Bioactive compounds and in vitro biological properties of Arthrospira platensis and Athrospira maxima: a comparative study

Abstract

Cyanobacteria, especially Arthrospira, are valuable resources of nutrients and natural pigments with many beneficial health-related properties. This study optimized the extraction conditions of Arthrospira to achieve high phenolic contents and antioxidant activities. Under optimized extraction conditions, the bioactive compounds (phenolics and pigment components), antioxidant activities, and inhibitions of the key enzymes relevant to some non-communicable diseases were compared between Arthrospira platensis and Arthrospira maxima. Optimized extraction conditions were determined as 2 h shaking time, 50 °C extraction temperature, and 1% (w/v) solid-to-liquid ratio, giving effective phenolic and phycocyanin contents using aqueous extraction, while 80% (v/v) aqueous ethanolic extraction provided high total chlorophyll content. Most antioxidant activities were higher using 80% (v/v) aqueous ethanolic extracts. Both Arthrospira species inhibited the key enzymes involved in controlling non-communicable diseases including hyperlipidemia (lipase), diabetes (α-amylase, α-glucosidase, and dipeptidyl peptidase-IV), Alzheimer’s disease (acetylcholinesterase, butyrylcholinesterase and β-secretase), and hypertension (angiotensin-converting enzyme). High inhibitory activities were detected against β-secretase (BACE-1), the enzyme responsible for β-amyloid plaque formation in the brain that acts as a significant hallmark of Alzheimer’s disease. Arthrospira extract and donepezil (Alzheimer’s disease drug) synergistically inhibited BACE-1, suggesting the potential of Arthrospira extracts as effective BACE-1 inhibitors. Interestingly, A. maxima exhibited higher bioactive compound contents, antioxidant activities, and key enzyme inhibitions than A. platensis, indicating high potential for future food and medicinal applications.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-74492-4.

Introduction

The interest in natural products as green medicine and sources of nutrients or bioactive ingredients with wide varieties of health benefits has been increasingly focused^1–3. Arthrospira, a highly nutritious blue-green algae, is an excellent source of protein and has numerous human health benefits⁴. Arthrospira is rich in nutrients that are essential for a balanced diet, such as proteins (55–70% with all essential amino acids), carbohydrates (12–25%), and lipids (4–8.2% with health-beneficial fatty acids, including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), 3,6 γ-linolenic acid, α-linolenic acid, stearidonic acid, and arachidonic acid)^5,6. Arthrospira has a high digestibility rate due to the absence of cellulose in its cell walls, which are composed of 86% digestible polysaccharides^7–9. Arthrospira is considered to be Generally Regarded As Safe (GRAS) for consumption and has been approved by the US Food and Drug Administration (FDA) as a dietary health supplement since 2003, with recommended consumption for adults between 3 and 10 g/day and a maximum daily intake limit of 30 g^10,11.

The natural pigments from Arthrospira such as phycocyanin, chlorophyll, and carotenoids are well documented for their corresponding health benefits¹². Bioactive compounds with different polarities can be extracted from Arthrospira using appropriate solvent systems. Extraction is commonly conducted using an aqueous buffer solvent to obtain phycocyanin, with major extraction factors consisting of temperature, pH, and light exposure^13–15. However, water-based extraction cannot maximize the extraction of functional compounds from Arthrospira biomass because an aqueous solvent is not suitable for extracting groups of health-related compounds such as phenolics, chlorophylls, and carotenoids, which are poorly dissolved in water. Carotenoids and chlorophylls are best extracted using less polar solvents such as acetone and alcohol, respectively^16,17. These pigment compounds have attracted wide investigations^4,12, while scant information is available on phenolics, the secondary metabolites mostly found in plants. Interest in microalgae as a source of phenolics began during the last decade. In 2012, the number of research papers with the keywords microalgae and phenolics was less than 10 but climbed to more than 60 in 2019¹⁸. Phenolics possess significant medicinal properties, and their antioxidant activities have been previously investigated, with a strong correlation between phenolic contents and antioxidant activities frequently reported^19,20. Recently, different techniques have been employed to extract phenolics from Arthrospira biomass, as well as identifying phenolic profiles and antioxidant activities^21–24. Environmentally friendly and affordable instrumental techniques are, however, required to further investigate high phenolic contents extracted from Arthrospira. Besides, previous studies focused only on A. platensis and paid less attention to other Arthrospira species, resulting in difficulty in accurate comparison of phenolic contents and biological properties between species as well as further selection of specific species for particular purpose.

A. platensis and A. maxima are well recognized as valuable resources that enhance food taste and nutrition, improve agricultural practices, and offer bioactive substances for nutraceuticals²⁵. Numerous studies have optimized the extraction conditions of A. platensis to achieve high phenolic contents and health-related properties; however, only one report has optimized the extraction condition of A. maxima and assessed the extraction of carotenoids and their antioxidant activities²⁶. Therefore, this study optimized the environmentally friendly extraction conditions of A. maxima using extraction temperature, solid-to-liquid ratio, shaking time, and ethanol concentration using affordable instrumental techniques and also compared the bioactive compounds (phenolics, phycobiliproteins, and chlorophylls) and antioxidant activities between A. platensis and A. maxima. The in vitro health-related properties including antioxidant activities and inhibition of the key enzymes relevant to the control of non-communicable diseases such as hyperlipidemia, diabetes, Alzheimer’s disease, and hypertension of both Arthrospira extracts were also examined. The comparison of these biological activities led to the selection of Athrospira species with potential health properties for further analysis of the synergistic effects of Arthrospira extract and synthetic drugs. The results of this study provide an approach to develop novel functional ingredients, functional foods, and other health-promoting products with a wider range of health benefits from A. maxima and A. platensis biomass.

Materials and methods

Sample preparation

Collection of Arthrospira species was done at Kasetsart University, Bangkok, Thailand and in compliance with the institutional, national, and international guidelines and legislation. Two species of cyanobacteria, A. platensis IFRPD 1182 and A. maxima IFRPD 1183, were obtained from the Institute of Food Research and Product Development (IFRPD), Kasetsart University, Bangkok, Thailand. A. platensis and A. maxima were cultured in Zarrouk medium²⁷ and maintained at a controlled temperature of 30 °C and a light intensity of 12 Klux with 32-watt white fluorescents. Continuous air bubbles mixed with 2% carbon dioxide at a flow rate of 0.67 vessel volumes per min (vvm) were passed through 0.22 μm polytetrafluorethylene (PTFE) membrane filters²⁸. The cells were grown to log phase and used as inoculum at 10% in an open raceway pond system containing 200 L of working volume, which was well mixed using a paddle wheel at 15 rpm. The cells were grown to the exponential phase, around 1 at an optical density (OD) of 560 nm. After that, they were harvested by filtration through a nylon cloth and washed with tap water to remove residual culture medium²⁹. The resulting A. platensis and A. maxima biomass were dried at 55 °C for 6 h until less than 5% moisture content was achieved. The dried biomass samples were then milled to 0.5 mm particle size for use in further experiments.

Optimization of extraction conditions

A single-factor experiment was employed to investigate the optimized extraction conditions of Arthrospira species to receive high phenolic contents and antioxidant activities. In this study, A. maxima was chosen to represent Arthrospira species due to its lack of previous information. Besides, A. maxima exhibited a higher yield than A. platensis during algae growth and sample preparation. Ferric ion reducing antioxidant power (FRAP) assay was selected as a representative for antioxidant determination due to its simple and less time-consuming protocol, as described in Sect. 2.4, while the protocol of total phenolic contents (TPCs) was described in Sect. 2.3.

The independent variables, including extraction temperature (30–90 °C), solid-to-liquid ratio (1–4% w/v), shaking time (1–6 h), and ethanol concentration (0–100% v/v), were varied. Each experiment was performed under other controlled variables, with dependent variables reported as TPCs and FRAP activities. The extraction was performed as previously reported³⁰. Briefly, the oven-dried sample was mixed with a selected solvent at a particular solid-to-liquid-ratio. The mixture was shaken in a WNE45 water bath shaker (Memmert GmBh, Eagle, WI, USA) for the selected temperature and time period. The supernatant was collected through centrifuged using a 3800× g Hettich® ROTINA 38R refrigerated centrifuge (Andreas Hettich GmbH, Tuttlingen, Germany) for 10 min and filtered through a 0.22 μm polyethersulfone (PES) syringe filter. The filtrate was kept − 20 °C until further analysis.

