Quantitative analysis of triterpenoids in different parts of Ilex hainanensis, Ilex stewardii and Ilex pubescens using HPLC–ELSD and HPLC–MS and antibacterial activity

An HPLC–ELSD method was first developed for the quantitative analysis of principle triterpenoids in Ilex hainanensis, Ilex stewardii and Ilex pubescens. The established method, with excellent precision, repeatability and recovery, was successfully applied to determine four triterpenoids in 24 samples from different species and medicinal parts of Ilex, and the changing trend was discussed. HPLC–ESI–MSⁿ was used for the identification of constituents in samples. The proposed method was simple, effective and suitable for investigations of these plants. Furthermore, the ethanol extracts from leaves of Ilex species, as well as their main components, were assessed for their antibacterial activities. The results indicated that the extracts of I. hainanensis, I. stewardii and I. pubescens could inhibit the growth of the tested oral pathogens, Streptococcus mutans and Actinomyces viscosu. Particularly, ilexgenin A was the most effective with MIC values of 7.8 and ?3.9 μg/ml, respectively.     

On-line HPLC–MS–DPPH assay for the analysis of phenolic antioxidant compounds in fruit wine: Antidesma thwaitesianum Muell.

A reversed phase high performance liquid chromatography (RP-HPLC) separation coupled with an electrospray ionisation mass spectrometry detection (ESI-MS) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was used for the screening of multiple antioxidant compounds in Antidesma thwaitesianum Muell. fruit wine. The active compounds were identified by comparison with authentic standards and published mass spectra. With the help of the multidimensional information of LC–ESI-MS/MS and DPPH assay, the compounds with different chemical structures could be determined in one run successfully. The antioxidant compounds were separated and identified as gallic acid, cyanidin-3-sophoroside, monogalloyl glucoside, delphinidin-3-sambubioside, catechin, caffeic acid, and pelargonidin-3-malonyl glucoside. This result shows that an on-line HPLC–MS–DPPH assay can be a powerful technique for the rapid characterisation of antioxidant compounds in plant extracts.     

Flavone-di-C-glycosides in citrus juices from Southern Italy

6,8-Di-C-glucopyranosylapigenin and 6,8-di-C-glucopyranosyldiosmetin were detected by HPLC–DAD–ESI–MS–MS in a variety of Southern Italian Citrus juices (orange, lemon, bergamot, citron, mandarin, clementine). In some juices, the amounts of C-glycosides detected were significant. 6,8-Di-C-glucopyranosylapigenin is characteristic of orange juice, while 6,8-di-C-glucopyranosyldiosmetin is the most important C-glycoside in lemon and citron juice. In bergamot juice the concentrations of 6,8-di-C-glucopyranosylapigenin and 6,8-di-C-glucopyranosyldiosmetin are similar. Clementine juice is distinctive as the amounts of both C-glycosides present are negligible.     

Organic acids,antioxidant capacity,phenolic content and lipid characterisation of Georgia-grown underutilized fruit crops

Four underutilized Georgia-grown fruit crops, namely loquat (Eriobotrya japonica), mayhaw (Crataegus sp.), fig (Ficus carica), and pawpaw (Asimina triloba), and their leaves were analysed for total polyphenols by Folin–Ciocalteau method, and antioxidant capacity by ferric-reducing antioxidant power (FRAP) and Trolox-equivalent antioxidant capacity (TEAC) assays. Organic acids and phenolic compounds were identified by RP-HPLC. For lipid profile, fruits were separated into two fractions – seed and fruit (i.e., without seed); lipid was extracted using the Folch method and analysed for fatty acids, phytosterols, tocopherols, and phospholipids. The major organic acid identified in all samples was malic acid (177–1918 mg/100 g FW). The predominant phenolic acids in all the fruits were gallic (1.5–6.4 mg/100 g FW) and ellagic (0.2–33.8 mg/100 g FW), and the most abundant flavonoid was catechin (12.2–37.8 mg/100 g FW). Total lipid content varied from 0.1% in mayhaw fruit to 21.5% in pawpaw seed. Linoleic acid was the predominant fatty acid in all of the samples (28.2–55.7%).     

