Semi-preparative Chromatography Research Articles

Efficient Enzymatic Enrichment of High-purity Nervonic Acid from Malania oleifera Seed Oil.

Nervonic acid (NA) is a monounsaturated fatty acid vital for brain health and is of emerging importance in various industrial applications, including therapeutics, food, and cosmetics. Given the growing demands of the food and pharmaceutical industries, there's a pressing need for high-purity NA. Previously, NA constituents in plant seed oils were chemically transformed into nervonic acid ethyl ester (NAEE) to facilitate extraction from seed oils. In this study, we present an enzymatic approach to convert NA constituents in Malania oleifera seed oil to NAEE. Combined with the utilization of the semi-preparative chromatography, we achieved a remarkable purity of 97.52% NAEE. Compared to conventional chemical preparations characterized by multiple steps, prolonged processing times, and low yields and purities, our enzymatic method stands out as a more efficient and advantageous alternative. On top of that, this innovative approach is environmentally friendly and circumvents health and safety issues associated with chemical processes.

Characterization of high value products in the side-stream of Laminaria hyperborea alginate production - Targeting the phenolic content

Sustainable production based on renewable biomass and efficient bioprocesses are important elements in the growing blue bioeconomy. The traditional Laminaria hyperborea alginate production disposes approximately 80 % of the raw material, ignoring large amounts of potential high-value products from the alga. Particularly, the leaf fraction of the seaweed is often disregarded. This study aimed to characterize high value products from the alginate production side-stream – focusing on the leaf biomass and particularly targeting the phenolic content. After extraction and solvent optimization, 60 % methanol was used for the extraction. The extract was further purified with PuriFlash and semi-preparative chromatography and increasing phenolic selectivity and purity was observed with TPC and qNMR, as well as antioxidant activity (ORAC). In the purified fractions, the LR LC-MS analyses displayed several masses, where 96 % (n = 1376) were of lower molecular weights (< 800 Da). Fifteen high value compounds were further identified using HR LC-MS (MS/MS) and/or NMR. This also included non-phenolics such as fucoxanthin, aliphatic acids and mannitol. Nonetheless, most compounds were identified as the targeted phenolics, consisting of lower molecular weight phenolic acids (salicylic acid, veratric acid, 5-caboxyvanillic acid, sinapic acid, 5-sulfosalicylic acid, vanillic acid 4-sulfate, and dihydrocaffeic acid 3-sulfate) and phlorotannins (trimer, tetramer, hexamer, and a sulfated dimer). None of the identified phenolics have previously been reported in L. hyperborea. In general, a high occurrence of sulfated phenolic compounds was observed and a sulfated diphlorethol/difucol was characterized for the first time. The isolation and characterization of high value components in the leaf biomass of L. hyperborea strongly supports the development of a total utilization of commercial alginate production. The characterization also adds information on the phenolic content of seaweeds at a molecular level, valuable to research on seaweed biosynthesis and development, chemical ecology, and ocean monitoring.

Toxicity of the 3,4-Methylenedioxymethamphetamine and Its Enantiomers to Daphnia magna after Isolation by Semipreparative Chromatography.

MDMA (3,4-methylenedioxymethamphetamine) is a chiral psychoactive recreational drug sold in illicit markets as racemate. Studies on the impact of MDMA on aquatic organisms are scarce. While enantioselectivity in toxicity in animals and humans has been reported, none is reported on aquatic organisms. This study aimed to investigate the ecotoxicological effects of MDMA and its enantiomers in Daphnia magna. For that, enantiomers (enantiomeric purity > 97%) were separated by liquid chromatography using a homemade semipreparative chiral column. Daphnids were exposed to three concentrations of (R,S)-MDMA (0.1, 1.0 and 10.0 µg L-1) and two concentrations of (R)- and (S)-enantiomers (0.1 and 1.0 µg L-1) over the course of 8 days. Morphophysiological responses were dependent on the substance form and daphnia development stage, and they were overall not affected by the (R)-enantiomer. Changes in swimming behaviour were observed for both the racemate and its enantiomers, but enantioselective effects were not observed. Reproductive or biochemical changes were not observed for enantiomers whereas a significant decrease in acetylcholinesterase and catalase activity was noted at the highest concentration of (R,S)-MDMA (10 µg L-1). Overall, this study showed that sub-chronic exposure to MDMA racemate and its enantiomers can interfere with morphophysiological and swimming behaviour of D. magna. In general, the (R)-enantiomer demonstrated less toxicity than the (S)-enantiomer.

