Radical scavenging activity and metabolomic profiling study of ylang-ylang essential oils based on high-performance thin-layer chromatography and multivariate statistical analysis.

Ylang-ylang (YY) essential oil (EO) is distilled from the fresh-mature flowers of the Annonaceae family tropical tree Cananga odorata [Lam.] Hook. f. & Thomson, and is widely used in perfume and cosmetic industries for its fragrant character. Herein, two different metabolomic profiles obtained using high-performance thin-layer chromatography (HPTLC), applying different stains, namely 2,2-diphenyl-1-picrylhydrazyl (DPPH·) and p-anisaldehyde, were used for discrimination of 52 YY samples across geographical origins and distillation grades. The first profile is developed using the DPPH· stain based on the radical scavenging activity (RSA) of YY EOs. Results of the HPTLC-DPPH· assay confirmed that RSA of YY EOs is in proportion to the length of distillation times. Major components contributing to the RSA of YY EOs were tentatively identified as germacrene D and α-farnesene, eugenol and linalool, by gas chromatography-mass spectrometry (GC-MS) and GC-flame ionisation detector (GC-FID). The second profile was developed using the general-purpose p-anisaldehyde stain based on the general chemical composition of YY EOs. Untargeted metabolomic discrimination of YY EOs from different geographical origins was performed based on the HPTLC-p-anisaldehyde profiles, followed by principal component analysis (PCA). A discrimination and prediction model for identification of YY distillation grade was developed using PCA and partial least squares regression (PLS) based on binned HPTLC-ultraviolet (254 nm) profiles, which was successfully applied to distillation grade determination of blended YY Complete EOs.

The Impact of Formulation on the Content of Phenolic Compounds in Snacks Enriched with Dracocephalum moldavica L. Seeds: Introduction to Receiving a New Functional Food Product.

A new type of multigrain snack has been designed containing varied additions of Moldavian dragonhead (Dracocephalum moldavica L.) seeds. The antioxidant properties and the general health benefits of this plant material have already been widely acknowledged. The research discussed herein aimed to investigate the influence of the formulation and expansion method (frying) on the content of polyphenolic compounds, individual phenolic acids, and antiradical properties of innovative snacks enriched with dragonhead seeds. The highest content of polyphenols (0.685 mg GAE/mL), free phenolic acids (47.052 µg/g of dry matter), and highest radical scavenging activity (96.23% towards DPPH) were found in the fried snacks enriched with 22% of seeds. In these samples, 11 phenolic acids were detected. Strong positive correlations were seen between the addition of dragonhead and the polyphenol content (r = 0.989) and between the quantity of the enriching additive and the content of free phenolic acids (r = 0.953). The research has shown that such innovative snacks have the potential to supply health-benefiting free phenolic acids, e.g., salicylic, isoferulic, ferulic, p-coumaric, vanillic. Our studies provide an introduction to the development of a new range of functional foods.

Targeted isolation of 1,1-diphenyl-2-picrylhydrazyl inhibitors from Saxifraga atrata using medium- and high- pressure liquid chromatography combined with online high performance liquid chromatography-1,1-diphenyl-2- picrylhydrazyl detection.

Traditional Tibetan medicine (TTM) is a valuable source of novel therapeutic lead molecules inspired by natural products (NPs). The health benefits of Saxifraga atrata are well documented in TTM, but reports on its chemical composition are limited, most likely due to the complicated purification process. Herein, target separation and identification of 4 main radical scavenging compounds from the methanolic extract of S. atrata was were performed using medium- and high-pressure liquid chromatography coupled with online HPLC-DPPH detection. The sample was pretreated using medium pressure liquid chromatography with MCI GELⓇ CHP20P styrene-divinylbenzene beads as a stationary phase, yielding 1.4 g of the target DPPH inhibitors (Fr4, 11.9% recovery). The compounds were further purified and isolated using HPLC on RP-C18 (ReproSil-Pur C18 AQ) followed by HILIC (Click XIon) column separation, resulting in 2.8 mg of fraction Fr4-1-1, 6.8 mg of fraction Fr4-2, 244.9 mg of the Fr4-3-1 sample, and 38.3 mg of Fr4-4-1. The structure and purity of the target compounds were determined, and four compounds (ethyl gallate, 11-O-galloylbergenin, rutin and isoquercitrin) were isolated with >95% purity. The developed methodology is efficient for targeted isolation of high-purity radical scavengers from NP extracts and could be used for rapid identification and isolation of DPPH inhibitors from various NPs.

