The original pyrogallol (1,2,3-trihydroxybenzene) method, which was developed specifically for superoxide dismutase, is now widely used for measuring superoxide-scavenging of other antioxidants. However, the strong pH effect has been ignored. In this study, the influencing factors have been systematically investigated for the first time, and a number of experiments have proved that the pH is of major importance. As major antioxidants contain carboxylic acid, ester, or lactone groups, pH 8.2 should be modified to physiological pH 7.4. The improved procedure is as follows. A pyrogallol solution (in 1 M HCl) is thoroughly mixed with pH 7.4 Tris-HCl buffer; A(325 nm) is measured every 30 s for 5 min at 37 °C. As the ΔA(325 nm, control) value reflects the initial concentration of substrate (•)O(2)(-), it should be well controlled to guarantee the accuracy of the method. The improved pyrogallol method is a reliable and cheap superoxide-scavenging assay suitable for all types of antioxidants.

Radix Angelicae Sinensisis (RAS) is one of the most popular traditional Chinese herbal medicines. In the present study, six RAS extracts (i.e., phenolic extract PE, petroleum ether extract PEE, ethyl acetate extract EAE, absolute ethanol extract AEE, 95% ethanol extract 95 EE, and water extract WE) were prepared and their antioxidant activities measured by DPPH (1,1-diphenyl-2-picrylhydrazyl radical), ABTS [2,2′-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid diammonium salt)], Reducing power, •O2– and lipid peroxidation assays. In general, PE, PEE and EAE had relatively high antioxidant activity, followed by AEE with moderate activity, as compared with 95 EE and WE that had low activity. Their phenolic contents (including total phenolic, ferulic acid, caffeic acid, same as below) were then determined by HPLC or spectrophotometry. The sequence of phenolic contents was roughly identical with that of antioxidant activity. When the values of 1/IC50 of various antioxidant assays were used to evaluate the level of antioxidant of the RAS extracts, (plot between 1/IC50 values and phenolic contents), the correlation coefficient (R) ranged from 0.642 to 0.941, with an average value of 0.839. Significant positive correlations demonstrated that the antioxidant effects of RAS might generally be considered a result of the presence of the phenolic compounds, especially ferulic acid and caffeic acid.

Background: Protocatechuic acid (PCA) is a natural phenolic acid widely distributed in plants and is considered as an active component of some traditional Chinese herbal medicines such as Cibotium barometz (L.) J.Sm, Stenoloma chusanum (L.) Ching, Ilex chinensis Sims. PCA was reported to possess various pharmacological effects which may be closely correlated with its antioxidant activities. However, the antioxidant of PCA has not been investigated systematically yet. Methods: In the study, the antioxidant activities of protocatechuic acid were measured in vitro using various antioxidant assays including 1,1-diphenyl-2-picryl-hydrazyl (DPPH•), 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS+•), superoxide anion radicals (•O2-) and hydroxyl radical (•OH) scavenging activity, ferric ions (Fe3+) and cupric ions (Cu2+) reducing power, ferrous ions (Fe2+) and cupric ions (Cu2+) chelating activity, compared with the positive controls Trolox or BHT.Results: In all assays, PCA along with positive controls exhibited dose-dependently antioxidant ability. Comparing to a standard antioxidant Trolox, the relative antioxidant activity of PCA (i.e. the ratio of IC50(Trolox)/IC50(PCA) ) was calculated as 2.8, 2.3, 3.7, 6.1, 4.2, 1.0, 2.7, 1.5, respectively, for DPPH, ABTS, reducing power (Fe3+), reducing power (Cu2+), superoxide anion radical-scavenging, hydroxyl radical-scavenging, chelating ability (Fe2+) and chelating ability (Cu2+). Conclusion: Comparing to Trolox, PCA shows much more effective antioxidant activity in vitro in both lipid and aqueous media. Hence, it could therefore be used in pharmacological or food industry as a natural antioxidant. It may exhibit antioxidant activity by both chelating metal transition ions as well as by scavenging free radicals via donating hydrogen atom (H•) or electron (e).Keywords: Protocatechuic acid, antioxidant, reducing power, free radical-scavenging, chelating ability.

BackgroundAs a typical Chinese herbal medicine, rhizoma Cimicifugae (RC, 升麻 in Chinese) possesses various pharmacological effects involved in antioxidant activity. However, its antioxidant activity has not been reported so far. The aim of the present study was to systematically evaluate the antioxidant ability of RC in vitro, then discuss the mechanism.MethodsFirstly, five RC extracts (i.e. petroleum ether extract PERC, ethyl acetate extract EARC, absolute ethanol extract AERC, 95% ethanol extract 95ERC, and water extract WRC) were prepared and determined by various antioxidant methods, including anti-lipidperoxidation, protection against DNA damage, ·OH scavenging, ·O2- scavenging, DPPH· (1,1-diphenyl-2-picryl-hydrazl radical) scavenging, ABTS+· (2,2’-azino-bis (3-ethylbenzo- thiazoline-6-sulfonic acid radical ion) scavenging, Cu2+-chelating, and Fe3+ reducing assays. Subsequently, we measured the chemical contents of five RC extracts, including total phenolics, total saponins, total sugars, caffeic acid, ferulic acid and isoferulic acid. Finally, we quantitatively analyzed the correlations between antioxidant levels (1/IC50 values) and chemical contents.ResultsIn the study, the antioxidant levels and chemical contents (including total phenolics, total saponins, total sugars, caffeic acid, ferulic acid and isoferulic acid) of five RC extracts were determined by various methods. In all antioxidant assays, five RC extracts increased the antioxidant levels in a dose-dependent manner. However, their antioxidant levels (IC50 values) and chemical contents significantly differed from each other. Quantitative analysis of the correlation showed that total phenolic was of significant positive correlations (average R value was 0.56) with antioxidant levels; In contrast, total sugars and total saponins had no positive correlation with antioxidant (the average R values were −0.20 and −0.26, for total sugars and total saponins, respectively); Among total phenolics, three phenolic acids (caffeic acid, ferulic acid and isoferulic acid) also displayed positive correlations (the average R values were 0.51, 0.50, and 0.51, for caffeic acid, ferulic acid and isoferulic acid, respectively).ConclusionsAs an effective antioxidant, Rhizoma Cimicifugae can protect DNA and lipids against oxidative damage. Its antioxidant ability can be responsible for its various pharmacological effects and may be mainly attributed to the existence of total phenolics, among which caffeic acid, ferulic acid and isoferulic acid are regarded as main bioactive components. Rhizoma Cimicifugae exerts its antioxidant effect through metal-chelating, and radical-scavenging which is via donating hydrogen atom (H·) and donating electron (e).

Current in vitro antioxidant assays have several limitations, which frequently cause inconsistent results. The study develops a new antioxidant assay using the 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO). After the investigation of various factors, the experimental protocol was briefly recommended as follows: PTIO and the sample solution were added to phosphate buffer (pH 7.4, 50 mM), incubated at 37 °C for 2 h, and then spectrophotometrically measured at 557 nm. The validation test based on 20 pure compounds and 30 lyophilized aqueous extracts suggested that PTIO scavenging had a good linear relationship, stability, and reproducibility. In the ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry analysis, PTIO was observed to give m/z 234 when encountering l-ascorbic acid. As an antioxidant assay, PTIO scavenging possesses four advantages, i.e., oxygen-centered radical, physiological aqueous solution, simple and direct measurement, and less interference from the tested sample. It can also satisfactorily analyze the antioxidant structure-activity relationship. PTIO scavenging has no stereospecificity and is at least involved in H transfer.