Preparative High-performance Liquid Chromatography Research Articles

Analysis of florfenicol in pig plasma using a validated PPT-HPLC-DAD method

High performance liquid chromatography (HPLC) has proven to be an effective tool for examining the disposition kinetics of florfenicol (FF), a synthetic broad-spectrum antibiotic used to treat infectious diseases in veterinary medicine. Modification and optimisation of the protein precipitation (PPT) sample preparation procedure and HPLC with diode array detector (DAD) instrumental method were carried out to ensure conditions suitable for FF analyses in pig plasma samples. Stable supernatants with good plasma mean recoveries of FF (99.8% ± 0.7%RSD) were achieved using 1% v/v phosphoric acid solution in methanol and 10% w/v sodium chloride in aqueous solution. The PPT-HPLCDAD method’s detection limit of 0.004 μg/mL and quantification limit of 0.013 μg/mL provides high sensitivity for analyses of FF in plasma samples. In addition, the optimisation of method conditions resulted in shorter extraction and analysis time and less solvent consumption, which stresses the sustainability of this method in analytical chemistry. The optimised and validated PPT-HPLC-DAD method was applied in a comparative study of FF in pig plasma after administration of veterinary medicinal products. The study was conducted on fattening pigs following repeated intramuscular administration of two similar solutions for injection at a dose of 20 mg FF/kg bodyweight (test groups 1 and 2). The solutions for injection contained the same FF concentration, i.e., 300 mg/mL, but differed in excipients. The aim was to examine the influence of administrated solutions for injection on the extent of exposure to FF in pig plasma. The dynamics of kinetic profiles of FF in pig plasma from both treatments correspond to the FF kinetic profiles published in similar studies. However, differences were observed in the concentrations of FF, which were constant throughout the study, and statistical differences between the test groups were confirmed (P<0.05). Though these findings suggest the possible influence of excipients, a full comprehensive conclusion on the influence of administrated solutions for injection on FF exposure in pig plasma requires additional research.

Phytochemical investigation of some active constituents from N-Butanol fraction of Tropaeolum majus L. cultivated in Iraq

Tropaeolum majus, Tropaeolaceae family, is anornamental plant and has a long history of use as a medicinal plant; it is cultivated currently in most countries of the world, including Iraq. The aim of this work was to isolate and identify three compounds from the leaves of Tropaeolum majus (N-butanol fraction) cultivated in Iraq. The constituents were isolated by preparative high-Performance Liquid Chromatography and their structures were determined using precise NMR spectroscopic techniques.

Inositol Derivatives with Anti-Inflammatory Activity from Leaves of Solanum capsicoides Allioni

Eight new inositol derivatives, solsurinositols A–H (1–8), were isolated from the 70% EtOH extract of the leaves of Solanum capsicoides Allioni. Careful isolation by silica gel column chromatography followed by preparative high-performance liquid chromatography (HPLC) allowed us to obtain analytically pure compounds 1–8. They shared the same relative stereochemistry on the ring but have different acyl groups attached to various hydroxyl groups. This was the first time that inositol derivatives have been isolated from this plant. The chemical structures of compounds 1–8 were characterized by extensive 1D nuclear magnetic resonance (NMR) and 2D NMR and mass analyses. Meanwhile, the in vitro anti-inflammatory activity of all compounds was determined using lipopolysaccharide (LPS)-induced BV2 microglia, and among the isolates, compounds 5 (IC50 = 11.21 ± 0.14 µM) and 7 (IC50 = 14.5 ± 1.22 µM) were shown to have potential anti-inflammatory activity.

A New Stilbene Glucoside from Biotransformation-Guided Purification of Chinese Herb Ha-Soo-Oh.

