HPLC-DAD-MS/MS-ESI screening of phenolic compounds in Pieris brassicae L. Reared on Brassica rapa var. rapa L

The phenolic profiles of cabbage white butterfly ( Pieris brassicae L.; Lepidoptera: Pieridae) at different development stages (larvae, exuviae, and butterfly), its excrements, and its host plant Brassica rapa var. rapa L. were determined by high performance liquid chromatography- diode-array detector-mass spectrometry/mass spectrometry-electrospray ionization (HPLC-DAD-MS/MS-ESI). Twenty-five acylated and nonacylated flavonoid glycosides and ferulic and sinapic acids were identified in host plant, from which only 12 compounds were found in the excrements. In addition, the excrements showed the presence of sulfate flavonoids and other flavonoid glycosides that were not detected in the leaves. In the larvae kept without food for 12 h, only 3 compounds common to the plant material and 2 others, also present in the excrements, were characterized. The results indicate that deacylation, deglycosylation, and sulfating steps are involved in the metabolic process of P. brassicae and that its excrements may constitute a promising source of bioactive compounds, which could be used to take profit of this common pest of Brassica cultures.     

Hydrolyzable tannins with the hexahydroxydiphenoyl unit and the m-depsidic link: HPLC-DAD-MS identification and model synthesis

This study was designed to develop efficient analytical tools for the difficult HPLC-DAD-MS identification of hydrolyzable tannins in natural tissue extracts. Throughout the study of the spectroscopic characteristics of properly synthesized stereodefined standards, it was observed that the UV-vis spectra of compounds with the m-depsidic link showed a characteristic shoulder at 300 nm, consistent with the simple glucogalloyl esters, whereas compounds with the hexahydroxydiphenoyl (HHDP) unit gave a diagnostic fragmentation pattern, caused by a spontaneous lactonization in the mass spectrometer. These observations were confirmed by HPLC-DAD-MS analyses of tannic acid and raspberry extracts, which are rich in hydrolyzable tannins with the m-depsidic link and the HHDP unit, respectively.     

Identification and quantification of coumarins in Peucedanum ostruthium (L.) Koch by HPLC-DAD and HPLC-DAD-MS

The rhizomes of Peucedanum ostruthium (L.) Koch (masterwort) are traditionally used in the alpine region as ingredient of liqueurs and bitters, and as a herbal drug. A sensitive and specific high-performance liquid chromatography-diode-array detection-mass spectrometry (HPLC-DAD-MS) method has been developed for the simultaneous identification and quantification of its main coumarins, oxypeucedanin hydrate, oxypeucedanin, ostruthol, imperatorin, osthole, isoimperatorin, and ostruthin. Fast HPLC separation could be achieved on an Acclaim C18 column (150 mm × 2.1 mm i.d., 3 μm) using a mobile phase gradient of acetonitrile-water modified with 0.01% acetic acid. The quantification by HPLC-DAD was performed with imperatorin as external standard and validated to demonstrate selectivity, linearity, precision, and accuracy. The content of the main coumarins was quantitated in various batches of commercial and field-collected rhizomes of Peucedanum ostruthium, as well as in beverages prepared thereof.     

Rapid HPLC screening of jasmonate-induced increases in tobacco alkaloids, phenolics, and diterpene glycosides in Nicotiana attenuata

A rapid, HPLC-based screening procedure for the main classes of secondary metabolites in Nicotiana attenuata leaves (alkaloids, phenolics, and diterpene glycosides) is reported. In a single step, leaves are extracted in aqueous acidified (0.5% acetic acid) methanol, and the extracted compounds are separated by reversed-phase HPLC with an acidic water/acetonitrile gradient in <30 min. The utility of the method in quantifying changes in the secondary metabolites after methyl jasmonate treatment of the plants, a treatment known to elicit resistance to herbivores in nature, is illustrated. Methyl jasmonate treatment elicited dramatic increases in some secondary metabolites (caffeoylputrescine, nicotine, and diterpene glycosides increased 12.5-, 1.4-, and 1.9-fold, respectively) but left others, such as rutin, unchanged. Such broad-based analytical screens will help characterize environmental and genetic changes in secondary metabolite profiles.     

