Enhanced Enzymatic Synthesis of Puerarin Palmitate with Different Acyl Donors for Lipid Solubility Improvement

Abstract

Puerarin is a flavonoid known as a natural antioxidant found in the root of Pueraria robata. Its antioxidant, anticancer, and anti-inflammatory effects have attracted attention as a potential functional ingredient in various bioindustries. However, puerarin has limited bioavailability owing to its low lipid solubility and stability. Acylation is proposed as a synthesis method to overcome this limitation. In this study, lipase-catalyzed acylation of puerarin and various acyl donors was performed, and the enzymatic synthetic condition was optimized. Under the condition (20 g/L of Novozym 435, palmitic anhydride, 1:15, 40 °C, tetrahydrofuran (THF)), the synthesis of puerarin ester achieved a significantly high conversion (98.97%) within a short time (3 h). The molecule of the synthesized puerarin palmitate was identified by various analyses such as liquid chromatography-mass spectrometry (LC-MS), Fourier-transform infrared spectroscopy (FT-IR), and carbon-13 nuclear magnetic resonance (¹³C NMR). The lipid solubility and the radical scavenging activity were also evaluated. Puerarin palmitate showed a slight decrease in antioxidant activity, but lipid solubility was significantly improved, improving bioavailability. The high conversion achieved for puerarin esters in this study will provide the foundation for industrial applications.

Keywords: antioxidant; flavonoid; flavonoid ester; lipase; lipid solubility; puerarin.

Figure 1

Effect of enzyme concentration on the conversion of puerarin ester (Novozym 435, palmitic acid as the acyl donor, 1:10 molar ratio of puerarin to acyl donor, reaction temperature of 40 °C, tert-amyl alcohol as the solvent, and reaction time of 48 h) (The optimal condition is indicated by a red box).

Figure 2

Effect of molar ratio of puerarin to acyl donor on the conversion of puerarin palmitate (20 g/L of Novozym 435, palmitic anhydride as the acyl donor, reaction temperature of 40 °C, tert-amyl alcohol as the solvent, and reaction time of 48 h) (The optimal condition is indicated by a red box).

Figure 3

Effect of reaction temperature on the conversion of puerarin palmitate (20 g/L of Novozym 435, palmitic anhydride as the acyl donor, 1:15 molar ratio of puerarin to acyl donor, tert-amyl alcohol as the solvent, and reaction time of 48 h) (The optimal condition is indicated by a red box).

Figure 4

Effect of organic solvent on the conversion of puerarin palmitate (20 g/L of Novozym 435, palmitic anhydride as the acyl donor, 1:15 molar ratio of puerarin to acyl donor, reaction temperature of 40 °C, and reaction time of 48 h) (The optimal condition is indicated by a red box).

Figure 5

Conversion of puerarin palmitate as a function of reaction time (20 g/L of Novozym 435, palmitic anhydride as the acyl donor, 1:15 molar ratio puerarin to acyl donor, reaction temperature of 40 °C, and THF as the solvent).

Figure 6

Mass spectra of puerarin palmitate in (a) scan mode and (b) SIM mode.

Figure 7

FT-IR spectra of (a) puerarin palmitate, (b) palmitic anhydride, and (c) puerarin.

Figure 8

¹³C NMR spectra of (a) puerarin and (b) puerarin palmitate. (The newly created C=O double bond in puerarin palmitate after esterification is indicated by a red shade).

Figure 9

Antioxidant activity of puerarin and puerarin palmitate using ABTS assay.

Figure 10

Reaction mechanism for the production of puerarin ester from puerarin and acyl donors via (a) esterification and (b) transesterification (The functional group of puerarin is shown in red, the functional group of the acyl donor is shown in blue, and the functional group newly created due to the reaction is shown in green).

Figure 11

Optimization procedure for puerarin palmitate synthesis (Optimal conditions are indicated in red).