Method Development for Purification of γ-oryzanol from Hydrolyzed Rice Bran Acid Oil by Semi-preparative Chromatography.

The objective of this study was to develop a method for isolation and purification of γ-oryzanol from hydrolyzed rice bran acid oil (RBAO) using semi-preparative chromatography by first applying silica coated-thin layer chromatography (TLC) to determine the suitable mobile phase. Subsequently, column chromatography was carried out to determine the effects of purification conditions such as the amount of and particle sizes of the sample silica gel, and elution modes, on the percentage of γ-oryzanol yield and recovery. The results from the TLC suggested that 75:25 (v/v) hexane to ethyl acetate mixture was a suitable mobile phase. The semi-chromatographic results indicated that the column containing 10 g of 25-40 µm silica gel with isocratic elution gave the highest yield (84%) of purified γ-oryzanol (> 95% purity). Further application of a step-gradient elution with 85:15 (v/v), followed by 75:25 (v/v) hexane to ethyl acetate mixture increased chromatographic resolution (Rs), resulting in enhanced separation efficiency, which in turn led to a higher yield of purified γ-oryzanol of 90%.

Estudio comparativo de la actividad anti-fúngica de extractos secuenciales de Fuchsia lycioides contra Candida sp / comparative study of the antifungal activity of sequencial extracts of Fuchsia lycioides against Candida sp

The genus Fuchsia is generally used in herbal preparations to treat conditions caused by microorganisms. Based on the popular use of this type of plants, the objective of this study was to obtain sequential extracts of increasing polarity from the branches of Fuchsia lycioides by maceration at room temperature and by the Soxhlet method at 60ºC, to later evaluate the antifungal capacity of the extracts against different clinical isolates of the Candida genus. The ethyl acetate extract exhibited strong anti-fungal activity, selectively inhibiting C. albicans strains with MIC and CMF values of 10 and 15 µg/mL, respectively; comparable with the drug itraconazole®. The analysis of the extract by GC-MS showed a high concentration of terpenoids (mainly phytol) and phenylpropanoids (mainly cinnamic acid), possibly responsible for the antifungal activity of the ethyl acetate extract of F. lycioides.

El género Fuchsia se usa generalmente en preparaciones de hierbas para tratar afecciones provocadas por microorganismos. En base al uso popular de este tipo de plantas, el objetivo de este estudio fue obtener los extractos secuenciales de polaridad creciente de las ramas de Fuchsia lycioides por maceración a temperatura ambiente y por el método Soxhlet a 60ºC, para luego evaluar la capacidad antifúngica de los extractos frente a diferentes aislados clínicos del genero Candida. El extracto de acetato de etilo exhibió una fuerte actividad antifúngica inhibiendo en forma selectiva las cepas de C. albicans con valores de CMI y de CMF de 10 y 15 µg/mL, respectivamente; comparables con el fármaco itraconazol®. El análisis del extracto por CG-EM mostró una alta concentración de terpenoides (principalmente fitol) y fenilpropanoides (principalmente ácido cinámico), posibles responsables de la actividad antifúngica del extracto de acetato de etilo de F. lycioides.

Functional Profiling and Future Research Direction of Rice Bran Oil in Bangladesh.

Rice bran oil (RBO) has been demonstrated to affect complex malfunctioned conditions such as oxidative stress, hyperlipidemia, hyperglycemia, hypertension, inflammation, abnormal cell growth (cancer), ulceration, immune and cognitive modulation. This unique effect of RBO is due to the presence of well-balanced fatty acid composition and several bioactive compounds, γ- oryzanol (cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, campesterol ferulate, and ß-sitosteryl ferulate), vitamin E (tocopherol and tocotrienol), phytosterols (ß-sitosterol, campesterol and stigmasterol) and other nutrients. The RBO composition of bioactive compounds varied geographically, thus the clear-cut mechanisms of action on complex disease cascades are still required. This review article summarized the RBO compositional profiling and compared it with other edible oils. This article also summarized Bangladesh RBO profiling and their proposed mechanism of action as well as the first line of defense in the prevention, management, and control of complex disease conditions. This review indicates how Bangladesh RBO increase their opportunity to be functional food for 21st century's ailment.

Synthesis, antitumor activity and in silico analyses of amino acid derivatives of artepillin C, drupanin and baccharin from green propolis.

