Purification and Partial Characterization of Bacillomycin L Produced by Bacillus amyloliquefaciens K103 from Lemon

Bacillus amyloliquefaciens K103 isolated from a lemon sample was used as a biocontrol agent to suppress Rhizoctonia solani Kühn and other fungal plant pathogens. Two antifungal compounds were purified from the culture broth using acid precipitation, gel permeation chromatography, and reversed-phase high-performance liquid chromatography. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis indicated that the antifungal compounds were two isomers similar to bacillomycin L. One of the predominant active fractions was subjected to quadrupole time-of-flight mass spectrometry and amino acid analysis to determine its structural characteristics, revealing that the antifungal compound with a molecular mass of 1,034.5464 was identical to bacillomycin L. This is the second report of lemon microflora producing bacillomycin L or any antifungal compound, suppressing the growth of R. solani Kühn. Meanwhile, the study provided insights into the enormous potential of food microbial resources and bacillomycin L antibiotics in biological control and sustainable agriculture.     

Normal phase high performance liquid chromatography for fractionation of organic acid mixtures extracted from crude oils

Crude oil contains such an extensive range of compounds that a complete analysis is impossible. Fractionation by chemical properties is often used to simplify analytical handling. This work presents a high performance liquid chromatography (HPLC) method using normal phase chromatography on a cyano-bonded phase column to separate acid extracts from crude oils into four fractions; non-polar compounds, saturated carboxylic acids, phenols and polyfunctional acids. The method has been developed both in analytical scale for characterisation of acid extracts, and in preparative scale to provide sufficient sample amounts for further analysis by complementary methods.     

Antioxidant activity guided separation of major polyphenols of marjoram (Origanum majorana L.) using flash chromatography and their identification by liquid chromatography coupled with electrospray ionization tandem mass spectrometry†

Marjoram extracts have been separated into polar and nonpolar parts using liquid–liquid extraction. Both polar and nonpolar parts of the extracts were further fractionated by flash chromatography. The obtained fractions (90 polar and 45 nonpolar fractions) were investigated for their antioxidant activities by 2,2‐diphenylpicrylhydrazyl and ferric ion reducing antioxidant power assays. A direct, positive, and linear relationship between antioxidant activity and total phenolic content of the fractions was observed. Based on antioxidant and total phenolic content data, the three fractions with the high antioxidant activities from polar and nonpolar part of the extract were analyzed for their constituent polyphenols by liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Compounds were identified by matching the mass spectral data and retention time with those of authentic standards. Identification of the compounds for which there were no “in‐house” standards available was carried out by accurate mass measurement of the precursor ions and product ions generated from collision‐induced dissociation. Rosmarinic acid was found to be the strongest antioxidant polyphenol conferring the highest antioxidant activity to fractions 47 and 17 of polar and nonpolar part of the extract, respectively. The identification of the rosmarinic acid was further confirmed by ¹H NMR spectroscopy.     

Determination of the volatile composition in brown millet, milled millet and millet bran by gas chromatography/mass spectrometry

The volatile compounds from brown millet (BM), milled millet (MM) and millet bran (MB) were extracted using simultaneous distillation/extraction with a Likens-Nickerson apparatus. The extracts were analysed using gas chromatography coupled with mass spectrometry (GC-MS). A total of 65 volatile compounds were identified in all of the samples. Among these compounds, 51, 51 and 49 belonged to BM, MM and MB, respectively. Aldehydes and benzene derivatives were the most numerous among all of the compounds. Three compounds (hexanal, hexadecanoic acid and 2-methylnaphthalene) were dominant in the BM and MM materials. Eight compounds (hexanal, nonanal, (E)-2-nonenal, naphthalene, 2-methylnaphthalene, 1-methylnaphthalene, hexadecanoic acid and 2-pentylfuran) were dominant in the MB materials. Apart from the aromatic molecules, which were present in all fractions, compounds present only in BM, MM or MB were also identified.     