New C-deoxyhexosyl flavones and antioxidant properties of Passiflora edulis leaf extract

The flavonoids present in passion fruit (Passiflora edulis) leaves were identified by a high-performance liquid chromatography-diode array detection-tandem mass spectrometry (HPLC-DAD-MS/MS) method. Sixteen apigenin or luteolin derivatives were characterized, which included four mono-C-glycosyl, eight O-glycosyl- C-glycosyl, and four O-glycosyl derivatives. With the exceptions of C-hexosyl luteolin and C-hexosyl apigenin, all the compounds exhibited a deoxyhexose moiety. Moreover, the uncommon C-deoxyhexosyl derivatives of luteolin and apigenin have been identified for first time in P. edulis by HPLC-DAD-MS/MS. The antioxidative capacity of passion fruit leaves was checked against DPPH radical and several reactive oxygen species (superoxide radical, hydroxyl radical, and hypochlorous acid), revealing it to be concentration-dependent, although a pro-oxidant effect was noticed for hydroxyl radical.     

A comparative study of flavonoid compounds, vitamin C, and antioxidant properties of baby leaf Brassicaceae species

A comparative study of antioxidant compounds, flavonoids and vitamin C, and also antioxidant activity was carried out in four species of Brassicaceae vegetables used for salads: watercress ( Nasturtium officinale R. Br.), mizuna [ Brassica rapa L. subsp. nipposinica (L.H. Bailey) Haneltand], wild rocket [ Diplotaxis tenuifolia (L.) DC.], and salad rocket [ Eruca vesicaria (L.) Cav.]. The characterization of individual phenolic compounds by HPLC-DAD-MS/MS-ESI in watercress and mizuna completes the polyphenol study previously reported for wild rocket and salad rocket. The qualitative study of flavonoids in watercress leaves showed a characteristic glycosylation pattern with rhamnose at the 7 position. Isorhamnetin 3,7-di- O-glucoside was identified in mizuna leaves and may be considered a chemotaxonomical marker in some B. rapa subspecies. Brassicaceae species showed differences in the quantitative study of flavonoids, and the highest content was detected in watercress leaves. Watercress and wild rocket leaves had the highest content of vitamin C. The antioxidant activity evaluated by different methods (ABTS, DPPH, and FRAP assays) showed a high correlation level with the content of polyphenols and vitamin C. In conclusion, the Brassicaceae leaves studied, watercress, mizuna, wild rocket, and salad rocket, presented a large variability in the composition and content of antioxidant compounds. These baby leaf species are good dietary sources of antioxidants with an important variability of bioactive compounds.     

Optimization, in-house validation, and application of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for the quantification of selected polyphenolic compounds in leaves of grapevine (Vitis vinifera L.)

Polyphenols in grapevine can be constitutive or induced, depending upon cultivar, plant organ, and environmental influences. The aim of the presented work was to develop and optimize a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to study the pattern and amount of selected polyphenols in leaves of Vitis vinifera L. The method is simple and does not require any sample cleanup. It covers representative metabolites of the structure classes cinnamic acids, flavonoids, and stilbenes and enables the simultaneous separation and quantification of 13 polyphenols within 9 min at concentration levels between 0.1 and 3 μg/g. We present the method performance characteristics and its application to the quantification of polyphenols in grapevine leaves of the cultivars Riesling and Pinot noir. A total of 7 of 13 target polyphenols were detected at concentrations above the limits of quantification. Interestingly, instead of the expected trans-resveratrol, the investigated leaf samples of both cultivars contained cis-resveratrol-3-O-glucoside. The measurements also showed that Riesling leaves tended to contain higher concentrations of the selected polyphenols than Pinot noir. In view of its intended future use, the developed method has been shown to be a powerful and fast tool to study polyphenols in grapevine leaves subjected to environmental stress conditions.     

Formation and accumulation of alpha-acids,beta-acids,desmethylxanthohumol, and xanthohumol during flowering of hops (Humulus lupulus L.)

Important secondary metabolites, present in hops (Humulus lupulus L.), include alpha-acids and beta-acids, which are essential for the brewing of beer, as well as the prenylated chalcones, desmethylxanthohumol, and xanthohumol, which exhibit interesting bioactive properties. Their formation and accumulation in five selected hop varieties, Wye Challenger, Wye Target, Golding, Admiral, and Whitbread Golding Variety, were quantitatively monitored by high-performance liquid chromatography using UV detection. All target compounds were present from the onset of flowering, not only in female hop cones but also in male inflorescences, albeit in low concentrations. During development from female inflorescences to cones, levels of alpha-acids, beta-acids, desmethylxanthohumol, and xanthohumol gradually increased, while each hop variety exhibited individual accumulation rates. Furthermore, these compounds were present in leaves of fully grown hops as well. The study demonstrated that key compounds for flavor and potential beneficial health effects associated with beer not only reside in the glandular lupulin structures but also are distributed over various parts of the hop plant.     