Ha-Soo-Oh is a traditional Chinese medicine prepared from the roots of Polygonum multiflorum Thunb. The herb extract has been widely used in Asian countries as a tonic agent and nutritional supplement for centuries. To identify new bioactive compounds in Chinese herbs, the biotransformation-guided purification (BGP) process was applied to Ha-Soo-Oh with Bacillus megaterium tyrosinase (BmTYR) as a biocatalyst. The result showed that a major biotransformed compound could be purified using the BGP process with preparative high-performance liquid chromatography (HPLC), and it was confirmed as a new compound, 2,3,5,3′,4′-pentahydroxystilbene-2-O-β-glucoside (PSG) following mass and nucleic magnetic resonance (NMR) spectral analyses. PSG was further confirmed as a biotransformation product from 2,3,5,4′-tetrahydroxystilbene-2-O-β-glucoside (TSG) by BmTYR. The new PSG exhibited 4.7-fold higher 1,1-diphenyl-2-picrylhydrazine (DPPH) free radical scavenging activity than that of TSG. The present study highlights the potential usage of BGP in herbs to discover new bioactive compounds in the future.

Identification of Cyclic Dipeptides and a New Compound (6-(5-Hydroxy-6-methylheptyl)-5,6-dihydro-2H-pyran-2-one) Produced by Streptomyces fungicidicus against Alternaria solani.

As an important microbial resource, Actinomycetes, especially Streptomyces, have important application values in medicine and biotechnology. Streptomyces fungicidicus SYH3 was isolated from soil samples in tomato-growing areas and showed good inhibitory effects on Alternaria solani in tomato. To obtain pure active compounds, SYH3 fermentation broth was subjected to XAD-16 macroporous resin and silica gel column chromatography. Combined with the repeated preparation and separation of preparative high-performance liquid chromatography (HPLC), a total of four monomer compounds were obtained after activity tracking. Compound 4 was identified as a new six-membered lactone ring compound named 6-(5-hydroxy-6-methylheptyl)-5,6-dihydro-2H-pyran-2-one by 1D and 2D nuclear magnetic resonance (NMR) data and mass spectrometry (MS). The other three active compounds belong to the cyclodipeptide, and their half maximal inhibitory concentration (IC50) values against A. solani were 43.4, 42.9, and 30.6 μg/mL, respectively. Compound 4 significantly inhibited the spore germination and induced swollen and deformed local hyphae of A. solani with an IC50 value of 24.9 μg/mL. Compound 4 also had broad-spectrum antifungal activity and had a good antifungal effect on the tested plant-pathogenic fungi. The modes of action of new compound (4) still require further investigation, representing a novel and effective anti-fungal agent for future application.

A review for discovering bioactive minor saponins and biotransformative metabolites in Panax quinquefolius L.

Panax quinquefolius L. has attracted extensive attention worldwide because of its prominent pharmacological properties on type 2 diabetes, cancers, central nervous system, and cardiovascular diseases. Ginsenosides are active phytochemicals of P. quinquefolius, which can be classified as propanaxdiol (PPD)-type, propanaxtriol (PPT)-type, oleanane-type, and ocotillol-type oligo-glycosides depending on the skeleton of aglycone. Recently, advanced analytical and isolated methods including ultra-performance liquid chromatography tandem with mass detector, preparative high-performance liquid chromatography, and high speed counter-current chromatography have been used to isolate and identify minor components in P. quinquefolius, which accelerates the clarification of the material basis. However, the poor bioavailability and undetermined bio-metabolism of most saponins have greatly hindered both the development of medicines and the identification of their real active constituents. Thus, it is essential to consider the bio-metabolism of constituents before and after absorption. In this review, we described the structures of minor ginsenosides in P. quinquefolius, including naturally occurring protype compounds and their in vivo metabolites. The preclinical and clinical pharmacological studies of the ginsenosides in the past few years were also summarized. The review will promote the reacquaint of minor saponins on the growing appreciation of their biological role in P. quinquefolius.

Isolation, structure elucidation, and high-performance liquid chromatography quantification of photolytic degradation impurities in acrivastine.

