Liquid Chromatography-diode Array Detection-electrospray Ionization Research Articles

Pot Aloe vera gel – a natural source of antioxidants

Aloe vera (Aloe barbadensis Miller) is widely spread around the world and it is used in the food and cosmetic industry and traditional medicine due to its high content in bioactive compounds. Thus, this article investigated the phenolic compounds of Romanian Aloe vera gel and its antioxidant capacity. The gel was extracted using 4 different solvents: methanol, ethanol, water, and acetone. Total phenolics were measured by the Folin-Ciocalteu method, total flavonoids by aluminium chloride reaction, and the antioxidant capacity by DPPH radical-scavenging activity. The profile of the phenolic compound was determined using Fourier transform infrared spectroscopy and Liquid Chromatography-Diode Array Detection–Electro-Spray Ionization Mass Spectrometry. The methanolic extract had a significantly higher antioxidant activity, followed by ethanolic, water, and acetone extracts. The methanolic extract had also the highest total polyphenol content, while ethanolic extract had the highest total flavonoid content. The extracts contained 14 compounds identified as 7 chromones (Aloesin, Neoaloesin, Aloinoside A, Aloinoside E, Aloe-emodin-glucoside, Isoaloeresin D, Methoxycoumaroyl-aloresin) 2 flavones (Luteolin-glucoside, Apigenin-glucoside), one hydroxycinnamic acid (Caffeic acid) and 4 to anthrones (Aloin A, Aloin B, Emodin, Aloe-emodin). The LC-MS results showed important quantities of Aloesin, Neoaloesin, Aloeresin E, and Aloe-emodin-glucoside while FTIR analysis showed the presence of polysaccharides, pectins, anthraquinones, and saponins.

Quality markers of Zingiberis Rhizoma Carbonisata before and after processing

Based on the previous research results of our group and literature research, the chemical components, mechanisms, pharmacodynamics, and pharmacokinetics of Zingiberis Rhizoma Carbonisata were summarized to determine the quality markers(Q-markers) of Zingiberis Rhizoma Carbonisata and Zingiberis Rhizoma. Our research group has clarified the differential components of Zingiberis Rhizoma Carbonisata and Zingiberis Rhizoma, the meridian-warming hemostatic effect of Zingiberis Rhizoma Carbonisata, the related targets and pathways of the effect, the endogenous biomarkers of Zingiberis Rhizoma Carbonisata, and the hemodynamic processes of Zingiberis Rhizoma Carbonisata and Zingiberis Rhizoma. Moreover, based on high-performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry(HPLC-DAD-ESIMS), a method for determining the content of Q-mar-kers was established. In conclusion, the study finally determined that gingerone, 6-shogaol, and diacetyl-6-gingerol were the Q-mar-kers of Zingiberis Rhizoma Carbonisata decoction pieces, and 6-gingerol, 8-gingerol, and 10-gingerol were Q-markers of Zingiberis Rhizoma decoction pieces. The result is expected to provide a reference for the establishment of quality standards for Zingiberis Rhizoma Carbonisata decoction pieces and Zingiberis Rhizoma decoction pieces.

The Influence of Ripeness on the Phenolic Content, Antioxidant and Antimicrobial Activities of Pumpkins (Cucurbita moschata Duchesne)

Cucurbita moschata Duchesne (Cucurbitaceae) is a plant food highly appreciated for the content of nutrients and bioactive compounds, including polyphenols and carotenoids, which contribute to its antioxidant and antimicrobial capacities. The purpose of this study was to identify phenolic acids and flavonoids of Cucurbita moschata Duchesne using high-performance liquid chromatography–diode array detection–electrospray ionization tandem mass spectrometry (HPLC–DAD–ESI-MS) at different ripening stages (young, mature, ripened) and determine its antioxidant and antimicrobial activities. According to the results, phenolic acids and flavonoids were dependent on the maturity stage. The mature fruits contain the highest total phenolic and flavonoids contents (97.4 mg GAE. 100 g−1 and 28.6 mg QE. 100 g−1).A total of 33 compounds were identified. Syringic acid was the most abundant compound (37%), followed by cinnamic acid (12%) and protocatechuic acid (11%). Polyphenol extract of the mature fruits showed the highest antioxidant activity when measured by DPPH (0.065 μmol TE/g) and ABTS (0.074 μmol TE/g) assays. In the antimicrobial assay, the second stage of ripening had the highest antibacterial activity. Staphylococcus aureus was the most sensitive strain with an inhibition zone of 12 mm and a MIC of 0.75 mg L−1. The lowest inhibition zone was obtained with Salmonella typhimurium (5 mm), and the MIC value was 10 mg L−1.