Breast cancer has the highest incidence and mortality in females, while prostate cancer has the second-highest incidence in males. Studies have shown that compounds from Brazilian green propolis have antitumor activities and can selectively inhibit the AKR1C3 enzyme, overexpressed in hormone-dependent prostate and breast tumors. Thus, in an attempt to develop new cytotoxic inhibitors against these cancers, three prenylated compounds, artepillin C, drupanin and baccharin, were isolated from green propolis to synthesize new derivatives via coupling reactions with different amino acids. All obtained derivatives were submitted to antiproliferative assays against four cancer cells (MCF-7, MDA MB-231, PC-3, and DU145) and two normal cell lines (MCF-10A and PNT-2) to evaluate their cytotoxicity. In general, the best activity was observed for compound6e, derived from drupanin, which exhibited half-maximal inhibitory concentration (IC50) of 9.6 ± 3 µM and selectivity index (SI) of 5.5 against MCF-7 cells.In silicostudies demonstrated that these derivatives present coherent docking interactions and binding modes against AKR1C3, which might represent a possible mechanism of inhibition in MCF-7 cells.

EPR imaging of sinapyl alcohol and its application to the study of plant cell wall lignification.

In bioimaging, bioorthogonal chemistry is most often used to visualize chemical reporters by fluorescence in their native environment. Herein, we show that TEMPO-based probes can be ligated to monolignol reporters by Diels-Alder chemistry in plant cell walls, paving the way for the study of lignification by EPR spectroscopy and imaging.

Comparison of flavonoids and phenylpropanoids compounds in Chinese water chestnut processed with different methods.

Different processing methods of Chinese water chestnut (CWC; Eleocharis dulcis (Burm.f.) Trin. ex Hensch.) steaming with skin (WPC), cooking with skin (WPS), steaming with peeling (PS), fresh cutting (FF) and cooking with peeling (PC) were compared. Liquid chromatography-mass spectrometry was used to analyze the metabolic profiles of the processed samples. A total of 454 metabolites, including 123 flavonoids and 57 phenylpropanoids, were characterized. The flavonoid and phenylpropanoid profiles were distinguished using PCA. Eighteen flavonoids and six phenylpropanoids were detected and quantitated in the WPC and WPS samples but not in the FF, PC and PS samples. In addition to the O-hexoside of tricin, kaempferol and luteolin were the predominant flavonoids in the WPC and WPS samples, and all three compounds were higher in the WPC and WPS samples than in the FF sample. This study provides new results regarding differences in the metabolite profile of CWC processed with different methods.

An environmentally friendly sodium dodecyl sulfate-synergistic microwave-assisted extraction followed by ultra-high-performance liquid chromatography with photodiode array method for the simultaneous extraction and determination of iridoids, phenylpropanoids, and lignans from Eucommiae Cortex.

A simple and green sodium dodecyl sulfate-synergistic microwave-assisted extraction method was developed to extract and determine the iridoids, phenylpropanoids, and lignans in Eucommiae Cortex followed by ultra-high-performance liquid chromatography with photodiode array detection. The biodegradable solution (sodium dodecyl sulfate) was used as a promising alternative to organic solvents. The response surface methodology provided the optimum extraction conditions (2 mg/mL sodium dodecyl sulfate, 1100 W microwave power, and 6 min extraction time). The recoveries of three types of components ranged from 95.0 to 105% (RSDs < 5%). The intra- and inter-day precision and accuracy were less than 3.40% and within the range of 97.1-105%, respectively. Compared with other extraction methods, this newly established method was more efficient and environmental friendly. The results demonstrated that sodium dodecyl sulfate-synergistic microwave-assisted extraction followed by ultra-high-performance liquid chromatography with photodiode array method was applicable for the simultaneous extraction and determination of these three types of compounds for quality evaluation of Eucommiae Cortex.

Dynamic analysis of secondary metabolites in various parts of Scrophularia ningpoensis by liquid chromatography tandem mass spectrometry.