Intra-specific biodiversity of Italian myrtle (Myrtus communis) through chemical markers profile and biological activities of leaf methanolic extracts

Methanolic extracts of Myrtus communis leaves from two Italian regions (Calabria and Sardinia) were processed to determine the content of myrtenol, linalool and eucalyptol. Among the Calabrian and Sardinian myrtle samples, linalool and eucalyptol chemotypes were prevalent. The extracts were also tested for antioxidant, antibacterial and antifungal activities. Myrtle leaves samples were dried and extracted through maceration. Partition chromatography was adopted to separate myrtenol, linalool and eucalyptol fractions. Analyses were performed through GC and GC-MS. Some of the samples showed a good scavenger activity evidenced by DPPH radical scavenging assay and beta-carotene bleaching test. Antibacterial and antifungal activities were generally weak. The phytochemical and biological characterization of all the extracts were determined with an aim to characterize the intra-specific biodiversity of myrtle populations.     

Molecular engineering of photoactive liquid crystalline polyester epoxies containing benzylidene moiety

A series of photoactive liquid crystalline polyester epoxies incorporating bisbenzylidene segments as photoactive mesogenic cores were synthesized by polyaddition of diepoxy monomers and terephthalic acid/trimesic acid. To investigate the influence of structural parameters such as, molecular architecture, structural rigidity of mesogenic unit and substituents on thermal, mesogenic, and photoactive properties, the bisbenzylidene segment was incorporated into one acyclic and two cycloalkanone units with two and four substituents, respectively in both linear and hyperbranched architectures. Degree of branching of hyperbranched polymers was found to be in the range of 0.49–0.62. All polymers exhibited nematic mesophase (nematic droplets). Photo induced (2π + 2π) cycloaddition reaction, upon exposure to light at 365 nm, was examined. Inter molecular photocycloaddition was confirmed by photoviscosity measurement of UV irradiated polymer solutions. Faster photo induced reactivity of polymers in hyperbranched architecture was observed when compared to linear structure. Acyclic units facilitated photocycloaddition, and five‐membered ring showed higher photoactivity compared to six‐membered ring. The steric hindrance caused by substituents decreased the photoactivity of polymers. Refractive index change was found to be in the range of 0.015–0.024. Substantial variation of refractive index indicates that these polymers could be used for optical recording. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 7637–7655, 2008     

Activity-Guided Characterization of COX-2 Inhibitory Compounds in Waltheria indica L. Extracts

Inflammation is the body’s response to infection or tissue injury in order to restore and maintain homeostasis. Prostaglandin E2 (PGE-2) derived from arachidonic acid (AA), via up-regulation of cyclooxygenase-2 (COX-2), is a key mediator of inflammation and can also be induced by several other factors including stress, chromosomal aberration, or environmental factors. Targeting prostaglandin production by inhibiting COX-2 is hence relevant for the successful resolution of inflammation. Waltheria indica L. is a traditional medicinal plant whose extracts have demonstrated COX-2 inhibitory properties. However, the compounds responsible for the activity remained unknown. For the preparation of extracts with effective anti-inflammatory properties, characterization of these substances is vital. In this work, we aimed to address this issue by characterizing the substances responsible for the COX-2 inhibitory activity in the extracts and generating prediction models to quantify the COX-2 inhibitory activity without biological testing. For this purpose, an extract was separated into fractions by means of centrifugal partition chromatography (CPC). The inhibitory potential of the fractions and extracts against the COX-2 enzyme was determined using a fluorometric COX-2 inhibition assay. The characterizations of compounds in the fractions with the highest COX-2 inhibitory activity were conducted by high resolution mass spectrometry (HPLC-MS/MS). It was found that these fractions contain alpha-linolenic acid, linoleic acid and oleic acid, identified and reported for the first time in Waltheria indica leaf extracts. After analyzing their contents in different Waltheria indica extracts, it could be demonstrated that these fatty acids are responsible for up to 41% of the COX-2 inhibition observed with Waltheria indica extract. Additional quantification of secondary metabolites in the extract fractions revealed that substances from the group of steroidal saponins and triterpenoid saponins also contribute to the COX-2 inhibitory activity. Based on the content of compounds contributing to COX-2 inhibition, two mathematical models were successfully developed, both of which had a root mean square error (RMSE) = 1.6% COX-2 inhibitory activity, demonstrating a high correspondence between predicted versus observed values. The results of the predictive models further suggested that the compounds contribute to COX-2 inhibition in the order linoleic acid > alpha linolenic acid > steroidal saponins > triterpenoid saponins. The characterization of substances contributing to COX-2 inhibition in this study enables a more targeted development of extraction processes to obtain Waltheria indica extracts with superior anti-inflammatory properties.     