Chiral Separation, Configuration Confirmation and Bioactivity Determination of the Stereoisomers of Hesperidin and Narirutin in Citrus reticulata Blanco.

Hesperidin and narirutin are a class of flavanone glycosides, which are the main active constituents in Citrus reticulata Blanco. In the present study, a chiral HPLC-UV method with amylose tris(3,5-dimethylphenylcarbamate) as a stationary phase under a normal-phase mode was used to achieve the stereoselective separation of the C-2 diastereomers of hesperidin and narirutin simultaneously. The single epimer was then successfully prepared by applying semi-preparative chromatography, whose absolute configuration (R/S) was characterized by combining the experimental electronic circular dichroism (ECD) detection with time-dependent density functional theory (TDDFT) calculations. The epimer composition of these two chiral flavanone glycosides in Citrus reticulata Blanco was then determined, which was found to be slightly different in the herbs from different production regions. The anti-inflammatory activity of each prepared single epimer was further evaluated, and some differences between one pair of epimers of hesperidin and narirutin were observed, which suggested that the presence of different epimers should be considered in the quality evaluation and control of natural medicine.

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%.

Study of 2,6-di(propan-2-yl)phenol stability in biomaterial

The aim of this study is to research the stability of 2.6-di(propan-2-yl)phenol in biomaterial. GC-MS (column DB-5MS EVIDEX (25 m×0.2 mm); stationary liquid phase of 5%-phenyl-95% dimethylpolysiloxane), TLC (Sorbfil plates, mobile phase of hexane-diethyl ether (9:1) and spectrophotometry (solvent medium - 95% ethanol) were used as methods of analysis. 2.6-di(propan-2-yl)phenol was isolated from the biomatrix (liver tissue) by infusion with a mixture of ethyl acetate-acetone (7:3). The analyte was purified by combining extraction (water-ethyl acetate system) and semi-preparative chromatography on a column of silica gel L 40/100 µm, eluent - hexane-acetone (7:3). It was found that at -22 °C, 0 °C, 12 °C, 20 °C and 30 °C 2.6-di(propan-2-yl)phenol can be present in the liver tissue for 119, 98, 70, 56 and 42 days, respectively. The possibility of mathematical description of analyte decomposition dynamics in biomaterial (liver tissue) at the considered temperatures on the basis of hyperbola equation has been studied. The experimentally calculated coefficients in the hyperbola equation (km) for temperatures -22 °C, 0 °C, 12 °C, 20 °C and 30 °C are equal to 1823, 1130, 697, 510, and 255, respectively. The dependence km on the conserving temperature (tо) was educed. The equation for the description of dependence is offered: km=30.61∙(50-to)-402.39. It is shown that this equation can be the basis for prediction of 2.6-di(propan-2-yl)phenol stability in biomaterial (liver tissue) in the temperature range from -22 °C to 30 °C.

Stereoselective separation, configuration determination, and quantification of flavanone glycosides in Citrus using chiral HPLC-UV in combination with CD spectra.

Flavanone glucosides, such as naringin and neohesperidin, are present in specific Citrus species and manifest a chiral center at the C-2 position of their flavanone moiety. This study successfully achieved the simultaneous stereoselective separation of the C-2 diastereomers of naringin, neohesperidin and hesperidin, as well as the partial separation of narirutin using a chiral high performance liquid chromatography with ultraviolet detection method with cellulose tris(3,5-dichlorophenylcarbamate) as the stationary phase under normal-phase mode. The mobile phase comprised n-hexane and ethanol (containing 0.25% formic acid) at a proportion of 65 : 35 (v/v) with a flow rate of 0.6 mL min-1. Each single epimer of chiral flavanone glycosides was prepared using chiral semi-preparative chromatography, and the absolute configuration was then characterized by combining the experimental electronic circular dichroism detection and time-dependent density functional theory calculations. The epimer composition of each chiral flavonoid glycoside in Fructus aurantii (Zhiqiao) and Fructus aurantii immaturus (Zhishi) was determined revealing variations among herbs collected from different production regions. Additionally, the epimer composition was found to be related to the harvesting time of the herbs. Considering the safety and efficacy, the existence of epimers of different stereo-configurations should be given more attention in the quality evaluation of natural drugs.

A Charge Variant of Bevacizumab Offers Enhanced FcRn-Dependent Pharmacokinetic Half-Life and Efficacy.