Antioxidant and antimicrobial extracts obtained from agricultural by-products: Strategies for a sustainable recovery and future perspectives

Durum wheat and rice brans are by-products deriving from the milling industry and source of important phytochemicals, mostly phenolics in bound form. The objectives of this work were to evaluate the most effective method for the extraction of phenolic acids from these by-products with antioxidant and antimicrobial activity and to standardize the obtained extracts to encourage their commercialization in health field. Free phenolics were extracted with ultrasound-assisted extraction (UAE) and bound phenolics with alkaline hydrolysis (AH) and ultrasound-assisted alkaline hydrolysis (UAAH). Extracts were analyzed with HPTLC (high-performance thin-layer chromatography) and RPHPLC-DAD-ESIMS (reversed-phase high-performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry); the content of trans-ferulic acid, p-coumaric acid, total phenolics were also quantified. The antioxidant activity was investigated using DPPH and ABTS methods. The antimicrobial activity was evaluated against Staphylococcus aureus and Staphylococcus epidermidis strains. Results obtained using UAAH showed the most interesting data, suggesting this method as the most effective to obtain active extracts from the by-products. Durum wheat, extracted with UAAH, exhibited the highest content of trans-ferulic acid (406.14 ± 0.65 μg FA/mg extract), total phenolics (610.58 ± 57.60 mg GAE/g dried extract) and good antioxidant and antimicrobial activities and it has been selected to formulate oil-in-water cosmetic products.

Comparison of Flavonoid Profiles in Sprouts of Radiation Breeding Wheat Lines (Triticum aestivum L.)

Wheat (Triticum aestivum Linn.; Poaceae), one of the most popular food crops worldwide, contains basic and essential nutrients and various health benefiting phytochemicals. Among them, flavonoids have attracted significant interest owing to their various health-promoting properties. In this study, 35 wheat mutant lines were developed via gamma-irradiated mutation breeding from the original cultivar. The effects of radiation breeding on the endogenous phytochemical production in the sprouts of these mutant lines were investigated using high performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry (HPLC-DAD-ESIMS) with multivariate analysis for the first time in this study. Fourteen characteristic peaks, including eleven flavone C-glycosides, two flavone O-glycosides, and one flavone, were identified. In addition, the contents of three flavone C-glycosides, namely, isoschaftoside, isoorientin, and isoscoparin, in 37 wheat sprout samples from the original cultivar, certificated cultivar, and the mutant lines were determined. A heat map combined with hierarchical clustering showed variation in the relative content for the flavonoids between the 37 wheat sprout samples, clustering into three groups. On principal component analysis scores scatter and loading plots, significant differences in the levels of flavonoids were found between the samples and several markers responsible for group separation were detected. These results provide a scientific reference for the phytochemical variation in wheat mutant lines, thereby aiding in further mutation mechanism studies and for the quality control of the improved wheat cultivars.

The use of thin-layer chromatography in the assessment of the quality of lutein-containing dietary supplements

Bivariant multiple development thin-layer chromatography technique (BMD–TLC) along with high-performance liquid chromatography–diode array detection–electrospray ionization–mass spectrometry (HPLC–DAD–ESI–MS) analysis was used in determination of lutein or lutein mixed with zeaxanthin in eight dietary supplements. The developed two-step TLC separation procedure combined purification, compaction of samples and separation of the analyzed compounds what significantly shortened and simplified samples preparation. Qualitative analysis was based on co-chromatography with reference substances and HPLC–DAD–ESI–MS analysis. It was revealed that three of eight dietary supplements did not contain lutein. In turn, quantitative analysis with the use of developed TLC conditions along with densitometry showed that the amount of lutein or its mixture with zeaxanthin in the others differed from that claimed by producers.

