Oral tamoxifen used in the prevention and treatment of ductal carcinoma in situ (DCIS) (estrogen-positive) patients has limited acceptance, due to its adverse side effects. The efficacy of tamoxifen is related to its major metabolite, 4-hydroxytamoxifen. Local transdermal therapy of 4-hydroxytamoxifen to the breast might avert the toxicity of oral tamoxifen, while maintaining efficacy. We aim to study the skin irritancy, as well as to evaluate the efficacy of the developed transfersome formulations, with/without emu oil, using a syngeneic mouse model of breast cancer. We also quantified tamoxifen/4-hydroxytamoxifen concentrations in blood plasma and performed histopathology. The skin irritancy test showed that the pure emu oil and transfersome formulations with or without the emu oil did not cause skin irritancy in the animals studied. A sensitive and specific LC-MS/MS method for the quantification of tamoxifen and 4-hydroxytamoxifen was developed and validated. Studies on tumor volume and necrosis (histopathology) using the breast cancer mouse model showed that the 4-OHT transfersomal formulations, with and without emu oil, showed comparable efficacy with that of orally administered tamoxifen. However, the transfersomal formulations, with and without emu oil, resulted in significantly lower (10.24 ± 0.07 and 32.45 ± 0.48 ng/mL, respectively) plasma concentrations of 4-hydroxytamoxifen, compared to the oral tamoxifen (TAMX) group (634.42 ± 7.54 ng/mL). This study demonstrated the potential use of emu oil in a local transdermal formulation for the treatment of breast cancer and its reduced adverse effects.
The proteome data of whole rice grain is considerably limited particularly for rice with pigmentations such as black and red rice. Hence, we performed proteome analysis of two black rice varieties (BALI and Pulut Hitam 9), two red rice varieties (MRM16 and MRQ100) and two white rice varieties (MR297 and MRQ76) using label-free liquid chromatography Triple TOF 6600 tandem mass spectrometry (LC-MS/MS). Our aim was to profile and identify proteins related to nutritional (i.e. antioxidant, folate and low glycaemic index) and quality (i.e. aromatic) traits based on peptide-centric scoring from the Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) approach. Both information dependent acquisition (IDA) and SWATH-MS run were performed in this analysis. Raw data was then processed using ProteinPilot software to identify and compare proteins from the six different varieties. In future, this proteomics data will be integrated with previously obtained genomics [1] and transcriptomics [2] data focusing on the above nutritional and quality traits, with an ultimate aim to develop a panel of functional biomarkers related to those traits for future rice breeding programme. The raw MS data of the pigmented and non-pigmented rice varieties have been deposited to ProteomeXchange database with accession number PXD018338.
The optimum combination of Baccaurea angulata fruit juice (X1: 15 - 85 ratio) and Trigona sp. honey (TH) (X2: 15 - 85 ratio) in developing a high antioxidant soft jelly was investigated based
on the antioxidant capacity (Y1), phenolic (Y2), and flavonoid (Y3) content. Response surface
methodology (RSM), via central composite design (CCD), was used to produce optimal combination effects of the two independent variables (B. angulata fruit juice and TH) for highest
recovery of antioxidant capacity (AC), total phenolic content (TPC), and total flavonoid content
(TFC). The polynomial models generated were satisfactory. The lack-of-fit test were higher
than p > 0.05 for all three analyses, signifying the suitability of the models in accurately predicting the variations. Predicted values of the analysis agreed with those of the experimental values.
An optimum combination of B. angulata fruit juice and TH was developed (ratio 40:40). The
sample also exhibited significant FRAP and DPPH radical scavenging activities. Several
polyphenols were identified for the samples through UHPLC-MS/MS. In conclusion, B. angulata and Trigona sp. honey have high potentials to be used in fortifying the soft jelly samples,
making them prospective food supplements due to their nutritional and health benefits.