The roots of Scrophularia ningpoensis are used as traditional medicines for thousands of years in China, nevertheless the stems and leaves were discarded as non-medicinal parts. Modern research have indicated the chemical constituents in the stems and leaves are similar to the identified in the roots, and the therapeutic effects of stems and leaves are superior to roots for some disease. In the study, the chemical constituents in roots, stems and leaves of S. ningpoensis were analyzed qualitatively by HPLC-Q-TOF-MS/MS. 40 compounds including 17 iridoid glycosides, 15 phenylpropanoids and 8 flavonoids were identified. Meantime, the dynamic accumulations of six index constituents in various parts were measured by HPLC-DAD. The results indicated the S. ningpoensis stems contained high content of aucubin (30.09 mg/g) and harpagide (28.4 mg/g) in August, and the leaves contained high content of harpagoside (12.02 mg/g) in July. The study provides the basis for the full development and utilization of the resource of stems and leaves from S. ningpoensis.

Characterization of the metabolites of TUG-891 in rat, dog, and human hepatocytes using ultra-high-performance liquid chromatography tandem mass spectrometry and nuclear magnetic resonance spectroscopy.

RATIONALE: TUG-891 is a potent and selective agonist of the long chain free fatty acid receptor 4. However, its metabolic profiles have not been revealed. The aim of this study was to investigate the in vitro metabolism of TUG-891 in hepatocytes. METHODS: TUG-891 at a concentration of 20 µM was incubated with rat, dog, and human hepatocytes at 37°C for 120 min. The samples were analyzed using ultra-high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry. The structures of the metabolites were proposed according to their MS/MS product ions. Furthermore, M4 and M5 were biosynthesized using human liver microsomes, and their structures were characterized using 13 C-NMR spectroscopy. RESULTS: Under the current conditions, eight metabolites were detected and structurally identified using high-resolution LC/MS and MS/MS spectra. The metabolites M4 and M5 were unambiguously confirmed to be TUG-891 alcohol and TUG-891 acid, respectively, using 13 C-NMR spectroscopy. Our results revealed that hydroxylation of methyl group at C-21 position to form TUG-891 alcohol (M5) followed by oxidation to yield TUG-891 aldehyde (M7) and carboxylic acid (M4) were the major metabolism processes. Phase II metabolism processes included glucuronidation and sulphation. CONCLUSIONS: Hydroxylation at the C-21 position was the primary metabolic site of TUG-891. This study provided an overview of the metabolic profiles of TUG-891 in hepatocytes.

A comparative study on root and bark extracts of Eleutherococcus senticosus and their effects on human macrophages.

BACKGROUND: Eleutherococcus senticosus or Siberian ginseng is a medicinal plant containing adaptogenic substances believed to regulate immune responses. Both, the root and stem bark are commonly used in traditional medicines. PURPOSE: The purpose of the present study is to chemically characterize E. senticosus root and bark extracts and to compare their effects on functions of human primary macrophages. STUDY DESIGN AND METHODS: HPLC-DAD-MS analysis was used to characterize chemical constituents of alcoholic extracts from E. senticosus root and bark. The data obtained and available databases were combined for network pharmacology analysis. Involvement of predicted pathways was further functionally confirmed by using monocyte-derived human macrophages and endotoxin-free E. senticosus root and bark extracts. RESULTS: Chemical analysis showed that the root extract contained more syringin, caffeic acid, and isofraxidin than the bark extract. At variance, bark extract contained more sesamin and oleanolic acid. Coniferyl aldehyde and afzelin were below the limit of quantification in both extracts. Network pharmacology analysis indicated that constituents of E. senticosus might affect the immune cell phenotype and signaling pathways involved in cell metabolism and cytoskeleton regulation. Indeed, both extracts promoted actin polymerization, migration, and phagocytosis of E. coli by macrophages pointing to macrophage polarization towards the M2 phenotype. In addition, treatment with E. senticosus root and bark extracts decreased phosphorylation of Akt on Ser473 and significantly reduced expression of the hemoglobin scavenger receptor CD163 by macrophages. Neither extract affected expression of CD11b, CD80, or CD64 by macrophages. In addition, macrophages treated with the bark extract, but not with the root extract, exhibited activated p38 MAPK and NF-κB and released increased, but still moderate, amounts of proinflammatory TNF-α and IL-6, anti-inflammatory IL-10, and chemotactic CCL1, which all together point to a M2b-like macrophage polarization. Differently, the root extract increased the IL-4-induced expression of anti-inflammatory CD200R. These changes in monocytes are in agreement with an increased M2a macrophage polarization. CONCLUSION: The ability of E. senticosus root and bark extracts to promote polarization of human macrophages towards anti-inflammatory M2a and M2b phenotypes, respectively, might underlay the immunoregulatory activities and point to potential wound healing promoting effects of this medicinal plant.

