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


Avinash A. K. Math1, Meenakshi Kaushik2, Elavarasan Krishnamoorthy3

1USM-KLE International Medical Programme, Nehru Nagar, Belagavi - 590010, Karnataka, India.

2ICMR-National Institute of Traditional Medicine, Nehru Nagar, Belagavi 590010, Karnataka, India.

3Central Institute of Fisheries Technology, CIFT Junction, CIFT Road Matsyapuri,

Willingdon Island, Kochi, Kerala 682029, India.

*Corresponding Author E-mail:



The development of resistance to multiple antibiotics by Plasmodium falciparum calls for the exploration of antibiotics from newer sources. Bioactive protein fragment obtained by controlled hydrolysis of the marine sources is being explored as potential antimalarial molecules. In vitro inhibitory activity of fish protein hydrolysates were determined against Plasmodium falciparum 3D7 cultures. Fish protein hydrolysates prepared from fish species [Pangasius (Pangasianodon hypophthalmus), Clam and White snapper (Macolor niger)] were used as a source to prepare six types of hydrolysates and were screened for the antiplasmodial activity using SYBR Green fluorescence Inhibition Assay. To determine the cytotoxic potential of hydrolysate samples MMT assay was performed on MCF-7 breast cancer cell lines. In the present investigation of six proteins hydrolysates samples, clam meat hydrolysate (Cm), fresh pangasius meat hydrolysate (Pm) and cook-wash processed pangasius meat hydrolysate (Pc) had more than 50% inhibition with EC50 values of 2.30, 4.87 and 5.98µg/ml respectively indicating high lethality at a lower concentration for Cm proteins. Except Pc sample, all hydrolysate had anti-proliferative effect across the concentration against MCF-7. Fish hydrolysates explored are highly active against Plasmodium in the preliminary investigation; present a candidature protein as promising source of antimalarial agents.


KEYWORDS: Plasmodium falciparum, MCF-7, protein hydrolysates, Pangasianodon hypophthalmus.




Malaria a vector-borne protozoan disease continues to be the prominent causes of mortality and morbidity in developing world effecting for about million people worldwide1,2. 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 millennial3,4. 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 properties5–8. 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 drug9,10. 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 sources11–14. 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 candidates14–16. 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.




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 90şC. 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% CO2 for 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.



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

S. No


% of inhibition

EC50 (µg/ml)


























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 bioactivities20–22.  Protein /peptide fractions based on their amino acid composition, length and biophysical interaction exerts bioactivities such as antioxidant anti-inflammatory antimicrobial and cytotoxicity23–25.


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 peptides17,26. 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 etc27,28


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 studies11,13,14 . In general, the protein hydrolysate exhibit their cytotoxic ability through various mechanism including cell cycle arrest, apoptosis induction and also cell membrane hydrolysis29,30


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.



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.



The authors have no conflicts of interest regarding this investigation.




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Received on 10.11.2021            Modified on 20.04.2022

Accepted on 14.07.2022           © RJPT All right reserved

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

DOI: 10.52711/0974-360X.2023.00283