Marine Algae Extract (Grateloupia Sparsa) for the Green Synthesis of Co3O4NPs: Antioxidant, Antibacterial, Anticancer, and Hemolytic Activities

The aqueous extract of red algae was used for bio-inspired manufacturing of cobalt oxide nanoparticles (Co3O4NPs) and for antioxidant, antibacterial, hemolytic potency, and anticancer activity. Typical, characterization techniques include UV-Vis, SEM, EDAX, TEM, FTIR, XRD, and TGA. Using an X-ray diffraction assay, the size of the Co3O4NPs crystal was determined to range from 23.2 to 11.8 nm. Based on TEM and SEM pictures, biosynthesized Co3O4NPs' had a homogeneous spherical morphology with a 28.8 to 7.6 nm average diameter. Furthermore, Co3O4NPs biological properties were investigated, including determining the antibacterial potency using the zone of inhibition (ZOI) method and determining the minimal inhibitory concentration (MIC). The antibacterial activity of Co3O4NPs was higher than that of the ciprofloxacin standard. Alternatively, scavenging of DPPH free radical investigation was carried out to test the antioxidant capacitance of Co3O4NPs, revealing significant antioxidant ability. The biosynthesized Co3O4NPs have a dose-dependent effect on erythrocyte viability, indicating that this technique is harmless. Furthermore, bioinspired Co3O4NPs effectively against HepG2 cancer cells (IC50: 201.3 μg/ml). Co3O4NPs would be a therapeutic aid due to their antioxidant, antibacterial, and anticancer properties.


Introduction
Diferent methods can be employed to synthesize metal oxide nanoparticles (MNPs). Each synthesis process has benefts and drawbacks. For instance, the chemical and physical approaches have several advantages, such as producing the desired size and number of nanoparticles. Still, it is eco-toxic, consumes energy, and is expensive and time-consuming [1,2]. Biological methods include plants, algae, microbes, and other natural substances, including starch, egg albumin, and gelatin, which are used in the biological approach to produce diverse types of MNPs. Tis biological method is called the "green method" [3][4][5][6].
Biological substances like starch and bovine albumin have also been employed in the synthesis of green MNPs [4,14,15].
Tese natural resources comprise biomolecules and metabolites that oxidize/reduce, stabilize, and produce specifc MNPs with less pollution, safer, and cheaper [16].
Current advances in marine bio nanotechnology enable and drive advancements in a wide range of industries, including nanomedicine, pharmaceuticals, environmental concerns, and agriculture. Marine organisms that can survive in extreme conditions are plants, algae, bacteria, fungi, actinomyces, yeast, invertebrates, and mammals. Among the phytochemicals/metabolites they can generate are peptides, polyphenols, proteins, carbohydrate polymers, polysaccharides, sulfated polysaccharides, and polysaccharide-protein complexes such as fucoidan, carrageenan, carboxymethyl cellulose, polyglutamic acid, melanin, and others. Tese substances have distinct characteristics that distinguish them as pioneers in the ecologically friendly production of MNPs such as Ag, Au, Ru, Cobalt Oxide, and ZnO in a single-phase system [17].
Algae are marine microorganisms that are heavily used to synthesize MNPs. Algae are bionanofactories because they produce stable nanomaterials that do not require cell upkeep [18]. Algae contain several bioactive substances like proteins, polysaccharides, and phytochemicals with -NH 2 , -OH, and -COOH functional groups used in MNP production [19]. Algae are classifed as microalgae or macroalgae [20]. Te green macroalgae Grateloupia sparsa was used by their function group that acts as reducing and stabilizing agents to manufacture MNPs [21,22].
Cobalt is a good transition metal for health [23]. It is a component of vitamin B12, which helps alleviate anemia by promoting the development of red blood cells [24]. Cobalt's unusual optical, magnetic, catalytic, and electrical properties make it ideal for nanosensors and nanoelectronics felds [25][26][27]. Cobalt is valuable in many sectors because of its CO 2+ , CO 3+ , and CO 4+ oxidation states [28].
Dozens of studies are directed toward using metal oxide nanoparticles in many applications [12,[29][30][31][32][33]. Te most often used metal oxide NPs are cobalt oxide (Co 3 O 4 NPs). Tese nanoparticles have recently gained popularity owing to their lower cost than noble metal nanoparticles. Teir vast surface area gave a unique electrical and magnetic property [34]. Co 3 O 4 NPs are nontoxic at low doses, exhibit high antibacterial and antifungal activity, and have fewer adverse efects than antibiotics [34][35][36].
Antibiotic resistance is currently a severe global health concern. So, an antibiotic agent that can kill harmful bacteria resistant to existing antibiotics is required [37]. Because MNPs are smaller and have more surface area than larger molecules, they exhibit strong antibacterial properties. Te MNPs disrupt the cell membrane and impede protein synthesis in bacteria [38]. MNPs such as cobalt oxide, iron oxide, and copper oxide all demonstrated antibacterial activity [39][40][41].
Te Co 3 O 4 NPs may potentially be antimicrobial; the disc difusion method was used to study the antibacterial activity of Co 3 O 4 NPs synthesized from Celosia argentea whole plant extract. Tese NPs were bactericidal against B. subtilis and E. coli [42]. Te antibacterial efcacy of green-mediated Co 3 O 4 NPs was studied using Hibiscus rosa-sinensis fower extract; the results revealed a promising activity against E. coli and S. aureus [43]. Two main points have been raised. Co 2+ and Co 3+ interact with the negative charge sections of the bacteria and cause cell death. Second, light irradiation in the conduction and valence bands may excite electrons on the surface of cobalt oxide, and excited electrons and oxygen molecules react to generate a superoxide radical anion [44].  [45]. Also, it was found that Co 3 O 4 NPs are cytotoxic to HeLa carcinoma cells [46]. Besides, the biogenic Co 3 O 4 NPs have improved radical scavenging and reducing power [47]. According to a recent study, the scavenging capability and antioxidant properties of bio-inspired Co 3 O 4 NPs are dose-dependent [42].
Hemolysis occurs when disrupted erythrocyte membranes, cause hemoglobin leakage and possibly jaundice or anemia. Te hemolytic potency of any newly synthesized pharmacological preparation must be tested [48]. Based on the hemolytic activity of green-synthesized Co 3 O 4 NPs, Shahzadi et al. results revealed that the bio-inspired Co 3 O 4 NPs had less hemolytic potency (2.95%) than the positive control triton-X-100 (95.25%) and less toxicity (1.02%) [42].
In this study, bioinspired Co 3 O 4 NPs synthesis was performed using red algae extract (Grateloupia sparsa) for Co 3 O 4 NPs synthesis. Tis metal oxide NPs characterization has been broadly done by UV, TEM, EDAX, SEM, XRD, FTIR, and TGA. Moreover, the antibacterial properties, anticancer potency, and hemolytic assay of Co 3 O 4 NPs have been studied in vitro.