Lysine variants of monoclonal antibodies (mAbs) result from incomplete clipping of the C-terminal lysine residues of the heavy chain. Although the structure of the lysine variants has been determined for several mAb products, a detailed study that investigates the impact of lysine charge variants on PK/PD and preclinical safety is yet to be published. An in-depth investigation of the impact of C- terminal lysine clipping of mAbs on safety and efficacy for bevacizumab charge variants. Charge variant isolation using semi-preparative chromatography is followed by a comparative analysis of FcRn binding, pharmacokinetics, and pharmacodynamics in relevant animal models. K1 variant exhibited improved FcRn binding affinity (4-fold), half-life (1.3-fold), and anti-tumor activity (1.3-fold) as compared to the K0 (main) product. However, the K2 variant, even though exhibited higher FcRn affinity (2-fold), displayed lower half-life (1.6-fold) and anti-tumor activity at medium and low doses. Differential proteomic analysis revealed that seven pathways (such as glycolysis, gluconeogenesis, carbon metabolism, synthesis of amino acids) were significantly enriched. Higher efficacy of the K1 variant is likely due to higher bioavailability of the drug, leading to complete downregulation of the pathways that facilitate catering of the energy requirements of the proliferating tumor cells. On the contrary, the K2 variant exhibits a shorter half-life, resulting only in partial reduction in the metabolic/energy requirements of the growing tumor cells. Overall, we conclude that the mAb half-life, dosage, and efficacy of a biotherapeutic product are significantly impacted by the charge variant profile of a biotherapeutic product.

Separation and identification of an abundant trigalloylglucose from special tea genetic resources

Galloylglucoses can extensively and intensively interact with proteins and possess many biological activities and exhibit perfect health benefits. A novel galloylglucose had been observed and separated from fresh shoots of some special tea genetic resources, and was characterized as a trigalloylglucose isomer with mass parent ion [M-H]^- at m/z 635.10 and daughter ions at m/z 483.08, 465.08, 313.02 and 168.98 and with UV absorbance peaks at 221nm and 278nm after being measured with UPLC-DAD-MS/MS. The trigalloylglucose was highly purified by semi-preparative chromatography and identified as 1,4,6-tri-O-galloyl-<italic>β</italic>-glucopyranose (1,4,6-TGG) by ¹H-NMR, ¹³C-NMR, H¹-H¹ COSY and HMQC. Antioxidant capacity of the 1,4,6-TGG was 2.68~5.77 μmol TEAC/μmol, and significantly higher (42.7−419.4%) than that of ascorbic acid, but similar to another well-known powerful antioxidant epigallocatechin gallate in tea, indicating the 1,4,6-TGG could be applied in the development of functional foods and pharmaceuticals in the future.

Multivariable optimization of ultrasound-assisted extraction for the determination of phenolic and antioxidants compounds from arrayan (Luma apiculata (DC.) Burret) leaves by microplate-based methods and mass spectrometry

The present study reports a multivariable optimization of ultrasound-assisted extraction of phenolic compounds from arrayan leaves. The parameters ethanol percentage in extraction solvent, temperature and extraction time, and mass to solvent ratio were optimized applying full factorial and central composite designs. According to the models, the optimal extraction conditions were: 42% of ethanol, extraction time of 27 min, extraction temperature of 50ºC and a mass to solvent ratio of 1:33.4 g mL−1. Under these conditions, a total phenolic content (TPC) of 128.16 ± 1.18 and 593.64 ± 6.49 mg of gallic acid equivalent per g of dry weight (DW) were determined in raw extract (RE) and polyphenols-rich extract (PRE), which was obtained by semi-preparative chromatography on XAD-7 column. Antioxidant capacity determined via FRAP analysis showed values of 1349.53 ± 28.99 and 6175.47 ± 127.64 µmol Trolox equivalent per g of DW for RE and PRE, and DPPH IC50 values of 831.40 ± 0.80 and 132.21 ± 2.51 µg mL−1, respectively. Polyphenols profile analyzed by liquid chromatography-mass spectrometry showed the presence of quercetin 3-ß-D-glucoside, myricetin, quercetin, kaempferol 3-glucoside and gallic acid; the latter compounds are reported for the first time in arrayan leaves. Due to the presence of polyphenols, antiviral activity was assayed over norovirus and hepatitis A showing a significant dose-dependent effect.

Usnic Acid and Usnea barbata (L.) F.H. Wigg. Dry Extracts Promote Apoptosis and DNA Damage in Human Blood Cells through Enhancing ROS Levels.