Comparative Analysis of Phytochemical Composition of Gamma-Irradiated Mutant Cultivars of Chrysanthemum morifolium

The flowers of chrysanthemum species are used as a herbal tea and in traditional medicine. In addition, members of the genus have been selected to develop horticultural cultivars of diverse floral colors and capitulum forms. In this research, we investigated the phytochemical composition of eight gamma-irradiation mutant cultivars of Chrysanthemum morifolium and their original cultivars. The mutant chrysanthemum cultivars were generated by treatment with various doses of 60Co gamma irradiation of stem cuttings of three commercial chrysanthemum cultivars as follows: ‘ARTI-Dark Chocolate’ (50Gy), ‘ARTI-Purple Lady’ (30 Gy), and ‘ARTI-Yellow Star’ (50 Gy) derived from ‘Noble Wine’; ‘ARTI-Red Star’ (50 Gy) and ‘ARTI-Rising Sun’ (30 Gy) from ‘Pinky’; ‘ARTI-Purple’ (40 Gy) and ‘ARTI-Queen’ (30 Gy) from ‘Argus’; and ‘ARTI-Rollypop’ (70 Gy) from ‘Plaisir d’amour’. Quantitative analysis of flavonoids, phenolic acids, anthocyanins, and carotenoids in the flowers of the 12 chrysanthemum cultivars was performed using high performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry (HPLC-DAD-ESIMS). Essential oils from the flowers of these cultivars were analyzed by gas chromatography–mass spectrometry (GC-MS). The mutant cultivars, ‘ARTI-Dark Chocolate’, ‘ARTI-Purple Lady’, ‘ARTI-Purple’, and ‘ARTI-Queen’ showed higher total amounts of flavonoid and phenolic acid compared with those of the respective original cultivars. The mutant cultivars, ‘ARTI-Dark Chocolate’, ‘ARTI-Purple Lady’ and ‘ARTI-Purple’, which produce purple to pink petals, contained more than two-times higher amounts of anthocyanins compared with those of their original cultivars. Of the mutant cultivars, ‘ARTI-Yellow Star’ in which petal color was changed to yellow, showed the greatest accumulation of carotenoids. Ninety-nine volatile compounds were detected, of which hydrocarbons and terpenoids were abundant in all cultivars analyzed. This is the first report that demonstrated the phytochemical analysis of novel chrysanthemum cultivars derived from C. morifolium hydrid using HPLC-DAD-ESIMS and GC-MS. These findings suggest that the selected mutant chrysanthemum cultivars show potential as a functional source of phytochemicals associated with the abundance of health-beneficial components, as well as good source for horticulture and pigment industries.

Phenolic compounds profile of different berry parts from novel Vitis vinifera L. red grape genotypes and Tempranillo using HPLC-DAD-ESI-MS/MS: A varietal differentiation tool

The detailed phenolic composition of different berry parts from two novel V. vinifera L. red grape genotypes (Moribel and Tinto Fragoso), together the well-known Tempranillo, was established using high performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS/MS) over two consecutive vintages (2016 and 2017). More than 50 phenolic compounds were identified and quantified: 25 anthocyanins, 17 flavonols, 7 hydroxycinnamic acid derivatives, 2 stilbenes, and several flavan-3-ols. As far as we know, some anthocyanin and flavonol dihexosides were reported for the first time in V. vinifera L. grapes. Application of Principal Component Analysis (PCA) to experimental data showed a good separation of the novel grape genotypes and Tempranillo according to the phenolic profile of skins and seeds, mainly based on the proportion of trisubstituted anthocyanin derivatives, flavonols and flavan-3-ols, being a useful tool to differentiate these grape varieties.