Collection of Red Algae.
Te crimson algae were collected during a trip to the Red Sea. To transport the collected algae to the laboratory, they were placed in a plastic bottle. Tus, samples are washed and cleansed with running water to remove salt, toxins, and epiphytes. Ten, it was dried and ground to a powder at room temperature using an electric blender.

Bio-Inspired Synthesis of Co 3 O 4 NPs.
Te algae-dried powder was utilized to prepare the extract. 5 gm of this powder was suspended in 50 mL DD water and heated the mixture to 60 C for 4 h. Ten, the extracts were cooled at room temperature (R.T), they were fltered through Whatman flter paper and kept at 4 C. After that, 10 ml of algae extracts were injected dropwise with 50 ml of cobalt nitrate at a concentration of 1 mg/ml as a source of cobalt, following which, at R.T, continual stirring was performed. Within 24 hours of incubation, the solution's color changes from pink to brown, indicating the creation of Co 3 O 4 NPs.

Co 3 O 4 NPs Characteristics.
Te UV-Vis spectrophotometry (dual beam, Shimadzu, 1900, Japan) was used to determine the production of Co 3 O 4 NPs at wavelengths between 300 and 600 nm. Te FTIR-6800 Spectrometer (JASCO, 500-4000 cm −1 ) was used to determine the functional moieties. All samples were subjected to X-ray diffraction (XRD, Philips X Pert difractometer, Te Netherlands) to validate the crystallinity and size of the Co 3 O 4 NPs. Additionally, the form and size distribution of the particles were investigated by scanning electron microscopy (SEM, FEI Quanta 200 FEG, Japan). Additionally, the elemental composition was identifed by an EDAX study. Further morphological images of Co 3 O 4 NPs were examined using 120 kV transmission electron microscopy (TEM, JEOL, and JEM 1400).