Phytochemical Characterization and In Vitro Antioxidant Properties of Four Brassica Wild Species from Italy.

In the present study, we evaluated for the first time the variability of antioxidant traits of four Brassica wild species: B. incana, B. macrocarpa, B. villosa, and B. rupestris. The content of the main water-soluble antioxidants (phenolics, ascorbic acid, and total biothiols) and the in vitro antioxidant potential (1,1-diphenyl-2-picrylhydrazil (DPPH) and superoxide anion scavenging capacity) were investigated. A total of 28 polyphenolic compounds were identified by LC/MS and quantitated by HPLC/DAD analysis. Kaempferol and quercetin derivatives were the most abundant phenolics compared to hydroxycinnamoyl gentiobiosides. In the ten populations, phenolics ranged from 163.9 to 533.9 mg/100 g dry weight (d.w.), ascorbic acid from 7.6 to 375.8 mg/100 g d.w., and total biothiols from 0.59 to 5.13 mg/100 g d.w. The different classes of phytochemicals were separated using solid-phase extraction at increasing methanol concentrations, and the antioxidant power of fractionated extracts was evaluated. The superoxide anion scavenging activity was significantly correlated to phenolics, particularly to flavonol derivatives, while DPPH was mainly related to ascorbic acid content. The present findings improve the knowledge of the phytochemical composition of Italian Brassica wild species by showing the great diversity of phytochemicals among populations and highlighting their importance as a valuable genetic resource for developing new cultivars with improved bioactive content.

Dissecting the Potential of Selenoproteins Extracted from Selenium-Enriched Rice on Physiological, Biochemical and Anti-Ageing Effects In Vivo.

Ageing is an irreversible phenomenon and the processes which can delay it are under consideration for a long time by the scientific community. Selenium is an important candidate for it, but the impact of selenoprotein on nutritional changes and ageing has not been reported well. In this regard, antioxidant activities and free radical scavenging effect of selenoproteins extracted from selenium-rich rice were studied. Mice were administered a subcutaneous abdominal injection of D-galactose to induce the ageing model and fed with different selenoprotein dosage diet. Deviations among biochemical activities (total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA)) in liver and serum of the mice were assessed. The degree of liver injury, antioxidant genes and protein relative expression were estimated. The protein content, selenium content, hydroxyl scavenging and DPPH radicals were accessed in selenoprotein components. The selenoprotein constituent had protein and selenium contents in different components as water-soluble proteins > alkali-soluble proteins > salt-soluble proteins > ethanol-soluble proteins. The enzymatic activity (total antioxidant capacity, GSH-Px and SOD) in liver and serum of mice was significantly enhanced in selenoprotein diet groups. D-Galactose-induced liver injury was significantly reduced by selenoprotein diet of 25 µg/(kg day). Real-time qPCR and Western blot disclosed the enhanced relative expression of antioxidant genes (SOD2, GPX1, TrxR2 and Nrf2) and HO-1 protein in the positive control (Vc) and selenoprotein diet groups. In conclusion, selenoprotein treatment was found to have a positive influence on liver hepatocytes and biochemical features in mice. It might be used as a potential diet in scavenging oxidative injury and supporting enzymatic antioxidant system.

Bioactive compounds and biological properties of Brazilian stingless bee honey have a strong relationship with the pollen floral origin.

Multivariate data analysis feasibility for the evaluation of Brazilian stingless bee honey (SBH) by pollen spectrum, bioactive compounds content, physicochemical, antioxidant and antimicrobial analysis was investigated. Levels of total and individual phenolics content were analyzed by HPLC-PDA. The antioxidant capacity was performed by 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH), oxygen radical absorption capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays. The total phenolic compounds from the thirty-two SBH was positively correlated with the antioxidant capacity. Bioactive compounds such as p-coumaric acid, quercetin, and hesperetin were identified in all the samples. Brazilian SBH shows more effective antibacterial activity against Gram-negative bacteria (E. coli and S. Typhimurium) compared to Gram-positive ones. Results also revealed that SBH could reach up to 45% higher antioxidant and biological activities than the traditional Apis mellifera honey. Chemometrics shows that chemical and biological properties of SBH have a strong relationship with the pollen botanical origin. Principal component analysis (PCA) grouped the honey into three categories with predominant pollen from Verbenaceae, Asteraceae and Sapindaceae families, confirming that SBH belonging to the same floral origin present similar characteristics.

