International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in BIOACTIVITIES OF PROTEIN DERIVED PEPTIDES FROM MARINE SANGUINOLENTUS

1MANJUSHA SAYD, 2NAFILA PP, 3ATHULYA M, 4KUNHI AAM

1,2,3,4Department of Biotechnology, SAFI Institute of Advanced Study, Vazhayoor, Malappuarm-673633

Abstract - Marine organisms are rich sources of structurally diverse bioactive compounds. Hence, a great interest has been developed nowadays to isolate bioactive compounds, which act as ACE inhibitors from marine resources because of their numerous health beneficial effects. Therefore, marine-derived bioactive peptides have a potential in use as functional ingredients in nutraceuticals and pharmaceuticals due to their effectiveness in both prevention and treatment of hypertension in addition to the nutritive value. Portunus sanguinolentus, three spotted crab is the commercially most important species of Portunus belongs to the family (swimming ) which includes most of the economic crab species throughout the world. The objective of the present study is to determine the bioactive effect such as antioxidant, antimicrobial , anti cancer and antihypertensive activities of protein derived peptides from crab protein hydrolysate fractions of red three spotted crab shell hydrolysate. Insilico analysis results indicated that Crab derived peptides have bioactive effect. ACE inhibition studies of the crab protein derived peptides further showed a percentage inhibition was obtained in 50.1% and 57.2% for pepsin and trypsin digests, respectively. This crab protein derived peptides was on SDS PAGE analysis revealed the shorter peptide fragments. Antioxidants study revealed showed that the undigested protein sample (crude protein) exhibited a maximum antioxidant potential of 69%. The crude protein from crab gills also has shown a high potential of 66% antioxidant activity. The protease hydrolysis studies has been revealed that 8% pepsin digestion fraction of protein hydrolysate of P. sanguinolentus has exhibited maximum 50% of antioxidant potential. Marine-derived ACE inhibitors and their inhibitory properties, emphasizing their potential application can be used as nutraceuticals in future in preventing hypertension.

Keywords - Bioactive peptide, ACE peptide, , Crab, Protein derived peptide.

I. INTRODUCTION proportions that affects 15-20% of all adults. Its treatment is one of the major risk factors for the is blessed with a rich biodiversity with high development of cardiovascular disease, stroke, and degree of endemism in hotspot areas like Western the end stage of renal disease.(Zhang et al.,2006). ghats (Dahanukar et al., 2004). Marine ecosystem is However, it was suggested that bioactive peptides the most diverse ecosystem in the world and special have higher tissue affinities and are subject to a attention is requisite enough to understand its slower elimination than captopril (Fujita and processes and function because it harbors diverse and Yoshikawa, 1999). The antihypertensive peptide unique floral and faunal community. India is blessed isolated from bonito fish hydrolysate product, has with a high degree of marine biodiversity, among found to be hydrolyzed by ACE to produce a smaller which the are predominant. The State of peptide than the initial one, which had 8-fold ACE Kerala situated in the South West part of peninsular inhibitory activity compared with the initial peptide India, has a slender stretch of land with a long surf (Fujita and Yoshikawa ;1999). Therefore, marine- beaten coast on the western side and a lush green derived bioactive peptides have a potential in use as mountain range on the eastern side. The crab fishery functional ingredients in nutraceuticals and in India is fast developing and there is a vast scope pharmaceuticals due to their effectiveness in both for the crab meat due to its delicacy and nutritional prevention and treatment of hypertension in addition richness. The crab meat contains rich amount of to the nutritive value. However, marine-derived protein, vitamins A and D, minerals, glycogen and bioactive peptides are well tolerated by the body, are free amino acids. Most of the marine crabs occurring not expected to exert any harmful side effects. along the Indian coasts are belonging to the family Angiotensin I-converting enzyme (ACE) (EC portunidae. The commercially important portunid 3.4.15.1) is a hypertension-responsible glycoprotein crabs found along Parangipettai coast are Scylla present both in biological fluids and many tissues serrata, S. tranquebarica, Portunus sanguinolentus, (Iwaniak et al., 2014 ). ACE inhibitors are the most- P. pelagicus, Podophthalamus vigil, C. feriata, C. studied food peptides and display different biological lucifera, C. natator, C. granulata and C. Truncate functions (Iwaniak et al.,2014; Minkiewicz et al., (Soundarapandian P and Shyamalendu Roy 2014). 2008). Since fish proteins are structurally diversified, Portunus sanguinolentus is a marine crab growing to they may serve as a substrate to produce peptides a size of 145mm carapace width. The potential health with multifunctional bioactivities (Harnedy and benefits related to crab consumption are because of FitzGerald.,2012 ). Using fish as a source of bioactive the presence of proteins, vitamins and unsaturated peptides provides a novel tool for the introduction of essential fatty acids (Pushparajan et al., 2012) potentially high-value bioactive products. Functional .Hypertension is a worldwide problem of epidemic foods are made from natural ingredients and are

