Assessment of anticancer, antibacterial, antifungal, and antioxidant activities of Micractinium reisseri (Chlorophyta, Trebouxiophyceae) methanolic extract

This study looked at the anticancer, antimicrobial, and antioxidant properties of a methanolic extract of Micractinium reisseri. In vitro antitumor activity of M. reisseri methanolic extract revealed an inhibitory effect against MCF-7 and HCT-116 (breast carcinoma and colon carcinoma) cell lines. Antimicrobial activity of M. reisseri methanolic extract was estimated against Gram-negative bacteria (Pseudomonas aeruginosa RCMB 010,049 and Escherichia coli RCMB 010,052), Gram-positive bacteria (Streptococcus pneumonia RCMB 010,010 and Bacillus subtilis RCMB 010,067) and fungi (Aspergillus fumigatus RCMB 02,568 and Candida albicans RCMB 05,036). The extract showed promising results against all the tested microbes except C. albicans which gave negative results. The algal extract showed 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing and hydroxyl radical scavenging activity in a concentration-dependent manner with maximum scavenging activity at concentrations (0.001, 0.001, and 5 mg/ml) for each assay, respectively. High-performance liquid chromatography (HPLC) revealed that the active compounds are phenols, alkaloids, and flavonoids. In conclusion, M. reisseri methanolic extract exhibited effective anticancer, antimicrobial, and antioxidant activities.


Introduction
Unicellular microalgae are microorganisms found in fresh or saltwater and have varied shapes with a diameter or length of about 3-10 µm (Ferreira et al. 2013). Many studies have been conducted to investigate the products of microalgal metabolism, not only to identify their nature but also to look for ingredients with potential applications to humans in a variety of important fields (De Morais et al. 2015). They contain amino acids, steroids, phenolic compounds, terpenoids, phlorotannins, halogenated ketones, alkenes, and cyclic polysulfides, all of which have different physiological (antioxidant, antibacterial, antiviral, antifungal, enzyme inhibiting, immunosuppressive, cytotoxic, and algicide activity) effects (Taskin et al. 2007 andRao et al. 2007). Trends in natural drug research suggest that algae are a promising source of new biochemically active substances (Mayer and Hamann 2005). Meanwhile, screening extracts or separation of metabolites from various microalgae is a collaborative method for expressing the biological activity of these components (Herrero et al. 2013). Micractinium 1 3 reisseri is a member of the Chlorellaceae family and is distinguished by spherical or oval cells with a single chloroplast that is parietal with a pyrenoid (Krienitz et al. 2004;Chae et al. 2019). This alga is well known for being a promising source of biofuels (El-Sheekh et al. 2020), but for unknown reasons, the vital activities of M. reisseri methanolic extract have not been tested yet. Therefore, for the first time, this work aims to evaluate the effectiveness of M. reisseri methanolic extract cytotoxicity against (MCF-7 & HCT-116) cell lines, antimicrobial activity with MIC against P. aeruginosa, E. coli, S. pneumonia, B. subtilis, C. albicans, A. fumigatus with the evaluation of its antioxidant power using 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and Hydroxyl radical scavenging assays.

Microalga and growth condition
Micractinium reisseri R. Hoshina, M. Iwataki, and N. Imamura, a unicellular green alga, was chosen for this study. A routine bacterial test, however, was performed on a regular basis by inoculating one drop of the culture at the start and end of the experiment into previously autoclaved 10 ml vials containing a bacterial growth medium, F. medium (Feeley et al. 1978). M. reisseri axenic cultures were grown in 500 ml Erlenmeyer flasks with 200 ml of the selected medium in Bold's Basal Medium (BBM). The experiment was carried out in a culturing chamber under controlled laboratory conditions (28 °C temperature, 80 µmol m −2 S −1 light intensity). This temperature was chosen based on the results of Ginzburg and Ginzburg (1981). Cultures were illuminated using a 16-h light/eight-hour dark cycle. All culture flasks were manually swirled daily to detach adhered algal cells from the flask walls. The research period lasted 18 days. To achieve suitable biomass for bioactivity measurements, large-scale cultivation was prepared under the same culture conditions.

Growth measurements
The optical density and pigment content of M. reisseri were used to estimate growth (chlorophyll a and carotenoids). These analyses were carried out every 2 days for the next 18 days. The optical density was determined using a Perkin Elmer (Lambda 1) Visible-UV (ultraviolet) spectrophotometer at 450 nm, as described by Robert (1979). Chlorophyll pigment absorption was measured at wavelengths of 664 nm, 647 nm, and 630 nm. The trichromatic equation of Jeffrey and Humphrey (1975) was used to calculate chlorophyll a and b. carotenoids at 450 nm were determined using the spectrophotometric method recommended by Jensen and Liaaen (1959).

