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Full Research Article

Phytochemical Screening, Antimicrobial, Anticancer Potentials and Molecular Docking of Chlorella Extract Targeting Caspase-9

N
Nahed Ahmed Hussien1,*
https://orcid.org/0000-0001-7019-9682
A
Aisha Ehab Tantawy2
1Department of Biology, College of Science, Taif University, Taif 21944, Saudi Arabia.
2Department of Computer Engineering, College of Computers and Information Technology, Taif University, Taif, Saudi Arabia.

ABSTRACT

Background: Chlorella, a green alga, is rich in nutrients and bioactive compounds that support health and has potential as a source of natural therapeutic agents.

Methods: Chlorella’s phytochemicals were analyzed using GC-MS and its antimicrobial and anticancer properties were evaluated. Molecular docking was performed with the major active compounds of Chlorella targeting caspase-9.

Result: Chlorella extract contains trimethylsilyl palmitate (20.22%), oleic acid (9.87%) and arachidic acid methyl ester (6.12%). It exhibits antimicrobial activity against Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Bacillus cereus and Candida albicans, with minimum inhibitory concentrations (MIC) and bactericidal concentrations (MBC) above 1000 µg/mL, indicating relatively low antimicrobial potency. Chlorella extract exhibited inhibitory activity against Huh-7 hepatocellular carcinoma (HCC) cells, with an IC50 above 100 μg/mL. After docking with caspase-9, methyl arachidate is the most effective compound, with a binding affinity of -4.5 kcal/mol. It meets Lipinski’s rule, shows drug-like properties via SwissADME and has low predicted toxicity (LD50≈5000 mg/kg). It is stabilized in the binding pocket with three hydrophobic and two hydrogen bonds. This initial finding suggests that Chlorella could be a promising candidate for HCC treatment.

INTRODUCTION

Microalgae primarily inhabit aquatic environments and have high growth rates due to efficient sunlight and CO2  use. They contain bioactive compounds like proteins, lipids and polyphenols, used in food, pharma and cosmetics (Barkia et al., 2019) and support health with antibacterial, anticancer, antioxidant, antifungal and antiviral activities (Haq et al., 2019). Algae extracts contain antioxidants that may reduce oxidative stress and lower the risk of chronic diseases (Sruthy and Baiju, 2025). Reactive oxygen species (ROS), generated internally or externally, can damage cells and DNA, leading to heart, brain diseases and cancers. Antioxidants can help mitigate these risks (Haq et al., 2019).
       
Chlorella species can be widely cultivated and supplements are now available globally. Chlorella cells contain nutrients and bioactives that promote health and may prevent diseases, making them potential sources for therapeutic compounds. These compounds’ levels vary by culture conditions and species (Rajeswari et al., 2026). Since their cell walls are cellulose, they are poorly digestible intact, requiring mechanical disruption in supplements. Commercial Chlorella products also include essential vitamins and minerals (Bito et al., 2020).
       
Hepatocellular carcinoma (HCC) causes high cancer mortality worldwide, with low survival rates despite advances in technology, screening and treatment (Mustafa et al., 2023). Challenges include late diagnosis, complex gene networks and drug side effects, leading to treatment failures. Effective care for end-stage liver disease and new anti-HCC drugs are vital. Molecular docking studies how phytochemicals interact with proteins at the atomic level, aiding in drug development (Kattan et al., 2020). Key protein targets in cancer therapy include transcription factors, apoptotic proteins, growth factor receptors, cell division kinases, MAP kinases and serine/threonine kinases. Caspases are crucial in apoptosis, initiating via two main pathways: the extrinsic and intrinsic pathways. The extrinsic pathway triggers when ligands bind to receptors, activating adaptor molecules that lead to caspase 8 activation. The intrinsic pathway involves mitochondrial signals, like membrane depolarization, causing caspase 9 to bind apoptosome complexes. Both pathways converge to activate caspases 3 and 7 (Anand et al., 2020).
       
This study identified phytochemical compounds in a commercial Chlorella supplement using GC-MS and examined its antimicrobial properties. While previous research has explored Chlorella’s health benefits, this work focuses on its anticancer effects against hepatocellular carcinoma in Huh-7 cells. In silico molecular docking was used to analyze how Chlorella’s active compounds bind to caspase-9, a key protein in apoptosis. This approach supports the compounds’ anticancer potential and predicts their interactions, enhancing understanding of their mechanisms.

MATERIALS AND METHODS

Crude extract preparation
 
The experiment at Taif University’s Biology Department in 2025 involved suspending 100 mg of Chlorella powder (Organic Traditions®, Canada) in 10 ml of 95% methanol. The mixture was vortexed on ice for 20 minutes, shaken at 25°C for 1 hour, then centrifuged at 4°C for 10 minutes at 1209 g. The supernatant was collected, re-extracted twice and combined supernatants filtered through No.1 Whatman paper. The extract was dried with a rotary evaporator (BÜCHI Rotavapor R-200, Switzerland) and stored at -20°C for future analysis (Ferdous et al., 2023).
 
Gas chromatography-mass spectroscopy analysis
 
The 95% methanolic Chlorella extract was analyzed using an RTX-5 MS capillary column (Restek) with a GC-MS-QP2010 Plus system (Shimadzu, Japan), covering 1.5-1000 m/z. Helium at 1 mL/min was the carrier gas. Detection employed an electron ionization source at 70 eV, with a split ratio of 1:10 to control helium flow at 14.1 mL/min and a 1.00 µL injection volume. The injector was at 250°C and the ion source at 200°C. Mass spectra from 45 to 450 Da were recorded at 70 eV during a 0.5-second scan. The run lasted 50 minutes with a solvent delay of 0 to 4.5 minutes. The oven started at 80°C for 4 minutes, then increased by 10°C per minute to 280°C. GC separates compounds by retention time, while MS identifies them. The software generates a chromatogram of peak abundance vs. retention time (Mohamed and Hussien, 2024). Comparing spectra with the Wiley Registry 8e library (similarity index ≥70%) helps identify compounds.
 
Antimicrobial evaluation
 
The antimicrobial activity of Chlorella extract was tested against five microbes: four bacteria and one fungus from Nawah Scientific Inc. (Mokatam, Cairo, Egypt): Escherichia coli ATCC 8739, Enterococcus faecalis ATCC 19433, Staphylococcus aureus ATCC 29213, Bacillus cereus ATCC 9634 and Candida albicans ATCC 10231. A two-fold serial dilution of Chlorella from 1000 to 1.953 ìg/mL in Mueller-Hinton broth was used for treatment, as described by Hussien et al., (2025). Ciprofloxacin (1.953-1000 ìg/mL) served as a positive control. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Chlorella were evaluated against various strains, with MIC representing the lowest concentration that prevents growth and MBC the concentration that kills 99.9% of bacteria. Three replicates were performed for each strain and treatment and a microplate ELISA reader (TECAN, Switzerland) was used to measure turbidity at an optical density (OD) of 600 nm.
 
Sulforhodamine B (SRB) cytotoxicity
 
The Huh-7 hepatocellular carcinoma (HCC) cell line was obtained from Nawah Scientific Inc. (Mokatam, Cairo, Egypt). Cells were cultured in DMEM containing 100 mg/mL streptomycin, 100 U/mL penicillin and 10% heat-inactivated fetal bovine serum and maintained in a humidified atmosphere with 5% CO2 at 37°C. Cells were treated with media containing various concentrations of Chlorella extract (100 μL/well; 0.001-100 μg/mL), as described by Allam et al., (2018). Media without Chlorella served as a negative control, while cisplatin (0.01-100 μg/mL) served as a positive control. Absorbance was measured at 540 nm and the extract was tested in triplicate at each concentration to ensure accuracy.
 
Statistical analysis
 
Data were presented as mean ± SE of replicates and analyzed using non-linear regression in OriginLab to generate a sigmoidal dose-response curve of Log10 Chlorella extract concentration versus cell viability. IC50 value was interpolated from the curve.
 
Molecular docking study
 
Based on GC-MS results, we selected the most abundant compounds for molecular docking with caspase-9 as the receptor. The 2D and 3D structures of these compounds (ligands) were obtained from the PubChem database. The 3D crystal structure of dimeric caspase-9 bound to ligand (D-Malate) (PDB ID: 2ar9, resolution 2.80 Å) was downloaded from RCSB PDB (http://www.rcsb.org/pdb/home/home.do). We used PyMOL to remove D-Malate and water molecules from the receptor. SwissADME was employed to predict their physicochemical properties, drug-likeness, pharmacokinetics and medicinal chemistry features (http://www.swissadme.ch/) (Daina et al., 2017). The top-docked compounds were selected according to Lipinski’s rule of five, a guideline for ideal drug candidates. Lipinski suggests that a compound demonstrates drug-like properties if it meets certain criteria: a molecular weight of 500 or less, no more than 5 hydrogen bond donors, no more than 10 hydrogen bond acceptors, lipophilicity below 5 and molar refractivity between 40 and 130 (Lipinski et al., 2001). Predicted LD50 values and toxicities for the seven compounds were determined using the ProTox-II web server (https://tox-new.charite.de/protox_II/) (Banerjee et al., 2024). Molecular docking of selected compounds into the receptor protein’s binding pockets was carried out with SMINA/Anaconda. Additionally, re-docking was performed to validate the accuracy of the docking protocol. Docking validation involved re-docking the co-crystallized ligand (D-Malate) into the active site using AutoDock’s fast scoring function. The most favorable conformations were chosen by comparing RMSD values between the protein and the co-crystallized ligand (Hevener et al., 2009). After docking, the resulting complexes were examined with the Protein Ligand Interaction Profiler (PLIP, https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index) to identify non-covalent interactions, such as hydrogen bonds, hydrophobic contacts, water bridges and ionic interactions and to generate their 2D visualizations (Adasme et al., 2021).