Bioactivities of Protein Derived Peptides from Marine Crab Portunus sanguinolentus

70 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in consumed as part of the normal diet. Marine generated according to the specificities of pepsin and organisms are rich sources of structurally diverse trypsin. Based on the in silico results, fragments with bioactive compounds. Hence, a great interest has ACE inhibitory activity were highlighted. ACE been developed nowadays to isolate bioactive inhibitory activity, expressed as a value of IC50, as an compounds, which act as ACE inhibitors from marine indicator of an enzyme’s biological response to a resources because of their numerous health beneficial dose of a biopeptide, was a criterion for selecting effects. According to the researches, it has reported peptides used in further stages of the studies that marine-derived bioactive peptides, chito (MałgorzataDarewicz et al.,(2014). The in silico oligosaccharide derivatives (COS) and phlorotannins analysis revealed that there exists several ‘hot spots’ have potent ACE inhibitory activity. Marine-derived of potential ACE inhibitory activity. Several of the ACE inhibitors and their inhibitory properties, predicted peptides overlap each other. The number of emphasizing their potential application as future peptides retrieved from In silico analysis may not pharmaceuticals to prevent hypertension was reported always correlate with the actual antihypertensive ( Fujita and Yokoyama, 2000). activity as it is based on the reported antihypertensive peptides in BIOPEP database that doesn’t consider Objective of the present study is to find the the potency (Cheung et al., 2009) . bioactivity of protein derived biopeptides from P.sanguinolentus . The study involves to find the The crab species Portunus sanguinolentus serve as a ACE inhibitory , antimicrobial and antioxidant source of functional materials, such as activity by insilico analysis followed by the polyunsaturated fatty acids, polysaccharides, minerals determination of different bioactivity of protein and vitamins, antioxidants, enzymes and bioactive derived peptides from crab P.sanguinolentus. The peptides. Bioactive peptides are short chains of amino effect of ACE inhibitory , antioxidant anticancer and acids are inactive within the sequence of the parent antimicrobial activities of fractions of three spotted protein, but can be released during gastrointestinal crab hydrolysate have also been tried This crab digestion, food processing, or fermentation. Bioactive protein derived peptides was on SDS PAGE analysis peptides that can reduce blood pressure are revealed the shorter peptide fragments.. antihypertensive peptides which act as ACE inhibitor. ACE inhibitor peptides, as a part of a food II. MATERIALS AND METHODS product or as a nutraceutical, especially when derived from a food protein of plant or origin, are of 2.1. Sample collection functional interest as they would have no harmful The sample used in this study was Portunus side effects (Hong et al., 2008). sanguinolentus purchased directly from a local market (Beypore,Calicut) and transported The objective of this study is to detect the ace immediately on ice to the laboratory and was stored inhibitory activity, antioxidant activity and in deep freezer. The chemicals used in the present antimicrobial activity of peptides derived from p. investigation were procured from the following sanguinolentus .This study was carried out in 3 sources: Pepsin, Trypsin, Acrylamide, N-N'- stages: in silico, ex vivo and in vitro Based on the in methylene bis-acrylamide, Tris(hydroxymethyl) silico results, fragments with ACE inhibitory activity amino methane (Tris), Coomassie Brilliant Blue R- were highlighted. ACE inhibitory activity, expressed 250, N-N, N'-N'-tetramethyl 1, 2-diaminoethane as a value of IC50, as an indicator of an enzyme’s (TEMED), Hippuryl-histidyl-leucine (HHL), biological response to a dose of a biopeptide, was a Hippuric acid (HA), Sodium tetraborate, and Triton criterion for selecting peptides used in further stages X-100, were purchased from Hi Media, Mumbai. - of the studies (MałgorzataDarewicz et al.,(2014). The Mercaptoethanol was purchased from Fluka, in silico analysis revealed that there exists several Switzerland and Amonium persulphate (APS) was ‘hot spots’ of potential ACE inhibitory activity. procured from SRL Limited. Several of the predicted peptides overlap each other. The number of peptides retrieved from In silico 2.2 In silico analysis for Antihypertensive analysis may not always correlate with the actual Peptides antihypertensive activity as it is based on the reported In the in silico stage, the present study involves the antihypertensive peptides in BIOPEP database that information annotated in BIOPEP database to doesn’t consider the potency (Cheung et al., 2009) . determine the potential biological activity profile to find ACE inhibitory peptides , antioxidant peptides 2.3. Extraction of Protein and antimicrobial peptides encrypted in selected Estimation of Protein Portunus sanguinolentus proteins. Since the Protein content of the extracts treated with different presence of “hidden” peptides does not indicate a solvents was estimated by the standared Lowry’s possible release of those biopeptides, computer method (Lowry et al., 1951). The extraction was software was used to simulate proteolysis to find standardised by using previously reported method ACE inhibitory peptides that can potentially be (Rosmilah M et al. 2008).