Biological Properties of Co 3 O 4 NPs
2.5.1. Antibacterial Potency. Antibacterial examinations against two G-negative and two G-positive bacteria (E-coli and P. aeruginosa) and (S. aureus and B. subtilis), respectively, were conducted in vitro using the zone of inhibition (ZOI) method by culturing the bacteria on Petri dish nutrient agar. Ten, 6 mm flter discs containing 20 μg/ml of Co 3 O 4 NPs were put on bacterial streaks, and discs with ciprofoxacin (30 μg/ml) were frequently placed in the same dish as standard antibiotics. Finally, all Petri plates were incubated for 24 h at 37 C to compute the inhibitory zone [8,49]. (MIC). MIC values were measured using Sarker's broth agar dilution method [50]. 100 ml of Co 3 O 4 NPs (2 mg/ml) were placed on the plate's initial row, and 50 μl of nutritional broth agar was applied to the other wells. After that, serial dilutions were conducted using sterilized pipettes in 1000 to 3.90 μg. Te resazurin solution was produced by mixing 260 mg in 50 μl of sterile distilled water. All wells were treated with the resazurin solution (10 μl). Also, 30 μl of nutritional broth was completed to a total capacity of 100 μl. Finally, 10 μl of culture suspensions were mixed with the contents of the wells, and then, the plate was incubated for 24 h at 37 C, and the color change was photometrically determined. Te color change from colorless purple to beginning purple was a desirable outcome. Te lowest MIC value in, which the solution becomes colorless [51].

Anticancer Potency.
We evaluated the antitumor activity of bioinspired Co 3 O 4 NPs utilizing the hepatic cancer cell line (HepG2) using the MTT test. Streptomycin and penicillin (1%) were added to DMEM for cell development at 37 C in a 5% CO 2 incubator. Additionally, diferent doses of Co 3 O 4 NPs (50-500 μg/ml) were incubated for 48 h at 37 C in a 96-well plate. Ten, each well was loaded with 20 μl MTT solution and incubated for 3 hours. Finally, 100 μl of DMSO was applied to the culture and incubated for 25 minutes; formazan production by live cells was determined using an Elisa reader set to 570 nm wavelength [52].

Hemolytic Activity.
A standard method was used to determine the hemolytic property of Co 3 O 4 NPs. 3 ml of freshly prepared K 3 -EDTA human blood was withdrawn and centrifuged for 5 minutes at 1500 rpm. Following that, the plasma was removed, and 2 ml of phosphate-bufered sterile saline (PBS) was added, followed by 5 minutes of centrifugation at 1500 rpm to remove any remaining PBS. Te frst human blood tube was flled with 100 μl of Co 3 O 4 NPs and incubated for 35 minutes at 37 C. Te tube was then placed in a cold bath for 5 minutes before centrifuging at 1500 rpm for 5 minutes. Te supernatant was diluted (1 : 10) with cooled PBS (4 C) [43]. Te same procedure was used in the tube with PBS and 0.1% Triton X-100 as a negative and positive control, respectively. Finally, each sample's optical density (OD) was measured using k 576 nm. Te following equation was used to measure the proportion of erythrocyte lysis in each sample: 2.5.5. Antioxidant Property. Spectrophotometric techniques were used to evaluate the acceptor activity of the DPPH. To make a stock solution, 25 ml of methanol was mixed with 2.5 mg of DPPH as a free radical. Individually, diferent amounts of Co 3 O 4 NPs ranging from (50-500 μg/ml) were added to a microplate. Ten, 100 μl of working solution were added to the microplate, covered, and incubated in the dark for 25 minutes. Te activity of the radical scavenger was then evaluated by measuring the OD at 517 nm with a spectrophotometer [53]. All measurements were taken in triplicate. Te following equation is used to measure antioxidant properties:

UV-Vis.
One of the main structural description methods for metallic oxide nanoparticles is UV-Vis spectroscopy. Figure 1 depicts the UV-Vis spectra of benign Co 3 O 4 NPs synthesized by an aqueous extract of red algae. Te surface plasmon resonance of Co 3 O 4 NPs is near 510 nm, lightly shifted from the broad to the long-wavelength area, confrming the Co 3 O 4 NPs formation. Tis tiny wavelength band was owing to transverse electrical oscillation. When Co 3 O 4 NPs particle size increased, the location and morphology of SPR were likewise shifted toward longer wavelengths [54]. Figure 2 were examined (FTIR). Te band at 3500 cm −1 represents the -OH, whereas the bands at 1525 cm −1 and 1060 cm −1 indicate the aromatic rings and the C�O group, respectively.

