Influence of extraction method and solvent system on the antioxidant activity of Scenedesmus parvus extract

Influence of extraction method and solvent system on the antioxidant activity of Scenedesmus parvus extract | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Influence of extraction method and solvent system on the antioxidant activity of Scenedesmus parvus extract Iqbal Iman Ruzi, Siti Nurfatimah Mohd Shahpudin, Sharlina Mohamad, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5288243/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Scenedesmus parvus , a Malaysian freshwater microalga, has garnered attention as a promising source of secondary metabolites with applications in various industries, including nutraceuticals, pharmaceuticals, food, and cosmetics. This study aimed to evaluate the extraction yield, total phenolic and flavonoid content, and antioxidant potential of S. parvus extracts obtained through different extraction methods (sonication, non-sonication, and soxhlet). Additionally, the fatty acid profile and key chemical constituents of S. parvus were analysed using GC-FID. Results revealed that the sonication method utilizing methanol as the extraction solvent yielded the highest extraction yield (14.5 ± 1.5%), whereas the non-sonication method employing hexane yielded the lowest (1.34 ± 0.2%). Furthermore, the sonicated ethanol extract exhibited the highest total phenolic content (66.32 ± 0.818 mg GAE g − 1 DW) and total flavonoid content (684.45 ± 28.928 mg QE g − 1 DW). Soxhlet extraction demonstrated superior antioxidant activity in both DPPH (IC50: 0.48 ± 0.035 mg mL − 1 ) and ABTS+ (IC50: 0.13 ± 0.003 mg mL − 1 ) scavenging assays compared to the others. These findings highlight the potential of different extraction methods from S. parvus , particularly those obtained with methanol as the extraction solvent, as valuable sources of natural antioxidants for application in nutraceuticals, pharmaceuticals, food, and cosmetics. Microalgae Scenedesmus parvus Fatty acid Extraction Antioxidant activity Figures Figure 1 Figure 2 Figure 3 1. Introduction Over the last few years, consumers are increasingly seeking natural antioxidants due to their associated health benefits and their applications across the food, cosmetic, and pharmaceutical sectors (Atta et al. 2017 ; Lourenço et al. 2019 ). Antioxidants play a crucial role in neutralising free radicals, which helps to avoid oxidative stress and lower the probability of some diseases, including cancer, autoimmune diseases, and neurodegenerative disorders (Lobo et al. 2010 ; Gupta et al. 2014 ; He et al. 2017 ; Malekmohammad et al. 2019 ; Lourenço et al. 2019 ; Didier et al. 2023 ; Didier et al. 2023 ). Microalgae have emerged as a significant natural source, recognised for their high concentrations of bioactive compounds exhibiting potent antioxidant activities (Safafar et al. 2015 ; Banskota et al. 2019 ; Del Mondo et al. 2021 ). There are several microalgae species, including Chlorella sp., Tetraselmis sp., Nannochloropsis sp., and Scenedesmus sp., have been reported to possess significant potential for producing secondary metabolites that can be utilised in the pharmaceutical industry (Tan and Lin 2011 ; Martin et al. 2014 ; Sharma et al. 2015 ; Pantami et al. 2020 ; Wali et al. 2020 ; Kumar et al. 2021 ; Montoya-Arroyo et al. 2022 ; Trentin et al. 2022 ; Ganeson et al. 2024 ). Within the diverse array of microalgae species, Scenedesmus parvus has garnered significant attention in Malaysia, particularly those isolated from acidic environments, because it can be considered a resilient strain. This extremophile microalga has generated substantial local interest because of its potential applications across various industries, especially the nutraceutical sector. It is postulated to have significant levels of polyphenols, flavonoids, and other bioactive compounds associated with antioxidant properties. These compounds act as reducing agents, hydrogen donors, singlet oxygen quenchers, and metal chelators and give multiple protection against oxidative damage in the human body (Wang et al. 2016 ; Sarian et al. 2017 ; Shalaby 2019 ; Coulombier et al. 2021a ; Coulombier et al. 2021b ). Extracting these valuable antioxidants from S. parvus is essential for determining the yield, purity, and bioactivity of the extracts Different techniques like maceration, ultrasonic-assisted extraction (UAE), and soxhlet extraction have been employed for the extraction of these compounds (Vongsak et al. 2013 ; Ameer et al. 2017 ; Łabowska et al. 2019 ; Mirzadeh et al. 2020 ; Monteiro et al. 2020 ). All methods have its own advantages and limitations affecting the performance of the extraction process and the stability of the identified bioactive compounds. Furthermore, the selection of solvent system is critical for the extraction process. It has been established that commonly used solvents such as water, ethanol, methanol, and their mixtures significantly affect the solubility and extraction efficiency of various antioxidant compounds.(Do et al. 2014 ; Chigayo et al. 2016 ; Lim et al. 2019 ; Muhamad et al. 2019 ; Nawaz et al. 2020 ). Hence, attributes like the polarity of the solvent, extraction temperature, and time also significantly affect the extracts' overall antioxidant activity. Thus, factors such as solvent polarity, extraction temperature, and duration play a crucial role in modulating the overall antioxidant activity of the extracts, as they directly influence the efficiency of bioactive compound isolation and stability. Optimizing these parameters is a key to maximizing the yield and potency of antioxidants while preserving their functional integrity. This study aims to comprehensively investigate the influence of various extraction methods and solvent systems on the antioxidant activity of S. parvus extracts. Through a comprehensive comparative analysis of various extraction parameters, this study aims to determine the optimal conditions for maximizing the antioxidant potential of S. parvus . Understanding these optimal conditions is essential for enhancing the practical applications of this microalga in developing natural antioxidant products, which can significantly contribute to both scientific knowledge and industrial advancements in the field of natural antioxidants. 2. Materials and Methods 2.1 Microalgae cultivation For this study, the chosen microalgae species was Scenedesmus parvus , which was identified and isolated by the Bioprocessing Division at the School of Industrial Technology, Universiti Sains Malaysia. The microalgae biomass was produced following a modified protocol adapted from a previous study (Hui et al. 2023a ). The modified algae growth (MLA) medium was used as both the seed culture and biomass production medium. The medium composition consisted of 0.49 g L − 1 magnesium sulphate (MgSO 4 .7H 2 O), 0.17 g L − 1 sodium nitrate (NaNO 3 ), 0.035 g L − 1 di-potassium phosphate (K 2 HPO 4 ), and 0.029 g L − 1 calcium chloride (CaCl 2 .2H 2 O). Sterilization of the medium was achieved using a 0.22 µm Millipore filter. Microalgal seeds were cultivated in 1 L Scott bottles containing 700 mL of the modified MLA medium. The initial microalgal cell concentration was set at 10% (v/v), equivalent to 0.03–0.05 g L − 1 (OD680 = 1.0). The cultures were incubated in an illuminated incubator with a controlled environment: a 0.3 L min − 1 flow of compressed air, light intensity of 450 µmol m² s − 1 , and a temperature of 24 ± 0.5°C. Both microalgal cultures were grown under identical conditions and harvested at the late logarithmic growth phase. The harvested cells were centrifuged at 4500 rpm for 15 minutes, rinsed twice with distilled water, and dried at 70°C for 24 hours. 2.2 Metabolite extraction methods 2.2.1 Ultrasound-assisted extraction (UAE) The extraction procedure was developed based on the previous study (Hui et al. 2023a ) with minor modifications. The microalgae biomass (2 g) was mixed with 20 mL of organic solvent in sealed conical flasks and sonicated in an ultrasonic bath (GT sonic P3, China) for 25 minutes at 60°C. The power and frequency of the ultrasonic bath were set at 100 watts and 40 kHz, respectively. After being treated with ultrasound, the mixture of solvent and biomass was placed in an incubation shaker set to 150 rpm and maintained at 35°C for 15 minutes. After this period, the sample was filtered and dried in an oven. The dried extract was weighed and stored at 4°C for the subsequent analyses. 2.2.2 Soxhlet extraction. Approximately 10g sample of S. parvus biomass was extracted with 400 mL of methanol over a period of 12 hours using a standard Soxhlet apparatus with a 500 mL boiler. After the extraction, the liquid extract was filtered and then evaporated to dryness under vacuum at 60°C using a rotary evaporator. The resulting dried extract was subsequently stored at 4°C for further analyses. 2.2.3 Determination of microalgae crude extraction method. The optimal extraction process for maximum yield and antioxidant activity was established based on different solvent systems and techniques. This study employed five solvents: methanol, ethanol, chloroform, hexane, and petroleum ether. Two extraction methods were assessed: i) ultrasound-assisted extraction (UAE) and ii) non-ultrasound-assisted extraction. The detailed test conditions are presented in Table 1 . A comparative analysis of all evaluated parameters will be performed against the soxhlet extraction method, which was regarded as the standard for extraction procedures Table 1 Details on tested conditions. Optimized parameter Tested condition Solvent system 100% Methanol 100% Ethanol 100% Chloroform 100% Hexane 100% Petroleum Ether Extraction method Ultrasound-assisted extraction method (UAE) Non-Ultrasound-assisted extraction method. 