INTRODUCTION:

Malaria a vector-borne protozoan disease continues to be the prominent causes of mortality and morbidity in developing world effecting for about million people worldwide^1,2. Chloroquine (CQ) most commonly used drug for decades reportedly failed due to increase in resistance to pathogen and has created a new challenge to the millennial^3,4. Lack of effective vaccines and development of resistance to multiple antibiotics by the pathogens has put us on toes to look for the new drug with promising mode of action as alternative source.

Traditional medicines and phytochemicals have always been primary source of molecules to be explored for antimalarial properties^5–8. Though many new candidates’ drugs are in clinical trials, recent studies have put forth the bioactive protein fractions or small peptides as effective contenders to overtake resistance drug^9,10. These protein fragments are of many classes; Antimicrobial peptides (AMPs), Cell-penetrating peptides (CPPs), Host-defence Peptides and are isolated from many plants, bacterize and animal sources^11–14. These bioactive protein fraction or peptides are front runners for the quest to new antibiotics. Bioactive peptides from various sources have been explored for their antimalarial activity which provide promising new candidates^14–16. A typical bioactive protein fragment is less than 100 amino acids long with a net positive charge and amphiphilic in nature predominately act by selectively disrupting pathogens cell membrane. Present study aimed to investigate whether protein fragments generated by hydrolyzing fish proteins possess any inhibitory capabilities against Plasmodium falciparum 3D7 under In-vitro conditions using Fluorescence-Based High-Throughput Antimalarial Drug Screening.

MATERIALS AND METHODS:

Materials:

Enzymes, Protamex and Papain were purchased from sigma alderich USA and Hi-media, Mumbai, India, respectively. SYBR Green I nucleic acid staining dye (10,000_ stock concentration) was purchased from Molecular Probes, Inc. (Eugene, Oreg.). Lysis buffer consisted of Tris (20 mM; pH 7.5), Triton X-100 (0.08%; vol/vol), Ethylenediaminetetraacetic acid (EDTA) (5mM) and saponin (0.008%;) wt/vol) was prepared and stored at room temperature. Human plasma and erythrocytes were obtained from healthy volunteer donors.

Method of preparation of fish protein hydrolysates:

The fish protein hydrolysates were prepared according to the methods described by Elavarasan et al. (2014)¹⁷ with slight modification. Fish protein hydrolysates were prepared from 3 species [Pangasius (Pangasianodon hypophthalmus), Clam and White snapper (Macolor niger)]. A total of six type of hydrolysates were used to screen the activity. (i) fresh pangasius meat hydrolysate (Pm), (ii) protein isolate of pangasius (alkali solubilization and iso-electric solubilization precipitation) hysrolysate (Pi), (iii) cook-wash processed pangasius meat hydrolysate (Pc), (iv) clam meat hydrolysate (Cm), (v) white snapper scale hydrolysate (WSc) and (vi) white snapper skin hydrolysate (WSk). Papain enzyme (30000 USP units/mg; Hi-media, Mumbai India) was used for hydrolyzing the substrates namely Pm. Pi, Pc, Cm (Enzyme to protein ratio - 1 %; temperature of 55°C, duration of hydrolysis-60 min, pH- 6.7±0.2). White snapper hydrolysates (WSk &WSc) were prepared using protamex, a protease from Bacillus sp with the declared minimum activity of 1.5 AU /g solid ((Enzyme to protein ratio - 1%; temperature of 60°C, duration of hydrolysis - 60min, pH- 6.7±0.2). For each batch of hydrolysis, 500g of the substrate was used. Before adding enzyme for hydrolysis, the distilled water was added at 1:2 ratio, homogenized and equilibrated to the required temperature. The hydrolysis process was carried out in a thermostatic water bath (Julabo TW20, Germany) with continuous stirring at 400rpm by an over head stirrer (Remi, Mumbai, India). The hydrolysis reaction was inactivated after 60min of hydrolysis by keeping the reaction mixture in a boiling water bath for 20min followed by cooling in an ice bath. The slurry was centrifuged in a laboratory centrifuge (Heraus MULTIFUGE X1R, Thermo scientific, USA) at 9500 rpm for 30min at the temperature of 4°C. The supernatant obtained was dried in a pilot scale spray dryer (S M. Science, Kolkata, India). The feeding rate was 21rpm. The inlet and outlet temperature were 155 and 90ºC. The fine powder obtained was transfered into a airtight glass container and stored under the dessicated condition.

