A New Flavonoid C-Glycoside from Celtis australis L. and Celtis occidentalis L. Leaves and Potential Antioxidant and Cytotoxic Activities

A major development over the past two decades has been the realization that free radical induced lipid peroxidation and DNA damage are associated with major health problems, e.g. cancer and ageing. Plant-derived antioxidants are increasingly found beneficial in protecting against these diseases. Celtis australis L. and Celtis occidentalis L. are two plants that have a variety of uses in folk medicine but have not been evaluated before for their antioxidant and cytotoxic properties. Therefore, the extracts of both plants’ leaves were investigated for these activities, as well as isolation of the bioactive compounds responsible for the activities. Molecular structures of the compounds were elucidated by UV, HRESIMS, 1D (¹H and ¹³C) and 2D (¹H-¹³C HSQC and ¹H-¹³C HMBC) NMR analyses. The ethanolic and aqueous extracts, n-butanol fractions and the isolated major compound were tested for their antioxidant activity using DPPH radical scavenging assay, xanthine oxidase-induced generation of superoxide radical and lipid peroxidation assay by thiobarbituric acid-reactive substances (TBARS) method using rat tissue homogenates. Cytotoxic activities were studied using standard MTT assay. A novel flavonoid C-triglycoside, 4‴-α-rhamnopyranosyl-2″-O-β-d-galactopyranosylvitexin, was isolated from both plants’ leaves, together with seven known flavonoids. The n-butanol fractions and the major compound 2″-O-β-galactopyranosylvitexin showed significant antioxidant activities, more pronounced than the tested standards BHT and dl-α-tocopherol in most tests. All extracts showed variable cytotoxic activities. This study provides strong evidence for the antioxidant and cytotoxic activities of the extracts of Celtis australis L. and Celtis occidentalis L. leaves, which were attributed to the polar n-butanol fractions and the major isolated flavonoid 2″-galactosylvitexin.     

Systematic screening and characterization of flavonoid glycosides in Carthamus tinctorius L. by liquid chromatography/UV diode-array detection/electrospray ionization tandem mass spectrometry

The traditional Chinese medicine (TCM) is a complex system, which always consists of numerous compounds with significant difference in the content and physical and chemical properties. In this paper, a screening method based on target molecular weights was developed to characterize the flavonoid glycosides in the flower of Carthamus tinctorius L. The screening tables of aglycone and glycan were designed, respectively, in order to select and combine freely. The multiple reaction monitoring (MRM) scan mode with higher sensitivity and selectivity was adopted in the screening, which benefit the characterization for the minor components. Seventy-seven flavonoid glycosides were screened out finally, and their structures were characterized by tandem mass spectrometric method in both positive and negative ion modes. The glycosylation mode, aglycone, sequence and/or the interglycosidic linkages of the glycan portion and glycosylation position were elucidated by the fragmentation rule in the MS. Numerous compounds screened out with this method showed the structure variety in secondary plant metabolites, and the purposeful screening systemically and subsequent structure characterization offered more information about the chemical constitutions of TCM.     

Isolation and analysis of bioactive diterpenoids in Salvia species (Salvia chionantha and Salvia kronenburgiii) by micellar electrokinetic capillary chromatography

In the present work, we isolated two bioactive diterpenoids, horminone and 7-O-acetylhorminone and developed a micellar electrokinetic chromatography (MEKC) method for the simultaneous quantitative analysis of them in Turkish Salvia species. The optimal separation electrolyte was 50 mmol/L SDS and 25% methanol at pH 11.5. The limits of detection (S/N = 3) were 3.269 and 4.518 μg/mL for horminone and 7-O-acetylhorminone, respectively. The method has been applied successfully to analyze these two components in Salvia chionantha and Salvia kronenburgii acetone extracts.     

