On-line HPLC-DPPH Assay Research Articles

Rapid screening and isolation of antioxidants from Eupatorium lindleyanum DC. using CCC target-guided by on-line HPLC-DPPH assay

Counter-current chromatography (CCC) target-guided by on-line HPLC with post-column DPPH assay was established for efficient screening and isolation of large amount of antioxidants from Eupatorium lindleyanum DC. On-line HPLC with post-column DPPH reaction was used to screen the antioxidants and optimize the biphasic solvent system of CCC, then the targeted peaks were purified using CCC. In the present study, three compounds, nepetin, cirsiliol and jaceosidin, were targeted and successively separated from n-butanol fraction of E. lindleyanum DC. by this strategy. All three compounds showed strong DPPH radical scavenging activity. These results confirmed that the strategy would be an efficient and effective method to isolate antioxidants from complex mixtures.

Preparative isolation of antioxidative compounds from Dracocephalum heterophyllum using off-line two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography guided by on-line HPLC-DPPH assay

Traditional Tibetan medicine (TTM) has been valuable for the identification of new therapeutic leads. Nevertheless, reports about the chemical constituents of TTM are meager owing to the lack of suitable purification techniques. In this study, an off-line two-dimensional reversed-phase/hydrophilic interaction liquid chromatography (2D RP/HILIC) technique guided by on-line HPLC-DPPH has been established for the isolation of pure antioxidants from the extract of Dracocephalum heterophyllum. According to the chromatographic recognition outcome of the HPLC-DPPH system, the first-dimensional (1D) separation on the Megress C18 preparative column yielded 6 antioxidative fractions (61.4% recovery) from the ethyl acetate fraction (6.1 g). In the second-dimensional (2D) separation, a HILIC XAmide preparative column was employed. In total, 8 antioxidants were isolated from D. heterophyllum with a purity of >95%, which indicated the efficiency of the developed method to prepare antioxidative compounds with high purity from plant extracts. In addition, this method was highly efficient for the preparation of structural analogues of the antioxidative polyphenols and could be applied for the purification of structural analogues from other resources.

Anti-Bacterial Activity and Online HPLC-DPPH Based Antiradical Kinetics of Medicinal Plant Extracts of High Relevance for Cosmetics Production

The present study provides an assessment of extracts of six medicinal plants for their applicability in cosmetic formulations aimed at anti-UV protection and anti-acne treatment. Ethanol extracts of Chelidonium majus L., Myrtus communis L., Hamamelis virginiana L., Juniperus communis L., Alchemilla vulgaris and Ilex paraguariensis are analysed for the phytochemical content, antimicrobial activity, antiradical activity and antiradical kinetics. The phytochemical composition of the plants is investigated by LC-MS analysis and the most active antioxidants in plant extracts are determined by online HPLC-DPPH assay. The kinetic parameters of the antiradical reaction are determined both for the studied plants and chlorogenic acid applying the mathematical treatment of the experimental kinetic data.The study shows that I. paraguariensis ethanolic extract possesses the highest potential in anti-UV and anti-aging cosmetology applications, while M. communis and J. communis reveal excellent antibacterial properties and high potential in anti-acne treatment.

HPLC와 DPPH radical 소거능 측정 방법의 결합에 의한 약용 식물 추출물의 항산화 활성 비교

Natural anti-oxidative compounds have important disease prevention and food preservation properties, in addition to anti-bacterial, anti-inflammation, anti-cancer, and skin whitening effects. High-performance liquid chromatography (HPLC), with an ultra vilolet (UV) detector coupled to a reverse phase C18 column and an online measurement system for 1, 1-diphenyl-2-picryl hydrazyl (DPPH) radicals, was used to search for potent antioxidative compounds in crude extracts. The online HPLCDPPH assay was then applied to confirm antioxidative compounds in water extracts from Radix of Pueraria lobata, Rhizoma of Zingiber officinale, Fructus of Chaenomeles sinensis, Cortex of Ulmus pumila, and Radix of Astragalus membranaceus. To determine the yields of the extracts, the Brix% of each extract solution was measured using a refractometer. When the relative DPPH radical scavenging ability values of the water extracts were compared with those of a positive control (ascorbic acid), the water extracts of P. lobata, C. sinensis, and U. pumila were 7.77%, 4.71%, and 4.19%, respectively. The results suggest that this method provides a useful assay for rapid measurement of DPPH radical scavenging abilities and conformation of antioxidative compounds in natural products. Moreover, it can reduce the time spent on the separation of active compounds from natural materials, such as medicinal plants, in addition to the use of reagents for separation.

Degradation of Caffeic Acid in Subcritical Water and Online HPLC-DPPH Assay of Degradation Products

Caffeic acid was subjected to degradation under subcritical water conditions within 160-240 °C and at a constant pressure of 5 MPa in a continuous tubular reactor. Caffeic acid degraded quickly at these temperatures; the main products identified by liquid chromatography-diode array detection/mass spectrometry were hydroxytyrosol, protocatechuic aldehyde, and 4-vinylcatechol. The reaction rates for the degradation of caffeic acid and the formation of products were evaluated. Online high-performance liquid chromatography/2,2-diphenyl-1-picryhydrazyl assay was used to determine the antioxidant activity of each product in the solution. It was found that the overall antioxidant activity of the treated solution did not change during the degradation process. This study showed a potential of formation of antioxidants from natural phenolic compounds under these subcritical water conditions, and this may lead to a discovering of novel antioxidants compounds during the extraction by this technique.

