Nm using a SpectraMax M2 plate reader.2, 2-Diphenyl-1-picryl-hydrazyl (DPPH: Sigma-Aldrich
Nm using a SpectraMax M2 plate reader.2, 2-Diphenyl-1-picryl-hydrazyl (DPPH: Sigma-Aldrich, Germany) is a stable free radical with a purple colour and upon scavenging, these free radicals turn to yellow. The free radical scavenging activity of the extract was evaluated using a modified method previously described [16]. Various concentrations of H. gordonii extracts were mixed with 90 M DPPH. Since DPPH is light sensitive, incubation was done in the dark at room temperature for 30 min. The absorbance of the resulting solution was measured using a plate reader at 520 nm. Vitamin C (ascorbic acid) was used as a positive control.Reducing power assayThe ability of H. gordonii extracts to reduce iron (III) was determined according to the Kadri’s method [23] with some modifications. Different concentrations of extracts were mixed with 2.5 mL of phosphate buffer (1 M, pH 6.6) and 2.5 mL of 1 potassium ferricyanide. The mixture was incubated at 40 for 20 min. After incubation, 2.5 mL of 10 trichloroacetic acid was addedKapewangolo et al. BMC Complementary and Alternative Medicine (2016) 16:Page 4 ofand centrifuged for 10 min at 3000 rpm. To 2.5 mL of this reaction mixture (upper layer), 0.5 mL of ferric chloride and 2.5 mL of water was added. Ascorbic acid was used as a reference standard. The absorbance was measured at 700 nm spectrophotometrically.Data analysisThe data is presented as mean plus or minus standard error of the mean (M ?SEM). The 50 inhibitory concentrations (IC50 values) for enzymes and DPPH assays were computed using Graphpad Prism 5 software (Graphpad Software Inc. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27689333 California, USA).ResultsQualitative and quantitative phytochemical analysis of H. gordonii extractsIC50 values of 73.55 ?0.04 and 69.81 ?9.45 g/mL, respectively (Table 2). Doxorubicin, a known RT inhibitor [16], was used as a positive control and inhibited HIV RT by 68 at 25 g/mL (IC50 < 25 g/mL). Both extracts also demonstrated inhibitory activity against HIV protease (PR) with IC50 values of 97.29 ?0.01 and 63.76 ?9.01 g/mL for PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26100631 ethanol and ethyl acetate extracts, respectively. MLN9708 biological activity Acetyl pepstatin was used as a known PR inhibitor and inhibited HIV PR by as much as 82 at 50 g/mL (IC50 < 50 g/mL). Both ethanol and ethyl acetate extracts had weak inhibition against HIV-1 integrase (IN) with <50 inhibition at the highest concentration tested of 400 g/mL. Sodium azide was used as a positive control compound for IN inhibition.DPPH (2, 2-Diphenyl-1-picryl-hydrazyl) assayPhytochemical results revealed the presence of a number of phytochemicals including phenolic components, alkaloids, terpenes, steroids and cardiac glycosides as shown in Table 1. It was noted that quinones and flavonoids were absent from H. gordonii extracts.Total phenolic and tannin contentsThe amount of total phenolic content of the extracts was found to be 420 ?0.17 and 319.9 ?0.2 mg GAE/g for ethanol and ethyl acetate extracts, respectively. Due to the absence of tannins in the ethyl acetate extract, as revealed by the phytochemical screening, the total tannin content was only determined for the ethanol extract of H. gordonii and the amount obtained was 330 ?0.2 mg TAE/g extract. These results indicate that H. gordonii could be a rich source of tannins and phenolic compounds.In vitro anti-HIV potential of H. gordoniiDPPH radical scavenging is one of the most widely used methods for assaying the antioxidant activity of compounds and plant extracts. The DPPH method is easy, rapid and sensitiv.