A biochemical characterization of the major peptides from the Venom of the giant Neotropical hunting ant Dinoponera australis

Venom from the “false tocandira” Dinoponera australis, a giant Neotropical hunting ant, paralyzes small invertebrate prey and induces a myriad of systemic effects in large vertebrates. HPLC/DAD/MS analyses revealed that the venom has over 75 unique proteinaceous components with a large diversity of properties ranging in size, hydrophobicity, and overall abundance. The six most abundant peptides, demonstrative of this diversity and hereafter referred to as Dinoponeratoxins, were de novo sequenced by exact mass precursor ion selection and Edman degradation. The smallest peptide characterized, Da-1039, is hydrophilic and has similarities to vasoactive peptides like kinin and bombesin. The two largest and most abundant peptides, Da-3105 and Da-3177, have a 92.9% identity in a 28 residue overlap and share ∼50 of their sequence with ponericin G2 (an antimicrobial from another ponerine ant Pachycondyla goeldii). One peptide, Da-1585, is a hydrophilic cleavage product of an amphipathic peptide, Da-2501. The most hydrophobic peptide, Da-1837, is amidated (a PTM observed in one half of the major peptides) and shares homology with poneratoxin, a sodium channel modifier found in the bullet ant Paraponera clavata. This study is the first examination of potential pharmacophores from venom of the genus Dinoponera (Order: Hymenoptera).     

Cylindrospermopsin induces alterations of root histology and microtubule organization in common reed (Phragmites australis) plantlets cultured in vitro

We aimed to study the histological and cytological alterations induced by cylindrospermopsin (CYN), a protein synthesis inhibitory cyanotoxin in roots of common reed (Phragmites australis). Reed is an ecologically important emergent aquatic macrophyte, a model for studying cyanotoxin effects. We analyzed the histology and cytology of reed roots originated from tissue cultures and treated with 0.5-40 μg ml⁻¹ (1.2-96.4 μM) CYN. The cyanotoxin decreased root elongation at significantly lower concentrations than the elongation of shoots. As general stress responses of plants to phytotoxins, CYN increased root number and induced the formation of a callus-like tissue and necrosis in root cortex. Callus-like root cortex consisted of radially swollen cells that correlated with the reorientation of microtubules (MTs) and the decrease of MT density in the elongation zone. Concomitantly, the cyanotoxin did not decrease, rather it increased the amount of β-tubulin in reed plantlets. CYN caused the formation of double preprophase bands; the disruption of mitotic spindles led to incomplete sister chromatid separation and disrupted phragmoplasts in root tip meristems. This work shows that CYN alters reed growth and anatomy through the alteration of MT organization.     

Separation and determination of ketoprofen, methylparaben and propylparaben in pharmaceutical preparation by micellar electrokinetic chromatography

Simple micellar electrokinetic chromatographic (MEKC) method was developed for the determination of ketoprofen as the active substance and methylparaben and propylparaben as preservatives in a semisolid pharmaceutical preparation. Separation was carried out with a fused silica capillary and UV detection at 200nm. Optimized background electrolyte was 50mM tricine buffer containing 30mM sodium dodecyl sulfate as surfactant and 15% (v/v) of methanol. Single separation took about 13min. No statistically significant differences were found when comparing the results with those of RP-HPLC method reported in literature.     

Nutritional and nutraceutical potential of rape (Brassica napus L. var. napus) and “tronchuda” cabbage (Brassica oleraceae L. var. costata) inflorescences

Two traditional cultivated vegetables highly consumed among Northern Portuguese regions were tested for their chemical composition, nutritional profile and in vitro antioxidant properties using four assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging activity, reducing power, inhibition of β-carotene bleaching and inhibition of lipid peroxidation by thiobarbituric acid reactive substances (TBARS) assay. The studied varieties of two Brassica species, locally known as “grelos” (rape) and “espigos” (“tronchuda” cabbage) are nutritionally well-balanced vegetables; particularly “tronchuda” cabbage revealed the highest levels of moisture, proteins, fat, energy, β-carotene and vitamin C; rape gave the highest contents of ash, carbohydrates, sugars (including fructose, glucose, sucrose and raffinose), essential n-3 fatty acid α-linolenic acid, and the best ratios of PUFA/SFA and n-6/n-3 fatty acids, tocopherols, lycopene, chlorophylls, phenolics, flavonoids, and also the highest antioxidant properties. The health benefits associated to the antioxidant properties reinforce their contribution to a healthy and balanced diet, highlight the interest of their consumption, validate the empirical use and add new values to traditional/regional products which have been used for a long time.     

