The team, publishing in the journal Pharmacia, collected fresh Sargassum polycystum from the east coast of Pangandaran at coordinates 7°42’22.5″S, 108°39’50.3″E, and had the material authenticated as Sargassaceae by the Herbarium Jatinangor at Universitas Padjadjaran. Forty kilograms of fresh algae were cleaned, dried at 40 °C for 24 hours, ground, and sieved, yielding just 2.6 kilograms of dried powder, a dry-material recovery of 6.50 percent. Crucially, a single common batch of this powder fed both extraction experiments, ensuring that any differences between the two methods reflected processing rather than raw-material variability.

Sargassum polycystum is a chemically rich target. Like other brown algae, it contains phlorotannins and other phenolic compounds capable of donating electrons or hydrogen atoms to neutralize free radicals, along with flavonoids, pigments, polysaccharides, and sterol-related metabolites. Brown algal cell walls, however, are formidable barriers: alginates, sulfated polysaccharides, cellulose, and proteins restrict solvent penetration and can slow the release of intracellular constituents. This structural complexity means that a high extraction yield, which simply measures total soluble mass, may largely reflect dissolved polysaccharides, salts, and pigments rather than the phenolic and flavonoid constituents that drive antioxidant quality.

To disentangle these effects, the researchers ran two independent three-factor, three-level Box–Behnken designs, each with 15 extraction runs including three independently prepared center points to estimate pure experimental error. Reflux extraction was tested across temperatures of 50 to 70 °C, durations of 1 to 3 hours, and solid-to-solvent ratios of 1:5 to 1:15, with particle size fixed at 60 mesh and 96 percent ethanol as the solvent. Maceration, the gentle room-temperature alternative, was tested across 24 to 72 hours, particle sizes of 40 to 80 mesh, and the same ratio range at 25 ± 2 °C. Four responses were tracked for every run: extraction yield, total phenolic content (TPC), total flavonoid content (TFC), and radical-scavenging potency expressed as the DPPH IC50, the concentration needed to quench half of the DPPH radicals in the assay.

The observed ranges told the first part of the story. Reflux produced yields of 5.46 to 15.38 percent, with the maximum at 60 °C, 2 hours, and a 1:10 ratio, while maceration spanned 2.01 to 15.30 percent, peaking after 72 hours at 60 mesh and 1:15. But the quality responses inverted the picture. Maceration achieved the highest phenolic content of the entire study, 62.98 mg gallic acid equivalents per gram of extract, and by far the strongest DPPH activity, with an IC50 of just 36.19 µg/mL, both at 24 hours, 80 mesh, and 1:10. Reflux peaked at 53.05 mg GAE/g and an IC50 of 98.66 µg/mL, notably at its mildest condition of 50 °C and 1 hour. In both designs, the recipe for maximum yield was a different recipe from the recipe for maximum antioxidant quality.

The statistical models behind these observations were robust. All eight response models, four per method, were significant, with p-values ranging from 0.001 to 0.006, and lack-of-fit tests were non-significant throughout, indicating the second-order models adequately represented the design space. R² values ranged from 81.78 to 99.16 percent. The dominant factors were revealing: extraction temperature carried the largest F-values among the reflux factors, while extraction time dominated every maceration response. The researchers also flagged an important caveat, noting that predicted R² values for the bioactive responses were substantially lower than adjusted R² values, particularly for reflux, signaling limited out-of-sample precision for TPC, TFC, and IC50 even when the models fit the observed data well.

Desirability-based multi-response optimization then translated the models into recommended operating points. For reflux, the optimum landed at 50.404 °C, 1.0404 hours, and a ratio of approximately 1:9.55, with a composite desirability of 0.663. Validation runs using three independently prepared extractions delivered a 10.32 percent yield, 39.68 mg GAE/g TPC, 33.24 mg QE/g TFC, and an IC50 of 109.25 µg/mL, all within the corresponding 95 percent confidence and prediction intervals. For maceration, the optimum was 24 hours, 80 mesh, and roughly 1:5.30, with a higher composite desirability of 0.704. Validation produced a 5.54 percent yield, 59.39 mg GAE/g TPC, 38.03 mg QE/g TFC, and an IC50 of 55.66 µg/mL, again within intervals, though the maceration IC50 prediction interval was wide, reflecting genuine uncertainty in that estimate.

The most consequential insight emerged when the team normalized results to the starting biomass rather than to the extract itself. Because the optimized maceration extract was more concentrated but obtained at lower yield, reflux actually recovered more total antioxidant-related material per unit of seaweed: an estimated 409.50 mg of gallic acid equivalents and 343.04 mg of quercetin equivalents per 100 grams of dry algae, versus 329.02 and 210.69 mg respectively for maceration. In other words, the choice between methods is not a simple ranking but a decision about the goal. If the priority is a concentrated, potent extract per gram, maceration wins. If the priority is stripping the maximum phenolic and flavonoid payload from a given harvest of biomass, reflux wins, provided temperature and duration are kept mild to protect thermolabile constituents.

