Use Of Bioactive Peptides Research Articles

Microbial- and Plant-Derived Bioactive Peptides and Their Applications against Foodborne Pathogens: Current Status and Future Prospects.

Bioactive peptides (BAPs) obtained from plants and microbes have been thoroughly explored and studied due to their prophylactic properties. The use of BAPs seems to be a promising substitute for several currently available antibiotics because of their antimicrobial properties against foodborne pathogens. BAPs have several other useful properties including antitumor, antihypertensive, antioxidant, antiobesity, and antidiabetic activities. Nowadays, scientists have attempted to recombinantly synthesize bioactive peptides to study their characteristics and potential uses, since BAPs are not found in large quantities in nature. Many pathogenic microorganisms including foodborne pathogens are becoming resistant to various antibiotics. To combat these pathogens, scientists are working to find novel, innovative, and safe antimicrobial agents. Plant- and microbe-based BAPs have demonstrated noteworthy antimicrobial activity against a wide range of pathogenic microorganisms, including foodborne pathogens. BAPs can kill pathogenic microorganisms by disrupting membrane integrity, inhibiting DNA and RNA synthesis, preventing protein synthesis, blocking protein activity, or interacting with certain intracellular targets. In addition, the positive effect of BAP consumption extends to gut microbiota modulation and affects the equilibrium of reactive oxygen species in the gut. This article discusses recombinant BAPs, BAPs generated from plants and microbes, and their antimicrobial applications and modes of action for controlling foodborne pathogens.

Membrane Separation of Chicken Byproduct Hydrolysate for Up-Concentration of Bioactive Peptides.

Membrane processes, such as microfiltration, ultrafiltration, and nanofiltration, are increasingly used for various applications in both upstream and downstream processing. Membrane-based processes play a critical role in the field of separation/purification of biotechnological products, including protein production/purification. The possibility of using membranes to separate peptides from a chicken byproduct hydrolysate and the effect of the performed downstream processing on the DPP-IV dipeptidyl peptidase IV (DPP-IV) inhibitory activity of mechanical deboning chicken residue (MDCR) has been investigated. The chicken byproduct hydrolysate was prepared by enzymatic hydrolysis followed by microfiltration (MF), ultrafiltration (UF), nanofiltration (NF), and reverse osmosis (RO) separation. Comparing all separation treatments, hydrolysates processed only by MF and UF show the best DPP-IV inhibition (59.5-60.0% at 1 mg/mL and 34.2-40.7% at 0.5 mg/mL). These samples show dose-responsive behavior. Bioactivity was correlated with molecular weight distribution profiles and average molecular weights. The nanofiltration process notably decrease the inhibitory activity, and these permeates show low DPP-IV inhibition (9.5-21.8% at 1 mg/mL and 3.6-12.1% at 0.5 mg/mL). The size-exclusion chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND) analysis confirms that NF and RO would retain the bioactive peptides in the concentrate in comparison to MF and UF. Bioactivity was correlated with molecular weight distribution profiles and average molecular weights. Permeates after ultrafiltration show an IC50 value of 0.75 mg/mL, comparable to other potent DPP-IV inhibitors derived from various food sources, and significantly more potent compared to the microfiltration sample, which shows an IC50 value of 1.04 mg/mL. The average molecular weight of the permeates calculated from the SEC chromatograms was 883 g/mol for UF and 1437 g/mol for MF. Of the four membranes studied, the UF membrane shows the best separation properties with respect to maximizing the yield and up-concentration of the bioactive peptides. Overall, UF was demonstrated to be a feasible technology for the removal of the undesired high-molecular-weight substances and up-concentration of small-molecular-weight bioactive peptides from chicken byproduct hydrolysate. These peptides might exhibit biological activity and could offer several health benefits. There is a high potential for the use of bioactive peptides, and more research in this field can lead to promising results that have significant effects in the food and medical industries.