Qualitative and quantitative analysis of steroidal saponins in crude extracts from Paris polyphylla var. yunnanensis and P. polyphylla var. chinensis by high performance liquid chromatography coupled with mass spectrometry

High performance liquid chromatography coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC–ESI-MSⁿ) and triple quadrupole mass spectrometric detection (HPLC–ESI-MS/MS), respectively, had been employed for the simultaneous identification and quantification of steroidal saponins in the rhizomes of Parispolyphylla var. yunnanensis and P. polyphylla var. chinensis, which are the qualified plants of “Chonglou” in Chinese. The HPLC experiments were performed by means of a reversed-phase C-18 column and a binary mobile phase system consisting of 0.1% aqueous formic acid and acetonitrile under gradient elution conditions. The characteristic fragmentation patterns of diosgenin- and pennogenin-type steroidal saponins were investigated using ESI-MSⁿ in negative ion mode. The MSⁿ data of the [M−H]⁻ ions provided structural information on the sugar sequence of the oligosaccharide chains and the aglycones of steroidal saponins. As a result, ten and seven saponins were determined in P. polyphylla var. yunnanensis and P. polyphylla var. chinensis, respectively, including four unknown compounds. One unknown compound was tentatively identified as diosgenin-3-O-α-l-rhamnopyranosyl(1 → 4) [α-l-rhamnopyranosyl(1 → 2)]-β-d-glucopyranoside and the aglycones of the other three new compounds were reported from Chonglou for the first time. The developed HPLC–ESI-MS/MS method was validated and found to be satisfactorily linear, selective and robust. The limits of detection (LODs) and quantitation (LOQs) ranged, respectively, from 0.5 to 10 ng/mL and 2 to 34 ng/mL depending on six various compounds. The intra- and inter-day precisions of the method were evaluated and were less than 5.0%. Recoveries ranged from 92% to 104% for all compounds. The established quality evaluation method was successfully used for simultaneous quantification of six predominant steroidal saponins in the rhizomes of these two Paris species.     

Acetylcholinesterase inhibitory and antioxidant properties of Cyclotrichium niveum, Thymus praecox subsp. caucasicus var. caucasicus, Echinacea purpurea and E. pallida

The dichloromethane, ethyl acetate, ethanol, and aqueous extracts of Cyclotrichium niveum (CN) and Thymus praecox subsp. caucasicus var. caucasicus (TP), Echinacea purpurea (EPU), and E. pallida (EPA) along with the essential oils of CN and TP were assessed for their anti-acetylcholinesterase (AChE) and antioxidant activities. AChE inhibition was estimated using spectrophotometric method of Ellman. Antioxidant activity was evaluated by 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and ferrous ion-chelating power tests. Ferric-reducing antioxidant power (FRAP) of CN and TP were also tested. CN essential oil was found to contain isomenthone (56.21%) and pulegone (19.76%). The ethyl acetate (83.11–87.98%) and dichloromethane (73.45–84.02%) extracts of CN showed the highest AChE inhibition. The ethyl acetate and ethanol extracts of TP exerted significant DPPH scavenger effect. The water extracts of CN and TP and the chloroform extract of the aerial parts of EPU displayed the highest ferrous ion-chelating effect. The leaf and flower essential oils of TP had the best FRAP.     

Determination of kava lactones and flavonoid glycoside in Scorzonera austriaca by capillary zone electrophoresis

A capillary zone electrophoretic method has been developed for the quantitative analysis of three active comppounds, 12-hydroxy-desmethoxyyangonin (HD), 12-beta-d-glucopyranoside-desmethoxyyangonin (GD) and luteolin 3'-(6-E-p-coumaroyl-beta-d-glucopyranoside) (LG) in Scorzonera austriaca with UV detection at 254 nm. The applied voltage was 25 kV and the capillary temperature was kept constant at 25 degrees C. The effect of buffer pH, the concentration of electrolyte and organic modifier on migration were studied systematically. Optimum separation was achieved with 20 mM borate buffer at pH 10.00 containing 10% (v/v) methanol. Daphnetin was used as internal standard for quantification. Regression equations revealed good linear relationship between the ratios of the peak area of each compound and its the ratios of concentration. All the correlation coefficients were higher than 0.9990. The relative standard deviations of migration time and the peak area were <1.46% and 5.13% (inter-day), and <1.65% and 5.16% (intra-day), respectively. The contents of the three compounds in S. austriaca were successfully determined with satisfactory repeatability and recovery.     