2.2.4 Crude Yield Calculation for Extraction The crude yield was determined by weighing the mass of the crude product obtained. The crude extract was subsequently then transferred into a pre-weighed aluminium plate, and the solvent was evaporated at 60°C in the dry oven until a constant weight was obtained. The yield of the crude extract The crude extract yield was then calculated using Eq. (1), as described in a previous study (Mahmood et al. 2017 ; Hui et al. 2023a ): $$\:\text{C}\text{r}\text{u}\text{d}\text{e}\:\text{y}\text{i}\text{e}\text{l}\text{d}\:\left(\text{%}\right)=\frac{\text{E}\text{x}\text{t}\text{r}\text{a}\text{c}\text{t}\text{e}\text{d}\:\text{c}\text{r}\text{u}\text{d}\text{e}\:\left(\text{m}\text{g}\right)}{\text{M}\text{i}\text{c}\text{r}\text{o}\text{a}\text{l}\text{g}\text{a}\text{e}\:\text{d}\text{r}\text{y}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\left(\text{m}\text{g}\right)}\text{x}\:100\text{%}$$ 2.3 Antioxidant capacity 2.3.1 Determination of total phenolic content (TPC) The total phenolic content assay was included in this study as it served as a standard measure for phenolics and was often utilised to optimize the experimental conditions. The total phenolic concentration was estimated using a modification of the method described in the previous study (Herald et al. 2012 ). Specifically, 25 µL of the extract or standard was mixed with 75 µL of distilled deionized water (DDW) and 25 µL of Folin–Ciocâlteu reagent. After approximately 6 minutes of reaction time, 100 µL of 75 g L − 1 Na 2 CO 3 was added to the mixture. The solution was kept in the dark at room temperature for 90 minutes for the reaction to occur, after which the absorbance was measured at 765 nm against a reagent blank. Gallic acid was used as a standard within the 0 − 1000 µg mL − 1 range to create a calibration curve (R 2 < 0.9900). The total phenolic concentration was expressed as mg gallic acid equivalent (GAE) per gram of dried sample. 2.3.2 Determination of total flavonoid content (TFC) Total flavonoid concentration was quantified using the spectrophotometric method described by the previous study (Herald et al. 2012 ). In detail, 25 µL of the extract was combined with 100 µL of distilled deionized water (DDW) and 10 µL of 50 g L − 1 NaNO 2 , and the mixture was vortexed for 5 minutes. Subsequently, 15 µL of 100 g L − 1 AlCl 3 was added. The solution was allowed to stand for 6 minutes, followed by adding 50 µL of 1 mol L − 1 NaOH and another 50 µL of DDW. The mixture was shaken for 30 seconds, and the absorbance was measured at 510 nm against a reagent blank. Total flavonoid concentration was expressed as mg quercetin equivalent (QE) per gram of sample. Quercetin was used as a standard in the 0 − 1000 µg mL − 1 range to generate a calibration curve (R 2 < 0.9900). 2.4 Antioxidant activity 2.4.1 DPPH scavenging activity The antioxidant scavenging activity was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical method, adhering to protocols established in prior studies with some adjustments (Herald et al. 2012 ; Xu 2017a ). Different dilutions of the samples (50 µL of ascorbic acid or extracts) were mixed with 195 µL of a 0.2 mM methanolic DPPH solution. After incubating for 30 minutes at 25°C, the absorbance at 520 nm was measured against a blank. The free radical scavenging activity of each solution was determined as a percentage of inhibition using the following formula: $$\:\text{F}\text{r}\text{e}\text{e}\:\text{r}\text{a}\text{d}\text{i}\text{c}\text{a}\text{l}\:\text{i}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n}\:\left(\text{%}\right)=\frac{\text{B}\text{l}\text{a}\text{n}\text{k}\:\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}-\text{S}\text{a}\text{m}\text{p}\text{l}\text{e}\:\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}}{\text{B}\text{l}\text{a}\text{n}\text{k}\:\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}}\:\text{X}\:100$$ The antioxidant activity of the extracts was expressed as IC50, which represents the concentration necessary to achieve a 50% reduction in the initial DPPH free radical concentration. IC50 values were calculated using linear regression, with ascorbic acid serving as the reference standard. 2.4.2 ABTS + scavenging activity The radical scavenging ability of the samples for ABTS+ (2,2'-azinobis-3-ethylbenzo-thiazoline-6-sulfonate) was evaluated using the method described in previous studies (Xu 2017b ; Moreira 2019 ; Gu et al. 2019 ). The preparation of the ABTS + solution began by mixing 5 mL of a 7 mM ABTS solution adjusted to pH 7.4 with 88 µL of a 140 mM potassium persulfate solution. This mixture was allowed to react in a dark environment at room temperature for 16 hours to ensure the complete formation of the ABTS + radical cation. A 0.5 mL portion of this mixture was diluted with 45 mL of ethanol to achieve an absorbance of 0.70 ± 0.05 units at 734 nm, measured by spectrophotometry. Different dilutions of the samples (20 µL of ascorbic acid or extracts) were then mixed with 280 µL of fresh ABTS + solution, and absorbance was measured 6 minutes after the initial mixing. Ascorbic acid served as a reference. The free radical scavenging activity was determined as IC50 (mg mL − 1 ), representing the concentration that reduced the ABTS + by 50%. 2.5 Fatty acid composition analysis The analysis of fatty acids in the microalgae extract was performed using a gas chromatography with flame ionization detector (GC-FID) system, using the Agilent model 7880B (Agilent Technologies, Santa Clara, California, USA). Separation of fatty acids was achieved utilizing an Agilent DB-Fast FAME column (60m × 250 µm × 0.25 µm). A sample volume of approximately 1 µL was injected into the GC column. Hydrogen gas served as the carrier gas, flowing at a rate of 40 mL min − 1 . The temperature program was initiated at 80°C for 1 minute, followed by a ramp to 165°C at a rate of 40°C min − 1 , maintained for 1 minute, and concluded with an increase to 230°C at 4°C min − 1 , held for 15 minutes. The detector temperature was set at 260°C, while the injector operated at 250°C with a split ratio 100:1. The Identification of each fatty acid within the extract was accomplished by comparing their retention times against a Supelco® standard. Quantification was performed using GC Solution® software, results were presented as percentages relative to the total identified fatty acids. 2.6 Statistical analysis The results are expressed as mean values accompanied by their respective standard deviations. Data compilation and analysis were facilitated by Microsoft Excel 2016 and GraphPad Prism version 9.1.0 for Windows (GraphPad Software based in San Diego, California, USA). To analyse the collected data, one-way ANOVA was employed, supplemented with Tukey's multiple comparison tests to determine statistical significance. 3. Results 3.1 Effect of sonication and solvent system on the extraction yield The extraction yields of S. parvus using various solvents under sonicated and non-sonicated conditions are shown in Fig. 1 . The results demonstrate significant variations in extraction efficiency depending on the types of solvents used and the implementation of sonication. Furthermore, the findings suggest that extracts produced via sonication result in considerably greater yields compared to those extracted without sonication, irrespective of the solvent employed. 3.2 The Impact of Different Extraction Methods on Total Phenolic and Flavonoid Content For future assessments of the antioxidant potential of S. parvus , this study will focus solely on the crude extracts of methanol and ethanol. These extracts were selected due to their higher yields compared to the other solvents tested in the study. This study believes higher yield extracts will likely contain a broader range of bioactive compounds, facilitating more comprehensive antioxidant evaluation. This increased diversity may enhance the sensitivity of assays, thereby yielding more accurate and reliable results. The summarised results in Table 2 and Fig. 2 show that extraction is significantly influenced by the method and solvent used. Table 2 Assessment of the total phenolic and flavonoid content and the antioxidant activity of S. parvus extract. Extract TPC (mg GAE g − 1 DW) TFC (mg QE g − 1 DW) DPPH (mg mL − 1 ) ABTS+ (mg mL − 1 ) Non-sonicated MeOH 23.58 ± 2.168 270.01 ± 19.174 0.57 ± 0.023 0.16 ± 0.002 Sonicated MeOH 14.82 ± 0.660 148.04 ± 43.758 0.84 ± 0.083 0.17 ± 0.002 Non-sonicated EtOH 20.15 ± 1.007 191.83 ± 17.646 0.69 ± 0.049 0.21 ± 0.009 Sonicated EtOH 66.32 ± 0.818 684.45 ± 28.928 1.57 ± 0.025 0.18 ± 0.004 Soxhlet 5.05 ± 0.632 39.96 ± 13.751 0.48 ± 0.035 0.13 ± 0.003 3.3 Effect of different extraction methods on the antioxidant activity The antioxidant activity of the S. parvus extract was evaluated using a radical scavenging assay targeting DPPH and ABTS + free radicals. The IC50 values, which represent the concentration required to inhibit 50% of the free radical activity, were used as indicators of antioxidant potency, with lower values corresponding to higher antioxidant activity. A summary of all data on antioxidant activity is summarized in Table 2 and Fig. 3 . 