RESULTS AND DISCUSSION

GC-MS analysis of the Chlorella extract identified phytochemicals, including fatty acids, hydrocarbons, phenolics, terpenes and their derivatives (Fig 1). The major phytochemical components of Chorella extract are high-oleic algal oil, fatty acids and their derivatives (including arachidic, palmitic, oleic and linolenic) (Table 1).

Fig 1: Chromatogram of Chlorella methanolic extract.



Table 1: Major compounds of Chlorella and their chemical structures.


       
This confirms that Chlorella’s commercial dietary supplement is as rich in fatty acids as cultivated (Pantami et al., 2020). The main phytochemicals in Chorella extract include trimethylsilyl palmitate (20.22%), oleic acid (9.87%), oleic acid trimethylsilyl ester (6.25%), arachidic acid methyl ester (6.12%), linoleoyl chloride (5.81%), monopalmitin (5.80%), 3Beta-hydroxy-5-cholestene 3-oleate (3.64%) and Algal oil (3.36%) (Table 1). Pantami et al., (2020) reported that C. vulgaris extract contains over 60% fatty acids, rich in omega-6, -7, -9 and -13, with omega-6 (notably 9,12-Octadecadienoic acid, Linoleic acid at 35.1%) being predominant, as determined through GC-MS analysis. Consequently, Chlorella species may serve as a valuable nutritional source when added to diets, highlighting its potential as an outstanding dietary supplement.
       
The study found that trimethylsilyl palmitate, a palmitic acid ester, is the main component, comprising 20.22% of the extract. This ester exhibits various effects, including antioxidant, hypocholesterolemic, nematicidal, hemolytic, 5-alpha-reductase inhibition, antipsychotic, anti-androgenic and anticancer activities (El-Fayoumy et al., 2021). Next are Oleic acid (9.87%) and its derivative (6.25%), which play beneficial roles in diseases such as coronary heart disease, rheumatoid arthritis and cancer (Piccinin et al., 2019). Arachidic acid methyl ester (6.12%) exhibits antifungal and antioxidant properties (Abdelillah et al., 2013) and helps prevent cholesterol gallstones by acting as a cholesterol solubilizer (Gilat et al., 2001). Phenolics in Chlorella sp. combat reactive oxygen and singlet oxygen species and decrease ROS production (Pradhan et al., 2021). 
       
Chlorella extract exhibits antimicrobial effects against Gram-negative bacteria, E. coli and Gram-positive bacteria, E. faecalis, S. aureus and B. cereus, as well as the fungus Candida albicans. Its MIC and MBC are over 1000 μg/mL, but it’s less potent than Ciprofloxacin (Table 2). As previously reported, MIC values for antibiotics range from 0.01 to 10 µg/mL, whereas plant extracts are considered antimicrobials if their MICs fall between 100 and 1000 µg/mL (Silva et al., 2013). This large gap between the antibiotic and the natural extract used could be attributed to several factors. The difference in their nature, solubility and size. Using a crude extract is less effective than using a single, isolated compound (Ben-Khalifa et al., 2021). 

Table 2: The minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) of Chlorella sp.


       
The current findings align with previous research reporting Chlorella sp.’s antibacterial effects against Gram-positive bacteria, S. epidermidis, S. aureus, Methicillin-resistant S. aureus (MRSA), E. faecalis and B. thuringiensis, as well as Gram-negative bacteria, E. coli, S. sonnei and S. marcescens (Shaima et al., 2022). They have reported that the inhibition zones ranged from 11 to 13.8 mm, with MIC values of 0.39-6.25 mg/mL. Additionally, antibiotics demonstrated stronger bacterial inhibition across various strains. The antimicrobial activity of Chlorella sp. is primarily attributed to its phytochemicals, especially polyphenols, which promote hydrogen peroxide production under aerobic conditions and are linked to hydroxyl groups (Dell’Anno et al., 2019).
       
Various chemical groups, such as phlorotannins, fatty acids, peptides, terpenes and polysaccharides, have been found to inhibit bacterial growth in algae. Nonetheless, the exact mechanisms behind this inhibition remain unclear (Shannon and Abu-Ghannam, 2016). Algal free fatty acids inhibit the bacterial electron transport chain and oxidative phosphorylation, disrupting ATP transfer. They inhibit enzymes like bacterial enoyl-acyl carrier protein reductase, crucial for fatty acid synthesis, causing cell lysis and degradation from peroxidation and auto-oxidation (Pradhan et al., 2014). Algal and sulfated polysaccharides interact with glycoprotein receptors, bind to bacterial walls, membranes and DNA and increase permeability, causing protein leakage and DNA binding (Pereira and Valado, 2025). Amphipathic peptides bind to polar and nonpolar membrane regions, disrupting bacterial processes and replication (Nguyen et al., 2011). All proposed antimicrobial mechanisms of algae are outlined in Fig 2.

Fig 2: Schematic of the proposed antibacterial mechanism of Chlorella extract’s active components. Created with figurelabs®.


       
HCC is among the most prevalent primary liver cancers and ranks as the fourth leading cause of cancer-related deaths worldwide (Mustafa et al., 2023). Results shown in Fig 3 indicate that Chlorella demonstrated a dose-dependent anticancer effect from 0.01 to 100 μg/mL. After 72 hours, cell viability was significantly reduced, with an IC50 exceeding 100 μg/mL, suggesting relatively lower cytotoxicity than cisplatin. These findings align with Shanab et al., (2012), who reported the anticancer potential of aqueous Chlorella extract against Ehrlich Ascites Carcinoma cells and HepG2 cells at 100 μg/mL. Chlorella’s anticancer effects are attributed to polar compounds such as phycobilins, phenolics and polysaccharides that induce apoptosis (Aboul-Enein et al., 2011). Oleic acid in Chlorella promotes apoptosis and differentiation in colorectal HT-29 cells (Llor et al., 2003) and decreases Her-2/neu expression in breast cancer cells (Menendez et al., 2005). Studies show Chlorella sp. has anticancer effects by activating apoptosis through increased P53, Bax and caspase-3, decreased Bcl-2 and suppressed AKT/mTOR pathways, causing DNA damage and apoptosis (Sawasdee et al., 2023).

Fig 3: Cell viability (%) of Huh-7 cell line treated with Chlorella.


       
Seven phytocomponents-trimethylsilyl palmitate; oleic acid; oleic acid trimethylsilyl ester; arachidic acid methyl ester (methyl arachidate); linoleic acid chloride (linoleoyl chloride); 2,3-Dihydroxypropyl hexadecanoate (monopalmitin) and 3Beta-hydroxy-5-cholestene 3-oleate (cholesteryl oleate)-were examined for Caspase-9 docking studies. Tables 3-6 present data on the pharmacokinetics, physicochemical properties, medicinal chemistry and drug-likeness of these compounds. Linoleoyl chloride was omitted because its calculated parameters were unavailable. Cholesteryl oleate does not meet Lipinski’s rules, with two violations: MW>500 and MLOGP>4.15. Only methyl arachidate and monopalmitin were selected for Caspase-9 docking because they meet these criteria, which indicate potential drug-like properties and lower toxicity, with a predicted LD50 of 5000 mg/kg. Both are expected to be non-mutagenic, non-tumorigenic and non-irritant. Methyl arachidate is highly lipophilic, resulting in low gastrointestinal absorption and limited blood-brain barrier permeability, whereas monopalmitin is less lipophilic, with good gastrointestinal absorption and blood-brain barrier permeability. These two compounds were docked into the Caspase-9 chain A docking pocket, replacing the co-crystallized ligand (Malate), with a binding affinity of -4.5 kcal/mol.

Table 3: Physicochemical properties of selected compounds found in Chlorella sp.



Table 4: Lipophilicity of the selected phytocomponents.



Table 5: Water solubility of selected compounds.



Table 6: Drug-likeness, pharmacokinetics, and medicinal chemistry of Chlorella selected compound.


       
Based on the data in Fig 4 and Table 7, methyl arachidate appears more stable within the docking site, forming three hydrophobic contacts and two hydrogen bonds with the receptor protein. In contrast, monopalmitin forms two hydrophobic interactions and three hydrogen bonds. Methyl arachidate interacts with caspase-9 at residues TRP (chain A):354, ARGA:355, PROA:357 and makes two hydrogen bonds at ARGA: 180 and ARGA: 355. Meanwhile, monopalmitin interacts with caspase-9 at VALA: 352, TRPA:354 and forms three hydrogen bonds at ARGA: 355 amino acids of the active pocket.

Fig 4: The non-covalent interactions (hydrogen and hydrophobic bonds) between caspase-9 and ligands.



Table 7: The non-covalent interactions (hydrogen and hydrophobic bonds) between Caspase 9 and ligands.