The quality of antivenom is governed by its safety and efficacy profiles. These quality characteristics are much influenced by the purity of antivenom content. Rigorous assessment and meticulous monitoring of antivenom purity at the preclinical setting is hence crucial. This study aimed to explore an integrative proteomic method to assess the physicochemical purity of four commercially available antivenoms in the region. The antivenoms were subjected to Superdex 200 HR 10/30 size-exclusion fast-protein liquid chromatography (SE-FPLC). The proteins in each fraction were trypsin-digested and analyzed by nano-ESI-liquid chromatography-tandem mass spectrometry (LC-MS/MS). SE-FPLC resolved the antivenom proteins into three major protein components of very high (>200 kDa), high (100-120 kDa) and medium (<60 kDa) molecular weights. The major components (80-95% of total proteins) in the antivenoms were proteins of 100-120 kDa consisting of mainly the light and partially digested heavy immunoglobulin chains, consistent with F(ab')2 as the active principle of the antivenoms. However, LC-MS/MS also detected substantial quantity of large proteins (e.g. alpha-2-macroglobulins), immunoglobulin aggregates and impurities e.g. albumins in some products. The method is practical and able to unveil the quantitative and qualitative aspects of antivenom protein compositions. It is therefore a potentially useful preclinical assessment tool of antivenom purity.
Strobilanthes crispus and Clinacanthus nutans are popular herbal plants in the Southeast
Asian region. The present work was aimed at determining the antioxidant activities and the
associated components in the leaf extracts of both species using polar and non-polar solvents
namely water, methanol, ethyl acetate, and hexane. The total phenolic content (TPC) and total
flavonoid content (TFC) were higher in the leaf extracts of S. crispus as compared to C.
nutans. Among the solvents, methanol was the best solvent in extracting the antioxidant
components for S. crispus (TPC: 159.85 ± 0.89 mg GAE/g extract and TFC: 955.47 ± 2.66 mg
RE/g extract). However, for C. nutans, its methanolic extract yielded the highest TPC (36.39
± 0.17 mg GAE/g extract), whereas ethyl acetate yielded the highest TFC (229.61 ± 7.81 mg
RE/g extract). The high levels of both TPC and TFC contributed to the antioxidant activities
of S. crispus extract as reflected in the methanolic extract attaining the highest level of
antioxidant activities, measured by ferric reducing antioxidant power (FRAP) (6.84 ± 1.12
mmol Fe2+/g extract), DPPH radical scavenging (IC50: 203.60 ± 7.28 μg/mL), and Trolox
equivalent antioxidant capacity (TEAC) (1.01 ± 0.01 mmol TE/g extract) assays. This
contrasted with C. nutans which showed lower antioxidant activities owing to its lower TPC
and TFC. Correlation analysis revealed significant correlations (p < 0.05, r = 0.915 - 0.985)
between both TPC and TFC in S. crispus and antioxidant activities. However, only TPC of C.
nutans showed a significant correlation with FRAP values (r = 0.934). Further tentative
identification of the constituents in the extracts using HPLC-ESI-QToF-MS/MS revealed the
existence of 20 polyphenolic compounds in both S. crispus and C. nutans, which were likely
responsible for their antioxidant activities. In addition, 15 polyphenolic compounds classified
as chalcones, isoflavanoids, flavones, and flavonols have not been previously reported in both
species. The methanolic extracts of both species yielded a higher content of antioxidants, with
S. crispus offering a richer source of dietary antioxidants as compared to C. nutans. However,
further study is needed to identify their bioactivities in relation to their bioactive components.