A method for the determination of minoxidil in hair-regrowth formulations by micellar electrokinetic capillary chromatography

A method based on micellar electrokinetic capillary chromatography (MEKC) was developed for determination of minoxidil in Rogaine and competing products. The original intent of the work was to offer an orthogonal means to HPLC for testing illicit imitations of Rogaine. However, because the patent has since expired, we offer the procedure as a confirmatory measure to HPLC for assay of generic minoxidil products. The MEKC procedure complements an earlier method based on free solution capillary electrophoresis (FSCE), designed to the same end. Validation was carried out on both a Dionex CES-1, which utilizes gravity injection, and a PE-ABI 270HT, which employs vacuum injection. The procedure was validated for both active pharmaceutical ingredient and for minoxidil solutions. The run buffer is pH 7.0, 20 mM sodium phosphate, 20 mM sodium dodecyl sulfate, with 10% isopropanol; the internal standard is dl-tryptophan. The method bears the attributes of simplicity, ease of use, and short analysis time (12 min). It is selective with respect to known process and degradation impurities. High efficiency was achieved on the CES-1, with a plate count exceeding 200,000 for minoxidil at an elution time of 9 min. Although slight differences in performance were noted across the two instruments, results on both were in conformance with modern day validation expectations. Comparison of MEKC with HPLC resulted in slightly higher values for the former, but all results met registration specifications and internal targets.     

Detection of monofluoroacetate in Palicourea and Amorimia species

Numerous plant species worldwide including Palicourea marcgravii and Tanaecium bilabiatum in Brazil cause sudden death and are known to contain monofluoroacetate (MFA). Other species in Brazil including some species traditionally assigned to Mascagnia but now properly called Amorimia species and other Palicourea species are reported to cause sudden death in livestock and are suspected to contain MFA due to the similarity of clinical signs. In this study, an HPLC-APCI-MS method to detect and quantify MFA was developed and was used to investigate plant material from field collections and/or herbarium specimens of Mascagnia, Amorimia, and Palicourea species suspected of causing sudden death. MFA was detected in Amorimia amazonica, Amorimia camporum, Amorimia exotropica, Amorimia pubiflora, Amorimia rigida, and Amorimia septentrionalis as well as Palicourea aeneofusca. MFA concentrations differ greatly between Palicourea species and Amorimia species, which may explain the incidence of poisoning and the amount of plant material required to cause sudden death between these taxa.     

Separation and determination of nimesulide related substances for quality control purposes by micellar electrokinetic chromatography

A micellar electrokinetic chromatography (MEKC) method has been developed and validated for the determination of nimesulide related compounds in pharmaceutical formulations. Electrophoretic separation of six European Pharmacopoeia (EP) impurities (A–F) was performed using a fused silica capillary (L_eff. = 50 cm, L_tot. = 57 cm, 50 μm i.d.) with a background electrolyte (BGE) containing 25 mM borate buffer (pH 9.5), 30 mM sodium dodecyl sulphate and φ = 3% (v/v) acetonitrile. The influence of several factors (surfactant and buffer concentration, pH, organic modifier, applied voltage, capillary temperature and injection time) was studied. The method was suitably validated with respect to linearity, limit of detection and quantification, accuracy, precision and selectivity. The calibration curves obtained for the six compounds were linear over the range 5–12 μg ml⁻¹ (0.05–0.12%). The relative standard deviations (s_r) of intra- and inter-day experiments were less than 5.0%. The detection limits ranged between 0.7 and 1.6 μg ml⁻¹ depending on the impurity. The proposed method was applied successfully to the quantification of nimesulide impurities in its pharmaceutical formulation.     

