Int J App Pharm, Vol 16, Issue 5, 2024, 133-138Original Article

COMBINING LIQUID CHROMATOGRAPHY WITH TANDEM MASS SPECTROMETRY (LC-MS/MS) TECHNIQUE, SPECTROPHOTOMETERVALIDATION AND AN IN SILICO STUDY OF 96% ETHANOL EXTRACT OF SPIRULINA PLATENSIS

TIWUK SUSANTININGSIH1,2, FADILAH FADILAH3*, ANI RETNO PRIJANTI4, NOVI SILVIA HARDIANY5

1Doctoral Program of Biomedical Science, Faculty of Medicine, Universitas Indonesia, Jakarta-10430, Indonesia. 2Department of Biochemistry Faculty of Medicine, UPN Veteran Jakarta, Jakarta-12450, Indonesia. 3Department of Medical Chemistry, Faculty of Medicine, University of Indonesia, Jakarta-10440, Indonesia. 4Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Indonesia, Jakarta-10430, Indonesia. 5Magister Program of Biomedical Science, Faculty of Medicine, University of Indonesia, Jakarta-10430, Indonesia
*Corresponding author: Fadilah Fadilah; *Email: fadilah.msi@ui.ac.id

Received: 07 May 2024, Revised and Accepted: 20 Jun 2024

ABSTRACT

Objective: This study was to analyze the component sofa 96% ethanol extract of Spirulina platensis by the LC-MS/MS technique, then validate them with the spectrophotometer technique using the C-phycocyanin standard and an in silico study approach as an antioxidant property of S. platensis against inflammatory.

Methods: Chromatographic resolution was attained with a Phenominex C18 (50 mm×2.6 mm, 3 µm) stationary column technique, validation using C-phycocyanin standard using the spectrophotometer technique, and an in silico study of c-phycocyanin using molecular docking analysis.

Results: Tentative active compounds such as flavonoid (Maltol and Morin), peptide (Cyclo Pro-Ala, Cyclo Pro-Pro, and Thymine), and phenol (m-Aminophenol, N-Methyltyramine, and Tyramine) have been identified from a 96% ethanol extract of S. platensis by LCMS/MS analysis. The concentration of c-phycocyanin in the 96% ethanol extract of S. platensis is 229, 2µg/ml. According to our in silico study, c-phycocyanin demonstrates potential as an anti-inflammatory agent.

Conclusion: The LC-MS/MS technique can detect flavonoid, peptide, and phenolic components in the 96% ethanol extract of S. platensis. A spectrophotometer can identify the validation equation of c-phycocyanin in a 96% ethanol extract of S. platensis. Based on our in silico study, c-phycocyanin demonstrate the capability to prevent inflammatory activity.

Keywords: C-phycocyanin, In silico study, LC-MS/MS, Spectrophotometer, Spirulina platensis

© 2024 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2024v16i5.51339 Journal homepage: https://innovareacademics.in/journals/index.php/ijap

INTRODUCTION

Spirulina platensis is a cyanobacterium filamentous microalga that contains various flavonoids, among other phytochemicals. These flavonoids enhance antioxidant activity, potentially offer protection against oxidative stress and inflammation [1, 2], and anticancer properties [3, 4]. Spirulina platens scan be used as a preventive supplement since it is high in protein, carbohydrates, polyunsaturated fatty acids, sterols, and other essential components [5, 6]. Spirulina platensis contains 60-70% dry-weight protein [7], essential amino acids, carotenoids, lipids, vitamins E, C, and selenium [8, 9]. It has been reported that the consumption of S. platensis could prevent or manage metabolic syndrome disorders [10-12]. Spirulina platensis has long been utilized for nutrition in Mexico, Africa, and Asia, especially in Indonesia. In Indonesia, S. platensis is cultivated using freshwater aquaculture.