Comprehensive analysis of Phyteuma orbiculare L., a wild Alpine food plant

Plants which have been traditionally eaten by the alpine population may provide new opportunities for agricultural development in mountain regions. In this context we have investigated the chemical composition of Phyteuma orbiculare (Campanulaceae), a perennial herb whose leaves have been eaten as salad by rural populations in Valais (Switzerland). Extracts of different polarities were subjected to comprehensive metabolite profiling using a dereplication platform combining HPLC–PDA-MS, and offline NMR analysis. Twenty-three compounds, including various phenolic glycosides, a new dimeric phenylpropanoid glucoside, saponins, and fatty acids were identified online, or after targeted isolation. Selected phenolic constituents were quantitatively assessed by HPLC–PDA analysis. In addition, substances relevant for nutrition, such as β-carotene, fatty acids, ascorbic acid and minerals were quantified in leaves and flowers. The antioxidant capacity was determined with an ORAC assay, and total phenolic compounds were quantified. Finally, the phytochemical profile was compared to that of the related species P. spicatum, P. hemisphaericum and P. ovatum.     

Screening natural antioxidants in peanut shell using DPPH–HPLC–DAD–TOF/MS methods

Peanut shell, a byproduct in oil production, is rich in natural antioxidants. Here, a rapid and efficient method using DPPH–HPLC–DAD–TOF/MS was used for the first time to screen antioxidants in peanut shell. The method is based on the hypothesis that upon reaction with 1, 1-diphenyl-2-picrylhydrazyl (DPPH), the peak areas of compounds with potential antioxidant activities in the HPLC chromatogram will be significantly reduced or disappeared, and the identity confirmation could be achieved by HPLC–DAD–TOF/MS technique. With this method, three compounds possessing potential antioxidant activities were found abundantly in the methanolic extract of peanut shell. They were identified as 5,7-dihydroxychromone, eriodictyol, and luteolin. The contents of these compounds were 0.59, 0.92, and 2.36 mg/g, respectively, and luteolin possessed the strongest radical scavenging capacity. DPPH–HPLC–DAD–TOF/MS assay facilitated rapid identification and determination of natural antioxidants in peanut shell, which may be helpful for value-added utilization of peanut processing byproducts.     

Qualitative and quantitative analysis of curcuminoids in herbal medicines derived from Curcuma species

A validated and sensitive HPLC–UV–MS method was developed for qualitative and quantitative analysis of curcuminoids in eight herbal medicines derived from four Curcuma species. The samples were separated on a YMC ODS-A C₁₈ column with a gradient elution of acetonitrile and 0.1% formic acid. Curcumin, demethoxycurcumin and bisdemethoxycurcumin showed good linearity (r > 0.9998) in the concentration ranges of 4.88–625, 4.29–550 and 3.98–510 μg/mL, respectively. The results suggested that the contents of three major curcuminoids in different herbal medicines varied significantly. Curcuminoids were only detected in Jianghuang, HuangsiYujin, and PengEzhu. Amongst them, Jianghuang contained the highest amounts of curcuminoids (40.36 mg/g), which were almost 20 times higher than HuangsiYujin (1.94 mg/g) and 400 times higher than PengEzhu (0.098 mg/g). Furthermore, amongst the Jianghuang samples collected from different areas, samples from Sichuan Province contained remarkably higher amounts of curcuminoids (22.21–40.36 mg/g) than other cultivation regions.     

Phenolic profile of Sercial and Tinta Negra Vitis vinifera L. grape skins by HPLC–DAD–ESI-MS: Novel phenolic compounds in Vitis vinifera L. grape

This study represents the first phytochemical research of phenolic components of Sercial and Tinta Negra Vitis vinifera L. The phenolic profiles of Sercial and Tinta Negra V. vinifera L. grape skins (white and red varieties, respectively) were established using high performance liquid chromatography–diode array detection–electrospray ionisation tandem mass spectrometry (HPLC–DAD–ESI-MSⁿ), at different ripening stages (véraison and maturity). A total of 40 phenolic compounds were identified, which included 3 hydroxybenzoic acids, 8 hydroxycinnamic acids, 4 flavanols, 5 flavanones, 8 flavonols, 4 stilbenes, and 8 anthocyanins. For the white variety, in both ripening stages, hydroxycinnamic acids and flavonols were the main phenolic classes, representing about 80% of the phenolic composition. For red variety, at véraison, hydroxycinnamic acids and flavonols were also the predominant classes (71%), but at maturity, anthocyanins represented 84% of the phenolic composition. As far as we know, 10 compounds were reported for the first time in V. vinifera L. grapes, namely protocatechuic acid-glucoside, p-hydroxybenzoyl glucoside, caftaric acid vanilloyl pentoside, p-coumaric acid-erythroside, naringenin hexose derivate, eriodictyol-glucoside, taxifolin-pentoside, quercetin-glucuronide-glucoside, malylated kaempferol-glucoside, and resveratrol dimer. These novel V. vinifera L. grape components were identified based on their MSⁿ fragmentation profile. This data represents valuable information that may be useful to oenological management and to valorise these varieties as sources of bioactive compounds.     

