iridoids and amarogentin

There is a need for the development of liposomes based nanomedicines formulation for better efficacy and safety of the available drugs in the market. Liposomes have various applications in the field of pharmaceutical and medical field for their drug target potential, diagnostic importance and imaging techniques. Natural plant based drugs and their derivatives have been used in the medicine, nutraceuticals, perfumery, cosmetic and beverages industry. More than half of the prescribed drugs in the worldwide are mainly derived from different natural sources. Development of plant derived product is an emerging field of food, pharmaceutical and health industries. Plants belonging to the Gentianaecae family are well known for their bitter taste and Swertia chirata is one of best plants among them. Various active phytochemical of Swertia chirata are bitter secoiridoids like gentiopicroside, amarogentin, swertiamarin, isovitexin and isogentisin. People use different species of Swertia in the form of decoction, infusion, paste and juice for the treatment of fever and enteric diseases. Swertia chirata possesses anticarcinogenic, antioxidative, hypoglycemic, antihepatotoxic, antimalarial, anti-inflammatory and antimicrobial activities. Amarogentin, a bitter secoiridoid glycoside present in Swertia chirata plant is an activator of human bitter taste receptor. Pharmacologically, amarogentin has antibacterial, antihepatitis, anticholinergic and chemopreventive activities, moreover, amarogentin has been proven for their anti-lieshmanial activity. Other studies also suggested that amarogentin acts on liver carcinogenesis, skin carcinogenesis and reduced tumour progression. In the present review, we have collected and compiled the data regarding biological sources, ethnomedicinal uses, phytochemistry, anticancer and anti-infective potential of amarogentin. For better understanding of various aspects of amarogentin, we have also discussed Swertia chirayita in a very concise manner. Further data related to various patents on amarogentin have also been discussed in this manuscript. However, we also admit that new advance biological research will also increase the medicinal and pharmacological value of amarogentin. Information regarding the chemistry of amarogentin, its biological sources, bioavailability as a pharmacological agent for the treatment and management of skin disorders and various forms of cancers will be beneficial to the scientists in the medicinal field.

Topics: Administration, Topical; Animals; Anti-Infective Agents; Antineoplastic Agents; Humans; Iridoids; Lipids; Patents as Topic; Plant Extracts; Plants, Medicinal; Swertia

Chirayata-the whole dried plant of Swertia chirayita-is an important traditional drug of Indian System of Medicines. A novel reverse-phase high performance liquid chromatography (RP-HPLC) method has been developed for the simultaneous determination and quantification of amaroswerin, amarogentin and andrographolide in a herbal drug "Chirayata," which is oftenly adulterated/substituted with herbal drug Kalmegh. The developed method is in accordance with International Council for Harmonization guidelines and is simple, precise, accurate, rapid, reproducible and specific to determine amarogentin, amaroswerin and andrographolide. Reverse-phase column (Water's X-bridge C18, 5 μm, 4.6 mm × 250 mm) with high resolution for all marker compounds was used with binary gradient elution (methanol:water) with a flow rate of 1 mL/min and detection at 235 nm. The developed method showed good linearity (R2 > 0.999) in a relatively wider range of concentration 2.968-95.00 ppm for amarogentin, amaroswerin and 5.625-180 ppm for andrographolide. The method is important for quality control analysis of drug Chirayata.

Topics: Chromatography, High Pressure Liquid; Iridoids

The active ingredients extracted from natural plants have anti-GC actions and can slow down gastric carcinoma (GC) progression. To investigate the impact of Amarogentin (AG) on GC cell multiplication, apoptosis and migration and the possible mechanisms.. qRT-PCR quantification of circKIF4A and miR-152-3p in GC tissues and normal counterparts as well as HGC-27 (human GC cell strain) and GES-1 (human gastric mucosal epithelial cell strain) was performed. HGC-27 cells were intervened by AG of various concentrations. si-NC, si-circKIF4A were further transfected into HGC-27 cells. Besides, pcDNA and pcDNA-circKIF4A were transfected into HGC-27 cells, after which 60 mmol/L AG was added for intervention. Cell multiplication, clone formation, as well as apoptosis and migration measurements were made by MTT, plate clone formation, flow cytometry and Transwell assays, respectively; Double luciferase reporter assay was performed for targeting relationship identification between circKIF4A and miR-152-3p; Western blots were carried out to measure Bax and Bcl-2 protein levels.. circKIF4A increased (P <0.05) and miR-152-3p decreased (P <0.05) in GC tissues and cell strains. Concentration-dependently, AG intervention contributed to enhanced cell multiplication inhibitory rate, apoptosis rate, miR-152-3p expression and Bax protein level (P <0.05), together with declined number of cell clones formed, migrating cells, circKIF4A expression and Bcl-2 protein level (P <0.05). After transfection of si-circKIF4A, cell multiplication inhibition rate, apoptosis rate and Bax protein level enhanced (P <0.05), while cell clones formed and migrating cells as well as Bcl-2 protein level reduced (P <0.05). miR-152-3p can be controlled by circKIF4A; pcDNA-circKIF4A transfection antagonized AG's effects on HGC-27 cell multiplication, clone formation, apoptosis and migration.. AG can decrease GC multiplication, clone formation and migration and induce apoptosis via modulating circKIF4A/miR-152-3p expression.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Iridoids; MicroRNAs; Proto-Oncogene Proteins c-bcl-2; Stomach Neoplasms