Spirulina platensis and its pigments such as c-phycocyanin, carotene, xanthophyll, and chlorophyll, exhibit exogenous antioxidant properties [13]. C-phycocyanin, rich in blue-green microalgae, is known to break the radical chain to inhibit reactive oxygen species and oxidative stress, act as a hepatoprotection, and help reduce blood glucose levels. C-phycocyanin can reduce free radicals, minimize nitrite production, suppress nitric oxide synthase (iNOS) expression, and inhibit lipid peroxidation [2, 14, 15]. Because c-phycocyanin is soluble in water, cells must first be disrupted using physical or chemical methods to extract the pigment [16-18]. C-phycocyanin prevents oxidative stress and cell damage in vitro in the hypoxia model employing the myoblast cell line H9c2 [19, 20].

This study aimed to explore and identify the most potent S. platensis compounds from the Indonesian medicinal algae. Using liquid chromatography coupled with mass spectrophotometry (LC-MS/MS) for precise identification and quantification, the study developed and validated an LC-MS/MS analytical procedure. The validation included a spectrophotometric analysis using a c-phycocyanin standard. Additionally, the in silico research was conducted to evaluate the antioxidant properties of c-phycocyanin from S. plarensis against oxidative stress and inflammation.

MATERIALS AND METHODS

Extraction of spirulina platensis

S. platensis powder from PT Algae park, Klaten-Indonesia (1000 g) was macerated using 96% ethanol and left to stand for 48 h at room temperature. The solution was then filtered to obtain precipitate, which was further macerated using 96% ethanol. A rotary evaporator was used to evaporate the solvent, which was then freeze-dried.

Materials

The tools used for the in silico study are a laptop (HP ENVY x360 13-AG0023AU). AMD Ryzen 7 2700 8GB 1TB Win 13 (Logical Processors 8) 8GB of memory RAM, a 64-bit operating system, an x64-based processor, and an Autodock 1.5.7 System. Micropipette 1000 µl**, 1.5 ml microtubes, tips, 96-well plate Biologyx, glassware, analytical balance (Sartorius). The chemicals used were Ethanol (BrataChem), Water proinjection (Sigma Aldrich), and a spectrophotometer (Varisocan, Thermo).

LCMS/MS analysis of a 96% ethanol extract of Spirulina platensis

The liquid chromatography-tandem mass spectrophotometry (LC-MS/MS) was conducted in PT Saraswanti INDO GENETECH, Bogor-Indonesia, under certificate number: SIG. LHP. VII.2023.211434171. the methodology employed was based on protocol 18-16/MU/SMM/-SIG (LCMS/MS), using a Quadrupole Time-of-Flight (QTOF) system. [21, 22]. Mass spectra were acquired using the AB Sciex 3200 Q-Trap LCMS/MS with the Perkin Elmer FX 15 UHPLC system (MA, USA). The negative ion mass spectra were obtained from the LC Q-Trap MS/MS detector in full ion scan mode (100 to 1200 m/z for the full scan and 50–1200 m/z for the MS/MS scan) at a scanrate of 0.5 Hz. The hyphenated system was supported with mass spectrometry software and a spectral library provided by ACD Labs (TO, CA). Analyte separation was carried out on a pre-packed C18 (4 × 250 mm, 5 μm, Phenomenex) column with a gradient mobile phase comprising water (solvent A) and ethanol with 1% acetonitrile (solvent B), each containing 0.1% formic acid and 5 mmol ammoniumformate. The gradient program commenced with 80% to 90% solvent B from 0.01 to 11.00 min with a flow rate of 1.0 ml/min. The injection volume was set to 20 µl**. All chromatographic procedures were performed at ambient temperature, and the corresponding peaks from the TOF Analyser of the 96% ethanol extract of S. platensis were identified by comparison with the literature data/ACD labs mass spectral library i. e., Flavonoid, Peptide, and Phenols.

Preparation of quality and calibration standard solutions

A 100 µg/ml solution of S. platensis extract in 96% ethanol was prepared using the mobile phase. Quality and calibration controls were processed with plasma blank samples containing flavonoid, peptide, and phenol standards. Eight calibration levels (1, 5, 35, 150, 350, 600, 900, and 1200 ng/ml) were created using the spike method on plasma blanks. Lower-QC (3 ng/ml), Medium-QC (6 ng/ml), and Higher-QC (9 ng/ml) solutions were also prepared. All processed solutions were stored at −20 °C until analysis.