The mechanistic explanation fits the physics of each process. Reflux accelerates mass transfer through controlled heating and continuous solvent condensation, but heat is a double-edged sword, since elevated temperatures and prolonged exposure can degrade phenolic and flavonoid structures. That is why reflux quality peaked at its gentlest setting. Maceration relies on passive diffusion, and grinding the seaweed to 80-mesh powder shortened diffusion pathways and increased contact area, allowing phenolics to be recovered within 24 hours. Extending maceration to 72 hours pulled more total soluble material into solution, inflating yield, but the additional mass was evidently not proportional phenolic content, and prolonged contact risks oxidation or degradation of compounds already released.

The authors are careful about the limits of their claims. Qualitative phytochemical screening of representative extracts confirmed reagent-reactive phenolics, flavonoids, tannins, saponins, and steroidal or triterpenoid constituents, with alkaloid reactions depending on the reagent used, but these colorimetric and precipitation tests are group-level indicators, not compound identifications. The Folin–Ciocalteu and aluminum chloride assays quantify equivalent classes rather than specific phlorotannins, and DPPH measures chemical radical scavenging under defined solvent conditions, not activity in living cells. The team recommends chromatographic profiling by HPLC-DAD or LC-MS to link process conditions to individual constituents, additional assays such as ABTS, FRAP, or ORAC to broaden the antioxidant picture, and quantification of residual solvent in future standardization work.

Even with those caveats, the study offers a practical blueprint for a growing industry. Indonesia’s extensive tropical coastline holds substantial Sargassum resources, and brown algae are increasingly pursued for antioxidant, anti-inflammatory, antidiabetic, and neuroprotective applications. The finding that reflux and maceration should be treated as complementary rather than interchangeable options, each with its own validated optimum, gives process developers a defensible starting point. The researchers suggest that future work should explore assisted extraction technologies and green-process metrics, including solvent consumption and energy demand, to push these conventional methods toward more sustainable production. For now, the message from Pangandaran is clear: in seaweed chemistry, what you extract and how much you extract are two different questions, and answering only one of them can leave the real prize in the pot.

Subject of Research: Multi-response optimization of reflux and maceration extraction of bioactive compounds from the brown alga Sargassum polycystum

Article Title: Parallel multi-response optimization of reflux and maceration extraction of Sargassum polycystum from the east coast of Pangandaran using Box–Behnken design

Article References: Susanti, S., Fisabilah, A., Kamil, I., Adlina, S., Rizkuloh, L. R., Mardianingrum, R., & Singgih, M. (2026). Parallel multi-response optimization of reflux and maceration extraction of Sargassum polycystum from the east coast of Pangandaran using Box–Behnken design. Pharmacia, 73, Article e209685. https://doi.org/10.3897/pharmacia.73.e209685

Image Credits: AI Generated

DOI: 10.3897/pharmacia.73.e209685

Keywords: Sargassum polycystum, brown algae, extraction optimization, response surface methodology, Box–Behnken design, antioxidant activity, total phenolic content, total flavonoid content, DPPH assay, reflux extraction, maceration, Pangandaran

The team focused on three widely distributed brown seaweeds: Sargassum polycystum, Turbinaria ornata, and Rosenvingea intricata. These species are abundant along Indian shorelines, yet they remain largely underexploited as raw materials for nutraceuticals, functional foods, and pharmaceuticals. Brown seaweeds in general are known to be reservoirs of valuable molecules, including phenolic compounds, flavonoids, sulfated polysaccharides such as fucoidan, and the carotenoid fucoxanthin, all of which have attracted intense scientific interest for their antioxidant, anti-inflammatory, and potential anticancer properties. The challenge has always been extraction: how to pull these compounds out of a tough, gelatinous algal matrix efficiently, cheaply, and without resorting to toxic chemicals that compromise the safety and sustainability of the final product.

Hot water extraction offers an elegant answer. Water is non-toxic, inexpensive, non-flammable, and universally available, making it arguably the greenest solvent that exists. The technique sits conceptually close to subcritical water extraction, a method in which heated liquid water becomes an increasingly capable solvent as its dielectric constant drops with rising temperature, allowing it to dissolve compounds that would normally require organic solvents. In this study, the researchers compared extraction performance at three temperatures: 28.5 degrees Celsius, representing ambient conditions, 70 degrees Celsius, and 90 degrees Celsius. The differences they observed were striking and statistically significant, underscoring how profoundly temperature shapes both the quantity and the quality of what can be recovered from seaweed biomass.