Acrivastine is a second-generation H1-receptor antagonist, and its structure is sensitive to ultraviolet. Four unknown and one reported degradation products can be detected in the ultraviolet radiation solutions of acrivastine. To improve the quality control of acrivastine, the photodegradation impurities were isolated and structurally elucidated. There are four new impurities (1-3 and 5), and one reported compound (4). The isolation strategy was designed as preparative high-performance liquid chromatography using a reversed phase column with volatile acid addition in the mobile phase, combined with preparative thin-layer chromatography using silica gel with alkaline addition in the mobile phase. Using the developed methods, five impurities (1-5) were efficiently purified after two or three chromatography runs with purities > 95%. The structures of compounds 1-5 are elucidated based on spectroscopy analysis of MS, and nuclear magnetic resonance spectroscopy. Using the impurity standard, the high-performance liquid chromatography method was developed and validated. The method was proved to be sensitive, accurate (Recovery% 96.1-107.7%), linear (0.15-0.75 μg/mL, R2 >0.996), robust, and specific, and it was successfully used to determine the degradation impurities of acrivastine and its formulation.

Isolation and characterization of novel peptides from fermented products of Lactobacillus for ulcerative colitis prevention and treatment

Ulcerative colitis (UC) is an incurable and highly complex digestive disease affecting millions of people worldwide. Compared to the current therapeutic drugs, bioactive peptides are more promising and safe substances as functional foods or drugs for the prevention and treatment of UC. The alcohol-soluble components from fermentation broth by fresh wheat germ and apple (AC-WGAF) were found to be effective in UC prevention in dextran sulfate sodium-induced mice in vivo. Herein, 4 novel peptides are identified from AC-WGAF by membrane ultrafiltration, recycling preparative high-performance liquid chromatography, and matrix-assisted laser desorption–ionization time-of-flight/time-of-flight mass spectrometry, possessing anti-colitis activity via using an in vitro model. One of those peptides named T24 (PVLGPVRGPFPLL) exhibited the most remarkable anti-colitis activity by preventing tight junction protein loss, maintaining epithelial barrier integrity, and promoting cell proliferation during in vitro and in vivo studies by regulating mitogen-activated protein kinase signaling pathways. Thus, T24 is a promising peptide as a functional food or novel drug for UC prevention and treatment.

In vitro dual activity of Aloe marlothii roots and its chemical constituents against Plasmodium falciparum asexual and sexual stage parasites

Ethnopharmacological relevanceAloe marlothii A.Berger (Xanthorrhoeaceae) is indigenous to southern African countries where its aqueous preparations are used in traditional medicine to treat several ailments including hypertension, respiratory infections, venereal diseases, chest pain, sore throat and malaria. Aim of the studyThe aims of this study were as follows: (i) isolate and identify the antiplasmodial active compounds in A. marlothii roots. As the water extract was previously inactive, the dichloromethane:methanol (DCM:MeOH) (1:1) was used, (ii) examine the activity of the isolated compounds against Plasmodium falciparum asexual blood stage (ABS) parasites as well as for transmission-blocking activity against gametocytes and gametes, and (iii) to use in silico tools to predict the target(s) of the active molecules. Materials and methodsThe crude DCM:MeOH (1:1) extract of A. marlothii roots was fractionated on a reverse phase C8 column, using a positive pressure solid-phase extraction (ppSPE) workstation to produce seven fractions. The resulting fractions and the crude DCM:MeOH extract were tested in vitro against P. falciparum (NF54) ABS parasites using the malaria SYBR Green I based-fluorescence assay. Flash silica chromatography and mass-directed preparative high-performance liquid chromatography were utilised to isolate the active compounds. The isolated compounds were evaluated in vitro against P. falciparum asexual (NF54 and K1 strains) and sexual (gametocytes and gametes) stage parasites. Molecular docking was then used for the in silico prediction of targets for the isolated active compounds in P. falciparum. ResultsThe crude extract and two SPE fractions displayed good antiplasmodial activity with >97% and 100% inhibition of ABS parasites proliferation at 10 and 20 μg/mL, respectively. Following UPLC-MS analysis of these active fractions, a targeted purification resulted in the isolation of six compounds identified as aloesaponol I (1), aloesaponarin I (2), aloesaponol IV (3), β-sorigenin-1-O-methylether (4), emodin (5), and chrysophanol (6). Aloesaponarin I (2) was the most bioactive, compared to other isolated constituents, against P. falciparum ABS parasites exhibiting equipotency against the drug-sensitive (NF54) (IC50 = 1.54 μg/mL (5 μM)) and multidrug-resistant (K1) (IC50 = 1.58 μg/mL (5 μM)) strains. Aloesaponol IV (3) showed pronounced activity against late-stage (>90% stage IV/V) gametocytes (IC50 = 6.53 μg/mL (22.6 μM)) demonstrating a 3-fold selective potency towards these sexual stages compared to asexual forms of the parasite (IC50 = 19.77 ± 6.835 μg/mL (68 μM)). Transmission-blocking potential of aloesaponol IV (3) was validated by in vitro inhibition of exflagellation of male gametes (94% inhibition at 20 μg/mL). In silico studies identified β-hematin and DNA topoisomerase II as potential biological targets of compounds 2 and 3, respectively. ConclusionThe findings from our study substantiate the traditional use of A. marlothii to treat malaria. To our knowledge, this study has provided the first report on the isolation and identification of antiplasmodial compounds from A. marlothii roots. Furthermore, our study has provided the first report on the transmission-blocking potential of one of the compounds from the genus Aloe, motivating for the investigation of other species within this genus for their potential P. falciparum transmission-blocking activity.