Amarogentin (AMA), a secoiridoid glycoside that is mainly derived from SwertiaandGentiana roots, has been confirmed to exhibit antioxidative, tumor-suppressive and anti-diabetic properties. This research intends to investigate the protective effect of AMA against sepsis-induced brain injury and its mechanism. NSC-34 and HT22 cells were treated with lipopolysaccharide (LPS) to induce an in-vitro sepsis model and then treated with varying concentrations (1, 5, 10 µM) of AMA. Cell proliferation and apoptosis were evaluated. The intensity of inflammation and oxidative stress were assessed by different methods. The AMPK/SIRT1/NF-κB pathway expression was determined by WB. An in-vitro sepsis model was set up with cecal ligation and puncture (CLP) in adult C57/BL6J mice, and different concentrations (25, 50, 100 mg/kg) of AMA were applied for treatment. Neurological function was evaluated using the modified neurological severity scores (mNSS), and the brain tissue damage was measured using hematoxylin-eosin (H&E) staining and Nissl staining. Tissue apoptosis was tested using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Then, the AMPK inhibitor Compound C (CC) was administered to confirm AMA-mediated mechanism. Our finding illustrated that AMA mitigated LPS-induced neuronal damage, inflammation and oxidative stress, activated the AMPK/SIRT1 pathway and choked NF-κB phosphorylation. Furthermore, AMA improved neurological functions of sepsis mice by reliving neuroinflammation and oxidative stress. Inhibition of AMPK attenuated the protective effect of AMA on neurons or the mice's brain tissues. In conclusion, AMA protected against sepsis-induced brain injury by modulating the AMPK/SIRT1/NF-κB pathway.

Topics: AMP-Activated Protein Kinases; Animals; Brain Injuries; DNA Nucleotidylexotransferase; Eosine Yellowish-(YS); Hematoxylin; Inflammation; Iridoid Glycosides; Iridoids; Lipopolysaccharides; Mice; NF-kappa B; Sepsis; Signal Transduction; Sirtuin 1

The coronavirus disease 2019 (COVID-19) pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents an unprecedented challenge to global public health with researchers striving to find a possible therapeutic candidate that could limit the spread of the virus. In this context, the present study employed an

Topics: Antiviral Agents; Coronavirus 3C Proteases; Coronavirus RNA-Dependent RNA Polymerase; COVID-19 Drug Treatment; Humans; Iridoids; Molecular Docking Simulation; Molecular Dynamics Simulation; Protease Inhibitors; SARS-CoV-2; Spike Glycoprotein, Coronavirus

Amarogentin (AG), a biologically active secoiridoid glycoside, is responsible for the efficacy of Gentianaceae based medications. Thus, qualitative and quantitative analyses of AG are of significance for batch to batch quality control purposes. By conjugating colloidal gold nanoparticles with the AG-specific monoclonal antibody, MAb 1E9, we were able to develop a single-step competitive immunochromatographic assay (ICA) for simple quantification of the AG content in plant samples. With a limit of detection of 250 ng/mL, the analytical results were obtained after immersing the ICA test strip in the detection mixture for 15 min. This new ICA is superior to conventional ICAs as it is considerably faster due to the speed with which the test strips can be produced and the omission of the time-consuming preparation phase that was previously required to make the fiber pad. Moreover, our ICA only needs a small amount of analyte (20 µL).The reliability of the reported test strip was confirmed by comparing its semi-quantitative results with those obtained via an indirect competitive enzyme-linked immunosorbent assay (icELISA). The positive correlation between these methods (R

Topics: Antibodies, Monoclonal; Chromatography, Affinity; Enzyme-Linked Immunosorbent Assay; Gentianaceae; Gold; Iridoids; Metal Nanoparticles; Molecular Conformation; Reagent Strips