       
Molecular docking of p53, caspase-9 and cyclin D1, which play roles in apoptosis and cell proliferation, is a target for inducing apoptosis in the MCF-7 breast cancer cells (Nurhayati et al., 2022) and HepG2 liver carcinoma cells (Anand et al., 2020). In silico and docking results indicate that methyl arachidate may inhibit caspase-9 and could serve as a promising liver anticancer agent. Our findings align with Mustafa et al., (2023), who docked the phytochemicals limonin and obamegine with caspase-9. Limonin interacted with amino acids ArgA:355, TrpA:354, GlyA:238, ArgA:178 and LeuA:177 at the active site. Obamegine bound to ProA:338, ProA:336, LeuA:335, AsnA:265, PheA:246 and LeuA:244 within the binding pocket. Additionally, Siddiqui et al., (2021) docked eight Moringa oleifera phytochemicals with caspase-3, where benzyl glucosinolate (binding affinity -8.4 kcal/mol) interacted with Phe265, Phe250, Arg207, Trp206, Tyr204 and Thr62. Suganya and Anuradha (2019) also docked astaxanthin, which interacted with amino acids Ile403, Cys402, Met400, Tyr397, Ile396, Phe351 and Phe348 of caspase-9.
       
Our findings agree with Wanandi et al., (2020), who studied the anti-cancer potential of a natural compound by examining its interactions with survivin, caspase-9 and caspase-3 via computational methods. In 2023, Mustafa et al. (2023) docked plant phytochemicals-liquoric acid (-9.8 kcal/mol), madecassic acid (-9.3 kcal/mol), limonin (-10.5 kcal/mol) and obamegine (-9.3 kcal/mol)-against EGFR and caspase-9. However, the molecular mechanisms behind HCC development, progression and metastasis are not well understood. In the current in silico analysis, we targeted caspase-9, a protease that triggers apoptosis via the intrinsic or mitochondrial pathway and is activated at sites involving multiple proteins (Kim et al., 2015). Extracellular signal-regulated kinases-1 and 2 (ERK1/2) are part of the mitogen-activated protein (MAP) kinase superfamily and regulate cell proliferation and survival. They inhibit apoptosis by deactivating proapoptotic proteins and supporting cell survival by blocking caspase-9 activity (Boston et al., 2011). Natural chemicals targeting caspase-9 could be promising for HCC treatment. Microalgae are rich in biocompatible compounds, making them valuable nutraceuticals (Sawasdee et al., 2023; Yu et al., 2024). Their phytochemicals may serve as future drug candidates against HCC.

CONCLUSION

This study assesses a commercial Chlorella supplement and its potential effects. In silico and docking studies suggest that methyl arachidate, a key component, may inhibit caspase-9, but further validation is needed. More research and trials are essential to confirm Chlorella’s biological properties. Overall, the findings highlight natural microalgae compounds’ potential for developing therapies for hepatocellular carcinoma (HCC) and encourage future medicinal research.

ACKNOWLEDGEMENT

The authors would like to acknowledge the Deanship of Graduate Studies and Scientific Research, Taif University for funding this work.
 
Research funding
 
The authors would like to acknowledge the Deanship of Graduate Studies and Scientific Research, Taif University for funding this work.

CONFLICT OF INTEREST

Authors state no conflict of interest.

REFERENCES


  1. Abdelillah, A., Houcine, B., Halima, D., Meriem, C.S., Imane, Z., Eddine, S.D., Abdallah, M. and Daoudi, C.S. (2013). Evaluation of antifungal activity of free fatty acids methyl esters fraction isolated from Algerian Linum usitatissimum L. seeds against toxigenic Aspergillus. Asian Pacific Journal of Tropical Biomedicine. 3(6): 443-448. https:/ /doi.org/10.1016/S2221-1691(13)60094-5.

  2. Aboul-Enein, A.M., Shalaby, E.A., Abul-Ela, F., Nasr-Allah, A.A., Mahmoud, A.M., El-Shemy, H.A. and Ahmed, M. (2011). Back to nature: Spotlight on cancer therapeutics. Vital Signs. 10: 8-9.

  3. Adasme, M.F., Linnemann, K.L., Bolz, S.N., Kaiser, F., Salentin, S., Haupt, V.J. and Schroeder, M. (2021). PLIP 2021: Expanding the scope of the protein-ligand interaction profiler to DNA and RNA. Nucleic Acids Research. 49(W1): W530- W534. https://doi.org/10.1093/nar/gkab294.

  4. Allam, R.M., Al-Abd, A.M., Khedr, A., Sharaf, O.A., Nofal, S.M., Khalifa, A.E., Mosli, H. A. and Abdel-Naim, A.B. (2018). Fingolimod interrupts the cross talk between estrogen metabolism and sphingolipid metabolism within prostate cancer cells. Toxicology Letters. 291: 77-85. https:// doi.org/10.1016/j.toxlet.2018.04.008.

  5. Anand, K., Abdul, N.S., Ghazi, T., Ramesh, M., Gupta, G., Tambuwala, M.M., Dureja, H., Singh, S.K., Chellappan, D.K., Dua, K., Pandi, B., Saravanan, M. and Chuturgoon, A.A. (2020). Induction of caspase-mediated apoptosis in HepG2 liver carcinoma cells using mutagen-antioxidant conjugated self-assembled novel carbazole nanoparticles and in silico modeling studies. ACS Omega. 6(1): 265-277. https://doi.org/10.1021/acsomega.0c04461.

  6. Banerjee, P., Kemmler, E., Dunkel, M. and Preissner, R. (2024). ProTox 3.0: A webserver for the prediction of toxicity of chemicals. Nucleic Acids Research. 52(W1): W513- W520. https://doi.org/10.1093/nar/gkae303.

  7. Barkia, I., Saari, N. and Manning, S.R. (2019). Microalgae for high- value products towards human health and nutrition. Marine Drugs. 17(5): 304. https://doi.org/10.3390/ md17050304.

  8. Ben-Khalifa, R., Gaspar, F.B., Pereira, C., Chekir-Ghedira, L. and Rodríguez-Rojo, S. (2021). Essential oil and hydrophilic antibiotic co-encapsulation in multiple lipid nanoparticles: Proof of concept and in vitro activity against Pseudomonas aeruginosa. Antibiotics. 10(11): 1300. https://doi.org/ 10.3390/antibiotics10111300.

  9. Bito, T., Okumura, E., Fujishima, M. and Watanabe, F. (2020). Potential of Chlorella as a dietary supplement to promote human health. Nutrients. 12(9): 2524. https://doi.org/ 10.3390/nu12092524.

  10. Boston, S.R., Deshmukh, R., Strome, S., Priyakumar, U.D., MacKerell, A.D. and Shapiro, P. (2011). Characterization of ERK docking domain inhibitors that induce apoptosis by targeting Rsk-1 and caspase-9. BMC Cancer. 11: 7. https://doi.org/10.1186/1471-2407-11-7.

  11. Daina A., Michielin, O. and Zoete, V. (2017). SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Scientific Reports. 7: 42717. https://doi.org/10.1038/ srep42717.

  12. Dell’Anno, M., Sotira, S., Rebucci, R., Reggi, S., Castiglioni, B. and Rossi, L. (2019). In vitro evaluation of antimicrobial and antioxidant activities of algal extracts. Italian Journal of Animal Science. 19(1): 103-113. https://doi.org/10.1080/ 1828051X.2019.1703563.

  13. El-Fayoumy, E.A., Shanab, S.M.M., Gaballa, H.S., Tantawy, M.A. and Shalaby, E.A., (2021). Evaluation of antioxidant and anticancer activity of crude extract and different fractions of Chlorella vulgaris axenic culture grown under various concentrations of copper ions. BMC Complementary Medicine and Therapies. 21(1): 51. https://doi.org/ 10.1186/s12906-020-03194-x.

  14. Ferdous, U.T., Nurdin, A., Ismail, S. and Balia Yusof, Z.N. (2023). Evaluation of the antioxidant and cytotoxic activities of crude extracts from marine Chlorella sp. Biocatalysis and Agricultural Biotechnology. 47: 102551. https:// doi.org/10.1016/j.bcab.2022.102551.

  15. Gilat, T., Leikin-Frenkel, A., Goldiner, I., Laufer, H., Halpern, Z. and Konikoff, F.M. (2001). Arachidyl amido cholanoic acid (Aramchol) is a cholesterol solubilizer and prevents the formation of cholesterol gallstones in inbred mice. Lipids. 36(10): 1135-1140. https://doi.org/10.1007/s11745-001- 0824-3.

  16. Haq, S.H., Al-Ruwaished, G., Al-Mutlaq, M.A., Naji, S. A., Al-Mogren, M., Al-Rashed, S., Ain, Q.T., Al-Amro, A.A. and Al- Mussallam, A. (2019). Antioxidant, anticancer activity and phytochemical analysis of green algae, Chaetomorpha collected from the Arabian gulf. Scientific Reports. 9(1): 18906. https://doi.org/10.1038/s41598-019-55309-1.

  17. Hevener, K.E., Zhao, W., Ball, D.M., Babaoglu, K., Qi, J., White, S.W. and Lee, R.E. (2009). Validation of molecular docking programs for virtual screening against dihydropteroate synthase. Journal of Chemical Information and Modeling. 49(2): 444-460. doi: 10.1021/ci800293n.