ХАРАКТЕРИСТИКА И ВЛИЯНИЕ МИКРОКАПСУЛЯЦИИ НА СТАБИЛЬНОСТЬ БИОПЕПТИДА В МОДЕЛИ IN VITRO

One of the ways to prevent the development of oxidative stress is the oral use of bioactive peptides. To ensure the targeted release of biologically active substances in the body, they are microcapsulated. The aim of the research is to characterize, predict, evaluate the antioxidant properties of a biopeptide and study the effect of microcapsulation on its stability in a model of the gastrointestinal tract. The molecular mass distribution of the peptide was evaluated by mass spectrometric method. Identification and characterization of the peptide was carried out using Protein NCBI and ARD databases. The antioxidant activity of the peptide was determined by DPPH, ABTS and FRAP methods. The microencapsulation of the peptide was carried out in a pseudo-boiling layer of 10 % maltodextrin solution. As a result of research, a peptide was isolated from trypsin hydrolysate of cow colostrum, its molecular weight, amino acid sequence, charge, hydrophobicity, isoelectric point, two- and three-dimensional structure were determined. Based on the obtained physicochemical characteristics of the peptide, a hypothesis about its antioxidant properties has been proposed, which has been confirmed in in vitro studies. It is proved that the microcapsulation of the peptide into a protective layer of maltodextrin ensures its stability in the model of the oral cavity and stomach and targeted release in the intestine in vitro.

Marine Organisms as Potential Sources of Inhibits ACE on Hypertension Patient

Abstract:
 Hypertension is a condition of chronic high blood pressure in the systemic arteries which is a major risk factor for cardiovascular disease, kidney disorders, and death. There are two types of hypertension, the primary hypertension is thought to be caused by hereditary factors, age, gender, race, and an unhealthy lifestyle. Secondary hypertension is caused by an obvious cause. The prevalence of hypertension in Indonesia is quite high with the highest incidence rate in South Kalimantan (44,1%) and the lowest in Papua (22,2%). The pathophysiology of hypertension involves the enzyme Angiotensin Converting Enzyme (ACE) which plays a role in regulating blood pressure. The process of converting Angiotensinogen to Angiotensin I, then Angiotensin II by ACE can cause vasoconstriction and fluid retention, which contribute to hypertension. The changes in circulation and blood vessel structure have played a role in the risk of hypertension complications, including stroke. The marine organisms such as fish, shellfish, cephalopods, seaweed, and algae contain peptides with antihypertensive, anticoagulant, antithrombotic, and hypercholesterolemic activities. For example, hydrolyzate of the jellyfish, Rhopilema esculentum can inhibit ACE with a high level of effectiveness. The current hypertension treatment often uses synthetic drugs such as ACE inhibitors and Angiotensin Receptor Blockers (ARBs), but these have undesirable side effects. Therefore, bioactive peptides from marine organisms can be an efficient and safe natural alternative. The research on protein hydrolysates from various sources has found ACE inhibitory activity and several peptides such as Trp-Pro-Met-Gly-Phe (WPMGF) and APP, KPLL, and VYPFPGPIPNSLPQNIPP were identified to have strong ACE inhibitory activity. These findings indicate the potential use of bioactive peptides from marine organisms as a natural alternative for the treatment and prevention of hypertension.

Biological stabilization of Arthrospira bioactive-peptides within biopolymers: Functional food formulation; bitterness-masking and nutritional aspects

Physicochemical/biological instability and bitterness are among the major challenges limiting the shelf life and direct use of bioactive-peptides in the production of functional foods. This study focused on the structural modification and improvement of biological, nutritional and techno-functional properties of blue-green algae protein by alcalase (30–180 min). Then, the optimal sample (H-120) was stabilized with maltodextrin (MD), gum-arabic (GA), WPC, alginate (A), and pectin (P) biopolymeric-carriers by the spray-drying process. The MD-WPC-encapsulated peptide presented the highest production yield (∼65%), retention of antioxidant activity, functional, flowability, and stability properties. Morphological (formation of wrinkled, irregular, and dented particles) and chemical (FTIR) evaluations respectively indicated the effect of feed composition on the structural properties and entrapment of peptides (through hydrogen bonds and hydrophobic reactions) in the polymer matrix. Finally, the optimal sample (SDP: MD-WPC) was used at different ratios (1, 2, and 4% w/w) for pan-bread fortification. The examination of physicochemical properties (moisture content, water activity, and specific volume), textural (porosity and compressive strength) indices, a color histogram (crust and crumb), antioxidant activity (DPPH• and ABTS• scavenging), and sensory analysis (color, texture, chewiness, taste, and overall acceptability) indicated biological stability, bitterness-masking, and the feasibility of functional bread production with 2%-SDP.

