Feasibility of leaf protein hydrolysates to reduce reactive oxygen species and cancer cell proliferation

The reactive oxygen species and health: Reactive oxygen species (ROS) arise from typical cellular metabolism and are essential for several cellular functions, as moderate amounts of ROS are required to support numerous physiological processes. Overproduction of ROS can lead to oxidative stress, compromising the structural integrity of essential biomolecules such as lipids and proteins DNA.1 Increased ROS levels, combined with the down-regulation of antioxidant enzyme systems, drive malignant transformation by targeting several molecules, including nuclear factor-κB and the nuclear factor erythroid 2-related factor 2. The signaling pathways these crucial elements regulate create an inflammatory setting that hinders apoptotic cell death while encouraging tumor growth, angiogenesis, and metastasis. These processes greatly supported malignant tumor initiation, growth, and advancement. Nonetheless, the impact of ROS on cancer depends on various factors, such as the genetic makeup of the cancer, the particular types of ROS involved (like O2•− and H2O2), and the levels and exposure time of ROS. Plant-derived natural antioxidants, including proteins, peptides, polyphenols, carotenoids, and vitamins, are a vital focus of scientific research. These compounds safeguard biomolecules from ROS, thereby alleviating oxidative stress. In particular, protein hydrolysates and bioactive peptides have attracted interest because of their widespread distribution, low toxicity, impressive free radical scavenging activity, and ability to bind metal ions. These properties are confirmed through clearly defined mechanisms, such as hydrogen atom transfer, single electron transfer, and/or the chelation of pro-oxidants metals.2,3 Leaf protein hydrolysates as antioxidants: Hydrolysates and peptides from plants such as sesame, rice, soy, potato, walnut, corn, hemp seed, and flaxseed have shown significant inhibition of ROS.3 However, the search for new plant-derived protein and peptide sources with antioxidant properties, especially from agro-industrial by-products, continues in augmenting. Green biomass, derived from leaves of industrial crops, serves as an economical and sustainable source of leaf protein concentrates and peptides for creating functional foods and biological applications. This approach meets consumers’ nutritional and cultural needs while addressing critical environmental issues. Leaf protein concentrate represents a viable alternative for edible protein sources exhibiting antioxidant properties, such as alfalfa,4 jackfruit,5 moringa,6 pumpkin,7 eggplant,1 mulberry,2 and amaranth.8 The hydrolysis of leaf protein concentrate exposes specific amino acid sequences within the primary structure that show improved antioxidative properties over the native form protein.8 Enzymatic hydrolysis involves using digestive, plant-derived, or microbial proteolytic enzymes to produce hydrolysates or peptides with antioxidant properties. The specificity of enzymes, along with hydrolysis conditions such as pH, temperature, and duration, greatly affects the molecular weight, hydrophobicity, and amino acid sequence of peptide chains and the concentration of free amino acids. Notably, the phenolic side chains of tyrosine and phenylalanine, the indole group found in tryptophan, the sulfhydryl group of cysteine, the imidazole group in histidine, and the pyrrolidine group present in proline can act as hydrogen or electron donors to free radicals. These interactions lead to the creation of more stable radical species.9 Protein hydrolysates derived from leaves demonstrate notable radical scavenging activity (RSA) and metal chelating activity, as shown by in vitro antioxidant assays (Figure 1). Protein hydrolysates from pumpkin leaves were produced with alcalase, trypsin, pepsin, and chymotrypsin. Among these, alcalase hydrolysates exhibited the lower value of EC50 (the concentration needed to scavenge 50% of free radicals). However, the peptide fractions < 1 kDa and 1–3 kDa showed greater activity than the hydrolysate.7 Similarly, hydrolysates from eggplant leaves produced by alcalase, trypsin, pepsin, and chymotrypsin exhibited strong RSA. Notably, chymotrypsin hydrolysates showed a lower EC50, and their peptide fractions under 1 kDa had enhanced activity compared to hydrolysate.1 For its part, pancreatin hydrolysates obtained from jackfruit leaves exhibited superior RSA compared to pepsin hydrolysates.5 Meanwhile, the neutrase hydrolysate derived from mulberry leaves,2 alcalase hydrolysate from alfalfa,4 and pepsin hydrolysate from amaranth8 also showed RSA (Figure 1).Figure 1: Antioxidant properties of leaf protein hydrolysates: alfalfa,4 jackfruit,5 moringa (Moringa oleifera),6 pumpkin,7 eggplant,1 mulberry,2 and amaranth.8Created with Sigma Plot software for Windows version 12.0. ABTS: 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid; DPPH: 