Fabrication and characterisation of silver nanoparticles using bract extract of Musa paradisiaca for its synergistic combating effect on phytopathogens, free radical scavenging activity, and catalytic efficiency.

This work explores the rapid synthesis of silver nanoparticles (AgNPs) from Musa paradisiaca (M. paradisiaca) bract extract. The bio-reduction of Ag+ ion was recorded using ultraviolet-visible spectroscopy by a surface plasmon resonance extinction peak with an absorbance at 420 nm. The phytoconstituents responsible for the reduction of AgNPs was probed using Fourier transform infrared spectroscopy. The X-ray diffraction pattern confirmed the formation of crystalline AgNPs that were analogous to selected area electron diffraction patterns. Morphological studies showed that the obtained AgNPs were monodispersed with an average size of 15 nm. The biologically synthesised AgNPs showed higher obstruction against tested phytopathogens. The synthesised AgNPs exhibited higher inhibitory zone against fungal pathogen Alternaria alternata and bacterial pathogen Pseudomonas syringae. Free radical scavenging potential of AgNPs was investigated using 1,1-diphenyl-2-picryl hydroxyl and 2,2-azinobis (3-ethylbenzothiazoline)-6-sulphonic acid assays which revealed that the synthesised AgNPs act as a potent radical scavenger. The catalytic efficiency of the synthesised AgNPs was investigated for azo dyes, methyl orange (MO), methylene blue (MB) and reduction of o-nitrophenol to o-aminophenol. The results portrayed that AgNPs act as an effective nanocatalyst to degrade MO to hydrazine derivatives, MB to leucomethylene blue, and o-nitro phenol to o-amino phenol.

Biosynthesis of Selenium Nanoparticles using yeast Nematospora coryli and examination of their anti-candida and anti-oxidant activities.

Selenium (Se) is a rare and essential element for the human body and other living organisms because of its role in the structure of several proteins and having anti-oxidant properties to reduce oxidative stress at cells. Some microorganisms can absorb Se oxyanions and convert them into zero-valent Se (Se0) in the nanoscale dimensions, which can be used for producing Se nanoparticles (SeNPs). In the present study, SeNPs were intracellularly biosynthesised by yeast Nematospora coryli, which is an inexpensive method and does not involve using materials hazardous for human and environment. The produced NPs were refined by a two-phase system and then characterised and identified by ultraviolet-visible, X-ray diffraction, X-ray fluorescence, transmission electron microscope, and Fourier transform infrared spectroscopy analyses. The structural analysis of biosynthesised SeNPs showed spherical-shaped NPs with size ranging from 50 to 250 nm. Also, extracted NPs were applied to explore their anti-candida and anti-oxidant activities. The results of this investigation confirm the biological properties of Se.

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.

Review of the Determination of the Antioxidant Activity of Foods, Food Ingredients, and Dietary Supplements by Thin Layer Chromatography-Direct Bioautography, Spectrometry, and the Dot-Blot Procedure.

This paper reviews a selection of the most important studies on antioxidants in foods (including beverages), food ingredients, and dietary supplements by effect-directed analysis using TLC with DPPH*, ABTS*+, and ß-carotene direct bioautography. Total antioxidant activity by visible mode spectrometry (colorimetry) with TLC used offline to obtain additional analytical results, mostly for identification and quantification of phenolic compounds in samples, is also discussed. Finally, dot-blot assay for total antioxidant activity, carried out on a TLC plate without mobile-phase development, is reviewed as an alternative with possible advantages compared with spectrometry.

Programmable electrochemical flow system for high throughput determination of total antioxidant capacity.