Determination of bioactive compound contents

The determination of bioactive compounds in the extracts of both Arthrospira species from Sect. 2.2 included phycocyanin, allophycocyanin, phycobiliproteins, chlorophyll a, chlorophyll b, total chlorophyll, and TPCs. These bioactive contents were spectrophotometrically measured utilizing a SpectraMax Plus 384 microplate reader (Molecular Devices, LLC., Sunnyvale, CA, USA) with a SoftMax® Pro analysis software (version 6.5.1). All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The contents of phycocyanin (PC), allophycocyanin (APC), and phycobiliproteins (PBPs) extracted using aqueous condition and 80% (v/v) aqueous ethanol were determined according to the previous reports³¹ using Eqs. (1)-(3).

1

2

3

where OD₆₁₅ is the absorbance of the extract at 615 nm, and OD₆₅₂ is the absorbance of the extract at 652 nm. The results were expressed as mg/L.

The contents of chlorophyll a, chlorophyll b, and total chlorophyll extracted using 80% (v/v) aqueous ethanol were examined according to the previous reports³² using Eqs. (4)-(6).

4

5

6

where OD₆₆₄ is the absorbance of the extract at 664 nm, and OD₆₄₈ is the absorbance of the extract at 648 nm. The results were expressed as mg/L. The contents of chlorophyll a, chlorophyll b, and total chlorophyll extracted under aqueous conditions were unavailable due to the lack of the calculated equations for aqueous extraction.

Investigation of TPCs was performed using Folin-Ciocalteu’s phenol reagent, as previously reported³³. A detection wavelength at 765 nm and different concentrations of gallic acid (0–200 µg/mL) were used to generate a standard curve for TPC determination. The results were, thus, expressed as mg gallic acid equivalent (GAE)/g dry weight (DW).

The phenolic profile of both Arthrospira species from Sect. 2.2 was examined utilizing a liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) following a well-established procedure as previously reported^34,35. The extractions were performed under acidic hydrolysis and non-acid conditions. Briefly, the powdery sample (100 mg) was mixed with 62.5% (v/v) aqueous methanol containing 0.5 g tert-butylhydroquinone (TBHQ) (10 mL) and formic acid (40 mL). The mixture was shaken in an 80 °C water bath shaker (TW20 series from Julabo GmbH, Seelbach, Germany) for 2 h before putting it on ice for 5 min. Ascorbic acid (1% (v/v), 100 µL) was added to the mixture, which was then subsequently sonicated for another 5 min using an ultrasonic bath (Branson Ultrasonics™ M series, Branson Ultrasonics Corp., Danbury, CT, USA). The final volume was made up to 50 mL using 62.5% (v/v) methanol containing 0.5 g TBHQ to achieve the final concentration of 100 mg/mL. For non-acid conditions, both Arthrospira extracts under optimized extraction conditions in Sect. 2.2 were vacuum evaporated using a DTC-22 diaphragm vacuum pump (EYELA CO., LTD., Shanghai, China) until dryness. The dry residues were weighted and redissolved in 62.5% (v/v) aqueous methanol to achieve 20 mg/mL final concentration. The samples underwent acidic hydrolysis and non-acid conditions were filtered through a 0.22 µM PTFE syringe filter. The samples under acid hydrolysis were viscous; therefore, they were diluted with 62.5% (v/v) aqueous methanol containing 0.5 g TBHQ to the final 80 mg/mL concentration. All filtrates (10 µL) were loaded onto the LC–ESI–MS/MS system (Thermo Fisher Scientific, Bremen, Germany) consisting of a 2.1 mm × 100 mm, 2.6 μm Accucore RP-MS column, a Dionex Ultimate 3000 series ultrahigh-performance liquid chromatography (UHPLC), a diode array detector (DAD) and a TSQ Quantis Triple Quadrupole mass spectrometer (MS). The gradient mobile phase consisting of solvent A (acetonitrile) and solvent B (Milli-Q water (18.2 MΩ·cm resistivity at 25 °C) containing 0.1% (v/v) formic acid) with a constant flow rate of 0.5 µL/min was set in sequence as follows; time 0 min: 10% solvent A and 90% solvent B, time 0.1 min: 10% solvent A and 90% solvent B, time 8 min: 80% solvent A and 20% solvent B, time 8.1 min: 10% solvent A and 90% solvent B, and time 10 min: 10% solvent A and 90% solvent B. The positive and negative fragment ions were generated under multiple scanning modes (full scanning (FS) and selective reaction monitoring (SRM)) utilizing a 50–1000 m/z mass range, 3500 V positive and negative ions, 350 °C vaporizer, 325 °C ion transfer tube, 15 Arb N₂ auxiliary gas, and 30 Arb N₂ sheath gas. The authentic standards of 24 phenolics including quercetin (> 98.0% HPLC, E), syringic acid (> 97.0% T), 4-hydroxybenzoic acid (> 99.0% GC, T), hesperidin (> 90.0% HPLC, T), naringenin (> 93.0% HPLC, T), chlorogenic acid (> 98.0% HPLC, T), (−)-epigallocatechin gallate (> 98.0% HPLC), kaempferol (> 97.0% HPLC), apigenin (> 98.0% HPLC), caffeic acid (> 98.0% HPLC, T), p-coumaric acid (> 98.0% GC, T), ferulic acid (> 98.0% GC, T), sinapic acid (> 99.0% GC, T), luteolin (> 98.0% HPLC), genistein (> 98.0% HPLC), myricetin (> 97.0% HPLC), cinnamic acid (> 98.0% HPLC), and 3,4-dihydroxybenzoic acid (≥ 97% T) from Tokyo Chemical Industry (Tokyo, Japan); galangin (≥ 98.0% HPLC), gallic acid (97.5–102.5% T), and rutin (≥ 94% HPLC) from Wuhan ChemFaces Biochemical Co., Ltd. (Hubei, China); rosmarinic acid (≥ 98% HPLC) and vanillic acid (≥ 97% HPLC) from Sigma-Aldrich (St. Louis, MO, USA); isorhamnetin (≥ 99.0% HPLC) from Extrasynthese (Genay, France) were used to plot the standard curves with validations and parameters as shown in Supplementary Table S1.

Determination of antioxidant activities

The antioxidant activities of both Arthrospira species extracted under optimized extraction conditions were determined using both single electron transfer (SET)-based and hydrogen atom transfer (HAT)-based assays, including FRAP, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, and oxygen radical absorbance capacity (ORAC) assays as previously reported³⁰. All antioxidant assays were performed utilizing a 96-well SpectraMax Plus 384 microplate reader (Molecular Devices, LLC., Sunnyvale, CA, USA) and a SoftMax® Pro analysis software (version 6.5.1). Briefly, the end-point FRAP assay composed of reactive reagent containing FeCl₃·6Н₂O and 2,4,6-tri(2-pyridyl)-S-triazine in acetate buffer, while its detection wavelength was set at 600 nm. The end-point DPPH radical scavenging assay composed of DPPH radical reagent and the detection wavelength at 520 nm. The kinetically fluorescent ORAC assay composed of reactive reagents as sodium fluorescein and 2,2’-azobis(2-amidinopropane) dihydrochloride, while its detection wavelength was set as an excitation wavelength at 485 nm and an emission wavelength at 528 nm. All antioxidant assays used Trolox at different concentrations to generate a standard curve, and the results were reported as µmol Trolox equivalent (TE)/g DW. All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Determination of in vitro enzyme inhibitory activities

The in vitro health properties of both Arthrospira species extracted under optimized extraction conditions were performed through enzyme inhibitory potentials including lipase, dipeptidyl peptidase-IV (DPP-IV), α-amylase, α-glucosidase, β-secretase (BACE-1), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and angiotensin-converting enzyme (ACE). The protocols for these enzyme inhibitory assays were followed the previous reports³⁵, as indicated in Table 1. For each particular enzyme assay (except for the end-point BACE-1 and ACE inhibitory assays), the substrate and indicator were mixed with the extract in the 96-well plate before adding enzyme and measured inhibitory activities immediately using the 96-well SpectraMax Plus 384 microplate reader (Molecular Devices, LLC., Sunnyvale, CA, USA) and a SoftMax® Pro analysis software (version 6.5.1) to receive initial velocity (V_i) of the enzyme reaction. The percentage of inhibition (% inhibition) was calculated using Eq. (7).