Protocol for sample preparation

Each spiked plasma sample of 50μl was mixed with 250μl of methyl alcohol having0.1%HCOOHtoprecipitatetheproteins present in the mixture. The resultant mixture was subjected to vortex mixing for 10 min. Then these sample solutions were centrifuged for20 min at 4.0 °C. After that, 150μl of supernatant liquid was relocated to polypropylene tubes, from which an aliquot of 5μl of samples was infused into the LC-MS/MS system.

C-phycocyanin

C-phycocyanin, sourced from PT Algaepark in Klaten, Indonesia, was used as the primary phytochemical standard at a concentration of 1 mg/ml. Water purification systems were used for all research involving water. The spectrophotometric quantification of c-phycocyanin was performed using a series of standard dilutions. A 1 mg/ml C-phycocyanin solution was serially diluted to 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml, 0.0625 mg/ml, and 0 mg/ml (blank) in 1.5 ml microtubes.

Ligand and protein preparation for an in silico study

C-phycocyanin as a ligand from phytoconstituents of S. platensis was downloaded from the PubChem database (http://pubchem.ncbi.nlm.nih.gov) in SDF structure format. The structure of c-phycocyanin (Structure2D_CID_6438349) was converted from the SDF to the PDB structure format using Marven Sketch software. The prepared PDB protein is used in the virtual screening docking method of PyRx tools.

The protein used for in silico study is the TNF-⍺ protein (PDB ID 7JRA). TNF-protein preparations were obtained from the UniProt website (https://www.uniprot.org/uniprotkb/P01375/entry#structure). This protein classification is a cytokine in Homo sapiens, and an expression system in Escherichia coli BL21 (DE3), with no mutation. TNF-⍺ has 3 chains: A, B, and C chains, with sequence lengths of 160, and has 2 native ligands, VGY and GOL [23].

The 3D protein crystal structures were retrieved from the RCSB Protein Data Bank with protein preparation. Using AutoDockTools version 1.5.7 software and protein optimization, the water was removed, hydrogen polar only was added, hydrogen non-polar was merged, and Gasteiger charge was added. The docking was performed using AutoDock4. The run Genetic Algorithm (GA) was set to 100 times. The docking analysis was performed using PyMOL version 2.4.1 and Discovery Studio Visualizer for 3D visualization.

The grid parameter file was prepared and optimized in several grid points with the lowest RMSD (<2Å). The compounds of the screening results were re-docked with selected grid boxes. Based on molecular docking validation with a re-docking method between 7JRA and its native ligand (VGY), the optimum grid box is 30x30x30 with a binding energy value of −1.78 Å, an RMSD value of 2.72 Å, and an inhibition constant value of 213.97 uM. The optimum grid center is x =-14.265, y =-2.177, and z =-26.652 with 0.375 Å spacing for the default setting. The native ligand VGY has bindings with Gly127, Tyr195, Leu196, Tyr227, Tyr135, Leu233, Gly198, and Ile123.

The docking analysis results displayed binding affinity values as several approximations were used to model protein-ligand interactions. RMSD with a zero value was used to predict the highest predicted total binding energy to a ligand and was then selected for analysis of 3D structures and 2D visualizations of ligand–residue interactions at their proper docking positions. The ligand-protein interactions were visualized using the software Discovery Studio Visualizer, and we observed the formation of hydrogen and hydrophobic bonds with the active site.

RESULTS AND DISCUSSION

LCMS/MS analysis of a 96% ethanol extract of Spirulina platensis

LCMS/MS analysis of a 96% ethanol extract of Spirulina platensisis shown in fig. 1 and table 1. A few compounds have been identified and enlisted based on the literature data and the Advanced Chemistry Development (ACD) Labs based mass spectral library i. e., flavonoid, peptide, and phenol.