Large-scale cultivation and culture harvesting
M. reisseri culture was previously grown in a 2000 ml Erlenmeyer flask containing 1400 ml of cultivation medium and a 50 ml known inoculum (with initial turbidity: 0.07 at 450 nm). This inoculum was divided into five groups of 280 ml each, and each inoculum was transferred to a sterilized 2000 ml Erlenmeyer flask containing 1400 ml sterilized cultivation medium, yielding five flasks. The indoor batch cultures were grown in the same manner as the indoor airlift cultures. The cultures were aerated using an air injection device connected to a glass pipe equipped with a sterilized bacterial filter (0.2 m), which released air bubbles from the flask bottoms. The air-flow rate was adjusted to ensure proper culture mixing through the upward movement of air bubbles. Centrifugation at 3606 g for 20 min was used to harvest the algal cells. The supernatants were removed, and the remaining pellets were used to make the methanolic extract.

M. reisseri methanolic extract
One hundred grams of M. reisseri biomass were extracted in one liter of 100% methanol. The mixtures were kept at room temperature in a shaking incubator for 3 days. After 3 days, the samples were centrifuged for 20 min at 7200 g. The clear extract was concentrated with a rotary evaporator at 40 °C under reduced pressure (72 mbar) and dried with a lyophilizer. The dried extract was collected and then dissolved in 5 mg/ml Di-methyl sulfoxide (DEMSO) (Chernane et al. 2014).

Phytochemical analysis of M. reisseri methanolic extract
The total flavonoid content was determined using the aluminum chloride colorimetric method with aluminum chloride hexahydrate (AlCl 3 ), and the results were expressed in milligram quercetin equivalents (QE)/g lyophilized powder (Chang et al. 2002). The total phenolic content was calculated using the Folin-Ciocalteu method and sodium carbonate (Na 2 CO 3 ). The data were converted to milligrams gallic acid equivalents (GAE) per 100 mL of algal extract. According to Ayoola et al. (2008), total alkaloid content was determined by reacting alkaloid with bromocresol green (BCG), resulting in a yellow-colored product. The method uses bromocresol green solution (110-4), NaOH/2N, distilled water, phosphate buffer solution (pH 4.7), and atropine standard solution to provide sensitivity and stability.

HPLC bio-screening of M. reisseri methanolic extract
The algal methanolic extracts were separated using an HPLC device Agilent Technologies1200 with an HP 1200 UV variable wavelength detector on a C18 Zorbax Hypersil column (150 mm 4.6 mm, i.d. 5 m). The extraction and HPLC analysis conditions were set according to (Rodríguez-Bernaldo et al. 2010); however, partial modifications were required to optimize the results.  (Mosmann 1983). Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated foetal bovine serum, 1% L-glutamine, HEPES buffer, and 50 g/ ml gentamycin was used to grow the cells. All cells were cultured twice a week and kept at 37 oC in a humidified atmosphere with 5% CO 2 .

Cytotoxicity evaluation using viability assay
Tumor cell lines were suspended in the medium at a concentration of 5 × 104 cell/well in Corning ® 96-well tissue culture plates, and then incubated for 24 h. After that, M. reisseri extract was added to 96-well plates in three replicates to achieve ten concentrations. As a control, six vehicle controls with media or 0.5% DMSO were run for each 96-well plate. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide test was used to determine the number of viable cells after 24 h of incubation (MTT test). The viability percentage was calculated as [(ODt/ODc)] × 100%, where ODt represents the mean optical density of wells treated with M. reisseri extract and ODc represents the mean optical density of untreated cells. The survival curve of each tumor cell line after M. reisseri extract treatment is obtained by plotting the relationship between surviving cells and drug concentration. The 50% inhibitory concentration (IC50), or the concentration required to cause toxic effects in 50% of intact cells, was calculated using Graphpad Prism software (San Diego, CA, USA) and graphic plots of the quantityresponse curve for each concentration (Mosmann 1983).

Evaluation of antimicrobial activity
Pseudomonas aeruginosa RCMB 010049, Escherichia coli RCMB 010052, Streptococcus pneumoniae RCMB 010010, Bacillus subtilis RCMB 010067, Aspergillus fumigatus RCMB 02568, and Candida albicans RCMB 05036 were the strains tested in this study. The standard disc diffusion assay (Balouiri et al. 2016) was used to test antimicrobial activity in vitro against the pathogenic bacteria and fungi mentioned. The experiment was repeated three times, and the average zone of inhibition was calculated. According to Doughari (2006), the minimum inhibitory concentration (MIC) of M. reisseri extract was determined in triplicate for each of the tested organisms.
The MIC values were calculated as the lowest dose of antibacterial substances that prevented the visible growth of microbes (EL-Saadony et al. 2021).

