Response surface design for ultrasonic assisted extraction process for calotropis procera seeds and evaluation of its antioxidant activity

Abstract

Calotropis Procera plant is a shrub commonly known as Sodom apple. It belongs to the Asclepiadaceae family (milkweed). C. procera is an original plant of North Africa and is widely dispersed in Asia. C. procera seed contains oil, protein, minerals, and soluble dietary fibers, and there are no reports on antioxidant concentration in the seeds. This study focuses on the development of the extraction conditions, namely, ultra-sonication power (percent amplitude), extraction time (minutes), and solvent to sample ratio (ml/g) to enhance antioxidant activity from C. procera seed. The defatting time screening was conducted to reduce processing time and energy. The best oil extraction time was one (1) hour using n-hexane (10 ml: 1 g), shaker (200 rpm) at room temperature. Screening of solvents was performed using chloroform, isopropanol, methanol, and water to maximize antioxidant activity using DPPH (1, 1-diphenyl-2-picrylhydrazyle) assay. The isopropanol extract gave the highest percentage inhibition in the DPPH assay, 57.731± 0.377 %, so it was used for optimization of the ultra-sonication-assisted extraction process. For the design of the optimization experiments, the response surface methodology (RSM) was adopted. With DPPH content as a response, three independent variables - ultra-sonication extraction time (10, 15, 20 minutes), solvent to sample ratio (10, 15, 20 ml/g), and ultra-sonication power (20, 60, 100 percentage amplitude), were optimized. The highest inhibition percentage of DPPH from C. procera seed extract was 68.81%, obtained at 20% amplitude of ultra-sonication, 10 minutes of ultra-sonication time, and 10 ml solvent per gram sample. The C. procera seed extracts were further investigated for their potential applications using in-vitro antioxidant assays like; total phenolic content (TPC) and ascorbic acid equivalent antioxidant potential (AEAC). The phenolic content of the seed extract was 26.324 mg of Gallic acid equivalent/g dry material. The AEAC antioxidant of C. procera seed extract was 28.707 mg of (Ascorbic Acid equivalent) AAeq/100 g. Gas chromatography mass spectrometry (GC-MS) was used to obtain a profile of the extracts and to identify potential bioactive compounds in the extracts. The GC-MS analysis indicated that β, β-carotene (21.94%), pentacontanoic acid propyl ester (14.93%), (E, E)-5-chloro-4-methyl-2,4-heptadiene (12.411%), (E)-2-nonenal (6.7174%), n-decanoic acid (6.1985%), pentanoic acid (5.9932%), (E)-2-octenal (1.6965%) and 1-methyl-4-nitroimidazole (1.3226%) were the major constituents of the C. procera seed extract. The antibacterial, antioxidant properties, anticancer, cardio-protective agents, skin protection from UV radiation, of phenolic compounds are well-studied. As such, this high phenolic content extract of C. procera seeds can be a promising source of alternative pharmaceutical products derived from natural sources to treat chronic diseases, decreasing future reliance on synthetic drugs.