In Vitro Analysis of Antioxidant, Anticancer, and Bioactive Components of Apocynum venetum Tea Extracts

Abstract

The dry leaf of Apocynum venetum tea extracts (AVTEs) belonging to the Apocynaceae family is a traditional Chinese medicine. The aim of this study is to identify the bioactive components of AVTE and analyse its antioxidant and anticancer activity in vitro. Method. Flavones and polyphenols in AVTE were determined by high-performance liquid chromatography (HPLC) assay. The scavenging capacity of tea extracts to 1,1-diphenyl-2-picrylhydrazyl (DPPH); 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS); hydroxyl (OH); and superoxide anion-free radicals were investigated by spectrophotometry. We also detailed the cytotoxicity assay of AVTE (50, 100, and 200 μg/mL) to human embryonic kidney 293T cells, the protective effect of AVTE on 293T cells induced by hydrogen peroxide (0.3 mmol/L), and the anticancer effect against the human hepatoma HepG2 cells via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. We investigated the antioxidative effects of AVTE in human embryonic kidney 293T cells and the anticancer mechanism in HepG2 human hepatoma cells via quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) assay. Results. HPLC analysis showed that AVTEs contain neochlorogenic acid, chlorogenic acid, rutin, isoquercetin, isochlorogenic acid B, astragalin, isochlorogenic acid C, rosmarinic acid, quercetin, and trans-cinnamic acid. These extracts have high antioxidant activity and dose-dependent relation through free radical scavenging experiments. The cell viability of 293T cells treated with hydrogen peroxide (0.3 mmol/L) was significantly lower than that of normal cells, and the cell viability of oxidatively stressed 293T cells after AVTE (50, 100, and 200 μg/mL) treatment was significantly improved (P<0.05). Moreover, cytotoxicity experiments showed that the survival rate of 293T cells was over 90%, but the proliferation of HepG2 cells was significantly inhibited in a dose-dependent manner by AVTE. Furthermore, cytoprotective effects in 293T cells were induced via upregulation of glutathione peroxidase (GSH-Px), GSH, superoxide dismutase (SOD), and catalase (CAT) antioxidant-related factors, as well as apoptosis in HepG2 cells was induced via upregulation of caspase-3, caspase-9, p21, and p53 apoptosis-associated factors, as assessed via mRNA expression levels after treatment with AVTE, which were consistent with the results of antioxidant gene detections. As a conclusion, AVTE appears to be an effectively functional drink, due to its rich functional components and antioxidant and anticancer activities.

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“…The antioxidant components in WTSNF can react with ABTS·+, thereby causing reaction systems to fade. 11 DPPH is a very stable nitrogen-centered free radical that can capture other free radicals. DPPH solution appears dark purple and turns light yellow after being combined with WTSNF.…”

“…Ethanol gradient dilution was then performed so the absorbance of the ABTS working solution at a wavelength of 732 nm was 0.7 ± 0.02. 11 Next, 0.5 mL of the WTSNF-ethanol solution at different concentrations (16 μg/mL and 32 μg/mL) was added to 4.0 mL of the ABTS working solution, shaken well for 10 seconds, and then kept in the dark for 10 minutes. Finally, 200 μL of this mixture was added to a 96-well plate, and the absorbance at 732 nm was determined.…”

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“…e amplification conditions (StepOne Plus, ermo Fisher Scientific) were as follows: denaturation at 95°C for 15 min, annealing at 60°C for 1 h, extension at 95°C for 15 min, 40 cycles. Taking GAPDH as the internal reference gene, the relative expression of each gene was calculated by 2 −ΔΔCT [22]. e corresponding gene primer sequence is shown in Table 1.…”