3.4 Fatty acid composition of the S. parvus extract The data in Table 3 shows a detailed comparison of the fatty acid composition of S. parvus extracts obtained using two different methods: soxhlet extraction and non-sonicated methanol extraction. The values are expressed in grams per 100 grams of extract sample. Table 3 Fatty acid profile of soxhlet and non-sonicated methanol S. parvus extract. Fatty Acid Soxhlet extract (g/100g) Non-sonicated MeOH extract (g/100g) Dodecanoic Acid (C12:0) 0.5 2.1 Tridecanoic Acid (C13:0) 0.7 2.8 Myristic Acid (C14:0) 0.5 1.7 Myristoleic Acid (C14:1) 0.9 3.3 Palmitic Acid (C16:0) 37.6 38.5 Palmitoleic Acid (C16:1) 1.0 1.7 Stearic Acid (C18:0) 4.1 10.2 Oleic Acid (C18:1 cis) 36.8 23.9 Linoleic Acid (C18:2 cis) 13.2 9.6 Linolenic Acid (C18:3n3) 4.8 4.6 Eicosanoic Acid (C20:0) - 1.7 4. Discussions 4.1 Effect of sonication and solvent system on the extraction yield Methanol and ethanol produced the highest extraction yields among all the solvents tested in the study, with methanol yielding approximately 10.3 ± 0.919% for the non-sonicated samples and around 14.65 ± 0.503% for the sonicated samples. Ethanol yielded 5.9 ± 0.230% in the absence of sonication, whereas the yield increased to 10.45 ± 1.202% with sonication. These findings aligned with previous findings that reported both methanol and ethanol as the most efficient solvents for the extraction the bioactive compounds from S. parvus (Ren et al. 2021 ; Tavakoli et al. 2021 ; Hui et al. 2023a ). Chloroform, being a less polar solvent than methanol and ethanol, yielded approximately 6.05 ± 0.474% for the non-sonicated samples and about 9.3 ± 0.354% for the sonicated samples. This indicates that chloroform extracts have lesser numbers of compounds compared to methanol and ethanol, but the application of sonication significantly increases the extraction yield. In the case of hexane and petroleum ether, both non-polar solvents, the extraction yields were very low, ranging from 1–2%, even with sonication. This suggests that the compounds in S. parvus . have limited solubility in non-polar solvents, resulting in low extraction yields. Sonication also influenced the extraction yield of most solvents, as demonstrated by the relatively higher extraction yields for sonicated samples compared to non-sonicated ones. Methanol and ethanol showed higher yield compared to the control, suggesting that sonication increases the efficiency of extraction by breaking down the cell walls and releasing the internal compound, as reported in many previous studies (Das and Eun 2018 ; Ren et al. 2021 ; Tavakoli et al. 2021 ; Hui et al. 2023b ). Sonication with chloroform also increased significantly, demonstrating its effectiveness in improving solvent’s penetration and extraction capability. Interestingly, the extraction yield using soxhlet extraction, despite being a lengthy process, was approximately 10.2 ± 0.362%, slightly lower than the sonicated methanol and ethanol extracts. This suggests that, while soxhlet extraction is effective, the combination of an appropriate solvent and sonication can yield better results, potentially outperforming conventional soxhlet extraction. Based on these findings, it can be concluded that solvent selection and the application of sonication are critical factors in the extraction of S. parvus . In comparison to the other solvents, methanol and ethanol used with sonication were the most effective solvents, producing significantly better results than conventional soxhlet extraction and the non-polar solvents. These results demonstrate that the extraction method and the type of solvent employed have a significant impact on the yield. The findings also support the sonication to enhance bioactive compound yield in microalgal research and related fields. Future studies should investigate the type of compounds extracted by each solvent and the biological activities associated with these extracts to further elucidate the benefits of sonication-assisted extraction. 4.2 The Impact of Different Extraction Methods on Total Phenolic and Flavonoid Content In the comparison between non-sonicate and sonicate methods for methanol extraction, the non-sonicate method yielded a higher phenolic content of 23.58 ± 2.168 mg GAE g − 1 DW and flavonoid content of 270.01 ± 19.174 mg QE g − 1 DW. In contrast, the sonicate method yielded a lower phenolic content of 14.82 ± 0.660 mg GAE g − 1 DW and a flavonoid content of 148.04 ± 43.758 mg QE g − 1 DW. These results are inconsistent with the previous studies that suggest sonication with methanol should enhance the TPC and TFC values rather than lowering the compound extracted from the S. parvus (Upadhyay et al. 2015 ; Kim and Lee 2017 ; Agarwal et al. 2018 ; Özcan et al. 2020 ; Ahmed et al. 2020 ). Therefore, further study is needed to elucidate this phenomenon. On the other hand, ethanol extraction depicted a different pattern of results. The sonication method was significantly more effective than the non-sonicate method, where the phenolic content increased from 20.15 ± 1.007 GAE g − 1 DW to 66.32 ± 0.818 mg GAE g − 1 DW, and the flavonoid content increased from 191.83 mg QE g − 1 DW to 684.45 ± 28.928 mg QE g − 1 DW. This significant increment further supports the effectiveness of sonication with ethanol in the disruption of the cell walls of the S. parvus to extract the phenolic and flavonoid compounds. This finding is consistent with previous studies showing that using sonication and ethanol during extraction enhances the recovery of phenolic and flavonoid compounds (Gogoi et al. 2019 ; Mousavi et al. 2022 ; Irfan et al. 2022 ). In contrast, the soxhlet extraction method, which has the longest extraction time and continuous solvent circulation, yielded the lowest yields of phenolic (5. 05 ± 0. 632 mg GAE g − 1 DW) and flavonoid (39. 96 ± 13. 751 mg QE g − 1 DW) contents. This reduced efficiency could be due to the degradation of bioactive compounds due to more prolonged exposure to heat compared to the other methods. In conclusion, this study demonstrates that selecting a proper extraction method and solvent will directly influence the quantity of phenolic and flavonoid compounds extracted from S. parvus . The results showed that sonication in ethanol was the most effective method for enhancing the extraction yield of these bioactive compounds. These findings could improve extraction processes in both research and industry, where maximizing the recovery of phenolic and flavonoid compounds is essential. Additionally, the results suggest that conventional Soxhlet extraction may not be ideal for such applications, particularly when targeting heat-sensitive bioactive compounds. 4.3 Effect of different extraction methods on the antioxidant activity The results of this study revealed significant variations in antioxidant activity depending on the extraction method and solvent used. For the methanol extract, the non-sonicated method yielded lower IC50 values for DPPH (0.57 ± 0.023 mg mL − 1 ), indicating greater antioxidant activity compared to the sonicated method, which had IC50 values of 0.84 ± 0.083 mg mL − 1 . A similar pattern was observed with the ethanol extract, where the non-sonicated method showed higher antioxidant activity against the DPPH free radical, with an IC50 value of 0.69 ± 0.049 mg mL − 1 , compared to 1.57 ± 0.025 mg mL − 1 for the sonicated method. These findings contrast with previous studies that reported increased antioxidant activity with sonication (Kim and Lee 2017 ; Özcan et al. 2020 ; Ranjha et al. 2020 ; Tavakoli et al. 2021 ; Mousavi et al. 2022 ; Irfan et al. 2022 ). It is hypothesized that the decrease in the antioxidant activity in sonicated extracts may result from the high energy during the sonication process affecting the structural integrity of antioxidant compounds, thereby reducing their activity. On the other hand, no significant differences in antioxidant activity were observed among different extraction methods and solvents for the ABTS + radical. These findings indicate that the choice of extraction method and solvent is crucial in determining the antioxidant activity of S. parvus extracts. Methanol emerged as a more effective solvent than ethanol, especially in non-sonicated extracts. Notably, the Soxhlet extraction method demonstrated significantly higher antioxidant activity against the DPPH radical (0.48 ± 0.035 mg mL − 1 ) compared to other methods, except for the non-sonicated methanol extract, despite having lower TPC and TFC values. These insights are essential for refining extraction techniques to maximize antioxidant content and are of great value to researchers and industries involved in the production of functional foods and nutraceuticals. 4.4 Fatty acid composition of the S. parvus extract The dodecanoic acid in the non-sonicated methanol extraction yielded a significantly higher concentration (2.1g 100g − 1 ) compared to the soxhlet extraction (0.5g 100g − 1 ), indicating that the non-sonicated methanol method is more effective for extracting shorter-chain saturated fatty acids. Similarly, the concentration of tridecanoic acid was higher in the non-sonicated methanol extract (2.8g 100g − 1 ) than in the soxhlet extract (0.7g 100g − 1 ). For myristic acid, both methods resulted in relatively low concentrations, but the non-sonicated methanol extraction method extracted slightly more (1.7g 100g − 1 ) than the soxhlet method (0.5g 100g − 1 ). Myristoleic acid concentrations were also higher in the non-sonicated methanol extract (3.3g 100g- 1 ) compared to the soxhlet extract (0.9 g 100g − 1 ). Palmitic acid was the most abundant fatty acid in both extracts, with similar concentrations in both methods (37.6g 100g − 1 in soxhlet and 38.5g 100g − 1 in non-sonicated methanol), suggesting that both methods are equally effective for extracting this fatty acid. The concentration of palmitoleic acid was higher in the non-sonicated methanol extract (1.7g 100g − 1 ) than in the soxhlet extract (1.0g 100g − 1 ). The non-sonicated methanol extract had more than double the concentration of stearic acid (10.2g 100g − 1 ) compared to the soxhlet extract (4.1g 100g − 1 ), highlighting the significant advantage of the non-sonicated methanol method for extracting this fatty acid. Conversely, oleic acid was more effectively extracted using the soxhlet method (36.8g 100g − 1 ) than the non-sonicated methanol method (23.9g 100g − 1 ), indicating that soxhlet extraction may be better for extracting monounsaturated fatty acids like oleic acid. Linoleic acid concentration was also higher in the soxhlet extract (13.2 g 100g − 1 ) compared to the non-sonicated methanol extract (9.6 g 100g − 1 ), similar to the trend observed with oleic acid. Both methods showed similar concentrations of linolenic acid (4.8 g 100g − 1 in soxhlet and 4.6 g 100g − 1 in non-sonicated methanol), indicating no significant difference between the methods for this fatty acid. Eicosanoic acid was detected only in the non-sonicated methanol extract (1.7 g 100g − 1 ), suggesting that the soxhlet method may not be suitable for extracting longer-chain saturated fatty acids. The fatty acid profile of S. parvus extracts varies significantly depending on the extraction method used. The non-sonicated methanol method generally yielded higher shorter-chain and saturated fatty acids concentrations. In contrast, the soxhlet method was more effective for extracting certain monounsaturated and polyunsaturated fatty acids. These findings suggest that the extraction method should be tailored to the specific fatty acids of interest in S. parvus extracts. 