Amarogentin is well known to be among the most bitter naturally occurring compound. Either as an individual one or extracts, amarogentin is used as a food additive and as a dietary supplement. The aim of the present investigation is to set-up a convenient process to selectively isolate amarogentin from the ethanolic roots extract of Gentiana lutea. The process consisted in the treatment of an aqueous suspension of such an extract with a panel of 21 solid inorganic / organic sorbents followed by filtration, desorption, and high performance liquid chromatography (HPLC) analyses. Among the solid materials tested, those containing Mg

Topics: Gentiana; Humans; Iridoids; Plant Roots; Taste

In this study, response surface methodology (RSM) and artificial neural network (ANN) was used to construct the predicted models of linear, quadratic and interactive effects of two independent variables viz. salicylic acid (SA) and chitosan (CS) for the production of amarogentin (I), swertiamarin (II) and mangiferin (III) from shoot cultures of Swertia paniculata Wall. These compounds are the major therapeutic metabolites in the Swertia plant, which have significant role and demand in the pharmaceutical industries.. Present study highlighted that different concentrations of SA and CS elicitors substantially influenced the % yield of (I), (II) and (III) compounds in the shoot culture established on modified ½ MS medium (supplemented with 2.22 mM each of BA and KN and 2.54 mM NAA). In RSM, different response variables with linear, quadratic and 2 way interaction model were computed with five-factor-three level full factorial CCD. In ANN modelling, 13 runs of CCD matrix was divided into 3 subsets, with approximate 8:1:1 ratios to train, validate and test. The optimal enhancement of (I) (0.435%), (II) (4.987%) and (III) (4.357%) production was achieved in 14 days treatment in shoot cultures of S. paniculata elicited by 9 mM and 12 mg L

Topics: Chitosan; Glycosides; Iridoid Glucosides; Iridoids; Neural Networks, Computer; Pyrones; Salicylic Acid; Swertia; Xanthones

In the present study, the replicative lifespan assay of yeast was used to guide the isolation of antiaging substance from

Topics: Aging; Antioxidants; Gentiana; Humans; Iridoids; Oxidative Stress

Whether and how amarogentin suppresses the angiogenesis effect in liver cancer cells after insufficient radiofrequency ablation (iRFA) are still poorly studied.. The number of liver cancer stem cells (LCSCs) and the level of vascular endothelial growth factor A (VEGFA) were assessed in liver cancer tissue after iRFA. Then, CD133-positive cells were detected in iRFA models of HepG2 and Huh7 cell lines treated with amarogentin. Tube formation assays were applied to observe the antiangiogenesis effects of amarogentin. In addition, the angiogenesis-related molecules p53, delta-like ligand 4 (Dll4), and Notch1 were detected in the iRFA cells and mouse models treated with amarogentin.. The mRNA and protein expression levels of CD133 and VEGFA were significantly higher in the residual liver cancer tissue than in the liver cancer tissues treated by hepatectomy. Amarogentin then markedly decreased the percentage of CD133-positive cells in the iRFA model in both HepG2 and Huh7 cell lines. The number of tubules formed by human umbilical vein endothelial cells (HUVECs) was significantly decreased by amarogentin. Inversely, the antiangiogenesis effect of amarogentin was counteracted after p53 silencing in the iRFA cell models.. Amarogentin prevents the malignant transformation of liver cancer after iRFA via affecting stemness and the p53-dependent VEGFA/Dll4/Notch1 pathway to inhibit cancer cell angiogenesis.

Topics: AC133 Antigen; Adaptor Proteins, Signal Transducing; Animals; Antineoplastic Agents, Phytogenic; Calcium-Binding Proteins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hepatectomy; Human Umbilical Vein Endothelial Cells; Humans; Iridoids; Liver Neoplasms; Male; Mice; Neoplastic Stem Cells; Neovascularization, Pathologic; Platelet Endothelial Cell Adhesion Molecule-1; Radiofrequency Ablation; Receptor, Notch1; Signal Transduction; Tumor Burden; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Amarogentin (AG) is one of the bitter secoiridoid glycosides, which exerts various pharmacological activities as a bitter stomachic. Recently, there is an increasing demand for AG-containing plants in Japan due to their use as folk medicines and food additives; hence, it is crucial to develop analytical techniques that are specific for AG. In this study, a new magnetic particles-based enzyme immunoassay (MPs-EIA) using a specific monoclonal antibody against AG (MAb 1E9) for the rapid determination of AG in plants of the family Gentianaceae was described. AG directly immobilized onto magnetic particles (MPs) was used as a competitor for free AG against MAb 1E9, thereby increasing the surface area of the solid phase and decreasing the immunoreaction time. In addition, the blocking step required in case of the conventional enzyme-linked immunosorbent assay could be avoided in the proposed MPs-EIA, which enables an even more rapid performance for the immunoassay. In the developed MPs-EIA, AG exhibited linearity in the range of 15.6-500 ng mL

