Gradient Solvent System Research Articles

Cytotoxic of Usnic Acid Isolated from Ramalina sp.

Ramalina sp. (Ramalinaceae) is a type of lichen known to contain many active secondary metabolite compounds that have the potential to be used as medicine or medicinal raw materials. One of the biological activities possessed by Ramalina sp. lichen is its anticancer activity. This research aims to isolate and identify active secondary metabolite compounds from the methanol extract of the Ramalina sp. lichen and to find out the cytotoxic activity of the isolated compound against MCF7 breast cancer cells. Compound 1 (usnic acid) was successfully isolated from fraction A. The isolation and purification process was carried out starting with a maceration process using acetone solvent, followed by silica gel column chromatography using a gradient solvent system consisting of n-hexane, n-hexane: EtOAc, EtOAc, EtOAc: MeOH, and MeOH with 5% increment of polarity to obtain 17 fractions (F-1 to F-17). From the 17 fractions obtained, fraction 3 (F-3) and fraction 4 (F-4) (eluted with n-hexane: EtOAc 30%), which had the same TLC profile, were combined and named as fraction A. Compound 1 (50 mg) is a yellow needle crystal that was formed in a bottle of fraction A, which was obtained after the process of combining fractions F-3 and F-4 and solvent evaporation process. The crystals were then separated and purified with CHCl3 and MeOH. Compound 1 was then characterized based on spectroscopic data. Various spectroscopic analysis data, including FTIR, 1D- and 2D-NMR, and LC-ESI-MS, show that Compound 1 is a dibenzofuran derivative compound with 18 carbons (3 from carbonyl groups (C=O) and 3 from methyl groups) and 2 hydroxyls (-OH). Cytotoxicity assay showed that at a low concentration of 18.75 ug/mL, Com-pound 1 caused a 67.06% decrease in MCF7 viability.

ISOLATION AND IDENTIFICATION OF APIGENIN, A FLAVONOID COMPOUND FROM MACARANGA HYPOLEUCA (REICHB.F. & ZOLL.)

The study on Macarang hypolueca (Reichb.f. & Zoll.), which was collected from secondary forests around Samarinda City, East Kalimantan, involved phytochemical investigations that led to the isolation of a flavone type compound of flavonoid from the ethyl acetate fraction. To separate the compounds, silica gel column chromatography was utilized with a gradient solvent system of n-hexane and ethyl acetate, along with the addition of 5%. Infrared analysis (FTIR), mass spectrum (LC-ESI-MS), and nuclear magnetic resonance (1D- and 2D-NMR) were used to identify and elucidate the structure. Based on spectroscopic data and comparison with appropriate references, the isolated compound was identified as apigenin

Optimization parameters for the production of dimer of epicatechin from an endophytic fungus Curvularia australiensis FC2AP using response surface methodology (RSM)

The search for natural therapeutic agents has intensified due to their potential to treat various diseases. Bioactive secondary metabolites from endophytes offer high therapeutic profiles and can be mass-produced after optimizing medium parameters and purification. This investigation aimed to maximize crude pigmented secondary metabolite (CPSM) production from Curvularia australiensis FC2AP by optimizing fermentation conditions statistically. The endophytic fungus produced a maximum yield of 8.81 UL/g from biomass using Sabouraud's Dextrose Broth. After screening essential factors, the Plackett-Burman design was used for factorial optimization, and the Box Behnken design was employed to investigate three significant factors. The final CPSM yield was 12.3 UL/g, approximately 4-fold higher than the preliminary growth medium. Chromatographic purification using a gradient solvent system resulted in six fractions, with the fourth fraction demonstrating the highest bioactivity profile. Structural characterization confirmed this fraction to be a dimer of epicatechin, which has anti-cancer properties, as confirmed through in vivo studies on Sprague Dawley rats. This is the first report of a epicatechin dimer produced from C. australiensis.