In this present study, possible active compounds such as flavonoid (Maltol and Morin), peptide (Cyclo Pro-Ala, Cyclo Pro-Pro, and Thymine), and phenol (m-Aminophenol, N-Methyltyramine, and Tyramine) have been identified from a 96% ethanol extract of S. platensis through LCMS/MS analysis. Until recently, the most common chemicals identified from S. platensis extract have been flavonoids, peptides, phenolic compounds, carotenoids, phycobiliproteins, chlorophyll, polyunsaturated fatty acids, sulphated polysaccharides, and sterols [24].

In fig. 2, the intensity of maltol is 3.83, cyclo (Pro-Pro) is 4.25 and morin is 14.00. These three main components have a high intensity of more than 20,000. This result is shown in table 2.

Fig. 1: The BPI Plot of 96% ethanol extract of Spirulina platensis

Table 1: Confirmed component summary of 96% ethanol extract of Spirulina platensis

Component name Formula Identification status Observed RT (min) Mass error (ppm) Total fragments found Isotope match Mz RMS PPM Isotope match intensity RMS percent Response Adducts
Biotin C10H16N2O3S Identified 7.03 0.2 12 0.86 2.75 18169 +H
Biotin C10H16N2O3S Identified 7.10 -0.6 14 0.93 2.83 18194 +H
Cyclo(Pro-Ala) C8H12N2O2 Identified 3.16 -0.9 2 2.22 8.55 8789 +H
Cyclo(Pro-Ala) C8H12N2O2 Identified 3.17 -0.8 4 1.24 8.44 7722 +H
Cyclo(Pro-Pro) C10H14N2O2 Identified 4.24 -0.8 5 1.07 9.70 20112 +H
Cyclo(Pro-Pro) C10H14N2O2 Identified 4.25 -0.8 10 2.12 9.75 19671 +H
Maltol C6H6O3 Identified 3.82 -0.4 1 1.21 5.54 30077 +H
Maltol C6H6O3 Identified 3.83 -0.8 1 0.98 4.48 30691 +H
m-Aminophenol C6H7NO Identified 0.95 0.4 3 1.98 5.18 3041 +H
m-Aminophenol C6H7NO Identified 0.94 -1.6 4 3.56 9.50 2588 +H
Morin C15H10O7 Identified 14.05 0.7 8 0.76 4.07 63525 +H
Morin C15H10O7 Identified 14.00 0.3 5 0.64 3.63 46487 +H
N-Methyltyramine C9H13NO Identified 8.04 -0.3 9 0.56 5.54 6198 +H
N-Methyltyramine C9H13NO Identified 8.05 -3.4 7 3.64 6.17 6281 +H
Thymine C5H6N2O2 Identified 2.03 4.3 3 4.55 8.53 368 +H
Thymine C5H6N2O2 Identified 2.03 1.0 1 1.83 9.29 403 +H
Tyramine C8H11NO Identified 4.62 -1.7 2 4.09 6.67 4509 +H
Tyramine C8H11NO Identified 4.66 -1.5 2 1.52 9.61 4567 +H

Fig. 2: Component plot of maltol, cyclo (Pro-Pro) and morin

Table 2: ESI mode with positive value

ESI mode Compound name Result
(+) Flavonoid (Maltol) Positive
(+) Flavonoid (Morin) Positive
(+) Peptide (Cyclo Pro-Ala) Positive
(+) Peptide (Cyclo Pro-Pro) Positive
(+) Peptide (Thymine) Positive
(+) Phenol (m-Aminophenol) Positive
(+) Phenol (N-Methyltyramine) Positive
(+) Phenol (Tyramine) Positive

In this present study, LC-MS/MS analysis of a 96% ethanol extract of S. platensis revealed flavonoid, peptide, and phenolic compounds. The most general compounds that have been reported in the 96% ethanol extract of S. platensis are flavonoids, peptide and phenolic compounds, carotenoids, phycobiliproteins, chlorophyll, polyunsaturated fatty acids, sulfated polysaccharides, and sterols. These reports strongly suggest that bioactive compounds such as the flavonoid present in the 96% ethanol extract of S. platensis are Maltol and Morin, and then peptide and phenol.