7

Table 1.

The components of enzyme inhibitory assays consist of types and quantities of enzymes, substrates, indicators, extracts, and detection wavelengths.

Assay	Assay Components
	Enzyme	Substrate	Indicator	Extract	Detection Wavelength
Lipase	100 µL of 20 µg/mL lipase 1	50 µL of 0.2 mM DMPTB	10 µL of 16 mM DTNB	40 µL	412 nm
AChE	100 µL of 0.25 µg/mL AChE 2	50 µL of 0.32 mM ACh
BChE	100 µL of 1.5 µg/mL BChE 3	50 µL of 0.4 mM BCh
ACE	3 µL of 0.5 U/mL ACE 4	30 µL of 3 mM HHL	15 µL of 20 mg/mL o-PDA	50 µL	λex = 360 nm λem = 485 nm
DPP-IV	50 µL of 0.02 U/mL DPP-IV 5	25 µL of 12 mM Gly-Pro-pNA + 100 µL Tris-HCl (pH 8)		25 µL	405 nm
α-Amylase	100 µL of 0.06 mg/mL α-amylase 6	50 µL of 1 mM pCNM		50 µL
α-Glucosidase	10 µL of 0.2 U/mL α-glucosidase 7	25 µL of 10 mM pNPG + 160 µL KPB (pH 7)		5 µL
BACE-1	BACE-1 FRET assay kit (Sigma-Aldrich, St. Louis, MO, USA) following manufacturer’s recommendations				λex = 320 nm λem = 405 nm

¹Candida rugosa lipase (type VII, ≥ 700 unit/mg); ²Electrophorus electricus AChE (1000 units/mg); ³ equine serum BChE (≥ 10 units/mg); ⁴ rabbit lung ACE (≥ 2 unit/mg); ⁵ recombinant human dipeptidyl peptidase-IV (≥ 10 units/mg); ⁶ porcine pancreatic α-amylase (type VII, ≥ 10 unit/mg); ⁷Saccharomyces cerevisiae α-glucosidase (type I, ≥ 10 U/mg protein). ACh, acetylthiocholine; AChE, acetycholinesterase; BACE-1, β-secretase; BCh, butyrylthiocholine; BChE, butyrylcholinesterase; pCNM, 2-chloro-4-nitrophenyl-α-D-maltotrioside; DMPTB, 2,3-dimercapto-1-propanol tributyrate; DPP-IV, dipeptidyl peptidase-IV; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); FRET, fluorescence resonance energy transfer; HHL, hippuryl-histidyl-leucine; KPB, potassium phosphate buffer; pNPG, p-nitrophenyl-α-D-glucopyranoside; o-PDA, o-phthaldialdehyde.

where A is V_i of an enzyme reaction without the extract (control), a is V_i of the enzyme solvent (control blank), B is V_i of the enzyme reaction with the extract (sample), and b is V_i of the plant extract (sample blank). A positive control of each enzyme inhibitory assay was a commercially available drug that acted as an inhibitor of that particular enzyme. Those drugs were orlistat (lipase inhibitory assay), saxagliptin (DPP-IV inhibitory assay), acarbose (α-amylase and α-glucosidase inhibitory assays), donepezil (AChE, BChE, and BACE-1 inhibitory assays), and lisinopril (ACE inhibitory assay). The half-maximal inhibitory concentration (IC₅₀) was determined utilizing a nonlinear regression fit on a GraphPad Prism program (version 9.0, La Jolla, CA) as follows:

8

where Y is inhibition (%), X is concentration, and Int_max is maximum inhibition (%). All enzymes, chemicals, and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Synergistic effect between Arthrospira extract and donepezil

Synergistic potential of Arthrospira extract and donepezil was studied as previously described³⁶. Arthrospira extract, donepezil, and their mixture were tested for BACE-1 inhibition. The synergistic effects were determined using the theoretical value (TV) and the actual experiment value (EV), as shown in Eq. (9).

9

Interpretation is as follows: synergistic when the TV is more than 5% (> 5%) below EV, antagonistic when the TV is > 5% above EV, and additive effect when TV and EV differed by < 5%.

Statistical analysis

All experiments were performed in triplicate using three independent sets of samples (n = 3), and the results were reported as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Duncan’s multiple comparison tests with significant differences at p < 0.05 was used for more than two sets of data, while an unpaired t-test with significant differences at p < 0.05 was used for comparison between two sets of data (a statistical package for the social sciences, version 18 for Windows, SPSS Inc., Chicago, IL, USA).

Results

Optimization of extraction conditions

The extraction conditions of Arthrospira species were optimized using a single-factor experiment with independent variables such as extraction temperature, solid-to-liquid ratio, shaking time, and ethanol concentration (Figs. 1, 2, 3 and 4 and Supplementary Table S2-S5). Each experiment was performed to study the effect of a single independent variable on the dependent variables (TPCs and FRAP activities). Due to its higher yield and lack of previous information, A. maxima was chosen to represent Arthrospira species in this study. Extraction temperature was varied between 30 and 90 °C and performed under aqueous conditions with a shaking time of 2 h and solid-to-liquid ratio of 1% w/v. Results indicated that TPC and FRAP activity were optimized at 10.89 mg GAE/g DW and 9.80 µmol TE/g DW, respectively under an extraction temperature of 50 °C (Fig. 1). TPC and FRAP activity declined under lower and higher extraction temperatures than this optimal point. Thus, an extraction temperature of 50 °C was chosen to further determine the best solid-to-liquid ratio.

Figure 1.

Effects of different temperatures on total phenolic contents (TPCs) and ferric ion reducing antioxidant power (FRAP) activities of Arthrospira maxima IFRPD 1183. The letters indicated statistically different TPCs and FRAP activities at p  < 0.05 of a sample prepared under different independent variable conditions using one-way analysis of variance (ANOVA) and Duncan’s multiple comparison test. GAE, gallic acid equivalent; DW, dry weight; TE, Trolox equivalent.

Figure 2.

Effects of different solid-to-liquid ratios on total phenolic contents (TPCs) and ferric ion reducing antioxidant power (FRAP) activities of Arthrospira maxima IFRPD 1183. The letters indicated statistically different TPCs and FRAP activities at p  < 0.05 of a sample prepared under different independent variable conditions using one-way analysis of variance (ANOVA) and Duncan’s multiple comparison test. GAE, gallic acid equivalent; DW, dry weight; TE, Trolox equivalent.

Figure 3.

Effects of different shaking periods on total phenolic contents (TPCs) and ferric ion reducing antioxidant power (FRAP) activities of Arthrospira maxima IFRPD 1183. The letters indicated statistically different TPCs and FRAP activities at p  < 0.05 of a sample prepared under different independent variable conditions using one-way analysis of variance (ANOVA) and Duncan’s multiple comparison test. GAE, gallic acid equivalent; DW, dry weight; TE, Trolox equivalent.

Figure 4.

Effects of different ethanol concentrations on total phenolic contents (TPCs) and ferric ion reducing antioxidant power (FRAP) activities of Arthrospira maxima IFRPD 1183. The letters indicated statistically different TPCs and FRAP activities at p  < 0.05 of a sample prepared under different independent variable conditions using one-way analysis of variance (ANOVA) and Duncan’s multiple comparison test. GAE, gallic acid equivalent; DW, dry weight; TE, Trolox equivalent.

The effect of the solid-to-liquid ratio (1–4% w/v) on TPC and FRAP activity of A. maxima was investigated under aqueous extraction with a shaking time of 2 h and extraction temperature of 50 °C (Fig. 2). The optimized TPC was achieved at 1% (w/v) solid-to-liquid ratio, while TPC started to decline when the solid-to-liquid ratio increased from 2 to 4% w/v. Statistically insignificant FRAP activity was observed under all investigated solid-to-liquid ratios. Thus, a solid-to-liquid ratio of 1% w/v was chosen to further investigate shaking time.