Identification and localization of morphological feature-specific metabolites in Reynoutria multiflora roots

Reynoutria multiflora roots are a classical herbal medicine with unique nourishing therapeutic effects. Anomalous vascular bundle (AVB) forming “cloudy brocade patterns” is a typical morphological feature of R. multiflora roots and has been empirically linked to its quality classification. However, scientific evidence, especially for AVB-specific specialised metabolites, has not been comprehensively revealed thus far. Herein, desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) analysis was applied to carry out an in situ analysis of specialised metabolites distributed specifically at the AVB and cork of R. multiflora roots. To enlarge the scope of compounds by DESI detection, various solvent systems including acetone, acetonitrile, methanol, and water were used to assist in the discoveries of 40 specialised metabolites with determined localization. A series of bioactive constituents including stilbenes, flavonoids, anthraquinones, alkaloids, and naphthalenes were found specifically around the brocade patterns. Notably, phospholipids were detected from R. multiflora roots by in situ analysis for the first time and were found mainly in the phloem of AVB (PAB). This is the first study to use gradient solvent systems in DESI-MSI analysis to locate the specialised metabolites distribution. The discovery of feature-specific compounds will bridge the empirical identification to precision quality control of R. multiflora roots.

Redispersible Reduced Graphene Oxide Prepared in a Gradient Solvent System.

We designed a gradient solvent strategy for the reduction of graphene oxide, matching the hydrophilic properties of graphene oxide (GO) and reduced graphene oxide (RGO), respectively. A third solvent was added dropwise to regulate the hydrophilic variation of the continuous gradient system which maintained the whole reduction process without aggregation, and the obtained RGO dispersions could maintain stability for a long time. The separated RGO solid powder can be directly ultrasonically redispersed in N-methyl-pyrrolidone (NMP) with an average particle size as low as 200 nm. Furthermore, RGO with a high C/O ratio of 13.75 was prepared on the basis of the gradient solvent system. Using different structures of dispersants and polymers as representatives, we employed successive solvent rinsing, thermal solvent extraction, and thermal treatment to study adsorption and desorption. It was found that the above measures differed significantly in the removal of surface sorbates. The selected fatty alcohol polyoxyethylene ether (AEO) series achieved a good balance between the system dispersion and surface adsorbate removal. The conductivity was originally 5236 S m−1, and it increased from 9024 to 18,000 S m−1 after thermal treatment at 300 and 500 °C, respectively.

A validated LC-MS/MS method for simultaneous quantification of simvastatin and simvastatin acid in beagle plasma: Application to an absolute bioavailability study.

A highly sensitive LC-MS/MS method for simultaneous detection of both simvastatin (SV) and simvastatin acid (SVA) in beagle plasma was developed and successfully applied to an absolute bioavailability study. Lovastatin (LV) was used as internal standard (IS). The analysis was performed using electrospray ionization and selective reaction monitoring in positive mode at m/z 441.0 → 325.0 for SV, 459.0 → 343.0 for SVA and 427.0 → 325.0 for the IS, respectively. The assay procedure involved a simple liquid-liquid extraction of SV, SVA and LV from beagle plasma into methyl tert-butyl ether. Separation of SV, SVA and the IS was achieved on a Shim-pack VP-ODS column (150 × 2.0mm, 5μm) with a binary gradient solvent system of 0.1% formic acid in water and methanol (15:85, v/v) as the mobile phase. The method was validated over the range of 0.25-500 ng/ml for SV (r2 ≥ 0.9923) and 0.24-481.23 ng/ml for SVA (r2 ≥ 0.9987). The results of method validation for accuracy, precision, extraction recovery, matrix effect and stability were within the acceptance criteria. The values of absolute bioavailability of SV and SVA in beagles were 2.97 and 25.40%, respectively. It is the first study developed for the measurement of absolute bioavailability of SV and SVA acid in beagles.

Application of Linear Gradient Solvent System in Centrifugal Partition Chromatography Facilitating Bioassay-Guided Fractionation of Yongdamsagan-Tang, Traditional Oriental Decoction.