Parasite Culture:

Cultivation of Parasites:

Prior to the experiment, the Plasmodium falciparum 3D7 were cultivated and maintained in fresh group B-positive human erythrocytes with 5% CO₂ at 37°C¹⁸. The culture media RPMI 1640 was supplemented with 3 g of glucose per liter, 45µg of hypoxanthine per liter, 50 µg of gentamicin per liter and 10% human serum. Culture media was changed every 3 to 4 days along with uninfected erythrocytes (subculture). The parasite stock culture was synchronized at late-ring stage with 5% sorbitol with no evident of schizonts. Initial parasitemias was determined by examining parasitized cells (N=500) on Giemsa stained thin blood smear under light microscope.

SYBR Green fluorescence Based Assay:

From the stock culture a working culture was obtained with 4% hematocrit and 0.5% parasitemia. For hydrolysate samples, plate for assay method was prepared in parallel with the same cells and medium. Hydrolysates were dispensed into triplicate test wells to yield desired concentrations with final well volume of 200µl. The plates were then incubated for 48 h, 100µl of SYBR Green I in lysis buffer (0.2µl of SYBR Green I/ml of lysis buffer) was introduced into each well. Thorough homogenization of contents was achieved until no visible erythrocyte sediment remained. Measurement of Fluorescence was read after 1 h of dark incubation with a multiwell plate reader at 485 and 530 nm, respectively.

Light Microscopy:

Pictures of Giemsa-stained blood smears of Test sample parasite cultures of strain 3D7 were taken with an OLUMPUS DP21 camera mounted on an OLUMPUS CX41 microscope. Individual images were cropped, and stored as a JPG file. The JPG file was used without any gamma, contrast, or colour adjustments.

Cytotoxic potential of fish hydrolysates:

a) Cell culture:

Human breast carcinoma cell lines, MCF-7 procured from National Centre for Cell Science [NCCS], Pune, India, were grown at 37°C in 95% humidity and 5% CO₂ atmosphere, in DMEM-Ham’s F12 medium (1:1, v:v, Gibco), supplemented with 10% heat inactivated fetalcalf serum (FCS, Dutscher) to which penicillin and streptomycin were added.

b) MTT Cytotoxic assay:

Antipoliferative activity of fish hydrolysate was assessed by incubating them at various concentrations with MCF-7 cancer cells grown in optimal conditions and assessed by spectrophotometric determination of conversion of MTT into “Formazan blue” by living cells at a wavelength of 492nm. The cell viability in percentage was calculated using the standard formula. The MCF-7 cells were seeded at a density of approximately 5 × 10³ cells/well in a 96 well flat-bottom micro plate and maintained at 37^°C in 95% humidity and 5% CO₂for overnight. Various concentrations of fish hydrolysate were used to treat the cells. The microplate was then incubated at 37^°C for 24 hours. The cells in well were washed twice with phosphate buffer solution, and 20µL of the MTT staining solution [5mg/ml in PBS] was added to each well and the plate was incubated at 37^°C. After 4h, 100µL of di- methyl sulfoxide [DMSO] was added to each well to dissolve the formazan crystals, and optical densities were recorded at 570nm in a micro plate reader¹⁹

The data were analyzed to calculate the percentage of growth inhibition induced by the presence of FPH in cell culture medium determined by the equation:

Surviving cells [%] = Mean OD of test compound / Mean OD of Negative control × 100

Statistical Analysis

Analyses were made in triplicate and expressed as mean ± SD; the data obtained were transformed and non-linear regression analysis using GraphPad Prism Version 7 Software was performed.

RESULTS AND DISCUSSION:

Table 1: Percentage (%) inhibition and EC(50) values of Plasmodium parasite inhibition by fish proteins hydrolysates sample using SYBR green fluorescence assay.

S. No	Sample	% of inhibition	EC50 (µg/ml)
1	WSk	42.80	10.33
2	Pi	47.32	6.34
3	WSc	45.00	9.54
4	Cm	62.55	2.30
5	Pm	64.70	4.87
6	Pc	56.20	5.98

Figure 1- Pictures of Giemsa-stained blood smears of sample treated Plasmodium parasite cultures of strain 3D7.