The effect of shaking time (1–6 h) on TPC and FRAP activity of A. maxima was investigated under the controlled variables of aqueous extraction with a solid-to-liquid ratio of 1% w/v and extraction temperature of 50 °C (Fig. 3). The lowest TPC was detected at 1 h shaking time, while a longer shaking time of 2–6 h yielded a statistically insignificant increase in TPC. The highest FRAP activity was observed under shaking time of 2–4 h, while shorter and longer shaking times showed decreased FRAP activity. To promote effective extraction, a shaking time of 2 h was chosen for further investigation on the effect of ethanol concentration.

The control variables of 2 h shaking time, 50 °C extraction temperature, and 1% (w/v) solid-to-liquid ratio were chosen to investigate the effect of ethanol concentration (0–100% (v/v) aqueous ethanol) on TPC and FRAP activity of A. maxima. Results indicated that aqueous extraction yielded the highest TPC, while TPC declined with increasing ethanol concentration (Fig. 4). The FRAP activity was optimized under 80% (v/v) aqueous ethanolic extraction, with lower FRAP activity observed at other ethanol concentrations.

Since TPC and FRAP activity were optimized using different ethanol concentrations (aqueous extraction for TPC and 80% (v/v) aqueous ethanolic extraction for FRAP activity), both solvent systems under 2 h shaking time, 50 °C extraction temperature, and 1% (w/v) solid-to-liquid ratio were further investigated regarding the contents of potential bioactive compounds, antioxidant activities, and enzyme inhibitory activities.

Contents of bioactive compounds

PCPs comprise a phycobilin (chromophore-like)-protein complex that captures light energy and sends it to chlorophylls for photosynthesis. PC, a member of the PCP family, is a blue-green water-soluble algal pigment protein complex that absorbs light at 620 nm (orange-red color) and emits fluorescence at 650 nm (blue color). APC is also a protein complex sharing structural similarity in the PCP family, which can absorb and emit longer wavelengths than PC. In this study, the PC, APC, and PCP contents of both Arthrospira species extracted under aqueous and 80% (v/v) aqueous ethanolic conditions were examined using spectrophotometric methods (Table 2). Results indicated that both Arthrospira species extracted under aqueous conditions contained statistically insignificant PC contents, while A. maxima possessed slightly but significantly higher PC content than A. platensis under 80% (v/v) aqueous ethanolic extraction. Aqueous method extracted higher PC from both Arthrospira species (10.2–12.4-fold higher) than 80% (v/v) aqueous ethanolic extraction. The opposite results were observed for APC contents, with 80% (v/v) aqueous ethanolic extraction a more suitable solvent system at 3.8–4.1-fold higher. Under both solvent systems, A. maxima possessed 1.3–1.4-fold higher APC contents than A. platensis. The PCP contents, as the sum of PC and APC, were 1.1–1.4-fold higher in A. maxima than under A. platensis under both solvent systems. The solvent system insignificantly affected extraction yields of PCP from A. platensis but 80% (v/v) aqueous ethanol was determined as a more appropriate solvent to extract PCP from A. maxima.

Table 2.

The bioactive compounds in Arthrospira platensis IFRPD 1182 and Arthrospira maxima IFRPD 1183 were extracted using different solvent systems.

Bioactive Compounds	Aqueous Extraction		80% (v/v) Ethanolic Extraction
	A. platensis	A. maxima	A. platensis	A. maxima
Phycocyanin (PC, mg/L)	222.74 ± 13.80 aA	230.66 ± 13.21 aA	18.03 ± 0.09 bB	22.69 ± 2.72 aB
Allophycocyanin (APC, mg/L)	67.30 ± 6.01 bB	87.27 ± 5.01 aB	256.19 ± 18.77 bA	355.19 ± 2.58 aA
Phycobiliprotein (PCP, mg/L)	290.04 ± 19.81 bA	317.92 ± 18.22 aB	274.22 ± 18.83 bA	378.59 ± 5.30 aA
Chlorophyll a (mg/L)	NA	NA	30.17 ± 1.03 a	31.95 ± 0.17 a
Chlorophyll b (mg/L)	NA	NA	3.28 ± 0.79 b	11.00 ± 0.55 a
Total chlorophyll (mg/L)	NA	NA	33.46 ± 1.81 b	42.94 ± 0.50 a
TPCs (mg GAE/g DW)	6.12 ± 0.35 bA	8.02 ± 0.73 aA	1.95 ± 0.13 bB	2.35 ± 0.05 aB

All data are displayed as mean ± standard deviation (SD) of triplicated experiments from three independent sets of samples (n = 3). The superscript lowercase letters indicated statistically different contents of bioactive compounds in different samples prepared under the same extraction solvent, while superscript uppercase letters indicated statistically different contents of bioactive compounds in the same sample extracted under different solvent systems of at p < 0.05 using unpaired t-test. DW, dry weight; GAE, gallic acid equivalent; NA, not available; TPCs, total phenolic contents.

Chlorophyll, a green pigment in algal thylakoid membrane for photosynthesis, consists of chlorophyll a and b. The carbonyl moiety of chlorophyll b is more soluble in polar solvents than chlorophyll a. In this study, chlorophyll a, chlorophyll b, and total chlorophyll contents of both Arthrospira species extracted under 80% (v/v) aqueous ethanol were examined using spectrophotometric methods (Table 2). The results showed statistically insignificant chlorophyll a contents in both Arthrospira species, while A. maxima exhibited 3.4-fold higher chlorophyll b content than A. platensis, leading to 1.3-fold higher total chlorophyll content in the former. In both species, chlorophyll a contents were greater than those of chlorophyll b (2.9- and 9.2-fold higher in A. maxima and A. platensis, respectively).

The aqueous condition extracted 3.1–3.4-fold higher TPC than 80% (v/v) aqueous ethanolic extraction from both Arthrospira species (Table 2). A. maxima provided slightly higher TPC (1.2–1.3-fold higher) than A. platensis under both solvent systems. To identify types and quantities of phenolics utilizing LC–ESI–MS/MS, the samples were prepared under both acidic hydrolysis (final extract concentration of 80 mg/mL) and non-acid condition (optimized extraction conditions with final extract concentration of 20 mg/mL). No phenolics were detected under our authentic standards (flavonoids as apigenin, (−)-epigallocatechin galangin, gallate, genistein, hesperidin, isorhamnetin, kaempferol, luteolin, myricetin, naringenin, quercetin, rutin and phenolic acids as caffeic acid, chlorogenic acid, cinnamic acid, p-coumaric acid, 3,4-dihydroxybenzoic acid, ferulic acid, gallic acid, 4-hydroxybenzoic acid, rosmarinic acid, sinapic acid, syringic acid, and vanillic acid). Even though the retention times matched those of the standards, the MS/MS parameters of the parent and quantitative daughter ions were incomparable (the chromatograms and integration results are shown in Supplementary Figure S1-S6).

Antioxidant activities

Antioxidant activities of A. platensis and A. maxima were determined using DPPH radical scavenging, FRAP, and ORAC assays under the optimized conditions for TPC extraction (aqueous extraction at 50 °C, 2 h shaking time, and 1% (w/v) solid-to-liquid ratio) and FRAP activity (80% (v/v) aqueous ethanolic extraction at 50 °C, 2 h shaking time, and 1% (w/v) solid-to-liquid ratio). The results are shown in Table 3. Under aqueous extraction, A. maxima possessed stronger antioxidant strength than A. platensis, as determined by all three antioxidant assays (1.1–1.8-fold higher). A. platensis exhibited 1.1–1.5-fold higher DPPH radical scavenging and FRAP activities than A. maxima under 80% (v/v) aqueous ethanolic extraction, while A. maxima provided 1.2-fold higher ORAC activity. Comparing extraction solvents, A. platensis extracted with 80% (v/v) aqueous ethanol exhibited 1.4–8.4-fold higher FRAP and ORAC activities than under aqueous extraction, while DPPH radical scavenging activity was higher for aqueous extraction (1.6-fold) than under 80% (v/v) aqueous ethanolic extraction. Similar results were observed in A. maxima, with FRAP activity 3.0-fold higher and DPPH radical scavenging activity 1.9-fold lower in aqueous than in 80% (v/v) aqueous ethanolic extractions. Different solvent extractions had no effect on ORAC activities, while A. maxima exhibited insignificantly different ORAC activities in both solvent systems.

