Ultrasonic extraction of Spirulina polysaccharides was optimized using orthogonal experimental design, considering key factors such as extraction time, pH, and temperature. An L25(3^5) orthogonal array was applied to systematically evaluate the impact of these variables on the extraction efficiency. A total of 25 samples were prepared, each containing 10.00 g of defatted Spirulina dry powder, placed in separate beakers. Distilled water was added according to the experimental design to adjust the pH and temperature, and ultrasonic treatment was carried out at 28 kHz for varying durations. After centrifugation and two repeated extractions, the supernatant was deproteinized using trichloroacetic acid, followed by ethanol precipitation and drying to obtain crude polysaccharide.
The total sugar content was determined using the anthrone-sulfuric acid method. The calculation formula used was: Total sugar content (%) = (polysaccharide concentration in solution / crude extract mass concentration) × 100%. The extraction rate was calculated as (crude extract mass / raw material mass) × 100%, and the extraction amount was determined by multiplying the crude extract mass with the total sugar content. Protein content in the crude product was measured using the Coomassie brilliant blue method.
A comparative study was conducted between the ultrasonic extraction method and the conventional water bath extraction method. The anti-tumor activity of the extracted Spirulina polysaccharides was evaluated on SKV3 ovarian cancer 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. Each well of a 96-well plate contained 1×10ⴠcells, with a final volume of 200 µL. After 12 hours of culture, the medium was replaced with different concentrations of Spirulina polysaccharide solutions ranging from 10 to 100 µg/mL. Following 48 hours of incubation, 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%. The results showed that the maximum polysaccharide yield was achieved under ultrasonic conditions at pH 11, 60°C, and for 60 minutes. Range analysis indicated that pH had the most significant effect on the extraction rate, followed by extraction time and temperature. The ultrasonic method yielded a higher polysaccharide content compared to the water bath method, though the difference was not statistically significant (P > 0.05).
Both methods 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. The composition of Spirulina polysaccharides is complex, consisting mainly of rhamnose, D-mannose, D-glucose, D-galactose, glucuronic acid, xylose, and fucose.
Common methods for determining sugar content include the anthrone-sulfuric acid and phenol-sulfuric acid colorimetric methods. However, discrepancies between measured and actual values are often encountered, especially in natural products. During ultrasonic extraction, excessive treatment time may lead to the degradation of polysaccharide molecules due to mechanical shearing, reducing the overall yield. Similarly, high temperatures can alter the molecular structure of polysaccharides, affecting extraction efficiency. The optimal pH for extraction was found to be 11, highlighting the importance of pH control in maximizing polysaccharide recovery.
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