Separation, characterization and anti-inflammatory activities of galactoglycerolipids from Perilla frutescens (L.) Britton

The study was to optimize the separation procedures, characterize the galactoglycerolipids and explore their anti-inflammatory activities. Two monogalactosyldiacylglycerols (MGDGs) and three digalactosyldiacylglycerols (DGDGs) from Perilla frutescens (L.) Britton were obtained through one-step silica gel column chromatography and preparative high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD). The presence of additional MGDG (1-O-9Z,12Z,15Z-octadecatrienoyl-2-O-7Z,10Z,13Z-hexadecatrienoyl-3-O-(β-D-galactopyranosyl)-sn-glycerol) and DGDG (1-O-9Z,12Z-octadecadienoyl-2-O-9Z,12Z,15Z-octadecatrienoyl-3-O-(β-D-galactopyranosyl-(1'→6'')-α-D-galactopyranosyl)-sn-glycerol) was concluded for the first time in perilla, by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). In lipopolysaccharide (LPS)-induced RAW264.7 cells, five galactoglycerolipids exhibited good inhibitory activities against nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression in a dose-dependent manner, suggesting that fatty acid chain length and unsaturation degree affected their anti-inflammatory activities.

Antibacterial Activity and Components of the Methanol-Phase Extract from Rhizomes of Pharmacophagous Plant Alpinia officinarum Hance.

The rhizomes of Alpinia officinarum Hance (known as the smaller galangal) have been used as a traditional medicine for over 1000 years. Nevertheless, little research is available on the bacteriostatic activity of the herb rhizomes. In this study, we employed, for the first time, a chloroform and methanol extraction method to investigate the antibacterial activity and components of the rhizomes of A. officinarum Hance. The results showed that the growth of five species of pathogenic bacteria was significantly inhibited by the galangal methanol-phase extract (GMPE) (p < 0.05). The GMPE treatment changed the bacterial cell surface hydrophobicity, membrane fluidity and/or permeability. Comparative transcriptomic analyses revealed approximately eleven and ten significantly altered metabolic pathways in representative Gram-positive Staphylococcus aureus and Gram-negative Enterobacter sakazakii pathogens, respectively (p < 0.05), demonstrating different antibacterial action modes. The GMPE was separated further using a preparative high-performance liquid chromatography (Prep-HPLC) technique, and approximately 46 and 45 different compounds in two major component fractions (Fractions 1 and 4, respectively) were identified using ultra-HPLC combined with mass spectrometry (UHPLC-MS) techniques. o-Methoxy cinnamaldehyde (40.12%) and p-octopamine (62.64%) were the most abundant compounds in Fractions 1 and 4, respectively. The results of this study provide data for developing natural products from galangal rhizomes against common pathogenic bacteria.