Table 3.

Antioxidant activities determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC) assays of Arthrospira platensis IFRPD 1182 and Arthrospira maxima IFRPD 1183 extracted under different solvent systems.

Antioxidant Activities (µmol TE/g DW)	Aqueous Extraction		80% (v/v) Ethanolic Extraction
	A. platensis	A. maxima	A. platensis	A. maxima
DPPH radical scavenging activities	11.16 ± 1.09 bA	12.76 ± 0.81 aA	7.15 ± 0.56 aB	6.73 ± 0.66 aB
FRAP activities	3.67 ± 0.32 bB	6.67 ± 0.46 aB	30.93 ± 0.66 aA	20.33 ± 1.25 bA
ORAC activities	65.58 ± 3.52 bB	103.70 ± 4.92 aA	90.45 ± 3.72 bA	104.25 ± 6.09 aA

All data are displayed as mean ± standard deviation (SD) of triplicated experiments from three independent sets of samples (n = 3). The superscript lowercase letters indicated statistically different antioxidant activities of different samples prepared under the same extraction solvent, while superscript uppercase letters indicated statistically different antioxidant activities of the same sample extracted under different solvent systems of at p < 0.05 using unpaired t-test. DW, dry weight; TE, Trolox equivalent.

Key enzyme inhibitory activities

Both Arthrospira species, A. maxima and A. platensis, extracted under aqueous and 80% (v/v) aqueous ethanolic systems were investigated for their potential in vitro health properties as inhibitors of the key enzymes relevant to hyperlipidemia (lipase), type II diabetes (α-amylase, α-glucosidase, and DPP-IV), Alzheimer’s disease (AChE, BChE, and BACE-1), and hypertension (ACE), with the results shown in Table 4. Inhibition of lipase, the enzyme that digests lipids to free fatty acids and glycerol before being absorbed into the body, leads to the control of hyperlipidemia. Results indicated that the 80% (v/v) aqueous ethanolic extracts of A. maxima and A. platensis exhibited lipase inhibitory activities of 39.92 and 47.44%, respectively while no activity was detected in aqueous extracts at a concentration of 2 mg/mL. Comparing between species, A. maxima exhibited 1.2-fold higher lipase inhibition than A. platensis.

Table 4.

Key enzyme inhibitory activities of Arthrospira platensis IFRPD 1182 and Arthrospira maxima IFRPD 1183 extracted under different solvent systems.

Key Relevant Enzymes	Enzyme Inhibition (% Inhibition)				Positive Controls (IC50)
	Aqueous Extraction		80% (v/v) Ethanolic Extraction
	A. platensis	A. maxima	A. platensis	A. maxima
Lipase 1	ND	ND	39.92 ± 3.41 b	47.44 ± 4.00 a	Orlistat (7.94 µM) *
α-amylase 1	ND	ND	ND	ND	Acarbose (14.58 µM) *
α-glucosidase 1	ND	ND	ND	ND	Acarbose (0.53 µM) $
DPP-IV 1	ND	ND	ND	ND	Saxagliptin (0.27 µM) *
AChE 1	ND	ND	ND	ND	Donepezil (3.12 µM) #
BChE 1	14.58 ± 0.92 a	7.25 ± 0.74 b	ND	ND	Donepezil (2.14 µM) #
BACE-1 2	54.23 ± 2.13 a	29.70 ± 2.76 b	60.35 ± 2.71 b	71.93 ± 3.20 a	Donepezil (0.14 mM) &
ACE 3	40.98 ± 0.86 bA	45.17 ± 0.69 aA	32.72 ± 2.06 bB	45.55 ± 3.30 aA	Lisinopril (1.79 nM)

All data are displayed as mean ± standard deviation (SD) of triplicated experiments from three independent sets of samples (n = 3). The superscript lowercase letters indicated statistically different enzyme inhibitory activities of different samples prepared under the same extraction solvent, while superscript uppercase letters indicated statistically different antioxidant activities of the same sample extracted under different solvent systems at p < 0.05 using an unpaired t-test. ACE, angiotensin-converting enzyme; AChE, acetylcholinesterase; BACE-1, β-secretase; BChE, butyrylcholinesterase; DPP-IV, dipeptidyl peptidase-IV; DW, dry weight; IC₅₀, half maximal inhibitory concentration; ND, not detected. ¹ Final extract concentration = 2 mg/mL; ² final extract concentration of aqueous extraction = 2 mg/mL and of 80% (v/v) ethanolic extraction = 0.5 mg/mL; ³ final extract concentration = 0.4 mg/mL. Information was obtained from * Chupeerach et al., 2022³⁵, ^$ Promyos et al., 2020³⁷, and ^# Temviriyanukul et al., 2022³⁸. ^& Experiment was performed in this study, in which donepezil exhibited IC₅₀ of 0.14 ± 0.01 mM or 53.86 ± 4.65 µg/mL against BACE-1, and lisinopril exhibited IC₅₀ of 1.79 ± 0.04 nM or 0.73 ± 0.02 µg/L against ACE.

The current medicinal treatments for type II diabetes emphasize on the inhibition of the carbohydrate hydrolyzing enzymes α-amylase and α-glucosidase and serum glucose involving enzyme, DPP-IV. No inhibitory activities against all three enzymes were observed in A. maxima and A. platensis extracted under both solvent systems using an extract concentration of 2 mg/mL.

The over-degradation of neurotransmitters by two cholinesterase enzymes, AChE and BChE, is hypothesized as one factor of Alzheimer’s disease occurrence. Alzheimer’s disease treatment involves the inhibition of cholinesterases by FAO-approved drugs. Inhibition of the amyloid-plaque formation enzyme, BACE-1, also leads to the control of Alzheimer’s disease and is used as a medicinal model for ligand-based drug design in Alzheimer’s disease treatment. Our results indicated that the two Arthrospira species extracted under both solvent systems exhibited no AChE inhibitory activity using an extract concentration of 2 mg/mL. However, under the same extract concentration, aqueous A. maxima and A. platensis extracts exhibited 7.25–14.58% inhibitions against BChE, while no inhibition was detected using 80% (v/v) aqueous ethanolic extracts. Under aqueous extraction, A. platensis possessed 2.0-fold higher BChE inhibitory activity than A. maxima. Interestingly, all extracts inhibited BACE-1, ranging from 29.70 to 71.93% inhibition. Aqueous-extracted A. platensis possessed 1.8-fold higher BACE-1 inhibitory activity than A. maxima extracted under the same solvent system using an extract concentration of 2 mg/mL. Opposite results were observed in 80% (v/v) aqueous ethanolic extraction, with A. maxima exhibiting 1.2-fold higher BACE-1 inhibitory activity than A. platensis using an extract concentration of 0.5 mg/mL. These results indicated that 80% (v/v) aqueous ethanolic extraction provided stronger BACE-1 inhibitory strength than aqueous extractions.

The inhibition of ACE, the enzyme that hydrolyzes the hormone angiotensin I (Ang I) to a vasoconstrictor angiotensin II (Ang II), causes restriction of vasopressin production as the primary treatment of hypertension. The results indicated that all Arthrospira species extracted under both solvent systems exhibited ACE inhibitory activities ranging from 32.72 to 45.55% using an extract concentration of 0.4 mg/mL. Under both solvent extractions, A. maxima exhibited 1.1–1.4-fold higher inhibitory activity than A. platensis. A. platensis extracted under aqueous solvent exhibited 1.3-fold higher ACE inhibitory activity than extractions under 80% (v/v) aqueous ethanol. However, the effect of solvent systems on ACE inhibition of A. maxima was minor, with insignificantly different inhibitory activities observed.

These in vitro health properties suggested that Arthrospira species contained effective anti-BACE-1 and ACE agents since all extracts exhibited inhibitory activities against these enzymes. The 80% (v/v) aqueous ethanolic extracts possessed stronger BACE-1 inhibitors than under aqueous extraction, while the latter contained more potent ACE inhibitors than the former. A. maxima extracted under 80% (v/v) aqueous ethanol provided BACE-1 inhibitory activity at more than 50%. Therefore, this extract was chosen to further investigate its synergistic effect with donepezil, an anti-Alzheimer’s disease drug.