The aim of this work was to develop a programmable flow system for rapid assessment of total antioxidant capacity (TAC). Novel features of the prototype include a single pressure-driven source, versatile manipulation of fluid flows, ability to adapt to different TAC assays, and compatibility with microfluidic design. Antioxidant activity was determined by electrochemical measurement of residual 2,2-diphenyl-2-picrylhydrayl hydrate (DPPH•) free radicals in the solution. The overall performance of the device was validated by the spectrophotometric method. The results indicated that dosing of reagents and samples could be controlled by pressure (R2 = 0.992) and time (R2 = 0.999) with high accuracy, and the mixing uniformity of the device was equivalent to that of the batch mixing (R2 = 0.994). TAC assays of a standard antioxidant, ascorbic acid, as well as selected samples such as orange and pomegranate juices, white wine, and green tea by the device were comparable to traditional measurement techniques. Due to the short incubation time, the approach may be more suitable for fast, rather than slow reacting antioxidant compounds. The developed system could be used for rapid TAC screening of food and biological samples.

ß-Cyclodextrin encapsulation elevates antioxidant capacity of tea: A closing chapter on non-epicatechins, atomistic insights from X-ray analysis, DFT calculation and DPPH assay.

Polyphenolic catechins prevalent in tea are powerful antioxidants for therapeutics of various free radical-related diseases. The non-epicatechins are thermally obtained from the naturally abundant epicatechins. In our study series on the structure-antioxidant property relationship of the CD inclusion complexes with tea catechins, this closing chapter presents the ß-CD encapsulation of three non-epicatechins, i.e., (-)-gallocatechin (GC) 1, (-)-catechin gallate (GC) 2, and (-)-gallocatechin gallate (GCG) 3 investigated by means of single-crystal X-ray diffraction, DFT calculation and DPPH radical scavenging activity assay. Detailed structural comparisons of the ß-CD inclusion complexes with both non-epi and epi type catechins reveal that a common host-guest hydrogen bonding scheme and the shielding of catechin OH groups inside the host circular wall play a prime role in flourishing antioxidant capacities in the order of 3 > 2 > 1. This is consistent with the relative thermodynamic stabilities derived from DFT energy minimization.

Rapid screening of brain-penetrable antioxidants from natural products by blood-brain barrier specific permeability assay combined with DPPH recognition.

Screening brain-penetrable antioxidants from natural products is a promising way for neuroprotective drug discovery. However, there is no screening methodology enables simultaneous investigation of antioxidant activity and blood-brain barrier (BBB) permeability of compounds from complex samples. Here we propose a novel strategy by combining BBB specific parallel artificial membrane permeability assay with 1,1-diphenyl-2-picrylhydrazyl recognition (BBB-PAMPA-DPPH) to achieve rapid multicomponent screening. First, BBB specific artificial membrane was constructed to separate the compounds with high BBB permeability in herbal extracts. The antioxidant activity of the isolated compounds could be optically recognized through the bleaching of the purple-colored DPPH. By off-line combined HPLC-UV/Q-TOF-MS analysis, the exact BBB-penetrable compounds responsible for the antioxidant activity could be rapidly screened. With this approach, compound 2,6,4'-trihydroxy-4-methoxybenzophenon in Rhizoma Anemarrhena was found to be an antioxidant with very high BBB permeability, which could also be detected in rat plasma and brain tissue after oral administration. Our findings suggested the BBB-PAMPA-DPPH method could be a powerful tool for neuroprotective drug discovery from natural products.

HPLC-DAD finger printing, antioxidant, cholinesterase, and α-glucosidase inhibitory potentials of a novel plant Olax nana.

