Antioxidant and Antimicrobial activity of Crude Sterol from Chlorella

Microalgae are minute algae found in both marine and freshwater ecosystems. Microalgae produce natural products and are being explored for biotechnological applications (1) Chlorella is one of the most well-known microalgae genera. Chlorella is a freshwater, unicellular organism with bioactive properties including colours, vitamins, proteins, sterols, and long-chain polyunsaturated fatty acids (2)

Plant sterols, also known as phytosterols, are naturally occurring chemicals present in plant-based foods such as Vegetable oils, Nuts, Seeds, Cereals, and Legumes in tiny quantities(3). Sterols are isoprenoid lipids present in eukaryotic membranes. They have important functions in regulating membrane Fluidity and Permeability. Plant oil is the primary source of Phytosterols nowadays. Microalgae might be a more sustainable source of Sterols compared to Plant and Shark Oils (4). In mammals, Cholesterol is the primary Sterol required for numerous biological functions. Ergosterol is the most common Sterol found in fungus. Plant sterols, also known as Phytosterols, share physiological and structural similarities with Cholesterol (4). Phytosterols are known for lowering Cholesterol, their Antioxidant properties are less studied. It states that, Phytosterol chemically works as Antioxidant and it is a moderate radical Scavenger(5). Chlorella spp. extracts exhibit Antibacterial activity against Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli. C. vulgaris' Antibacterial activity is linked to its Cyclic Peptides, Terpenoids, Alkaloids, Steroids, and Tannins. Algae are a valuable source of Antimicrobial compounds used in laboratory experiments to suppress disease-causing microorganisms(2). Antibiotic resistance in pathogens is rising due to overuse, leading to multidrug-resistant microorganisms. There is a significant need to identify novel antipathogenic compounds with minimal negative effects (2) The growing number of documented cases of Antibiotic resistance is a global issue. Researchers have explored the antibacterial properties of few microalgae extracts (6). The purpose of the current study to address this issue, by screening new Antibiotics or Chemicals with Antibacterial characteristics from Chlorella.

MATERIAL AND METHODS

Extraction of Crude Sterols from Chlorella pyrenoidosa and Chlorella vulgaris

Weight the 1 gm Chlorella spp fine powder harvested from lab cultivation. Saponification of Sterols was achieved by adding 30 ml of 0.2 M Methanolic Potassium hydroxide (KOH) solution and reflux it for next 30 minutes on same temperature. Then add 50 ml Hexane and shake vigorously for 1 minute.  The hexane layer will be at upper level in separating funnel. Separate the hexane in 250 ml Crucible and allow it to evaporate.

DPPH Anti-Oxidant Assay

100μl of the test concentration (Crude Sterol) was incubated with 100μl DPPH solution. The mixture was incubated at room temperature under dark conditions for 30 minutes. After the incubation period, the absorbance of the mixture was read Spectrophotometrically at 517nm. Appropriate color blanks were prepared which contained 100μl of test concentration and 100μl of Methanol. Negative control or Control consisted of 100μl DPPH reagent and 100μl of Methanol. The Absorbance of Control is used to calculate DPPH free radical Scavenging antioxidant potential of samples. IC50 was calculated from Dose response curve.

Note: Lower the IC50 Value better is the antioxidant potential of sample under study

Antimicrobial Activity:

Preparation of E. coli and S. aureus culture: -

E. coli and S. aureus cultures were prepared by inoculating a loopful of each organism from their respective Sterile Nutrient Agar Slants into Sterile Nutrient Broth. The inoculated broths were incubated at 37°C overnight. The Minimum Inhibitory Concentrations (MICs) of crude Sterols extracted from Chlorella pyrenoidosa and Chlorella vulgaris were determined against both bacterial species.

Agar Cup Method:

Antibiotics are placed in wells on agar plates seeded with bacteria like E. coli or S. aureus. After incubation at 37°C for 24 hours, the inhibition zones diameters are measured. Sterile Nutrient Agar is used, with cultures inoculated, and 0.1 ml of sample solutions or controls placed in wells. Pre-diffusion occurs for 30 minutes before incubation and zone measurements.

Paper Disc Method:

The disc diffusion method tests antibiotic effectiveness by placing antibiotic-soaked discs on agar plates inoculated with E. coli or S. aureus. As the antibiotic diffuses, a concentration gradient forms, creating a zone of inhibition. Disc soaked in Cefotaxime and test samples, along with controls (Antibiotic and Ethanol), are placed on Nutrient agar. After 24 hours of incubation at 37°C, the inhibition zones' diameters are measured and interpreted to assess Antimicrobial activity.

