Natural Product Extracts Research Articles

Acetylcholinesterase affinity-based screening assay on Lippia gracilis Schauer extracts

The use of affinity-based protein assay produced by covalently linking acetylcholinesterase to magnetic beads, followed by chemical characterization of the selective binders using Liquid Chromatography with tandem High-Resolution Mass Spectrometry (LC–HRMS) is herein described for profiling crude aqueous natural product extracts. The fishing assay was first modulated using galanthamine as a reference ligand and then, the assay condition was adjusted for the aqueous leaves extracts obtained from Lippia gracilis Schauer (genotype 201) that was used as the natural combinatory library. From the experiments, a selective binder has been undisclosed with an accurate mass of 449.1131 m/z and identified as eriodictyol 2′-O-glucoside or eriodictyol 3′-O-glucoside. The selectivity of the binding assay was demonstrated, as much as, that erydictiol 7-O-glucoside was not fished, although it was present in the crude aqueous extract. The binding assay platform exhibited high specificity and did not require any sample pretreatment, making it appropriate for profiling binders at natural libraries.

Identification of cyclosporin C from Amphichorda felina using a Cryptococcus neoformans differential temperature sensitivity assay.

We used a temperature differential assay with the opportunistic fungal pathogen Cryptococcus neoformans as a simple screening platform to detect small molecules with antifungal activity in natural product extracts. By screening of a collection extracts from two different strains of the coprophilous fungus, Amphichorda felina, we detected strong, temperature-dependent antifungal activity using a two-plate agar zone of inhibition assay at 25 and 37°C. Bioassay-guided fractionation of the crude extract followed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) identified cyclosporin C (CsC) as the main component of the crude extract responsible for growth inhibition of C. neoformans at 37°C. The presence of CsC was confirmed by comparison with a commercial standard. We sequenced the genome of A. felina to identify and annotate the CsC biosynthetic gene cluster. The only previously characterized gene cluster for the biosynthesis of similar compounds is that of the related immunosuppressant drug cyclosporine A (CsA). The CsA and CsC gene clusters share a high degree of synteny and sequence similarity. Amino acid changes in the adenylation domain of the CsC nonribosomal peptide synthase's sixth module may be responsible for the substitution of L-threonine compared to L-α-aminobutyric acid in the CsA peptide core. This screening strategy promises to yield additional antifungal natural products with a focused spectrum of antimicrobial activity.

The Bioactive Lipid (S)-Sebastenoic Acid Impacts Motility and Dispersion in Vibrio cholerae.

Although Gram-negative bacterial pathogens continue to impart a substantial burden on global healthcare systems, much remains to be understood about aspects of basic physiology in these organisms. In recent years, cyclic-diguanylate (c-di-GMP) has emerged as a key regulator of a number of important processes related to pathogenicity, including biofilm formation, motility and virulence. In an effort to discover chemical genetic probes for studying V. cholerae we have developed a new motility-based high-throughput screen to identify compounds that modulate c-di-GMP levels. Using this new screening platform, we tested a library of microbially-derived marine natural products extracts, leading to the discovery of the bioactive lipid (S)-sebastenoic acid. Evaluation of the effect of this new compound on bacterial motility, vpsL expression and biofilm formation implied that (S)-sebastenoic acid may alter phenotypes associated to c-di-GMP signaling in V. cholerae.

Vaccinium angustifolium (lowbush blueberry) leaf extract increases extravillous trophoblast cell migration and invasion in vitro.