Synergistic effect between Arthrospira maxima IFRPD 1183 extract and donepezil

As shown in Table 4, the ethanolic extract of A. maxima revealed promising anti- Alzheimer’s disease properties via BACE-1 inhibition. Several approaches have been proposed to treat Alzheimer’s disease including developing new pharmaceuticals or adding potential compounds, such as phytochemicals or small peptides, to increase the therapeutic efficacy of existing Alzheimer’s disease drugs. Consequently, the synergistic potential of donepezil (Alzheimer’s disease drug) and A. maxima ethanolic extract was assessed. Interestingly, the ethanolic extract of A. maxima exhibited a synergistic effect with donepezil in all combinations, especially when the IC₂₀ of donepezil was combined with the IC₃₀ of A. maxima extract, resulting in inhibition of BACE-1 by nearly 50% (Table 5). This result implied that the donepezil dose could be reduced by approximately 2.5-fold when combined with A. maxima extract, while BACE-1 inhibition remained at IC₅₀.

Table 5.

Synergistic effect of Arthrospira maxima IFRPD 1183 extracted under 80% (v/v) ethanolic extraction and donepezil (Alzheimer’s disease drug).

Samples		% Inhibition of BACE-1
Donepezil	A. maxima	Experimental Value (EV)	Theoretical Value (TV)	Interpretation
IC10 (5 µg/mL)		12.65 ± 0.27
IC20 (10 µg/mL)		20.02 ± 2.23
IC30 (20 µg/mL)		29.00 ± 0.35
	IC10 (3 µg/mL)	12.20 ± 0.51
	IC20 (6 µg/mL)	19.41 ± 0.13
	IC30 (16 µg/mL)	31.65 ± 1.94
IC10 (5 µg/mL)	IC10 (3 µg/mL)	29.56 ± 1.29	12.43	synergism
IC10 (5 µg/mL)	IC20 (6 µg/mL)	29.81 ± 0.35	16.03	synergism
IC10 (5 µg/mL)	IC30 (16 µg/mL)	38.03 ± 1.15	22.15	synergism
IC20 (10 µg/mL)	IC10 (3 µg/mL)	24.99 ± 0.41	16.11	synergism
IC20 (10 µg/mL)	IC20 (6 µg/mL)	30.85 ± 1.90	19.72	synergism
IC20 (10 µg/mL)	IC30 (16 µg/mL)	42.47 ± 0.03	25.84	synergism
IC30 (20 µg/mL)	IC10 (3 µg/mL)	26.14 ± 0.37	20.83	synergism
IC30 (20 µg/mL)	IC20 (6 µg/mL)	37.92 ± 0.71	24.21	synergism
IC30 (20 µg/mL)	IC30 (16 µg/mL)	46.36 ± 1.07	30.33	synergism

Experimental values are displayed as mean ± standard deviation (SD) of triplicated experiments (n = 3).

Discussion

Exploring the use of Arthrospira biomass in food production represents a compelling approach to developing novel functional food products. The two species of Arthrospira, A. platensis and A. maxima, differ in helical shape, distribution of gas vacuoles, coil diameter, and architecture^25,39. The trichomes of A. maxima are larger and not constricted at the cross-walls, while A. platensis is characterized by short trichomes with a constant diameter of the loose coils⁴⁰. Both species have been widely investigated in many aspects but only optimized extraction conditions with specific instrumental requirements aiming for high phenolics have been reported in A. platensis²¹, while no information is available on A. maxima. Thus, this study performed extraction conditions of A. maxima using a simple method. A. platensis and A. maxima as closely related species were subjected to the same extraction conditions, and the extracts were compared for their bioactive compounds, antioxidant activities, and key enzyme inhibitions.

In this study, TPC extraction was optimized using aqueous conditions, 2 h shaking time, 50 °C extraction temperature, and 1% (w/v) solid-to-liquid ratio using A. maxima as a representative study. Even though most literatures had reported the conventional method of phenolic extraction from plants using a mixture of water and organic solvents (ethanol, methanol, acetone, etc.), the decoction technique (hot water extraction) was suitable for heat-stable and water-soluble phenolics with high polarity index⁴¹. As for Arthrospira, it was found that most literatures had used a mixture of water and organic solvents to extract phenolics^22,24. However, aqueous extraction of A. platensis at high temperature (80 °C) and a short time (10 min) was also reported²⁰, while none on A. maxima was available. It was possible that phenolics in aqueous extracted A. maxima were the phenolic glycosides (rather than phenolic aglycones) or other water-soluble phenolics. Besides, the optimal temperature at 50 °C was selected since higher temperatures (70–90 °C) might degrade most phenolics, while lower temperature (30 °C) might not be able to break bacterial cell walls to release phenolics. Besides, 2–6 h of shaking time yielded optimal TPCs; therefore, 2 h shaking time was selected to extract TPCs at 50 °C. It was possible that phenolics extracted at this particular temperature was stabled up to 6 h in the aqueous solution. Interestingly, the low solid-to-liquid ratio of 1% (w/v) yielded greater TPCs than the higher ones (2–4% w/v). According to the mass transfer principle⁴², the increased interactions between the solid surface of A. maxima and water at a low solid-to-liquid ratio were achieved, causing a driving force to be elevated during the mass transfer of phenolics through bacterial cell walls into the water. Even though TPCs were optimized in aqueous extraction, FRAP activity was optimized using 80% (v/v) aqueous ethanolic extraction, suggesting the existence of bioactive compounds (other than phenolics) with high antioxidant activities or phenolics with lower polarity index (than the ones extracted with water) in ethanolic fractions. Due to the variations in phenolic contents and antioxidant activities, both solvent systems were selected for further investigation on bioactive compounds, antioxidant activities, and key enzyme inhibitions.

Under these optimized extraction conditions, the aqueous condition extracted higher PC than 80% (v/v) aqueous ethanol, while the latter extracted higher APC. This result concurred with previous literature detailing that PC was frequently extracted using aqueous conditions^16,31,32,43. Our results gave PC contents of 0.22–0.23 mg/mL or 22.27–23.37 mg/g DW, slightly lower than previous reports of aqueous extraction of A. platensis with PC content of 0.64–0.70 mg/mL extracted by repeated freezing and thawing⁴³. Another study reported that A. platensis aqueous extraction at 0.08 g/mL concentration, 25 °C, and 4 h exhibited higher PC content of 3.68 mg/mL³¹. Our results showed 11-fold lower PC content than the aqueous extraction of A. platensis (4 times of sonication, each for 30 min), with PC content of 251.2 mg/g¹⁶, and 2-fold lower than dichloromethane/methanol (1:1, v/v) extracted A. maxima (homogenization), which exhibited PC content of 45.3 mg/g⁴⁴. Lower PC contents detected in our experiment might be due to our optimized extraction conditions, which were more suitable for extracting phenolics rather than PC. Besides, the buffer used in the previous reports is more suitable for extracting PC than water used in our experiment since the salt in the buffer helps stabilize proteins^16,31,43. Thermal treatment at 50 °C for 2 h might also degrade proteins in our experiment, while others performed PC extraction at lower temperature^31,43. Interestingly, we found that APC contents were higher in 80% (v/v) aqueous ethanolic extraction than in aqueous condition, even though ethanolic condition could degrade most proteins. Our APC contents (0.36–0.56 mg/mL) in aqueous ethanolic extraction were also higher than reported by aqueous extraction (0.29–0.33 mg/mL)⁴³. It was possible that the interference of chlorophyll detected using a nearby wavelength could cause an overestimation of APC contents⁴⁵. Besides, the interference of phycocyanobilin, a chromophore of PC and APC that was previously reported to be effectively extracted using ethanol, was also possible⁴⁶. Thus, the contents of PC, APC, and PCP in 80% (v/v) aqueous ethanolic extracts might be overestimated and could not be used to explain the biological activities of these ethanolic fractions.