As important pharmaceutical resources, traditional herbal medicines retain continuous attention. To do that, isolation and identification of bioactive molecules from traditional herbal decoction are important. However, conventional fractionation through octadecyl silica column faces irreversible sample adsorption that causes a bias in bioactivity assessment. However, liquid-liquid chromatographic system suffers tedious K value calculation as well as insufficient capacity in separation power when crude extract composed of widely ranging polarities. Here, we developed a comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) to aid bioassay-guided isolation. The lower aqueous phase of the n-hexane-acetonitrile-water (10:2:8, v/v) was used as the stationary, whereas its upper organic phase followed by the upper phase of ethyl acetate-acetonitrile-water and water-saturated n-butanol-acetonitrile-water in the same ratio were eluted in a linear gradient mode, thereby increasing polarity in the mobile phase. The HPLC profiling of CPC fraction showed that proposed gradient CPC was suitable to separate metabolites from Yongdamsagan-Tang, a traditional medicinal decoction made of ten herbal plants. Exhibiting a high recovery yield of 98.3%, antioxidant response element (ARE) luciferase-inducing assay in HepG2 cells indicated that the fractions composed of baicalein and wogonin, the marker natural products of Scutellaria baicalensis, were to be the most effective molecules from Yongdamsagan-Tang. The presented results demonstrated that bioassay-guided separation that assisted with a linear gradient CPC is an incomparable alternative to HPLC and biphasic CPC in terms of higher yield rate and redundant K value calculation, respectively, which led to an unbiased/time-saving separation and identification of bioactive molecules from the complex crude extract of natural products.

Development & validation of LC–MS/MS assay for 5-amino-1-methyl quinolinium in rat plasma: Application to pharmacokinetic and oral bioavailability studies

5-Amino-1-methyl quinolinium (5-AMQ) is a potent Nicotinamide N-methyl transferase (NNMT) inhibitor. NNMT is an enzyme that catalyzes the N-methylation of the endogenous substrate nicotinamide, as well as exogenous xenobiotics. NNMT is fundamental to cellular metabolism; NNMT is overexpressed in select tissues (e.g., adipose tissue, skeletal muscle, etc.) in pathophysiological conditions, making it a clinically relevant target for drug development in several chronic diseases including obesity and diabetes. The objective of this study was to develop and validate a simple, sensitive, and reproducible liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the quantification of 5-AMQ in rat plasma and urine samples. 5-AMQ was extracted from plasma and urine by protein precipitation. Chromatographic separation was achieved using an ACE® Excel™ C18 column (2 μm, 50 × 2.1 mm) with a binary gradient solvent system comprising of water (A) and acetonitrile (B) containing 0.1 % formic acid as the mobile phase. Analysis was performed using an API 4000 QTRAP hybrid triple quadruple mass spectrometer and multiple reaction monitoring (MRM) in positive mode at m/z transitions of 159.100 → 90.00 and 162.200 → 117.200 for 5-AMQ and the internal standard, respectively. The standard curves of 5-AMQ in rat urine and plasma samples were linear in the concentration range of 10–2500 ng/mL. The intra-day and inter-day precisions and accuracies for 5-AMQ at four concentration levels in rat plasma and urine samples were found to be within the 15 % FDA acceptance range. Similarly, the accuracy and precision of 5-AMQ quantification in samples diluted up to 20-fold using blank plasma were within the 15 % acceptable range. Furthermore, the extraction recoveries and matrix effects at three concentration levels of rat plasma samples ranged from 99.5 %–110.6 % and -6.1 %–14.1 %, respectively. 5-AMQ was stable in rat plasma samples subjected to standard storage, preparation, and handling conditions, with less than 15 % variation noted at two concentration levels. The validated, sensitive, and reproducible LC–MS/MS method for 5-AMQ in rat plasma and urine samples was effectively applied to a pharmacokinetic study in rats with IV and oral administration of 5-AMQ. 5-AMQ displayed substantial plasma exposures via IV and oral route, with a mean maximum plasma concentration of 2252 ng/mL after oral administration, mean area under the curve (AUC0-∞) of 3708 h.ng/mL and 14431 h.ng/mL for the IV and oral groups, respectively, mean terminal elimination half-life of 3.80 ± 1.10 h and 6.90 ± 1.20 h respectively after intravenous and oral dose, and a good oral bioavailability (F % = 38.4).