2,2-diphenyl-1-picrylhydrazyl; MCA: metal chelating activity; RSA: radical scavenging activity.Nevertheless, the chemical and spectrophotometric techniques employed to evaluate their RSA or metal chelating activity are insufficient to completely capture their potential. Antioxidant tests conducted in cellular models provide complete insight into how these antioxidants function across different human cell types. Cellular antioxidant activity of leaf protein hydrolysates: The cellular antioxidant activity of different molecules enhances the understanding of how these compounds or extracts interact with complex enzymatic reactions in biological systems.10 The potential antioxidant of leaf protein hydrolysates (LPHs) obtained from jackfruit5,11 and moringa6,10 in cellular models of human epithelial colorectal adenocarcinoma (Caco-2) and human keratinocytes (HaCaT) cells, have been explored. Pepsin and pancreatin hydrolysates from jackfruit leaf,5 which exhibited good RSA, were assessed on H2O2-stressed HaCaT cells because the skin is exposed to stressors (endogenous and external) that produce ROS. The levels of intracellular ROS in HaCaT cells were decreased by 18.0–25.8% after treatment with pepsin hydrolysates. Although pancreatin hydrolysates demonstrated better RSA, only pepsin hydrolysate exhibited a significant protective effect against oxidative stress.11 Differences arise due to factors like the structure and size of peptides, the charge of amino acid side groups, and the critical matter of cellular bioavailability. The way peptides interact with free radicals, including electronic interactions, hydrogen-bonding properties, and the positioning and steric features of amino acid residues, plays a significant role in determining the overall protective effect. Furthermore, moringa LPHs obtained by simulated gastrointestinal digestion showed cellular antioxidative activity on Caco-2 cells of 73.45–83.62% at 250 and 500 µg/mL.10 The mechanism of antioxidant peptides is still not fully understood; however, some potential actions of LPHs may be linked to certain mechanisms, as shown in Figure 2. Studies have evidenced that LPHs can stop lipid peroxidation by disrupting the oxidation chain reaction of fats (Figure 2b),1,8 which may be due to deactivation of ROS and neutralization free radicals (Figure 2a)8 and metal chelation ability (Figure 2d).1,8 Another possible mechanism of LPHs could involve stimulating the production of defense enzymes, including superoxide dismutase and glutathione peroxidase catalase, promoting defensive actions against ROS (Figure 2c),12 which suggests activating the transcription factor known as nuclear factor erythroid 2-related factor 2. Furthermore, antioxidant peptides may facilitate the intracellular conversion of cysteine into glutathione, thereby helping protect cells from ROS-induced damage.Figure 2: Possible antioxidant mechanism of leaf protein hydrolysates, based on previous studies.3,13Created with GraphPad Prism (version 8.2.1). a: Free radicals; b: fats; c: reactive oxygen species; d: metal chelation. A•: Radical formed when antioxidant donates an electron (less reactive than R•); AH: antioxidant; CAT: catalase; GPx: glutathione peroxidase; Nrf2: nuclear factor erythroid 2-related factor 2; R•: free radical; RH: reduced radical formed when the R• radical captures a hydrogen or electron from the antioxidant; SOD: superoxide dismutase.Antiproliferative activity of leaf protein hydrolysates: Some LPHs with RSA also have shown some antiproliferative activity; among them are the LPHs of moringa and jackfruit (Figure 1). Moringa LPHs showed antiproliferative activity for Caco-2 cells was 78.19–90.20% at 200–500 µg/mL.10 Pancreatin hydrolysates of jackfruit demonstrated varying effects on cancer cells, such as human cervical (HeLa), ovarian (SKOV-03), and liver (Hep G2) cancer cells. Antiproliferative activity reached 25.3% at 1000 mg/mL from hydrolysate at 240 minutes. At 30 and 60 minutes, hydrolysates showed 13.2–14.5% (500 µg/mL) activity in SKOV-03 cells. In Hep G2, 60 minutes hydrolysates reduced activity by 17% at 1000 µg/mL.11 Meanwhile, jackfruit pepsin hydrolysate showed a more substantial protective effect against oxidative stress but lacked antiproliferative properties in cancer cells. These differences were attributed to higher concentrations of hydrophobic amino acids in pancreatin hydrolysates, which have a low net charge. The affinity for the hydrophobic region of the cell membrane facilitates internalization and translocation, which may destabilize the membrane and induce the formation of pores that could ultimately result in cell death. These studies highlight the importance of analysis in cellular models since protein hydrolysates have a complex composition, and some peptides possibly interact in ways that negate each other’s effects, influencing their efficacy and mechanisms within cells. Then, the findings showed that LPHs modulate oxidative stress biomarkers, including intracellular levels