  18. Hussien, N.A., Khalil, M.A.E.F., Schagerl, M. and Ali, S.S. (2025). Green synthesis of zinc oxide nanoparticles as a promising nanomedicine approach for anticancer, antibacterial and anti-inflammatory therapies. International Journal of Nanomedicine. 20: 4299-4317. https://doi.org/ 10.2147/IJN.S507214.

  19. Kattan, S.W., Nafie, M.S., Elmgeed, G.A., Alelwani, W., Badar, M. and Tantawy, M.A. (2020). Molecular docking, anti- proliferative activity and induction of apoptosis in human liver cancer cells treated with androstane derivatives: Implication of PI3K/AKT/mTOR pathway. Journal of Steroid Biochemistry and Molecular Biology. 198: 105604. https://doi.org/10.1016/j.jsbmb.2020.105604.

  20. Kim, B., Srivastava, S.K. and Kim, S.H. (2015). Caspase-9 as a therapeutic target for treating cancer. Expert Opinion on Therapeutic Targets. 19(1): 113-127. https://doi.org/ 10.1517/14728222.2014.961425.

  21. Lipinski, C.A., Lombardo, F., Dominy, B.W. and Feeney, P.J. (2001). Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Reviews. 46(1-3): 3- 26. https://doi.org/10.1016/s0169-409x(00)00129-0.

  22. Llor, X., Pons, E., Roca, A., Alvarez, M., Mane, J., Fernandez- Banares, F. and Gassull, M. A. (2003). The effects of fish oil, olive oil, oleic acid and linoleic acid on colorectal neoplastic processes. Clinical Nutrition. 22(1): 71-79. https://doi.org/10.1054/clnu.2002.0627.

  23. Menendez, J.A., Vellon, L., Colomer, R. and Lupu, R. (2005). Oleic acid, the main monounsaturated fatty acid of olive oil, suppresses Her-2/neu (erbB-2) expression and synergistically enhances the growth inhibitory effects of trastuzumab (Herceptin) in breast cancer cells with Her-2/neu oncogene amplification. Annals of Oncology. 16(3): 359-371. https://doi.org/10.1093/annonc/mdi090.

  24. Mohamed, H.R.H. and Hussien, N.A. (2024). GC-MS analysis and molecular docking studies of Lavandula dentata leaves extract of Taif Region, Saudi Arabia. Indian Journal of Animal Research. 58(10): 1677-1687. doi: 10.18805/IJAR.BF-1808.

  25. Mustafa, G., Younas, S., Mahrosh, H. S., Albeshr, M.F. and Bhat, E.A. (2023). Molecular docking and simulation-binding analysis of plant phytochemicals with the hepatocellular carcinoma targets epidermal growth factor receptor and caspase-9. Molecules (Basel, Switzerland). 28(8): 3583. https://doi.org/10.3390/molecules28083583.

  26. Nguyen, L.T., Haney, E.F. and Vogel, H.J. (2011). The expanding scope of antimicrobial peptide structures and their modes of action. Trends in Biotechnology. 29(9): 464-472. https://doi.org/10.1016/j.tibtech.2011.05.001.

  27. Nurhayati, A.P.D., Rihandoko, A., Fadlan, A., Ghaissani, S.S., Jadid, N. and Setiawan, E. (2022). Anti-cancer potency by induced apoptosis by molecular docking P53, caspase, cyclin D1, cytotoxicity analysis and phagocytosis activity of trisindoline 1,3 and 4. Saudi Pharmaceutical Journal: SPJ: The Official Publication of the Saudi Pharmaceutical Society. 30(9): 1345-1359. https://doi.org/10.1016/ j.jsps.2022.06.012.

  28. Pantami, H.A., Ahamad, B.M.S., Lee, S.Y., Ismail, I.S., Mohd, F.S.M., Nakakuni, M. and Shaari, K. (2020). Comprehensive GCMS and LC-MS/MS metabolite profiling of Chlorella vulgaris. Marine Drugs. 18(7): 367. https://doi.org/ 10.3390/md18070367.

  29. Pereira, L. and Valado, A. (2025). Beyond nutrition: The therapeutic promise of seaweed-derived polysaccharides against bacterial and viral threats. Marine Drugs. 23(10): 407. https://doi.org/10.3390/md23100407.

  30. Piccinin, E., Cariello, M., De Santis, S., Ducheix, S., Sabbà, C., Ntambi, J.M. and Moschetta, A. (2019). Role of oleic acid in the gut-liver axis: From diet to the regulation of its synthesis via stearoyl-CoA desaturase 1 (SCD1). Nutrients. 11(10): 2283. https://doi.org/10.3390/nu11102283.

  31. Pradhan, B., Patra, S., Dash, S.R., Nayak, R., Behera, C. and Jena, M. (2021). Evaluation of the anti-bacterial activity of methanolic extract of Chlorella vulgaris beyerinck with special reference to antioxidant modulation. Future Journal of Pharmaceutical Sciences. 7(1): 17. https:// doi.org/10.1186/s43094-020-00172-5.

  32. Pradhan, J., Das, S. and Das, B.K. (2014). Antibacterial activity of freshwater microalgae: A review. African Journal of Pharmacy and Pharmacology. 8(10): 809-818. https:// doi.org/10.5897/AJPP2013.0002.

  33. Rajeswari, C., Manikandavelu, D., Ahilan, B., Aruna, S., Muralidharan, N., Ruby, P. and Joshna, M. (2026). Growth and biochemical characteristics of microalgae Chlorella vulgaris grown on various combinations of fish waste hydrolysate and seaweed hydrolysate. Indian Journal of Animal Research. 1-7. doi: 10.18805/IJAR.B-5438. 

  34. Sawasdee, N., Jantakee, K., Wathikthinnakon, M., Panwong, S., Pekkoh, J., Duangjan, K., Yenchitsomanus, P. T. and Panya, A. (2023). Microalga Chlorella sp. extract induced apoptotic cell death of cholangiocarcinoma via AKT/ mTOR signaling pathway. Biomedicine and Pharmacotherapy = Biomedecine and Pharmacotherapie. 160: 114306. https://doi.org/10.1016/j.biopha.2023.114306.

  35. Shaima, A.F., Mohd Yasin, N.H., Ibrahim, N., Takriff, M.S., Gunasekaran, D. and Ismaeel, M.Y.Y. (2022). Unveiling antimicrobial activity of microalgae Chlorella sorokiniana (UKM2),  Chlorella sp. (UKM8) and Scenedesmus sp. (UKM9).  Saudi Journal of Biological Sciences. 29(2): 1043-1052. https://doi.org/10.1016/j.sjbs.2021.09.069.

  36. Shanab, S.M., Mostafa, S.S., Shalaby, E.A. and Mahmoud, G.I. (2012). Aqueous extracts of microalgae exhibit antioxidant and anticancer activities. Asian Pacific Journal of Tropical Biomedicine. 2(8): 608-615. https://doi.org/ 10.1016/S2221-1691(12)60106-3.

  37. Shannon, E. and Abu-Ghannam, N. (2016). Antibacterial derivatives of marine algae: An overview of pharmacological mechanisms and applications. Marine Drugs. 14(4): 81. https://doi.org/10.3390/md14040081.

  38. Siddiqui, S., Upadhyay, S., Ahmad, I., Hussain, A. and Ahamed, M. (2021). Cytotoxicity of Moringa oleifera fruits on human liver cancer and molecular docking analysis of bioactive constituents against caspase-3 enzyme. Journal of Food Biochemistry. 45: e13720. https://doi.org/10.1111/jfbc. 13720.

  39. Silva, A.C.O., Santana, E.F., Saraiva, A.M., Coutinho, F.N., Castro, R.H.A., Pisciottano, M.N.C., Amorim, E.L.C. and Albuquerque, U.P. (2013). Which approach is more effective in the selection of plants with antimicrobial activity? Evidence- Based Complementary and Alternative Medicine. pp 308980. https://doi.org/10.1155/2013/308980.

  40. Sruthy, E.S. and Baiju, E.K.C. (2025). Green treasures: Phytochemical screening and antioxidant potential of freshwater species of Oedogonium, Ulothrix and  Mougeotia  (Chlorophyceae). Applied Phycology. 6(1): 74-95. https:/ /doi.org/10.1080/26388081.2024.2441148.

  41. Suganya, V. and Anuradha, V. (2019). In silico molecular docking of astaxanthin and sorafenib with different apoptotic proteins involved in hepatocellular carcinoma. Biocatalysis and Agricultural Biotechnology. 19: 101076. https:// doi.org/10.1016/j.bcab.2019.101076.

  42. Wanandi, S.I., Limanto, A., Yunita, E., Syahrani, R.A., Louisa, M., Wibowo, A.E. and Arumsari, S. (2020). In silico and in vitro studies on the anti-cancer activity of andrographolide targeting survivin in human breast cancer stem cells.  PloS One. 15(11): e0240020. https://doi.org/10.1371/ journal.pone.0240020.

  43. Yu, H.Y., Cho, D.H., Seo, D., Yoo, C., Park, S.B., Jung, W.K., Jung, J.E. and Kim, H.S. (2024). Microalga Chlorella sp. biomass containing high lutein prevents light-induced photooxidation and retinal degeneration in mice. Algal Research. 82: 103620. https://doi.org/10.1016/j.algal.2024.103620.
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    Phytochemical Screening, Antimicrobial, Anticancer Potentials and Molecular Docking of Chlorella Extract Targeting Caspase-9

    N
    Nahed Ahmed Hussien1,*
    https://orcid.org/0000-0001-7019-9682
    A
    Aisha Ehab Tantawy2
    1Department of Biology, College of Science, Taif University, Taif 21944, Saudi Arabia.
    2Department of Computer Engineering, College of Computers and Information Technology, Taif University, Taif, Saudi Arabia.