气质联用法分析野西瓜果实的化学成分

目的 采用气质联用法(GC-MS)对刺山柑(Capparis spinosa)果实的化学成分进行分析。 方法 野西瓜药材经粉碎破壳、70%乙醇回流提取、石油醚(沸程60~90 ℃)萃取后,采用VF-5ms石英毛细管柱(30 m×0.25 mm,0.25 μm)进行气相色谱分析;柱温从80 ℃开始,保持2 min,以15 ℃/min的速度升到300 ℃,并保持10 min,气化温度为250 ℃;载气为高纯度氦气,流量为1.0 ml/min;质谱检测器为EI离子源,电子能量70 eV,离子源温度200 ℃,分流比30:1,进样量为1.0 μl。各成分的相对含量分析应用色谱峰面积归一法。 结果 共检测出53个峰,确认出其中48种成分,含量较高的物质有棕榈酸21.82%,硬脂酸7.49%,油酸42.93%,甘油单油酸酯2.39%,维生素E 1.67%等。化合物的类型主要为饱和脂肪酸酯、不饱和脂肪酸和烃类化合物。 结论 通过气质联用技术,为鉴定野西瓜果实中的化学成分建立起了一种快速、准确、灵敏的分析方法。     

Metabolic profiling and biological capacity of Pieris brassicae fed with kale (Brassica oleracea L. var. acephala)

Phenolic and organic acid profiles of aqueous extracts from Pieris brassicae material and the host kale (Brassica oleracea L. var. acephala) leaves were determined by HPLC/UV–DAD/MSⁿ-ESI and HPLC–UV, respectively. The identified phenolics included acylated and nonacylated flavonoid glycosides, hydroxycinnamic acyl gentiobiosides, and sulphate phenolics. Kale exhibited the highest content (11 g/kg lyophilized extract), while no phenolics were identified in the butterflies or exuviae. Nine different organic acids were characterized in the materials, with kale showing the highest amount (112 g/kg lyophilized extract). With the exception of the exuviae extract, the rest were screened for bioactivity. Using spectrophotometric microassays, all exhibited antiradical capacity against DPPH and NO in a concentration-dependent way, whereas only kale and excrement extracts were active against superoxide. All displayed activity on intestinal smooth muscle, albeit with distinct relaxation–contraction profiles. Larvae and butterfly extracts were more efficacious for intestinal relaxation than was kale extract, whereas excrement extract evoked only contractions, thus evidencing their different compositions. Collectively, these results show that P. brassicae sequesters and metabolizes kale’s phenolic compounds. Moreover, the extract’s bioactivities suggest that they may constitute an interesting source of bioactive compounds whose complex chemical structures preclude either synthesis or isolation.     

Application of simple on-line sweeping sample concentration technique coupled micellar electrokinetic chromatography for simultaneous analysis of estrogen and androgen epimer

A reliable, convenient, and sensitive on-line sweeping-MEKC sample concentration technique has been applied for the simultaneous separation of six steroids including two pairs of epimer with 10 mM phosphate buffer (pH 7.0) that contains 80 mM sodium dodecyl sulfate (SDS), 14 mM β-cyclodextrin (β-CD), and 4% (v/v) methanol. The column length was 105 cm (effective length, 90 cm). Samples were hydrostatically injected for 600 s. The separation was performed at ambient temperature under an applied voltage of 25 kV. The external standard calibration curves of the six steroidal hormones proved good linearity (r² = 0.9785-0.9941) within the concentration range 0.025-1.0 μg mL⁻¹. The limit of detections of the on-line sweeping-MEKC with the ultraviolet detector at 220 nm for estrone, α-estradiol, β-estradiol, androstenedione, epitestosterone, and testosterone were 10, 24, 28, 53, 73, and 11 ng mL⁻¹ and were 240, 125, 93, 47, 32, and 200 times more sensitive than MEKC, respectively. The Saccharomyces cerevisiae mediated simultaneous stereoselective reduction of estrone and androstenedione exhibited a 100% stereoselectivity toward β-estradiol and testosterone. The accuracy and precision achieved for the spiking experiments of the sweeping-MEKC were 95-98% and less than 3.8% (RSD), respectively.     