From In Silico to a Cellular Model: Molecular Docking Approach to Evaluate Antioxidant Bioactive Peptides

The increasing need to counteract the redox imbalance in chronic diseases leads to focusing research on compounds with antioxidant activity. Among natural molecules with health-promoting effects on many body functions, bioactive peptides are gaining interest. They are protein fragments of 2-20 amino acids that can be released by various mechanisms, such as gastrointestinal digestion, food processing and microbial fermentation. Recent studies report the effects of bioactive peptides in the cellular environment, and there is evidence that these compounds can exert their action by modulating specific pathways. This review focuses on the newest approaches to the structure-function correlation of the antioxidant bioactive peptides, considering their molecular mechanism, by evaluating the activation of specific signaling pathways that are linked to antioxidant systems. The correlation between the results of in silico molecular docking analysis and the effects in a cellular model was highlighted. This knowledge is fundamental in order to propose the use of bioactive peptides as ingredients in functional foods or nutraceuticals.

Therapeutic Effect of a Latent Form of Cortistatin in Experimental Inflammatory and Fibrotic Disorders.

Cortistatin is a cyclic neuropeptide that recently emerged as an attractive therapeutic factor for treating inflammatory, autoimmune, fibrotic, and pain disorders. Despite of its efficiency and apparent safety in experimental preclinical models, its short half-life in body fluids and its potential pleiotropic effects, due to its promiscuity for several receptors expressed in various cells and tissues, represent two major drawbacks for the clinical translation of cortistatin-based therapies. Therefore, the design of new strategies focused on increasing the stability, bioavailability, and target specificity of cortistatin are lately demanded by the industry. Here, we generated by molecular engineering a new cortistatin-based prodrug formulation that includes, beside the bioactive cortistatin, a molecular-shield provided by the latency-associated protein of the transforming growth factor-β1 and a cleavage site specifically recognized by metalloproteinases, which are abundant in inflammatory/fibrotic foci. Using different models of sepsis, inflammatory bowel disease, scleroderma, and pulmonary fibrosis, we demonstrated that this latent form of cortistatin was a highly effective protection against these severe disorders. Noteworthy, from a therapeutic point of view, is that latent cortistatin seems to require significantly lower doses and fewer administrations than naive cortistatin to reach the same efficacy. Finally, the metalloproteinase-cleavage site was essential for the latent molecule to exert its therapeutic action. In summary, latent cortistatin emerges as a promising innovative therapeutic tool for treating chronic diseases of different etiologies with difficult clinical solutions and as a starting point for a rational development of prodrugs based on the use of bioactive peptides.

Tripeptide IRW Protects MC3T3-E1 Cells against Ang II Stress in an AT2R Dependent Manner.

Multiple strategies including the use of bioactive peptides and other nutraceuticals are being adopted to maintain bone health. This study provides an improved and deeper understanding of the pharmacological effects that a bioactive peptide IRW (Ile-Arg-Trp) extends on bone health. Our results showed that IRW treatment protects osteoblasts against Ang II induced decline in cell proliferation and restores protein levels of collagen type I alpha 2 chain (COL1A2) and alkaline phosphatase (ALP) levels in MC3T3-E1 cells (p < 0.05). Apart from augmentation of these mineralization factors, the angiotensin II (Ang II) induced apoptotic stress in osteoblasts was mitigated by IRW as well. At the molecular level, IRW abolished the cytochrome-c release via modulation of pro-and anti-apoptotic genes in MC3T3-E1 cells (p < 0.05). Interestingly, IRW also increased cellular levels of cytoprotective local RAAS factors such as MasR, Ang (1–7), ACE2, and AT2R, and lowered the levels of Ang II effector receptor (AT1R). Further, our results indicated a lower content of inflammation and osteoclastogenesis biomarkers such as cyclooxygenase 2 (COX2), nuclear factor kappa B (NF-κB), and receptor activator of nuclear factor kappa-B ligand (RANKL) following IRW treatment in MC3T3-E1 cells (p < 0.05). The use of an antagonist-guided cell study indicated that IRW contributed to the process of cytoprotection and proliferation of osteoblasts via Runt-related transcription factor 2 (RUNX2) in face of Ang II stress in an AT2R dependent manner. The key findings of our study showed that IRW could potentially have a therapeutic role in the treatment and/or prevention of bone disorders.

