Production Optimization and In Vitro Evaluation of Anti-Proliferative, Anti-Oxidant and Anti-Inammatory Potential of the Antibacterial Peptide MAFP9

Peptides are important natural bioactive agents obtained from various sources including microbes, plants and animals. The present study involved the optimization and physicochemical characterization of an antibacterial peptide MFAP9 produced by a marine fungus Aspergillus fumigatus BTMF9 and its vitro evaluation of antiproliferative, antioxidant and anti-inammatory potential. Antiproliferative activity was performed using A549 lung carcinoma and normal L929 broblast cell lines employing MTT assay method. Antioxidant activity was assessed using DPPH assay and Cox2 inhibition assay of peptide treated LPS stimulated THP1 cell lines was used for anti-inammatory studies. The results of in vitro antiproliferative activity assessment showed that MFAP9 was cytotoxic with an IC 50 value of 29 µg/mL towards A549 cells. Fluorescence microscopic analysis revealed apoptotic features such as chromatin condensation and alterations in the size and the shape of cells after treatment with MFAP9 at a concentration 50 µg/mL. The DPPH radical scavenging activity of MFAP9 exhibits reasonably higher scavenging activity compared to that of standard antioxidant at same concentration level. Moreover, MFAP9 showed signicant anti-inammatory activities. Time course experiment was conducted with the Czapek- Dox medium and optimized the rate of MFAP9 production. The study on various physicochemical parameters on the stability revealed that the maximum active temperature of MAFP9 is 50 o C, stable in basic pH and 1 mM concentration of Ni 2+ , Cd 2+ and Mg 2+ promotes the peptide activity. Hopefully, by employing the advancements in proteomics, bioinformatics, and modication strategies, MFAP9 could emerge as novel promising therapeutic drug in future for various clinical applications.


Introduction
The biodiversity of the marine environment and the associated chemical diversity constitute a practically inexhaustible resource of bioactive lead compounds for the development of therapeutic agents with anticancer, anti-in ammatory and antioxidant potential. Peptides play a vital role in inter and intra-cellular communications and cellular function. Considering the bene cial characteristics of high a nity, speci city, and low toxicity, peptides are effective substitutes for chemical drugs (Xueqin et al. 2017). There is a large variety of physiological activities induced by bioactive peptides which are mainly determined by the type, number, sequence and properties of amino acids in the peptide (Agyei and Danquah, 2012). The marine peptides have potential applications in the pharmaceutical industry owing to their bioactivities such as antimicrobial, antiviral, antitumor, antioxidative, cardioprotective, immunomodulatory, analgesic, anti-diabetic, appetite suppressing and neuroprotective activities (Cheung et al. 2015).
Bioactive peptides from various marine sources have been studied for their anticancer activity (Carniato et al. 2019). Cancer is one of the most life-threatening diseases and becomes a major growing health problem worldwide due to the lack of comprehensive early detection methods and proper medicines (Akindele et al. 2015; Khalil et al. 2018; Chanda and Nagani, 2013). Despite the signi cant development in biomedical eld, cancer remains ambiguous especially from the therapeutic perspective and one of the greatest challenges to mankind (Silva et al. 2019). It is evident that AMPs derived from marine fungi have signi cant pharmacological activities and act as a cancer suppressant (Eghtedari et al. 2021).
Bioactive peptides exhibit immunomodulatory, anticancer potential as well as antioxidant potential. (Chakrabarti et al. 2014;Suarez-Jimenez et al. 2012). Antioxidants protect the body against reactive oxygen species (ROS), which exert oxidative damage to membrane lipids, protein and DNA. The association of chronic in ammation with the initiation and progression of various diseases such as cancers, cardiovascular diseases, fatty liver and Alzheimer's disease involve a cascade of events comprising the recruitment of in ammatory cells to the site of action, release of in ammatory mediators and resultant tissue damage. Recently, chemical and bioengineering strategies have been proposed to develop potent, selective and metabolically stable peptide preparations with low cost and reduced tendency to trigger undesired side-effects. These novel ndings together with innovative techniques make antimicrobial peptides as an emerging category for clinical applications (Luong et al. 2020).