Bioactivities of Protein Derived Peptides from Marine Crab Portunus sanguinolentus

71 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in 2.4 Preparation of Goat lung Acetone powder water bath for 5 min. These hydrolysates were used Goat lung was minced and homogenized in 5 as the source of ACE inhibitor peptides. volumes of ice cold 0.1 M Tris HCl buffer, pH 8.2. Ten volumes of ice cold acetone was added and the Table 2. List of Enzymes used for hydrolysis of Crab homogenate filtered. The residue was washed once hydrolysate again with acetone. The residue was air dried at 25 ± SI 2 °C. The lung acetone powder was stored at -20 ± 2 Enzyme Buffer pH NO °C until used. 2.5. Preparation of goat lung ACE ACE was extracted from Goat lung acetone powder. 1 Pepsin 10mM HCl 2 One gram of acetone powder was extracted with 10 mL of 0.1 M TrisHCl buffer, pH 8.2 containing 0.3 M 2 Trypsin 50 mM TrisHCl 8 NaCl and 0.5% Triton X-100 at 4 ˚C for 16-18 h. The extract was centrifuged at 14,100 × g for 60 min at 4 2.9. SDS Poly Acrylamide Gel Electrophoresis °C. The supernatant was precipitated by the addition SDS-PAGE was carried out to evaluate the protein of solid (NH ) SO to a final concentration of 50% 4 2 4 profile after each digestion step. The assay was (w/v). The precipitate obtained after centrifugation performed according to standard protocols ( (6000 rpm, for 10 at 4⁰C). The precipitated enzyme Laemmli et al.,1970 ) using 10% and separating and were dialyzed against the same buffer minus Triton 5% stacking acrylamide gels. The molecular weight X-100 (1 L x 3) for 24 h and stored at -20 ˚C until markers usedwere purchased from BIO-RADPvt. used. Ltd., Bangalore, India. were the Low MW standard 2.6. In vitro colorimetric assay of ACE and ACE kit (97,000–14,400 Da, GE Healthcare Life Sciences, inhibitor activity Little Chalfont, Buckinghamshire, UK). Staining was ACE activity was assayed by monitoring the release performed according to the standard Coomassie of Hippuric Acid from the substrate HHL as brilliant blue procedure . described (Zhang et al. (2009). ACE inhibitory activity was determined according to the method of 2.10. Antioxidant Assay was measured to measure Zhang et al. (2009). the antioxidant activity of crude protein extract and

the digested peptides of crab P.sanguinolentus, 0.1ml Inhibition was calculated from the equation: of the sample was taken in a vial and then adds 0.5ml

of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (Girgihet ACE inhibitory activity (%) = [(A-B) ⁄ (A)]* 100 al., 2011). Then these sample mixture was incubated

for 30min in a dark place. After the incubation period where A is the absorbance with ACE and HHL the DPPH scavenging activity was measured by using without ACE inhibitory sample, B is the absorbance the spectrophotometer in 620nm. with ACE, HHL and ACE inhibitory sample.