BACKGROUND: The medicinal importance of a novel plant Olax nana Wall. ex Benth. (family: Olacaceae) was revealed for the first time via HPLC-DAD finger printing, qualitative phytochemical analysis, antioxidant, cholinesterase, and α-glucosidase inhibitory assays. METHODS: The crude methanolic extract of O. nana (ON-Cr) was subjected to qualitative phytochemical analysis and HPLC-DAD finger printing. The antioxidant potential of ON-Cr was assessed via 1,1-diphenyl,2-picrylhydrazyl (DPPH), 2,2-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid (ABTS) and hydrogen peroxide (H2O2) free radical scavenging assays. Furthermore, acetylcholinesterase (AChE) & butyrylcholinesterase (BChE) inhibitory activities were performed using Ellman's assay, while α- glucosidase inhibitory assay was carried out using a standard protocol. RESULTS: The qualitative phytochemical analysis of ON-Cr revealed the presence of secondary metabolites like alkaloids, flavonoids, tannins, sterols, saponins and terpenoids. The HPLC-DAD finger printing revealed the presence of 40 potential compounds in ON-Cr. Considerable anti-radical activities was revealed by ON-Cr in the DPPH, ABTS and H2O2 free radical scavenging assays with IC50 values of 71.46, 72.55 and 92.33 µg/mL, respectively. Furthermore, ON-Cr showed potent AChE and BChE inhibitory potentials as indicated by their IC50 values of 33.2 and 55.36 µg/mL, respectively. In the α-glucosidase inhibition assay, ON-Cr exhibited moderate inhibitory propensity with an IC50 value of 639.89 µg/mL. CONCLUSIONS: This study investigated Olax nana for the first time for detailed qualitative phytochemical tests, HPLC-DAD finger printing analysis, antioxidant, anticholinesterase and α-glucosidase inhibition assays. The antioxidant and cholinesterase inhibitory results were considerable and can provide scientific basis for further studies on the neuroprotective and anti-Alzheimer's potentials of this plant. ON-Cr may further be subjected to fractionation and polarity guided fractionation to narrow down the search for isolation of bioactive compounds.

Genoprotective, antioxidant, antifungal and anti-inflammatory evaluation of hydroalcoholic extract of wild-growing Juniperus communis L. (Cupressaceae) native to Romanian southern sub-Carpathian hills.

BACKGROUND: Juniperus communis L. represents a multi-purpose crop used in the pharmaceutical, food, and cosmetic industry. Several studies present the possible medicinal properties of different Juniperus taxa native to specific geographical area. The present study aims to evaluate the genoprotective, antioxidant, antifungal and anti-inflammatory potential of hydroalcoholic extract of wild-growing Juniperus communis L. (Cupressaceae) native to Romanian southern sub-Carpathian hills. METHODS: The prepared hydroethanolic extract of Juniperus communis L. was characterized by GC-MS, HPLC, UV-Vis spectrometry and phytochemical assays. The antioxidant potential was evaluated using the DPPH assay, the antifungal effect was studied on Aspergillus niger ATCC 15475 and Penicillium hirsutum ATCC 52323, while the genoprotective effect was evaluated using the Allium cepa assay. The anti-inflammatory effect was evaluated in two inflammation experimental models (dextran and kaolin) by plethysmometry. Male Wistar rats were treated by gavage with distilled water (negative control), the microemulsion (positive control), diclofenac sodium aqueous solution (reference) and microemulsions containing juniper extract (experimental group). The initial paw volume and the paw volumes at 1, 2, 3, 4, 5 and 24 h were measured. RESULTS: Total terpenoids, phenolics and flavonoids were estimated to be 13.44 ± 0.14 mg linalool equivalent, 19.23 ± 1.32 mg gallic acid equivalent, and 5109.6 ± 21.47 mg rutin equivalent per 100 g of extract, respectively. GC-MS characterization of the juniper extract identified 57 volatile compounds in the sample, while the HPLC analysis revealed the presence of the selected compounds (α-pinene, chlorogenic acid, rutin, apigenin, quercitin). The antioxidant potential of the crude extract was found to be 81.63 ± 0.38% (measured by the DPPH method). The results of the antifungal activity assay (for Aspergillus niger and Penicillium hirsutum) were 21.6 mm, respectively 17.2 mm as inhibition zone. Test results demonstrated the genoprotective potential of J. communis undiluted extract, inhibiting the mitodepressive effect of ethanol. The anti-inflammatory action of the juniper extract, administered as microemulsion in acute-dextran model was increased when compared to kaolin subacute inflammation induced model. CONCLUSION: The hydroalcoholic extract obtained from wild-growing Juniperus communis native to Romanian southern sub-Carpathian hills has genoprotective, antioxidant, antifungal and anti-inflammatory properties.