XRD.
Te XRD values are depicted in Figure 3.  Figure 4(a). As explained, the nanoparticles clump together and form massive particles. Te aggregation of nanoparticles has been described as an indication of metallic nanoparticle production processes [55,56]. (Figure 4(b)). Te particle size distribution on the TEM graphic showed that the Co 3 O 4 NPs were 28.8 to 7.6 nm in size. Te particles seem to be spherical as well SEM and TEM investigations produced similar fndings for a wide nanoparticle size range. Based on our fndings, recent studies revealed that cobalt ferrite nanoparticles made from aqueous extracts of sesame ranged from 3.0 to 20.0 nm in size [57]. Nerium Indicum and Conocarpus erectus methanol extracts were also employed to biosynthesize Co 3 O 4 NPs ranging from 20 to 60 nm in particle sizes [58]. Furthermore, the size of Moringa oleifera extractbiosynthesized cobalt nanoparticles ranges from 20 to 50 nm [59]. According to energy dispersion analysis, the elemental contents of materials were established by high-resolution EDAX (Figure 4(c)). EDAX of Co 3 O 4 NPs was performed in the 0 to 20 keV range. It revealed a 7 keV Co 3 O 4 NPs peak [42]. Te EDAX profle had a strong cobalt signal and several short peaks [60].

TEM and EDAX. A TEM of Co 3 O 4 NPs surface morphology was displayed in
3.1.6. TGA. Te Co 3 O 4 NPs' thermal stability is essential in evaluating their ability to survive various utilities such as fuel cells and conductor-based applications. As a result, thermogravimetric analysis was performed up to 800 C. Te pristine composition declines exponentially, reaching approximately 260 C to lose about 6.3% of its original weight, which could be attributable to the release of organic solvents and water, as seen in Figure 5. A brief plateau distinguishes the second phase of weight loss between 260 and 410 C, followed by a high breakdown rate and the losses, which in this case, was ∼17.6%. Co 3 O 4 NPs appear more thermally stable than the former Co 3 O 4 NPs/algae, particularly in the frst phase of thermal degradation due to the decomposition of organic species than reported in the literature [43,61]. It is worth noting that Co 3 O 4 NPs/algae are slightly more stable below 420 C. As a result, the presence of algae may provide a good thermal scafold for preserving stability at temperatures below 420 C.

Antibacterial Potency.
Bacterial infection is the main serious problem in infectious illnesses in terms of death and morbidity and treatment costs [62]. Antibiotic usage has also been related to many issues, including bacterial resistance, among others. As a result, researchers strive to develop novel strategies to lower the likelihood of  Bioinorganic Chemistry and Applications infectious diseases starting and spreading [32]. Te rapid advancement of nanotechnology will provide tools for creating new substances with novel antibacterial properties [63,64]. Several investigations into the antibacterial potency of biogenic metal nanoparticles have been published, with promising fndings against various bacteria strains [4,12]. At a dose of (30 μg/ml), bio-inspired Co 3 O 4 NPs were tested against various bacterial species. G-positive bacteria were (B. subtilis and S. aureus), whereas G-negative bacteria were (P. aeruginosa and E. coli) compared to the standard antibiotic ciprofoxacin disk (30 μg/ml). We found that Co 3 O 4 NPs were efective on candidate bacterial species but still lower than the efect of ciprofoxacin based on ZOI measurements. On the other hand, P. aeruginosa with low sensitivity at a MIC of 23.0 ± 5.3 μg/ml and B. subtilis are highly sensitive to bio-inspired Co 3 O 4 NPs, with MIC values of 18.6 ± 3.8 μg/ml. Table 1 depicts MIC values and the ZOI for antibacterial activity are compared to efective ciprofoxacin. A G-positive bacteria cell wall is composed of peptidoglycan layered with (∼70 nm thick), which permits Co 3 O 4 NPs to interface directly with the outer membrane of bacteria more readily than G-negative bacteria, which have a layer of lipopolysaccharides (1-2 mm thick) [65]. Tis variety in bacterial cell wall structure and thickness makes G-positive bacteria's membrane rupture faster and leads to their death [66]. As a result, the antibacterial activity of Co 3 O 4 NPs can be compact in size, and a high surface-tovolume ratio allows them to interface with the bacterial cell membrane. Figure 6 depicts how green Co 3 O 4 NPs work against bacteria by attaching to the bacterial cell wall and modifying its permeability [67]. Te penetration of reactive oxygen species (ROS) into the cytoplasm damaged the nucleus and plasmid, causing a shift in cell signaling and, eventually, death [68].