Purification and Identification of a Novel Angiotensin Converting Enzyme Inhibitory Peptide from the Enzymatic Hydrolysate of Lepidotrigla microptera.

In this study, Lepidotrigla microptera were hydrolyzed with four different proteolytic enzymes (Papain, neutrase, flavourzyme, and alcalase), and their distribution of molecular weights and ACE-inhibitory activity were tested. The alcalase hydrolysates showed the maximum ACE-inhibitory activity. A novel ACE-inhibitory peptide was isolated and purified from Lepidotrigla microptera protein hydrolysate (LMPH) using ultrafiltration, gel filtration chromatography, and preparative high performance liquid chromatography (prep-HPLC). The amino acid sequence of the purified peptide was identified as Phe-Leu-Thr-Ala-Gly-Leu-Leu-Asp (DLTAGLLE), and the IC50 value was 0.13 mg/mL. The ACE-inhibitory activity of DLTAGLLE was stable across a range of temperatures (<100 °C) and pH values (3.0–11.0) and retained after gastrointestinal digestion. DLTAGLLE was further identified as a noncompetitive inhibitor by Lineweaver–Burk plot. The molecular docking simulation showed that DLTAGLLE showed a high binding affinity with ACE sites by seven short hydrogen bonds. As the first reported antihypertensive peptide extracted from alcalase hydrolysate of Lepidotrigla microptera, DLTAGLLE has the potential to develop functional food or novel ACE-inhibitor drugs.

Isolation, Characterization, and Toxicity Study of Stress Degradation Products of Pranlukast Hydrate.

Pranlukast hydrate (PRN), a cysteinyl leukotriene receptor antagonist (CysLT1), is used to treat bronchial asthma. The objective of this study is to perform the isolation, characterization, and toxicity analysis of stress degradation products of PRN. In high-performance liquid chromatography (HPLC), the separation was achieved using a Phenomenex Gemini C18 (250 × 4.6 mm, 5 μ) column; the ammonium format buffer (50 mM), pH 4, with formic acid: acetonitrile (50:50, v/v) was used as a mobile phase at a flow rate of 1.25 mL/min; and the photodiode array detector was used for detection at 230 nm. The drug was subjected to stress degradation as per ICH Q1A (R2) and ICH Q1B guidelines. The drug was found to be labile in alkaline (62.48% degradation) and photolytic (liquid state) (7.67% degradation) conditions, whereas the drug was found to be stable in acidic, peroxide, photolytic (solid state), and thermal conditions. The characterization of the drug and its degradation products was achieved using liquid chromatography-electrospray ionization-quadrupole time of flight tandem mass spectrometry (LC-ESI-QTOF-MS/MS), and the degradation mechanism was proposed. There were two degradation products observed in alkaline conditions (DP6 and DP9), whereas six novel degradation products were observed in photolytic degradation products (DP1, DP3, DP4, DP5, DP7, and DP10). The developed method was successfully validated as per the ICH Q2 (R1) guideline. The isolation of the alkaline degradation product DP9 was performed using preparative HPLC, and it was found to be 96.8% pure degradation product. The characterizations of the isolated degradation product (DP9) and procured impurity were performed using MS/MS, NMR, and FTIR. The mass of the procured impurity and DP9 were observed to be 404 and 500 Da, respectively. The in vitro cytotoxicity study of the procured impurity and DP9 was conducted using a 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay using an A549 cell line, and they were found to be cytotoxic at concentrations above 62.5 and 250 μg/mL, respectively. Furthermore, an in silico toxicity study was performed to predict the toxicity of all the major characterized degradation products of PRN using admetSAR software version 2.0. DP1, DP2, DP6, and DP10 were found to be hepatotoxic, mutagenic according to the micronucleus test, and aquatic toxic. We can conclude that the drug should be kept away from the direct exposure of light and the toxicity levels of DP1, DP2, DP6, and DP10 should be reduced below 0.1% to avoid their toxic effect.