5. Conclusion In conclusion, this study concludes that sonication is crucial in maximizing for optimizing the extraction yield and bioactive compound content of Scenedesmus parvus . Methanol and ethanol, particularly especially when combined with sonication, proved to be the most effective solvents for extracting phenolic and flavonoid compounds and enhancing antioxidant activity. Therefore, future research should explore the specific compounds extracted by each solvent and evaluate their associated biological activities. Declarations Competing interests The authors declare no conflict of interest. Funding This research was funded by Fundamental Research Grant Scheme grant from the Malaysian Ministry of Higher Education 203.CIPPT.6712051 Author Contribution I.R., S.M., and M.K. made significant contributions throughout this study, including writing the manuscript, conducting experiments, and analyzing the data to its conclusion. Acknowledgement The authors express their sincere gratitude to the staff and faculty of the School of Industrial Technology at Universiti Sains Malaysia for their invaluable assistance in completing this study. References Agarwal C, Máthé K, Hofmann T, Csóka L (2018) Ultrasound‐Assisted Extraction of Cannabinoids from Cannabis Sativa L. Optimized by Response Surface Methodology. J Food Sci 83(3):700–710. https://doi.org/10.1111/1750-3841.14075 Ahmed M, Ramachandraiah K, Jiang G-H, Eun JB (2020) Effects of Ultra-Sonication and Agitation on Bioactive Compounds and Structure of Amaranth Extract. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5288243","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":371067085,"identity":"bf81b97c-362b-4e5e-954d-a04c4ad0239d","order_by":0,"name":"Iqbal Iman Ruzi","email":"","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Iqbal","middleName":"Iman","lastName":"Ruzi","suffix":""},{"id":371067087,"identity":"1f3571c1-da19-4632-854c-00f85a7d66d7","order_by":1,"name":"Siti Nurfatimah Mohd Shahpudin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYDACZgaGA0CKn4GBsQGEGBjYGwhpYQZrkWxgg2nhOUCENRAtDAwQLRIJ+DXIt/MfPFzYdk+Cf35z64afOxii5We+Mf50g2FbIi73GRxmZjg8s61YQuIYY9vN3jMMuRtu55hJ5zDcxq0F6JfDvG0JdQxALTd424BapHPMmPFpkW+GaJGQB9nyF6hl/swzxp/xaWE4DNViANRyG2RLww0eA/wOO8xscJjnXIKE4bHEttuybRK5G86klUnnGNw2xumw/oOPP/OUJUjIHT7+7ObbNpvc+e2HN3/Oqbgti9NhaEACZjuDI7FaEMCeZB2jYBSMglEwXAEA2zZcYyKsLOIAAAAASUVORK5CYII=","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Siti","middleName":"Nurfatimah Mohd","lastName":"Shahpudin","suffix":""},{"id":371067089,"identity":"a26a9e01-30fe-4513-926d-7e544136b3b9","order_by":2,"name":"Sharlina Mohamad","email":"","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Sharlina","middleName":"","lastName":"Mohamad","suffix":""},{"id":371067092,"identity":"a2a9800c-4b22-41e5-a93f-029acd4ae984","order_by":3,"name":"Mohd Asyraf Kassim","email":"","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Mohd","middleName":"Asyraf","lastName":"Kassim","suffix":""}],"badges":[],"createdAt":"2024-10-18 09:23:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5288243/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5288243/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":67923750,"identity":"45923766-e311-40e3-b627-d095c22b157e","added_by":"auto","created_at":"2024-10-31 08:32:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":29308,"visible":true,"origin":"","legend":"\u003cp\u003eExtraction yield produced from different extraction procedures\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5288243/v1/51a1f136b391bd507648d13d.png"},{"id":67923528,"identity":"d72c822d-ed29-4038-a934-70a8439392d6","added_by":"auto","created_at":"2024-10-31 08:24:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":82956,"visible":true,"origin":"","legend":"\u003cp\u003eThe total phenolic and flavonoid content and the antioxidant activity of S. \u003cem\u003eparvus\u003c/em\u003e extracts.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5288243/v1/4d583968893b3585674537e7.png"},{"id":67923751,"identity":"71223b15-30ee-4557-8f68-c5306b444631","added_by":"auto","created_at":"2024-10-31 08:32:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":25887,"visible":true,"origin":"","legend":"\u003cp\u003eExtract IC50 against DPPH and ABTS+ free radical.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5288243/v1/06776dec0575bf5255b1b875.png"},{"id":77109801,"identity":"441e8ba3-7df8-4128-8968-302061f7cb85","added_by":"auto","created_at":"2025-02-25 08:47:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1179528,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5288243/v1/e7cef6a2-7663-4ce6-8fa2-ae452869a0e8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Influence of extraction method and solvent system on the antioxidant activity of Scenedesmus parvus extract","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOver the last few years, consumers are increasingly seeking natural antioxidants due to their associated health benefits and their applications across the food, cosmetic, and pharmaceutical sectors (Atta et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Louren\u0026ccedil;o et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Antioxidants play a crucial role in neutralising free radicals, which helps to avoid oxidative stress and lower the probability of some diseases, including cancer, autoimmune diseases, and neurodegenerative disorders (Lobo et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Gupta et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; He et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Malekmohammad et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Louren\u0026ccedil;o et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Didier et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Didier et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Microalgae have emerged as a significant natural source, recognised for their high concentrations of bioactive compounds exhibiting potent antioxidant activities (Safafar et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Banskota et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Del Mondo et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). There are several microalgae species, including Chlorella sp., Tetraselmis sp., Nannochloropsis sp., and Scenedesmus sp., have been reported to possess significant potential for producing secondary metabolites that can be utilised in the pharmaceutical industry (Tan and Lin \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Martin et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Pantami et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wali et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Montoya-Arroyo et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Trentin et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ganeson et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Within the diverse array of microalgae species, \u003cem\u003eScenedesmus parvus\u003c/em\u003e has garnered significant attention in Malaysia, particularly those isolated from acidic environments, because it can be considered a resilient strain. This extremophile microalga has generated substantial local interest because of its potential applications across various industries, especially the nutraceutical sector. It is postulated to have significant levels of polyphenols, flavonoids, and other bioactive compounds associated with antioxidant properties. These compounds act as reducing agents, hydrogen donors, singlet oxygen quenchers, and metal chelators and give multiple protection against oxidative damage in the human body (Wang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sarian et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Shalaby \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Coulombier et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e; Coulombier et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExtracting these valuable antioxidants from S. \u003cem\u003eparvus\u003c/em\u003e is essential for determining the yield, purity, and bioactivity of the extracts Different techniques like maceration, ultrasonic-assisted extraction (UAE), and soxhlet extraction have been employed for the extraction of these compounds (Vongsak et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ameer et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Łabowska et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mirzadeh et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Monteiro et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). All methods have its own advantages and limitations affecting the performance of the extraction process and the stability of the identified bioactive compounds. Furthermore, the selection of solvent system is critical for the extraction process. It has been established that commonly used solvents such as water, ethanol, methanol, and their mixtures significantly affect the solubility and extraction efficiency of various antioxidant compounds.