Perturbations to extravillous trophoblast (EVT) cell migration and invasion are associated with the development of placenta-mediated diseases. Phytochemicals found in the lowbush blueberry plant (Vaccinium angustifolium) have been shown to influence cell migration and invasion in models of tumorigenesis and noncancerous, healthy cells, however never in EVT cells. We hypothesized that the phenolic compounds present in V.angustifolium leaf extract promote trophoblast migration and invasion. Using the HTR-8/SVneo human EVT cell line and Boyden chamber assays, the influence of V.angustifolium leaf extract (0 to 2×104 ng/ml) on trophoblast cell migration (n=4) and invasion (n=4) was determined. Cellular proliferation and viability were assessed using immunoreactivity to Ki67 (n=3) and trypan blue exclusion assays (n=3), respectively. At 20ng/ml, V.angustifolium leaf extract increased HTR-8/SVneo cell migration and invasion (p<.01) and did not affect cell proliferation or viability. Chlorogenic acid was identified as a major phenolic compound of the leaf extract and the most active compound. Evidence from Western blot analysis (n=3) suggests that the effects of the leaf extract and chlorogenic acid on trophoblast migration and invasion are mediated through an adenosine monophosphate-activated protein (AMP) kinase-dependent mechanism. Further investigations examining the potential therapeutic applications of this natural health product extract and its major chemical compounds in the context of placenta-mediated diseases are warranted.

Cathepsin D immobilized capillary reactors for on-flow screening assays

The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (KM = 81.9 ± 7.49 μmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts.

The Great Chemistry Race

The Great Chemistry Race

An in vitro affinity-based method for studying herb-drug interactions for direct identification of cytochrome P450 1A2, 3A4, and 2C9 specific ligands from herbal extracts using ultrafiltration-high performance liquid chromatography.

A novel in vitro strategy for affinity-based ultrafiltration-high performance liquid chromatography (HPLC) was developed for the direct identification of cytochrome P450 (CYP) 1A2, 3A4, and 2C9 specific ligands from Danshen extracts, in which human liver microsome (HLM) was used as the source of CYP enzymes. The Danshen extract was incubated without HLM, with HLM, or with HLM where the active site of the target enzyme was blocked with a specific competitive probe before ultrafiltration to isolate ligand–enzyme complexes from unbound compounds. Subsequently, HPLC analysis was performed on the released ligands from the complexes. α-Naphthoflavone, ketoconazole, and sulfaphenazole were used as specific competitive probes for CYP1A2, 3A4, and 2C9, respectively. The signal-to-noise ratio (S/N) and specific-signal-to-noise ratio (S-S/N) of each compound were calculated using the obtained peak areas. Finally, two criteria were applied to select putative ligands for each specific target: (1) S/N > 1; (2) S-S/N > 0. Finally, dihydrotanshinone was identified as a specific ligand for CYP1A2 and tanshinone I, cryptotanshinone, and tanshinone IIA were identified as specific ligands for CYP1A2, 2C9, and 3A4. It was demonstrated that the newly developed method can be used to rapidly and directly detect specific ligands from natural product extracts in multi-target systems.

HISTAMINE DETERMINATION IN FISHERY PRODUCTS: ALTERNATIVE METHODS FOR EXTRACTION AND QUANTIFICATION

HISTAMINE DETERMINATION IN FISHERY PRODUCTS: ALTERNATIVE METHODS FOR EXTRACTION AND QUANTIFICATION

Tailoring Properties of Acidic Types of Natural Deep Eutectics Solvents (NADES): Enhanced Solubility of Curcuminoids from Curcuma zeodaria

Recently Natural Deep Eutectic Solvents (NADES) show their potential as a promising green solvents at 21th century for extraction of natural products. Rutin, a poorly water soluble flavonoid, was reported better solubilized in NADES than in water as well as a paclitaxel and ginkgolide B, a completely water-insoluble compound. In case of curcuminoids, phenolic compounds from powder of Curcuma Zeodaria shown better solubilized in acidic type of NADES such malic acid-sucrose-water (MAS-H2O = 1:1:11, mole ratio) and citric acid-sucrose-water (CAS-H2O = 1:2:15, mole ratio) compare to water and ethanol. Indicating that NADES, a water-based solvent, appropriate for extracting curcuminoids. However, the inherent high viscosity of NADES hamper the process extraction. Lowering the viscosity, water content of NADES is varied, adding a certain amount of water up to 60% of water content. All the varied water content of both CAS-H2O and MAS-H2O were successfully extracted curcuminoids. Yielded 0.06-0.16 mg curcuminoids/g dry weight. However, 60% of water content of both CAS-H2O and MAS-H2O gave more or less similar value of curcuminoids to CAS-H2O (20% of water) and MAS-H2O (30% of water). Curcuminoids is more stable in CAS-H2O compare to MAS-H2O for 96 h of time duration.