Statistical optimization of an RP-HPLC method for the determination of selected flavonoids in berry juices and evaluation of their antioxidant activities.

An isocratic RP-HPLC method for the separation and identification of selected flavonoids (quercetin, rutin, luteolin-7-O-glucoside, kaempferol and kaempferol-3-O-glucoside) in commercial berry juices (blackcurrant, blueberry, red raspberry and cherry) was developed with the aid of central composite design and response surface methodology. The optimal separation conditions were a mobile phase of 85:15 (% v/v) water-acetonitrile, pH 2.8 (adjusted with formic acid), flow rate 0.5 mL min-1 and column temperature 35°C. The obtained levels of bioflavonoids (mg per 100 mL of juice) were as follows: for quercetin, ca. 0.21-5.12; for kaempferol, ca. 0.05-1.2; for rutin, ca. 0.4-6.5; for luteolin-7-O-glucoside, ca. 5.6-10.2; and for kaempferol-3-O-glucoside, ca. 0.02-0.12. These are considerably lower than the values in fresh fruits. Total phenolic, flavonoid and anthocyanin contents were determined spectrophotometrically. Total flavonoid content varied as follows: blackcurrant > blueberry > red raspberry > cherry. The antioxidant activity of juice extracts (DPPH and ABTS methods) expressed as IC50 values varied from 8.56 to 14.05 mg L-1 . These values are ~2.5-3 times lower than quercetin, ascorbic acid and Trolox®, but compared with rutin and butylhydroxytoluene, berries show similar or better antioxidant activity by both the DPPH and ABTS methods.

Quantitative fingerprinting based on the limited-ratio quantified fingerprint method for an overall quality consistency assessment and antioxidant activity determination of Lianqiao Baidu pills using HPLC with a diode array detector combined with chemometric methods.

Lianqiao Baidu pills are widely used herbal medicinal preparation that were analyzed to develop a quality consistency technique. The characteristic fingerprints of 28 batches of Lianqiao Baidu pill samples were established at five wavelengths and simultaneously assessed by using a limited-ratio quantified fingerprint method using 15 marker compounds. The principal component analysis and fingerprinting results were compared, and the qualitative classification of the samples by principal component analysis agreed with their quantitative evaluation by the limited-ratio quantified fingerprint method. Furthermore, the antioxidant activities of the samples were surveyed and determined using a 2,2-diphenyl-1-picrylhydrazyl radical-scavenging approach. A relationship between the common peaks in the fingerprints and the antioxidant activities was established using a partial least squares model. The relationship can be used both to determine the antioxidant activities of the Lianqiao Baidu pill preparations in vitro and as a reference for the selection of active constituents for sample quality classification. The classification results for the samples based on principal component analysis agreed with the quantitative evaluation by the limited-ratio quantified fingerprint method, which demonstrated that the method can be applied to the holistic quality control of traditional Chinese medicine and herbal preparations.

LC-MS/MS profiling of polyphenol-enriched leaf, stem and root extracts of Korean Humulus japonicus Siebold & Zucc and determination of their antioxidant effects.

Polyphenols from ethyl acetate extracts from the leaves, stems and roots of Korean Humulus japonicus were comprehensively profiled using liquid chromatography-electrospray ionization-tandem mass spectrometry. A total of 36 polyphenols were detected, of which 26 were structurally characterized based on their [M - H]- peak, tandem mass spectrometry fragmentation pattern, UV-vis absorption and published data. Validation data provided satisfactory results for the evaluated parameters. The determination coefficients were ≥0.9812. The limits of detection and quantification were 0.017-0.573 and 0.056-1.834 mg/L, respectively, indicating good performance limits. The accuracy (expressed as percentage recovery) at 50 and 100 mg/L was 71.4-99.7 and 75.1-105.1%, with precisions (expressed as relative standard deviation) of 1.5-7.3 and 0.8-4.1%, respectively, indicating acceptable accuracy and precision values. The leaves were rich in total polyphenols (3089.9 ± 6.4 mg/kg of fresh sample) followed by the stems (1313.9 ± 6.4 mg/kg of fresh sample) and roots (655.2 ± 2.7 mg/kg of fresh sample). Antioxidant activity, determined by α,α-diphenyl-ß-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity and ferric reducing antioxidant power assay, revealed the lowest EC50 value for the leaf extracts, indicating a higher scavenging activity in this tissue followed by the roots and stems. Overall, the results indicated that H. japonicus is rich in polyphenols and could be a potential alternative to Humulus lupulus (hop plant) in the brewery industry.