One unit of ACE activity is defined as the amount of III. RESULTS AND DISCUSSIONS enzyme, which releases 1 mole of HA per min at 37 °C and pH 8.2. Studies on brachyuran fauna of Indian seas were Goat lung ACE was pre-incubated with different initiated by Milne Edwards (1834). Most of the concentrations of the peptide fractions and the earlier workers concentrated mostly on the taxonomic residual activity determined as described above. The aspects of brachyuran crabs of Indian waters (Milne IC50 value is defined as the concentration required Edwards, 1834; Henderson, 1893) .The crabs rank decreasing the ACE activity by 50%. The percent third after shrimps and lobsters for their esteemed inhibition curves were plotted using a minimum of seafood delicacy and also the value of fishery they five determinations for each inhibitor concentration support. and IC50 values computed from the semi logarithmic plots. A linear regression analysis was performed A wide range of bioactivities have been described , using Origin 6.1. including antimicrobial and antifungal properties, 2.7.General protease assay cholesterol lowering ability, antithrombotic effects, Activity of proteases was quantified by monitoring enhancement of mineral absorption, immune the cleavage of Bovine Serum Albumin as described modulatory effects and antihypertensive effect earlier (Ohtsuki et al., 1995). (Rutherfurd-Markwick and Moughan, 2005). 2.8 Enzymatic hydrolysis of Protein Moreover, some peptides are multifunctional and can One gram of isolated proteins were suspended in 5 exert more than one of the effects listed (Meisel and mL of buffer as listed in (Table 2.) for pepsin, and Walsh, 2006). In recent years, natural products from trypsin, and digested using an enzyme substrate ratio marine samples have a wide spectrum of biological of 0.5, 1, 2, 4, 6, 8 and 10 % (w/w) at 37 °C for 4 h. l. activities, numerous therapeutic applications include The hydrolysis was terminated by heating in a boiling

Bioactivities of Protein Derived Peptides from Marine Crab Portunus sanguinolentus

72 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in antiviral, antibacterial, antitumor activity, and very gastrointestinal enzymes like pepsin and trypsin at different kinds of substances have been obtained.. different concentration (0.5% -10%). Therefore the ability of gastrointestinal proteases to release ACE Fig.1 Sequence of Portunus sanguinolentus meat inhibitory peptides from P.sanguinolentus flesh proteins showing the potent antihypertensive peptides protein was studied. All the digests showed the as analysed using the bioactive potency prediction of inhibitory activity; the maximum inhibition was BIOPEP have been obtained from BIOPEP obtained in 50.1% and 57.2% for pepsin and trypsin (www.uwm. edu.pl/biochemia). Red: dipeptides, digests, respectively. According to previous reports Underlined: tripeptides and Bold: tetrapeptides. the ACE inhibition from Crab derived proteins is little being studied. 2. Insilico Analysis For Antioxidant Peptides Anti oxidant activity is the fundamental property and 2.4. Ace Inhibitory activity of Pepsin digested much important for life. Many of the biological sample. functions such as anti-mutagenicity, anti- Generally, pepsin is secreted from gastric mucosa in carcinogenicity and anti-aging, among others the stomach and had preferential specificity for the originate from this property. In recent years, peptides aromatic amino acids, phenylalanine, tyrosine, and from many marine animal sources such as sea urchin tryptophan. Pepsin is an endopeptidase with broad gonad , smooth hound protein , loach protein , specificity produced in the mucosal lining of the sardinelle by-products proteins , jumbo squid skin , stomach and degrades proteins. Pepsin is one of three grass carp muscle , horse mackerel viscera protein , principal protein-degrading, or proteolytic, enzymes sea cucumber , alaska pollack skin and pacific hake in the digestive system, the other two being have been found to possess antioxidant activity. In chymotrypsin and trypsin (Cooper et al., 1990). addition, fish frame proteins such as Hoki frame Recent advances in biotechnology have proved the protein ,Alaska pollack frame protein and Tuna ability of enzymes to produce novel food products, backbone protein have also been proven to be the modified food composition, or improved waste good sources of antioxidant peptides. Based on the In processing (Lee et al., 2011). Ace Inhibitory activity silico results, fragments with the antioxidantactivity of Pepsin digested sample is presented in Fig. 2. were highlighted. The in silico analysis revealed that there exists several ‘hot spots’ of potential antioxidant activity and are shown in Fig.2. This results were analogous to the previous in silico studies for the antioxidant activities from other marine sources (Kim etal.,2011)

3. 1. Standardisation and extraction of Protein from Portunus sanguinolentus Protein extracted from the meat of Portunus sanguinolentus using different solvents under Fig.2 SDS-PAGE profile of Portunus sanguinolentus varying pH and molar concentration. The amount of protein/pepsin digests. protein estimated by Lowry’smethod (Lowry et al.,1951) were presented in Table.1. Among the Lane C is P. sanguinolentus meat protein control and various solvents for treating the extraction of Lane 1-10 is Jackfruit seed protein digested using proteins , the higher amount of protein extracted was enzyme to protein ratio (w/w) of 0.5, 1, 2, 4, 6, 8 and found to be higher in 0.1M Phosphate buffered saline 10% respectively. of pH 7.2 .Hence this solvent was further used for the total isolation of protein . The results were in comparable with already reported marine proteins from other sources. Premarathna et al., (2015) analysed the meat of male P.pelagicus for protein, fat, moisture and ash contents by proximate analysis.