Figure 2– Cytotoxic activity and determination of the EC(50) value of fish protein hydrolysates against the MCF-7 cell lines. (A) WSc, (B)WSk, (C) Cm, (D) Pc, (E) Pi, (F) Pm.

The antimalarial potential of six fish proteins hydrolysates is shown (Table no 1). In present investigation Cm and Pm was found to have more than 50% of inhibition (62.55 and 64.70µm/ml) respectively. Pc also exhibited 56.20% of inhibition whereas WSk, Pi, and WSc hydrolysates had inhibition in range of 40- 50%. In terms of EC(50) values Cm, Pm and Pc hydrolysates had lowest EC-50 values (2.30, 4.87 and 5.98) respectively indicating high lethality. Proteins hydrolysates differ in their biological properties based on the nature of proteases used as the sequence of released peptides are differed chiefly based on the type of enzyme used for cleavage and hydrolysate condition employed. All the hydrolysates were found to differ from each other in their inhibition ability.

Parasite morphological evaluation was carried out by incubating all the protein hydrolysates with synchronized ring stage parasites for 24hours (Fig.1). Parasites appear to be condensed round structures some showed segmented trophozoits and rings, where as in control cultures all the parasites developed into trophozoits.

The morphological study demonstrated that all the protein hydrolysates had no deteriorating effect on RBC membranes. Both in control and test samples, no morphological deformities of RBCs were observed indicating the safety of samples for future in-vivo evaluation.

Our study demonstrates the possibility and scope of the production of bioactive proteins fractions from fish source as antiparasitic therapeutics. Protein fractions and peptides have been isolated from a variety of animal and plant sources for their range of bioactivities^20–22. Protein /peptide fractions based on their amino acid composition, length and biophysical interaction exerts bioactivities such as antioxidant anti-inflammatory antimicrobial and cytotoxicity^23–25.

Bioactive protein fragments can be derived from fish proteins by hydrolyzing with proteolytic enzymes. By optimizing the conditions of hydrolysis, using different enzymes and substrates (fish species), a wide range of hydrolysate with the desired physical, chemical, functional and biological properties can be produced. The nature of peptides released by an proteolytic enzyme controlled by various factors including nature and specificity of protease, the state of proteins in substrate (native and denatured), presence of other metabolites (lipid oxidation products etc.) and primarily on the process parameters such as temperature of hydrolysis, duration of hydrolysis, pH, E/S ratio and drying process employed for concentrating the peptides^17,26. In the present study, six different hydrolysate substrate were explored (3 are from Pangasius sp; One from clam meat; two are from white snapper) and results indicate the nature of their individual proteins hydrolysate were different. The reason for using different enzymes to obtain hydrolysate owed to the efficiency of protamex to hydrolyze the white snapper fish, scale and skin substrates which is predominantly rich in collagen and papain enzyme on other hand acts majorly on muscle proteins in which the myosin and actin present in high amount along with troponin, tropomyosin etc^27,28

To evaluate cytotoxic or proliferative potential of the samples MMT assay was performed on MCF-7 breast cancer cell lines. Except for Pc sample all hydrolysate had anti-proliferative effect across concentration (Fig. 2). Whereas Pc sample inversely had proliferating potential at higher concentration; this could be due to presence of peptides which act as nutrient agent in the culture media. The concentration of active and inactive peptides and also potency of active peptide determines the possible bioactivity in whole of the protein hydrolysate. Application of bioactive guided purification enhances the bioactive ability of bioactive peptides. Cytotoxic potential exhibited by our samples complement the results of antimalarial activity as mechanism of activity may be same as propose by previous studies^11,13,14 . In general, the protein hydrolysate exhibit their cytotoxic ability through various mechanism including cell cycle arrest, apoptosis induction and also cell membrane hydrolysis^29,30

Present study demonstrates bioactive activity of fish hydrolysates suggesting the potential of fish for being interesting source of new chemotherapeutic agents. Results also give insight in the variation of activity seen in fish samples prepared by various techniques. Further investigations including inflammation membranolytic and isolation are beyond the scope of the current study. Standardized isolation process of producing fish hydrolysate wills enable isolation of active agents with reproducible activity.