Determination of voriconazole in human serum and plasma by micellar electrokinetic chromatography

The micellar electrokinetic capillary chromatography (MEKC) separation and analysis of voriconazole and UK 115794 (internal standard) were examined and an assay for determination of voriconazole in human plasma and serum was developed. The MEKC medium comprises a 2:15 (v/v) mixture of methanol and a pH 9.3 buffer composed of 5 mM Na₂B₄O₇, 7 mM Na₂HPO₄ and 54 mM SDS. Sample preparation is based upon liquid/liquid extraction with ethylacetate and dichloromethane (75%/25%) at physiological pH. Using this approach with 250 μl serum or plasma and reconstitution of the dried extract into 100 μl of a buffer composed of 0.5 mM Na₂B₄O₇ and 0.7 mM Na₂HPO₄ (pH 9.3), the detection and quantitation limits were determined to be 0.1 and 0.2 μg/ml, respectively, a sensitivity that is suitable for therapeutic drug monitoring of voriconazole (provisional therapeutic range: 1–6 μg/ml) in human plasma and serum samples. The method was validated and compared to an HPLC method, showing excellent agreement between the two for a set of 91 samples that stemmed from patients being treated with voriconazole. The MEKC assay is also demonstrated to be suitable to explore pharmacokinetic data of voriconazole.     

Determination of patulin in commercial apple juice by micellar electrokinetic chromatography.

A novel and validated micellar electrokinetic capillary chromatography (MEKC) method using ultraviolet detection (UV) has been applied to the quantitative analysis of patulin (PAT) in commercial apple juice. Patulin was extracted from samples with an ethylacetate solution. The micellar electrokinetic capillary chromatography (MECK) parameters studied for method optimization were buffer composition, voltage, temperature, and a separation between PAT and 5-hydroxymethylfurfural (HMF) (main interference in apple juice PAT analysis) peaks until reaching baseline. The method passes a series of validation tests including selectivity, linearity, limit of detection and quantification (0.7 and 2.5 microgL(-1), respectively), precision (within and between-day variability) and recovery (80.2% RSD=4%), accuracy, and robustness. This method was successfully applied to the measurement of 20 apple juice samples obtained from different supermarkets. One hundred percent of the samples were contaminated with a level greater than the limit of detection, with mean and median values of 41.3 and 35.7 microgL(-1), respectively.     

毛果枳椇子的黄酮类成分研究

目的 研究毛果枳椇子的黄酮类成分.方法 用硅胶柱色谱、ODS柱色谱、Sephadex LH-20柱色谱、高效液相色谱法进行分离纯化,依据理化性质和光谱数据鉴定化合物结构.结果 从毛果枳椇子的70％乙醇提取物中分离鉴定了9种黄酮类化合物,分别为:槲皮素(quecetin,1)、杨梅素(myricitin,2)、双氢槲皮素(dihydroquercetin,3)、(2R,3R)-二氢杨梅素[(2R,3R)-dihydromyricitin,4]、(-)-没食子儿茶素[(-)-gallocatechin,5]、当药黄素(swertish,6)、牡荆素2〃-O-β-D-葡萄糖苷(vitexin 2〃-O-β-D-glucopyranoside,7)、斯皮诺素(spinosin,8)、异牡荆素2〃-O-3-D-葡萄糖苷(isovitexin 2〃-O-β-D-glucopyranoside,9).结论 化合物1,3,5～9为首次从毛果枳椇子中分离得到.     

Toxicity testing of saponin-containing Yucca schidigera Roetzl. juice in relation to hepato- and nephrotoxicity of Narthecium ossifragum (L.) Huds

Yucca schidigera juice in doses of 1.5 g (63 mg sapogenin) and 3.0 g (126 mg sapogenin) per kg live weight was administered intraruminally to 30 lambs for 21 days to investigate whether the saponins in Y. schidigera were toxic to lambs and whether they could cause hepatogenous photosensitisation. Twelve lambs died or had to be euthanised. The main pathological findings in the diseased lambs were acute tubular necrosis in the kidneys, dehydration and watery content in the gastrointestinal tract. Fifteen lambs were euthanised at the end of the study, and the main pathological findings in dosed animals were accumulation of homogeneous pale PAS-positive material in the hepatocytes. There was a rise in serum creatinine and urea concentrations in the lambs with renal lesions the day before they died. Major Y. schidigera-related saponins were found in the liver and kidney samples from all lambs that were dosed with Y. schidigera juice. The results of the present study demonstrate that un-hydrolysed saponins can be absorbed from the gastrointestinal tract. The possible role of saponins in causing nephrotoxicity is discussed.