Our results also indicated that 80% (v/v) aqueous ethanol could extract higher chlorophyll a content than chlorophyll b in both Arthrospira species, and A. maxima provided higher chlorophyll contents than A. platensis. A previous study on different solvent systems supported our findings that ethanol was suitable for extracting chlorophyll¹⁷. Our chlorophyll a contents (30.17–31.95 mg/L or 3.02–3.20 mg/g DW) were higher than previously reported. A 90% (v/v) methanolic extraction of A. platensis (repeated freezing and thawing) exhibited chlorophyll a content ranging from 5.93 to 6.04 mg/L⁴³, while 50% (v/v) aqueous ethanolic A. platensis extract exhibited chlorophyll a content of 0.57 mg/g DW²⁴. The 80% acetone extraction of A. platensis exhibited chlorophyll a content of 2.54–3.29 mg/L³². However, our chlorophyll a contents were lower than A. maxima under ultrasonication extraction (0.52 kHz frequency, 32.59 °C, and 4.91 h), exhibiting chlorophyll a content of 17.98 mg/g, while conventional 70% (v/v) ethanolic extraction at 80 °C for 24 h yielded 13.81 mg/g⁴⁷. Dichloromethane/methanol (1:1, v/v) extracted A. maxima (homogenization) exhibited total chlorophyll content of 7.69 mg/g⁴⁴, while the aqueous extraction of A. platensis (4 times of sonication, each for 30 min) exhibited chlorophyll a of 10.8 mg/g¹⁶. Differences in chlorophyll contents resulted from the diverse extraction conditions (methods, solvents, temperatures, times, etc.) as well as the internal and external factors of Arthrospira biomass (such as strains, growth, and post-harvest conditions). Nevertheless, the chlorophyll contents were undetermined in our extracts using aqueous condition due to lack of calculated equations, while the previous literature used high performance liquid chromatography (HPLC) technique to quantify chlorophyll contents¹⁶.

The aqueous conditions also extracted higher TPC (6.12–8.02 mg GAE/g DW) than 80% (v/v) aqueous ethanol in our experiment, similar to previous research results. Aqueous extraction (80 °C, 10 min) of A. platensis yielded TPC of 43.2 mg GAE/g²⁰, while extraction with acidic methanol (20 °C, 2 h) and 50% (v/v) aqueous ethanol exhibited TPC of 0.14-6.04 mg GAE/g DW^22,24. Under high pressure/temperature extraction (HPTE), A. platensis exhibited TPC of 3.32 mg GAE/g dry biomass (DB)²¹. However, under optimized extraction conditions using response surface methodology based on the HPTE technique (aqueous ethanol ranging 20–60% (v/v) at 180 °C), TPC increased to 28.4 mg GAE/g DB²¹. Only two previous reports detailed the extraction of phenolics from A. maxima and neither was performed under aqueous conditions. The ethanolic extract (homogenization) of A. maxima exhibited TPC of 4.51–16.96 mg GAE/g DW²³, while extraction under dichloromethane/methanol (1:1, v/v) (homogenization) exhibited TPC of 1.32 mg GAE/g⁴⁴. TPC also exhibited strong correlations with chlorophyll a and b contents²⁴, with better correlation with chlorophyll b than with chlorophyll a contents. No phenolics were detected by LC-ESI-MS/MS analysis in our experiments; however, the previous research suggested acacetin, pinocembrin, catechin, epicatechin, pyrocatechol, vanillic acid, gallic acid, and syringic acid in A. platensis^20–22, while gallic acid, p-OH-benzoic acid, chlorogenic acid, cinnamic acid, and pinostrobin were detected in A. maxima²³. Different extraction conditions and detection methods (ethanolic, acidic methanol or HPTE extractions with HPLC analysis)^20–23 impacted these findings. Besides, TPCs of less than 10 mg GAE/g DW were considered low compared to other plants. Our extracts might also contain some phenolics excluded from our standard list (most are phenolic aglycones). Since greater TPCs were detected in the aqueous than in 80% (v/v) aqueous ethanolic extracts, it was possible that phenolics might be in a form of phenolic glycoside. Therefore, according to these reasons, even though we can detect phenolic contents using spectrophotometric methods (TPCs), the types and quantities of particular phenolic were not detected using LC-ESI-MS/MS method.

Under different extraction conditions, antioxidant activities determined by FRAP (20.33–30.96 µmol TE/g DW) and ORAC (90.45–104.25 µmol TE/g DW) assays were optimized under 80% (v/v) aqueous ethanolic extraction, while high DPPH radical scavenging activities (11.16–12.76 µmol TE/g DW) were achieved under aqueous conditions. A. platensis and A. maxima extracted under different extraction solvents possessed various antioxidant strengths⁴⁸. Aqueous extraction of A. platensis (4 times of sonication, each for 30 min) exhibited DPPH radical scavenging activity of 18.5 µmol TE/g¹⁶, while extraction with acidic methanol (20 °C, 2 h) exhibited DPPH radical scavenging activity of 1.64 µmol TE/g DW²². A. platensis extracted under 50% (v/v) aqueous ethanol exhibited ORAC activity of 295.87-393.24 µmol TE/g DW depending on extraction time (0–180 min)²⁴. The ethanolic extract of A. maxima (homogenization) exhibited DPPH radical scavenging activity with IC₅₀ values ranging from 23.22 to 35.62 µg/mL²³, while 80% (v/v) aqueous ethanolic extraction (50 °C, 24 h) exhibited ORAC activity of 396 µmol TE/g extract²⁶. A. maxima at 100 mg/mL under ultrasonication extraction with 20.52 kHz frequency at 32.59 °C for 4.91 h exhibited DPPH radical scavenging activity of 69.38%, which was 1.2-fold higher than conventional extraction (70% (v/v) aqueous ethanol, 80 °C, and 24 h)⁴⁷. Animal studies and clinical trials have shown that Arthrospira has positive effects on improving disease-related symptoms through the antioxidation capacity of PC¹⁵, which scavenges hydroxyl, alkoxyl, and peroxyl radicals as well as suppressing inducible nitric oxide synthase (iNOS) expression, reducing nitrite production, and inhibiting lipid peroxidation^49–51. The purified PC possessed DPPH radical scavenging activity as strong as ascorbic acid (a control standard), while lower activities than ascorbic acid were observed in FRAP, superoxide-radical scavenging (SRSA), and reducing power (RP) assays⁵². These previous data supported our findings that aqueous extracts contained higher PC contents and DPPH radical scavenging activities than 80% (v/v) aqueous ethanolic extraction. Chlorophyll a was responsible for the high FRAP and ORAC activities found in 80% (v/v) aqueous ethanolic extracts. A previous report suggested that the DPPH radical scavenging assay was not a suitable method for unusually highly polar chlorophylls⁵³. Despite this, most reported antioxidant activities of chlorophyll a were performed using the DPPH radical scavenging assay, while FRAP and ORAC activities were not reported⁵⁴.

Arthrospira species under particular extraction conditions exhibited enzyme inhibitory activities, with 80% (v/v) aqueous ethanolic extracts exhibiting lipase inhibitory activities, suggesting its role in the control of obesity or hyperlipidemia. Our results concurred with previous research, suggesting that Arthrospira (unknown species) intake (50 mg/kg/day for 4 weeks) decreased total serum cholesterol and triglyceride levels as well as malondialdehyde content by 22, 31, and 56%, respectively in streptozotocin (STZ)-induced diabetic rats²², while an A. platensis-fed rat possessed reduced serum triacylglycerol incretion after 2 h of oral administration of lipid emulsion⁵⁵. A clinical study in obese patients with A. platensis consumption (2 g/day for 12 weeks) also indicated significantly lower levels of body mass index (BMI), body fat, triglycerides, waist circumference, and high sensitivity C reactive protein⁵⁶. Interestingly, chlorophyll a could reduce the release rate of free fatty acids and pancreatic lipase activity during in vitro intestinal digestion⁵⁷. Thus, chlorophyll a that was predominantly detected in our 80% (v/v) aqueous ethanolic extracts might be responsible for lipase inhibitory property in Arthrospira extracts.

In our study, no inhibitory activities were detected in the key enzymes related to type II diabetes. However, previous research indicated a 20% reduction in serum glucose in Arthrospira (unknown species) fed STZ-induced diabetic rats²². Combining this result with our findings suggested that Arthrospira possessed anti-hyperglycemic activity through molecular pathways other than inhibiting the carbohydrate degrading enzymes (α-amylase and α-glucosidase) and glucose homeostasis involving the DPP-IV enzyme.