4a-methyl-dodecahydro-1H-pyrrolo[3,4-b]quinoline-6-one produced by Endophytic Fungi Aspergillus niger E12 obtained from Dodonaea viscosa Plant Leaves as a Novel Antibacterial Compound.

Endophytic fungi were isolated from forty plant leaf samples from Gudiyam forest. The potent antibacterial strain Aspergillus niger E12 isolated from the plant Dodonaea viscosa showed maximal antibacterial activity against all the test organisms, viz., Staphylococcus aureus, Bacillus coagulans, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The production of the antibacterial compound was optimized using the yeast extract sucrose medium (2% YES) using response surface methodology (RSM). For the production, the optimal parameters were carbon/nitrogen (C:N) ratio, 9:1; temperature, 25°C; pH, 5.7; incubation time, 240h; and rpm, 30. A zone of inhibition of 19.33mm was observed as maximal bioactivity against Pseudomonas aeruginosa. The antibacterial compound was purified by extraction with ethyl acetate, activity-guided fractionation, and preparative high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), Fourier transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) studies showed that the Aspergillus niger E12 bioactive substance is 4a-methyl-dodecahydro-1H-pyrrolo [3,4-b] quinoline-6-one.

Coumarins from the Roots of Angelica archangelica and Antibacterial Activity Against Methicillin Resistant Staphylococcus aureus

Angelica archangelica (Fam. Apiaceae) roots were extracted sequentially with n-hexane, dichloromethane (DCM) and methanol (MeOH) using a Soxhlet apparatus. The n-hexane and DCM extracts were fractionated by Vacuum Liquid Chromatography (VLC) over silica gel followed by Preparative Thin Layer Chromatography (PTLC) to yield two coumarins, osthol (1) and osthenol (2). However, the methanol extract was fractionated by solid-phase extraction using C18 cartridge followed by analysis of components by analytical High Performance Liquid Chromatography (HPLC) and purification by preparative High Performance Liquid Chromatography (HPLC) to give two coumarins, bergapten (3) and heraclenol (4) along with sucrose (5). Compounds were identified by a series of 1D (1H and 13C) and 2D (COSY, HSQC and HMBC) NMR spectroscopy and mass spectrometric analysis. The crude extracts (n-hexane, DCM, and methanol) and compound 1, were screened for antimicrobial activity against Gram-positive bacteria (Methicillin resistant Staphylococcus aureus, MRSA and Micrococcus luteus), Gram-negative bacteria, Pseudomonas aeruginosa, and a fungus, Candida albicans. The antimicrobial assays showed that the crude DCM and n-hexane extracts, alongside compound 1, largely inhibited the growth of the microorganisms at high concentrations. Compound 1, which was further tested for antibacterial activity against a series of clinical isolates of methicillin resistance Staphylococcus aureus, showed moderate level of activity against only one strain. Dhaka Univ. J. Pharm. Sci. 20(3): 275-281, 2022 (June) Centennial Special Issue

[Lignans from Agrimonia pilosa].

Thirteen lignans were isolated from 60% ethanol extract of Agrimonia pilosa by column chromatography over silica gel, ODS, and MCI and preparative high performance liquid chromatography(HPLC). Their chemical structures were identified by physiochemical properties and spectral data as(7S,8S)-threo-4,7,9,9'-tetrahydroxy-3,3',5'-trimethoxy-8-O-4'-neolignan(1),(+)-4,9,9'-trihydroxy-3-methoxy-3',7-epoxy-8,4'-oxyneolignan(2), dihydrodehydro-diconiferyl alcohol(3), 4,9,9'-trihydroxy-3,3',5-trimethoxy-4',7-epoxy-8,5'-neolignan(4),(-)-secoisolariciresinol(5), 4,7,9,9'-tetrahydroxy-3,3',5'-trimethoxy-8-O-4'-neolignan(6),(+)-isolariciresinol(7), 4,7,9,9'-tetrahydroxy-3,3'-dimethoxy-8-O-4'-neolignan(8), burselignan(9),(-)-evofolin B(10), icariol A2(11), ciwujiatone(12), and(+)-4″,4-dihydroxy-3,3',3″,3,5,5'-hexamethoxy-7,9';7',9-diepoxy-4,8″;4',8-bisoxy-8,8'-dineolignan-7″,7,9″,9-tetraol(13). Compound 1 was a new compound, and compounds 1-13 were isolated from Agrimonia plant for the first time. This study can enrich the chemical components in A. pilosa and provide material conditions for the follow-up study of its biological activity and the elucidation of its pharmacodynamic substances.