A simple protocol for the isolation of amentoflavone from two species of Biophytum DC. (Oxalidaceae) and evaluation of its antiproliferative potential

The biflavonoid, amentoflavone, isolated earlier from the medicinally important species, Biophytum sensitivum, is reportedly a potent bio-active compound. In the present study, extraction protocols were standardized for the isolation of amentoflavone from two other species of Biophytum viz. B. reinwardtii (Zucc.) Klotzsch and B. veldkampii Shanavas et al and subsequent characterization of the same (using various chromatographic and spectroscopic methods) followed by screening of its bioactive potential using selected in vitro cytotoxicity assays (MTT assay, Trypan Blue Exclusion Assay and Flow cytometry).The shade-dried plant parts of the two species of Biophytum were Soxhlet extracted with ethyl alcohol (70%) and used as the raw material for the isolation of amentoflavone. Column chromatography, with a gradient solvent system of chloroform, methanol and water, followed by the thin layer chromatography of the pooled fractions with a solvent combination of chloroform, acetone and formic acid was adopted. A single fraction in both species, showed a clear prominent band in TLC, [a distinct fluorescent yellow band under short UV (100 nm) and blackish brown colour under long UV (320 nm)]. The band crystallized into a yellow powdery substance. Co-TLC (Rf value- 0.6) and High Performance Liquid Chromatography (Rt- 5.3 min) was used to confirm the isolated compound as amentoflavone. Yield, physical (solubility and melting point) and chemical properties Ultraviolet- Visible spectroscopy (UV–vis), High Resolution Mass Spectroscopy (HRMS) and Nuclear Magnetic Resonance Spectroscopy (1H, 13C, DEPT) of amentoflavone were also noted for both species. The yield of amentoflavone was higher in B. reinwardtii (86.66 mg /100 g dried plant powder) than in B. veldkampii (77.50 mg/100 g). The data generated from the different spectral techniques corresponded well with the chemical configuration of amentoflavone in the database. Therefore it could be confirmed that the isolated pure compound from both species of Biophytum was amentoflavone (C30H18O10). The isolated compound, amentoflavone was tested against cancer and normal cell lines. Amentoflavone was not cytotoxic to Human Peripheral Lymphocytes (normal cell lines) in trypan blue exclusion assay. MTT assay clearly showed that the exposure of amentoflavone at different concentrations resulted in decrease of MCF- 7(cancer cell lines) cell proliferation in a dose- dependent manner. Apoptosis was found to be dose-dependent, which was further confirmed by flow cytometry. Significant cell cycle arrest occurred in S phase and G2/M phase. Thus, anticancer studies (trypan blue exclusion assay, MTT assay and flow cytometry) proved useful for the scientific validation of the anticancer activity of the isolated pure compound, amentoflavone. The present study is the first report on the isolation of amentoflavone from Biophytum reinwardtii and B. veldkampii. Since the amentoflavone content in the two species of Biophytum is high, it can be extracted in sufficient amounts and both species can be considered potential source of the compound.

Characterization of copolymers of polycarbonate and polydimethylsiloxane by 2D chromatographic separation, MALDI-TOF mass spectrometry, and FTIR spectroscopy

The structure and composition of polycarbonate polydimethylsiloxane copolymer (PC-co-PDMS) was investigated by applying various analytical approaches including chromatographic separation methods, spectrometric, and spectroscopic detection techniques. In particular, size exclusion chromatography (SEC) and liquid adsorption chromatography operating at different conditions (e.g. using gradient solvent systems) were used to achieve separations according to molar mass and functionality distribution. The coupling of both techniques resulted in fingerprint two-dimensional plots, which could be used to easily compare different copolymer batches. Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry was applied for structural investigations. The different ionization behavior of both comonomers, however, strongly limited the applicability of this technique. In contrast to that, Fourier-transform Infrared (FTIR) spectroscopy could be used to quantify the amount of PDMS in the copolymer at different points in the chromatogram. The resulting methodology was capable of distinguishing PC-co-PDMS copolymer from PC homopolymer chains present in the material.

Analysis, formation and inhibition of polycyclic aromatic hydrocarbons in foods: An overview