Bioactive Peptides: From Basic Research to Clinical Trials and Commercialization.

Chronic diseases, including metabolic diseases, have become a worldwide public health issue. Research regarding the use of bioactive peptides or protein hydrolysates derived from food, as the diet-based strategies for the prevention and mitigation of chronic diseases, has increased exponentially in the past decades. Numerous in vitro and in vivo studies report the efficacy and safety of food-derived bioactive peptides and protein hydrolysates as antihypertensive, anti-inflammatory, antidiabetic, and antioxidant agents. However, despite promising preclinical results, an inadequate understanding of their mechanisms of action and pharmacokinetics restrict their clinical translation. Commercialization of bioactive peptides can be further hindered due to scarce information regarding their efficacy, safety, bitter taste, as well as the lack of a cost-effective method of production. This review provides an overview of the current clinical evidence and challenges to commercial applications of food-derived bioactive peptides and protein hydrolysates for the prevention and alleviation of chronic diseases.

Novel bioactive peptides of Achillea eriophora show anticancer and antioxidant activities

Despite the limitations of current methods in cancer treatment, the use of bioactive peptides can be as an alternative to treat today. Therefore, isolation and relative purification of bioactive peptides was carried out form Achillea eriophora using a Sep-Pak C18 SPE cartridge and Amicon® Ultra Centrifugal Filters. The presence of desired peptides was checked using RP-HPLC and confirmed using LC-MS. The results of anticancer assay showed that the peptide mixture inhibits the growth of MCF-7 cancerous cell line with the values of IC50, GI50, and LC50 equal to 18.73 ± 0.22, 7.52 ± 0.15, and 56.73 ± 0.18 µg/mL, respectively. It also showed DPPH radical scavenging activity and cupric-ion reducing power with the IC50 value of 5.095 ± 0.23 and 63.3 ± 0.44 µg/mL, respectively. Although flavonoids were present in the sample along with the peptides, their amount was trivial (18.097 ± 1.36 μg/mL). Nevertheless, the results of the LC-MS showed mass-to-charge ratios of 301.17, 261.22, and 243.25, which was a dipeptide or tripeptide in compression to enzyme-digested BSA as a standard. In addition, SEM analysis of the purified peptide mixture showed that it kills the MCF-7 cancerous cell line by creating pores in the membrane. Therefore, it might be valuable to these peptides sequenced and be studied for physicochemical properties. Animal and clinical studies could help its application in drug development.

Marine Bioactive Peptides in Supplements and Functional Foods - A Commercial Perspective.

Many bioactive peptides have been described from marine sources and much marine biomass is still not explored or utilized in products. Marine peptides can be developed into a variety of products, and there is a significant interest in the use of bioactive peptides from marine sources for nutraceuticals or functional foods. We present here a mini-review collecting the knowledge about the value chain of bioactive peptides from marine sources used in nutraceuticals and functional foods. Many reports describe bioactive peptides from marine sources, but in order to make these available to the consumers in commercial products, it is important to connect the bioactivities associated with these peptides to commercial opportunities and possibilities. In this mini-review, we present challenges and opportunities for the commercial use of bioactive peptides in nutraceuticals and functional food products. We start the paper by introducing approaches for isolation and identification of bioactive peptides and candidates for functional foods. We further discuss market-driven innovation targeted to ensure that isolated peptides and suggested products are marketable and acceptable by targeted consumers. To increase the commercial potential and ensure the sustainability of the identified bioactive peptides and products, we discuss scalability, regulatory frameworks, production possibilities and the shift towards greener technologies. Finally, we discuss some commercial products from marine peptides within the functional food market. We discuss the placement of these products in the larger picture of the commercial sphere of functional food products from bioactive peptides.

Detection of bioactive peptides including gonadotrophin-releasing factors (GnRHs) in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS).