The biodiversity of marine microorganisms and their peptides enable them to be a good choice to develop commercial products which are yet to be explored. However, as more peptides originating from marine microbes are evaluated at the preclinical and clinical stages, optimization of microbial fermentation broth and development of high-throughput culture methodology will expedite the present investigation on the marine peptides for further application in pharmaceutical and nutraceutical industries (Kawanishi et al. 2011;Jaiganesh and Kumar, 2012). A recent study reported the isolation, puri cation and characterization of an antibacterial peptide MFAP9 from a marine A. fumigatus BTMF9 (NCBI GenBank accession No.HQ285882) as an excellent lead compound with strong antibacterial and antibio lm potential (Raghavan et al. 2021). In the current study, optimization of medium components and culture conditions for maximum peptide production was analyzed followed by stability. Further the study evaluated the biological features, including the antiproliferative activities toward A549 cancer cell lines, antioxidant capacity based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and determined anti-in ammatory potential employing cycloxygenase 2 inhibition assay of peptide treated LPS stimulated THP1 cell lines.

Materials And Methods
Antibacterial peptide production: study on the effect of bioprocess variables by 'one-factor at-a-time method' Various bioprocess parameters affecting maximum peptide production under submerged fermentation were studied by one-factor at-a-time method using CD medium. The parameters included temperature, inorganic nitrogen source, organic nitrogen sources, carbon source, initial pH, sodium chloride, incubation period, inoculum concentration, agitation, metal ions and additional inorganic nitrogen source. The effect of each parameter for maximal peptide production was evaluated and incorporated the same variable at its optimized level in the subsequent experiment to optimize next parameter. In each case, samples were analyzed for antibacterial activity and protein concentration using standard protocol unless otherwise mentioned (Enan et al. 1996;Bradford 1976). All the experiments were carried out in triplicates.
Time course experiment was conducted with the Czapek-Dox medium and optimized medium after onefactor-at -a -time method to ascertain the rate of peptide production. The optimized medium with selected conditions included 30 o C, sodium nitrate (0.3%) as inorganic nitrogen source, sucrose (3%) as carbon source, initial pH 7, sodium chloride (0.5%), inoculum concentration (0.4%), Ca 2+ as metal ion (2mM), ammonium phosphate (1%) as additional inorganic nitrogen source incubated at 150 rpm for 168 h. Samples were taken at a regular interval of 24 h for antibacterial activity analysis.
Physico-chemical parameters on the stability and activity of peptide The stability of the MAFP9 has been studied using various physico-chemical parameters. Effect of various non-ionic and ionic detergents such as triton X-100, tween 80, tween 20, SDS and CTAB (0.1% each w/v) on peptide activity was determined by incubating the puri ed peptide in each detergent for 1 h.
Effect of various metal ions was evaluated by incubation along with 1 mM concentrations of various metals ions such as Na + , Ca 2+ , Mg 2+ , Fe 3+ , Mn 2+ , Ni 2+ , , Ba 2+ , Cd 2+ , Zn 2+ , Cu 2+ , Co 2+ , Cr 2+ and Al 3+ respectively for 1 h. The effect of reducing agents on the activity was studied by incubating the MAFP9 In vitro antiproliferative studies of peptide on cultured cell lines Cell culture maintenance L929 broblast (Normal cell line) and A549 (lung carcinoma cell line) were the cell lines used for this study and were purchased from National Centre for Cell Sciences (NCCS), Pune, India. Dulbecco's modi ed Eagles media (HiMedia) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, USA) was used for cell line maintenance and grown to con uency at 37°C in 5 % CO 2 (NBS, Eppendorf, Germany) in a humidi ed atmosphere in a CO 2 incubator.

Procedure
The cells were trypsinized (500 µL of 0.025% Trypsin in PBS/ 0.5 mM EDTA solution (HiMedia)) for 2 min and passaged to T asks in complete aseptic conditions. Peptide sample was added to grown cells at a nal concentration of 6.25 µg/ml, 12.5 µg/mL, 25 µg/mL, 50 µg/mL and 100 µg/mL from a stock of 0.1 mg/mL and incubated for 24 h. The percentage difference in cell viability was determined by standard MTT assay after 24 h of incubation (Arung et al. 2006). The morphological features of cells were imaged using inverted phase contrast microscope (Olympus CKX41, Japan) with Optika Pro5 CCD camera.