Polycyclic aromatic hydrocarbons (PAHs), formed through incomplete combustion or pyrolysis of wood or gasoline, represents an important class of toxicological compounds because of its wide distribution in the environment and possible contamination of foods. This paper deals with an overview of analysis, formation and inhibition of PAHs in foods. Extraction of PAHs from food is routinely conducted by saponification of lipid with Soxhlet method, followed by purification with a Sep-Pak Florisil cartridge and partition. This method can remove more impurities than the sonication method. With HPLC, all 16 PAHs can be simultaneously separated by a gradient solvent system and detected by UV at 254 nm or fluorescence employing programmable wavelength with seven settings of excitation/emission. With GC, all 16 PAHs can also be simultaneously separated by a temperature programming method and detected by flame ionization detector (FID) or ion-trap mass detector (ITD). Although HPLC can provide adequate separation of 16 PAHs, nevertheless, the presence of impurities in food sample can interfere with the subsequent identification and separation. For GC, a number of isomeric PAHs partially overlap. Nonetheless, the application of GC-ITD can readily identify the various PAHs in foods through reconstructed ion chromatograms even in the presence of fat- or PAH-like impurities. Charcoal-grilling and smoking are the two major processing methods which can result in the formation of high amount of total carcinogenic PAHs. No carcinogenic PAHs are detected in steaming and liquid smoke flavoring. The amount of PAHs in food samples can also be greatly reduced by sorption into low-density polyethylene bags. Further research in necessary to determine how the formation of PAHs during processing can be directly retarded or eliminated.

Analysis of soy isoflavones in foods and biological fluids: An overview

Soy isoflavones are phytoestrogens mainly present in soybean and soybean products. Isoflavones have been reported to possess physiological activities such as inhibition of cancer cell proliferation, reduction of cardiovascular risk, prevention of osteoporosis and alleviation of postmenopausal syndrome. This paper gives an overview of soy isoflavone analysis in foods and biological fluids. Extraction of isoflavones is often carried out by polar solvents such as methanol, ethanol, acetone or acetonitrile, or in combination with acid-containing water. The separation, identification and quantification of isoflavones are usually conducted by gas chromatography coupled with flame ionization detector or mass spectrometer (GC/FID or GC/MS), high-performance liquid chromatography with UV or mass spectrometer (HPLC/UV or HPLC/MS) and immunoassay. GC methods provide high sensitivity and adequate separation of only few soy isoflavones; however, a time-consuming derivatization step is needed before analysis. HPLC with a gradient solvent system with or without acid has been applied to analyze soy isoflavones, but most HPLC methods failed to separate all 12 isoflavones in a single run or the separation time is lengthy. Nevertheless, an appropriate choice of mobile phase and gradient condition in a C18 column with UV detection could provide a simultaneous separation of 12 soy isoflavones within short time. For immunoassay, it is convenient, fast, highly sensitive and can analyze a large number of samples at the same time. Yet, the drawback is that only a few soy isoflavones can be determined.

Analysis, Formation and Inhibition of Heterocyclic Amines in Foods: An Overview

Heterocyclic amines (HAs), an important class of toxicological compounds, can be formed through heating of four precursors, amino acids, creatinine, creatine and sugars. Numerous studies have shown that most HAs are mutagenic and carcinogenic, and the safety of HAs-containing foods has become a major concern for the public. This paper deals with an overview of analysis, formation and inhibition of HAs in foods. Analysis of HAs from foods is routinely conducted by protein precipitation, followed by acid-base solvent partition, purification with column chromatography and identification and quantification by high-performance liquid chromatography (HPLC), or gas chromatography-mass spectrometry (GC-MS). However, most of these methods result in low recovery. With HPLC, all 16 HAs can be simultaneously separated by a binary or ternary gradient solvent system and detected by UV. Using fluorescence detection, only nine HAs can be detected. For GC-MS, only a number of HAs are separated and identified by employing a temperature programming method. GC-MS provides rapid identification and quantification of HAs in foods, however, this technique requires a time-consuming derivatization and the number of HAs separation is limited. Also, some HAs may degrade and result in peak tailing. The application of HPLC with photodiode-array detection (HPLC-DAD) can not only permit rapid identification and quantification of HAs, but also enhance sensitivity and selectivity. Broiling and frying are two major processing methods causing the formation of high amount of HAs. The formation of HAs in foods during cooking can be inhibited by incorporating additives such as antioxidants and amino acids. However, the results have been controversial. Further research is necessary to study the effect of various additives on inhibition of HAs formation in foods.