Identification Markers of Adulteration in Korean Red Ginseng (Panax Ginseng) Products Using High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography–Mass Spectrometry (LC-MS)

ABSTRACTThe commercial products of Panax ginseng have increasing market demand and high prices due to the pharmacological activities. To obtain high profits, P. ginseng may be adulterated with lower priced morphologically similar species such as Platycodon grandiflorum, Codonopsis lanceolata, and Pueraria lobata. This study was designed to validate accurate methods for the analysis of adulteration in P. ginseng products. High-performance liquid chromatography (HPLC) and ultraperformance liquid chromatography–diode array detector–electrospray ionization–ion trap–time of flight–mass spectrometry (UPLC–DAD–ESI-IT-TOF-MS) were validated to analyze the raw plant materials, self-prepared formulations, and commercial products of P. ginseng, C. lanceolata, P. grandiflorum, and P. lobata. The developed analytical methods were confirmed by quality assurance parameters such as linearity, sensitivity, precision, and accuracy. Lobetyolin and ononin were identified as marker compounds by HPLC and confirmed by accurate mass measurement with ESI-IT-TOF-MS. HPLC analysis of self-prepared formulations indicated that by increasing the ratio of C. lanceolata, P. grandiflorum, and P. lobata in P. ginseng extracts, the peak area is increased at the same retention time. The limits of detection and quantification for lobetyolin and ononin were 0.098 and 0.171, and 0.108 and 0.726 mg/kg, respectively. Furthermore, the intraday precision (<1.0%) measurements confirmed that the developed analytical methods fulfill the required criteria for characterization of these products. The results demonstrated that the developed liquid chromatographic and mass spectrometric methods accurately characterized adulteration in P. ginseng commercial products.

Phytochemical characterization of polyphenolic compounds with HPLC–DAD–ESI–MS and evaluation of lipid-lowering capacity of aqueous extracts from Saharan plant Anabasis aretioides (Coss & Moq.) in normal and streptozotocin-induced diabetic rats

ObjectiveAnabasis aretioides (Coss & Moq.), a Saharan plant belonging to Chenopodiaceae family, is widely distributed in semi-desert areas from the Tafilalet region of Morocco. This plant is extensively used by local population against diabetes and cardiovascular disorders. The purpose of the study was to investigate the effect of the aqueous A. aretioides extract on lipid metabolism in normal and streptozotocin (STZ)-induced diabetic rats and to identify the polyphenolic compounds present. In addition, the in vitro antioxidant activity of the aqueous A. aretioides extract was also evaluated. MethodsThe effect of an aerial part aqueous extract (APAE) of A. aretioides (5 mg/kg of lyophilized A. aretioides APAE) on plasma lipid profile was investigated in normal and STZ-induced diabetic rats (n = 6) after once daily oral administration for 15 days. The aqueous extract was tested for its 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity. Polyphenolic compounds in the extracts were definitively characterized by high-performance liquid chromatography–diode array detection–electrospray ionization–mass spectrometry. ResultsIn diabetic rats, oral administration of A. aretioides APAE provoked a significant decrease in both plasma cholesterol and triglyceride levels from the first to the second week (P < 0.01). A significant decrease on plasma triglyceride levels was also observed in normal rats (P < 0.01), where the reduction was 53%. In addition, the phytochemical analysis revealed the presence of 12 polyphenolic compounds. Moreover, according to the DPPH radical-scavenging activity, the aqueous extract showed an in vitro antioxidant activity. ConclusionAqueous A. aretioides APAE exhibits lipid-lowering and in vitro antioxidant activities. Many polyphenols were present in this extract and these phytoconstituents may be involved in the pharmacological activity of this plant.

Quantification of Antioxidant Phenolic Compounds in a New Chrysanthemum Cultivar by High-Performance Liquid Chromatography with Diode Array Detection and Electrospray Ionization Mass Spectrometry.