Further research verified the antioxidant capacity of Spirulina, establishing a basis for its development as a functional food. Spirulina has antioxidant peptides [25]. Our study found that a 96% ethanol extract of S. platensis contains peptides such as Cyclo Pro-Ala, Cyclo Pro-Pro, and Thymine, which may have antioxidant properties.

C-phycocyanin standard of 96% ethanol extract of Spirulina platensis

To determine whether or notc-phycocyanin is bound in the 96% ethanol extract of Spirulina platensis, a calibration curve was constructed by comparing the c-phycocyanin standard curve. As we know, c-phycocyanin is cultivated in Indonesia. Standard values for calibration using 1 mg/ml of c-phycocyanin range from serial dilution to 1 mg/ml of c-phycocyanin. Calibration curves were usedin Hycult Biotech 4-parameter logistic (4PL) across the operating range, and calibration was employed for quantitative analysis using 4PL regression. The concentration of c-phycocyanin in the 96% ethanol extract of Spirulina platensis is 229.2µg/mlas shown in fig. 3. This study shows that a 96% ethanol extract of S. platensis containsc-phycocyanin, a phycobiliprotein-like peptide that acts as an antioxidant and anti-inflammatory[15,19,26,27]. A study of s-phycocyanin in an animal model of Acute Myocardial Infarct showed that it prevents oxidative stress and inflammation because c-phycocyanin down-regulates iNOS, COX2, and phospho-NFκB p65, reducing the mRNA synthesis of IL1b and TNF-⍺ [15, 19].

 

 

Fig. 3: Calibration curve of C-phycocyanin

In silico results of molecular docking analysis

C-Phycocyanin has a binding affinity of-4.46 kcal/mol and an inhibition constant of 536.16 uM, with 1 atom in H-bond TYR195, compared to the native ligand, in fig. 4. The result of amino acid mapping in the c-phycocyanin complex areTyr195, Tyr227, Tyr135, Ile131, Leu133, Gly192, Ile134, Gly193, Leu133, and Leu233.

Fig. 4: Results of the complex interaction c-phycocyanin-7JRA

The molecular docking pose for the native ligand with the TNF-⍺ receptor binding site showed several important amino acid residues, such as His91, Tyr135, Tyr227, Ile231, Leu133, Tyr195, Gly198, Ile134, and Gly193. Through comparison with the c-phycocyanin conformation in the binding sites of TNF,Tyr195, Tyr227, Tyr135, Ile131, Leu133, Gly192, Ile134, Gly193, Leu133, and Leu233 with 1H-bond play an important role in enhancing ligand’s affinity. This complex shows an H-bond interaction with Tyr195 that can interact with the TNF-⍺ protein for inflammation.

C-phycocyanin, an antidiabetic inhibitor, inhibits α-amylase and α-glucosidase by binding to the active site and disrupting substrate-enzyme interaction [28]. C-phycocyanin has a binding energy of-4.46 kcal/mol to TNF, and it is the most common phytoconstituent that interacts with TNF-⍺. Our molecular docking investigation revealed that c-phycocyanin had the best amino acid bond interaction with the 7JRA protein. The findings suggest a significant connection between c-phycocyanin and 7JRA. It could be hypothesized that c-phycocyanin has the potential to prevent the activity of inflammatory agents. C-phycocyanin, known as one of the bioactive phytoconstituents with important biological functions in natural plant compounds, is available on Spirulina platensis [29, 30].