    ABSTRACT

    Background: Chlorella, a green alga, is rich in nutrients and bioactive compounds that support health and has potential as a source of natural therapeutic agents.

    Methods: Chlorella’s phytochemicals were analyzed using GC-MS and its antimicrobial and anticancer properties were evaluated. Molecular docking was performed with the major active compounds of Chlorella targeting caspase-9.

    Result: Chlorella extract contains trimethylsilyl palmitate (20.22%), oleic acid (9.87%) and arachidic acid methyl ester (6.12%). It exhibits antimicrobial activity against Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Bacillus cereus and Candida albicans, with minimum inhibitory concentrations (MIC) and bactericidal concentrations (MBC) above 1000 µg/mL, indicating relatively low antimicrobial potency. Chlorella extract exhibited inhibitory activity against Huh-7 hepatocellular carcinoma (HCC) cells, with an IC50 above 100 μg/mL. After docking with caspase-9, methyl arachidate is the most effective compound, with a binding affinity of -4.5 kcal/mol. It meets Lipinski’s rule, shows drug-like properties via SwissADME and has low predicted toxicity (LD50≈5000 mg/kg). It is stabilized in the binding pocket with three hydrophobic and two hydrogen bonds. This initial finding suggests that Chlorella could be a promising candidate for HCC treatment.

    INTRODUCTION

    Microalgae primarily inhabit aquatic environments and have high growth rates due to efficient sunlight and CO2  use. They contain bioactive compounds like proteins, lipids and polyphenols, used in food, pharma and cosmetics (Barkia et al., 2019) and support health with antibacterial, anticancer, antioxidant, antifungal and antiviral activities (Haq et al., 2019). Algae extracts contain antioxidants that may reduce oxidative stress and lower the risk of chronic diseases (Sruthy and Baiju, 2025). Reactive oxygen species (ROS), generated internally or externally, can damage cells and DNA, leading to heart, brain diseases and cancers. Antioxidants can help mitigate these risks (Haq et al., 2019).
           
    Chlorella     species can be widely cultivated and supplements are now available globally. Chlorella cells contain nutrients and bioactives that promote health and may prevent diseases, making them potential sources for therapeutic compounds. These compounds’ levels vary by culture conditions and species (Rajeswari et al., 2026). Since their cell walls are cellulose, they are poorly digestible intact, requiring mechanical disruption in supplements. Commercial Chlorella products also include essential vitamins and minerals (Bito et al., 2020).
           
    Hepatocellular carcinoma (HCC) causes high cancer mortality worldwide, with low survival rates despite advances in technology, screening and treatment (Mustafa et al., 2023). Challenges include late diagnosis, complex gene networks and drug side effects, leading to treatment failures. Effective care for end-stage liver disease and new anti-HCC drugs are vital. Molecular docking studies how phytochemicals interact with proteins at the atomic level, aiding in drug development (Kattan et al., 2020). Key protein targets in cancer therapy include transcription factors, apoptotic proteins, growth factor receptors, cell division kinases, MAP kinases and serine/threonine kinases. Caspases are crucial in apoptosis, initiating via two main pathways: the extrinsic and intrinsic pathways. The extrinsic pathway triggers when ligands bind to receptors, activating adaptor molecules that lead to caspase 8 activation. The intrinsic pathway involves mitochondrial signals, like membrane depolarization, causing caspase 9 to bind apoptosome complexes. Both pathways converge to activate caspases 3 and 7 (Anand et al., 2020).
           
    This study identified phytochemical compounds in a commercial Chlorella supplement using GC-MS and examined its antimicrobial properties. While previous research has explored Chlorella’s health benefits, this work focuses on its anticancer effects against hepatocellular carcinoma in Huh-7 cells. In silico molecular docking was used to analyze how Chlorella’s active compounds bind to caspase-9, a key protein in apoptosis. This approach supports the compounds’ anticancer potential and predicts their interactions, enhancing understanding of their mechanisms.

    MATERIALS AND METHODS

    Crude extract preparation
     
    The experiment at Taif University’s Biology Department in 2025 involved suspending 100 mg of Chlorella powder (Organic Traditions®, Canada) in 10 ml of 95% methanol. The mixture was vortexed on ice for 20 minutes, shaken at 25°C for 1 hour, then centrifuged at 4°C for 10 minutes at 1209 g. The supernatant was collected, re-extracted twice and combined supernatants filtered through No.1 Whatman paper. The extract was dried with a rotary evaporator (BÜCHI Rotavapor R-200, Switzerland) and stored at -20°C for future analysis (Ferdous et al., 2023).
     
    Gas chromatography-mass spectroscopy analysis
     
    The 95% methanolic Chlorella extract was analyzed using an RTX-5 MS capillary column (Restek) with a GC-MS-QP2010 Plus system (Shimadzu, Japan), covering 1.5-1000 m/z. Helium at 1 mL/min was the carrier gas. Detection employed an electron ionization source at 70 eV, with a split ratio of 1:10 to control helium flow at 14.1 mL/min and a 1.00 µL injection volume. The injector was at 250°C and the ion source at 200°C. Mass spectra from 45 to 450 Da were recorded at 70 eV during a 0.5-second scan. The run lasted 50 minutes with a solvent delay of 0 to 4.5 minutes. The oven started at 80°C for 4 minutes, then increased by 10°C per minute to 280°C. GC separates compounds by retention time, while MS identifies them. The software generates a chromatogram of peak abundance vs. retention time (Mohamed and Hussien, 2024). Comparing spectra with the Wiley Registry 8e library (similarity index ≥70%) helps identify compounds.
     
    Antimicrobial evaluation
     
    The antimicrobial activity of Chlorella extract was tested against five microbes: four bacteria and one fungus from Nawah Scientific Inc. (Mokatam, Cairo, Egypt): Escherichia coli ATCC 8739, Enterococcus faecalis ATCC 19433, Staphylococcus aureus ATCC 29213, Bacillus cereus ATCC 9634 and Candida albicans ATCC 10231. A two-fold serial dilution of Chlorella from 1000 to 1.953 ìg/mL in Mueller-Hinton broth was used for treatment, as described by Hussien et al., (2025). Ciprofloxacin (1.953-1000 ìg/mL) served as a positive control. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Chlorella were evaluated against various strains, with MIC representing the lowest concentration that prevents growth and MBC the concentration that kills 99.9% of bacteria. Three replicates were performed for each strain and treatment and a microplate ELISA reader (TECAN, Switzerland) was used to measure turbidity at an optical density (OD) of 600 nm.
     
    Sulforhodamine B (SRB) cytotoxicity
     
    The Huh-7 hepatocellular carcinoma (HCC) cell line was obtained from Nawah Scientific Inc. (Mokatam, Cairo, Egypt). Cells were cultured in DMEM containing 100 mg/mL streptomycin, 100 U/mL penicillin and 10% heat-inactivated fetal bovine serum and maintained in a humidified atmosphere with 5% CO2 at 37°C. Cells were treated with media containing various concentrations of Chlorella extract (100 μL/well; 0.001-100 μg/mL), as described by Allam et al., (2018). Media without Chlorella served as a negative control, while cisplatin (0.01-100 μg/mL) served as a positive control. Absorbance was measured at 540 nm and the extract was tested in triplicate at each concentration to ensure accuracy.
     
    Statistical analysis
     
    Data were presented as mean ± SE of replicates and analyzed using non-linear regression in OriginLab to generate a sigmoidal dose-response curve of Log10 Chlorella extract concentration versus cell viability. IC50 value was interpolated from the curve.
     
    Molecular docking study
     
    Based on GC-MS results, we selected the most abundant compounds for molecular docking with caspase-9 as the receptor. The 2D and 3D structures of these compounds (ligands) were obtained from the PubChem database. The 3D crystal structure of dimeric caspase-9 bound to ligand (D-Malate) (PDB ID: 2ar9, resolution 2.80 Å) was downloaded from RCSB PDB (http://www.rcsb.org/pdb/home/home.do). We used PyMOL to remove D-Malate and water molecules from the receptor. SwissADME was employed to predict their physicochemical properties, drug-likeness, pharmacokinetics and medicinal chemistry features (http://www.swissadme.ch/) (Daina et al., 2017). The top-docked compounds were selected according to Lipinski’s rule of five, a guideline for ideal drug candidates. Lipinski suggests that a compound demonstrates drug-like properties if it meets certain criteria: a molecular weight of 500 or less, no more than 5 hydrogen bond donors, no more than 10 hydrogen bond acceptors, lipophilicity below 5 and molar refractivity between 40 and 130 (Lipinski et al., 2001). Predicted LD50 values and toxicities for the seven compounds were determined using the ProTox-II web server (https://tox-new.charite.de/protox_II/) (Banerjee et al., 2024). Molecular docking of selected compounds into the receptor protein’s binding pockets was carried out with SMINA/Anaconda. Additionally, re-docking was performed to validate the accuracy of the docking protocol. Docking validation involved re-docking the co-crystallized ligand (D-Malate) into the active site using AutoDock’s fast scoring function. The most favorable conformations were chosen by comparing RMSD values between the protein and the co-crystallized ligand (Hevener et al., 2009). After docking, the resulting complexes were examined with the Protein Ligand Interaction Profiler (PLIP, https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index) to identify non-covalent interactions, such as hydrogen bonds, hydrophobic contacts, water bridges and ionic interactions and to generate their 2D visualizations (Adasme et al., 2021).