(Do et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Chigayo et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lim et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Muhamad et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nawaz et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Hence, attributes like the polarity of the solvent, extraction temperature, and time also significantly affect the extracts' overall antioxidant activity. Thus, factors such as solvent polarity, extraction temperature, and duration play a crucial role in modulating the overall antioxidant activity of the extracts, as they directly influence the efficiency of bioactive compound isolation and stability. Optimizing these parameters is a key to maximizing the yield and potency of antioxidants while preserving their functional integrity. This study aims to comprehensively investigate the influence of various extraction methods and solvent systems on the antioxidant activity of S. \u003cem\u003eparvus\u003c/em\u003e extracts. Through a comprehensive comparative analysis of various extraction parameters, this study aims to determine the optimal conditions for maximizing the antioxidant potential of S. \u003cem\u003eparvus\u003c/em\u003e. Understanding these optimal conditions is essential for enhancing the practical applications of this microalga in developing natural antioxidant products, which can significantly contribute to both scientific knowledge and industrial advancements in the field of natural antioxidants.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Microalgae cultivation\u003c/h2\u003e \u003cp\u003eFor this study, the chosen microalgae species was \u003cem\u003eScenedesmus parvus\u003c/em\u003e, which was identified and isolated by the Bioprocessing Division at the School of Industrial Technology, Universiti Sains Malaysia.\u003c/p\u003e \u003cp\u003eThe microalgae biomass was produced following a modified protocol adapted from a previous study (Hui et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). The modified algae growth (MLA) medium was used as both the seed culture and biomass production medium. The medium composition consisted of 0.49 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e magnesium sulphate (MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO), 0.17 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sodium nitrate (NaNO\u003csub\u003e3\u003c/sub\u003e), 0.035 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e di-potassium phosphate (K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e), and 0.029 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e calcium chloride (CaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO). Sterilization of the medium was achieved using a 0.22 \u0026micro;m Millipore filter. Microalgal seeds were cultivated in 1 L Scott bottles containing 700 mL of the modified MLA medium. The initial microalgal cell concentration was set at 10% (v/v), equivalent to 0.03\u0026ndash;0.05 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (OD680\u0026thinsp;=\u0026thinsp;1.0). The cultures were incubated in an illuminated incubator with a controlled environment: a 0.3 L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e flow of compressed air, light intensity of 450 \u0026micro;mol m\u0026sup2; s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and a temperature of 24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C. Both microalgal cultures were grown under identical conditions and harvested at the late logarithmic growth phase. The harvested cells were centrifuged at 4500 rpm for 15 minutes, rinsed twice with distilled water, and dried at 70\u0026deg;C for 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Metabolite extraction methods\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Ultrasound-assisted extraction (UAE)\u003c/h2\u003e \u003cp\u003eThe extraction procedure was developed based on the previous study (Hui et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e) with minor modifications. The microalgae biomass (2 g) was mixed with 20 mL of organic solvent in sealed conical flasks and sonicated in an ultrasonic bath (GT sonic P3, China) for 25 minutes at 60\u0026deg;C. The power and frequency of the ultrasonic bath were set at 100 watts and 40 kHz, respectively. After being treated with ultrasound, the mixture of solvent and biomass was placed in an incubation shaker set to 150 rpm and maintained at 35\u0026deg;C for 15 minutes. After this period, the sample was filtered and dried in an oven. The dried extract was weighed and stored at 4\u0026deg;C for the subsequent analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Soxhlet extraction.\u003c/h2\u003e \u003cp\u003eApproximately 10g sample of S. \u003cem\u003eparvus\u003c/em\u003e biomass was extracted with 400 mL of methanol over a period of 12 hours using a standard Soxhlet apparatus with a 500 mL boiler. After the extraction, the liquid extract was filtered and then evaporated to dryness under vacuum at 60\u0026deg;C using a rotary evaporator. The resulting dried extract was subsequently stored at 4\u0026deg;C for further analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Determination of microalgae crude extraction method.\u003c/h2\u003e \u003cp\u003eThe optimal extraction process for maximum yield and antioxidant activity was established based on different solvent systems and techniques. This study employed five solvents: methanol, ethanol, chloroform, hexane, and petroleum ether. Two extraction methods were assessed: i) ultrasound-assisted extraction (UAE) and ii) non-ultrasound-assisted extraction. The detailed test conditions are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A comparative analysis of all evaluated parameters will be performed against the soxhlet extraction method, which was regarded as the standard for extraction procedures\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetails on tested conditions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOptimized parameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTested condition\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSolvent system\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100% Methanol\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100% Ethanol\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100% Chloroform\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100% Hexane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100% Petroleum Ether\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExtraction method\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUltrasound-assisted extraction method (UAE)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNon-Ultrasound-assisted extraction method.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4 Crude Yield Calculation for Extraction\u003c/h2\u003e \u003cp\u003eThe crude yield was determined by weighing the mass of the crude product obtained. The crude extract was subsequently then transferred into a pre-weighed aluminium plate, and the solvent was evaporated at 60\u0026deg;C in the dry oven until a constant weight was obtained. The yield of the crude extract The crude extract yield was then calculated using Eq.\u0026nbsp;(1), as described in a previous study (Mahmood et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hui et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e):\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{C}\\text{r}\\text{u}\\text{d}\\text{e}\\:\\text{y}\\text{i}\\text{e}\\text{l}\\text{d}\\:\\left(\\text{%}\\right)=\\frac{\\text{E}\\text{x}\\text{t}\\text{r}\\text{a}\\text{c}\\text{t}\\text{e}\\text{d}\\:\\text{c}\\text{r}\\text{u}\\text{d}\\text{e}\\:\\left(\\text{m}\\text{g}\\right)}{\\text{M}\\text{i}\\text{c}\\text{r}\\text{o}\\text{a}\\text{l}\\text{g}\\text{a}\\text{e}\\:\\text{d}\\text{r}\\text{y}\\:\\text{w}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t}\\:\\left(\\text{m}\\text{g}\\right)}\\text{x}\\:100\\text{%}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Antioxidant capacity\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Determination of total phenolic content (TPC)\u003c/h2\u003e \u003cp\u003eThe total phenolic content assay was included in this study as it served as a standard measure for phenolics and was often utilised to optimize the experimental conditions. The total phenolic concentration was estimated using a modification of the method described in the previous study (Herald et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Specifically, 25 \u0026micro;L of the extract or standard was mixed with 75 \u0026micro;L of distilled deionized water (DDW) and 25 \u0026micro;L of Folin\u0026ndash;Cioc\u0026acirc;lteu reagent. After approximately 6 minutes of reaction time, 100 \u0026micro;L of 75 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e was added to the mixture. The solution was kept in the dark at room temperature for 90 minutes for the reaction to occur, after which the absorbance was measured at 765 nm against a reagent blank. Gallic acid was used as a standard within the 0 \u0026minus;\u0026thinsp;1000 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range to create a calibration curve (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.9900). The total phenolic concentration was expressed as mg gallic acid equivalent (GAE) per gram of dried sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Determination of total flavonoid content (TFC)\u003c/h2\u003e \u003cp\u003eTotal flavonoid concentration was quantified using the spectrophotometric method described by the previous study (Herald et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In detail, 25 \u0026micro;L of the extract was combined with 100 \u0026micro;L of distilled deionized water (DDW) and 10 \u0026micro;L of 50 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaNO\u003csub\u003e2\u003c/sub\u003e, and the mixture was vortexed for 5 minutes. Subsequently, 15 \u0026micro;L of 100 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e AlCl\u003csub\u003e3\u003c/sub\u003e was added. The solution was allowed to stand for 6 minutes, followed by adding 50 \u0026micro;L of 1 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaOH and another 50 \u0026micro;L of DDW. The mixture was shaken for 30 seconds, and the absorbance was measured at 510 nm against a reagent blank. Total flavonoid concentration was expressed as mg quercetin equivalent (QE) per gram of sample. Quercetin was used as a standard in the 0 \u0026minus;\u0026thinsp;1000 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range to generate a calibration curve (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.9900).