Separation and purification of four phenolic compounds from persimmon by high-speed counter-current chromatography.

An efficient method was established by high-speed counter-current chromatography (HSCCC) for preparation of four phenolic compounds from the depolymerization products of persimmon tannin. Using the two solvent systems of n-hexane/ethyl acetate/water (3:17:20, v/v/v) and ethyl acetate/methanol/water (50:1:50, v/v/v), the preparative isolation was successfully performed by a two-step separation. The yields of one run (150mg crude sample) for gallic acid, methyl gallate, and epigallocatechin-3-gallate-(4ß→8, 2ß→O→7)-epigallocatechin-3-gallate dimer (A-type EGCG dimer) were 4.7, 44.2 and 5.9mg, respectively. In addition, 4.6mg epicatechin-3-gallate-(4ß→8, 2ß→O→7)-epicatechin-3-gallate dimer (A-type ECG dimer) was obtained by further preparative high-performance liquid chromatography (prep-HPLC). The purities of these compounds were all above 95.0% and their structures were identified by HPLC/ESI-MS. We found that HSCCC had definite advantages for the preparation of dimeric procyanidins compared with previous methods. Furthermore, it was shown that the four phenolic compounds possessed greater antioxidant activities than Trolox.

Isolation and characterization of the compounds responsible for the antimutagenic activity of Combretum microphyllum (Combretaceae) leaf extracts.

BACKGROUND: Mutations play a major role in the pathogenesis and development of several chronic degenerative diseases including cancer. It follows, therefore that antimutagenic compound may inhibit the pathological process resulting from exposure to mutagens. Investigation of the antimutagenic potential of traditional medicinal plants and compounds isolated from plant extracts provides one of the tools that can be used to identify compounds with potential cancer chemopreventive properties. The aim of this study was to isolate and characterise the compounds responsible for the antimutagenic activity of Combretum microphyllum. METHODS: The methanol leaf extract of C. microphyllum was evaluated for antimutagenicity in the Ames/microsome assay using Salmonella typhimurium TA98. TA100 and TA102. Solvent-solvent fractionation was used to partition the extracts and by using bioassay-guided fractionation, three compounds were isolated. The antimutagenic activity of the three compounds were determined in the Ames test using Salmonella typhimurium TA98, TA100 and TA102. The antioxidant activity of the three compounds were determined by the quantitative 2,2-diphenyl-1-picrylhydrazyl (DPPH)-free radical scavenging method. The cytotoxicity was determined in the MTT assay using human hepatocytes. RESULTS: A bioassay-guided fractionation of the crude extracts for antimutagenic activity led to the isolation of three compounds; n-tetracosanol, eicosanoic acid and arjunolic acid. Arjunolic acid was the most active in all three tested strains with a antimutagenicity of 42 ± 9.6%, 36 ± 1.5% and 44 ± 0.18% in S. typhimurium TA98, TA100 and TA102 respectively at the highest concentration (500 µg/ml) tested, followed by eicosanoic acid and n-tetracosanol. The antioxidant activity of the compounds were determined using the quantitative 2,2 diphenyl-1-picryhydrazyl (DPPH)-free radical scavenging method. Only arjunolic acid had pronounced antioxidant activity (measured as DPPH-free scavenging activity) with an EC50 value of 0.51 µg/ml. The cytotoxicity of the isolated compounds were determined in the MTT assay using human hepatocytes. The compounds had low cytotoxicity at the highest concentration tested with LC50 values >200 µg/ml for n-tetracosanol and eicosanoic acid and 106.39 µg/ml for arjunolic acid. CONCLUSIONS: Based on findings from this study, compounds in leaf extracts of C. microphyllum protected against 4-NQO and MMC induced mutations as evident in the Ames test. The antimutagenic activity of arjunolic acid may, at least in part, be attributed to its antioxidant activity resulting in the detoxification of reactive oxygen species produced during mutagenesis.