Determination of apoptosis and necrosis by acridine orange (AO) and ethidium bromide (EB) double staining method DNA-binding dyes AO and EB (Sigma, USA) were used for the morphological detection of apoptotic and necrotic cells (Attari et al. 2009). AO is taken up by both viable and non-viable cells and emits green uorescence if intercalated into double stranded nucleic acid (DNA). Ethidium bromide is taken up only by non-viable cells and discharges red uorescence by intercalation into DNA. A549 cells were washed by cold PBS and then stained with a mixture of AO (100 μg/mL) and EB (100 μg/mL) at room temperature for 10 min. The stained cells were washed twice with 1X PBS and analyzed using a uorescence microscope in blue lter (Olympus CKX41 with Optika Pro5 camera). The cells were segregated into living cells (normal green nucleus), late apoptotic (orange-stained nuclei with chromatin condensation or fragmentation), early apoptotic (bright green nucleus with condensed or fragmented chromatin), and necrotic cells (uniformly orange-stained cell nuclei). Cell line was cultured in RPMI 1640 [HiMedia] media, supplemented with 10% heat inactivated FBS, antibiotics (Penicillin and Streptomycin) and 1.5% sodium bicarbonate. The media was ltered by 0.2 µm pore sized cellulose acetate lter (Sartorius) in totally aseptic conditions. The cells were then grown till 60% con uency followed by activation with 1 µl LPS (1 µg/mL). LPS stimulated THP 1 cells were exposed with different concentrations of peptide samples (6.25 µg/ml, 12.5 µg/mL 25 µg/mL, 50 µg/mL and 100 µg/mL) from a stock of 1mg/ml dissolved in 1% DMSO and incubated for 24 hours.
The crude enzyme isolation was done by spinning at 6000 rpm for 10 minutes. Supernatant was discarded and 200 µl of cell lysis buffer (1MTris HCl, 0.25M EDTA, 2M NaCl, 0.5% Triton) was added .The incubation was done for 30 minutes at 4°C and enzyme assay was done in pellet suspended in a small amount of supernatant.

Assay of cycloxygenase (COX-2) activity
The assay mixture contained 100 mM Tris-HCl buffer (pH. 8), 5 mM glutathione, 5 μM hemoglobin and crude enzyme supernatant. The reaction was started by the addition of 200 μM arachidonic acid and incubated at 37ºc for 20 min. Reaction was terminated by adding 0.2 mL of 10% TCA in 1N HCl, mixed and 0.2 mL of 1 % thiobarbituric acid was added. Reaction mixture kept in a boiling water bath for 20 min, cooled and centrifuged at 1000 rpm for 3 min. The absorbance was measured at 632 nm for COX activity.

MAFP9 production optimization by Aspergillus fumigatus BTMF9
The incubation period at which maximum production noticed when the culture was incubated for 72 h and antibacterial activity (1600 AU/mL) remained stable throughout the incubation time. Agitation plays a major role by providing proper aeration for MFAP9 production during submerged fermentation.
Maximum peptide production of 1600 AU/mL with speci c activity, log 10 4.71 ± 0.05 AU/mg was obtained when the culture was incubated with an agitation of 150 rpm. The effect of various metal ions on the peptide production was evaluated and among all metal ions, activity was observed when Ca 2+ , Ba 2+ , Cr 2+ , Mn 2+ ,Cu 2+ and Al 3+ were added in the media. However a drastic increase in activity was obtained in presence of Ca 2+ with activity of 3200 AU/mL and speci c activity of log 10 4.83 ± 0.02 AU/mg. The effect of additional inorganic nitrogen source indicated that ammonium phosphate exhibited maximum activity, 3200 AU/mL. The inoculum concentration that supports MFAP9 production was found to be ranged from 0.3-1% (1600 AU/mL). However, the optimal inoculum concentration for maximum production was 0.4% at which antibacterial activity, 3200 AU/mL and speci c activity (log 10 4.53 ± 0.12 AU/mg).