Bioassay-Guided Separation of Centipeda minima Using Comprehensive Linear Gradient Centrifugal Partition Chromatography

A comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) was developed for the bioassay-guided isolation of natural compounds. The gradient solvent system consisted of three different ternary biphasic solvents types: n-hexane–acetonitrile–water (10:2:8, v/v), ethyl acetate–acetonitrile–water (10:2:8, v/v), and water-saturated n-butanol–acetonitrile–water (10:2:8, v/v). The lower phase of the n-hexane–acetonitrile–water (10:2:8, v/v) was used as the stationary phase, while its upper phase, as well as ethyl acetate–acetonitrile–water (10:2:8), and water-saturated n-butanol–acetonitrile–water (10:2:8, v/v) were pumped to generate a linear gradient elution, increasing the mobile phase polarity. We used the gradient CPC to identify antioxidant response elements (AREs), inducing compounds from Centipeda minima, using an ARE-luciferase assay in HepG2 cells, which led to the purification of the active molecules 3-methoxyquercetin and brevilin A. The developed CPC solvent systems allow the separation and isolation of compounds with a wide polarity range, allowing active molecule identification in the complex crude extract of natural products.

Differential adsorption of analyte and deuterated analogue onto the rotor seal of the injector in high-performance liquid chromatography/electrospray ionisation mass spectrometry.

Samples were injected onto the HPLC system via either a Valco C14W.1 internal sample injector or a Rheodyne 7125 injector, both with dissimilar Vespel rotor seals, and linked to a quadrupole time-of-flight (QTOF) mass spectrometer. Alternatively, samples were infused directly into the mass spectrometer via a syringe-driver. Electrospray ionisation in positive mode was used in both cases. Experiments demonstrated significant differential adsorption of the analyte (RAC inhibitor, NSC23766) and its tri-deuterated internal standard onto the Vespel rotor seal, with the latter seeming to be preferentially adsorbed. The deuterated analogue also showed lower electrospray sensitivity, as demonstrated by syringe infusion of a mixture of the compounds, compared with their separate infusion at the same concentration. This study has demonstrated the problem of differential adsorption onto HPLC Vespel rotor seals, and that the use of a stable-isotope-labelled analogue as internal standard does not guarantee the constancy of the analyte/internal response ratio in quantitative methods. The partial solution was to work at much higher concentration where adsorption, whilst still apparent, was relatively insignificant.

Identification and quantification of phenolic acid compounds of twenty-six mushrooms by HPLC–DAD

Phenolic acids are found in different foods in the human diet, for example mushrooms. Determination of phenolic acids is important because of their relationship to their role in disease prevention due to their bioactive properties. In this study, the phenolic acid profile of 26 mushroom species was analyzed by using high-performance liquid chromatography method coupled with photodiode array detector (HPLC–DAD) and 16 phenolic acid compounds were identified. The chromatographic separation was performed using Intertsil ODS-3 reverse phase C18 column (5 µm, 250 mm × 4.6 mm i.d), gradient solvent system with 1.5 mL/min flow rate and detected at 280 nm. The coefficient of determination (R2) was in the range of 0.9965–0.9999. Limit of detection and quantification ranged from 0.001–0.970 to 0.001–2.940 µg/L, respectively. The phenolic compounds were characterized according to their retention times and UV data were compared with commercial standards. S. granulatus (71.79 µg/g) and L. nuda (68.38 µg/g) revealed the highest concentration of total phenolic compounds among the studied mushrooms. Gallic acid was found as the major phenolic compound in R. aurora (2.96 ± 0.56 µg/g) while 6,7-dihydroxy coumarin was identified as major phenolic compounds in A. tabescens (2.07 ± 0.25 µg/g) and L. leucothites (9.02 ± 0.87 µg/g). Fumaric acid was found as the most abundant compounds in 16 out of 26 mushrooms. Catechin hydrate was identified as major phenolic compounds in the rest of mushrooms. This method provided a beneficial standardization procedure of phenolic acid compounds in mushroom samples.

Quantitative Determination of Andrographolide and Related Compounds in Andrographis paniculata Extracts and Biological Evaluation of Their Anti-Inflammatory Activity.

Extraction, isolation and characterization of Andrographis paniculata (A.p.) products were developed. Three natural diterpenes compounds were obtained and one was used for chemical modifications. Evaluation of their inhibition of TNFα induced NFκB transcriptional activity. A rapid analytical method for the determination and quantitation of three diterpenoid lactones (andrographolide 1, didehydroandrographolide 2, neoandrographiside 3) found in A. paniculata extracts was investigated. After some optimizations on column type and injection solvent, the separation was achieved in 9 min on a monolithic Chromolith Performance RP18e column (100 mm × 4.6 mm ID, 2 µm), with a gradient solvent system of water and methanol, UV detection at 220 nm and ELSD detection. The method was proved to be suitable for the quantitation of these three diterpenes in four different commercial Andrographis dietary supplements. The anti-inflammatory activities of a mixture of known composition have been evaluated showing differences in activity depending on the relative ratio of various diterpenes and also a possible synergic activity for some of them.

