Identification and Quantification of β-Sitosterol β-d-Glucoside of an Ethanolic Extract Obtained by Microwave-Assisted Extraction from Agave angustifolia Haw

Abstract

β-sitosterol β-d-glucoside (BSSG) was extracted from "piña" of the Agave angustifolia Haw plant by microwave-assisted extraction (MAE) with a KOH solution such as a catalyst and a conventional maceration method to determine the best technique in terms of yield, extraction time, and recovery. The quantification and characterization of BSSG were done by high-performance thin layer chromatography (HPTLC), Fourier-transform infrared spectroscopy (FT-IR), and high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). With an extraction time of 5 s by MAE, a higher amount of BSSG (124.76 mg of β-sitosterol β-d-glucoside/g dry weight of the extract) than those for MAE extraction times of 10 and 15 s (106.19 and 103.97 mg/g dry weight respectively) was shown. The quantification of BSSG in the extract obtained by 48 h of conventional maceration was about 4-5 times less (26.67 mg/g dry weight of the extract) than the yields reached by the MAE treatments. MAE achieved the highest amount of BSSG, in the shortest extraction time while preserving the integrity of the compound's structure.

Keywords: Agave angustifolia Haw; MAE; β-sitosterol β-d-glucoside.

Figure 1

Qualitative high-performance thin layer chromatographic (HPTLC) plate derivatized with Komarovsky reagent; Track 1 standard of β-sitosterol β-d-glucoside (BSSG), Tracks 2–4 ethanolic extracts by microwave-assisted extraction (MAE) in extraction time of 5 s, Tracks 5–7 ethanolic extracts by MAE in extraction time of 10 s, Tracks 8–10 ethanolic extracts by MAE in extraction time of 15 s, Tracks 11–13 ethanolic extracts by MAE in extraction time of 5 s with KOH solution, Tracks 14–16 ethanolic extracts by MAE in extraction time of 10 s with KOH solution, Tracks 17–18 ethanolic extracts by MAE in extraction time of 15 s with KOH solution, and Tracks 16–18 ethanolic extracts of A. angustifolia, with white light and mobile phase toluene:ethyl acetate:formic acid (5:5:0.7 v/v/v).

Figure 2

HPTLC plate derivatized with Komarovsky reagent; Tracks 1–6 calibration curve of BSSG, Tracks 7–9 ethanolic extracts by MAE in extraction time of 5 s with KOH solution, Tracks 10–12 ethanolic extracts by MAE in extraction time of 10 s with KOH solution, Track 13–15 ethanolic extracts by MAE in extraction time of 15 s with KOH solution, and Tracks 16–18 ethanolic extracts by maceration method in extraction time of 48 h of A. angustifolia, with white light and mobile phase toluene:ethyl acetate:formic acid (5:5:0.7 v/v/v).

Figure 3

3D densitograms; track 6 represents the standard solution of BSSG, tracks 7–9 show extracts obtained by MAE in 5 s with KOH solution, tracks 10–12 are the extracts obtained by MAE in 10 s with KOH solution, tracks 13–15 show the extracts obtained by MAE in 15 s with KOH solution, and tracks 16–18 represent maceration at 48 h. All measurements were taken at λ 540 nm.

Figure 4

Spectral comparison of HPTLC chromatograms obtained by the BSSG standard, and ethanolic extracts obtained by MAE and by maceration of A. angustifolia. Wavelengths from 200 to 600 nm. (1) BSSG standard, (2) ethanolic extracts by MAE in extraction time of 5 s with KOH solution, (3) ethanolic extracts by MAE in extraction time of 10 s with KOH solution, (4) ethanolic extracts by MAE in extraction time of 15 s with KOH solution, and (5) ethanolic extracts by maceration at 48 h.

Figure 5

(A) FT-IR spectra of the BSSG standard (BSSG) and the ethanolic extracts obtained by MAE from A. angustifolia (5, 10, 15 s) with KOH solution; (B) ampliation of Figure 5A in the region from 600 to 1800 cm⁻¹.

Figure 5

(A) FT-IR spectra of the BSSG standard (BSSG) and the ethanolic extracts obtained by MAE from A. angustifolia (5, 10, 15 s) with KOH solution; (B) ampliation of Figure 5A in the region from 600 to 1800 cm⁻¹.

Figure 6

High-performance liquid chromatography−electrospray ionization−mass spectrometry (HPLC-ESI-MS) positive ion of the mass spectrum of ethanolic extract of 5 s obtained by MAE with KOH solution of A. angustifolia.