    RESULTS AND DISCUSSION

    GC-MS analysis of the Chlorella extract identified phytochemicals, including fatty acids, hydrocarbons, phenolics, terpenes and their derivatives (Fig 1). The major phytochemical components of Chorella extract are high-oleic algal oil, fatty acids and their derivatives (including arachidic, palmitic, oleic and linolenic) (Table 1).

    Fig 1: Chromatogram of Chlorella methanolic extract.



    Table 1: Major compounds of Chlorella and their chemical structures.


           
    This confirms that Chlorella’s commercial dietary supplement is as rich in fatty acids as cultivated (Pantami et al., 2020). The main phytochemicals in Chorella extract include trimethylsilyl palmitate (20.22%), oleic acid (9.87%), oleic acid trimethylsilyl ester (6.25%), arachidic acid methyl ester (6.12%), linoleoyl chloride (5.81%), monopalmitin (5.80%), 3Beta-hydroxy-5-cholestene 3-oleate (3.64%) and Algal oil (3.36%) (Table 1). Pantami et al., (2020) reported that C. vulgaris extract contains over 60% fatty acids, rich in omega-6, -7, -9 and -13, with omega-6 (notably 9,12-Octadecadienoic acid, Linoleic acid at 35.1%) being predominant, as determined through GC-MS analysis. Consequently, Chlorella species may serve as a valuable nutritional source when added to diets, highlighting its potential as an outstanding dietary supplement.
           
    The study found that trimethylsilyl palmitate, a palmitic acid ester, is the main component, comprising 20.22% of the extract. This ester exhibits various effects, including antioxidant, hypocholesterolemic, nematicidal, hemolytic, 5-alpha-reductase inhibition, antipsychotic, anti-androgenic and anticancer activities (El-Fayoumy et al., 2021). Next are Oleic acid (9.87%) and its derivative (6.25%), which play beneficial roles in diseases such as coronary heart disease, rheumatoid arthritis and cancer (Piccinin et al., 2019). Arachidic acid methyl ester (6.12%) exhibits antifungal and antioxidant properties (Abdelillah et al., 2013) and helps prevent cholesterol gallstones by acting as a cholesterol solubilizer (Gilat et al., 2001). Phenolics in Chlorella sp. combat reactive oxygen and singlet oxygen species and decrease ROS production (Pradhan et al., 2021). 
           
    Chlorella     extract exhibits antimicrobial effects against Gram-negative bacteria, E. coli and Gram-positive bacteria, E. faecalis, S. aureus and B. cereus, as well as the fungus Candida albicans. Its MIC and MBC are over 1000 μg/mL, but it’s less potent than Ciprofloxacin (Table 2). As previously reported, MIC values for antibiotics range from 0.01 to 10 µg/mL, whereas plant extracts are considered antimicrobials if their MICs fall between 100 and 1000 µg/mL (Silva et al., 2013). This large gap between the antibiotic and the natural extract used could be attributed to several factors. The difference in their nature, solubility and size. Using a crude extract is less effective than using a single, isolated compound (Ben-Khalifa et al., 2021). 

    Table 2: The minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) of Chlorella sp.


           
    The current findings align with previous research reporting Chlorella sp.’s antibacterial effects against Gram-positive bacteria, S. epidermidis, S. aureus, Methicillin-resistant S. aureus (MRSA), E. faecalis and B. thuringiensis, as well as Gram-negative bacteria, E. coli, S. sonnei and S. marcescens (Shaima et al., 2022). They have reported that the inhibition zones ranged from 11 to 13.8 mm, with MIC values of 0.39-6.25 mg/mL. Additionally, antibiotics demonstrated stronger bacterial inhibition across various strains. The antimicrobial activity of Chlorella sp. is primarily attributed to its phytochemicals, especially polyphenols, which promote hydrogen peroxide production under aerobic conditions and are linked to hydroxyl groups (Dell’Anno et al., 2019).
           
    Various chemical groups, such as phlorotannins, fatty acids, peptides, terpenes and polysaccharides, have been found to inhibit bacterial growth in algae. Nonetheless, the exact mechanisms behind this inhibition remain unclear (Shannon and Abu-Ghannam, 2016). Algal free fatty acids inhibit the bacterial electron transport chain and oxidative phosphorylation, disrupting ATP transfer. They inhibit enzymes like bacterial enoyl-acyl carrier protein reductase, crucial for fatty acid synthesis, causing cell lysis and degradation from peroxidation and auto-oxidation (Pradhan et al., 2014). Algal and sulfated polysaccharides interact with glycoprotein receptors, bind to bacterial walls, membranes and DNA and increase permeability, causing protein leakage and DNA binding (Pereira and Valado, 2025). Amphipathic peptides bind to polar and nonpolar membrane regions, disrupting bacterial processes and replication (Nguyen et al., 2011). All proposed antimicrobial mechanisms of algae are outlined in Fig 2.

    Fig 2: Schematic of the proposed antibacterial mechanism of Chlorella extract’s active components. Created with figurelabs®.


           
    HCC is among the most prevalent primary liver cancers and ranks as the fourth leading cause of cancer-related deaths worldwide (Mustafa et al., 2023). Results shown in Fig 3 indicate that Chlorella demonstrated a dose-dependent anticancer effect from 0.01 to 100 μg/mL. After 72 hours, cell viability was significantly reduced, with an IC50 exceeding 100 μg/mL, suggesting relatively lower cytotoxicity than cisplatin. These findings align with Shanab et al., (2012), who reported the anticancer potential of aqueous Chlorella extract against Ehrlich Ascites Carcinoma cells and HepG2 cells at 100 μg/mL. Chlorella’s anticancer effects are attributed to polar compounds such as phycobilins, phenolics and polysaccharides that induce apoptosis (Aboul-Enein et al., 2011). Oleic acid in Chlorella promotes apoptosis and differentiation in colorectal HT-29 cells (Llor et al., 2003) and decreases Her-2/neu expression in breast cancer cells (Menendez et al., 2005). Studies show Chlorella sp. has anticancer effects by activating apoptosis through increased P53, Bax and caspase-3, decreased Bcl-2 and suppressed AKT/mTOR pathways, causing DNA damage and apoptosis (Sawasdee et al., 2023).

    Fig 3: Cell viability (%) of Huh-7 cell line treated with Chlorella.


           
    Seven phytocomponents-trimethylsilyl palmitate; oleic acid; oleic acid trimethylsilyl ester; arachidic acid methyl ester (methyl arachidate); linoleic acid chloride (linoleoyl chloride); 2,3-Dihydroxypropyl hexadecanoate (monopalmitin) and 3Beta-hydroxy-5-cholestene 3-oleate (cholesteryl oleate)-were examined for Caspase-9 docking studies. Tables 3-6 present data on the pharmacokinetics, physicochemical properties, medicinal chemistry and drug-likeness of these compounds. Linoleoyl chloride was omitted because its calculated parameters were unavailable. Cholesteryl oleate does not meet Lipinski’s rules, with two violations: MW>500 and MLOGP>4.15. Only methyl arachidate and monopalmitin were selected for Caspase-9 docking because they meet these criteria, which indicate potential drug-like properties and lower toxicity, with a predicted LD50 of 5000 mg/kg. Both are expected to be non-mutagenic, non-tumorigenic and non-irritant. Methyl arachidate is highly lipophilic, resulting in low gastrointestinal absorption and limited blood-brain barrier permeability, whereas monopalmitin is less lipophilic, with good gastrointestinal absorption and blood-brain barrier permeability. These two compounds were docked into the Caspase-9 chain A docking pocket, replacing the co-crystallized ligand (Malate), with a binding affinity of -4.5 kcal/mol.

    Table 3: Physicochemical properties of selected compounds found in Chlorella sp.



    Table 4: Lipophilicity of the selected phytocomponents.



    Table 5: Water solubility of selected compounds.



    Table 6: Drug-likeness, pharmacokinetics, and medicinal chemistry of Chlorella selected compound.


           
    Based on the data in Fig 4 and Table 7, methyl arachidate appears more stable within the docking site, forming three hydrophobic contacts and two hydrogen bonds with the receptor protein. In contrast, monopalmitin forms two hydrophobic interactions and three hydrogen bonds. Methyl arachidate interacts with caspase-9 at residues TRP (chain A):354, ARGA:355, PROA:357 and makes two hydrogen bonds at ARGA: 180 and ARGA: 355. Meanwhile, monopalmitin interacts with caspase-9 at VALA: 352, TRPA:354 and forms three hydrogen bonds at ARGA: 355 amino acids of the active pocket.

    Fig 4: The non-covalent interactions (hydrogen and hydrophobic bonds) between caspase-9 and ligands.



    Table 7: The non-covalent interactions (hydrogen and hydrophobic bonds) between Caspase 9 and ligands.