HPLC–PDA–ESI–MS/MS profiling and chemopreventive potential of Eucalyptus gomphocephala DC

An HPLC–PDA–ESI/MS/MS method was developed to identify the phytoconstituents of the EtOAc fraction of Eucalyptus gomphocephala DC. The antioxidant effect of the EtOAc fraction together with its sub-fractions was determined in vitro. The cytotoxicity was evaluated on different cell lines. The EtOAc fraction exhibited strong antioxidant activity, reduced the viability of all cell lines and was more active on MCF-7 and HepG-2 cell lines. Subsequently, the cytotoxicity of the sub-fractions and the isolated compounds were tested on MCF-7, HepG-2. The EtOAc fraction possessed potential antitumour promoting properties. It inhibited the stimulated NO (20%), 5-LOX (48.0%) and COX-2 (49.7%) respectively (at concentration of 20 μg/ml). This study suggests that this fraction is a source of different antioxidant and cytotoxic compounds with potential chemopreventive properties that might prevent different stages of the carcinogenesis process.     

Effects induced by the nodulation with Bradyrhizobium japonicum on Glycine max (soybean) metabolism and antioxidant potential

This study was conducted in order to assess the effect of the inoculation of Glycine max (soybean) with Bradyrhizobium japonicum on the metabolite profile and antioxidant potential of its aerial parts. Extracts containing phenolic compounds and organic acids were analysed by HPLC–DAD and HPLC–UV, respectively. Volatiles profile was determined by HS-SPME/GC–IT-MS. The antioxidant activity of acidic and methanolic extracts was assessed against DPPH. Nodulation caused by B. japonicum led to an increase in the contents of phenolic compounds and organic acids. The same was observed for volatile compounds, with compounds like linalyl acetate, menthyl acetate and α-farnesene being detected only in inoculated plants. The phenolic extracts showed stronger antioxidant capacity than the organic acid extracts. In addition, extracts from plants nodulated with B. japonicum exhibited significantly higher antioxidant activity than control samples. These findings suggest that the inoculation with nodulating B. japonicum strains may be employed to manipulate the content of interesting metabolites in G. max aerial parts.     

Flavonol glucosides in Allium species: A comparative study by means of HPLC–DAD–ESI-MS–MS

Cultivars and consumption typologies of some Allium species can significantly vary from a chemical point of view and even small differences can be important for their characterization and differentiation. Bulbs of three varieties and four consumption typologies of onion (Allium cepa L.) and two varieties of shallot (Allium ascalonicum Hort.) were subjected to HPLC–DAD–ESI-MS–MS analysis. Seven flavonol glucosides were identified in all the samples, two of which, quercetin 3,4′-diglucoside and quercetin 4′-glucoside, represent about the 90% of the overall contents. Cultivars and consumption typologies of the Allium species under study show significant differences in flavonol contents, from the very low quantity of antioxidant compounds in white onion, about 7 mg/kg against 600–700 mg/kg that were found in red and gold varieties, to the enormous content of flavonols that are present in onions of prompt consumption, where quercetin 4′-glucoside exceeds 1 g/kg and quercetin 3-glucoside is present in a ratio higher then 10:1 with respect to its value in the other onion typologies. Shallots are very rich in the two major flavonols.     

Comparison of phenolic composition and antioxidant properties of two native Chilean and one domestic strawberry genotypes

