from 3.42% to 24.62%, 6.14% to 20.16%, and 2.03% to 21.99%, respectively. These findings suggest that these plant protein extracts can be used as natural resources for developing food supplements with neuroprotective activity.

A Novel Kunitz Trypsin Inhibitor from Enterolobium gummiferum Seeds Exhibits Antibiofilm Properties against Pathogenic Yeasts.

Plant peptidase inhibitors play crucial roles in plant defence mechanisms and physiological processes. In this study, we isolated and characterised a Kunitz trypsin inhibitor from Enterolobium gummiferum seeds named EgPI (E. gummiferum peptidase inhibitor). The purification process involved two chromatography steps using size exclusion and hydrophobic resins, resulting in high purity and yield. EgPI appeared as a single band of ~20 kDa in SDS-PAGE. Under reducing conditions, the inhibitor exhibited two polypeptide chains, with 15 and 5 kDa. Functional characterisation revealed that EgPI displayed an inhibition stoichiometry of 1:1 against trypsin, with a dissociation constant of 8.4 × 10-9 mol·L-1. The amino-terminal sequencing of EgPI revealed the homology with Kunitz inhibitors. Circular dichroism analysis provided insights into the secondary structure of EgPI, which displayed the signature typical of Kunitz inhibitors. Stability studies demonstrated that EgPI maintained the secondary structure necessary to exhibit its inhibitory activity up to 70 °C and over a pH range from 2 to 8. Microbiological screening revealed that EgPI has antibiofilm properties against pathogenic yeasts at 1.125 µmol·L-1, and EgPI reduced C. albicans biofilm formation by 82.7%. The high affinity of EgPI for trypsin suggests potential applications in various fields. Furthermore, its antibiofilm properties recommended its usefulness in agriculture and antimicrobial therapy research, highlighting the practical implications of our research.

Chemical modification, structure elucidation and antifungal mechanism studies of a peptide extracted from garlic (Allium sativum L.).

BACKGROUND: Garlic is a promising source of antimicrobial peptide separation, and chemical modification is an effective method for activity improvement. The present study aimed to improve the antifungal activity of a peptide extracted from garlic. Chemical modifications were conducted, and the structure-activity relationship and antifungal mechanism were investigated. RESULTS: The results indicated that the cationic charge induced by Lys residue at the N-terminal was important for the antimicrobial activity, and the modified sequence exhibited significant antifungal activity with low mammalian toxicity and a low tendency of drug resistance (p < 0.05). The structure-activity relationship analysis revealed that the modified active peptide had a predominant α-helical structure and an inner cyclic correlation. Transcriptomic analysis showed that peptide KMLKKLFR (Lys-Met-Leu-Lys-Lyse-Leu-Phe-Arg) affected the rRNA processing and carbon metabolism process of Candida albicans. In addition, the membrane potential study indicated a non-membrane destruction mechanism, and molecular docking analysis and a DNA interaction assay suggested promising inner targets. CONCLUSION: The results of the present study indicate that chemical modification by amino acid substitution was effective for antimicrobial activity improvement. The present study would benefit future antimicrobial peptide development and suggests that garlic is a great source of antibacterial peptides and peptide template separations for coping with antibiotic resistance. © 2024 Society of Chemical Industry.

Characterization, Bioaccesibility and Antioxidant Activities of Phenolic Compounds Recovered from Yellow pea (Pisum sativum) Flour and Protein Isolate.

This study focused on studying the bioaccesible phenolic compounds (PCs) from yellow pea flour (F) and protein isolate (I). Total phenolic contents (TPC), PCs composition and antioxidant activities were analysed in ethanol 60% extracts obtained by applying ultrasound assisted extraction (UAE, 15 min/40% amplitude). The preparation of I under alkaline conditions and the elimination of some soluble components at lower pH produced a change of PCs profile and antioxidant activity. After simulated gastrointestinal digestion (SGID) of both ingredients to obtain the digests FD and ID, notable changes in the PCs concentration and profiles could be demonstrated. FD presented a higher ORAC activity than ID (IC50 = 0.022 and 0.039 mg GAE/g dm, respectively), but lower ABTS•+ activity (IC50 = 0.8 and 0.3 mg GAE/g dm, respectively). After treatment with cholestyramine of extracts from FD and ID in order to eliminate bile salts and obtain the bioaccesible fractions FDb and IDb, ROS scavenging in H2O2-induced Caco2-TC7 cells was evaluated, registering a greater activity for ID respect to FD (IC50 = 0.042 and 0.017 mg GAE/mL, respectively). These activities could be attributed to the major bioaccesible PCs: OH-tyrosol, polydatin, trans-resveratrol, rutin, (-)-epicatechin and (-)-gallocatechin gallate for FD; syringic (the most concentrated) and ellagic acids, trans-resveratrol, and (-)-gallocatechin gallate for ID, but probably other compounds such as peptides or amino acids can also contribute.