The results obtained for the time course experiment for MFAP9 production after medium optimization is shown in Fig. 1 and it is evident that a signi cant increase in activity was occurred in optimized medium compared to Czapek -Dox medium. The peptide production was started at 72 h for both media, however the activity obtained in optimized medium (6400 AU/mL) was far higher than in unoptimized medium (1600 AU/mL). In optimized medium, the activity decreased after 120 h whereas in other case activity was steady throughout the incubation time. Maximum speci c activity was recorded on 72 h for both media even though speci c activity was higher in optimized medium (log 10 4.63 ± 0.00 AU/mg).
Physico-chemical parameters on the stability and activity of MAFP9 The effect of various physico-chemical parameters on the activity of MAFP9 is depicted in the Table 1.
Maximum active temperature of MAFP9 was found to be 50 o C, stable in basic pH and 1mM concentration of Ni 2+ , Cd 2+ and Mg 2+ promoted the antibacterial activity. A549 adenocarcinomic human alveolar basal epithelial cells were used to study antiproliferative activity of MFAP9 and Fig. 2.A shows the percentage viability of cells after peptide treatment. The results revealed that the peptide exhibited signi cantly higher cytotoxicity on cancer cells and the IC 50 value was determined as 29 µg/mL. The cell viability decreased considerably in a concentration-dependent manner.
The morphological image of A549 cells after peptide MFAP9 treatment showed distorted cells with globular shape. Also cells got fragmented into membrane-bound particles producing irregular shapes that are smaller in size than the untreated cells (Fig. 2.B & C). It was also found that the quantities of the cells decreased when treated with MFAP9.
The type of cell death induced by MFAP9 in A549 cells was further assessed by investigating the morphological changes after double staining with acrydine orange (AO) and ethidium bromide (EB). As in ( The DPPH radical scavenging activity of the antibacterial peptide MFAP9 compared with that of standard antioxidant ascorbic acid. The results revealed that MFAP9 had a little higher scavenging activity compared to standard antioxidant at same concentration level. From the Fig. 3A, it was apparent that activity of both standard and MFAP9 was based on a concentration dependent manner. The in vitro antiin ammatory effect of MFAP9 was determined using THP1 cell lines. LPS stimulated THP1 cells were exposed with different concentrations of peptide sample. The spectroscopic analysis showed loss of activity of in ammatory enzyme COX-2 when culture supernatant incubated with peptide MFAP9 in the presence of the substrate, arachidonic acid revealed the anti-in ammatory potential of MFAP9 by competing with the substrate (Fig. 3. B).

Discussion
The fungal producers may be capable of changing the nature of the end synthesized products depending on the environmental conditions, moreover an increase in amount of bioactive compounds may be found after optimization of cultural conditions. A well understanding of the effect of culture conditions on biosynthesis may direct to better utilization of microbial sources. The availability of reports are fewer regarding the studies on effect of nutritional and cultural conditions on mycelial growth and antimicrobial metabolite production by the antagonistic fungal strains (Jain and Pundir, 2011; Ottoni et al. 2017). From the result, it was noted that the MFAP9 production was started on the 3rd day of incubation (72 h) and remained in a stationary mode till the last incubation time studied (168 h). As the speci c activity was highest at 72 h, that particular incubation time was considered as optimum for peptide production. Atalla et al. (2008) showed that the antibiotic compound production by a marine fungal isolate Varicosporina ramulosa increased gradually until reached the maximum after 8 days and then decreased. The requirement of 9 days incubation for the production of diketopiperazines was reported by Zheng et al. In shake ask cultures agitation is an important factor to get a growth and production of the compounds. It was observed that agitation at almost all rotation rates ranging from 50 to 200 rpm gave a stable yield but it was higher for 150 rpm. Parameters such as temperature, agitation, culture and time of synthesis vary on the fungus used and shall be regulated in order to obtain the desired nanoparticle characteristics (Birla et al. 2013). The effect of agitation, temperature and initial pH on the growth rate of the shiitake mushroom Lentinula edodes (Berk) Pegler showed an increase under agitation and the generation time was found to be 1.5 days (Hiroko Hassegawa et al. 2005).
