Ultrasonic extraction of Spirulina polysaccharides was optimized using orthogonal experiments, considering key factors such as extraction time, pH, and temperature. An L25(3^5) orthogonal array was employed to design the experimental parameters. A total of 25 samples were prepared, each containing 10.00 g of defatted Spirulina dry powder placed in separate beakers. Distilled water (200 mL) was added according to the experimental design, adjusting the pH and temperature accordingly. The samples were then subjected to ultrasonic treatment at a frequency of 28 kHz for varying durations. After sonication, the extracts were centrifuged, and the supernatant was collected. This process was repeated twice, followed by deproteinization with trichloroacetic acid, precipitation with ethanol, and drying to obtain crude polysaccharides.
The total sugar content was determined using the anthrone-sulfuric acid method. The formula used was: Total sugar content (%) = (concentration of polysaccharide in solution / crude extract mass concentration) × 100. The extraction rate was calculated as (crude extract mass / raw material mass) × 100%, while the extraction amount was calculated as (crude extract mass × total sugar content). Protein content in the crude product was measured using the Coomassie blue method.
A comparison between the ultrasonic extraction method and the conventional water extraction method was conducted based on the orthogonal experiment results. The anti-tumor activity of the extracted Spirulina polysaccharide was evaluated against ovarian cancer SKV3 cells in vitro. Cells in logarithmic growth phase were seeded at a density of 5 × 10³ cells/mL in DMEM with 10% fetal bovine serum. A 96-well plate was used, with 1 × 10ⴠcells per well and a final volume of 200 µL. After 12 hours of incubation, the medium was replaced with Spirulina polysaccharide solutions at concentrations ranging from 10 to 100 µg/mL. Following 48 hours of treatment, cell viability was assessed using the MTT assay.
The inhibition rate was calculated as: Inhibition rate (%) = [(average OD of control group - average OD of experimental group) / average OD of control group] × 100%. Results showed that the maximum polysaccharide yield was achieved under conditions of pH 11, 60°C, and 60 minutes of ultrasonic extraction. Range analysis indicated that pH had the most significant effect on the extraction efficiency, followed by extraction time and temperature.
Comparing the two methods, the ultrasonic extraction method yielded higher polysaccharide content than the traditional water bath method. Both extracts exhibited anti-tumor activity against SKV3 cells, with inhibition rates of 43.01%–33.497% and 39.26%–53.1293%, respectively. A dose-dependent relationship was observed, but no significant difference was found between the two methods (P > 0.05).
The composition of Spirulina polysaccharides is complex, consisting mainly of rhamnose, D-mannose, D-glucose, D-galactose, glucuronic acid, xylose, and fucose. Commonly used methods for sugar content determination include the anthrone-sulfuric acid and phenol-sulfuric acid colorimetric methods. However, in practice, especially when analyzing natural products, discrepancies between measured values and actual conditions often occur.
When using the ultrasonic method, excessive extraction time may lead to degradation of polysaccharide molecules due to mechanical shearing, resulting in reduced yield during post-processing. High temperatures can also alter the structure of polysaccharide macromolecules, affecting extraction efficiency. The pH significantly influences the extraction process, with optimal results observed at pH 11 in this study.
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