Identification of active compounds from medicinal plant extracts using gas chromatography-mass spectrometry and multivariate data analysis

Conventional methods of drug discovery from natural products include bioassay-guided fractionation, which is tedious and has low efficiency. The aim of this work is to develop a platform method to rapidly identify bioactive compounds from crude plant extracts and their partially purified fractions using multivariate data analysis (MVDA). Soxhlet extraction and liquid-liquid fractionation were used to prepare different extracts and fractions from the leaves of a medicinal plant, Ardisia elliptica. The extracts and fractions were analysed chemically using GC-MS, and their ability to inhibit platelet aggregation was investigated. Two MVDA methods were developed and optimised to analyse the results. In the first method, compounds with the highest contribution scores for biological activity calculated by different models were listed as potential antiplatelet compounds. For the second MVDA method, a correlation of the concentrations of constituents and biological activities in the various extracts and fractions for each compound was done. Compounds with the highest correlation coefficients were identified as potential antiplatelet compounds. One of the predicted components was isolated, purified and confirmed to possess antiplatelet effects. This platform method can be developed and optimised for other plant extracts and biological activities, thus reducing time and cost of drug discovery while improving efficiency.     

Oat (<Emphasis Type="Italic">Avena sativa</Emphasis>) Seed Extract as an Antifungal Food Preservative Through the Catalytic Activity of a Highly Abundant Class I Chitinase

Extracts from different higher plants were screened for the ability to inhibit the growth of Penicillium roqueforti, a major contaminating species in industrial food processing. Oat (Avena sativa) seed extracts exhibited a high degree of antifungal activity and could be used directly on rye bread to prevent the formation of P. roqueforti colonies. Proteins in the oat seed extracts were fractionated by column chromatography and proteins in fractions containing antifungal activity were identified by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and database searches. Identified antifungal candidates included thaumatin-like proteins, 1,3-beta-glucanase, permatin precursor, pathogenesis-related protein type 1, and chitinases of class I and II. Class I chitinase could be specifically removed from the extracts and was found to be indispensable for 50% of the P. roqueforti inhibiting activity. The purified class I chitinase has a molecular weight of approximately 34 kDa, optimal chitinase activity at pH 7, and exists as at least two basic isoforms (pI values of 7.6 and 8.0). Partial sequencing of the class I chitinase isoforms by LC-MS/MS revealed a primary structure with high similarity to class I chitinases of wheat (Triticum aestivum), barley (Hordeum vulgare), and rye (Secale cereale). Oat, wheat, barley, and rye seed extracts were compared with respect to the abundance of the class I chitinase and decrease in antifungal activity when class I chitinase is removed. We found that the oat seed class I chitinase is at least ten times more abundant than the wheat, barley, and rye homologs and that oat seed extracts are highly active toward P. roqueforti as opposed to extracts of other cereal seeds.     