The use of bioactive peptides as a doping agent in both human and animal sports has become increasingly popular in recent years. As such, methods to control the misuse of bioactive peptides in equine sports have received attention. This paper describes a sensitive accurate mass method for the detection of 40 bioactive peptides and two non-peptide growth hormone secretagogues (< 2 kDa) at low pg/mL levels in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS). A simple mixed-mode cation exchange solid-phase extraction (SPE) cartridge was employed for the extraction of 42 targets and/or their in vitro metabolites from horse urine. The final extract was analyzed using UHPLC/HRMS in positive electrospray ionization (ESI) mode under both full scan and data independent acquisition (DIA, for MS2 ). The estimated limits of detection (LoD) for most of the targets could reach down to 10 pg/mL in horse urine. This method was validated for qualitative detection purposes. The validation data, including method specificity, method sensitivity, extraction recovery, method precision, and matrix effect were reported. A thorough in vitro study was also performed on four gonadotrophin-releasing factors (GnRHs), namely leuprorelin, buserelin, goserelin, and nafarelin, using the S9 fraction isolated from horse liver. The identified in vitro metabolites have been incorporated into the method for controlling the misuse of GnRHs. The applicability of this method was demonstrated by the identification of leuprorelin and one of its metabolites, Leu M4, in urine obtained after intramuscular administration of leuprorelin to a thoroughbred gelding (castrated horse).

Tuna red meat hydrolysate as core and wall polymer for fish oil encapsulation: a comparative analysis.

Microencapsulation by spray drying is a well-accepted technique for fish oil stabilization. However, severe operational conditions during atomisation destabilise the emulsion, leading to capsule collapse and induction of auto-oxidation. Hence, use of food grade cross-linkers to strengthen the wall material and antioxidants to prevent lipid oxidation has been suggested. A promising option in this line is the use of bioactive peptides, which ensure oxidative stability through structural and chemical stabilisation. Present study attempted to compare the efficacy of yellowfin tuna red meat hydrolysate in protecting the core sardine oil, when used as wall and core polymer during encapsulation. Encapsulates were characterised based on morphological and physical parameters, as well as by in vitro digestibility studies. Their storage stability was also compared under accelerated (60°C), chilled (4°C) and ambient conditions (28°C). Tuna protein hydrolysate exhibited significantly higher protective efficacy when used as core polymer rather than in the wall matrix of encapsulates.

Health Implications of Bioactive Peptides: A Review.

Today, due to immobility, improper food habits, and changes in lifestyle, communities are faced with an increase in health problems such as blood pressure, cholesterol, diabetes, and thrombosis. Bioactive peptides are considered as being the main products of protein hydrolysis which exert high effects on the nervous, immune, and gastrointestinal systems. Unlike synthetic drugs, bioactive peptides have no side effects and this advantage has qualified them as an alternative to such drugs. Due to the above-mentioned properties, this paper focuses on the study of health-improving attributes of bioactive peptides such as anti-oxidative, anti-hypertensive, immunomodulatory, anti-microbial, anti-allergenic, opioid, anti-thrombotic, mineral-binding, anti-inflammatory, hypocholesterolemic, and anti-cancer effects. We also discuss the formation of bioactive peptides during fermentation, the main restrictions on the use of bioactive peptides and their applications in the field of functional foods. In general, food-derived biologically active peptides play an important role in human health and may be used in the development of novel foods with certain health claims.

Antioxidant Peptides from Terrestrial and Aquatic Plants Against Cancer.

Cancer is the leading cause of morbidity and mortality worldwide. Therefore, the search for new and less aggressive treatments is currently the focus of the anticancer research. An attractive alternative for this purpose is the use of bioactive peptides from plants. Plants live everywhere on Earth, both on land and in water, and they are a major source of diverse molecules with pharmacological potential as antioxidant peptides. Hence, this review focuses on the importance of the antioxidant activity of terrestrial and aquatic plant peptides against cancer throughout several mechanisms. The influence of the antioxidant activity of peptides by different factors such as molecular weight and amino acid composition as a crucial factor for anticancer activity is also revised. Furthermore, the relation of antioxidant activity with anticancer property as well as safety and legal aspects of protein hydrolysates and bioactive peptides for their use in cancer treatments is discussed.

The use of bioactive peptides to modify materials for bone tissue repair.

It has been well recognized that the modification of biomaterials with appropriate bioactive peptides could further enhance their functions. Especially, it has been shown that peptide-modified bone repair materials could promote new bone formation more efficiently compared with conventional ones. The purpose of this article is to give a general review of recent studies on bioactive peptide-modified materials for bone tissue repair. Firstly, the main peptides for inducing bone regeneration and commonly used methods to prepare peptide-modified bone repair materials are introduced. Then, current in vitro and in vivo research progress of peptide-modified composites used as potential bone repair materials are reviewed and discussed. Generally speaking, the recent related studies have fully suggested that the modification of bone repair materials with osteogenic-related peptides provide promising strategies for the development of bioactive materials and substrates for enhanced bone regeneration and the therapy of bone tissue diseases. Furthermore, we have proposed some research trends in the conclusion and perspectives part.