2.3. Protease Hydrolysis of P. sanguinolentus flesh Protein with Proteases and ACE Inhibition The protein hydrolysis of crab flesh was studied to analyze the release of shorter peptides and subsequent inhibiton with ACE inhibiton. The mean contents of the crude extracts was obtained from P .sanguinolentus using 0.1M phosphate buffered saline of pH 7.2 were 377.6 mg. The crab protein hydrolysates were prepared by hydrolysis using

Bioactivities of Protein Derived Peptides from Marine Crab Portunus sanguinolentus

73 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in 2.3 Ace Inhibitory Activity of Trypsin Digested Trypsin digested protein hydrolysate shown Sample maximum antioxidant potential of 65% at an enzyme ACE inhibitory activity was assayed by measuring concentration of 0.5% and is represented in Fig.9. the release of HA from the substrate HHL according Numerous antioxidant studies also have been reported to Jimsheena and Gowda (2009). The results are as and is concurrent with findings. The findings of shown in Fig.3 and Table.4. numerous studies have confirmed that protein hydrolysates from enzymatic hydrolysis of animal and plant proteins can act as direct scavengers of diverse free radicals or behave as antioxidants in

model systems (Girgihet al. , 2011).

0.5 1 2 4 20% 6 33% 8 10

Fig.3 SDS-PAGE profiling of Portunus sanguinolentus 16% protein/Trypsin digests. 0.79% 12% 9.2% Lane C is Portunus sanguinolentus flesh protein 9.9% control and Lane 1-10 is protein digested using enzyme to protein ratio (w/w) of 0.5, 1, 2, 4, 6, 8 and 10% respectively. Fig. 4. Antioxidant activity of trypsin digested from P.sanguinolentus

CONCLUSION

Crabs are the most interesting groups of crustaceans. The present study reveals that the protein content and moisture content of P. sanguinolentus are 37.7% and 83.3% respectively. The release of ACE inhibitor peptides from the precursor sequence is a prerequisite to validate the previous in silico prediction system. The protein digested fractions from the Portunus sanguinolentus was showed prominent ACE inhibitory activity; the maximum of 50.1% and 57.2% on pepsin and trypsin digests, respectively. Fig.4. ACE inhibition profiles of Portunus sanguinolentus protein/Trypsin digestion The crab derived peptides have shown a prominent antioxidant effect. The antioxidant activity is highest It was revealed from the Biochemical ACE Inhibition in the native protein of Portunus sanguinolentus , assays and SDS Protein profiling that there was 69%. The crab gills also having greater antioxidant significant ACE inhibition was obtained. The release activity (66%). Protein/ pepsin digest have shown a of shorter peptides was released on enzyme maximum, of 50%, antioxidant activity at an enzyme hydrolysis of protein hydrolysate was evident from concentration of 8%. The trypsin digest of crude SDS PAGE analysis as well. There were no similar protein has been observed a maximum a 55% of reports were obtained from the Portunus crab was antioxidant activity at 0.5% as well. Thus it can be obtained till now. The results of our study is the first concluded that the protein derived peptides from report of ACE inhibitory effect of the crab derived marine crab Portunus sanguinelentus have both peptides from the Marine crab Portunus antioxidant and ACE inhibitory activity and it can be sanguinolentus can be a good potential as ACE one of the good nutraceuticals. Significant ACE inhibitory and further antihypertensive in future. inhibitory and Antioxidant results have been obtained in the present study. This preliminary work on Crab 2.5. Antioxidant Studies of Portunus derived peptides leads to further bio active anticancer sanguinolentus protein hydrolysate effects and animal models in detail to establish a In this present antioxidant study of the crab protein, good nutraceuticals. the undigested protein sample (crude protein) exhibited a maximum of 69% antioxidant activity. ACKNOWLEDGEMENTS The protein hydrolysate of P. sanguinolentus on treating with 8% of has shown a maximum 50% of The authors acknowledged to the management, SIAS, antioxidant activity and is represented in Fig.5. Malappuram.

Bioactivities of Protein Derived Peptides from Marine Crab Portunus sanguinolentus

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Bioactivities of Protein Derived Peptides from Marine Crab Portunus sanguinolentus

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