Ambrisentan use in a HIV-1 infected patient with end-stage renal disease and pulmonary hypertension: minimal removal by hemodialysis - a case report.

BACKGROUND: Ambrisentan is a selective endothelin receptor antagonist used for the treatment of pulmonary arterial hypertension (PAH). Little is known about ambrisentan removal by hemodialysis in patients with end-stage renal disease (ESRD). CASE PRESENTATION: A 53-year-old woman with HIV/hepatitis C virus (HCV) co-infection, PAH and ESRD on regular hemodialyis was admitted in our hospital due to refractory heart failure while on treatment with bosentan (125 mg twice daily) and tadalafil (20 mg once daily) for PAH and antiretroviral treatment (cART) including darunavir/cobicistat (800/150 mg once daily). Excessive exposure to bosentan due to drug interactions between bosentan and darunavir/cobicistat was suspected. Bosentan was replaced by ambrisentan, with progressive improvement in her clinical condition. Pre- and postdialyzer cocentrations of ambrisentan in plasma were determined and hemodialysis extraction ratio for ambrisentan was 2%. CONCLUSIONS: Our results suggest that hemodialysis results in minimal ambrisentan removal, and therefore no specific ambrisentan dosage adjustment seems to be required in ESRD patients undergoing hemodialysis.

Quantification of Tocopherols, Tocotrienols and γ-Oryzanol Contents of Local Rice Varieties in Northeastern Thailand.

A total of 101 local rice varieties, composted of 85 glutinous and 16 non-glutinous varieties grown in wet season 2016, were analyzed for tocopherols (TOC), tocotrienols (T3) and γ-oryzanol (Orz). Two popular varieties, RD6 and KDML105, were used as standard checks for glutinous and non-glutinous varieties, respectively. γ-TOC was found in all glutinous varieties (0.47-9.78 mg/kg), which were higher than RD6 (0.16 mg/kg). α-TOC was found in 40 varieties (1.02-6.29 mg/kg), only 6 varities were higher than RD6 (3.95 mg/kg). δ-T3 was found in 81 varieties (0.57-7.00 mg/kg), mostly varities were higher than RD6 (0.67 mg/kg). γ-T3 was found in all glutinous varieties (8.00-22.1 mg/kg), while RD6 contained 13.3 mg/kg. For α-T3, it was found in 39 glutinous varieties (1.52-9.94 mg/kg), mostly varities were higher than RD6 (2.62 mg/kg). Orz was found in all glutinous rice varieties (276-638 mg/kg), while RD6 contained 423 mg/kg. γ-TOC was found in all non-glutinous varieties (0.67-5.21 mg/kg), which were higher than KDML105. α-TOC was found in only 5 varieties (1.92-2.83 mg/kg), while KDML105 contained 2.23 mg/kg. δ-T3 was found in all non-glutinous varieties (1.64-8.87 mg/kg), which were higher than KDML105 (0.71 mg/kg). γ-T3 was also found in all non-glutinous varieties (8.80-17.58 mg/kg), while KDML105 contained 15.8 mg/kg. α-T3 was found in 9 non-glutinous varieties (4.56-8.93 mg/kg),while KDML105 contained only 0.86 mg/kg. Orz was also found in all those non-glutinous varieties (272-469 mg/kg), while KDML105 contained 469 mg/kg. These results indicate that γ-T3 was the highest vitamin E isomer present in all rice samples, while γ-TOC, α-TOC, δ-T3 and α-T3 were present in trace amounts. Orz was found in all local rice varieties.

Determination of γ-Oryzanol in Refined Rice Bran Oil by Nuclear Magnetic Resonance Method.

