Development and Validation of Conditions for Extracting Flavonoids Content and Evaluation of Antioxidant and Cytoprotective Activities from Bougainvillea x buttiana Bracteas (var. Rose)

Abstract

In this study the effect of the ethanol concentration of Bougainvillea x buttiana extracts on the flavonoids content, and its antioxidant and cytoprotective activities in vitro were determined and compared. For the elucidation of the chemical constituents, the high-performance liquid chromatography method (HPLC) was used, and verification of the antioxidant activity was carried out using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical method. The cytoprotective effects of extracts were determined by exposure to hydrogen peroxide. The HPLC analysis showed the presence of rutin, quercetin-3-glucoside and quercetin rhamnoside. Among the extracts investigated the best recuperation of the rutin content was observed in extracts with 80% ethanol (83 ± 5 mg/mL). The amounts of rutin present in all extracts contribute to the antioxidant capacity and the IC₅₀ was 427.49 (0%), 275.41 (50%), 271.61 (80%), and 272.14 (100%) µg/mL. The lowest percentage of viability was found in the cultures exposed to 100% ethanol (92%). In cultures exposed to hydrogen peroxide the percentages of protection were 25%, 33%, 78%, and 65% for cultures treated for 72 h at 0%, 50%, 80%, and 100% ethanol, respectively. The ethanolic extract of B. x buttiana was confirmed to have high rutin content with potent antioxidant activity, low cytotoxic and strong cytoprotective effects.

Keywords: Bougainvillea x buttiana; Rutin; antioxidant activity; cytoprotective activity; extraction; quercetin-O-glucoside; quercetin-rhamnoside.

Figure 1

Comparison of total flavonoids content in ethanolic extracts. Samples of Bougainvillea x buttiana extract ethanol 0%, 50%, 80%, and 100% at 26 °C for 24 h were assayed to determine the concentration of total phenolic content as described in Materials and Methods. Each bar corresponds to the results (mean ± standard deviation (SD)) from three different preparations.

Figure 2

HPLC of Rutin. Chromatographic profile of the routine concentration curve (standard) and extracts of EtOH-0%, EtOH-50%, EtOH-80%, and EtOH-100%.

Figure 3

Flavonoid determination. The yield of rutin, quercetin glucoside and quercetin rhamnoside in different ethanol extracts was determine. Individual samples of B. x buttiana extract ethanol 0%, 50%, 80%, and 100% were investigated to determine the yield of rutin as described in Materials and Methods. Each bar corresponds to the results (mean ± standard deviation (SD)) from three different preparations.

Figure 4

Antioxidant activity percentage. Individual samples of B. x buttiana extract ethanol 0%, 50%, 80%, and 100% were investigated to determine the antioxidant activity as described in Materials and Methods. Quercetin was used as reference antioxidants. Each bar corresponds to the results (mean ± standard deviation (SD)) from three different preparations.

Figure 5

IC₅₀ values and antioxidant activity index (AAI). Individual samples of B. x buttiana extract ethanol 0%, 50%, 80%, and 100% were investigated to evaluate the IC₅₀ (A) and antioxidant activity index (B) as described in Materials and Methods. Each bar corresponds to the results (mean ± standard deviation (SD)) from three different preparations.

Figure 6

Ethanol concentration effect on the cellular viability. Individual samples of B. x buttiana extract with 1 mg/mL of each extract 0%, 50%, 80%, and 100% for 24, 48, and 72 h were investigated to determine the viability percentage by MTT test as described in Materials and Methods. Each bar corresponds to the results (mean ± standard deviation (SD)) from three different preparations.

Figure 7

Extract ethanol concentration effect on cell survival. Groups of cells were treated with 1 mM of H₂O₂ to induce oxidative stress for 24 h at 37 °C with 5% CO₂. After this incubation, the cells were treated with 1 IC₅₀ individual extract ethanol of 0% (427.49 µg/mL), 50% (275.41 µg/mL), 80% (271.61 µg/mL), and 100% (272.14 µg/mL) and incubated for 24 h to determine the viability percentage as described in Materials and Methods. Each bar corresponds to the results (mean ± standard deviation (SD)) from three different preparations.

Figure 8

Extract 80% EtOH effect on cell survival. Groups of cells were treated with 1 mM of H₂O₂ to induce oxidative stress for 24 h at 37 ºC with 5% CO₂. After this incubation, the cells were treated with extract 80% EtOH at different IC₅₀ values were assayed to determine the viability percentage as described in Materials and Methods. Each point corresponds to the results (mean ± standard deviation (SD)) from three different preparations.