CONCLUSION:

Fish hydrolysates of present study are highly active against Plasmodium in blood-stage. With raise in drug resistance and to combat other tropical diseases it is need of the hour to explore new drugs with new mechanisms. Our study demonstrates the application of protein hydrolysates from fish sources to be explored for their possible bio prospecting especially in the field of Pharmaceutical Sciences. This preliminary investigation into the ability of fish peptides to act as promising antimalarial and anticancer agents gives new scope for further studies on the promising peptide to be fractionated to isolate the active peptide and further evaluate the mechanism of their activity.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

REFERENCE:

1. Programme WHOGM. World Malaria Report 2017. 2017. doi:10.1071/EC12504

2. Zambare KK, Thalkari AB, Tour NS. A Review on Pathophysiology of Malaria: A Overview of Etiology, Life Cycle of Malarial Parasite, Clinical Signs, Diagnosis and Complications. Asian Journal of Research in Pharmaceutical Science. 2019;9(3):226. doi:10.5958/2231-5659.2019.00035.3

3. Wellems TE, Plowe C V. Chloroquine-resistant malaria. The Journal of infectious diseases. 2001;184(6):770–776. doi:10.1086/322858

4. Thillainayagam M, Ramaiah S. Mosquito, malaria and medicines – A review. Research Journal of Pharmacy and Technology. 2016;9(8):1268–1276. doi:10.5958/0974-360X.2016.00241.9

5. Francis P, Suseem S. Antimalarial Potential of Isolated Flavonoids-A Review. Research Journal of Pharmacy and Technology. 2017;10(11):4057. doi:10.5958/0974-360x.2017.00736.3

6. Wardani AK, Wahid AR, Rosa NS. In vitro antimalarial activity of ashitaba root extracts (Angelica keiskei k.). Research Journal of Pharmacy and Technology. 2020;13(8):3771–3776. doi:10.5958/0974-360X.2020.00667.8

7. Mutiah R, Badiah R, Hayati EK, Widyawaruyanti A. Activity of Antimalarial Compounds from Ethyl Acetate Fraction of Sunflower Leaves ( Helianthus annuus L.) against Plasmodium falciparum Parasites 3D7 Strain . Asian Journal of Pharmacy and Technology. 2017;7(2):86. doi:10.5958/2231-5713.2017.00015.0

8. Suruse P, Duragkar N, Shivhare U, Raut S, Warokar A. Contribution of Traditional Medicines to the Development of Modern Medicine for Malaria. Research Journal of Pharmacology and Pharmacodynamics. 2011;3(1):1-4–4.

9. Bell A. Antimalarial Peptides: The Long and the Short of It. Current Pharmaceutical Design. 2012;17(25):2719–2731. doi:10.2174/138161211797416057

10. Reddy M, Reddy S, Chandra A, Santhosh B, Surekha C, Naveen K. Novel Approaches for Delivery of Proteins and Peptides – A Review. Research Journal of Pharmaceutical Dosage Forms and Technology. 2013;5(1):7–11.

11. Vale N, Aguiar L, Gomes P. Antimicrobial peptides: A new class of antimalarial drugs? Frontiers in Pharmacology. 2014;5(DEC):275. doi:10.3389/fphar.2014.00275

12. Aguiar L, Machado M, Sanches-Vaz M, Prudêncio M, Vale N, Gomes P. Coupling the cell-penetrating peptides transportan and transportan 10 to primaquine enhances its activity against liver-stage malaria parasites. MedChemComm. 2019;10(2):221–226. doi:10.1039/c8md00447a

13. El Chamy Maluf S, Dal Mas C, Oliveira EB, Melo PM, Carmona AK, Gazarini ML, Hayashi MAF. Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom. Peptides. 2016;78:11–16. doi:10.1016/j.peptides.2016.01.013

14. Mather MW, Ke H. Novel Defense Peptides from Platelets Kill Malaria Parasites. Trends in Parasitology. 2018;34(9):729–731. doi:10.1016/j.pt.2018.07.013

15. Bianchin A, Bell A, Chubb AJ, Doolan N, Leneghan D, Stavropoulos I, Shields DC, Mooney C. Design and evaluation of antimalarial peptides derived from prediction of short linear motifs in proteins related to erythrocyte invasion. PLoS ONE. 2015;10(6). doi:10.1371/journal.pone.0127383

16. Linington RG, Clark BR, Trimble EE, Almanza A, Ureña LD, Kyle DE, Gerwick WH. Antimalarial peptides from marine cyanobacteria: Isolation and structural elucidation of gallinamide A. Journal of Natural Products. 2009;72(1):14–17. doi:10.1021/np8003529