γ-Oryzanol contained as a minor component in rice bran oil (RBO) is a group of phytosterol ferulates. The existence of γ-oryzanol in rice bran oil is very interesting because of the special biological functions. The methods to determine γ-oryzanol in crude RBO include the absorptiometry, thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). However, those methods need much solvent and long operating time. Moreover, some of those methods are not able to apply to refined RBO because of the low sensitivity. We have developed the method to determine γ-oryzanol in refined RBO using proton nuclear magnetic resonance (1H-NMR). Refined RBO had a specific chemical shift due to the methoxy group of ferulate in γ-oryzanol at 3.9 ppm on the 1H-NMR spectrum. The intensity of the chemical shift due to the methoxy group of ferulate was consistent with the amounts of γ-oryzanol in refined RBO. This NMR method was able to quickly and easily determine γ-oryzanol in refined RBO.

Interference of hydroxyphenylpyruvic acid, hydroxyphenyllactic acid and tyrosine on routine serum and urine clinical chemistry assays; implications for biochemical monitoring of patients with alkaptonuria treated with nitisinone.

OBJECTIVES: We have assessed the effect of elevated concentrations of hydroxyphenylpyruvic acid (HPPA), hydroxyphenyllactic acid (HPLA) and tyrosine, on a range of chemistry tests in serum and urine to explore the potential for chemical interference on routine laboratory analyses in patients with alkaptonuria (AKU) treated with nitisinone and similarly implications for patients with hereditary tyrosinemia type 1 (HT-1). MATERIALS AND METHODS: HPPA, HPLA and tyrosine were added separately to pooled serum from subjects without AKU in a range of assays with Roche Modular chemistries. Effects on urine were assessed by changes in urine strip chemistries after mixing a positive control urine with various amounts of the test compounds and reading on a Siemens urine strip meter. RESULTS: No significant effect (p > 0.1) was observed up to 225 µmol/L of HPPA and HPLA, and up to 5000 µmol/L tyrosine, on any of the serum-based assays including those with peroxidase-coupled reaction systems of enzymatic creatinine, urate, total cholesterol, HDL cholesterol and triglyceride. Both the monohydroxy HPPA, and the dihydroxy homogentisic acid (HGA), at increased urine concentrations typical of nitisinone-treated AKU and non-treated AKU respectively, did however show marked negative interference in strip assays for glucose and leucocytes; i.e. those with peroxide-linked endpoints. The effect of increased HPLA was less marked. CONCLUSIONS: In patients with AKU or on nitisinone treatment and HT-1 patients on nitisinone, urine strip chemistry testing should be used sparingly, if at all, to avoid false negative reporting. It is recommended that urine assays should be organised with a suitable specialist laboratory.

Evaluation of γ-oryzanol Accumulation and Lipid Metabolism in the Body of Mice Following Long-Term Administration of γ-oryzanol.

γ-Oryzanol (OZ), abundant in rice bran oil, has gained attention due to its physiological activities (e.g., lipid-lowering effects). However, the absorption and metabolism of orally ingested OZ have not yet been fully elucidated. In this study, diets containing normal or high contents of OZ were fed to obesity model mice for 8 weeks, and OZ concentrations in plasma and organs were analyzed by HPLC-MS/MS. To evaluate the relationship between OZ accumulation and lipid metabolism in vivo, lipid concentrations in the mice plasma and liver were also measured. As a result, the accumulation of intact OZ in plasma and organs was seen in mice fed diets containing OZ, where mice fed diets containing higher OZ contents demonstrated higher levels of OZ accumulation and lower amounts of plasma lipids. These results, in combination with our additional data from a single oral administration test, suggest the possibility that intact OZ, along with its metabolites (e.g., ferulic acid), is biologically-active.

Comparative study on amount of nutraceuticals in by-products from solvent and cold pressing methods of rice bran oil processing.

Rice bran oil (RBO) has become a popular oil globally. However, the RBO extraction process leaves various residue products, which contain bioactive substances of varying potency which could be significant sources of functional ingredients for both food production and pharmaceutical manufacture. The objective of our study was to compare the bioactive substances in various by-products derived from the two rice bran oil processing methods; solvent extraction and cold pressing. The residues from solvent extraction processing contained up to 97.37 mg/100 g of γ-aminobutyric acid in defatted rice bran, and the rice acid oil contained high levels of vitamin E (tocopherols, tocotrienols), up to 120.59 mg/100 g, as well as γ-oryzanol (3829.65 mg/100 g), phytosterol (599.40 mg/100 g), and policosanol compounds (332.79 mg/100 g). All of these values are higher than in the residues derived from cold pressing. Importantly, high amounts of total nutraceuticals (8.3 kg/100 kg) were found in residues from both processing methods, indicating the commercial potential of these residues as a source of functional ingredients for food production, as dietary supplements, and in pharmaceutical manufacture.

