Novel Extraction Method Using Excipients to Enhance Yield of Genistein and Daidzein in Trifolium pratensis L

Abstract

Isoflavones can be found in different chemical forms, but the health beneficial effects mainly appear in their free forms-aglycones. Their yield in red clover (Trifolium pratensis L.) extracts differs due to different extraction and hydrolysis methodologies. The main aim of this study was to obtain the highest yields of daidzein and genistein from red clover blossoms through the various extraction and hydrolysis methods and to increase their quantities using additional excipients. Extracts were obtained by ultrasound-assisted, heat-reflux and maceration methods combining them with acidic, alkaline, and thermal hydrolysis. Using ultrasound-assisted extraction with optimal conditions and heat-reflux method highest yields of isoflavones were obtained in UTE510 (393.23 ± 19.66 µg/g daidzein and 171.57 ± 8.58 µg/g genistein); UTE530 (415.07 ± 20.75 µg/g daidzein and 150.57 ± 7.53 µg/g genistein) and HNE5 (432.30 ± 21.61 µg/g daidzein and 154.50 ± 7.72 µg/g genistein) samples. These conditions were used with excipients: magnesium aluminometasilicate, croscarmellose sodium, sodium carboxymethyl starch and vinylpyrrolidone-vinyl acetate copolymer. This is the first study reporting the ability of the vinylpyrrolidone-vinyl acetate copolymer to promote solubilization and availability of active compounds from a herbal extract, resulting in enhanced isoflavones yield. The results of the present study showing increased solubility and availability provided by the vinylpyrrolidone-vinyl acetate copolymer suggest that this preparation could in principle also reduce variability due to limited water solubility of isoflavones.

Keywords: Trifolium pratensis L.; aglycones; excipients; extractions; isoflavones; red clover.

Figure 1

Chemical structures of genistin, genistein, daidzin and daidzein.

Figure 2

Decoding of the samples provided in Table 1.

Figure 3

Decoding of the samples provided in Table 2.

Figure 4

Influence of UAE treatment time and solvent concentration without hydrolysis on the maximal extraction yield of genistein and daidzein in extracts. ^a p < 0.05 vs maceration with 50% ethanol (MNE5); ^b p < 0.05 vs maceration with 70% ethanol (MNE7). Sample codes and preparation conditions are displayed in Table 1.

Figure 5

Influence of UAE treatment time and solvent concentration with acidic hydrolysis on the maximal extraction yield of genistein and daidzein in extracts. ^a p < 0.05 vs. maceration with 50% ethanol (MNE5); ^b p < 0.05 vs. maceration with 70% ethanol (MNE7). Sample codes and preparation conditions are displayed in Table 1.

Figure 6

Influence of UAE treatment time and solvent concentration with alkaline hydrolysis on the maximal extraction yield of genistein and daidzein in extracts. ^a p < 0.05 vs. maceration with 50% ethanol (MNE5); ^b p < 0.05 vs. maceration with 70% ethanol (MNE7). Sample codes and preparation conditions are displayed in Table 1.

Figure 7

UAE treatment with thermal hydrolysis, HRE, ME and ME with natural hydrolysis on the maximal extraction yield of genistein and daidzein in extracts. ^a p < 0.05 vs. maceration with 50% ethanol (MNE5). Sample codes and preparation conditions are displayed in Table 1.

Figure 8

Quantitative yield of isoflavone aglycones using excipients (1%). Control samples without excipients (HNW; UTW10; UTW30), samples with magnesium aluminometasilicate (HNEMO; UTEMO10; UTEMO30), croscarmellose sodium (HNWCO; UHWCO10; UHWCO30), sodium carboxymethyl starch (HNWSO; UTWSO10; UTWSO30) and vinylpyrrolidone-vinyl acetate copolymer (HNWVO; UTWVO10; UTWVO30). ^a p < 0.05 vs. HNW, ^b p < 0.05 vs. UTW10, ^c p < 0.05 vs. UTW30. Sample codes and preparation conditions are displayed in Table 2.

Figure 9

Quantitative yield of isoflavone aglycones using different amounts (1, 2.5 and 5%) of vinylpyrrolidone-vinyl acetate copolymer for extractions. Control samples without excipients (HNW; UTW10; UTW30), 1% of excipient (HNWVO; UTWVO10; UTWVO30), 2.5% (HNWVT; UTWVT10; UTWVT30) and 5% (HNWVF; UTWVF10; UTWVF30). * p < 0.05 vs. HNE5 sample prepared HRE method. Sample codes and preparation conditions are displayed in Table 2.

Figure 10

Quantitative yield of isoflavone aglycones using 1 of vinylpyrrolidone-vinyl acetate copolymer for extractions in 50% ethanol. Control samples without excipients (HNE5; UTE510; UTE530), 1% of excipient (HNEVO; UTEVO10; UTEVO30), ^a p < 0.05 vs. HNE5 sample; ^b p < 0.05 vs. UTE510, ^c p < 0.05 vs. UTE530. Sample codes and preparation conditions are displayed in Table 2.