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Antioxidant activity\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 DPPH scavenging activity\u003c/h2\u003e \u003cp\u003eThe antioxidant scavenging activity was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical method, adhering to protocols established in prior studies with some adjustments (Herald et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Xu \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e). Different dilutions of the samples (50 \u0026micro;L of ascorbic acid or extracts) were mixed with 195 \u0026micro;L of a 0.2 mM methanolic DPPH solution. After incubating for 30 minutes at 25\u0026deg;C, the absorbance at 520 nm was measured against a blank. The free radical scavenging activity of each solution was determined as a percentage of inhibition using the following formula:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\text{F}\\text{r}\\text{e}\\text{e}\\:\\text{r}\\text{a}\\text{d}\\text{i}\\text{c}\\text{a}\\text{l}\\:\\text{i}\\text{n}\\text{h}\\text{i}\\text{b}\\text{i}\\text{t}\\text{i}\\text{o}\\text{n}\\:\\left(\\text{%}\\right)=\\frac{\\text{B}\\text{l}\\text{a}\\text{n}\\text{k}\\:\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}-\\text{S}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}\\:\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}}{\\text{B}\\text{l}\\text{a}\\text{n}\\text{k}\\:\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}}\\:\\text{X}\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe antioxidant activity of the extracts was expressed as IC50, which represents the concentration necessary to achieve a 50% reduction in the initial DPPH free radical concentration. IC50 values were calculated using linear regression, with ascorbic acid serving as the reference standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 ABTS\u0026thinsp;+\u0026thinsp;scavenging activity\u003c/h2\u003e \u003cp\u003eThe radical scavenging ability of the samples for ABTS+ (2,2'-azinobis-3-ethylbenzo-thiazoline-6-sulfonate) was evaluated using the method described in previous studies (Xu \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e; Moreira \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The preparation of the ABTS\u0026thinsp;+\u0026thinsp;solution began by mixing 5 mL of a 7 mM ABTS solution adjusted to pH 7.4 with 88 \u0026micro;L of a 140 mM potassium persulfate solution. This mixture was allowed to react in a dark environment at room temperature for 16 hours to ensure the complete formation of the ABTS\u0026thinsp;+\u0026thinsp;radical cation. A 0.5 mL portion of this mixture was diluted with 45 mL of ethanol to achieve an absorbance of 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 units at 734 nm, measured by spectrophotometry. Different dilutions of the samples (20 \u0026micro;L of ascorbic acid or extracts) were then mixed with 280 \u0026micro;L of fresh ABTS\u0026thinsp;+\u0026thinsp;solution, and absorbance was measured 6 minutes after the initial mixing. Ascorbic acid served as a reference. The free radical scavenging activity was determined as IC50 (mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), representing the concentration that reduced the ABTS\u0026thinsp;+\u0026thinsp;by 50%.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Fatty acid composition analysis\u003c/h2\u003e \u003cp\u003eThe analysis of fatty acids in the microalgae extract was performed using a gas chromatography with flame ionization detector (GC-FID) system, using the Agilent model 7880B (Agilent Technologies, Santa Clara, California, USA). Separation of fatty acids was achieved utilizing an Agilent DB-Fast FAME column (60m \u0026times; 250 \u0026micro;m \u0026times; 0.25 \u0026micro;m). A sample volume of approximately 1 \u0026micro;L was injected into the GC column. Hydrogen gas served as the carrier gas, flowing at a rate of 40 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The temperature program was initiated at 80\u0026deg;C for 1 minute, followed by a ramp to 165\u0026deg;C at a rate of 40\u0026deg;C min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, maintained for 1 minute, and concluded with an increase to 230\u0026deg;C at 4\u0026deg;C min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, held for 15 minutes. The detector temperature was set at 260\u0026deg;C, while the injector operated at 250\u0026deg;C with a split ratio 100:1. The Identification of each fatty acid within the extract was accomplished by comparing their retention times against a Supelco\u0026reg; standard. Quantification was performed using GC Solution\u0026reg; software, results were presented as percentages relative to the total identified fatty acids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe results are expressed as mean values accompanied by their respective standard deviations. Data compilation and analysis were facilitated by Microsoft Excel 2016 and GraphPad Prism version 9.1.0 for Windows (GraphPad Software based in San Diego, California, USA). To analyse the collected data, one-way ANOVA was employed, supplemented with Tukey's multiple comparison tests to determine statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effect of sonication and solvent system on the extraction yield\u003c/h2\u003e \u003cp\u003eThe extraction yields of S. \u003cem\u003eparvus\u003c/em\u003e using various solvents under sonicated and non-sonicated conditions are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The results demonstrate significant variations in extraction efficiency depending on the types of solvents used and the implementation of sonication. Furthermore, the findings suggest that extracts produced via sonication result in considerably greater yields compared to those extracted without sonication, irrespective of the solvent employed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 The Impact of Different Extraction Methods on Total Phenolic and Flavonoid Content\u003c/h2\u003e \u003cp\u003eFor future assessments of the antioxidant potential of S. \u003cem\u003eparvus\u003c/em\u003e, this study will focus solely on the crude extracts of methanol and ethanol. These extracts were selected due to their higher yields compared to the other solvents tested in the study. This study believes higher yield extracts will likely contain a broader range of bioactive compounds, facilitating more comprehensive antioxidant evaluation. This increased diversity may enhance the sensitivity of assays, thereby yielding more accurate and reliable results. The summarised results in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e show that extraction is significantly influenced by the method and solvent used.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAssessment of the total phenolic and flavonoid content and the antioxidant activity of S. \u003cem\u003eparvus\u003c/em\u003e extract.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTPC \u003c/p\u003e \u003cp\u003e(mg GAE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTFC \u003c/p\u003e \u003cp\u003e(mg QE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDPPH \u003c/p\u003e \u003cp\u003e(mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eABTS+ \u003c/p\u003e \u003cp\u003e(mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNon-sonicated MeOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e23.58\u0026thinsp;\u0026plusmn;\u0026thinsp;2.168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e270.01\u0026thinsp;\u0026plusmn;\u0026thinsp;19.174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSonicated MeOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e14.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e148.04\u0026thinsp;\u0026plusmn;\u0026thinsp;43.758\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.083\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNon-sonicated EtOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e20.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e191.83\u0026thinsp;\u0026plusmn;\u0026thinsp;17.646\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSonicated EtOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e66.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.818\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e684.45\u0026thinsp;\u0026plusmn;\u0026thinsp;28.928\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoxhlet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.632\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e39.96\u0026thinsp;\u0026plusmn;\u0026thinsp;13.751\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effect of different extraction methods on the antioxidant activity\u003c/h2\u003e \u003cp\u003eThe antioxidant activity of the S. \u003cem\u003eparvus\u003c/em\u003e extract was evaluated using a radical scavenging assay targeting DPPH and ABTS\u0026thinsp;+\u0026thinsp;free radicals. The IC50 values, which represent the concentration required to inhibit 50% of the free radical activity, were used as indicators of antioxidant potency, with lower values corresponding to higher antioxidant activity. A summary of all data on antioxidant activity is summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Fatty acid composition of the S.\u003cem\u003eparvus\u003c/em\u003e extract\u003c/h2\u003e \u003cp\u003eThe data in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows a detailed comparison of the fatty acid composition of S. \u003cem\u003eparvus\u003c/em\u003e extracts obtained using two different methods: soxhlet extraction and non-sonicated methanol extraction. The values are expressed in grams per 100 grams of extract sample.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFatty acid profile of soxhlet and non-sonicated methanol S. \u003cem\u003eparvus\u003c/em\u003e extract.