Microbial transformation of ambrisentan to its glycosides by Cunninghamella elegans.

The purpose of this paper is to describe the glycosylation of ambrisentan (AMB) by cultures of Cunninghamella elegans ATCC 9245. AMB is an endothelin receptor antagonist, which is used to treat pulmonary arterial hypertension. Filamentous fungi are morphologically complex and may exhibit different forms depending on the species and the nature of the culture medium. A biotransformation study was conducted to investigate the ability of C. elegans to metabolize AMB. Parameters were optimized by testing on different culture media and concentrations, pH, drug concentration, static and shaking conditions. Ambrisentan's metabolite, obtained after 240 h of incubation as a result of glycosylation pathway, was separated by HPLC and determined by high-resolution mass spectrometry. The method showed linearity over 300-1000 µg mL-1 (r = 0.998). Accuracy, precision, robustness and stability studies agree with international guidelines. Results are consistent in accordance with the principles of green chemistry as the experimental conditions had a low environmental impact, and used little solvent.

Phenolic composition, antioxidant and antinociceptive activities of Syringa vulgaris L. bark and leaf extracts.

Metabolite profile, antioxidant and antinociceptive activities of Syringa vulgaris bark and leaf methanolic extracts were investigated. By means of HPLC-DAD-ESI-TOF and HPLC-DAD-ESI-MS/MS, a total of 33 phenolics were identified, including 15 secoiridoids, 6 phenylpropanoids, 3 flavonoids, 3 lignans and 6 low molecular weight phenols. Validated quantitative analysis show that syringin (2.52%) and rutin (1.13%) are the main phenolic compounds in bark and leaf, respectively. Notable radical scavenging and antinociceptive activities of the bark and leaf extracts were confirmed by in vitro DPPH● and ABTS●+ assays and by in vivo hot-plate method in mice, respectively. Our results could lay the scientific basic of future clinical perspectives of lilac bark and leaf.

Thermal rice oil degradation evaluated by UV-Vis-NIR and PARAFAC.

Thermal rice oil degradation was evaluated by UV-Vis and NIR in three-way arrays assembled with samples, different temperatures, and the absorbance at different wavelengths by applying PARAFAC chemometric method. The loadings matrix of the mode corresponding to the samples (scores) contains the information related to the samples. The loadings on the temperature mode resemble kinetics profiles. These profiles change with the nature of the component responsible for the factor and evolve with the heating temperature. The loadings on the spectral mode reveal the antioxidants γ-oryzanol and tocopherol, and oxidation products are the components responsible for the two factors. The results achieved showed that the antioxidants concentration decreases starting at 70 °C while oxidation products start to increase more pronounced after 90 °C. The proposed method is shown to be a simple and fast way to obtain information about the oxidative stability of rice oils.

Simultaneous determination of tocols, γ-oryzanols, phytosterols, squalene, cholecalciferol and phylloquinone in rice bran and vegetable oil samples.

In this study, a simultaneous analytical method of tocols, γ-oryzanols, phytosterols, squalene, cholecalciferol and phylloquinone were developed using HPLC-DAD-FLD. The developed method allowed the quantification of 18 compounds in 30 min. Method validation showed linearity of calibration curves (α = 0.05). RSD of intra-day, inter-day and inter-laboratory precision were less than 4.88%. The limit of detections (LODs) and limit of quantifications (LOQs) were low (0.009-2.166 µg g-1) with recoveries around 96.0-102.9%. Results derived from the established method demonstrated a wide variation of detected compounds in rice bran and vegetable oil samples (22.4-1774.6 µg g-1 tocols, ND-26484 µg g-1 γ-oryzanols, ND-12655 µg g-1 phytosterols, ND-3189 µg g-1 squalene, ND-105.3 µg g-1 cholecalciferol, and ND-54.4 µg g-1 phylloquinone). Thus, the developed HPLC-DAD-FLD method is a powerful analytical tool for the above mentioned compounds useful in food and pharmaceutical application.