Chemical composition of essential oil and headspace-solid microextracts from fruits of Myrica gale L. and antifungal activity

The essential oil and the volatile compounds of Myrica gale fruits were analysed by gas chromatography (GC) and GC-mass spectrometry (GC-MS). The volatile compounds were detected using two different fibres for headspace-solid phase microextraction (HS-SPME), Carboxen/PDMS and PDMS. Sixty two compounds were identified, which represented more than 90% of the total extracts. Major components of fruit essential oil are alpha-pinene (22.6%), 1,8-cineole (18.9%) and germacrone (14.2%), whereas they are germacrone (25.1%), alpha-pinene (12.2%), limonene (8.1%) and alpha-phellandrene (8.0%) for the leaf essential oil. Major volatile fruit compounds detected in HS-SPME were alpha-pinene, 1,8-cineole, p-cymene and eth-cadinene. As M. gale fruits are traditionally used in brewery for flavouring beer or as a spice in soups or stews, the antifungal properties of these essential oils were investigated on a panel of foodborne fungi, namely Aspergillus flavus, Cladosporium cladosporioides and Penicillium expansum. A complete antifungal activity was observed at 1000 ppm against C. cladosporioides. Both essential oil and entire fruits could thus be used as an additive in food or cosmetic preparations for their flavour, odour and their conservative properties.     

Antioxidant activity of lignin phenolic compounds extracted from kraft and sulphite black liquors

The antioxidant activity of the phenolic compounds present in industrial black liquors obtained from the two cooking processes (kraft and sulphite) used in Portugal to produce Eucalyptus globulus pulp was evaluated. The black liquors treated at several pH values were extracted with ethyl acetate. Phenolic fractions were further separated by liquid chromatography of the crude extracts of kraft liquor at pH = 6 and sulphite liquor at the original pH. Total phenolic content was determined in terms of gallic acid equivalents (Folin-Ciocalteu colorimetric method), and the antioxidant activity in the crude extracts at several pH values and in the separated fractions was measured using the DPPH test for radical scavenging capacity. The total phenolic content of crude extracts and separated fractions ranged from 92.7 to 181.6 and from 91.6 to 1,099.6 mg GAE/g, respectively, while the antioxidant activity index (AAI) ranged from 2.20 to 3.41 and from 2.21 to 11.47 respectively, showing very strong antioxidant activity in all studied cases. The fractions separated by column chromatography were submitted to mass spectrometry analysis and the results were compared to others in the literature of natural products, mainly from Eucalyptus, and the characteristic bands of functional groups were identified by 1H-NMR and FTIR. These methods allowed the identification of 17 phenolic compounds.     

Preparative isolation and purification of senkyunolide-I, senkyunolide-H and ferulic acid from Rhizoma Chuanxiong using counter-current chromatography

Three active compounds, senkyunolide-I, senkyunolide-H and ferulic acid (FA), were successfully isolated and purified from the extracts of Rhizoma Chuanxiong by counter-current chromatography (CCC). Based on the principle of the partition coefficient values (k) for target compounds and the separation factor (α) between target compounds, the two-phase solvent system that contains n-hexane-ethyl acetate-methanol-water at an optimized volume ratio of 3:7:4:6 v/v was selected for the CCC separation, and the lower phase was employed as the mobile phase in the head-to-tail elution mode. In a single run, 400 mg of the crude extract yielded pure senkyunolide-I (6.4 mg), senkyunolide-H (1.7 mg) and FA (4.4 mg) with the purities of 98, 93 and 99%, respectively. The CCC fractions were analyzed by high-performance liquid chromatography, and the structures of the three active compounds were identified by MS and (1)H NMR.     

Control of charcoal rot fungus Macrophomina phaseolina by extracts of Datura metel

Methanolic leaf and fruit extracts of Datura metel were found highly effective in suppressing against Macrophomina phaseolina, the cause of charcoal rot disease. These extracts were further subjected to successive fractionation with n-hexane, chloroform, ethyl acetate and n-butanol. All the concentrations (3.125-200?mg?mL?1) of chloroform, ethyl acetate and n-butanol fractions of leaf extract, and n-hexane fraction of fruit extract completely inhibited the target fungal growth. Two compounds A and B from the n-hexane fraction of fruit extract and compound C from n-butanol fraction of leaf extract were obtained by TLC. Compound B exhibited the best antifungal activity with an MIC value of 7.81?μg?mL?1 that was at par with that of commercial fungicide mancozeb (80% w/w). This study concludes that M. phaseolina can be effectively controlled by natural antifungal compounds in n-hexane fraction of methanolic fruit extract of D. metel.     