Generation of Flavor-Active Compounds by Electrochemical Oxidation of (R)-Limonene.

Terpenes may be converted by electrochemical oxidation to various oxidized products with appealing aroma properties. In this study, (R)-limonene was anodically oxidized in the presence of ethanol, and the resulting mixture exhibited a pleasing fruity, herbal, citrus-like, and resinous odor. The aroma-active compounds were purified by means of preparative high-performance liquid chromatography, and their structures were elucidated by means of gas chromatography (GC)-mass spectrometry and nuclear magnetic resonance spectroscopy. In addition, the odor of the isolated compounds was determined by means of GC-olfactometry. Seventeen compounds were isolated, and for only four of them, analytical data had been reported previously in the literature. Furthermore, only for two of the compounds, an odor description had been available in the literature.

Synergistic effects of trans-p-coumaric acid isolated from the ethanol extract of Gynura procumbens in promoting intestinal absorption of chlorogenic acid and reversing alcoholic fatty liver disease

Ethnopharmacological relevanceOur previous studies found that the ethanol extract of Gynura procumbens (EEGS) reduced hepatic steatosis in alcoholic fatty liver disease (AFLD). Aim of the studyTo explore the active ingredients from EEGS and their relevant mechanism of action in alleviating alcoholic liver injuries. Aim of the studyTo explore the active ingredients from EEGS and their intestinal absorption characteristics as an approach for understanding mechanism of action in alleviating alcoholic liver injuries. Materials and methodsMonitored by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), chemical constituents from the prepared EEGS were isolated by means of solvent extraction, repeated column chromatography, preparative HPLC and other methods, and their structures were identified based on spectroscopic methods. The in vivo intestinal absorption rate of chlorogenic acid (CA), the active component of the EEGS, both in a single form and in the EEGS were monitored by the single-pass intestinal perfusion (SPIP) method in rats. The protective effect of EEGS and its active components on alcoholic liver injuries was evaluated in the alcoholic liver injury model of C57BL/6J male mice induced by Lieber-DeCarli alcohol liquid feed. ResultsThree noncaffeoyl quinic acid components were isolated and identified from the EEGS, namely, 3-trans-p-coumaroyl quinic acid (0.9%), 3-cis-p-coumaroyl quinic acid (2.7%), and trans-p-coumaric acid (0.6%). In vivo intestinal absorption of CA decreased with the increase of pH value of perfusion solution in the range of 5.5–7.8. The maximum absorption percentage of CA alone was 6.7 ± 2.4%, while the maximum absorption percentage of CA in the EEGS was 16.0 ± 2.2%, which was 2.4 times higher than that of CA alone. The results of animal experiments showed that the degree of fatty liver of mice treated with EEGS was significantly lower than that of the CA, trans-p-coumaric acid, and the combination group of CA and trans-p-coumaric acid alone. ConclusionThe above results indicated that trans-p-coumaric acid isolated from the dried stems of Gynura procumbens assisted CA being absorbed into the body and worked together with CA to improve the function of liver lipid metabolism, reduce hepatic lipid accumulation in a mouse model of AFLD and effectively counteract alcohol-induced fatty liver disease.

Meroterpenoids Possibly Produced by a Bacterial Endosymbiont of the Tropical Basidiomycete Echinochaete brachypora.