Identification of hydroxycinnamoylquinic acids of arnica flowers and burdock roots using a standardized LC-DAD-ESI/MS profiling method

A screening method using LC-DAD-ESI/MS was developed for the identification of common hydroxycinnamoylquinic acids based on direct comparison with standards. A complete standard set for mono-, di-, and tricaffeoylquinic isomers was assembled from commercially available standards, positively identified compounds in common plants (artichokes, asparagus, coffee bean, honeysuckle flowers, sweet potato, and Vernonia amygdalina leaves) and chemically modified standards. Four C18 reversed phase columns were tested using the standardized profiling method (based on LC-DAD-ESI/MS) for 30 phenolic compounds, and their elution order and retention times were evaluated. Using only two columns under standardized LC condition and the collected phenolic compound database, it was possible to separate all of the hydroxycinnamoylquinic acid conjugates and to identify 28 and 18 hydroxycinnamoylquinic acids in arnica flowers (Arnica montana L.) and burdock roots (Arctium lappa L.), respectively. Of these, 22 are reported for the first time.     

Further knowledge on the phenolic profile of Colocasia esculenta (L.) Shott

Colocasia esculenta (L.) Shott, commonly called taro, is an ancient species selected for its edible tuber. Its huge "elephant ear" like leaves are also consumed in sauces and stews or as soups. Forty-one phenolic metabolites (11 hydroxycinnamic acid derivatives and 30 glycosylated flavonoids) were identified by high-performance liquid chromatography-diode array detection-electrospray ionization/mass spectrometry (HPLC-DAD-ESI/MS(n)) in the leaves of two C. esculenta varieties cultivated in Azores Islands. To our knowledge, 34 of the 41 phenolic compounds are being reported for the first time in this species. Phenolics quantification was achieved by an HPLC-DAD accurate and sensitive validated method. Although the qualitative profile of the two varieties is quite similar, quantitative differences were observed between them. "Giant white" and "red" varieties (local denomination) contain, respectively, ca. 14 and 21% of phenolic acids, 37 and 28% of flavones mono-C-glycosides, 42 and 43% of flavones di-C-glycosides, 3 and 4% of flavones mono-C-(O-glycosyl)glycosides, and both of them ca. 2% of flavones di-C-(O-glycosyl)glycosides and 2% of flavones-O-glycosides. Luteolin-6-C-hexoside was the compound present in higher amounts in both varieties. The established phenolic profile is an added value for the authenticity and quality control of C. esculenta and may be useful in the discrimination of its varieties.     

Identification of (poly)phenolic compounds in concord grape juice and their metabolites in human plasma and urine after juice consumption

Analysis of Concord grape juice by HPLC with ESI-MS(n), PDA, and fluorescence detection resulted in the identification and quantification of 60 flavonoids and related phenolic compounds, which were present at an overall concentration of 1508 ± 31 μmol/L. A total of 25 anthocyanins were detected, which were mono- and di-O-glucosides, O-acetylglucosides, O-p-coumaroyl-O-diglucosides, and O-p-coumaroylglucosides of delphinidin, cyanidin, petunidin, peonidin, and malvidin. The anthocyanins represented 46% of the total phenolic content of the juice (680 μmol/L). Tartaric esters of hydroxycinnamic acids, namely, trans-caftaric and trans-coutaric acids, and to a lesser extent trans-fertaric acid accounted for 29% of the phenolic content, with a total concentration of 444 μmol/L, of which 85% comprised trans-caftaric acid. Free hydroxycinnamic acids were also quantified but contributed to <1% of the total phenolic content (8.4 μmol/L). The other groups of polyphenolic compounds present in the juice, accounting for 24% of the total, comprised monomeric and oligomeric units of (epi)catechin and (epi)gallocatechin (248 μmol/L), flavonols (76 μmol/L), gallic acid (51 μmol/L), and trans-resveratrol (1.5 μmol/L). The bioavailability of the (poly)phenolic compounds in 350 mL of juice was investigated following acute intake by healthy volunteers. Plasma and urine were collected over 0-24 h and analyzed for parent compounds and metabolites. In total, 41 compounds, principally metabolites, were identified.     