Chemical Composition,Antifungal and Anti-Biofilm Activities of Volatile Fractions of Convolvulus althaeoides L. Roots from Tunisia

The antifungal drugs currently available and mostly used for the treatment of candidiasis exhibit the phenomena of toxicity and increasing resistance. In this context, plant materials might represent promising sources of antifungal agents. The aim of this study is to evaluate for the first time the chemical content of the volatile fractions (VFs) along with the antifungal and anti-biofilm of Convolvulus althaeoides L. roots. The chemical composition was determined by gas chromatography coupled to a flame ionization detector and mass spectrometry. In total, 73 and 86 chemical compounds were detected in the n-hexane (VF1) and chloroform (VF2) fractions, respectively. Analysis revealed the presence of four main compounds: n-hexadecenoic acid (29.77%), 4-vinyl guaiacol (12.2%), bis(2-ethylhexyl)-adipate (9.69%) and eicosane (3.98%) in the VF extracted by hexane (VF1). n-hexadecenoic acid (34.04%), benzyl alcohol (7.86%) and linoleic acid (7.30%) were the main compounds found in the VF extracted with chloroform (VF2). The antifungal minimum inhibitory concentrations (MICs) of the obtained fractions against Candida albicans, Candida glabrata and Candida tropicalis were determined by the micro-dilution technique and values against Candida spp. ranged from 0.87 to 3.5 mg/mL. The biofilm inhibitory concentrations (IBF) and sustained inhibition (BSI) assays on C. albicans, C. glabrata and C. tropicalis were also investigated. The VFs inhibited biofilm formation up to 0.87 mg/mL for C. albicans, up to 1.75 mg/mL against C. glabrata and up to 0.87 mg/mL against C. tropicalis. The obtained results highlighted the synergistic mechanism of the detected molecules in the prevention of candidosic biofilm formation.     

Distribution of inorganic arsenic and methylated arsenic in marine organisms

Inorganic arsenic and methylated arsenic compounds in 60 specimens of marine organisms were investigated by hydride generation derivatization and cold-trap gas chromatography–mass spectrometry (GC MS). Chloroform–methanol extracts from seaweeds, shellfish, fish, crustaceans and other marine organisms were separated into water-soluble and lipid-soluble fractions. The arsenic compounds in each fraction were identified and analysed as arsine, methylarsine, dimethylarsine and trimethylarsine. Trimethylarsenic compounds were distributed mainly in the water-soluble fraction of muscle of carnivorous gastropods, crustaceans and fish. The amounts of dimethylated arsenic compounds were found to be larger than that of trimethylated arsenic in the lipid-soluble fraction of fish viscera. Dimethylated arsenic compounds were distributed in the water-soluble fraction of Phaeophyceae.     

Utilization of O-phthalaldehyde-sulphuric acid as a spray reagent in thin-layer chromatographic detection of some indolealkylamines and application to cutaneous secretion extracts of toad species

An expansion of the utilisation of o-phthalaldehyde in sulphuric acid medium as spray reagent was carried out when tryptophan and some tryptophan-derived indole alkylamines such as tryptamine, serotonin, bufotenine, dehydrobufotenine and bufotenidine were examined by thin-layer chromatography. Rf-values and limits of detection ranging from 20 (serotonin) to 100 (dehydrobufotenine) ng per spot were found. Application of this reagent for the detection of some of these compounds was carried out, using either methanolic extracts or column chromatographic fractions of the skin secretion of the toads Bufo ictericus and Odontophrynus cultripes.     

Gel chromatography of steroid oestrogens on sephadex LH-20.

The behavior of 23 steroid oestrogens Sephadex LH-20 using six solvent systems was investigated. The fractions eluted by gel column chromatography were analysed by thin-layer chromatography and spectrometry. The method was used for the radiochemical purity of labelled compounds by isotopic dilution.     