Nowadays, numerous biomedical studies performed on natural compounds and plant extracts aim to obtain highly selective pharmacological activities without unwanted toxic effects. In the big world of medicinal plants, Usnea barbata (L) F.H. Wigg (U. barbata) and usnic acid (UA) are well-known for their therapeutical properties. One of the most studied properties is their cytotoxicity on various tumor cells. This work aims to evaluate their cytotoxic potential on normal blood cells. Three dry U. barbata extracts in various solvents: ethyl acetate (UBEA), acetone (UBA), and ethanol (UBE) were prepared. From UBEA we isolated usnic acid with high purity by semipreparative chromatography. Then, UA, UBA, and UBE dissolved in 1% dimethyl sulfoxide (DMSO) and diluted in four concentrations were tested for their toxicity on human blood cells. The blood samples were collected from a healthy non-smoker donor; the obtained blood cell cultures were treated with the tested samples. After 24 h, the cytotoxic effect was analyzed through the mechanisms that can cause cell death: early and late apoptosis, caspase 3/7 activity, nuclear apoptosis, autophagy, reactive oxygen species (ROS) level and DNA damage. Generally, the cytotoxic effect was directly proportional to the increase of concentrations, usnic acid inducing the most significant response. At high concentrations, usnic acid and U. barbata extracts induced apoptosis and DNA damage in human blood cells, increasing ROS levels. Our study reveals the importance of prior natural products toxicity evaluation on normal cells to anticipate their limits and benefits as potential anticancer drugs.

Evaluation of the Seasonality and Extraction Method on the Polar Extracts of Croton grewioides Baill. by Chromatogram Fingerprinting and Isolation of a New Triglycosylated Flavonoid

Croton grewioides Baill. popularly known as “canelinha” or “canelinha-de-cheiro” has been used for the treatment of influenza, antitussive, febrifuge and headache; however, the study of its phytochemical composition is limited. The aim of this study was to investigate the effect of the extraction method and seasonality through leaf extracts of four accessions of Croton grewioides by fingerprint chromatograms aided by principal component analysis to analyze the differences and similarities among the samples. We aimed also to provide chemical characterization of isolated secondary metabolites using semi-preparative liquid chromatography. The results showed that only the chemical profile of the methanolic extracts of accessions 101 and 113 were influenced by the seasonality. For the first time, four flavonoids were isolated through semi-preparative chromatography in this species, characterized as quercetin 3-O-β-D-galactopyranosyl-(1→2)-α-L-rhamnopyranoside-(1→6)-α-L-rhamnopyranoside (1), quercetin 3-O-β-D-galactopyranosyl-(1→2)-α-apiopyranoside-(1→6)-α-L-rhamnopyranoside (2), quercetin 3-O-glucopyranoside (3) and 3-O-methyl-quercetin (4), the flavonoid (2) has been recognized as a new triglycosylated derivative.

Development of methods for determining and studying the persistence of 2,4-dimethylhydroxybenzene and 2,6-dimethylhydroxybenzene in biological material

To study the features of the determination and preservation of 2.4-dimethylhydroxybenzene and 2.6-dimethylhydroxybenzene in biological material. Extraction, semi-preparative chromatography, TLC, GC-MS and UV spectrophotometry are considered as methods of analysis. The 2.4- and 2.6-dimethylhydroxybenzenes were isolated from the biomaterial by double infusion (30 minutes each) with a mixture of ethyl acetate-acetone (7: 3) at a weight ratio of the insulating liquid and biomaterial of 2:1. Purification was carried out by extraction and chromatography in a semi-preparative (190×10 mm) column of silica gel L 40/100 µm using the eluent hexane-dioxane-propanol-2 (80: 5: 1). Analytes were determined by TLC (Sorbfil plates, mobile phase hexane-dioxane-propanol-2 (120: 5: 1)), GC-MS (DB-5MS EVIDEX column (25 m × 0.2 mm) with a stationary phase (5%-phenyl) - methylpolysiloxane), UV spectrophotometry (solvent - 95% ethanol). The developed methods for the determination of 2.4- and 2.6-dimethyl derivatives of hydroxybenzene in biomaterial (liver tissue) are validated according to the criteria of linearity, selectivity, correctness and precision. The study of the dynamics of decomposition of 2.4- and 2.6-dimethyl hydroxybenzene derivatives in model mixtures with liver tissue, carried out using the developed techniques showed that with an increase in temperature the duration of preservation of analytes in biological material decreases. Moreover, the 2.4-isomer is more stable during storage than the 2.6-isomer. At temperatures of -25 °C, 0-2 °C, 8-10 °C, 20-22 °C, 36 °C the duration of retention of 2.4-dimethylhydroxybenzene is 402, 379, 358 and 224 days, respectively, the duration of retention of 2.6-dimethylhydroxybenzene is 356, 312, 224 and 136 days, respectively.