Review peptide‐targeted liposomes for selective drug delivery: Advantages and problematic issues

We describe the use of bioactive peptides to drive liposomal drugs on target receptors overexpressed in cancer cells. A detailed description for several targeting liposomes derivatized with different peptides and thus able to target relevant receptors for cancer therapy is reported. Even if the development of peptide-targeted liposomes represents a great advance in the use of liposomal drugs for cancer therapy, many critical issues are still unsolved in the development of these innovative drug delivery systems. Advantages and drawbacks of the peptide-mediated active targeting are discussed.

Ceramic granules enhanced with B2A peptide for lumbar interbody spine fusion: an experimental study using an instrumented model in sheep

New generations of devices for spinal interbody fusion are expected to arise from the combined use of bioactive peptides and porous implants. The purpose of this dose-ranging study was to evaluate the fusion characteristics of porous ceramic granules (CGs) coated with the bioactive peptide B2A2-K-NS (B2A) by using a model of instrumented lumbar interbody spinal fusion in sheep. Instrumented spinal arthrodesis was performed in 40 operative sites in 20 adult sheep. In each animal, posterior instrumentation (pedicle screw and rod) and a polyetheretherketone cage were placed in 2 single-level procedures (L2-3 and L4-5). All cages were packed with graft material prior to implantation. The graft materials were prepared by mixing (1:1 vol/vol) CGs with or without a B2A coating and morselized autograft. Ceramic granules were coated with B2A at 50, 100, 300, and 600 microg/ml granules (50-B2A/CG, 100-B2A/CG, 300-B2A/CG, and 600-B2A/CG, respectively), resulting in 4 B2A-coated groups plus a control group (uncoated CGs). Graft material from each of these groups was implanted in 8 operative sites. Four months after arthrodesis, interbody fusion status was assessed with CT, and the interbody site was further evaluated with quantitative histomorphometry. All B2A/CG groups had higher CT-confirmed interbody fusion rates compared with those in controls (CGs only). Seven of 8 sites were fused in the 50-B2A/CG, 100-B2A/CG, and 300-B2A/CG groups, whereas 5 of 8 sites were fused in the group that had received uncoated CGs. New woven and lamellar bone spanned the fusion sites with excellent osseointegration. There was no heterotopic ossification or other untoward events attributed to the use of B2A/CG in any group. Each B2A/CG treatment produced more new bone than that in the CG group. Bioactive treatment with B2A effectively enhanced the fusion capacity of porous CGs. These findings suggest that B2A/CG may well represent a new generation of biomaterials for lumbar interbody fusion and indicate that additional studies are warranted.

Synthetic Peptides in the Form of Dendrimers Become Resistant to Protease Activity

In previous papers, we observed that dendrimers of peptide mimotopes of the nicotinic receptor ligand site are strong antidotes against the lethality of the nicotinic receptor ligand alpha-bungarotoxin. Although their in vitro activity is identical to that of dendrimers, the corresponding monomeric peptide mimotopes are not effective in vivo. Because the higher in vivo efficiency of dendrimers could not in this case be related to polyvalent interaction, the stability to blood protease activity of monomeric versus tetrabranched dendrimeric mimotope peptides was compared here by incubating three different mimotopes with human plasma and serum. Unmodified peptides and cleaved sequences were followed by high pressure liquid chromatography and mass spectrometry. Tetrabranched peptides were shown to be much more stable in plasma and also in serum. To assess the notable stability of multimeric peptides, different bioactive neuropeptides, including enkephalins, neurotensin and nociceptin, were synthesized in monomeric and tetrabranched forms and incubated with human plasma and serum and with rat brain membrane extracts. All the tetrabranched neuropeptides fully retained biological activity and generally showed much greater stability to blood and brain protease activity. Some tetrabranched peptides were also resistant to trypsin and chymotrypsin. Our findings provide new insights into the possible therapeutic use of bioactive peptides.