Drawbacks associated with the use of chemical fungicides to control plant pathogenic fungi such as Botrytis cinerea stimulate the need for alternatives. Therefore, the present study was carried out to determine the antifungal potentials of Moringa oleifera extracts against B. cinerea. Phytochemical analysis using qualitative chemical tests revealed the presence of huge amount of crucial phytochemicals compounds like phenolic compounds, alkaloids and saponins in the M. oleifera leaf extract. Antifungal bioassay of the crude extracts indicated better mycelial growth inhibition by methanol leaf extract (99%). The minimum inhibitory concentration (MIC) was 5 mg/ml with 100% spore germination inhibition and minimum fungicidal concentration (MFC) was 10 mg/ml with 98.10% mycelial growth inhibition using broth micro dilution and poisoned food techniques. Gas chromatography-mass spectrometry (GC-MS) analysis led to the identification of 67 volatile chemical compounds in the leaf extract with 6-decenoic acid (Z)- (19.87%) was the predominant compound. Further chemical elucidation of the crude extracts performed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) showed the presence of non-volatile chemical compounds, mostly flavones, flavonoids and phenolic acids (i.e. quercetin and kaempferol). Scanning electron microscopy and transmission electron microscopy analysis showed positive effect of M. oleifera leaf extract on the treated conidia and mycelium of B. cinerea. Findings revealed that irreversible surface and ultra-structural changes with severe detrimental effects on conidia and mycelium morphology compared to control treatment. Overall findings suggested that M. oleifera leaf extract is a promising candidate for biological control of fungal pathogens, thus limiting overdependence on chemical fungicides.
A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous quantification of apomorphine and its metabolites apomorphine sulfate and norapomorphine in human plasma for supporting clinical development of a novel apomorphine sublingual thin film (APL) for the treatment of Parkinson's disease. Analytes and internal standards (IS) were extracted from human plasma by Oasis HLB SPE cartridge, followed by a reversed phase LC-MS/MS analysis using multiple reaction monitoring (MRM) in positive mode (m/z 268 → 237 for apomorphine, 348 → 237 for apomorphine sulfate, and 348 → 237 for norapomorphine). Stable isotope-labeled compounds were used as IS for respective analytes. The validated curve ranges were 0.02-20 ng/mL, 10-1000 ng/mL, and 0.5-20 ng/mL for apomorphine, apomorphine sulfate and norapomorphine, respectively. Extraction recoveries were found to be 73.4 % (apomorphine), 81.1 % (apomorphine sulfate), and 58.6 % (norapomorphine). Established long-term plasma frozen storage stabilities were 504 days at -20 °C and276 days at -60 °C, respectively. The method has been successfully used for analyzing pharmacokinetics (PK) samples collected from a comparative bioavailability study of APL and the marketed apomorphine subcutaneous (s.c.) product Apo-go®. The results demonstrated that the 15-mg APL film administrated via sublingual produced comparable PK characteristics of apomorphine when compared to the commercial product Apo-go (2-mg) via s.c. administration, hence establishing the dose regimen for this sublingual formulation. It was also noticed that the sublingual 15-mg APL film produced a significantly higher apomorphine sulfate metabolite level than the 2-mg s.c. Apo-go, and both treatments yielded a negligible level of norapomorphine metabolite in humans.
Selected Australian native fruits such as Davidson's plum, finger lime and native pepperberry have been reported to demonstrate potent antioxidant activity. However, comprehensive metabolite profiling of these fruits is limited, therefore the compounds responsible are unknown, and further, the compounds of nutritional value in these native fruits are yet to be described. In this study, untargeted and targeted metabolomics were conducted using the three fruits, together with assays to determine their antioxidant activities. The results demonstrate that targeted free and hydrolysed protein amino acids exhibited high amounts of essential amino acids. Similarly, important minerals like potassium were detected in the fruit samples. In antioxidant activity, Davidson's plum reported the highest activity in ferric reducing power (FRAP), finger lime in antioxidant capacity (ABTS), and native pepperberry in free radical scavenging (DPPH) and phosphomolybdenum assay. The compounds responsible for the antioxidant activity were tentatively identified using untargeted GC×GC-TOFMS and UHPLC-QqQ-TOF-MS/MS metabolomics. A clear discrimination into three clusters of fruits was observed using principal component analysis (PCA) and partial least squares (PLS) analysis. The correlation study identified a number of compounds that provide the antioxidant activities. GC×GC-TOFMS detected potent aroma compounds of limonene, furfural, and 1-R-α-pinene. Based on the untargeted and targeted metabolomics, and antioxidant assays, the nutritional potential of these Australian bush fruits is considerable and supports these indigenous fruits in the nutraceutical industry as well as functional ingredients for the food industry, with such outcomes benefiting Indigenous Australian communities.
