Two-Dimensional High-Performance Thin-Layer Chromatography with Bioautography for Distinguishing Angelicae Dahuricae Radix Varieties: Chemical Fingerprinting and Antioxidant Profiling.

Angelicae Dahuricae Radix (ADR) holds a prominent place in traditional medicine for its remarkable antioxidative, anti-allergic, and antiproliferative capabilities. Recognized within the Korean Pharmacopoeia (KP 12th), Angelica dahurica (Hoffm.) Benth. and Hook.f. ex Franch. and Sav. (AD) and Angelica dahurica var. formosana (H. Boissieu) Yen (ADF) serve as the botanical origins for ADR. Differentiating these two varieties is crucial for the formulation and quality control of botanical drugs, as they are categorized under the same medicinal label. This research utilized two-dimensional high-performance thin-layer chromatography (2D-HPTLC) to effectively distinguish AD from ADF. Additionally, a quantitative analysis reveals significant differences in the concentrations of key active constituents such as oxypeucedanin, imperatorin, and isoimperatorin, with AD showing higher total coumarin levels. We further enhanced our investigative depth by incorporating a DPPH bioautography, which confirmed known antioxidant coumarins and unearthed previously undetected antioxidant profiles, including byakangelicin, byakangelicol, falcarindiol in both AD and ADF, and notably, 2-linoleoyl glycerol detected only in AD as an antioxidant spot. This comprehensive approach affords a valuable tool set for botanical drug development, emphasizing the critical need for accurate source plant identification and differentiation in ensuring the efficacy and safety of herbal medicine products.

Antioxidant Activity and Phenolic Compound Identification and Quantification in Western Australian Honeys.

This study reports on the total phenolic content and antioxidant activity as well as the phenolic compounds that are present in Calothamnus spp. (Red Bell), Agonis flexuosa (Coastal Peppermint), Corymbia calophylla (Marri) and Eucalyptus marginata (Jarrah) honeys from Western Australia. The honey's total phenolic content (TPC) was determined using a modified Folin-Ciocalteu assay, while their total antioxidant activity was determined using FRAP and DPPH assays. Phenolic constituents were identified using a High Performance Thin-Layer Chromatography (HTPLC)-derived phenolic database, and the identified phenolic compounds were quantified using HPTLC. Finally, constituents that contribute to the honeys' antioxidant activity were identified using a DPPH-HPTLC bioautography assay. Based on the results, Calothamnus spp. honey (n = 8) was found to contain the highest (59.4 ± 7.91 mg GAE/100 g) TPC, followed by Eucalyptus marginata honey (50.58 ± 3.76 mg GAE/100 g), Agonis flexuosa honey (36.08 ± 4.2 mg GAE/100 g) and Corymbia calophylla honey (29.15 ± 5.46 mg GAE/100 g). In the FRAP assay, Calothamnus spp. honey also had the highest activity (9.24 ± 1.68 mmol Fe2+/kg), followed by Eucalyptus marginata honey (mmol Fe2+/kg), whereas Agonis flexuosa (5.45 ± 1.64 mmol Fe2+/kg) and Corymbia calophylla honeys (4.48 ± 0.82 mmol Fe2+/kg) had comparable FRAP activity. In the DPPH assay, when the mean values were compared, it was found that Calothamnus spp. honey again had the highest activity (3.88 ± 0.96 mmol TE/kg) while the mean DPPH antioxidant activity of Eucalyptus marginata, Agonis flexuosa, and Corymbia calophylla honeys were comparable. Kojic acid and epigallocatechin gallate were found in all honeys, whilst other constituents (e.g., m-coumaric acid, lumichrome, gallic acid, taxifolin, luteolin, epicatechin, hesperitin, eudesmic acid, syringic acid, protocatechuic acid, t-cinnamic acid, o-anisic acid) were only identified in some of the honeys. DPPH-HPTLC bioautography demonstrated that most of the identified compounds possess antioxidant activity, except for t-cinnamic acid, eudesmic acid, o-anisic acid, and lumichrome.

Rapid Identification and Comparison of Compounds with Antioxidant Activity in Coreopsis tinctoria Herbal Tea by High-Performance Thin-Layer Chromatography Coupled with DPPH Bioautography and Densitometry.

