Ultrasound-Assisted Extraction of Squalene and 2-Acetyl-1-Pyrroline from Pandan Leaf: The Effects of Drying Methods and Extraction Conditions

Abstract

Pandan, a tropical crop, is rich in squalene (SQ), known for its antioxidant and hypoglycemic properties, and 2-acetyl-1-pyrroline (2-AP), which imparts a characteristic aroma. This study focuses on the extraction of the two bioactive compounds from Pandan leaves and investigates the effects of drying methods, extraction solvents, and conditions on the yield of SQ and 2-AP. Results show that hot air-dried Pandan leaves when extracted using the binary solvent system of ethanol and n-hexane (EH), yield higher SQ content while maintaining an adequate content of 2-AP. To further optimize the extraction process, a single-factor experiment was followed by optimization using Box-Behnken design (BBD) and response surface methodology (RSM). The optimal extraction conditions were determined as follows: ultrasound time of 60 min, a temperature of 50 °C, power of 300 W, and a solid-to-liquid ratio of 1:5 g/mL. Under these conditions, an SQ yield of 1229.98 ± 13.09 μg/DW 1 g Pandan leaves and a 2-AP yield of 80.72 ± 0.88 μg/DW 1 g Pandan leaves were achieved, representing increases of 3.30% and 9.82% compared to pre-optimization values. Additionally, the antioxidant activities of EH extracts were evaluated through various in vitro assays. The extracts demonstrated significant DPPH and ABTS free radical scavenging activity (12.46 μmol TE/g DW and 22.14 μmol TE/g DW, respectively), along with ferric and cupric ion reducing power (10.629 μmol TE/g DW and 14.275 μmol TE/g DW, respectively). The extracts also exhibited notable inhibitory effects on α-amylase and α-glucosidase. The findings suggest that these extracts are a promising natural source of antioxidants with potential applications in health and nutrition.

Keywords: Pandan leaf; antioxidant; extraction solvents; hypoglycemic; squalene; ultrasound-assisted extraction.

Figure 1

(a) HPLC chromatogram of Squalene (SQ) standard and Pandan leaf extracts; (b) SQ content: the effect of extraction solvents and drying methods; (c) LC-MS/MS chromatogram of 2-acetyl-1-pyrroline (2-AP) standard and Pandan leaf extracts; (d) 2-AP content: the effect of extraction solvents and drying methods. The lowercase letters on the line were expressed as the significance between various extraction systems. The uppercase letters (A and B) were expressed as the significance between hot air drying and freeze drying. Retention time (RT).

Figure 2

SEM images of leaf cell residues of hot air-dried Pandan powder and freeze-dried Pandan powder before and after ultrasound-assisted extraction at 10,000 magnifications disrupted. Differences can be seen where the red arrows point. (a) hot air-dried and (b) freeze-dried Pandan powder before extraction; (c) hot air-dried and (d) freeze-dried Pandan powder after extraction.

Figure 3

Effects of (a) ultrasound time, (b) ultrasound temperature, (c) ultrasound power, and (d) solid-to-liquid ratio on the extraction content of SQ and 2-AP from Pandan leaf extracts. Different letters on the same line mean statistically different (p < 0.05).

Figure 4

Three-dimensional response surface: (a) and (d) the interaction between ultrasound time and temperature for the content of SQ and 2-AP, respectively; (b) and (e) the interaction between ultrasound time and power for the content of SQ and 2-AP, respectively; (c) and (f) the interaction between ultrasound power and temperature for the content of SQ and 2-AP, respectively.

Figure 5

(a) the total phenolic content (TPC), (b) the total flavonoid content (TFC), and (c) the total Triterpenes content (TTC) in EH Pandan leaf extract before and after optimization. Different letters on the same figure mean statistically different (p < 0.05).

Figure 6

Antioxidant activities in the Pandan leaf extracts of pre-optimization and after optimization determined using DPPH, ABTS radical scavenging assays (a), FRAP, and CUPRAC assays (b). Different letters on the same color figure mean statistically different (p < 0.05).

Figure 7

(a) α-amylase and (b) α-glucosidase inhibition (IC₅₀ values in mg/mL, data were obtained from triplicate experiments and expressed as mean ± STD) of acarbose and Pandan leaf extracts before optimization and after optimization. Different letters on the same figure mean statistically different (p < 0.05).