ABSTRACT
<p><b>OBJECTIVE</b>To investigate the antioxidant activities as well as phytochemical constituents of Antidesma thwaitesianum Müll. Arg. leaf extracts.</p><p><b>METHODS</b>The leaves of A. thwaitesianum were extracted using three different methods: blending with distilled water, maceration with ethanol and decoction. The chemical antioxidant activity of the plant leaf extracts was evaluated using 2,2-diphenyl-1-picryhydrazyl (DPPH) radical and 2,2'-azinobis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS⁺) radical scavenging assays, as well as the ferric reducing antioxidant power assay. Cellular antioxidant activity was determined by superoxide and nitric oxide scavenging assays. The cytotoxicity of the leaf extracts in RAW 264.7 and differentiated HL-60 cells was tested in parallel using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays, respectively. The total phenolic and flavonoid contents were also assessed by spectrophotometric analysis. Phytochemical constituents of the most potent extract were investigated by liquid chromatography with an electrospray ionization quadrupole time-of-flight mass spectrometer (LC-ESI-QTOF-MS/MS).</p><p><b>RESULTS</b>The ethanolic (ME) and decoction (LW) extracts of dried leaves had the highest chemical scavenging activity against DPPH and ABTS⁺ free radicals with half maximal effective concentration (EC) values ranging from 3.54 to 6.44 μg/mL. ME and LW exerted moderate ferric reducing activity, with ferric reducing antioxidant power values of 847.41 and 941.26 mg Fe/g extract, respectively. Similarly, ME showed potent cellular scavenging activity against superoxide and nitric oxide radicals with ECvalues of 58.12 and 71.90 μg/mL, respectively. However, LW exhibited only strong nitric oxide scavenging activity with an ECvalue of 91.20 μg/mL. The cell viability of RAW 264.7 and HL-60 cells was greater than 70% in all tested concentrations of both extracts, thus confirming the absence of their cytotoxicity. ME and LW contained high total phenolic contents of 231.14 and 274.42 mg gallic acid equivalents per gram, respectively, as well as high total flavonoid contents of 18.82 and 22.17 mg quercetin equivalents per gram, respectively. LC-ESI-QTOF-MS/MS analysis revealed the presence of 52 structurally characterized compounds in ME, 43 of which were tentatively identified. Hydroxycinnamic acids such as caffeic acid and its derivatives were the predominant phenolic compounds.</p><p><b>CONCLUSION</b>This is the first report describing potent chemical and cellular antioxidant effects of the ethanolic leaf extract of A. thwaitesianum. The extract contained high total phenolic and flavonoid contents. LC-ESI-QTOF-MS/MS analysis further revealed an abundance of caffeic acid derivatives and flavonoids. These data support its potential use as dietary supplements in oxidative stress prevention.</p>
ABSTRACT
Adulterations with synthetic drugs are common problems with herbal medicines and this can potentially cause serious adverse effects. In this paper, a robust LC-MS/MS method for the sensitive and reliable determination of 33 illegal adulterations from herbal medicines and dietary supplements was established. The drugs were isolated from herbal sample using 70% (v/v) methanol. Chromatographic separation was done using C18 column (150x4.6 mm id., 5 µM) with gradient elution of 0.1% (v/v) formic acid and methanol at flow rate of 0.6 ml/min. For most drugs, two transitions were monitored (multiple reaction monitoring; MRM) using protonated or deprotonated as precursor ions under their respective optimal collision energy for each compound. The detection limit was in the level of picogram and the separation of at least 30 compounds can be done in 30 min. The application of this method was used in the sample of herbal medicines, dietary food supplements, coffee for weight controlling and cola drink, totally 49 samples. The adulterate found was dexamethasone in a sample of herbal medicines used for muscle ache, sibutramine was found in 2 samples of dietary food supplements and caffeine was found in 3 samples of dietary food supplements that did not claim on product. With this developed method for analysis of adulterants in herbal medicine, the product quality can be better controlled and regulated to ensure consumers’ safety.