The phenolic composition of extracts from the forms chiloensis and patagonica of Fragaria chiloensis were compared with that of the commercial strawberry Fragaria × ananassa cv. Chandler by high performance liquid chromatography with diode-array detector (HPLC–DAD) and high performance liquid chromatography with electrospray ionization mass spectroscopy detector (HPLC–ESI–MS). The phenolic constituents in the three species were mainly proanthocyanidins, hydrolysable tannins, anthocyanins and flavonol glycosides. In both native strawberry species the main flavonol glycoside was quercetin 3-O-glucuronide and the minor anthocyanins identified were cyanidin-malonyl-glucoside and pelargonidin-malonyl-glucoside. The highest anthocyanin content was found in the commercial red strawberry while ellagic acid was the main phenolic in the native white strawberry. From the methanolic fruit extract of the native Chilean strawberry Fragaria chiloensis ssp. chiloensis f. chiloensis four known antioxidants were isolated by selective fractionation using the bleaching of the free radical scavenger 1,1-diphenyl-2-picrylhydrazyl (DPPH) as the guiding assay. The antioxidant properties (measured as the bleaching of the DPPH radical) were determined for methanol extracts of the three fruits, for fractions of F. chiloensis ssp. chiloensis f. chiloensis, and for the isolated compounds cyanidin-3-glucoside, pelargonidin-3-glucoside, quercetin-3-glucuronide and ellagic acid. This study allows a clear chemical differentiation between the commercial strawberry and the Chilean white strawberry.     

A rapid method for simultaneous determination of triterpenoid saponins in Pulsatilla turczaninovii using microwave-assisted extraction and high performance liquid chromatography–tandem mass spectrometry

In the present study, a microwave-assisted extraction (MAE) and high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for the simultaneous determination of seven triterpenoid saponins in Pulsatilla turczaninovii. Operational conditions of MAE were optimized using a central composite design. Multiple-reaction monitoring was employed for quantification while switching the electrospray ion source polarity between positive and negative modes. Full validation of the method was carried out (linearity, precision, accuracy, limit of detection and limit of quantification). The results indicated that the method was rapid, specific and reliable. The developed method was successfully applied to determine the investigated compounds in nine batches of P. turczaninovii. These samples were collected in different seasons from the Liaoning province and varied greatly. The total triterpenoid saponins content was the highest in April, the seedling stage, and decreased from May to August. The contents of total triterpenoid saponins decreased significantly after the blooming period.     

Sensitive determination of domoic acid in shellfish by on-line coupling of weak anion exchange solid-phase extraction and liquid chromatography–diode array detection–tandem mass spectrometry

An automated method based on the use of on-line solid-phase extraction (SPE) coupled to liquid chromatography with diode array detection and tandem mass spectrometry (LC–DAD–MS/MS) has been developed for the determination of domoic acid in shellfish. The on-line coupling of SPE and liquid chromatography was accomplished by a column switching approach. A weak anion exchange (WAX) sorbent was selected for the SPE procedure, allowing a selective cleanup of shellfish extracts. High sensitivity was achieved by on-line pre-concentration of large volume injections (50–1000 μL). A simple protein precipitation cleanup with acetone was used to efficiently remove proteins from shellfish extracts, preventing possible column clogging during chromatographic separation.     

Chemical composition and antioxidant activities of Myrtus communis L. berries extracts

Myrtle (Myrtus communis L.) berries extracts were prepared with solvents at different polarity (water, ethanol, and ethyl acetate) and analysed using different in vitro tests in order to evaluate their antioxidant properties. Antiradical and total antioxidant activities were measured with DPPH and FRAP tests, respectively. Their ability to protect biological molecules was assessed using the cholesterol and LDL oxidation assays. In addition, phenolic compounds and unsaturated fatty acids composition was analysed by HPLC–DAD and HPLC–MS/MS. Ethanol and water extracts showed the highest amount of extracted compounds, but the highest antiradical and antioxidant activities were found in ethanol and ethyl acetate extracts. These extracts were also the ones with the highest content of phenolic compounds. In addition, our results showed a highly significant correlation between the amount of total phenols and antiradical (R² = 0.9993) or antioxidant activities (R² = 0.9985) in these extracts. HPLC–DAD and HPLC–MS analyses showed significant quantitative and qualitative differences among these three extracts. The ethyl acetate extract had the highest protective effect in assays of thermal (140 °C) cholesterol degradation and Cu²⁺-mediated LDL oxidation, inhibiting the reduction of polyunsaturated fatty acids and cholesterol, and the increase of their oxidative products. These results suggest that because of these properties, myrtle berries could be used in dietary supplements preparations or as food additives.     