A Review on-Herbs in Anticancer

Cancer is the abnormal growth of cells in our bodies that can lead to death. Cancer is a leading cause of mortality in the world and it's the cause of more than 20% of all deaths. The major problem in the cancer chemotherapy is the toxicity of the established drugs. Some of the natural product extracts are capable of preventing or inhibiting the process of carcinogenesis. Thousands of herbal and traditional compounds are being examine by many searchers worldwide to validate their use as anti-cancerous drugs. Medicinal plants and their use in various traditional systems of medicine are indeed awe-inspiring. General public consider herbal remedies to be safe, as it causes less side-effects and less likely to cause dependency. Various dietary agents that interfere with these stages of cancer, thus blocking malignancy and thus Cancer induction, growth, and progression are multi-step events and numerous studies have beendemonstrated. Fruits and vegetables contains untapped reservoir ofvarious nutritive and non-nutritive phytochemicals with potential cancer chemo-preventiveactivity. Plants have been used for treating diseases since time immemorial. More than 50% of modern drugs in clinical use are of natural products. Study is required to determine the efficacy of these plant products in treating cancers in humans.

Ionic liquid-based microwave-assisted extraction: Fast and green extraction method of secondary metabolites on medicinal plant

Background: Ionic liquid-based microwave-assisted extraction (IL-MAE) is one of the non-conventional extraction methods that has been developed and applied in recent years. Some studies have reported the success of this approach for extracting the target compound (secondary metabolites) from medicinal plants optimally. Objective: This review paper aimed to provide detail information about the application of the IL-MAE method as a fast and green extraction of a secondary metabolite from the medicinal plant. Materials and Methods: The literature published on IL-MAE was searched and collected using online resources from the electronic databases including Google Scholar, DOAJ, PubMed, ScienceDirect, and Scopus. Results: This review highlights the role of IL as a green solvent, the basic principles, mechanisms of MAE, and its utilization of natural product extraction. Furthermore, this review explained about the application of the IL-MAE method to extract secondary metabolite (particularly the targeted compound) from a medicinal plant, and a brief extraction mechanism of IL-MAE using Fourier-transform infrared spectroscopy and scanning electron microscopy. Conclusion: The application of IL-MAE method has successfully performed to extract the targeted secondary metabolite from a natural product, where the extraction process to be rapid, efficient, and green.

Modification and validation of Folin-Ciocalteu assay for faster and safer analysis of total phenolic content in food samples

Folin-Ciocalteu (F-C) is a routine assay in several laboratories around the world and has been widely employed for total phenolic content (TPC) quantification from food samples. F-C assay was modified to obtain a system reaction safer, as well as decrease the use of reagents, energy expenditure and time-consuming. After evaluating alternatives solvents and supports, and the effect of the binomial time/temperature, the modified F-C assay was as follows: 40% ethanol as solvent, 20 minutes at room temperature (25±2°C) using 50% F-C reagent and 5% sodium carbonate solution. The modified F-C assay was linear in 2.5-50.0 µg/mL range to gallic acid. Limit of detection and limit of quantification were 0.195 and 0.591 µg/mL, respectively. Intra-day and inter-day precision (relative standard deviation (RSD) 0.06-3.28%) and accuracy (93.28-104.28%) were also demonstrated. The assay was robust for F-C reagent (45-55%) and sodium carbonate (4.5-5.5%) changes. The modified assay was employed to analyse food samples containing phenolic compounds and the results corroborated with conventional assay. The modified F-C assay demonstrated to be reproducible, robust, fast, easy, inexpensive, safe and reliable for quantify phenolic compounds in food samples. The employment of ethanol in F-C assay decreases the environmental impact and, consequently, makes the analysis safer than conventional F-C assay. Furthermore, the modified F-C assay is conducted under milder conditions (time/temperature), which is particularly helpful for numerous analyses. Thus, the modified and validated F-C assay can be used as a routine assay in quality control and chemical profiling for natural product extracts and foods.