陆地棉法尼基焦磷酸合酶基因的克隆及表达分析

棉酚是棉花重要的次生代谢产物之一,具有抗虫及抗病害的作用。法尼基焦磷酸合酶是杜松烯合酶的上游酶,催化合成法尼基焦磷酸,该物质是棉酚等次生代谢物生物合成的前体。本文依据亚洲棉法尼基焦磷酸合酶基因序列,采用同源RT-PCR技术,克隆了陆地棉法尼基焦磷酸合酶基因,对其序列和编码产物结构进行了分析。荧光定量PCR分析表明,该基因在棉花植株叶片表达量最高,根部最低,在花、铃、蕾、茎中的表达水平接近。以上工作为棉花异戊二烯基途径的分析及相关次生代谢的途径工程研究奠定了一定的理论基础。     

Polyphenols as chemotaxonomic markers in Italian "long-storage" tomato genotypes

"Long-storage" tomato ( Solanum lycopersicum L.) is a niche product typical of the Mediterranean area, traditionally cultivated under no water supply, the fruits of which combine a good taste with excellent nutritional properties. High-performance liquid chromatography coupled with diode array detection and electron spray-mass spectrometry (HPLC/DAD/ESI-MS) was used to identify the phenolic profile in 10 landraces of long-storage tomato, grown under a typical semiarid climate, as compared to a processing tomato hybrid cultivated in the same environment, under both well-irrigated and unirrigated conditions. Sixteen different secondary metabolites, belonging to the classes of cinnamoylquinic acids and flavonoids, were identified. Quantitative analyses were also performed to monitor the changes in the phenolic content along the batch. The results highlighted that landraces originating from the same area exhibit different fruit morphologies but own a similar biochemical profile. Moreover, the two controls (well irrigated and unirrigated) are placed into the same cluster, suggesting that these secondary metabolites in tomato fruits may be more genetics-dependent than environment-dependent. Given the analysis of phenols nowadays represents a useful tool to assess the genetic variability in tomato, these compounds could be adopted as chemotaxonomic markers in the traceability of this niche product.     

Antioxidant Activity and Characterization of Phenolic Compounds from Bacaba ( Oenocarpus bacaba Mart.) Fruit by HPLC-DAD-MS(n)

The phytochemicals in fruits have been shown to be major bioactive compounds with regard to health benefits. Bacaba ( Oenocarpus bacaba Mart.) is a native palm fruit from the Brazilian savannah and Amazon rainforest that plays an important role in the diet of rural communities and is also a source of income for poor people. This paper reports the characterization and analyses of phenolics from bacaba fruit extract. The total phenolic content of bacaba fruit amounted to 1759.27 ± 1.01 mg GAE/100 g, the flavonoid content was 1134.32 ± 0.03 mg CTE/100 g, and the anthocyanin content was 34.69 ± 0.00 mg cyn-3-glc/100 g. The antioxidant activity was evaluated through different assays [ORAC, FRAP, DPPH, TEAC, and cellular antioxidant assay (CAA) assays] and revealed a significant antioxidant capacity for bacaba in comparison to the data available in the literature. The assignment of the phenolic compounds using HPLC-DAD-MS(n) was based on the evaluation of their UV-vis absorption maxima (λ(max)) and mass spectral analyses, and 14 compounds were tentatively identified. The results suggest that bacaba fruits are a promising source of phenolics.     

Phenolic acids in foods: an overview of analytical methodology

Phenolic acids are aromatic secondary plant metabolites, widely spread throughout the plant kingdom. Existing analytical methods for phenolic acids originated from interest in their biological roles as secondary metabolites and from their roles in food quality and their organoleptic properties. Recent interest in phenolic acids stems from their potential protective role, through ingestion of fruits and vegetables, against oxidative damage diseases (coronary heart disease, stroke, and cancers). High performance liquid chromatography (HPLC) as well as gas chromatography (GC) are the two separation techniques reviewed. Extraction from plant matrixes and cleavage reactions through hydrolysis (acidic, basic, and enzymatic) are discussed as are the derivatization reagents used in sample preparation for GC. Detection systems discussed include UV-Vis spectroscopy, mass spectrometry, electrochemical, and fluorometric detection. The most common tandem techniques are HPLC/UV and GC/MS, yet LC/MS is becoming more common. The masses and MS fragmentation patterns of phenolic acids are discussed and tabulated as are the UV absorption maxima.     