17. Elavarasan K, Naveen Kumar V, Shamasundar BA. Antioxidant and functional properties of fish protein hydrolysates from fresh water carp (Catla catla) as influenced by the nature of enzyme. Journal of Food Processing and Preservation. 2014;38(3):1207–1214. doi:10.1111/jfpp.12081

18. Trager W, Jensen JB. Human malaria parasites in continuous culture. Science (New York, N.Y.). 1976;193(4254):673–675. doi:10.1126/science.781840

19. Jadhav K, Deore S, Dhamecha D, Hr R, Jagwani S, Jalalpure S, Bohara R. Phytosynthesis of Silver Nanoparticles: Characterization, Biocompatibility Studies, and Anticancer Activity. ACS Biomaterials Science and Engineering. 2018;4(3):892–899. doi:10.1021/acsbiomaterials.7b00707

20. Zasloff M. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proceedings of the National Academy of Sciences of the United States of America. 1987;84(15):5449–53. doi:10.1097/00043764-198806000-00004

21. Carter V, Underhill A, Baber I, Sylla L, Baby M, Larget-Thiery I, Zettor A, Bourgouin C, Langel Ü, Faye I, et al. Killer Bee Molecules: Antimicrobial Peptides as Effector Molecules to Target Sporogonic Stages of Plasmodium. PLoS Pathogens. 2013;9(11):1–13. doi:10.1371/journal.ppat.1003790

22. Abu-salem FM, Mahmoud MH, Gibriel a Y, Abou-arab A. Characterization of Antioxidant Peptides of Soybean Protein Hydrolysate. World Academy of Science, Engineering and Technology. 2013;79(7):249–253.

23. Sánchez A, Vázquez A. Bioactive peptides: A review. Food Quality and Safety. 2017;1(1):29–46. doi:10.1093/fqsafe/fyx006

24. Yadav AR, Mohite SK. Potential role of peptides for development of cosmeceutical skin products. Research Journal of Topical and Cosmetic Sciences. 2020;11(2):77–82. doi:10.5958/2321-5844.2020.00014.x

25. Nair TS, Meghana R, Shlini P. Antimicrobial Activity of the protein fraction obtained in the extraction of Curcumin. Asian Journal of Research in Chemistry. 2019;12(4):199. doi:10.5958/0974-4150.2019.00037.3

26. Elavarasan K, Shamasundar BA, Badii F, Howell N. Angiotensin I-converting enzyme (ACE) inhibitory activity and structural properties of oven- and freeze-dried protein hydrolysate from fresh water fish (Cirrhinus mrigala). Food Chemistry. 2016;206(July):210–216. doi:10.1016/j.foodchem.2016.03.047

27. Elavarasan K, Shamasundar BA. Angiotensin-I-Converting Enzyme Inhibitory Activity and Antioxidant Properties of Cryptides Derived from Natural Actomyosin of Catla catla Using Papain. Journal of Food Quality. 2018;2018. doi:10.1155/2018/9354829

28. Dara PK, Elavarasan K, Shamasundar BA. Improved Utilization of Croaker Skin Waste and Freshwater Carps Visceral Waste: Conversion of Waste to Health Benefitting Peptides. International Journal of Peptide Research and Therapeutics. 2020;(0123456789). doi:10.1007/s10989-020-10053-3

29. Nwachukwu ID, Aluko RE. Anticancer and antiproliferative properties of food-derived protein hydrolysates and peptides. Journal of Food Bioactives. 2019;7:18–26. doi:10.31665/jfb.2019.7194

30. Song R, Wei R, Zhang B, Yang Z, Wang D. Antioxidant and antiproliferative activities of heated sterilized pepsin hydrolysate derived from half-fin anchovy (Setipinna taty). Marine Drugs. 2011;9(6):1142–1156. doi:10.3390/md9061142

Received on 10.11.2021 Modified on 20.04.2022

Research J. Pharm. and Tech 2023; 16(4):1721-1726.

DOI: 10.52711/0974-360X.2023.00283

Preliminary Screening for Cytotoxicity and Antiplasmodial activity of Fish Protein Hydrolysates on Erythrocytes infected with Plasmodium falciparum 3D7