The flowers of Chrysanthemum morifolium Ramat. have been used as an herbal tea and in traditional medicine, and the plant has been developed to produce horticultural cultivars of various colors and shapes. In this study, a new chrysanthemum cultivar with dark purple petals (C. morifolium cv. ARTI-Dark Chocolate; ADC) was developed by radiation-induced mutation breeding of its original cultivar with purple striped white petals (C. morifolium cv. Noble Wine, NW). The phenolic profile and antioxidant property of ADC were investigated and compared with NW and the commercially available medicinal herb, C. morifolium with yellow petals (CM), in order to find a scientific support to produce a new source of natural antioxidant. Flavonoid and phenolic acid profiles of the ethanol extracts of the three flowers were analyzed by high-performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry (HPLC-DAD-ESIMS), while antioxidant properties were evaluated using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assay. Among the tested flowers, ADC possessed the strongest antioxidant capacity and the highest phenolic contents. Flavonoids (acacetin, apigenin, luteolin, acacetin-7-O-β-glucoside, apigenin-7-O-β-glucoside, luteolin-7-O-β-glucoside, and linarin) and phenolic acids (chlorogenic acid and mixture of 1,4-, 1,5-, and 3,5-dicaffeoylquinic acids) were identified and quantified.

Rapid Screening of Potential Phosphodiesterase Inhibitors from the Roots of Ilex pubescens Hook. et Arn. Using a Combination of Ultrafiltration and LC-MS.

The cyclic nucleotide phosphodiesterase (PDE) plays an important role in regulating the levels of second messenger molecules cAMP and cGMP. Various PDE inhibitors have been successfully developed into drugs for targeted diseases. In addition, PDE inhibitors can also be found in different foods and natural medicines. In this study, ultrafiltration liquid chromatography–diode-array detector–electrospray ionization–ion-trap–time-of-flight–mass spectrometry (ultrafiltration LC–DAD–ESI–IT–TOF–MS) was applied to screen PDE inhibitors from the roots of Ilex pubescens Hook. et Arn. As a result, 11 major compounds were identified in I. pubescens roots, with nine compounds as potential PDE inhibitors, among which five were further confirmed to be active against PDEI and PDE5A dose-dependently in vitro, with ilexsaponin A1 and ilexsaponin B2 being the strongest. HPLC quantification of these bioactive compounds suggested that they are major components in the plant. The results demonstrate that ultrafiltration LC–DAD–ESI–IT–TOF–MS is an efficient method for rapid screening of PDE inhibitors from natural medicines.

Determination of Phenolic Content in Different Barley Varieties and Corresponding Malts by Liquid Chromatography-diode Array Detection-Electrospray Ionization Tandem Mass Spectrometry.

A simple and reliable method for the simultaneous determination of nine phenolic compounds in barley and malted barley was established, using liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS/MS). The phenolic compounds can be easily detected with both systems, despite significant differences in sensitivity. Concentrations approximately 180-fold lower could be achieved by mass spectrometry analysis compared to diode array detection, especially for the flavan-3-ols (+)-catechin and (−)-epicatechin, which have poor absorptivity in the UV region. Malt samples were characterized by higher phenolic content comparing to corresponding barley varieties, revealing a significant increase of the levels of (+)-catechin and (−)-epicatechin during the malting process. Moreover, the industrial malting is responsible for modification on the phenolic profile from barley to malt, namely on the synthesis or release of sinapinic acid and epicatechin. Accordingly, the selection of the malting parameters, as well as the barley variety plays an important role when considering the quality and antioxidant stability of beer.

Characterization of flavonoid glycosides from rapeseed bee pollen using a combination of chromatography, spectrometry and nuclear magnetic resonance with a step-wise separation strategy

To identify the structures of flavonoid glycosides in bee pollen collected from rapeseed plants (Brassica napus L.), we utilised an approach that combined liquid chromatography–diode array detector–electrospray ionization–mass spectrometry (LC–DAD–ESI–MS) and nuclear magnetic resonance (NMR) technology with a step-wise separation strategy. We identified four constituents of high purity in rape bee pollen samples: (1) quercetin-3-O-β-D-glucosyl-(2→l)-β-glucoside, (2) kaempferol-3, 4′-di-O-β-D-glucoside, (3) 5, 7, 4′-trihydroxy-3′-methoxyflavone-3-O-β-D-sophoroside and (4) kaempferol-3-O-β-D-glucosyl-(2→l)-β-D-glucoside. This study will also provide useful reference standards for qualification and quantification of four flavonoid glycosides in natural products.