Anticancer Potency of Co 3 O 4 NPs.
Cancer remains the world's most prominent cause of mortality. Te number of cancer cases has been steadily increasing, and it is expected to reach around 21 million by 2030 [69,70]. Hepatic cancer is the 2nd common cause of mortality in males and the 6th common cause of death in females. Excessive alcohol intake over a long period of time, as well as HCV and HBV infections, and other toxins, all increase the risk [71]. Using the HepG2 cell line, the anticancer efects of Co 3 O 4 NPs were also studied. Cancer cells were applied to various doses of Co 3 O 4 NPs (50-500 μg/ml) over 24 h. In this study, Co 3 O 4 NPs were discovered to have signifcant anticancer potential, with an IC 50 value of 201.3 μg/ml. Figure 7 revealed an estimated 80% fatality rate at 500 μg/ml. Our fndings show that Co 3 O 4 NPs generate ROS that interacts with cells and causes cellular oxidative stress, leading to DNA destruction and cell death. Tis is because the microscopic nanoparticles are soluble in the internal acid medium, which has a pH of 4.5. Te Co 3 O 4 NPs can create pores in the membrane and dissolve in the cells, and eventually, the cell dies [72]. Cancer cells were also suppressed by Co 3 O 4 NPs, suggesting their anticancer potential. Our fndings consistently show that metal nanoparticles have a signifcant anticancer potential [73].   (Figure 8(a)), Triton-X-100 having 97.3% toxicity (Figure 8(b)), and PBS having 1.01% toxicity (Figure 8(c)).

Antioxidant Activity.
A DPPH-free radical scavenging test was used to evaluate the free radical scavenging capability of green-produced Co 3 O 4 NPs. Tese results revealed four diferent Co 3 O 4 NP concentrations; the free radical scavenging capability increased as the Co 3 O 4 NP concentration rose (Figure 9). Te peak of DPPH radical scavenging  (88.2%) was observed at 500 mg/ml. Te lowest DPPH radical scavenging was observed at 50.1 mg/ml (35.0%), whereas the highest DPPH radical scavenging was obtained at 500 mg/ml (88.2%). Co 3 O 4 NPs are hypothesized to operate as electron donors, interacting with free radicals and converting them into more stable molecules capable of stopping the radical chain reaction [48].

Conclusions
Red algae were employed to create eco-friendly cobalt oxide nanoparticles (Co 3 O 4 NPs). Te Co 3 O 4 NPs were formed in inhomogeneous spheres with diameters ranging from 28.8 to 7.6 nm. Te antibacterial activity of Co 3 O 4 NPs was examined, and it was revealed that nanoparticle concentrations of 30 μg/ml widened the inhibition zone against candidate species from 11.7 to 17.6 mm, still lower than standard antibiotics with a ZOI of 18.1 mm in its higher efcacy. Furthermore, the minimal inhibitory concentrations for (P. aeruginosa, E. coli, S. aureus, and B. subtilis) were adjusted to be around 23.0, 21.1, 20.6, and 18.6 for each bacterial species. Furthermore, Co 3 O 4 NPs were investigated for anticancer activity in vitro against the HepG2 cell line. Cell mortality for 500 μg/ml was reported to be more than 80% after 24 hours of exposure. Furthermore, the antioxidant activity was studied, and it was observed that the maximum radical scavenging of DPPH was attained at 500 mg/ml of Co 3 O 4 NPs (88.2%).

Data Availability
Te data supporting this study's results are available upon request from the corresponding author.

Conflicts of Interest
Te authors declare that they have no conficts of interest regarding the publication of this paper.