Evaluation of antioxidant activity
The antioxidant compound that prevents the oxidation of DPPH was used to test the free radical scavenging activities (Blois 1958). The following equation was used to calculate the ability of diet extract to scavenge DPPH radicals: Hydroxyl radical scavenging activity was assayed as described by Elizabeth and Rao (1990). The inhibition percentage was estimated as follows: The antioxidant capacity of M. reisseri extract is measured using ferric reducing antioxidant power (FRAP) according to Banerjee and Maulik (2002) and Sutharsingh et al. (2011). The reducing power is due to the conversion of ferric cyanide into ferrous form via the donation of an efrom the antioxidant molecule found in M. reisseri extract. After blank subtraction, the reducing power was expressed as an increase in absorbance at 700 nm.

Growth measurements
Figures 1 and 2 depict M. reisseri growth measurements by optical density, growth rate, chlorophyll a, chlorophyll b, and carotenoid content. The maxima of these DPPH radical scavenging activity = A control − A sample A control × 100 The inhibition percentage (%) = (test − control)∕control × 100 measurements were observed on the 14th, 6th, 12th, 14th, and 12th days of cultivation, respectively. As a result, the harvesting date was set for the 14th day of cultivation to obtain the highest yield of algal cells. Table 1 demonstrates the cytotoxic activity of M. reisseri extract against MCF-7 and HCT-116 cell lines, the results showed that M. reisseri extract exhibits cytotoxicity in all concentrations (ranging from 1.56 to 50 g/ml), the cytotoxic activity increased by increasing the extract concentration in a concentration-dependent manner, showing inhibition percentages ranging from (1.78 ± 1.34%-83.68 ± 1.57%) and (10.55 ± 2.05%-86.33 ± 0.35%). Thus, M. reisseri methanolic extract was more effective against colon cancer than breast cancer. According to Gabriel et al. (2022), geraniol has been widely investigated as a potential anticancer agent, and the same is true for M. reisseri extract ( Table 2, Fig. 3). Gallic acid and quercetin, which are found in the extract, have also been linked to the prevention and progression of various types of cancer (Zhang et al. 2019 andHong et al. 2021).

Antimicrobial activity
With the evaluation of their MIC, a strong and remarkable activity of M. reisseri methanolic extract was detected against all tested Gram-positive, Gram-negative bacterial pathogens and fungi (Table 3). Antibacterial activity was classified as no activity (−: inhibition diameter 10 mm), low (+ : inhibition diameter between 10 and 15 mm), moderate (+ + : inhibition diameter between 15 and 20 mm), and high activity (+ + + : inhibition diameter 20 mm). However, as shown in Table 3 and Fig. 4, the majority of inhibition zones produced by M. reisseri extract against all tested pathogens were highly promising, ranging from moderate to high activity levels. The highest antibacterial activity levels were reported against B. subtilis RCMB 010,067 and E. coli RCMB 010,052, with mean inhibition zones and standard deviations of (20.60.58, 22.41.2) and MICs of 1.95 g/ml and 0.98 g/ml, respectively. According to Table 3 and Fig. 4, the extract has moderate antibacterial activity against P. aeruginosa RCMP 010,049 and S. pneumonia RCMP 010,010, with a mean inhibition zone and SD (18.3 0.72-18.3 0.63) and MIC 7.81 for the two bacterial strains. These findings were supported by the findings of Mc Gee et al. (2020), who examined extracts from 80 newly isolated marine and freshwater microalgae strains such as Micractinium and tested them for antimicrobial activity against 6 pathogens (Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, and Candida albicans). Our findings also show that M. reisseri extract has moderate antifungal activity against Aspergillus fumigatus but no activity against Candida albicans.

Antioxidant activity
The antioxidant activity of M. reisseri methanolic extract in DPPH scavenging and FRAP is shown in Fig. 5. The activity was highest at the lowest concentrations (0.001 mg/ml) and decreased as the algal extract concentration increased. The algal extract demonstrated the greatest antioxidant activity in the hydroxyl radical scavenging test at the highest tested concentration (5 mg/ml). The extract's reducing power is primarily due to the presence of flavonoids, alkaloids, and phenolic compounds (Table 2), which is supported by previous findings of Prasad and Muralidhara (2017)

Conclusion
M. reisseri could be a promising treatment for cancer, microbes, and oxidative damage diseases after the necessary clinical studies. According to the findings, M. reisseri methanolic extract has promising cytotoxicity against MCF-7 and HCT-116 cell lines, as well as antioxidant, antibacterial, and antifungal properties. Because each tool used for algal extraction has unique properties, optimizing growth factors such as pH, culture media component, and solvent type may increase the amount and effectiveness of the extracted compounds.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Conflict of interest
No funding was received from any organization for the completion of this research article.
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