Physiological effects of extraction juices from apple, grape, and red beet pomaces in rats

In comparison to classical fruit juice processing, polyphenols and dietary fiber can be extracted from pomace by means of pectinases and cellulases. In the present study, rats were fed with such produced extraction juices from apples, grapes, and red beets as drinking fluids instead of water for 4 weeks to evaluate their physiological effects. In all test groups, the intake of extraction juices was greater as compared to control (water intake), resulting in a higher urine excretion. In the apple and grape group, pH values in feces was lower than control. Administration of extraction juices from apples increased fecal counts of Lactobacillus and Bifidobacterium. More acetate and total short-chain fatty acids appeared in intestinal contents of the apple and red beet group. Furthermore, the intestinal contents of test groups contained higher concentrations of primary bile acids, cholesterol, and cholesterol metabolites but lower concentrations of secondary bile acids. The total amount of steroids excreted by these groups was also greater than control. Quercetin and isorhamnetin appeared in urine of rats fed extraction juices from apples and grapes; in urine of the former group, phloretin was found also. Administration of the extraction juices, enriched in secondary plant metabolites and dietary fiber, resulted in beneficial nutritional effects in rats.     

Natural abundance of 15N in nitrate,ureides, and amino acids from plant tissues

The natural ¹⁵N abundances (δ¹⁵N values) were measured for nitrate and free and bound amino acids from the leaves of field-grown spinach (Spinacia oleracea L.) and komatsuna (Brassica campestris L.), as well as ureides and free and bound amino acids in the leaves and roots of hydroponically grown soybean (Glycine max L.) totally depending on dinitrogen. Nitrate from the spinach and komatsuna leaves and ureides from leaves and roots of soybean showed higher δ¹⁵N values than the total tissue N and N in free or bound amino acid fractions. The δ¹⁵N values of individual free and bound amino acids, determined by GC/C/MS using their acetylpropyl derivatives, were similar in leaf tissues except for proline but varied in soybean root tissues. The order of ¹⁵N enrichment was similar in the four samples: aspartic acid > glutamic acid > threonine, proline, valine > glycine + alanine +serine, γ-amino butyric acid, and phenylalanine.     

超声波微波协同提取沙棘叶黄酮及其组成和活性研究

沙棘叶中富含黄酮类化合物等多种活性成分,为提高其提取率和利用率,本研究采用常规溶剂萃取、超声辅助、微波辅助、超声波微波协同提取4种方法对沙棘叶黄酮进行提取,测定沙棘叶黄酮得率并观察沙棘叶的微观组织结构,比较筛选沙棘叶黄酮的最佳提取方法。并采用响应面法对最佳提取方法进行工艺优化,同时测定沙棘叶黄酮组成和体外抗氧化活性。结果表明,超声波微波协同提取法是提取沙棘叶黄酮的最佳方法,黄酮得率较常规溶剂萃取法提高了42.54%(P<0.05),沙棘叶细胞损伤最严重。超声波微波协同提取沙棘叶黄酮的最佳工艺为乙醇体积分数61%、提取时间18 min、微波功率446 W,此时黄酮得率为42.09 mg·g^-1。沙棘叶黄酮提取液中共鉴定出6种黄酮类成分,分别为儿茶素、丁香酸、山萘酚、槲皮素、异鼠李素、杨梅素,其中儿茶素含量最高,为1.474 8 mg·g^-1,其余5种的含量均在0.1~0.3 mg·g^-1之间,山萘酚最低,为0.125 2 mg·g^-1;沙棘叶黄酮提取液具有较强的还原力及较高的1,1-二苯基-2-三硝基苯肼(DPPH)、2,2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)和羟基自由基清除率,抗氧化活性较高。本研究为沙棘叶黄酮的工业化生产提供了科学依据。     

Chemical characterization of Cuban propolis by HPLC-PDA, HPLC-MS, and NMR: the brown, red, and yellow Cuban varieties of propolis

Sixty-five samples of propolis were collected from eleven regions of Cuba; methanolic extracts of propolis were prepared from all samples, and a classification method was developed using a combination of NMR, HPLC-PDA, and HPLC-ESI/MS techniques. The analysis of (1)H and (13)C NMR spectra and chromatographic profiles of all propolis extracts allowed the definition of three main types of Cuban propolis directly related to their secondary metabolite classes: brown Cuban propolis (BCP), rich in polyisoprenylated benzophenones, red Cuban propolis (RCP), containing isoflavonoids as the main constituents, and yellow Cuban propolis (YCP), probably with aliphatic compounds. Subsequently, the principal compounds of the brown and red types were characterized by HPLC-ESI/MS analysis. Instrumental techniques used are complementary; NMR was shown to be a quick and informative tool for the rapid analysis of crude propolis polar extracts and allowed the identification of the main class of secondary metabolites, while LC-PDA and LC-MS techniques were useful tools for qualitative and quantitative analysis of marker compounds of Cuban propolis.