C-phycocyanin can reduce inflammation and enhance the antioxidant capacity of the liver and kidney via the gut microbiota and their metabolites [28]. Its antioxidant effect on the liver may be mediated by GSH and related enzymes, while in renal tissue, it works by activating the NRF2 pathway [31]. In silico studies with molecular docking showed the important residues involved in Hydrogen bonds and hydrophobic interactions [12, 32, 33]. In this study, we found that the C-phycocyanin-7JRA complex has a Hydrogen bond, in line with several studies in silico. Thus, C-phycocyanin was predicted to be ananti-inflammatory candidate. The analysis of the correlation between the Hydrogen bond and some hydrophobic interactions predicted they had good activity as anti-inflammatory activators.

CONCLUSION

AnLC-MS/MS technique was developed to measurea 96% ethanol extract of S. platensis. In this present study, LC-MS/MS analysis of a 96% ethanol extract of S. platensis revealed flavonoid, peptide, and phenolic compounds. The validation equation for c-phycocyanin in a 96% ethanol extract of S. platensis was found to be 229.2µg/ml. C-Phycocyanin has a binding affinity of-4.46 kcal/mol and an inhibition constant of 536.16 uM, with 1 atom in H-bond TYR195. C-phycocyanin shows potential in inhibiting the activity of an inflammatory agent in molecular docking analysis.

ACKNOWLEDGMENT

The researcher would like to thank PT Algaepark, Klaten-Indonesia for the S. platensis and C-phycocyanin provided to us.

FUNDING

This research has received funding from Hibah Riset UI 2022 PUTI Q2 Universitas Indonesia (contract no…).

AUTHORS CONTRIBUTIONS

TS gathers, analyzes data, and finalizes the manuscript. FF, ARP, and NSH conceptualized the study, supervised data collection and analysis, and contributed to manuscript preparation and review. All authors contributed to completing the manuscript.

CONFLICT OF INTERESTS

The authors report no financial or other conflicts of interest in this work.

REFERENCES

  1. Br R. A review on spirulina (Arthrospira platensis) for its antioxidant and neuroprotective effect. IJCAAP. 2020;4(4):116-9. doi: 10.18231/j.ijcaap.2019.024.

  2. Abadi KC, Iswanti FC, Jusman SW, Fadilah F, Prijanti AR. Spirulina platensis effect on oxidative stress of rat’s offspring brain exposed to cigarette smoke during pregnancy and lactation. Acta Biochim Indones. 2022;5(1):64. doi: 10.32889/actabioina.64.

  3. Ai X, Yu P, Li X, Lai X, Yang M, Liu F. Polysaccharides from Spirulina platensis: extraction methods, structural features and bioactivities diversity. Int J Biol Macromol. 2023 Mar;231:123211. doi: 10.1016/j.ijbiomac.2023.123211, PMID 36632963.

  4. Akbarizare M, Ofoghi H, Hadizadeh M. Dual effect of sapogenins extracted from Spirulina platensis on telomerase activity in two different cell lines. Mol Biol Res Commun. 2021 Mar;10(1):1-4. doi: 10.22099/mbrc.2020.38230.1537, PMID 33681391.

  5. Abouzed TK, Althobaiti F, Omran AF, Eldomany EB, El-Shazly SA, Alharthi F. The chemoprevention of Spirulina platensis and garlic against diethylnitrosamine induced liver cancer in rats via amelioration of inflammatory cytokines expression and oxidative stress. Toxicol Res (Camb). 2022 Feb;11(1):22-31. doi: 10.1093/toxres/tfab118, PMID 35237408.

  6. Agrawal M, Perumal Y, Bansal S, Arora S, Chopra K. Phycocyanin alleviates ICV-STZ induced cognitive and molecular deficits via PI3-kinase dependent pathway. Food Chem Toxicol. 2020;145:111684. doi: 10.1016/j.fct.2020.111684, PMID 32805344.

  7. Ahda M, Suhendra, Permadi A. Spirulina platensis microalgae as high protein-based products for diabetes treatment. Food Rev Int. 2024;40(6):1796-804. doi: 10.1080/87559129.2023.2238050.

  8. Chei S, Oh HJ, Song JH, Seo YJ, Lee K, Kim KJ. Spirulina maxima extract prevents activation of the NLRP3 inflammasome by inhibiting ERK signaling. Sci Rep. 2020;10(1):2075. doi: 10.1038/s41598-020-58896-6, PMID 32034213.