HPLC Method for Simultaneous Determination of Six Isoflavones in Soybean Yoghurt

Objective: To establish an HPLC method for simultaneous determination of six isoflavones (daidzin, glycitin, genistin, daidzein, glycitein, and genistein) in soybean yoghurt, providing a reference for its development of quality standards and functional evaluation. Methods: The six kinds of isoflavones were measured on a C18 column (250 mm×4.6 mm, 5μm) with a gradient solvent system of acetonitrile - trifluoroacetic acid solution as mobile phase at a flow rate of 1mL·mL-1 and the column temperature was kept at 40°C. The detection wavelength was 255nm. Results: Excellent chromatographic separation and good linearity with peak areas were achieved for 6 kinds of soybean isoflavones composition. The experiment was reproducible. Within the studied concentration ranges, the RSD were less than 2.50% and the average recovery were between 94.10% and 99.70%. Conclusion: This HPLC method is simple, feasible, stable and reliable. More importantly, it can detect the content of six soybean isoflavones simultaneously.

Application of the natural deep eutectic solvent choline chloride-sorbitol to extract chlorogenic acid and caffeine from green coffee beans (Coffea canephora)

This study aimed to determine the optimum method for extracting caffeine and chlorogenic acid (CGA) from green coffee beans (GCB) of Coffea canephora using choline chloride-sorbitol, a natural deep eutectic solvent (NADES). Three different preparations of choline chloride-sorbitol and choline chloride-sorbitol-urea (2:1, 4:1, and 6:1) were used for the extraction. The most effective preparation was used to evaluate the effect of dilution with water and extraction time. Reverse-phase High-performance liquid chromatography (HPLC), with a gradient solvent system of 0.1% acetic acid (90%) and acetonitrile (10%), was used to quantify the CGA and caffeine. Choline chloride-sorbitol at a ratio of 4:1 was the most effective for extracting chlorogenic and caffeine, with caffeine and CGA yield of 4.49 and 16.59 mg/g dry weight, respectively. The optimum water concentration was not found. Using a higher NADES dilution for extraction corresponded to an increased yield of caffeine and CGA. The effective time for extraction was found to be 30 min, which yielded the most caffeine and CGA. Based on these results, choline chloride-sorbitol could be an alternative green solvent for extracting caffeine and CGA from C. canephora GCB.

ISOLATION, CHARACTERIZATION AND ANTIFERTILITY ACTIVITY OF THE ACTIVE MOIETY FROM THE STEM OF MUSA PARADISIACA L.

Bioactive principles from the hyroalcoholic (50%) extract of Musa paradisiaca L. stem were isolated and characterized to evaluate antifertility activity in female albino rats. Oral acute toxicity study was done with crude extract for 24h. The hydro alcoholic extract of M. paradisiaca stem was subjected to silica gel column chromatography using gradient solvent system DCM: Eth, Mth: Eth and Mth., ten different fraction were collected. The yield of fraction Mu-HA- Mps was 580 mg, further fractionated for purification by using solvent DCM-MeOH (3:2) to yield compound Mu-HA-Mps (20.22 %w/w). Isolated compound Mu-HA-Mps was subjected to evaluation of its antifertility potential by antiovulatory and estrogenic activity. The isolated compound Mu-HA-Mps was found to exhibit significant antiovulatory and antiestrogenic activity at doses of 100 and 200 mg/kg body weight. Isolated compound Mu-HAMps was subjected to structure elucidation by UV, IR, NMR and MASS spectral analytical methods. The results of the present study provide evidence of anti-fertility activity of isolated compound Mu-HA-Mps as claimed in the traditional use. The hydroalcoholic extract of Musa paradisiaca L. furnished a compound whose structure was established as 4’-methoxy-7-hydroxyisoflavone on the basis of physical and spectral basis and could be a good source of drug for birth control.