Effect of various metal ions on production of MFAP9 was studied and calcium was observed to produce a higher yield and this was incorporated into the optimized medium. In contrast to enzymes and its subunits, metal ion is a limiting factor, but for peptides this is not a critical factor. An eight-fold decrease in the activity of microplusin was observed when the medium was supplemented with Cu 2+ , nonetheless, the antimicrobial activity of microplusin didn't affect when other metal ions such as Ca 2+ , Co 2+ , Fe 2+ , Mg 2+ , Mn 2+ , and Zn 2+ were added to the media. Overall analysis of the data indicated that the peptide exerts its activity by chelating copper ions. As a consequence, the disruption of copper homeostasis by microplusin results in deleterious changes of the bacterial respiratory pro le (Silva et al., 2009). Inoculum concentration is one of the important factors in the growth of the fungal mycelia and production of the compound. If inoculum concentration is very much higher this may lead to competition and the culture may attain a stable phase and this may lead to death of the fungus, thus production e ciency may get reduced. The optimum inoculum concentration of 0.4% was observed to be favourable for production of peptide MFAP9.
Time course experiment with optimized and unoptimized media was performed and it was noted that a several fold increases in production of peptide occurred in optimized medium. The maximal antibacterial activity as well as speci c activity was observed on the 3rd day of incubation in both media. The time course for antimicrobial agent production varies according to the strain and cultivation conditions. As evidenced that the maximum antimicrobial agent production was achieved after 4 days of incubation of Cladosporium sp (Miao and Qian, 2005), 5 days of incubation of Penicillium corylophilum (Silva et al. 2004) and for a fungal isolate SS2, production seems to be stable on the 5th and 6th days of the incubation with maximum production on the 5th day (Rajasekar et al. 2012). A typical time course study between statistically optimized and unoptimized medium was compared in case of exopolysaccharide (EPS) production by Schizophyllum commune AGMJ-1 for 18 days in shake ask conditions and found that under statistically optimized medium there was a 4 fold increase in EPS production was obtained (Joshi et al. 2013).
Nonionic detergents are generally considered as mild detergents and that they do not interact extensively with the protein surface, whereas non-speci c binding of ionic detergents, particularly SDS to the protein surface may lead to protein unfolding (Mogensen et al. 2005). Tween 20, tween 80 and triton X-100 were the non-ionic detergents used to evaluate the effect on peptide. The study revealed that Triton X in uences the peptide by 50% reduction in activity when compared to other mild detergents. In contrast, treatment with ionic detergents like SDS and CTAB resulted in complete inactivation of peptide. In case of Plantaricin C19 produced by Lactobacillus plantarum C19 lost its activity after treatment with SDS or Triton X-100 (Atrih et al. 2001). According to Wen and Chen, (2014) the antifungal activity of PcPAF was 35% inhibited by 0.5% SDS compared with the control whereas no change was observed on the activity of PcPAF when treated with 0.5% Triton X-100 and 1% Tween-20.
Antimicrobial peptides need to be stable in physiological conditions for therapeutic use against local or systemic infections due to the reason that the antagonism between the peptides and ionic strength in their environment may inhibit the development as novel antibiotics. In present study, the activity of peptide was enhanced by the addition of metal ions Ni 2+ , Cd 2+ and Mg 2+ , whereas activity decreased in presence of Cu 2+ , Al 3+ and Na + . Furthermore it was noted that the activity of peptide was not at all affected by the other ions such as Ba 2+ , Fe 3+ , Zn 2+ , Co 2+ , Ni 2+ and Ca 2+ . AFP of Aspergillus giganteus was found to act by binding to the membrane or cell wall of sensitive fungi, and it was revealed that Both DTT and β-mercaptoethanol as reducing agents had a prominent in uence on the activity of peptide MFAP9. It was evidenced that DTT caused signi cant reduction of activity, whereas complete inactivation of peptide occurred using 80 mM concentration of β-mercaptoethanol. The sensitivity of peptide towards β-mercaptoethanol con rmed the presence of intra disul de bond and its vital role for the stability of peptide. It was noted that DTT reduced the activity of the bacteriocin produced by B. subtilis LFB112 (Xie et al. 2009). Effect of oxidizing agents was studied by incubating the peptide with DMSO that acts as a mild oxidant and another strong oxidant sodium hypochlorite. The results shown that DMSO had no in uence on the activity of peptide, whereas high concentration of sodium hypochlorite caused a decline in the activity of MFAP9. The effect of oxidizing agents on inhibitory activity of protease inhibitor isolated from Pleurotus oridanus revealed that the activity declined in a concentration dependent manner for both oxidizing agents DMSO and H 2 O 2 (Pannippara et al. 2020).