of ROS and apoptosis, within various cell lines.3 However, it is imperative to verify that the new hydrolysates or peptides, do not adversely affect healthy tissues within the human body. Cytotoxicity of leaf protein hydrolysates: Normal cell lines are utilized to demonstrate the cytotoxic effects of various molecules. Jackfruit pancreatin hydrolysates showed no cytotoxicity on HaCaT and macrophage cells at 1000 µg/mL, while pepsin hydrolysates showed none up to 500 µg/mL. Macrophages showed a 23% increase in viability after pepsin hydrolysate treatment at 250 μg/mL. The effect was amplified with pancreatin hydrolysate at 125 μg/mL, leading to a 41.9% increase in cell proliferation compared to untreated cells.11 The findings reveal that protein hydrolyzate from pancreatin is non-cytotoxic, not affecting normal cells but moderately inhibiting cancer cell proliferation. Conversely, moringa LPHs did not exhibit toxicity on RAW 264.7 cells at 1–100 µg/mL6 and on CCD18-Co (human colon fibroblasts) cells at 250–500 µg/mL.10 The findings emphasize the low toxicity and safety use of LPHs, even in larger quantities. This feature is vital for developing anticancer drugs, primarily because amino acids or low molecular weight peptides released during hydrolysis serve as nutrients and support cellular promotion so cells can easily utilize growth.10 Future research directions and limiting factors: Since LPHs can stimulate the proliferation of normal cells, they have the potential to be safe ingredients for novel functional foods. In fact, jackfruit pancreatin hydrolysates have been used to microencapsulate lipophilic compounds via emulsions, indicating their potential as a plant-based alternative for clean-label functional products. Integrating protein hydrolysates into bioactive molecule encapsulation can enhance their antioxidant and anticancer effects, leading to therapeutic microstructures. In that sense, the fractionating jackfruit hydrolysate may enhance the potential for anticancer or antioxidant activity, as these effects are associated with specific peptides that demonstrate direct cytotoxicity against cancer cells or protection against oxidative stress. Furthermore, it remains imperative to investigate other hydrolysates and peptides derived from green biomasses in cellular models, which have been analyzed using spectrophotometry (Figure 1). This exploration aims to diversify and augment the spectrum of antioxidant and antiproliferative molecules available for human applications. Likewise, the small size of these peptides contributes to their low immunogenicity, suggesting their potential as immunomodulators and scar treatment agents repair.10 Likewise, due to the using green biomass as a source of LPHs and peptides is a relatively recent trend, most of the antioxidant and antiproliferative characterization has thus far been restricted to in vitro studies, which could be a limiting factor for future applications. Further investigation using in vivo models is essential for a comprehensive understanding of the effects of LPHs on biological systems. In vivo studies are essential to validate the effects of LPHs under realistic physiological conditions before their use in pharmaceutical or nutraceutical applications. This allows us to understand the complex biological interactions, absorption and metabolism, efficacy, safety, and toxicity of these compounds. Despite, LPHs and peptides derived from green biomasses represent promising alternatives that warrant further exploration as antioxidants or antiproliferative agents. Furthermore, LPHs and peptides can provide a cost-effective and sustainable source of plant proteins, a factor of particular importance within a circular economy and biorefinery framework. Such an approach underscores the potential benefits of leveraging agricultural by-products to enhance health outcomes and promote environmental sustainability. Conclusions: LPHs from various sources show great potential as antioxidants or antiproliferative agents. These hydrolysates reduce ROS and slow down the proliferation of cancer cells without causing significant cytotoxicity on normal cells. Additionally, their proliferative effect on normal cells indicates that hydrolysates may serve as immunomodulatory and/or wound-healing agents, suggesting their safe use as innovative alternative ingredients or additives derived from plant proteins to produce biofunctional foods. Green biomass undoubtedly represents a novel source of proteins, hydrolysates, and bioactive peptides with antioxidant and antiproliferative properties. Due to its great availability, it deserves attention and consideration in future industrial scaling. Nonetheless, further investigation utilizing in vivo models is essential for a comprehensive understanding of the effects of LPHs on biological systems. This research would confirm their safety profile and provide evidence of their cytotoxic, antioxidant, and antiproliferative effects in a more realistic context.