  9. Chen H, Qi H, Xiong P. Phycobiliproteins-a family of algae-derived biliproteins: productions, characterization and pharmaceutical potentials. Mar Drugs. 2022 Jul 9;20(7). doi: 10.3390/md20070450, PMID 35877743.

  10. Bobescu E, Balan A, Moga MA, Teodorescu A, Mitrica M, Dima L. Are there any beneficial effects of spirulina supplementation for metabolic syndrome components in postmenopausal women? Mar Drugs. 2020 Dec 17;18(12). doi: 10.3390/md18120651, PMID 33348926.

  11. Chen H, Zeng F, Li S, Liu Y, Gong S, Lv X. Spirulina active substance mediated gut microbes improve lipid metabolism in high-fat diet fed rats. Journal of Functional Foods. 2019;59:215-22. doi: 10.1016/j.jff.2019.04.049.

  12. Dagnino Leone J, Figueroa CP, Castaneda ML, Youlton AD, Vallejos Almirall A, Agurto Munoz A. Phycobiliproteins: structural aspects, functional characteristics, and biotechnological perspectives. Comput Struct Biotechnol J. 2022;20:1506-27. doi: 10.1016/j.csbj.2022.02.016, PMID 35422968.

  13. Agustina S, Aidha NN, Oktarina E. Ekstraksi antioksidan spirulina sp. dengan menggunakan metode ultrasonikasi dan aplikasinya untuk krim kosmetik. J Kimia Kemasan. 2018;40(2). doi: 10.24817/jkk.v40i2.4100.

  14. Abdelghany AK, Gamal A, Abdel Wahab A, Abdel Razik AH, El-Samannoudy SI, Ibrahim MA. Evaluating the neuroprotective effect of Spirulina platensis–loaded niosomes against Alzheimer’s disease induced in rats. Drug Deliv Transl Res. 2023 Oct;13(10):2690. doi: 10.1007/s13346-023-01301-2, PMID 36790720.

  15. Hao S, Yan Y, Huang W, Gai F, Wang J, Liu L. C-phycocyanin reduces inflammation by inhibiting NF-κB activity through downregulating PDCD5 in lipopolysaccharide-induced RAW 264.7 macrophages. J Funct Foods. 2018;42:21-9. doi: 10.1016/j.jff.2018.01.008.

  16. Aoki J, Sasaki D, Asayama M. Development of a method for phycocyanin recovery from filamentous cyanobacteria and evaluation of its stability and antioxidant capacity. BMC Biotechnol. 2021 Jun 16;21(1):40. doi: 10.1186/s12896-021-00692-9, PMID 34134665.

  17. Braune S, Kruger Genge A, Kammerer S, Jung F, Kupper JH. Phycocyanin from Arthrospira platensis as potential anti-cancer drug: review of in vitro and in vivo studies. Life (Basel). 2021 Jan 27;11(2). doi: 10.3390/life11020091, PMID 33513794.

  18. Brito AF, Silva AS, de Oliveira CV, de Souza AA, Ferreira PB, de Souza IL. Spirulina platensis prevents oxidative stress and inflammation promoted by strength training in rats: dose-response relation study. Sci Rep. 2020 Apr 14;10(1):6382. doi: 10.1038/s41598-020-63272-5, PMID 32286405.

  19. Blas Valdivia V, Moran Dorantes DN, Rojas Franco P, Franco Colin M, Mirhosseini N, Davarnejad R. C-Phycocyanin prevents acute myocardial infarction-induced oxidative stress, inflammation and cardiac damage. Pharm Biol. 2022;60(1):755-63. doi: 10.1080/13880209.2022.2055089, PMID 35373708.

  20. Bulimaga V, Efremova N, Zosim L, Balan BL. The application of some coordination compounds in regulation of the content of antioxidants in Spirulina platensis; 2023.