           
    Molecular docking of p53, caspase-9 and cyclin D1, which play roles in apoptosis and cell proliferation, is a target for inducing apoptosis in the MCF-7 breast cancer cells (Nurhayati et al., 2022) and HepG2 liver carcinoma cells (Anand et al., 2020). In silico and docking results indicate that methyl arachidate may inhibit caspase-9 and could serve as a promising liver anticancer agent. Our findings align with Mustafa et al., (2023), who docked the phytochemicals limonin and obamegine with caspase-9. Limonin interacted with amino acids ArgA:355, TrpA:354, GlyA:238, ArgA:178 and LeuA:177 at the active site. Obamegine bound to ProA:338, ProA:336, LeuA:335, AsnA:265, PheA:246 and LeuA:244 within the binding pocket. Additionally, Siddiqui et al., (2021) docked eight Moringa oleifera phytochemicals with caspase-3, where benzyl glucosinolate (binding affinity -8.4 kcal/mol) interacted with Phe265, Phe250, Arg207, Trp206, Tyr204 and Thr62. Suganya and Anuradha (2019) also docked astaxanthin, which interacted with amino acids Ile403, Cys402, Met400, Tyr397, Ile396, Phe351 and Phe348 of caspase-9.
           
    Our findings agree with Wanandi et al., (2020), who studied the anti-cancer potential of a natural compound by examining its interactions with survivin, caspase-9 and caspase-3 via computational methods. In 2023, Mustafa et al. (2023) docked plant phytochemicals-liquoric acid (-9.8 kcal/mol), madecassic acid (-9.3 kcal/mol), limonin (-10.5 kcal/mol) and obamegine (-9.3 kcal/mol)-against EGFR and caspase-9. However, the molecular mechanisms behind HCC development, progression and metastasis are not well understood. In the current in silico analysis, we targeted caspase-9, a protease that triggers apoptosis via the intrinsic or mitochondrial pathway and is activated at sites involving multiple proteins (Kim et al., 2015). Extracellular signal-regulated kinases-1 and 2 (ERK1/2) are part of the mitogen-activated protein (MAP) kinase superfamily and regulate cell proliferation and survival. They inhibit apoptosis by deactivating proapoptotic proteins and supporting cell survival by blocking caspase-9 activity (Boston et al., 2011). Natural chemicals targeting caspase-9 could be promising for HCC treatment. Microalgae are rich in biocompatible compounds, making them valuable nutraceuticals (Sawasdee et al., 2023; Yu et al., 2024). Their phytochemicals may serve as future drug candidates against HCC.

    CONCLUSION

    This study assesses a commercial Chlorella supplement and its potential effects. In silico and docking studies suggest that methyl arachidate, a key component, may inhibit caspase-9, but further validation is needed. More research and trials are essential to confirm Chlorella’s biological properties. Overall, the findings highlight natural microalgae compounds’ potential for developing therapies for hepatocellular carcinoma (HCC) and encourage future medicinal research.

    ACKNOWLEDGEMENT

    The authors would like to acknowledge the Deanship of Graduate Studies and Scientific Research, Taif University for funding this work.
     
    Research funding
     
    The authors would like to acknowledge the Deanship of Graduate Studies and Scientific Research, Taif University for funding this work.

    CONFLICT OF INTEREST

    Authors state no conflict of interest.

    REFERENCES


    1. Abdelillah, A., Houcine, B., Halima, D., Meriem, C.S., Imane, Z., Eddine, S.D., Abdallah, M. and Daoudi, C.S. (2013). Evaluation of antifungal activity of free fatty acids methyl esters fraction isolated from Algerian Linum usitatissimum L. seeds against toxigenic Aspergillus. Asian Pacific Journal of Tropical Biomedicine. 3(6): 443-448. https:/ /doi.org/10.1016/S2221-1691(13)60094-5.

    2. Aboul-Enein, A.M., Shalaby, E.A., Abul-Ela, F., Nasr-Allah, A.A., Mahmoud, A.M., El-Shemy, H.A. and Ahmed, M. (2011). Back to nature: Spotlight on cancer therapeutics. Vital Signs. 10: 8-9.

    3. Adasme, M.F., Linnemann, K.L., Bolz, S.N., Kaiser, F., Salentin, S., Haupt, V.J. and Schroeder, M. (2021). PLIP 2021: Expanding the scope of the protein-ligand interaction profiler to DNA and RNA. Nucleic Acids Research. 49(W1): W530- W534. https://doi.org/10.1093/nar/gkab294.

    4. Allam, R.M., Al-Abd, A.M., Khedr, A., Sharaf, O.A., Nofal, S.M., Khalifa, A.E., Mosli, H. A. and Abdel-Naim, A.B. (2018). Fingolimod interrupts the cross talk between estrogen metabolism and sphingolipid metabolism within prostate cancer cells. Toxicology Letters. 291: 77-85. https:// doi.org/10.1016/j.toxlet.2018.04.008.

    5. Anand, K., Abdul, N.S., Ghazi, T., Ramesh, M., Gupta, G., Tambuwala, M.M., Dureja, H., Singh, S.K., Chellappan, D.K., Dua, K., Pandi, B., Saravanan, M. and Chuturgoon, A.A. (2020). Induction of caspase-mediated apoptosis in HepG2 liver carcinoma cells using mutagen-antioxidant conjugated self-assembled novel carbazole nanoparticles and in silico modeling studies. ACS Omega. 6(1): 265-277. https://doi.org/10.1021/acsomega.0c04461.

    6. Banerjee, P., Kemmler, E., Dunkel, M. and Preissner, R. (2024). ProTox 3.0: A webserver for the prediction of toxicity of chemicals. Nucleic Acids Research. 52(W1): W513- W520. https://doi.org/10.1093/nar/gkae303.

    7. Barkia, I., Saari, N. and Manning, S.R. (2019). Microalgae for high- value products towards human health and nutrition. Marine Drugs. 17(5): 304. https://doi.org/10.3390/ md17050304.

    8. Ben-Khalifa, R., Gaspar, F.B., Pereira, C., Chekir-Ghedira, L. and Rodríguez-Rojo, S. (2021). Essential oil and hydrophilic antibiotic co-encapsulation in multiple lipid nanoparticles: Proof of concept and in vitro activity against Pseudomonas aeruginosa. Antibiotics. 10(11): 1300. https://doi.org/ 10.3390/antibiotics10111300.

    9. Bito, T., Okumura, E., Fujishima, M. and Watanabe, F. (2020). Potential of Chlorella as a dietary supplement to promote human health. Nutrients. 12(9): 2524. https://doi.org/ 10.3390/nu12092524.

    10. Boston, S.R., Deshmukh, R., Strome, S., Priyakumar, U.D., MacKerell, A.D. and Shapiro, P. (2011). Characterization of ERK docking domain inhibitors that induce apoptosis by targeting Rsk-1 and caspase-9. BMC Cancer. 11: 7. https://doi.org/10.1186/1471-2407-11-7.

    11. Daina A., Michielin, O. and Zoete, V. (2017). SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Scientific Reports. 7: 42717. https://doi.org/10.1038/ srep42717.

    12. Dell’Anno, M., Sotira, S., Rebucci, R., Reggi, S., Castiglioni, B. and Rossi, L. (2019). In vitro evaluation of antimicrobial and antioxidant activities of algal extracts. Italian Journal of Animal Science. 19(1): 103-113. https://doi.org/10.1080/ 1828051X.2019.1703563.

    13. El-Fayoumy, E.A., Shanab, S.M.M., Gaballa, H.S., Tantawy, M.A. and Shalaby, E.A., (2021). Evaluation of antioxidant and anticancer activity of crude extract and different fractions of Chlorella vulgaris axenic culture grown under various concentrations of copper ions. BMC Complementary Medicine and Therapies. 21(1): 51. https://doi.org/ 10.1186/s12906-020-03194-x.

    14. Ferdous, U.T., Nurdin, A., Ismail, S. and Balia Yusof, Z.N. (2023). Evaluation of the antioxidant and cytotoxic activities of crude extracts from marine Chlorella sp. Biocatalysis and Agricultural Biotechnology. 47: 102551. https:// doi.org/10.1016/j.bcab.2022.102551.

    15. Gilat, T., Leikin-Frenkel, A., Goldiner, I., Laufer, H., Halpern, Z. and Konikoff, F.M. (2001). Arachidyl amido cholanoic acid (Aramchol) is a cholesterol solubilizer and prevents the formation of cholesterol gallstones in inbred mice. Lipids. 36(10): 1135-1140. https://doi.org/10.1007/s11745-001- 0824-3.

    16. Haq, S.H., Al-Ruwaished, G., Al-Mutlaq, M.A., Naji, S. A., Al-Mogren, M., Al-Rashed, S., Ain, Q.T., Al-Amro, A.A. and Al- Mussallam, A. (2019). Antioxidant, anticancer activity and phytochemical analysis of green algae, Chaetomorpha collected from the Arabian gulf. Scientific Reports. 9(1): 18906. https://doi.org/10.1038/s41598-019-55309-1.

    17. Hevener, K.E., Zhao, W., Ball, D.M., Babaoglu, K., Qi, J., White, S.W. and Lee, R.E. (2009). Validation of molecular docking programs for virtual screening against dihydropteroate synthase. Journal of Chemical Information and Modeling. 49(2): 444-460. doi: 10.1021/ci800293n.