ISOLATION AND IDENTIFICATION OF EUPHORBIA TRIODONTA PLANT POLYPHENOLS BY CHROMATO-MASS SPECTROMETRY

The aim of this work is to study the composition of polyphenols contained in plants of Euphorbia triodontus Prokh., growing throughout the Republic of Uzbekistan, in particular the Ferghana Valley region, using a high-performance liquid chromatograph with a diode-matrix detector (HPLC-DAD) and a tandem chromato-mass spectrometer (HPLC – Q-TOF-MS/MS).&#x0D; The roots of the plant Euphorbia triodontus Prokh., growing on the territory of Uzbekistan, collected in the period after flowering, studied the polyphenolic composition. For the first time from this type of Euphorbia, the method of stepwise hydrophobic chromatography was used to isolate polyphenolic fractions. By HPLC, as a result of semi-preparative chromatography, 6 individual compounds were isolated from the fraction eluted with a 20% methanol solution in the amount of: E-20-1 – 7 mg, E-20-2 – 22 mg, E-20-3 – 14.2 mg, E-20-4 – 41 mg, E-20-5 – 11.4 mg and E-20-6 – 32 mg. As a result of mass spectrometric analyzes, it was established that the polyphenols isolated in an individual state consist of gallic acid and glucose linked by an ester bond: O-galloyl-β-D-glucose. The structure of individual compounds was determined: E-20-1 – 1-O-meta-trigalloyl-β-D-glucopyranosyl-(4-O-β-D)-1-O-meta-trigalloyl-β-D-glucose, E-20-2 – 1-O-meta-trihalloyl-β-D-glucose, E-20-3 – 1-O-meta-tetragalloyl-β-D-glucose, E-20-4 – 1-O-meta-trigalloyl-4-O-haloyl-β-D-glucose, E-20-5 – 2,3,6-tri-O-galloyl-β-D-glucose and E-20-6 – 1,2,3,4,6-penta-O-galloyl-β-D-glucose.

Identification of Novel Low-Weight Sulfhydryl Conjugates of Oxidized 5-O- and 6-O-Substituted Betanidin Pigments.

The formation of conjugates of oxidized betacyanin pigments with selected low-weight sulfhydryl scavengers was studied. Short-lived quinonoids, quinone methides, and aminochromes derived from oxidized betacyanins are able to form adducts with different efficiencies. In this report, mass spectrometric and NMR identifications of CS-linked conjugates of cysteine, cysteamine, N-acetylcysteine, and dl-dithiolthreitol with quinonoid forms generated through oxidation of betanidin, betanin, and gomphrenin is presented. An adduct that formed between cysteine and quinonoid generated from betanin by its oxidation and decarboxylation (2-decarboxy-xanbetanin) was detected and reported for the first time. The most stable gomphrenin CS-conjugate, N-acetylcysteinylated gomphrenin, was isolated by semipreparative chromatography and its structure was established by NMR analysis. This enabled to confirm the conjugation position at carbon C-4 and to indicate the presence of a dopachromic intermediate during oxidation of gomphrenin. Conjugation of betacyanins with thiol-bearing moieties may generate new molecules with modified chemical and biological properties. Obtained results confirm that gomphrenin is capable of forming CS-conjugates with higher efficiency than betanin.

Enhanced process integration for the extraction, concentration and purification of di-acylated cyanidin from red cabbage

Anthocyanins are currently one of the most important pigments in the plants with great proven health-promoting benefits, however, their unstable chemical nature makes difficult their purification process. This work proposes the integration of two stages for the separation and purification of the pigments from red cabbage leaves. For the first stage, the acidified extract, containing 32.05 mg cyanidin-3-glucoside per milliliter (mg ECyn-3-glu•L−1), was subjected to micro, ultrafiltration and adsorption processes, allowing to concentrate up to 3221.45 mg ECyn-3-glu•L−1 of the pigments after an adsorption process. In the second stage, the pigments were fully fractionated by molecular exclusion chromatography, reverse-phase vacuum liquid chromatography and semi-preparative chromatography, isolating the stable di-acylated cyanidin with antioxidant activity. Finally, purified fractions were also analyzed by NMR analysis.