Development and Implementation of an HTS-Compatible Assay for the Discovery of Selective Small-Molecule Ligands for Pre-microRNAs

microRNAs (miRNAs) are small gene regulatory RNAs, and their expression has been found to be dysregulated in a number of human diseases. To facilitate the discovery of small molecules capable of selectively modulating the activity of a specific miRNA, we have utilized new high-throughput screening technology targeting Dicer-mediated pre-miRNA maturation. Pilot screening of ~50,000 small molecules and ~33,000 natural product extract libraries against pre-miR-21 processing indicated the potential of our assay for this goal, yielding a campaign Z′ factor of 0.52 and an average plate signal-to-background (S/B) ratio of 13. Using two-dimensional screening against a second pre-miRNA, pre-let-7d, we evaluated the selectivity of confirmed hits. The results presented demonstrate how high-throughput screening can be used to identify selective small molecules for a target RNA.

Solvents for Extraction of Antidiabetic, Iron Chelating, and Antioxidative Properties from Bottle Gourd Fruit

ABSTRACTBottle Gourd, Lagenaria siceraria (Molina) Standley (LS), fruit contain antidiabetic and antioxidant properties. The effect of solvents on extraction of bioactive components has not been studied. The choice of solvent is a major factor for extraction of natural products. The research compared alpha-amylase, alpha-glucosidase, and beta-carotene bleaching inhibitory and iron chelating potential of LS fruit extracts in the solvents water, acetone, ethanol, ethyl acetate, chloroform, and methanol and their ability to extract phenolics and flavonoids from the fruit. The methanolic extract was further fractionated into solvents of increasing polarity to separate phytochemicals based on their polarity. Ethyl acetate proved to be the best solvent to extract phenolics; acetone was best to extract flavonoids. The aqueous decoction was the most potent alpha-glucosidase inhibitor (IC50 0.28 mg∙mL−1) and ethyl acetate extract was the most effective alpha-amylase inhibitor (IC50 0.2 mg∙mL−1) and had the highest beta-carotene bleaching inhibitory potential (IC50 3.16 mg∙mL−1). The iron chelating potential of the ethyl acetate extract was second to the chloroform extract (IC50 82.11 and 199.2 µg∙m−1, respectively). The alpha-glucosidase inhibitory activities of the ether and ethyl acetate fractions were IC50 0.32 and 0.33 mg∙mL−1, respectively. The ethyl acetate fraction had IC50 0.26 mg∙mL−1 in alpha-amylase inhibitory activity compared to acarbose with IC50 0.23 mg∙mL−1. The ethyl acetate extract and aqueous decoction exhibited the best antidiabetic and antioxidative activities indicating the effectiveness of these solvents for extraction of bioactive substances.

Gram‐Scale Synthesis of the (−)‐Sparteine Surrogate and (−)‐Sparteine

AbstractAn 8‐step, gram‐scale synthesis of the (−)‐sparteine surrogate (22 % yield, with just 3 chromatographic purifications) and a 10‐step, gram‐scale synthesis of (−)‐sparteine (31 % yield) are reported. Both syntheses proceed with complete diastereocontrol and allow access to either antipode. Since the syntheses do not rely on natural product extraction, our work addresses long‐term supply issues relating to these widely used chiral ligands.

Gram-Scale Synthesis of the (-)-Sparteine Surrogate and (-)-Sparteine.