Chemical fingerprinting of Gardenia jasminoides Ellis by HPLC–DAD–ESI-MS combined with chemometrics methods

A fingerprint analysis method has been developed for characterization and discrimination of Gardenia jasminoides Ellis from different areas. The chemometrics methods including similarity evaluation, principal components analysis (PCA) and hierarchical clustering analysis (HCA) were introduced to identify more useful chemical markers for improving the quality control standard of dried ripe fruits of G. jasminoides Ellis. Then the selected chemical markers were analyzed by high performance liquid chromatography–diode array detection–electrospray ionization mass spectrometry (HPLC–DAD–ESI-MS) qualitatively and quantitatively. 23 characteristic peaks were assigned while 19 peaks of them were identified by comparing retention times, UV and MS spectra with authentic compounds or literature data. Moreover, 14 of them were determined quantitatively which could effectively evaluate the quality of G. jasminoides Ellis. This study was expected to provide comprehensive information for the quality evaluation of G. jasminoides Ellis, which would be a valuable reference for further study and development of this herb and related medicinal products.

Fortification of Cookies with Peanut Skins: Effects on the Composition, Polyphenols, Antioxidant Properties, and Sensory Quality

Food fortification may be carried out to improve the health status of consumers. In this study, peanut skins were added at 1.3, 1.8, and 2.5% to cookies to increase their polyphenol content. Insoluble fiber was increased by up to 52%. In addition, total phenolic content and the corresponding antioxidant capacities also increased as evidenced by increases of epicatechin and procyanidin dimers A and B. In addition, trimers and tetramers of procyanidins were identified only in peanut skin-fortified cookies. Addition of 2.5% peanut skins rendered an increase of up to 30% in the total polyphenols as evaluated by high-performance liquid chromatography-diode array detection-electrospray ionization multistage mass spectrometry (HPLC-DAD-ESI-MS(n)). Sensory evaluation results demonstrated that peanut skin-fortified cookies were well accepted, which suggests that the present formulation may lend itself for commercial exploitation.

MICROWAVE-ASSISTED HYDROLYZATION OF THE FLAVONOIDS IN EPIMEDIUM KOREAMUM NAKAI

In the present paper, icariside I has been prepared by hydrolyzing the flavonoids extract of Epimedium Koreamum Nakai in acid solution. Two heating methods, reflux and microwave irradiation, were used in the hydrolysis reaction and hydrolyzed products were analyzed by high performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectometry (HPLC-DAD-ESI-MSn). The four flavonoids in the extracts have been identified as epimedin A, epimedin B, epimedin C, and icariin, and the hydrolysis product was icariside I. Icariside I has been evaluated by comparing with the peak areas of HPLC-DAD spectra obtained using different heating methods. The experimental results demonstrate that the advantage of microwave irradiation over conventional reflux is validated, and the hydrolysis time is dramatically reduced, and the yield of icariside I is effectively improved. Also, optimum conditions for microwave hydrolysis are established. As a result, the optimum yield of icariside I can be reached under the following conditions: the reaction temperature is 70°C; the microwave irradiation power is 600 W; and the microwave irradiation time is 10 min. The optimum yield by this method was (300.46 ± 0.41 µg), which was 2.3 times more than the classical way (128 ± 0.86 µg). In view of the efficiency, energy saving, and environmental protection in the procedures of microwave-assisted hydrolyzation, this method will have a bright future in the industrial application.