Identification and Molecular Mechanism of Anti-inflammatory Peptides Isolated from Jack Bean Protein Hydrolysates: in vitro Studies with Human Intestinal Caco-2BBe Cells.

Jack bean (JB), Canavalia ensiformis (L.) DC, is a commonly cultivated legume in Indonesia. It is rich in protein, which can be hydrolyzed, making it potentially a good source of bioactive peptides. Intestinal inflammation is associated with several diseases, and the production of interleukin-8 (IL-8) in intestinal epithelial cells induced by tumor necrosis factor (TNF)-α has an important role in inflammatory reaction. The present study investigated the anti-inflammatory effects of peptides generated from enzymatic hydrolysis of JB protein on human intestinal Caco-2BBe cells. Additionally, in silico approaches were used to identify potential bioactive peptides. JB protein hydrolysate (JBPH) prepared using pepsin and pancreatin reduced the IL-8 expression at protein and mRNA levels in Caco-2BBe cells stimulated with TNF-α. Immunoblot analysis showed that the JBPH reduced the TNF-α-induced phosphorylation of c-Jun-NH(2)-terminal kinase, nuclear factor kappa B (NF-κB), and p38 proteins. Anti-inflammatory activity was observed in the 30% acetonitrile fraction of JBPH separated on a Sep-Pak C18 column. An ultrafiltration method revealed that relatively small peptides (< 3 kDa) had a potent inhibitory effect on the IL-8 production. Purification of the peptides by reversed-phase and anion-exchange high performance chromatography produced three peptide fractions with anti-inflammatory activities. A combination of mass spectrometry analysis and in silico approaches identified the potential anti-inflammatory peptides. Peptides derived from JB protein reduces the TNF-α-induced inflammatory response in Caco-2BBe cells via NF-κB and mitogen-activated protein kinase signaling pathways. Our results may lead to a novel therapeutic approach to promote intestinal health.

Jackfruit Leaf Protein Hydrolysates Obtained by Enzymatic Hydrolysis of Leaf Protein Concentrate with Pepsin and Pancreatin: Molecular Weight, Cytotoxicity, Antiproliferative Activity, and Oxidative Stress.

Jackfruit leaf protein hydrolysates obtained from the enzymatic hydrolysis of leaf protein concentrate with gastrointestinal enzymes have shown good techno-functional properties and high antioxidant capacity. However, molecular weight, antiproliferative activity, cytotoxicity and the ability to reduce reactive oxygen species (ROS) are still unknown. Therefore, this study aimed to evaluate the effect of jackfruit leaf protein hydrolysates obtained by enzymatic hydrolysis with pepsin and pancreatin at different hydrolysis times (30-240 min) on molecular weights, cytotoxicity, antiproliferation of cancer cells, and the reduction of reactive oxygen species in H2O2-induced HaCaT cells. The electrophoretic profile indicated that H-Pep contains peptides with molecular weights between 25 - 20 kDa. Meanwhile, H-Pan is composed of molecular weight products between 25 - 20 kDa and < 20 kDa. H-Pan and H-Pep (125-500 µg/mL) did not show significant cytotoxicity on HaCaT (human keratinocytes) and J774A.1 (murine macrophage cells). Antiproliferative activity was achieved in human cervical, ovarian, and liver cancer cells. H-Pan-240 min (1000 µg/mL) reduced the cell viability of cervical cancer cells by 23% while H-Pan-60 min significantly reduced cell viability of ovarian and liver cancer cells by 14.5 (500 µg/mL) and 17% (1000 µg/mL), respectively (P < 0.05). The protective effect against oxidative stress on H2O2-stressed HaCaT cells was obtained with H-Pep-60 min, which reduced 25% of ROS at 250 µg/mL (P < 0.05). The findings demonstrate the safe use of green biomass as a source of plant protein hydrolysates.

In Vitro Studies of Pumpkin (Cucurbita moschata var. Kashi Harit) Seed Protein Fraction(s) to Evaluate Anticancer and Antidiabetic Properties.

Chronic diseases like cancer and diabetes are the major public health concerns of India and worldwide. Nowadays, plant-derived products are in great demand for the treatment of these diseases. Pumpkin seeds are traditionally implicated for their pharmacological properties, as exemplified by benign prostatic hyperplasia. Earlier, pumpkin seed proteins were extracted by the Osborne method, and their functional and nutritional qualities were evaluated. Here, the aim is to assess in vitro, the anticancer and antidiabetic properties of seed protein fractions. HepG2, MDA-MB-231, and MCF-7 cell lines were treated with water-soluble (WF) and alkali-soluble fractions (AF) to assess cytotoxicity, while pancreatic ß-cells and insulin resistance (IR) - HepG2 cell lines were treated with WF to evaluate the antidiabetic potential. WF and AF showed cytotoxic effects towards HepG2 and MDA-MB-231 cell lines, suggesting apoptosis-mediated anticancerous activity. WF potentiates glucose-stimulated insulin secretion in pancreatic ß-cells, in a dose-dependent manner. In IR-HepG2 cell line studies, control, metformin, and WF-treated groups showed uptake of glucose, when compared to the diabetic group, which is well-correlated with the upregulated expressions of GLUT2 and GLUT4 transporters in these groups. These results indicate that proteins from WF and AF may have anticancerous and antidiabetic properties and thus have the potential to utilize pumpkin proteins in the management of cancer and diabetes.