  21. Ghosh D, Mondal S, Ramakrishna PK. Phytochemical profiling using Lc-Q-Tof-Ms analysis and in vitro antioxidant activity of a rare salt-secreting mangrove Aegialitis rotundifolia Roxb. leaves extract. Int J Pharm Pharm Sci. 2019;11(3):37-47. doi: 10.22159/ijpps.2019v11i3.29985.

  22. Nimmakayala MR, Kolli D, Durga Bhavani PN. Bioanalytical method development and validation of maralixibat in rat plasma by lc-ms/ms detection and its application to a pharmacokinetic study. Int J App Pharm. 2023;15(4):166-72. doi: 10.22159/ijap.2023v15i4.47768.

  23. Xiao HY, Li N, Duan JJ, Jiang B, Lu Z, Ngu K. Biologic-like in vivo efficacy with small molecule inhibitors of TNFα identified using scaffold hopping and structure-based drug design approaches. J Med Chem. 2020;63(23):15050-71. doi: 10.1021/acs.jmedchem.0c01732, PMID 33261314.

  24. Pantami HA, Ahamad Bustamam MS, Lee SY, Ismail IS, Mohd Faudzi SM, Nakakuni M. Comprehensive GCMS and LC-MS/MS metabolite profiling of Chlorella vulgaris. Mar Drugs. 2020 Jul 17;18(7). doi: 10.3390/md18070367, PMID 32709006.

  25. Shydlovska O, Zhang S. Preparation and identification of antioxidant peptides from Spirulina platensis [thesis]. Ukraine; 2023.

  26. Hikal MS, El-Sayed SM, Gabr GA. Antioxidant activities of phycocyanin: a bioactive compound from Spirulina platensis. J Pharm Res Int. 2020:73-85.

  27. Deepa J, Aleykutty NA, Jyoti H. Effect of combination of two plant extracts on diabetes mellitus. Int J Pharm Pharm Sci. 2018;10(4). doi: 10.22159/ijpps.2018v10i4.24100.

  28. Munawaroh SH, Gumilar GG, Nurjanah F. In vitro molecular docking analysis of microalgae extracted phycocyanin as an antidiabetic candidate. Biochem Eng J. 2020;161.

  29. Li X, Zhao M, Ding Z, Wang X, Zhang Z. C-phycocyanin ameliorates cyclophosphamide-induced liver injury and metabolic disorder through the modulation of the gut microbiota in mice. Algal Res. 2023;71. doi: 10.1016/j.algal.2023.103093.

  30. Li Z, Gan L, Yan S, Yan Y, Huang W. Effect of C-phycocyanin on HDAC3 and miRNA-335 in Alzheimer’s disease. Transl Neurosci. 2020;11(1):161-72. doi: 10.1515/tnsci-2020-0101, PMID 33312721.

  31. Han P, Li J, Zhong H, Xie J, Zhang P, Lu Q. Anti-oxidation properties and therapeutic potentials of spirulina. Algal Res. 2021;55. doi: 10.1016/j.algal.2021.102240.

  32. Zhang Y, Li L, Qin S, Yuan J, Xie X, Wang F. C-phycocyanin alleviated cisplatin-induced oxidative stress and inflammation via gut microbiota-metabolites axis in mice. Front Nutr. 2022;9:996614. doi: 10.3389/fnut.2022.996614, PMID 36225866.

  33. Azminah A, Erlina L, Radji M, Munim A, Syahdi RR, Yanuar A. In silico and in vitro identification of candidate SIRT1 activators from Indonesian medicinal plants compounds database. Comput Biol Chem. 2019;83:107096. doi: 10.1016/j.compbiolchem.2019.107096, PMID 31377446.

  34. Fadilah F, Kezia I, Erlina L. Uncovering potential neuroprotective flavonoids for Alzheimer’s disease using cutting-edge molecular simulation and in vitro SHSY-5Y analysis. J Pharm Pharmacogn Res. 2024;12(2):204-17. doi: 10.56499/jppres23.1715_12.2.204.