Comparison among Activities and Isoflavonoids from Pueraria thunbergiana Aerial Parts and Root

Abstract

: Kudzu (Pueraria thunbergiana Benth.) has long been used as a food and medicine for many centuries. The root is the most commonly used portion of the plant, but the aerial parts are occasionally used as well. In this study, we investigated the constituent compounds and biological activities of the aerial parts, leaves, stems, and sprouts, and compared their constituents and activities with those of roots. Leaf extract showed a significantly higher TPC level at 59 ± 1.6 mg/g and lower free radical scavenging (FRS) values under 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and NO inhibition at 437 ± 11, 121 ± 6.6 μg/mL and 107 ± 4.9 μg/mL, respectively, than those of sprout, stem, and root extract. Leaf extract also significantly suppressed lipopolysaccharide (LPS)-mediated gene expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). The main components of leaf extract were found to be genistin and daidzin. This study suggests that the leaves of kudzu are a good source of biological activities and isoflavones that can be used in functional or medicinal foods and cosmetics for the prevention or treatment of diseases related to inflammation and oxidative stress.

Keywords: COX-2; Kudzu leaves; NO; Phenolic content; Pueraria thunbergiana; iNOS.

Figure 1

Effects by extraction parts of kudzu on the viability and NO levels of RAW 264.7 macrophages. A density of 1 × 10⁴ cells/well of macrophages were seeded in a 96-well plate and incubated with various concentrations of each extract for 24 h and cell viability was determined by MTT assay. A density of 1 × 10⁴ cells/well of macrophages were seeded in a 96-well plate and the various concentrations of sample and LPS (1 μg/mL) were treated with DMEM (without penicillin and fetal bovine serum (FBS)) to 0.2 mL/well and incubated for 24 h. Untreated samples without LPS treatment were negative controls. Each value is expressed as mean ± SD of three independent experiments. *; p < 0.01, **; p < 0.005, ***; p < 0.001 and ****; p < 0.0005 versus culture media without sample which acts as a control.

Figure 2

Inhibitory effects of KL on iNOS and COX-2 production in LPS-stimulated Raw 264.7 cells. Equal amounts of cell lysates (30 μg) were subjected to electrophoresis and analysed for inducible nitrix oxide synthase (iNOS) and COX-2 production by Western blot. Raw 264.7 cells were incubated with LPS (1 μg/mL) and KL extract for 24 h. Each value is expressed as mean ± SD of three independent experiments. **** p < 0.0005 as analyzed by Duncan’s multiple range test. Nor: normal; Con: control.

Figure 3

Inhibitory effects of KR on iNOS and COX-2 production in LPS-stimulated Raw 264.7 cells. Equal amounts of cell lysates (30 μg) were subjected to electrophoresis and analysed for iNOS and COX-2 production by Western blot. Raw 264.7 cells were incubated with LPS (1 μg/mL) and KR extract for 24 h. Each value is expressed as mean ± SD of three independent experiments. **** p < 0.0005 as analyzed by Duncan’s multiple range test.

Figure 4

Chemical structures of seven isoflavonoids identified from each part of kudzu.

Figure 5

Ultra Performance Liquid Chromatography with Diode-Array Detection (UPLC-DAD) chromatogram for the quantification of the compounds 1–7 in each extract of the part of kudzu at 245 nm. A BEH column (Waters, USA), C18 (2.1 × 100 mm i.d.; 1.7 µm) was used. The gradient program with 0.1% formic acid with water (A) and acetonitrile (B) flowing at a rate of 0.5 mL/min was: 5–15% B (0–10min), 15–20% B (10–15 min), 20–30% B (15–20 min), 30–100% B (20–22 min), 100–5% B (22–23 min), 5–5% B (23–25 min). The volume of injection was 5 µL. 1: Puerarin (6.6 min), 2: Daidzin (7.9 min), 3: Schaftoside (8.6 min), 4: Genistin (9.8 min), 5: Ononin (15.5 min), 6: Daidzein (16.0 min), 7: Genistein (18.1 min). (A) Reference standards, (B) KL, (C) KSP, (D) KST, (E) KR.