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFatty Acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSoxhlet\u003c/p\u003e \u003cp\u003eextract (g/100g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNon-sonicated MeOH\u003c/p\u003e \u003cp\u003eextract (g/100g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDodecanoic Acid (C12:0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTridecanoic Acid (C13:0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMyristic Acid (C14:0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMyristoleic Acid (C14:1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePalmitic Acid (C16:0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePalmitoleic Acid (C16:1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStearic Acid (C18:0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOleic Acid (C18:1 cis)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLinoleic Acid (C18:2 cis)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLinolenic Acid (C18:3n3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEicosanoic Acid (C20:0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussions","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Effect of sonication and solvent system on the extraction yield\u003c/h2\u003e \u003cp\u003eMethanol and ethanol produced the highest extraction yields among all the solvents tested in the study, with methanol yielding approximately 10.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.919% for the non-sonicated samples and around 14.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.503% for the sonicated samples. Ethanol yielded 5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.230% in the absence of sonication, whereas the yield increased to 10.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.202% with sonication. These findings aligned with previous findings that reported both methanol and ethanol as the most efficient solvents for the extraction the bioactive compounds from S. \u003cem\u003eparvus\u003c/em\u003e (Ren et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tavakoli et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hui et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). Chloroform, being a less polar solvent than methanol and ethanol, yielded approximately 6.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.474% for the non-sonicated samples and about 9.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.354% for the sonicated samples. This indicates that chloroform extracts have lesser numbers of compounds compared to methanol and ethanol, but the application of sonication significantly increases the extraction yield. In the case of hexane and petroleum ether, both non-polar solvents, the extraction yields were very low, ranging from 1\u0026ndash;2%, even with sonication. This suggests that the compounds in S. \u003cem\u003eparvus\u003c/em\u003e. have limited solubility in non-polar solvents, resulting in low extraction yields.\u003c/p\u003e \u003cp\u003eSonication also influenced the extraction yield of most solvents, as demonstrated by the relatively higher extraction yields for sonicated samples compared to non-sonicated ones. Methanol and ethanol showed higher yield compared to the control, suggesting that sonication increases the efficiency of extraction by breaking down the cell walls and releasing the internal compound, as reported in many previous studies (Das and Eun \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ren et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tavakoli et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hui et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). Sonication with chloroform also increased significantly, demonstrating its effectiveness in improving solvent\u0026rsquo;s penetration and extraction capability. Interestingly, the extraction yield using soxhlet extraction, despite being a lengthy process, was approximately 10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.362%, slightly lower than the sonicated methanol and ethanol extracts. This suggests that, while soxhlet extraction is effective, the combination of an appropriate solvent and sonication can yield better results, potentially outperforming conventional soxhlet extraction.\u003c/p\u003e \u003cp\u003eBased on these findings, it can be concluded that solvent selection and the application of sonication are critical factors in the extraction of S. \u003cem\u003eparvus\u003c/em\u003e. In comparison to the other solvents, methanol and ethanol used with sonication were the most effective solvents, producing significantly better results than conventional soxhlet extraction and the non-polar solvents. These results demonstrate that the extraction method and the type of solvent employed have a significant impact on the yield. The findings also support the sonication to enhance bioactive compound yield in microalgal research and related fields. Future studies should investigate the type of compounds extracted by each solvent and the biological activities associated with these extracts to further elucidate the benefits of sonication-assisted extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e4.2 The Impact of Different Extraction Methods on Total Phenolic and Flavonoid Content\u003c/h2\u003e \u003cp\u003eIn the comparison between non-sonicate and sonicate methods for methanol extraction, the non-sonicate method yielded a higher phenolic content of 23.58\u0026thinsp;\u0026plusmn;\u0026thinsp;2.168 mg GAE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW and flavonoid content of 270.01\u0026thinsp;\u0026plusmn;\u0026thinsp;19.174 mg QE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW. In contrast, the sonicate method yielded a lower phenolic content of 14.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.660 mg GAE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW and a flavonoid content of 148.04\u0026thinsp;\u0026plusmn;\u0026thinsp;43.758 mg QE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW. These results are inconsistent with the previous studies that suggest sonication with methanol should enhance the TPC and TFC values rather than lowering the compound extracted from the S. \u003cem\u003eparvus\u003c/em\u003e (Upadhyay et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kim and Lee \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Agarwal et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; \u0026Ouml;zcan et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ahmed et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, further study is needed to elucidate this phenomenon.\u003c/p\u003e \u003cp\u003eOn the other hand, ethanol extraction depicted a different pattern of results. The sonication method was significantly more effective than the non-sonicate method, where the phenolic content increased from 20.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.007 GAE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW to 66.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.818 mg GAE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW, and the flavonoid content increased from 191.83 mg QE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW to 684.45\u0026thinsp;\u0026plusmn;\u0026thinsp;28.928 mg QE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW. This significant increment further supports the effectiveness of sonication with ethanol in the disruption of the cell walls of the S. \u003cem\u003eparvus\u003c/em\u003e to extract the phenolic and flavonoid compounds. This finding is consistent with previous studies showing that using sonication and ethanol during extraction enhances the recovery of phenolic and flavonoid compounds (Gogoi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mousavi et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Irfan et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In contrast, the soxhlet extraction method, which has the longest extraction time and continuous solvent circulation, yielded the lowest yields of phenolic (5. 05\u0026thinsp;\u0026plusmn;\u0026thinsp;0. 632 mg GAE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW) and flavonoid (39. 96\u0026thinsp;\u0026plusmn;\u0026thinsp;13. 751 mg QE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW) contents. This reduced efficiency could be due to the degradation of bioactive compounds due to more prolonged exposure to heat compared to the other methods.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrates that selecting a proper extraction method and solvent will directly influence the quantity of phenolic and flavonoid compounds extracted from S. \u003cem\u003eparvus\u003c/em\u003e. The results showed that sonication in ethanol was the most effective method for enhancing the extraction yield of these bioactive compounds. These findings could improve extraction processes in both research and industry, where maximizing the recovery of phenolic and flavonoid compounds is essential. Additionally, the results suggest that conventional Soxhlet extraction may not be ideal for such applications, particularly when targeting heat-sensitive bioactive compounds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Effect of different extraction methods on the antioxidant activity\u003c/h2\u003e \u003cp\u003eThe results of this study revealed significant variations in antioxidant activity depending on the extraction method and solvent used. For the methanol extract, the non-sonicated method yielded lower IC50 values for DPPH (0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), indicating greater antioxidant activity compared to the sonicated method, which had IC50 values of 0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.083 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. A similar pattern was observed with the ethanol extract, where the non-sonicated method showed higher antioxidant activity against the DPPH free radical, with an IC50 value of 0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.049 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, compared to 1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the sonicated method. These findings contrast with previous studies that reported increased antioxidant activity with sonication (Kim and Lee \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; \u0026Ouml;zcan et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ranjha et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tavakoli et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mousavi et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Irfan et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It is hypothesized that the decrease in the antioxidant activity in sonicated extracts may result from the high energy during the sonication process affecting the structural integrity of antioxidant compounds, thereby reducing their activity. On the other hand, no significant differences in antioxidant activity were observed among different extraction methods and solvents for the ABTS\u0026thinsp;+\u0026thinsp;radical.