Extraction and characterization of phlorotannin-enriched fractions from the Atlantic seaweed Bifurcaria bifurcata and evaluation of their cytotoxic activity in murine cell line

Phlorotannins, a group of polymeric polyphenols composed of phloroglucinol monomers, were obtained from the brown alga Bifurcaria bifurcata by solvent extraction (acetone/water 70:30 v/v) and further semipreparative chromatography. Characterization of several polyphenolic fractions with different polymerization degrees was carried by HPLC-MS. Cytotoxic activity of phlorotannin fractions were tested in ATDC5 murine cell line and analyzed by MTT assay. A fraction eluted between 14 and 16 min showed the highest cytotoxicity against ATDC5 murine cells in a dose-responsive manner. Hydroxytetrafuhalol (tetrafuhalol B) was the main component of this fraction. Fractions with the highest molecular size, corresponding with oligomeric phlorotannins with a polymerization degree > 6, did not show the same cytotoxicity, likely due to their size. Cytotoxicity for the fraction containing the higher polarity and lower molecular size was not observed.

New aurone epoxide and auronolignan from the heartwood of Cotinus coggygria Scop

New aurone epoxide, 2,10-oxy-10-methoxysulfuretin (14), and new auronolignan (15), named cotinignan A, were isolated by silica gel column and semipreparative HPLC chromatography from the methylene chloride/methanol extract of Cotinus coggygria Scop. heartwood. In addition, thirteen known secondary metabolites namely sulfuretin, 2,3-trans-fustin, fisetin, butin, butein, taxifolin, eriodictyol, 3',5,5',7–tetrahydroxyflavanone, 3',4',7-trihydroxyflavone, 3-O-methyl-2,3-trans-fustin, 3-O-galloyl-2,3-trans-fustin, β-resorcylic acid and 3-O-β-sitosterol glucoside were isolated as well. Their structures were elucidated by 1D and 2D NMR, HR-ESI-MS, IR and UV. Ten out of eleven isolated flavonoids possess 7, 3' and 4' hydroxy groups. These structural features could be considered as chemotaxonomic characteristic of flavonoids from C. coggygria. Cotinignan A (15) represents new subclass of secondary metabolites - auronolignans.

The application of multidimensional NMR analysis to cis/trans isomers study of menaquinone-7 (vitamine K2MK-7), identification of the (E,Z3,E2,ω)-menaquinone-7 isomer in dietary supplements

The aim of this paper was to determine the chemical structure of cis/trans isomers of vitamin K2MK-7 found in dietary supplements. Vitamin K2MK-7 was extracted, along with contamination in the form of cis/trans isomers of menaquinone-7, from dietary supplements through tetrahydrofuran and then subjected to fractionation using the semi-preparative chromatography column with cholesteryl group phase (strong stereoselectivity for hydrophobic compounds). The obtained fractions of cis/trans isomers of menaquinone-7 underwent analysis with the use of multidimensional NMR methods optimized for maximum signal separation. NMR analysis made it possible to identify the first geometric isomer of (E,Z3,E2,ω)-menaquinone-7, which underwent NMR comparative analysis with a known “full trans” isomer. It was also possible to confirm the presence of other cis/trans isomers in menaquinone-7 in the collected fractions. Due to the fact that the other fractions indicate a mixture of at least two geometric isomers of menaquinone-7, it was not possible to conduct full structural identification.

Peptides derived from in vitro gastrointestinal digestion of germinated soybean proteins inhibit human colon cancer cells proliferation and inflammation

The aim was to investigate the potential of germinated soybean proteins asa source of peptides with anticancer and anti-inflammatory activities produced after simulated gastrointestinal digestion. Protein concentrate from germinated soybean was hydrolysed with pepsin/pancreatin and fractionated by ultrafiltration. Whole digest and fractions>10, 5–10, and<5kDa caused cytotoxicity to Caco-2, HT-29, HCT-116 human colon cancer cells, and reduced inflammatory response caused by lipopolysaccharide in macrophages RAW 264.7. Antiproliferative and anti-inflammatory effects were generally higher in 5–10kDa fractions. This fraction was further purified by semi-preparative chromatography and characterised by HPLC-MS/MS. The most potent fraction was mainly composed of β-conglycinin and glycinin fragments rich in glutamine. This is the first report on the anti-cancer and anti-inflammatory effects of newly isolated and identified peptides from germinated soybean released during gastrointestinal digestion. These findings highlight the potential of germination as a process to obtain functional foods or nutraceuticals for colon cancer prevention.