The acyclotide ribe 31 from Rinorea bengalensis has selective cytotoxicity and potent insecticidal properties in Drosophila.

Cyclotides and acyclic versions of cyclotides (acyclotides) are peptides involved in plant defense. These peptides contain a cystine knot motif formed by three interlocked disulfide bonds, with the main difference between the two classes being the presence or absence of a cyclic backbone, respectively. The insecticidal activity of cyclotides is well documented, but no study to date explores the insecticidal activity of acyclotides. Here, we present the first in vivo evaluation of the insecticidal activity of acyclotides from Rinorea bengalensis on the vinegar fly Drosophila melanogaster. Of a group of structurally comparable acyclotides, ribe 31 showed the most potent toxicity when fed to D. melanogaster. We screened a range of acyclotides and cyclotides and found their toxicity toward human red blood cells was substantially lower than toward insect cells, highlighting their selectivity and potential for use as bioinsecticides. Our confocal microscopy experiments indicated their cytotoxicity is likely mediated via membrane disruption. Furthermore, our surface plasmon resonance studies suggested ribe 31 preferentially binds to membranes containing phospholipids with phosphatidyl-ethanolamine headgroups. Despite having an acyclic backbone, we determined the three-dimensional NMR solution structure of ribe 31 is similar to that of cyclotides. In summary, our results suggest that, with further optimization, ribe 31 could have applications as an insecticide due to its potent in vivo activity against D. melanogaster. More broadly, this work advances the field by demonstrating that acyclotides are more common than previously thought, have potent insecticidal activity, and have the advantage of potentially being more easily manufactured than cyclotides.

The heat-stable protein fraction from Opuntia ficus-indica seeds exhibits an enzyme protective effect against thermal denaturation and an antibacterial activity.

Desiccation tolerance in developing seeds occurs through several mechanisms among which, a common group of proteins named dehydrins has received considerable attention. So far, there is no information dealing with the accumulation of dehydrins in seeds of Opuntia ficus-indica. We have initiated here an extraction protocol based on two critical steps: heat and acid treatments, and the purity of this fraction was analyzed by FTIR spectroscopy. Western blot analysis of the heat-stable protein fraction (HSF) revealed two main bands of approximately 45 and 44 kDa, while three others of ∼40, 32, and 31 kDa were faintly visible, which were recognized by anti-dehydrin antibodies. This fraction exhibited a Cu2+ -dependent resistance to protease treatments. Next, we performed a series of assays to compare the functional properties of the HSF with those of the previously characterized wheat dehydrin (DHN-5). Antibacterial assays revealed that HSF exhibits only moderate antibacterial activities against gram-negative and gram-positive bacteria, with a minimum inhibition concentration ranging from 0.25 to 1 mg/ml. However, in vitro assays revealed that compared to DHN-5, HSF exhibits higher protective activities of the lactate dehydrogenase (LDH) when exposed to heat, freezing, and dehydration stresses. The protective role of HSF seems to be linked to its best ability to minimize protein aggregation.

Antifungal symbiotic peptide NCR044 exhibits unique structure and multifaceted mechanisms of action that confer plant protection.

In the indeterminate nodules of a model legume Medicago truncatula, ∼700 nodule-specific cysteine-rich (NCR) peptides with conserved cysteine signature are expressed. NCR peptides are highly diverse in sequence, and some of these cationic peptides exhibit antimicrobial activity in vitro and in vivo. However, there is a lack of knowledge regarding their structural architecture, antifungal activity, and modes of action against plant fungal pathogens. Here, the three-dimensional NMR structure of the 36-amino acid NCR044 peptide was solved. This unique structure was largely disordered and highly dynamic with one four-residue α-helix and one three-residue antiparallel ß-sheet stabilized by two disulfide bonds. NCR044 peptide also exhibited potent fungicidal activity against multiple plant fungal pathogens, including Botrytis cinerea and three Fusarium spp. It inhibited germination in quiescent spores of B. cinerea In germlings, it breached the fungal plasma membrane and induced reactive oxygen species. It bound to multiple bioactive phosphoinositides in vitro. Time-lapse confocal and superresolution microscopy revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci, followed by gradual loss of turgor, subsequent accumulation in the cytoplasm, and elevated levels in nucleoli of germlings. Spray-applied NCR044 significantly reduced gray mold disease symptoms caused by the fungal pathogen B. cinerea in tomato and tobacco plants, and postharvest products. Our work illustrates the antifungal activity of a structurally unique NCR peptide against plant fungal pathogens and paves the way for future development of this class of peptides as a spray-on fungistat/fungicide.