Characterization of the components present in the active fractions of health gingers (Curcuma xanthorrhiza and Zingiber zerumbet) by HPLC–DAD–ESIMS

Curcuma xanthorrhiza and Zingiber zerumbet are two of the most commonly used ingredients in Indo-Malaysian traditional medicines, health supplements and tonics. Recently, a number of products derived from the aqueous extracts of these species have appeared in the market in the form of spray-dried powder packed in sachet or bottle. On-line high performance liquid chromatography, coupled with diode array detection and electrospray ion trap tandem mass spectroscopy (HPLC–DAD–ESI–MSⁿ), was used to analyze the components in the antioxidant-active fractions from the rhizomes of these species. Three components were identified from C. xanthorrhiza, including bisdemethoxycurcumin (1), demethoxycurcumin (2) and curcumin (3). The active fraction from Z. zerumbet consisted of five components, including kaempferol 3-O-rhamnoside (4), compound 5 [kaempferol 3-O-(2″-O-acetyl)rhamnoside (5a) or kaempferol 3-O-(3″-O-acetyl)rhamnoside (5b)], kaempferol 3-O-(4″-O-acetyl)rhamnoside (6), kaempferol 3-O-(3″,4″-O-diacetyl)rhamnoside (7) and kaempferol 3-O-(2″,4″-O-diacetyl)rhamnoside (8). To confirm their identities, the components from Z. zerumbet were isolated conventionally and were analyzed by spectroscopic techniques as well as by comparison with literature data.     

Identification and quantification of leaf surface flavonoids in wild-growing populations of Dracocephalum kotschyi by LC–DAD–ESI-MS

Dracocephalum kotschyi Boiss. (Lamiaceae) is an aromatic and perennial herb endemic to Iran with interesting pharmacological and biological properties. The flavonoids luteolin-7-O-glucoside, apigenin-7-O-glucoside (cosmosiin), luteolin 3′-O-β-d-glucuronide, luteolin, apigenin, cirsimaritin, isokaempferide, penduletin, xanthomicrol, calycopterin and the polyphenol rosmarinic acid were identified among 13 natural populations of the plant by ESI–MS, LC–DAD and LC–DAD–ESI-MS. The plant extracts containing the identified compounds showed significant antioxidant activity, which was correlated with the flavonoid content. Additionally, leaf and stem size and geographical variability among the studied populations were correlated with flavonoid accumulation. Canonical correlation analysis was used to find a relationship between plant dimensions and phytochemical composition, and the plants with the lowest growth indices were found to have the highest levels of methoxylated flavonoids.     

Selenium species in leaves of chicory,dandelion, lamb’s lettuce and parsley

Lamb’s lettuce, dandelion, parsley and four cultivars of chicory were cultivated aeroponically for 41 days with nutrient solution containing 7  mg Se/L in the form of Na₂SeO₄. Se compounds were determined by high performance liquid chromatography–ultraviolet treatment–hydride generation atomic fluorescence spectrometry (HPLC–UV–HG-AFS) in the green parts of the selected plants. Se species were extracted by water and by enzymatic hydrolysis with Protease XIV. Separation of Se^IV, Se^VI, SeMet, SeMeSeCys and SeCys₂ was made by a combination of anion and cation exchange chromatography in which the columns were connected on-line to a UV–HG-AFS detection system. Se accumulated efficiently in plant leaves up to 480 μg/g dry mass, mostly as Se^VI, i.e. the form of Se in the nutrient solution. Beside inorganic Se, selenomethionine (6–21%), selenomethylselenocysteine (0.5–4.4%) and selenocistine (<DL-0.8%) were determined in the extracts after enzymatic hydrolysis. Some unidentified peaks were also observed in the chromatograms.     

Analysis of mycotoxin fumonisins in corn products by high-performance liquid chromatography coupled with evaporative light scattering detection

Fumonisins are mycotoxins produced by the fungus Fusarium verticillioides, which is a widespread pathogen of corn. The mycotoxins are known to cause fatal diseases in some domestic animals and have been linked to human esophageal cancer in China and South Africa. Here, we describe a simple method for direct and quantitative analysis of the toxins in food products. The method is based on high-performance liquid chromatography coupled with an evaporative laser scattering detector (HPLC–ELSD) without any prior derivatization of the samples. Using this method, we have analyzed corn-based food samples from central markets in eastern China. The results showed that FB₁ was the main contaminant in the samples. The overall level of fumonisin contamination was relatively low, with a range of 0.25–1.80 μg/g (mean 0.74 μg/g) in 66.7% (16 of 24) of corn samples from Middle-eastern Area, 0.21–0.29 μg/g (mean 0.24 μg/g) in 28.6% (6 of 21) of corn samples from Northeastern Area, and 0.30–3.13 μg/g (mean 0.47 μg/g) in 30.0% (6 of 20) of corn samples from Southeastern Area.