Minimal Inhibitory Concentration:

MIC is the Minimum Concentration of the antibiotic that inhibits the growth of the organism.

Addition table for MIC

Table I. Experiment setup for Minimum Inhibitory Concentration

Three sample were used for each organism, three sets for E. Coli culture and three sets for S. aureus culture. Incubate the tubes at 37°C for 24 hours. MIC is interpreted as the least dilution which inhibits growth judged by lack of turbidity in the tube.

Minimal Lethal Concentration:

The Minimum Lethal Concentration (MLC) is the lowest drug concentration that kills a pathogen. The positive control tube, with no sample, is sub-cultured and incubated at 37°C for 24 hours. Tubes from the MIC test showing no growth are sub-cultured, and results are observed after 24 hours. Outcomes may include similar colonies (Bacteriostasis), fewer colonies (partial Bactericidal activity), or no growth (complete kill). The highest dilution showing ≥99% inhibition is the MLC.

Results and Discussion:

Extraction of Crude Sterols:

Figure I & II. Extract of Crude Sterols from C. Pyrenoidosa (Fig. I) and C. vulgaris (Fig. II)

Table II. Yield of Crude sterol Extract from both C. pyrenoidosa and C. vulgaris

Crude sterols were extracted from C. pyrenoidosa and C. vulgaris using the saponification method. C. pyrenoidosa showed a higher yield (9%) compared to C. vulgaris (5%), with both considered superior yields for commercial applications.

Antioxidant Activity through DPPH Assay:

Figure III: Scavenging Activity of crude sterols from C. pyrenoidosa (Fig III)

Figure IV: Scavenging Activity of crude sterols from C. vulgaris

The crude sterol extracted from Chlorella’s both strains have low Scavenging activity as per DPPH biochemical assay. Each extract shows scavenging activity below 50% which restricts to calculate the IC₅₀ value.

Anti-Microbial Activities:

Agar Cup Method:

Figure V. and VI. E. Coli Agar cup method Fig VII and VIII S. aureus Agar Cup Method

The antimicrobial activity of crude Sterols from C. pyrenoidosa and C. vulgaris was tested against E. coli and S. aureus using the agar cup method. Against E. coli, the inhibition zones were 26 mm for C. pyrenoidosa and 30 mm for C. vulgaris, while ethanol (positive control) showed 14 mm and 11 mm, respectively. For S. aureus, the inhibition zones were 19 mm for C. pyrenoidosa and 12 mm for C. vulgaris, with ethanol showing 13 mm and 12 mm. Both samples showed moderate Antibacterial activity, C. vulgaris more effective against E. coli and C. pyrenoidosa more effective against S. aureus.

Disc Diffusion Method:

Table IV. Results of Disc Diffusion Methods

Figure IX, X, XI. Results of E. coli (IX), S. aureus (X) and Control (XI) Disc Diffusion Method

The Antimicrobial activity of crude Sterols from C. pyrenoidosa and C. vulgaris was tested against E. coli and S. aureus using the disc diffusion method. Against E. coli, the inhibition zones were 26 mm and 27 mm for C. pyrenoidosa and C. vulgaris, respectively, while the positive control (Cefotaxime) showed 36 mm for both strains. Ethanol showed no inhibition (0 mm). Against S. aureus, the inhibition zones were 7 mm and 6 mm, with the positive control showing 30 mm. Both strains demonstrated moderate Antibacterial activity. Higher inhibition in the agar cup method compared to the disc diffusion method suggests more efficient diffusion or higher concentrations of antimicrobial agents in the wells. The findings indicate that C. vulgaris crude bioactive compounds effectively inhibited both Gram-negative and Gram-positive bacteria.

Minimum Inhibitory Concentration (MIC):

The MIC results for C. pyrenoidosa and C. vulgaris against E. coli and S. aureus showed turbidity at 0.1 to 0.4 concentrations, while the 0.5 concentration had clear broth without turbidity, indicating effective antimicrobial activity at 0.5 concentration.

Figure XII-XV: E.coli (Fig. XII & XIII) and S. aureus (Fig. XIV & XV) results on Minimum Inhibition Concentrations (MIC)

Minimum Lethal Concentration (MLC):

As per MIC results the 0.5 concentrations are non-turbid and clear. Therefore, we conducted MLC on 0.5 concentrations of C. pyrenoidosa and C. vulgaris against E.coli and S. aureus. The microbial growth was observed on both extracts against E.coli and S.aureus.

Figure XVI-XIX: Results of Minimum Lethal Concentration on E.coli (Fig. XVI & XVII) and S. aureus (Fig. XVIII & XIX)