Results obtained for the effect of chemical amino acid modi ers on the activity of MFAP9 indicated the presence of tryptophan, cysteine and histidine residues in the peptide and also their importance in the function of antibacterial activity. Modi cation of histidine residues with DEPC resulted in a sudden reduction in the activity of peptide and also observed insigni cant role of higher concentrations as the inhibition percentage remained same at all the concentrations. Similar effect was observed when modi cation of cysteine residues with iodoacetamide was done. On the contrary, the modi cation with PMSF had no effect on the activity of the peptide suggested that the amino acid serine may either not present or having no role in the activity of peptide. There were reports suggesting the process of excess lecithin synthesis by A549 cells resulted in pulmonary surfactant synthesis. Those surfactants may impede therapeutics during pulmonary disease treatment (Hohlfeld et al. 1997). It pauses challenge on anticancer drugs e cacy on A549 cell lines. In the present study, the intervention of peptide with the surfactants resulted activity against cell lines. It was also observed that the peptide acts in a concentration dependent mode. In vitro anticancer effect of fucoidan isolated from brown seaweed Turbinaria conoides was studied on A549 cell line. Similar to MFAP9, fucoidan also inhibited the growth of cancer cells in a dose-dependent manner and potent anticancer activities were 24.9-73.5% in the concentrations of 31.25-500 g/ml. The CTC50 value against the cancer cell was found to be 45 g/ml and the CTC50 value of normal Vero cell line was 325 g/ml (Kumari et al. 2018). Aspergillamide A, which is a peptide isolated from the mycelium of a marine sediment-derived Aspergillus strain found to demonstrate modest cytotoxicity (IC 50 , 16µg/mL) to human colon carcinoma HCT-116 cells (Lee et al. 2013). Calcaelin is a ribosome inactivating protein (RIP) from the puffball mushroom Calvatia caelata with an N-terminal sequence resembling those of American ginseng and Chinese ginseng RIPs Calcaelin is a ribosome inactivating protein (RIP) from the puffball mushroom Calvatia caelata with an N-terminal sequence resembling those of American ginseng and Chinese ginseng RIPs, expressed anti-mitogenic activity toward spleen cells and antiproliferative activity towards tumor cells (Ng 2005  Current study demonstrates the free radical scavenging activity of MFAP9 in comparison with ascorbic acid at different concentrations and showed that the peptide had a comparable activity with that of standard antibiotic and the activity followed a concentration dependent mode.
Nonsteroidal Anti-In ammatory Drugs (NSAIDs) are among the most primarily used therapeutic invention for the treatment of pain and in ammation. It was observed that consumption of NSAIDs can lead to high incidence of gastrointestinal irritation due to the development of life-threatening gastrointestinal ulcers and also causes abnormal renal physiology. COX-2 is overexpressed in numerous premalignant and malignant lesions con rmed the signi cant correlation between the COX-2 inhibition activity of non steroidal anti-in ammatory drugs (NSAIDs) and their antitumor e cacies (

Conclusion
The antibacterial peptide MAFP9 demonstrated signi cant inhibition on tumor cell proliferation and free radical scavenging activity in comparison with ascorbic acid. In addition to that MFAP9 induced cell death through apoptosis. The media conditions were optimized and increased the production to several fold. Hence, the antibacterial peptide MAFP9 will have a guiding signi cance in pharmaceutics and therapeutics.

Con ict of interest
The authors declared that they have no con icts of interest to this work.