Stingless bee (Meliponini) honey has long been considered a high-value functional food, but the perceived therapeutic value has lacked attribution to specific bioactive components. Examination of honey from five different stingless bee species across Neotropical and Indo-Australian regions has enabled for the first time the identification of the unusual disaccharide trehalulose as a major component representing between 13 and 44 g per 100 g of each of these honeys. Trehalulose is an isomer of sucrose with an unusual α-(1 → 1) glucose-fructose glycosidic linkage and known acariogenic and low glycemic index properties. NMR and UPLC-MS/MS analysis unambiguously confirmed the identity of trehalulose isolated from stingless bee honeys sourced across three continents, from Tetragonula carbonaria and Tetragonula hockingsi species in Australia, from Geniotrigona thoracica and Heterotrigona itama in Malaysia and from Tetragonisca angustula in Brazil. The previously unrecognised abundance of trehalulose in stingless bee honeys is concrete evidence that supports some of the reported health attributes of this product. This is the first identification of trehalulose as a major component within a food commodity. This study allows the exploration of the expanded use of stingless bee honey in foods and identifies a bioactive marker for authentication of this honey in associated food standards.
Amoebiasis, caused by Entamoeba histolytica, is one of the leading parasitic infections in the world. This study was aimed at profiling antigenic membrane proteins of a virulent variant of E. histolytica HM-1:IMSS. The membrane proteins were extracted using ProteoExtract® kit (Merck, Darmstadt, Germany) or conventional method, separated using OFFGEL 3100 fractionator (Agilent Technologies, Santa Clara, California), followed by SDS-PAGE and Western blot analysis. Selected antigenic membrane proteins were identified using LC-ESI-MS/MS. Subsequently, the proteins were classified according to their biological processes and predictions were made on membrane and membrane-associated proteins. When the proteins were probed with pooled sera from amoebic liver abscess (ALA) patients, 10 and 15 antigenic proteins with molecular weights 25 to 200 kDa were identified using the ProteoExtract® kit and conventional method, respectively. LC-ESI-MS/MS identified 13 antigenic proteins, and both extraction methods predicted six of them as membrane and membrane-associated proteins. The topmost biological processes which comprised of six proteins were involved in cellular processes.. These antigenic membrane proteins merit further investigations as potential candidates for vaccine studies.
This article summarises the current methods for total malachite green (MG) detection which is known as a sum of MG and leuco-malachite green (LMG) that has been used extensively in aquaculture as fungicide, dye color in textile and other purposes in food industries. LMG is a reducing form of MG, where the MG is easily reduced due to the photo-oxidative de-methylation process. Nevertheless, the use of MG had become an issue due to its toxicity effects. Many analytical instruments such as HPLC, LC—MS/MS, GC—MS, and spectrometry have been widely used for detection of MG. However, these methods require long time sample preparation and analysis, expensive, use hazardous reagents and indirect measurements. Hence, other analytical methods which are more sensitive, safe, rapid, inexpensive and portable are required. Alternatively, biosensors promise a more sensitive and rapid detection method for MG and LMG.
Background:Helicobacter pylori colonizes the gastric mucosa of more than half of the world's population. There is increasing evidence H. pylori protects against the development of obesity and childhood asthma/allergies in which the development of these diseases coincide with transient dysbiosis. However, the mechanism underlying the association of H. pylori eradication with human metabolic and immunological disorders is not well-established. In this study, we aimed to investigate the local and systemic effects of H. pylori eradication through untargeted fecal lipidomics and plasma metabolomics approaches by liquid chromatography mass spectrometry (LC-MS). Results: Our study revealed that eradication of H. pylori eradication (i.e., loss of H. pylori and/or H. pylori eradication therapy) changed many global metabolite/lipid features, with the majority being down-regulated. Our findings primarily show that H. pylori eradication affects the host energy and lipid metabolism which may eventually lead to the development of metabolic disorders. Conclusion: These predictive metabolic signatures of metabolic and immunological disorders following H. pylori eradication can provide insights into dynamic local and systemic metabolism related to H. pylori eradication in modulating human health.