Interestingly, both A. platensis and A. maxima exhibited different degrees of ACE inhibition under aqueous and 80% (v/v) aqueous ethanolic extractions. A previous report on the anti-hypertensive effect of A. maxima was unavailable but administration of A. platensis (4.5 g for 12 weeks) significantly reduced systolic blood pressure, sE-selectin, soluble vascular cell adhesion molecule-1 (sVCAM-1), and endothelin-1 levels in systemic arterial hypertensive (SAH) patients under treatment of ACE inhibitors⁵⁸. When utilizing in silico digestion and molecular docking, four digestive peptides from PC exhibited several hydrogen bondings and hydrophobic interactions at the active site of ACE⁵⁹, suggesting their potential as strong ACE inhibitors. Hydrolyzing A. platensis PCP by five proteases (alcalase, bromelain, papain, pepsin, and trypsin) yielded active peptides with ACE inhibitory potentials⁶⁰, while the chlorophyllic fraction extracted from Gongronema latifolium and Vernonia amygdalina leaves also expressed ACE inhibition, with IC₅₀ values ranging from 0.21 to 0.41 mg/mL⁶¹. A literature search suggested that the ACE inhibitory activities detected in both aqueous and 80% (v/v) aqueous ethanolic extracts in our study might result from their predominant bioactive ingredients including PC, PCP, and chlorophylls.

Arthrospira species under aqueous and 80% (v/v) aqueous ethanolic extractions effectively inhibited the amyloid formation enzyme, BACE-1, rather than the cholinergic neurotransmitter degrading enzymes, AChE and BChE. These are all key enzymes controlling the occurrence of Alzheimer’s disease. In amyloidogenic pathway of Alzheimer’s disease (β-amyloid formation hypothesis), transmembrane amyloid precursor protein (APP) was cleaved by BACE-1 to produce soluble N-terminal APP (sAPPβ), while the remaining membrane-tethered C-terminal fragments β (CTFβ or C99) was further hydrolyzed by γ-secretase to form APP intracellular domain (AICD) and extracellular β-amyloid (Aβ) protein with a molecular weight of approximately 4 kDa (Fig. 5)⁶². Accumulation of Aβ misfolding leads to the formation of senile plaques. Our study indicated that both aqueous and ethanolic extracts could inhibit BACE-1 activities, with ethanolic fractions exhibiting stronger inhibitory strength than the aqueous ones. Even though the effect of Arthrospira on the β-amyloid formation hypothesis of Alzheimer’s disease through inhibition of BACE-1 activity has yet to be investigated, the previous literature reported that 70% (v/v) ethanolic extract of A. maxima could lower the contents of APP and BACE-1 as well as prevent cell death caused by Aβ-induced neurotoxicity in PC12 cells⁶³. Besides, Arthrospira (unknown species) possessed a neuroprotective effect, which reduced cerebral infarction volume and increased post-stroke locomotor activity in Sprague–Dawley rats fed on an Arthrospira diet (2%, 20–30 g/day) for 4 weeks⁶⁴, while other studies emphasized oxidative damage mechanism in neuronal cells^65–67. Thus, our result was the first to propose anti-Alzheimer’s disease through the β-amyloid formation hypothesis by the inhibitory effect of Arthrospira extract on BACE-1 activity (Fig. 5).

Figure 5.

The proposed mechanism of Arthrospira species on anti-Alzheimer’s disease through the amyloidogenic pathway of β-amyloid formation. In this hypothesis, amyloid precursor protein (APP) is hydrolyzed by β-secretase (BACE-1) to produce soluble N-terminal APP (sAPPβ) and membrane-tethered C-terminal fragments β (CTFβ or C99). The later is further hydrolyzed by γ-secretase to produce APP intracellular domain (AICD) and extracellular β-amyloid (Aβ) proteins, which subsequently accumulate to form amyloid plaques. Inhibition of BACE-1 by Arthrospira species could lead to retardment of Alzheimer’s disease development.

Approved Alzheimer’s disease drugs have minimal side effects, such as nausea and vomiting, to severe side effects, including brain swelling⁶⁸. Therefore, combinations between drugs and phytochemicals or small peptides have been extensively studied. Several investigations showed that the co-administration of Alzheimer’s disease drugs and small bioactive molecules resulted in reduced medication dosage without compromising effectiveness^36,69,70. In this study, the 80% (v/v) aqueous ethanolic extract of A. maxima exhibited higher potential BACE-1 inhibition than A. platensis. Hence, only the 80% (v/v) aqueous ethanolic extract of A. maxima was used to investigate the synergistic effect with donepezil. Results showed that A. maxima extract and donepezil synergistically inhibited BACE-1, especially the mixture between the IC₂₀ of donepezil and the IC₃₀ of A. maxima extract, which led to BACE-1 inhibition of nearly 50% (Table 5). Unlike plant extracts which contain excessive phytochemicals, blue-green algae are rich in proteins, peptides, and chlorophylls. PC improved cognitive dysfunction in intracerebroventricular (ICV)-STZ-induced cognitive declined Wistar rats⁷¹. One of the ICV-STZ inductive functions increased hippocampal cholinesterase activity, and PC significantly prevented this increase⁷¹, suggesting its role in the cholinergic hypothesis of Alzheimer’s disease. X-ray crystallographic analysis and molecular docking of PC into BACE-1 indicated stronger interactions than BACE-1 and its previously reported inhibitor⁷². PC was also previously reported to hinder the fibrillation of bovine serum albumin (BSA) through several hydrogen bonding interactions⁷³. Our results indicated that PC was predominantly present in aqueous extracts, with high BACE-1 inhibitions (more than 60%) observed in Arthrospira species extracted under 80% (v/v) aqueous ethanol. These results suggested the possibility of other bioactive ingredients in Arthrospira as effective BACE-1 inhibitors. Our results also showed that chlorophyll a were predominant in the 80% (v/v) aqueous ethanolic extracts; thus, it was possible that this compound might contribute anti-Alzheimer’s disease property though β-amyloid formation hypothesis. It was previously reported that chlorophyll a exhibited a greater strength to reduce amyloid precursor protein and BACE-1 levels than 70% (v/v) ethanolic extracted A. maxima and its PC⁶³. However, no previous literature has reported the effect of chlorophyll a on BACE-1 activity. The synergistic effect observed in this study might result from donepezil binding to BACE-1, causing an enzyme conformational change that facilitated favorable interactions with Arthrospira bioactive compounds such as chlorophyll a or vice versa.

Conclusions

The optimized conditions for extracting phenolics and antioxidants from Arthrospira were 2 h shaking time, 50 °C extraction temperature, and 1% (w/v) solid-to-liquid ratio. TPC and PC were highly extracted under aqueous conditions, while chlorophyll a were obtained under 80% (v/v) aqueous ethanolic extraction. Antioxidants with FRAP and ORAC activities were maximized under 80% (v/v) aqueous ethanolic extraction, while aqueous extraction was suitable for antioxidants with DPPH radical scavenging activities. The 80% (v/v) aqueous ethanolic extracts exhibited higher key enzyme inhibitions in lipase, BChE, BACE-1, and ACE inhibitory assays than those extracted under aqueous conditions, especially in the BACE-1 inhibitory assay, where more than 60% inhibition was detected. Arthrospira extract also showed a synergistic effect with donepezil, an Alzheimer’s disease drug. Interestingly, A. maxima extracts exhibited higher bioactive compounds, antioxidant activities, and key enzyme inhibitions than A. platensis extracts, suggesting their potential as a promising source of bioactive compounds with medicinal properties. Further investigations are required to determine the bioactive compounds in A. maxima responsible for BACE-1 inhibition to promote future drug design development for preventing and treating Alzheimer’s disease.

Author contributions

S.T. conducted the laboratory experiments as well as collected and analyzed the experimental results. W.I. analyzed and validated the experimental results. P.T. supervised the study and wrote the manuscript. Y.S. contributed to the conceptualization, methodology, formal analysis, and manuscript writing. P.Tr. and W.P. contributed to sample preparation and manuscript writing. D.S. and U.S. contributed to the conceptualization, methodology, data validation, supervision, and manuscript writing. All authors have read and agreed to the published version of the manuscript.

Funding

The APC was paid by Mahidol University, Thailand.

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.