\u003c/p\u003e \u003cp\u003eThese findings indicate that the choice of extraction method and solvent is crucial in determining the antioxidant activity of S. \u003cem\u003eparvus\u003c/em\u003e extracts. Methanol emerged as a more effective solvent than ethanol, especially in non-sonicated extracts. Notably, the Soxhlet extraction method demonstrated significantly higher antioxidant activity against the DPPH radical (0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) compared to other methods, except for the non-sonicated methanol extract, despite having lower TPC and TFC values. These insights are essential for refining extraction techniques to maximize antioxidant content and are of great value to researchers and industries involved in the production of functional foods and nutraceuticals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Fatty acid composition of the S. \u003cem\u003eparvus\u003c/em\u003e extract\u003c/h2\u003e \u003cp\u003eThe dodecanoic acid in the non-sonicated methanol extraction yielded a significantly higher concentration (2.1g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) compared to the soxhlet extraction (0.5g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), indicating that the non-sonicated methanol method is more effective for extracting shorter-chain saturated fatty acids. Similarly, the concentration of tridecanoic acid was higher in the non-sonicated methanol extract (2.8g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) than in the soxhlet extract (0.7g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). For myristic acid, both methods resulted in relatively low concentrations, but the non-sonicated methanol extraction method extracted slightly more (1.7g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) than the soxhlet method (0.5g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Myristoleic acid concentrations were also higher in the non-sonicated methanol extract (3.3g 100g-\u003csup\u003e1\u003c/sup\u003e) compared to the soxhlet extract (0.9 g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003ePalmitic acid was the most abundant fatty acid in both extracts, with similar concentrations in both methods (37.6g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in soxhlet and 38.5g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in non-sonicated methanol), suggesting that both methods are equally effective for extracting this fatty acid. The concentration of palmitoleic acid was higher in the non-sonicated methanol extract (1.7g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) than in the soxhlet extract (1.0g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The non-sonicated methanol extract had more than double the concentration of stearic acid (10.2g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) compared to the soxhlet extract (4.1g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), highlighting the significant advantage of the non-sonicated methanol method for extracting this fatty acid. Conversely, oleic acid was more effectively extracted using the soxhlet method (36.8g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) than the non-sonicated methanol method (23.9g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), indicating that soxhlet extraction may be better for extracting monounsaturated fatty acids like oleic acid.\u003c/p\u003e \u003cp\u003eLinoleic acid concentration was also higher in the soxhlet extract (13.2 g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) compared to the non-sonicated methanol extract (9.6 g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), similar to the trend observed with oleic acid. Both methods showed similar concentrations of linolenic acid (4.8 g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in soxhlet and 4.6 g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in non-sonicated methanol), indicating no significant difference between the methods for this fatty acid. Eicosanoic acid was detected only in the non-sonicated methanol extract (1.7 g 100g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), suggesting that the soxhlet method may not be suitable for extracting longer-chain saturated fatty acids.\u003c/p\u003e \u003cp\u003eThe fatty acid profile of S. \u003cem\u003eparvus\u003c/em\u003e extracts varies significantly depending on the extraction method used. The non-sonicated methanol method generally yielded higher shorter-chain and saturated fatty acids concentrations. In contrast, the soxhlet method was more effective for extracting certain monounsaturated and polyunsaturated fatty acids. These findings suggest that the extraction method should be tailored to the specific fatty acids of interest in S. \u003cem\u003eparvus\u003c/em\u003e extracts.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, this study concludes that sonication is crucial in maximizing for optimizing the extraction yield and bioactive compound content of \u003cem\u003eScenedesmus parvus\u003c/em\u003e. Methanol and ethanol, particularly especially when combined with sonication, proved to be the most effective solvents for extracting phenolic and flavonoid compounds and enhancing antioxidant activity. Therefore, future research should explore the specific compounds extracted by each solvent and evaluate their associated biological activities.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by Fundamental Research Grant Scheme grant from the Malaysian Ministry of Higher Education 203.CIPPT.6712051\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eI.R., S.M., and M.K. made significant contributions throughout this study, including writing the manuscript, conducting experiments, and analyzing the data to its conclusion.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors express their sincere gratitude to the staff and faculty of the School of Industrial Technology at Universiti Sains Malaysia for their invaluable assistance in completing this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgarwal C, M\u0026aacute;th\u0026eacute; K, Hofmann T, Cs\u0026oacute;ka L (2018) Ultrasound‐Assisted Extraction of Cannabinoids from \u003cem\u003eCannabis Sativa\u003c/em\u003e L. 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Oxid Med Cell Longev 2016:1\u0026ndash;13. https://doi.org/10.1155/2016/5692852\u003c/li\u003e\n\u003cli\u003eXu J (2017a) DPPH radical scavenging capacity measurement. https://doi.org/ 10.17504/protocols.io.jiqckdw\u003c/li\u003e\n\u003cli\u003eXu J (2017b) ABTS radical scavenging capacity measurement v1. https://doi.org/ 10.17504/protocols.io.jirckd6\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Microalgae, Scenedesmus parvus, Fatty acid, Extraction, Antioxidant activity","lastPublishedDoi":"10.21203/rs.3.rs-5288243/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5288243/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eScenedesmus parvus\u003c/em\u003e, a Malaysian freshwater microalga, has garnered attention as a promising source of secondary metabolites with applications in various industries, including nutraceuticals, pharmaceuticals, food, and cosmetics. This study aimed to evaluate the extraction yield, total phenolic and flavonoid content, and antioxidant potential of S. \u003cem\u003eparvus\u003c/em\u003e extracts obtained through different extraction methods (sonication, non-sonication, and soxhlet). Additionally, the fatty acid profile and key chemical constituents of S. \u003cem\u003eparvus\u003c/em\u003e were analysed using GC-FID. Results revealed that the sonication method utilizing methanol as the extraction solvent yielded the highest extraction yield (14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5%), whereas the non-sonication method employing hexane yielded the lowest (1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2%). Furthermore, the sonicated ethanol extract exhibited the highest total phenolic content (66.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.818 mg GAE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW) and total flavonoid content (684.45\u0026thinsp;\u0026plusmn;\u0026thinsp;28.928 mg QE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW). Soxhlet extraction demonstrated superior antioxidant activity in both DPPH (IC50: 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and ABTS+ (IC50: 0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) scavenging assays compared to the others. These findings highlight the potential of different extraction methods from S. \u003cem\u003eparvus\u003c/em\u003e, particularly those obtained with methanol as the extraction solvent, as valuable sources of natural antioxidants for application in nutraceuticals, pharmaceuticals, food, and cosmetics.\u003c/p\u003e","manuscriptTitle":"Influence of extraction method and solvent system on the antioxidant activity of Scenedesmus parvus extract","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-31 08:24:45","doi":"10.21203/rs.3.rs-5288243/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"358d5430-51c1-48ca-91a4-d8989c3ea49f","owner":[],"postedDate":"October 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-25T08:39:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-31 08:24:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5288243","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5288243","identity":"rs-5288243","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}