Fruit ripening is a complex phenomenon involving a series of biochemical, physiological and organoleptic changes. Ripening process in mangosteen (Garcinia mangostana Linn.) is unique of which the fruit will only ripen properly if harvested during its middle stage (emergence of purple/pink colour) but not earlier (green stage). The knowledge on the molecular mechanism and regulation behind this phenomenon is still limited. Hence, electrospray ionization liquid chromatography mass spectrometry (ESI-LC-MS) based metabolomics analysis was applied to determine the metabolome of mangosteen ripening. Specifically, mangosteen pericarp, aril and seed were collected at four different ripening stages (stage 0: green, stage 2: yellowish with pink patches, stage 4: brownish red and stage 6: dark purple) and subjected to metabolite profiling analysis. The data provided in this article have been deposited to the EMBL-EBI MetaboLights database (DOI: 10.1093/nar/gks1004. PubMed PMID: 23109552) with the identifier MTBLS595. The complete dataset can be accessed here https://www.ebi.ac.uk/metabolights/MTBLS595.
With a diverse host range, Meloidogyne incognita (root-knot nematode) is listed as one of the most economically important obligate parasites of agriculture. This nematode species establishes permanent feeding sites in plant root systems soon after infestation. A compatible host-nematode interaction triggers a cascade of morphological and physiological process disruptions of the host, leading to pathogenesis. Such disruption is reflected by altered gene expression in affected cells, detectable using molecular approaches. We employed a high-throughput proteomics approach to elucidate the events involved in a compatible banana- M. incognita interaction. This study serves as the first crucial step in developing natural banana resistance for the purpose of biological-based nematode management programme. We successfully profiled 114 Grand naine root proteins involved in the interaction with M. incognita at the 30th- and 60th- day after inoculation (dai). The abundance of proteins involved in fundamental biological processes, cellular component organisation and stress responses were significantly altered in inoculated root samples. In addition, the abundance of proteins in pathways associated with defence and giant cell maintenance in plants such as phenylpropanoid biosynthesis, glycolysis and citrate cycle were also implicated by the infestation.
The aim of this study was to investigate the cytotoxic, antiproliferative activity and the induction of apoptosis by L-amino acid oxidase isolated from Calloselasma rhodostoma crude venom (CR-LAAO) on human colon cancer cells. CR-LAAO was purified using three chromatographic steps: molecular exclusion using G-50 gel filtration resin, ion-exchange by MonoQ column and desalted on a G25 column. The purity and identity of the isolated CR-LAAO was confirmed by SDS-PAGE and LC-MS/MS. CR-LAAO demonstrated time- and dose-dependent cytotoxic activity on SW480 (primary human colon cancer cells) and SW620 (metastatic human colon cancer cells) with an EC50 values of 6 μg/ml and 7 μg/ml at 48 hr, respectively. Quantification of apoptotic cells based on morphological features demonstrated significant increase in apoptotic cell population in both SW480 and SW620 cells which peaked at 48 hr. Significant increase in caspase-3 activity and reduction in Bcl-2 levels were demonstrated following CR-LAAO treatment. These data provide evidence on the potential anticancer activity of CR-LAAO from the venom of C. rhodostoma for therapeutic intervention of human colon cancer.