A simple and efficient method based on high-performance thin-layer chromatography coupled with 2,2-diphenyl-1-picrylhydrazyl (DPPH) bioautography (HPTLC-DPPH) was established for the screening and comparison of antioxidants in different parts of Coreopsis tinctoria herbal tea from different origins and other related herbal tea materials, which used Chrysanthemum morifolium cv. "Gongju" and "Hangju" in this study. Scanning densitometry after DPPH derivatization was applied for the determination of antioxidant capacities of isolated compounds in each sample. It is considered that ethanol extracts of C. tinctoria had stronger antioxidant activity and more characteristic bands than those of 2 compared samples, C. morifolium cv. "Gongju" and "Hangju." Chemometric analysis results showed that the combination of hierarchical clustering analysis and principal component analysis based on determined antioxidant capacities could be used for the discrimination of different parts of C. tinctoria and C. morifolium. Results showed that 7 compounds made up the major contributions of antioxidant activity in C. tinctoria, including okanin, isookanin, marein, flavanomarein, 5,7,3',5'-tetrahydroxyflavanone-7-O-glucoside, 3,5-dicaffeoylquinic acid, and chlorogenic acid. Therefore, 7 compounds were identified as major antioxidant biomarkers for quality control of C. tinctoria. Results demonstrated that the established method could be applied for the identification of C. tinctoria, and were beneficial for the bioactivity-based quality control of C. tinctoria.

In vitro antioxidant and anti-inflammatory activities of extracts from Potentilla recta and its main ellagitannin, agrimoniin.

ETHNOPHARMACOLOGICAL RELEVANCE: Potentilla recta is one of the numerous cinquefoil species growing in Poland. It is used in traditional medicine e.g. in the treatment of skin inflammation. AIM OF THE STUDY: The purpose of the present study is to evaluate antioxidant and anti-inflammatory activities of extracts and subfractions of the P. recta herb (obtained by using solvents of different polarity) in in vitro systems as well as to examine their chemical composition. MATERIALS AND METHODS: Antioxidant activities of the extracts, subfractions and agrimoniin were evaluated using DPPH and three other radicals (O2(-), H2O2, and HClO) generated in cell-free systems. Anti-hyaluronidase activity was measured by using the turbidimetric method. Inhibition of lipoxidase activity was measured spectrophotometrically, using linoleic acid as a substrate. The composition of the most active subfraction was determined using the HPLC-DAD-MS(n) method. RESULTS: All tested samples showed scavenging activity against all the examined reactive species in a concentration-dependent manner. The highest scavenging activity against DPPH, H2O2 and HClO was observed in the ethyl acetate subfraction (PRE3) (SC50 ± SEM [µg/mL]: 25.39 ± 2.49, 1.79 ± 0.25 and 8.52 ± 1.16 respectively). It was only in the xanthine/xanthine oxidase system that the antioxidation potential of the diethyl ether subfraction (PRE2) (SC50 ± SEM [µg/mL]: 6.59 ± 1.33) was higher than that of the subfraction PRE3 (SC50 ± SEM [µg/mL]: 8.57 ± 1.37). Also, in the studies of lipoxidase and hyaluronidase inhibition activity the strongest effect was observed for PRE3, with IC50 [µg/mL] = 86.31 ± 5.46, and 12.99 ± 1.31, respectively. The chromatographic method (HPTLC-DPPH) revealed that the principal substance responsible for the activity, is a tannin like compound. Isolated agrimoniin showed significant reactive oxygen species scavenging activity and significant enzyme inhibition activity (including xanthine oxidase inhibition activity). Agrimoniin exerted the strongest scavenging activity against H2O2 (SC50 ± SEM [µM]: 0.20 ± 0.01). This compound also significantly inhibited the enzymatic activity of lipoxidase (IC50 [µM] = 36.47 ± 1.29), and, particularly, of hyaluronidase (IC50 [µM] = 2.65 ± 0.40). CONCLUSIONS: The strong scavenging activity against H2O2, and the inhibition of the enzymatic activity of lipoxidase, and particularly, hyaluronidase observed for the tested subfractions and agrimoniin, partly explain the beneficial effects of P. recta in treatment of skin inflammation.