Evaluation of antioxidant activity of ten compounds in different tea samples by means of an on-line HPLC–DPPH assay

Tea (Camellia sinensis), a well-known traditional beverage in China, has drawn growing attention due to its various benefits to health. In this study, an on-line assay of coupling high performance liquid chromatography separation with 1,1-diphenyl-2-picrylhydrazyl free radical reaction system (HPLC–DPPH) was applied to evaluate the antioxidant activity of different teas. Twelve main chromatographic peaks were detected in tea, and they were identified as gallic acid, 3-galloyl-quinic acid, theobromine, (−)-gallocatechin, (−)-epigallocatechin, caffeine, procyanidin dimer, (−)-epicatechin, (−)-epigallocatechin gallate, (−)-gallocatechin gallate, 1,2,6-tri-galloyl-glucose and (−)-epicatechin gallate by comparing their retention times and DAD spectra with standard compounds respectively or with reference to our previous study. DPPH assay showed that ten out of twelve investigated compounds have free radical scavenging activity, and their contents were determined or estimated. Trolox equivalent antioxidant capacities (TEACs) of the ten active components were also determined. EGCG was the most potent antioxidant with a TEAC value of 5.822. Catechin components were the major contributors to the antioxidant activity of tea; they decreased during tea fermentation and led to the reduction of antioxidant activity. Different tea samples were scientifically classified and their antioxidant activities were comprehensively evaluated by on-line HPLC–DPPH assays coupled with principal component analysis and hierarchical clustering analysis. The results indicated that the combination of the on-line HPLC–DPPH assay, principal component analysis and hierarchical clustering analysis could be suitable for the bioactivity assessment and variety characterization of tea and its derived products. Additionally, a simple and rapid method for simultaneous capture of chemical quantitative analysis and bioactivity evaluation by determining contents of the bioactive compounds was firstly established. This method could provide a comprehensive evaluation of tea.

Screening of free radical scavengers from Erigeron breviscapus using on-line HPLC-ABTS/DPPH based assay and mass spectrometer detection

Erigeron breviscapus is a well-known traditional Chinese herbal medicine. In this study, on-line HPLC-ABTS/DPPH assay coupled with MS detection were applied to screen and identify the free radical scavengers in 70% methanol extracts of E. breviscapus. Using on-line HPLC-ABTS-MS and HPLC-DPPH-MS assay, 13 radical scavengers (including 4-O-caffeoylquinic acid (4-CQA) (1), 9-caffeoyl-2,7-anhydro-2-octulosonic acid (9-COA) (2), 3-caffeoyl-2,7-anhydro-3-deoxy-2-octulopyranosonic acid (3-CDOA) (3), erigeside I (4), quercetin-3-O-glucuronide (5), eriodictyol-7-O-glucuronide (6), scutellarin (7), 1,4-di-O-caffeoylquinic acid (1,4-di-CQA) (8), 3,5-di-CQA (9), 1-malonyl-3,5-di-CQA (10), erigoster B (11), 4,5-di-CQA (12) and 4,9-di-CDOA (13)) and 9 radical scavengers (including 1, 4, 7, 8, 9, 10, 11, 12 and 13) were discovered, respectively. Furthermore, the anti-oxidative activities of 4 compounds, including 7, 9, 11 and 12 were evaluated. Reverse anti-oxidative activity order of scutellarin and 3,5-di-CQA was observed in on-line HPLC-ABTS assay and on-line HPLC-DPPH assay. To validate their anti-oxidative activities, the off-line ABTS and DPPH assays were performed. Given sufficient reaction time, 3,5-di-CQA showed higher activity than scutellarin, which was consistent with the order obtained in on-line HPLC-ABTS assay. These results revealed that on-line HPLC-ABTS assay is a more sensitive method for screening and determining free radical scavengers, especially more suitable for those compounds with slower reaction kinetics.

Identification and quantification of free radical scavengers in the flower buds of Lonicera species by online HPLC‐DPPH assay coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Flos Lonicerae, derived from the flower buds of several medicinal Lonicera species, is a commonly used herbal medicine with multiple pharmacological activities, one of the major ones being antioxidant activity. In this study, free radical scavengers in the flower buds of six Lonicera species were screened, identified and quantified by online HPLC-DPPH (1,1-diphenyl-2-picrylhydrazyl) assay coupled with LC quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). The antioxidants were firstly screened from the complex plant matrix by the online HPLC-DPPH assay. Then the active compounds were identified by LC Q-TOF MS/MS, and the possible fragmentation pathways were proposed. The reactivity of antioxidants available was investigated using an internal standard method by online LC assay. The contents of 12 antioxidants were also determined or estimated by HPLC coupled with diode array detector. The total antioxidant capability determined by the online method was used as the marker to evaluate the quality of Flos Lonicerae. The results were important to clarify the material basis and therapeutic mechanism of Flos Lonicerae.

Achillea millefolium L. s.l. herb extract: Antioxidant activity and effect on the rat heart mitochondrial functions

An extract of Achillea millefolium herb (YE) was investigated for antioxidant activity using chemical and biological assays. Qualitative and quantitative analysis of some major phenolics was carried out by HPLC. An on-line HPLC-DPPH assay showed that YE possesses significant antiradical activity which is due to the presence of active components amongst phenolic compounds. Furthermore, direct effects of YE and a mixture of its identified phenolic compounds (MPC) on isolated rat heart mitochondrial function were investigated. We found that YE in concentration-dependent manner induce a decrease in State 3 respiration rate without any changes in the integrity of inner mitochondrial membrane. Thus, pyruvate oxidation was affected only by the highest used YE concentrations; meanwhile succinate oxidation was reduced even at lower YE concentrations. MPC had no effect on mitochondrial State 3 respiration rate. Fluorimetric measurements demonstrated that YE at concentrations that had no effect on the State 3 respiration rate significantly decreased H 2O 2 production in mitochondria.