Assessment of Lipase Inhibitory Efficiency, Total Phenolic Content and Antioxidant Activity of Spirogyra sp. Extract from Closed-Circulation Cultivation System

Abstract

Background and Objectives : Obesity is a lipid homeostasis dysfunction and hyperlipidemia condition, leading to the occurrence of the other chronic diseases such as cardiovascular disease, diabetes and hypertension. The enzyme which is importance in lipid digestion is lipase. Inhibition of lipase enzyme decreases the digestion and the absorption of lipid to the cells, causing in lower level of the lipid in blood. The studies of many natural lipase inhibitors may help the reduction of synthetic drug which causes long-term adverse side effects. Thus, the objectives of this research were to assess the lipase inhibitory efficiency, total phenolic content and antioxidant activity in Spirogyra sp. extract. Spirogyra sp. was the green-fresh water macroalgae with high nutrition values and antioxidants content such as phenolic and flavonoids compounds. But the phytochemicals containing in algae were variation because of the different aquatic habitat of algae. It made the unstable quality and productivity of algae from natural sources.  In this study, Spirogyra sp. sample was cultivated from closed-circulating system, which the culture conditions were controlled for both productivity and quality of algae. Moreover, algae were non-contaminated from any toxic substances because there was the water treatment process both before and after cultivation. There has been no previous study on the assessment of lipase inhibitory efficiency, total phenolic content and antioxidant activity in Spirogyra sp., which was cultivated in the closed-circulation cultivation system.

Methodology : Spirogyra sp. sample was cultured in 1,000 L closed-circulating raceway pond type in transparent acrylic roof greenhouse. Water treatment was done using precipitation method coupled with the aerator before and after each cultivation cycle. Algae sample was picked, washed, dried at 50^OC for 2 days and blended. Dried powder sample was macerated with 70% ethanol at a ratio of dried powder : solvent was 1:5 weight by volume for 48 h, and evaporated to produce the crude extract. Afterthat, crude extract was determined the lipase inhibitory efficiency by measuring the rate of oleic acid released from triolein breakdown by pancreatic lipase enzyme, compared with the drug named Xenical (standard Orlistat). Total phenolic content was performed using Folin–Ciocalteu phenol reagent method. The antioxidant activity was conducted using the DPPH radical scavenging activity method. The enzyme inhibition kinetic was evaluated using the Lineweaver-Burg plot derived from the Michaelis-Menten equation. The relationship between lipase inhibition efficiency and, total phenolic content and DPPH antioxidant activity was also investigated using Pearson correlation coefficient.

Main Results : The results showed that lipase inhibition efficiency increased with increasing the extract concentration (p0.05). The inhibitory efficiency of Spirogyra sp. extract and the anti-obesity drug named Xenical at the same concentration of 40 mg/mL were 23.81±0.97% and 85.71±2.33 %, respectively, and the IC₅₀ value (the 50% lipase inhibition concentration) were 90.91±1.83 and 16.67±1.87 mg/mL, respectively. In comparison, the percentage of lipase inhibition of Xenical was 3.59 times higher than that of Spirogyra sp. extract while the IC₅₀ value of Xenical was 5.45 times lower than that of the extract. The lower IC₅₀ value showed the higher enzyme inhibition. Although the enzyme inhibitory efficiency of Spirogyra sp. extract was lower than Xenical, it is still counted that Spirogyra sp. extract had the lipase inhibition power. The behavior of lipase inhibition of Spirogyra sp. extract was non-competitive manner, with consideration to V_max, K_m, K_i and K_i' values derived from the Lineweaver-Burg plot. The V_max  values (maximum reaction rate) were significantly decreased (p0.05), the K_m values (Michaelis-Menten constant) were significantly unchanged (p>0.005) and the K_i value was more than the K_i' value (Inhibition constant). This inhibition behavior meant that the inhibitor bound to both enzyme or enzyme-substrate complex at the allosteric sites, resulting in the decrease of the enzyme activity. In addition, the total phenolic content of Spirogyra sp. extracts was 36.65±0.25 mg gallic acid/g extract and the DPPH antioxidant activity was 3.13±0.03 mg Trolox/g extract. The percentage of DPPH radical scavenging was 70.30±0.64. Pearson correlation coefficient showed that the percentage of lipase inhibition was strong correlation with total phenolic content (r=0.8224), and moderate correlation with DPPH radical scavenging activity (r=0.7145) of the extract. It indicated that phenolic compounds and antioxidant activity in the extract play an important role in the efficiency of lipase inhibition. Generally, natural extract that contained high content of phenolic and flavonoid compounds, and high antioxidant activity, also showed the high lipase inhibition efficiency.

Conclusions  : The results of this research provided that the extract of Spirogyra sp. cultivated in closed-circulating system, which the culture conditions were controlled for both quantity and quality of algae, was potential used as a lipase inhibitor. The extract had high content of total phenolic and antioxidant activity, influenced on the lipase enzyme inhibition capacity. Therefore, Spirogyra sp. extract has the potential development to the heathy product. However, this study was the assessment of lipase inhibitory potential of Spirogyra sp. extract only in test tube (in vitro), the studies on lipase inhibitors from natural extract should be done in cultured cells (in vitro) or a laboratory animal (in vivo) in order to confirm the exactly efficiency, possible side effects and the use safe of the extract.