Identification of Mushroom and Murine Tyrosinase Inhibitors from Achillea biebersteinii Afan. Extract

Growing scientific evidence indicates that Achillea biebersteinii is a valuable source of active ingredients with potential cosmetic applications. However, the data on its composition and pharmacological properties are still insufficient. This study aims to optimize the extraction procedure of the plant material, evaluate its phytochemical composition, and compare anti-tyrosinase potential of A. biebersteinii extracts obtained by various methods. In order to identify compounds responsible for the tyrosinase inhibitory activity of A. biebersteinii, the most active anti-tyrosinase extract was fractionated by column chromatography. The fractions were examined for their skin lightening potential by mushroom and murine tyrosinase inhibitory assays and melanin release assay. HPLC-ESI-Q-TOF-MS/MS analysis of the total extract revealed the presence of several phenolic acids, flavonoids, flavonoid glucosides, and carboxylic acid. Among them, fraxetin-8-O-glucoside, quercetin-O-glucopyranose, schaftoside/isoschaftoside, gmelinin B, 1,3-dicaffeoylquinic acid (1,3-DCQA), and ferulic acid were found in the fractions with the highest skin lightening potential. Based on obtained qualitative and quantitative analysis of the fractions, it was assumed that the caffeoylquinic acid derivatives and dicaffeoylquinic acid derivatives are more likely responsible for mushroom tyrosinase inhibitory activity of A. biebersteinii extracts and fractions. Ferulic acid was proposed as the most active murine tyrosinase inhibitor, responsible also for the reduced melanin release from B16F10 murine melanoma cells.     

Phytochemical Fingerprinting and In Vitro Antimicrobial and Antioxidant Activity of the Aerial Parts of Thymus marschallianus Willd. and Thymus seravschanicus Klokov Growing Widely in Southern Kazakhstan

The chemical composition of the hydroethanolic extracts (60% v/v) from the aerial parts of Thymus marschallianus Willd (TM) and Thymus seravschanicus Klokov (TS) from Southern Kazakhstan flora was analyzed together with their hexane fractions. Determination of antibacterial, antifungal and antioxidant activities of both extracts was also performed. RP-HPLC/PDA and HPLC/ESI-QTOF-MS showed that there were some differences between the composition of both extracts. The most characteristic components of TM were rosmarinic acid, protocatechuic acid, luteolin 7-O-glucoside, and apigenin 7-O-glucuronide, while protocatechuic acid, luteolin 7-O-glucoside, luteolin 7-O-glucuronide, and eriodictyol predominated in TS. The content of polyplenols was higher in TS than in TM. The GC-MS analysis of the volatile fraction of both examined extracts revealed the presence of thymol and carvacrol. Additionally, sesquiterpenoids, fatty acids, and their ethyl esters were found in TM, and fatty acid methyl esters in TS. The antioxidant activity of both extracts was similar. The antibacterial activity of TS extract was somewhat higher than TM, while antifungal activity was the same. TS extract was the most active against Helicobacter pylori ATCC 43504 with MIC (minimal inhibitory concentration) = 0.625 mg/mL, exerting a bactericidal effect. The obtained data provide novel information about the phytochemistry of both thyme species and suggest new potential application of TS as a source of bioactive compounds, especially with anti-H. pylori activity.     

LC-MS analysis of oils of Monodora myristica and Monodora tenuifolia and isolation of a novel cyclopropane fatty acid

Abstract

Seeds of Monodora myristica and M. tenuifolia were extracted with hexane and the extracts were subjected to column chromatography, LC-MS and NMR analysis. In addition to masses of previously isolated compounds, other masses corresponding to unidentified compounds from the plants were detected. Using 2?D NMR techniques, one of the fractions from M. tenuifolia was characterised as a novel 13-(2-butylcyclopropyl)-6,9-dodecadienoic acid. However, none of the compounds detected in LC-MS corresponded to the ones previously identified by GC-MS.