Multi-Response Optimization of Ultrasonic Assisted Enzymatic Extraction Followed by Macroporous Resin Purification for Maximal Recovery of Flavonoids and Ginkgolides from Waste Ginkgo biloba Fallen Leaves

Abstract

In the present study, the process of ultrasonic assisted enzymatic extraction (UAEE), followed by macroporous resin purification, was successfully developed to achieve maximal recovery of flavonoids and ginkgolides from Ginkgo biloba fallen leaves (GBFL). Three effective extracted factors, including UAE power, EtOH%, and the amount of cellulase were screened by Plackett⁻Burman design (PBD). The important variables were further optimized by rotatable central composite design (RCCD). After the combination of PBD and RCCD, the resulting optimal UAEE conditions were as follows: UAE power of 218 W; EtOH% of 68%; the amount of cellulase of 8.4 mg; UAE temperature of 40 °C; UAE time of 20 min; pH of 5.0; and, sample particle size of 40 mesh. Under the optimum conditions; the yields of flavonoids were 0.74 ± 0.05% (n = 3) and ginkgolides was 0.42 ± 0.06% (n = 3), which were close to the predicted values. Moreover, the further enriching flavonoids and ginkgolides from the obtained GBFL extracts using the above optimum UAEE condition was successfully achieved by macroporous resin DA-201. After column adsorption and desorption on DA-201; the percentage of total flavonoids was (25.36 ± 1.03)%; ginkgolides was (12.43 ± 0.85)% and alkylphenols was (0.003 ± 0.0005)% from the obtained dry extracts of GBFL which were complied with Chinese pharmacopoeias. Therefore, the present study provided a convenient and efficient method for extraction and purification of flavonoids and ginkgolides from waste GBFL.

Keywords: Ginkgo biloba fallen leaves; Plackett–Burman design; macroporous resin; response surface methodology; ultrasonic–assisted enzymatic extraction.

Figure 1

Pareto chart showing evaluated seven variables on the yields of flavonoids (A) and ginkgolides (B) from G. biloba fallen leaves. Variables with t-values higher than the critical value (2.776) were regarded as being statistically significant.

Figure 2

Diagnostic plots for model adequacy. (A) predicted versus actual of the yields of flavonoids; (B) predicted versus actual of the yields of ginkgolides; (C) internally studentized residuals of the yields of flavonoids; and, (D) internally studentized residuals of the yields of ginkgolides.

Figure 3

Three-dimensional contour plots showing the experimental factors and their mutual interactions: (A) effect of ultrasound-assisted extraction (UAE) power and EtOH% on the yield of flavonoids; (B) effect of UAE power and the amount of cellulase on the yield of flavonoids, and (C) effect of EtOH% and the amount of cellulase on the yield of flavonoids. Two-dimensional contour plots showing the experimental factors and their mutual interactions: (a) effect of ultrasound-assisted extraction (UAE) power and EtOH% on the yield of flavonoids; (b) effect of UAE power and the amount of cellulase on the yield of flavonoids, and (c) effect of EtOH% and the amount of cellulase on the yield of flavonoids.

Figure 3

Three-dimensional contour plots showing the experimental factors and their mutual interactions: (A) effect of ultrasound-assisted extraction (UAE) power and EtOH% on the yield of flavonoids; (B) effect of UAE power and the amount of cellulase on the yield of flavonoids, and (C) effect of EtOH% and the amount of cellulase on the yield of flavonoids. Two-dimensional contour plots showing the experimental factors and their mutual interactions: (a) effect of ultrasound-assisted extraction (UAE) power and EtOH% on the yield of flavonoids; (b) effect of UAE power and the amount of cellulase on the yield of flavonoids, and (c) effect of EtOH% and the amount of cellulase on the yield of flavonoids.

Figure 4

Three-dimensional contour plots showing the experimental factors and their mutual interactions: (A) effect of UAE power and EtOH% on the yield of ginkgolides; (B) effect of UAE power and the amount of cellulase on the yield of ginkgolides; and, (C) effect of EtOH% and UAE power on the yield of ginkgolides. Two-dimensional contour plots showing the experimental factors and their mutual interactions: (a) effect of UAE power and EtOH% on the yield of ginkgolides; (b) effect of UAE power and the amount of cellulase on the yield of ginkgolides; and, (c) effect of EtOH% and UAE power on the yield of ginkgolides.

Figure 4

Three-dimensional contour plots showing the experimental factors and their mutual interactions: (A) effect of UAE power and EtOH% on the yield of ginkgolides; (B) effect of UAE power and the amount of cellulase on the yield of ginkgolides; and, (C) effect of EtOH% and UAE power on the yield of ginkgolides. Two-dimensional contour plots showing the experimental factors and their mutual interactions: (a) effect of UAE power and EtOH% on the yield of ginkgolides; (b) effect of UAE power and the amount of cellulase on the yield of ginkgolides; and, (c) effect of EtOH% and UAE power on the yield of ginkgolides.

Figure 5

Elution conditions of flavonoids and ginkgolides from DA-201 macroporous resin. (A) Effect of ethanol concentration on desorption of total target compounds; and, (B) effect of elution volume on desorption of total target compounds. Results were expressed as the mean value ± standard deviation (n = 3).