Liquid chromatography–diode array detection–mass spectrometry for compositional analysis of low molecular weight heparins

Low molecular weight heparins (LMWHs) are important artificial preparations from heparin polysaccharide and are widely used as anticoagulant drugs. To analyze the structure and composition of LMWHs, identification and quantitation of their natural and modified building blocks are indispensable. We have established a novel reversed-phase high-performance liquid chromatography–diode array detection–electrospray ionization–mass spectrometry approach for compositional analysis of LMWHs. After being exhaustively digested and labeled with 2-aminoacridone, the structural motifs constructing LMWHs, including 17 components from dalteparin and 15 components from enoxaparin, were well separated, identified, and quantified. Besides the eight natural heparin disaccharides, many characteristic structures from dalteparin and enoxaparin, such as modified structures from the reducing end and nonreducing end, 3-O-sulfated tetrasaccharides, and trisaccharides, have been unambiguously identified based on their retention time and mass spectra. Compared with the traditional heparin compositional analysis methods, the approach described here is not only robust but also comprehensive because it is capable of identifying and quantifying nearly all components from lyase digests of LMWHs.

Detoxification of Toxic Phorbol Esters from Malaysian Jatropha curcas Linn. Kernel by Trichoderma spp. and Endophytic Fungi

The presence of phorbol esters (PEs) with toxic properties limits the use of Jatropha curcas kernel in the animal feed industry. Therefore, suitable methods to detoxify PEs have to be developed to render the material safe as a feed ingredient. In the present study, the biological treatment of the extracted PEs-rich fraction with non-pathogenic fungi (Trichoderma harzianum JQ350879.1, T. harzianum JQ517493.1, Paecilomyces sinensis JQ350881.1, Cladosporium cladosporioides JQ517491.1, Fusarium chlamydosporum JQ350882.1, F. chlamydosporum JQ517492.1 and F. chlamydosporum JQ350880.1) was conducted by fermentation in broth cultures. The PEs were detected by liquid chromatography-diode array detector-electrospray ionization mass spectrometry (LC-DAD-ESIMS) and quantitatively monitored by HPLC using phorbol-12-myristate 13-acetate as the standard. At day 30 of incubation, two T. harzianum spp., P. sinensis and C. cladosporioides significantly (p < 0.05) removed PEs with percentage losses of 96.9%–99.7%, while F. chlamydosporum strains showed percentage losses of 88.9%–92.2%. All fungal strains could utilize the PEs-rich fraction for growth. In the cytotoxicity assay, cell viabilities of Chang liver and NIH 3T3 fibroblast cell lines were less than 1% with the untreated PEs-rich fraction, but 84.3%–96.5% with the fungal treated PEs-rich fraction. There was no inhibition on cell viability for normal fungal growth supernatants. To conclude, Trichoderma spp., Paecilomyces sp. and Cladosporium sp. are potential microbes for the detoxification of PEs.

Simultaneous Screening and Identifying Four Categories of Particular Flavonoids in the Leaves of Murraya exotica L. by HPLC-DAD-ESI-MS-MS

Polymethoxylated flavonoids (PMFs), the particular flavonoid subclass in which all or almost all hydroxyls are capped by methylation, have high oral bioavailability and various activities. A sensitive high-performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS-MS) method was established to screen and identify the PMFs in the leaves of Murraya exotica. Eight PMF standards, including two polymethoxylated flavones, two polymethoxylated flavanones, two polymethoxylated chalcones and two PMF glycosides, were first to be analyzed in positive mode by collision-induced dissociation MS-MS. On the basis of the ESI-MS, characterizations were deduced, and in the results of the extracted ion chromatogram MS-MS experiment, 26 PMFs, including 18 flavones, five flavanones or chalcones and three PMF glycosides, were screened out from the complex extract of the leaves of M. exotica. Among them, 24 PMFs were hydroxylated polymethoxyflavonoids, whereas the rest were all permethoxylated PMFs. This was the first systematic report on the presence of PMFs in the leaves of M. exotica. The results indicated that the established analytical method could be adopted as a rapid, effective technique for the structural characterization of PMFs from the complex extracts of traditional Chinese medicines.