The yield data tell the first part of the story. At 90 degrees Celsius, extraction yielded 9.18 percent for Sargassum polycystum, 10.14 percent for Turbinaria ornata, and 8.78 percent for Rosenvingea intricata, each figure significantly higher than what was obtained at the lower temperatures. In practical terms, roughly a tenth of the dried seaweed biomass was converted into soluble extract simply by treating it with hot water. For an industry built on margins, that recovery rate matters. It means that a coastal biorefinery could valorize seaweed harvests with minimal solvent costs, minimal hazardous waste streams, and straightforward equipment, since the process requires nothing more technically demanding than controlled heating and filtration.

Yield alone, however, says nothing about biological activity, and this is where the study delivers its most compelling evidence. Across all three species, extracts obtained at 90 degrees Celsius showed significantly higher antioxidant properties than extracts prepared at lower temperatures, as measured by total phenolic content, total flavonoid content, and ferric reducing antioxidant power assays. The researchers attribute this to the thermal disruption of the seaweed matrix at higher temperatures. Heat softens and breaks down cell walls and liberates bound phenolic compounds that remain locked inside the algal tissue under ambient conditions. In effect, boiling water does mechanically and chemically what harsher solvents do, but without the environmental baggage.

The antioxidant story was reinforced by two free radical assays. Extracts from the 90 degree Celsius treatments showed significantly greater scavenging activity against 2,2-diphenyl-1-picrylhydrazyl, the stable free radical commonly known as DPPH that serves as a standard benchmark for antioxidant capacity. They also performed significantly better in the deoxyribose assay, a test that evaluates both site-specific and non-site-specific radical scavenging by measuring the protection of deoxyribose sugar from degradation by hydroxyl radicals. Because hydroxyl radicals are among the most reactive and damaging species generated in living tissues, strong performance in this assay hints at genuine protective potential in biological systems rather than mere laboratory chemistry.

Perhaps the most visually dramatic evidence came from the DNA nicking assay. In this experiment, plasmid DNA is exposed to Fenton’s reagent, a mixture that generates hydroxyl radicals and converts the intact supercoiled DNA into fragmented, nicked forms that migrate differently on a gel. When the seaweed extracts were added, they shielded the DNA from this oxidative damage, preserving its structural integrity. The result demonstrates that the compounds recovered by hot water extraction are not just chemically active in solution but capable of protecting a biologically central molecule, DNA, from radical-induced breakage. That protective effect strengthens the case for these extracts as candidates for dietary supplements, nutraceuticals, and functional food ingredients aimed at reducing oxidative stress.

The implications extend well beyond human nutrition. The authors point to the potential of these seaweed extracts as immune boosters and growth enhancers for animals, fish, and shellfish, a direction that could matter enormously for India’s vast aquaculture sector. Natural antioxidant additives derived from marine algae could reduce reliance on synthetic antioxidants in feed, improve animal health, and add value to seaweed harvests that currently command low prices. Given that global interest in seaweed farming is rising rapidly, both as a food source and as a tool for climate mitigation, methods that convert raw biomass into high-value products are exactly the kind of technology that could accelerate the so-called blue economy.

There are, of course, caveats and next steps. The study reports crude extracts rather than purified single compounds, so further work will be needed to identify precisely which molecules drive the observed antioxidant and DNA-protective effects, and to confirm that the activity survives digestion, storage, and processing at industrial scale. Temperature optimization will also need balancing against the thermal sensitivity of individual compounds, since prolonged high heat can degrade some bioactives even as it releases others. Nevertheless, the central message stands: for three abundant Indian brown seaweeds, ordinary hot water outperformed milder conditions on every measure of yield and antioxidant potency tested, all with a solvent that costs essentially nothing and harms nothing. In a field often enamored with exotic solvents and high-tech equipment, the humble kettle may turn out to be the biorefinery’s most underrated tool.

Subject of Research: Hot water extraction of antioxidant bioactive compounds from Indian brown seaweeds

Article Title: Optimization of Hot Water Extraction as a Green Approach to Valorise Bioactive Compounds from Indian Brown Seaweeds: A Comparative Study of Sargassum polycystum, Turbinaria ornata, and Rosenvingea intricata

Article References: Optimization of Hot Water Extraction as a Green Approach to Valorise Bioactive Compounds from Indian Brown Seaweeds: A Comparative Study of Sargassum polycystum, Turbinaria ornata, and Rosenvingea intricata. (n.d.). https://doi.org/10.1007/s12649-026-03789-4

Image Credits: AI Generated

DOI: 10.1007/s12649-026-03789-4

Keywords: brown seaweeds, hot water extraction, green extraction, antioxidant activity, bioactive compounds, Sargassum polycystum, Turbinaria ornata, Rosenvingea intricata, DNA protection, nutraceuticals, phenolic compounds, waste valorization