A mycelial culture of the African basidiomycete Echinochaete cf. brachypora was studied for biologically active secondary metabolites, and four compounds were isolated from its crude extract derived from shake flask fermentations, using preparative high-performance liquid chromatography (HPLC). The pure metabolites were identified using extensive nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). Aside from the new metabolites 1-methoxyneomarinone (1) and (E)-3-methyl-5-(-12,13,14-trimethylcyclohex-10-en-6-yl)pent-2-enoic acid (4), the known metabolites neomarinone (2) and fumaquinone (4) were obtained. Such compounds had previously only been reported from Actinobacteria but were never isolated from the cultures of a fungus. This observation prompted us to evaluate whether the above metabolites may actually have been produced by an endosymbiontic bacterium that is associated with the basidiomycete. We have indeed been able to characterize bacterial 16S rDNA in the fungal mycelia, and the production of the metabolites stopped when the fungus was sub-cultured on a medium containing antibacterial antibiotics. Therefore, we have found strong evidence that compounds 1–4 are not of fungal origin. However, the endofungal bacterium was shown to belong to the genus Ralstonia, which has never been reported to produce similar metabolites to 1–4. Moreover, we failed to obtain the bacterial strain in pure culture to provide final proof for its identity. In any case, the current report is the first to document that polyporoid Basidiomycota are associated with endosymbionts and constitutes the first report on secondary metabolites from the genus Echinochaete.

Novel Glycosylation by Amylosucrase to Produce Glycoside Anomers.

Simple SummaryAll livings are composed of cells, which contain lipid, proteins, nuclei acids, and saccharides. Saccharides include polysaccharides, oligo saccharides, disaccharides, which are linked by monosaccharides. Monosaccharides such as glucose exist in two forms, named α and β anomer, in solution. In addition, monosaccharides could be linked with lipid, proteins, nuclei acids or other saccharide to form glycosides through glycosylation. In nature, glycosylation is catalyzed by enzymes. Until now, all enzymes catalyzed glycosylation to form glycosides with either α or β form but not both. This study found an enzyme, amylosucrase from Deinococcus geothermalis (DgAS), could catalyze glycosylation of a kind of lipid named ganoderic acids triterpenoids from a medicinal fungus Ganoderma lucidum to form both α and β anomer of glycosides. This is the first report that enzymes could catalyze such glycosylation and a possible reaction mechanism was proposed.Glycosylation occurring at either lipids, proteins, or sugars plays important roles in many biological systems. In nature, enzymatic glycosylation is the formation of a glycosidic bond between the anomeric carbon of the donor sugar and the functional group of the sugar acceptor. This study found novel glycoside anomers without an anomeric carbon linkage of the sugar donor. A glycoside hydrolase (GH) enzyme, amylosucrase from Deinococcus geothermalis (DgAS), was evaluated to glycosylate ganoderic acid F (GAF), a lanostane triterpenoid from medicinal fungus Ganoderma lucidum, at different pH levels. The results showed that GAF was glycosylated by DgAS at acidic conditions pH 5 and pH 6, whereas the activity dramatically decreased to be undetectable at pH 7 or pH 8. The biotransformation product was purified by preparative high-performance liquid chromatography and identified as unusual α-glucosyl-(2→26)-GAF and β-glucosyl-(2→26)-GAF anomers by mass and nucleic magnetic resonance (NMR) spectroscopy. We further used DgAS to catalyze another six triterpenoids. Under the acidic conditions, two of six compounds, ganoderic acid A (GAA) and ganoderic acid G (GAG), could be converted to α–glucosyl-(2→26)-GAA and β–glucosyl-(2→26)-GAA anomers and α-glucosyl-(2→26)-GAG and β-glucosyl-(2→26)-GAG anomers, respectively. The glycosylation of triterpenoid aglycones was first confirmed to be converted via a GH enzyme, DgAS. The novel enzymatic glycosylation-formed glycoside anomers opens a new bioreaction in the pharmaceutical industry and in the biotechnology sector.