    18. Hussien, N.A., Khalil, M.A.E.F., Schagerl, M. and Ali, S.S. (2025). Green synthesis of zinc oxide nanoparticles as a promising nanomedicine approach for anticancer, antibacterial and anti-inflammatory therapies. International Journal of Nanomedicine. 20: 4299-4317. https://doi.org/ 10.2147/IJN.S507214.

    19. Kattan, S.W., Nafie, M.S., Elmgeed, G.A., Alelwani, W., Badar, M. and Tantawy, M.A. (2020). Molecular docking, anti- proliferative activity and induction of apoptosis in human liver cancer cells treated with androstane derivatives: Implication of PI3K/AKT/mTOR pathway. Journal of Steroid Biochemistry and Molecular Biology. 198: 105604. https://doi.org/10.1016/j.jsbmb.2020.105604.

    20. Kim, B., Srivastava, S.K. and Kim, S.H. (2015). Caspase-9 as a therapeutic target for treating cancer. Expert Opinion on Therapeutic Targets. 19(1): 113-127. https://doi.org/ 10.1517/14728222.2014.961425.

    21. Lipinski, C.A., Lombardo, F., Dominy, B.W. and Feeney, P.J. (2001). Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Reviews. 46(1-3): 3- 26. https://doi.org/10.1016/s0169-409x(00)00129-0.

    22. Llor, X., Pons, E., Roca, A., Alvarez, M., Mane, J., Fernandez- Banares, F. and Gassull, M. A. (2003). The effects of fish oil, olive oil, oleic acid and linoleic acid on colorectal neoplastic processes. Clinical Nutrition. 22(1): 71-79. https://doi.org/10.1054/clnu.2002.0627.

    23. Menendez, J.A., Vellon, L., Colomer, R. and Lupu, R. (2005). Oleic acid, the main monounsaturated fatty acid of olive oil, suppresses Her-2/neu (erbB-2) expression and synergistically enhances the growth inhibitory effects of trastuzumab (Herceptin) in breast cancer cells with Her-2/neu oncogene amplification. Annals of Oncology. 16(3): 359-371. https://doi.org/10.1093/annonc/mdi090.

    24. Mohamed, H.R.H. and Hussien, N.A. (2024). GC-MS analysis and molecular docking studies of Lavandula dentata leaves extract of Taif Region, Saudi Arabia. Indian Journal of Animal Research. 58(10): 1677-1687. doi: 10.18805/IJAR.BF-1808.

    25. Mustafa, G., Younas, S., Mahrosh, H. S., Albeshr, M.F. and Bhat, E.A. (2023). Molecular docking and simulation-binding analysis of plant phytochemicals with the hepatocellular carcinoma targets epidermal growth factor receptor and caspase-9. Molecules (Basel, Switzerland). 28(8): 3583. https://doi.org/10.3390/molecules28083583.

    26. Nguyen, L.T., Haney, E.F. and Vogel, H.J. (2011). The expanding scope of antimicrobial peptide structures and their modes of action. Trends in Biotechnology. 29(9): 464-472. https://doi.org/10.1016/j.tibtech.2011.05.001.

    27. Nurhayati, A.P.D., Rihandoko, A., Fadlan, A., Ghaissani, S.S., Jadid, N. and Setiawan, E. (2022). Anti-cancer potency by induced apoptosis by molecular docking P53, caspase, cyclin D1, cytotoxicity analysis and phagocytosis activity of trisindoline 1,3 and 4. Saudi Pharmaceutical Journal: SPJ: The Official Publication of the Saudi Pharmaceutical Society. 30(9): 1345-1359. https://doi.org/10.1016/ j.jsps.2022.06.012.

    28. Pantami, H.A., Ahamad, B.M.S., Lee, S.Y., Ismail, I.S., Mohd, F.S.M., Nakakuni, M. and Shaari, K. (2020). Comprehensive GCMS and LC-MS/MS metabolite profiling of Chlorella vulgaris. Marine Drugs. 18(7): 367. https://doi.org/ 10.3390/md18070367.

    29. Pereira, L. and Valado, A. (2025). Beyond nutrition: The therapeutic promise of seaweed-derived polysaccharides against bacterial and viral threats. Marine Drugs. 23(10): 407. https://doi.org/10.3390/md23100407.

    30. Piccinin, E., Cariello, M., De Santis, S., Ducheix, S., Sabbà, C., Ntambi, J.M. and Moschetta, A. (2019). Role of oleic acid in the gut-liver axis: From diet to the regulation of its synthesis via stearoyl-CoA desaturase 1 (SCD1). Nutrients. 11(10): 2283. https://doi.org/10.3390/nu11102283.

    31. Pradhan, B., Patra, S., Dash, S.R., Nayak, R., Behera, C. and Jena, M. (2021). Evaluation of the anti-bacterial activity of methanolic extract of Chlorella vulgaris beyerinck with special reference to antioxidant modulation. Future Journal of Pharmaceutical Sciences. 7(1): 17. https:// doi.org/10.1186/s43094-020-00172-5.

    32. Pradhan, J., Das, S. and Das, B.K. (2014). Antibacterial activity of freshwater microalgae: A review. African Journal of Pharmacy and Pharmacology. 8(10): 809-818. https:// doi.org/10.5897/AJPP2013.0002.

    33. Rajeswari, C., Manikandavelu, D., Ahilan, B., Aruna, S., Muralidharan, N., Ruby, P. and Joshna, M. (2026). Growth and biochemical characteristics of microalgae Chlorella vulgaris grown on various combinations of fish waste hydrolysate and seaweed hydrolysate. Indian Journal of Animal Research. 1-7. doi: 10.18805/IJAR.B-5438. 

    34. Sawasdee, N., Jantakee, K., Wathikthinnakon, M., Panwong, S., Pekkoh, J., Duangjan, K., Yenchitsomanus, P. T. and Panya, A. (2023). Microalga Chlorella sp. extract induced apoptotic cell death of cholangiocarcinoma via AKT/ mTOR signaling pathway. Biomedicine and Pharmacotherapy = Biomedecine and Pharmacotherapie. 160: 114306. https://doi.org/10.1016/j.biopha.2023.114306.

    35. Shaima, A.F., Mohd Yasin, N.H., Ibrahim, N., Takriff, M.S., Gunasekaran, D. and Ismaeel, M.Y.Y. (2022). Unveiling antimicrobial activity of microalgae Chlorella sorokiniana (UKM2),  Chlorella sp. (UKM8) and Scenedesmus sp. (UKM9).  Saudi Journal of Biological Sciences. 29(2): 1043-1052. https://doi.org/10.1016/j.sjbs.2021.09.069.

    36. Shanab, S.M., Mostafa, S.S., Shalaby, E.A. and Mahmoud, G.I. (2012). Aqueous extracts of microalgae exhibit antioxidant and anticancer activities. Asian Pacific Journal of Tropical Biomedicine. 2(8): 608-615. https://doi.org/ 10.1016/S2221-1691(12)60106-3.

    37. Shannon, E. and Abu-Ghannam, N. (2016). Antibacterial derivatives of marine algae: An overview of pharmacological mechanisms and applications. Marine Drugs. 14(4): 81. https://doi.org/10.3390/md14040081.

    38. Siddiqui, S., Upadhyay, S., Ahmad, I., Hussain, A. and Ahamed, M. (2021). Cytotoxicity of Moringa oleifera fruits on human liver cancer and molecular docking analysis of bioactive constituents against caspase-3 enzyme. Journal of Food Biochemistry. 45: e13720. https://doi.org/10.1111/jfbc. 13720.

    39. Silva, A.C.O., Santana, E.F., Saraiva, A.M., Coutinho, F.N., Castro, R.H.A., Pisciottano, M.N.C., Amorim, E.L.C. and Albuquerque, U.P. (2013). Which approach is more effective in the selection of plants with antimicrobial activity? Evidence- Based Complementary and Alternative Medicine. pp 308980. https://doi.org/10.1155/2013/308980.

    40. Sruthy, E.S. and Baiju, E.K.C. (2025). Green treasures: Phytochemical screening and antioxidant potential of freshwater species of Oedogonium, Ulothrix and  Mougeotia  (Chlorophyceae). Applied Phycology. 6(1): 74-95. https:/ /doi.org/10.1080/26388081.2024.2441148.

    41. Suganya, V. and Anuradha, V. (2019). In silico molecular docking of astaxanthin and sorafenib with different apoptotic proteins involved in hepatocellular carcinoma. Biocatalysis and Agricultural Biotechnology. 19: 101076. https:// doi.org/10.1016/j.bcab.2019.101076.

    42. Wanandi, S.I., Limanto, A., Yunita, E., Syahrani, R.A., Louisa, M., Wibowo, A.E. and Arumsari, S. (2020). In silico and in vitro studies on the anti-cancer activity of andrographolide targeting survivin in human breast cancer stem cells.  PloS One. 15(11): e0240020. https://doi.org/10.1371/ journal.pone.0240020.

    43. Yu, H.Y., Cho, D.H., Seo, D., Yoo, C., Park, S.B., Jung, W.K., Jung, J.E. and Kim, H.S. (2024). Microalga Chlorella sp. biomass containing high lutein prevents light-induced photooxidation and retinal degeneration in mice. Algal Research. 82: 103620. https://doi.org/10.1016/j.algal.2024.103620.
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