An 8-step, gram-scale synthesis of the (-)-sparteine surrogate (22 % yield, with just 3 chromatographic purifications) and a 10-step, gram-scale synthesis of (-)-sparteine (31 % yield) are reported. Both syntheses proceed with complete diastereocontrol and allow access to either antipode. Since the syntheses do not rely on natural product extraction, our work addresses long-term supply issues relating to these widely used chiral ligands.

A high-throughput screening campaign to identify inhibitors of DXP reductoisomerase (IspC) and MEP cytidylyltransferase (IspD)

The rise of antibacterial resistance among human pathogens represents a problem that could change the landscape of healthcare unless new antibiotics are developed. The methyl erythritol phosphate (MEP) pathway represents an attractive series of targets for novel antibiotic design, considering each enzyme of the pathway is both essential and has no human homologs. Here we describe a pilot scale high-throughput screening (HTS) campaign against the first and second committed steps in the pathway, catalyzed by DXP reductoisomerase (IspC) and MEP cytidylyltransferase (IspD), using compounds present in the commercially available LOPAC1280 library as well as in an in-house natural product extract library. Hit compounds were characterized to deduce their mechanism of inhibition; most function through aggregation. The HTS workflow outlined here is useful for quickly screening a chemical library, while effectively identifying false positive compounds associated with assay constraints and aggregation.

Analysis of small molecule compounds in complex mixture system by LC-MS

It has been a challenging task of analytical chemistry to qualitatively and quantitatively analyze the diverse compounds in the complex mixture. Liquid chromatography-mass spectroscopy (LC-MS), especially, ultra-high performance liquid chromatography (UPLC) coupled with high resolution mass spectrometry (HRMS), is a powerful tool for the analysis of complex samples. UPLC can greatly enhance the ability and efficiency of chromatographic separation. On the other hand, HRMS can provide more precise molecular weight and can thus improve the reliability and accuracy of qualitative and quantitative analysis. In this paper, we summarized the application of LC-MS technology to analysis of small molecule compounds in typical complex mixtures (urine, blood, cell metabolites and natural product extracts), concentrating on sample preparation, data acquisition, data (pre)processing, qualitative and quantitative analysis, multivariate analysis. Furthermore, technical challenges and developments in the analysis of complex samples by LC-MS were summarized and prospected.

Lactoferrin, chitosan and Melaleuca alternifolia-natural products that show promise in candidiasis treatment.

The evolution of microorganisms resistant to many medicines has become a major challenge for the scientific community around the world. Motivated by the gravity of such a situation, the World Health Organization released a report in 2014 with the aim of providing updated information on this critical scenario. Among the most worrying microorganisms, species from the genus Candida have exhibited a high rate of resistance to antifungal drugs. Therefore, the objective of this review is to show that the use of natural products (extracts or isolated biomolecules), along with conventional antifungal therapy, can be a very promising strategy to overcome microbial multiresistance. Some promising alternatives are essential oils of Melaleuca alternifolia (mainly composed of terpinen-4-ol, a type of monoterpene), lactoferrin (a peptide isolated from milk) and chitosan (a copolymer from chitin). Such products have great potential to increase antifungal therapy efficacy, mitigate side effects and provide a wide range of action in antifungal therapy.

Supercritical Fluid Extraction of Bioactive Compounds from Plant Materials.

There has been growing interest in the application of supercritical solvents over the last several years, many of the applications industrial in nature. The purpose of plant material extraction is to obtain large amounts of extract rich in the desired active compounds in a time-sensitive and cost-effective manner. The productivity and profitability of a supercritical fluid extraction (SFE) process largely depends on the selection of process parameters, which are elaborated upon in this paper. Carbon dioxide (CO2) is the most desirable solvent for the supercritical extraction of natural products. Its near-ambient critical temperature makes it suitable for the extraction of thermolabile components without degradation. A new approach has been adopted for SFE in which the solubility of nonpolar supercritical CO2 can be enhanced by the addition of small amounts of cosolvent.