Natural antioxidants in spices and herbs have attracted considerable attention as potential inhibitors against the formation of mutagenic heterocyclic amines (HCAs) in heat-processed meat. In this study, the inhibitory activity of four spices/herbs and their mixtures on HCAs formation in grilled beef were examined. A simplex centroid mixture design with four components comprising turmeric, curry leaf, torch ginger and lemon grass in 19 different proportions were applied on beef samples before grilling at 240 ºC for 10 min. The HCAs were extracted from the samples using solid phase extraction (SPE) method and analysed using Liquid chromatography mass spectrometry LC-MS/MS. All spices/herbs in single or mixture forms were found to reduce total HCA concentrations in marinated grilled beef ranging from 21.2% for beef marinated with curry leaf to 94.7% for the combination of turmeric and lemon grass (50:50 w/w). At the optimum marinade formula (turmeric: lemon grass 52.4%: 47.6%), concentration of 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), Harman, Norharman and AαC were 2.2, 1.4, 0.5, 2.8 and 1.2 ng/g, respectively. The results of the mutagenic activity demonstrated that this optimised marinade formula significantly (p
Mitragyna speciosa is a psychoactive plant known as "ketum" in Malaysia and "kratom" in Thailand. This plant is distinctly known to produce two important alkaloids, namely mitragynine (MG) and 7-hydroxymitragynine (7-OH-MG) that can bind to opioid receptors [1]. MG was reported to exhibit antidepressant properties in animal studies [2]. These compounds were also proposed to have the potential to replace opioid analgesics with much lower risks of side effects [3]. To date, there are only over 40 metabolites identified in M. speciosa [4,5]. To obtain a more complete profile of secondary metabolites in ketum, we performed metabolomics study using mature leaves of the green M. speciosa variety. The leaf samples were extracted using methanol prior to liquid chromatography-electrospray ionization-time of flight-mass spectrometry (LC-ESI-TOF-MS) analysis. This data can be useful to for the identification of unknown metabolites that are associated with alkaloid biosynthesis pathway in M. speciosa.
Snake venoms are complex mixtures of proteins and peptides that play vital roles in the survival of venomous snakes. As with their diverse pharmacological activities, snake venoms can be highly variable, hence the importance of understanding the compositional details of different snake venoms. However, profiling venom protein mixtures is challenging, in particular when dealing with the diversity of protein subtypes and their abundances. Here we described an optimized strategy combining a protein decomplexation method with in-solution trypsin digestion and mass spectrometry of snake venom proteins. The approach involves the integrated use of C18 reverse-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and nano-electrospray ionization tandem mass spectrometry (nano-ESI-LC-MS/MS).
With varied, brightly patterned wings, butterflies have been the focus of much work on the evolution and development of phenotypic novelty. However, the chemical structures of wing pigments from few butterfly species have been identified. We characterized the orange wing pigments of female Elymnias hypermnestra butterflies (Lepidoptera: Nymphalidae: Satyrinae) from two Southeast Asian populations. This species is a sexually dimorphic Batesian mimic of several model species. Females are polymorphic: in some populations, females are dark, resemble conspecific males, and mimic Euploea spp. In other populations, females differ from males and mimic orange Danaus spp. Using LC-MS/MS, we identified nine ommochrome pigments: six from a population in Chiang Mai, Thailand, and five compounds from a population in Bali, Indonesia. Two ommochromes were found in both populations, and only two of the nine compounds have been previously reported. The sexually dimorphic Thai and Balinese populations are separated spatially by monomorphic populations in peninsular Malaysia, Singapore, and Sumatra, suggesting independent evolution of mimetic female wing pigments in these disjunct populations. These results indicate that other butterfly wing pigments remain to be discovered.
Wax apple is one of the underutilized fruits that is considered a good source of fibers, vitamins, minerals as well as antioxidants. In this study, a comparative analysis of the developments of wax fruit ripening at the proteomic and metabolomic level was reported. 2D electrophoresis coupled with MALDI-TOF/TOF was used to compare the proteome profile from three developmental stages named immature, young, and mature fruits. In general, the protein expression profile and the identified proteins function were discussed for their potential roles in fruit physiological development and ripening processes. The metabolomic investigation was also performed on the same samples using quadrupole LC-MS (LC-QTOF/MS). Roles of some of the differentially expressed proteins and metabolites are discussed in relation to wax apple ripening during the development. This is the first study investigating the changes in the proteins and metabolites in wax apple at different developmental stages. The information obtained from this research will be helpful in developing biomarkers for breeders and help the plant researchers to avoid wax apple cultivation problems such as fruit cracking.