Topics: Antibodies, Immobilized; Antibodies, Monoclonal; Immunoenzyme Techniques; Iridoids; Limit of Detection; Magnets; Time Factors

AMP-activated protein kinase (AMPK) is a drug target for treatment of metabolic and cardiovascular complications. Extracts of Gentianaceace plants exhibit anti-diabetic and anti-atherosclerotic effects, however, whether their phyto-constitutents activate AMPK remains to be determined.. Molecular docking of Gentiana lutea constituents was performed with crystal structure of human α. AG interacts with the α. In conclusion, AG exerts beneficial vasculo-metabolic effects by activating AMPK.. Amarogentin, a naturally occurring secoiridoid glycoside, is a promising lead for design and synthesis of novel drugs for treatment and management of dyslipidemia and cardiovascular diseases.

Topics: AMP-Activated Protein Kinases; Animals; Atherosclerosis; Calorimetry; Diabetes Mellitus, Experimental; Endothelium, Vascular; Enzyme Activation; Glucose; Human Umbilical Vein Endothelial Cells; Humans; Iridoids; Lipids; Male; Mice; Mice, Inbred C57BL; Molecular Docking Simulation; Rats; Signal Transduction; Tumor Necrosis Factor-alpha

Topics: Animals; Biomarkers; Carbon Tetrachloride; Discriminant Analysis; Disease Models, Animal; Drugs, Chinese Herbal; Gas Chromatography-Mass Spectrometry; Humans; Iridoids; Liver; Liver Cirrhosis; Male; Medicine, Chinese Traditional; Metabolic Networks and Pathways; Metabolome; Metabolomics; Mice; Mice, Inbred C57BL; Principal Component Analysis; Protective Agents; Tandem Mass Spectrometry

In this study, antitumor activity of epigallocatechin gallate (EGCG; major component of green tea polyphenol), eugenol (active component of clove), and amarogentin (active component of chirata plant) either alone or in combination were evaluated in Hela cell line. It was evident that EGCG with eugenol-amrogentin could highly inhibit the cellular proliferation and colony formation than individual treatments. Induction of apoptosis was also higher after treatment with EGCG in combination with eugenol-amrogentin than individual compound treatments. The antiproliferative effect of these compounds was due to downregulation of cyclinD1 and upregulation of cell cycle inhibitors LIMD1, RBSP3, and p16 at G1/S phase of cell cycle. Treatment of these compounds could induce promoter hypomethylation of LimD1 and P16 genes as a result of reduced expression of DNA methyltransferase 1 (DNMT1). Thus, our study indicated the better chemotherapeutic effect of EGCG in combination with eugenol-amarogentin in Hela cell line. The chemotherapeutic effect might be due to the epigenetic modification particularly DNA hypomethylation through downregulation of DNMT1.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Catechin; Cell Proliferation; DNA (Cytosine-5-)-Methyltransferase 1; Drug Synergism; Eugenol; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Iridoids; LIM Domain Proteins; Tea; Tumor Suppressor Proteins; Uterine Cervical Neoplasms

Amarogentin is an efficacious Chinese herbal medicine and a component of the bitter apricot kernel. It is commonly used as an expectorant and supplementary anti-cancer drug. β-Glucosidase is an enzyme that hydrolyzes the glycosidic bond between aryl and saccharide groups to release glucose. Upon their interaction, β-glucosidase catalyzes amarogentin to produce considerable amounts of hydrocyanic acid, which inhibits cytochrome C oxidase, the terminal enzyme in the mitochondrial respiration chain, and suspends adenosine triphosphate synthesis, resulting in cell death. Hydrocyanic acid is a cell-cycle-stage-nonspecific agent that kills cancer cells. Thus, β-glucosidase can be coupled with a tumor-specific monoclonal antibody. β-Glucosidase can combine with cancer-cell-surface antigens and specifically convert amarogentin to an active drug that acts on cancer cells and the surrounding antibodies to achieve a killing effect. β-Glucosidase is injected intravenously and recognizes cancer-cell-surface antigens with the help of an antibody. The prodrug amarogentin is infused after β-glucosidase has reached the target position. Coupling of cell membrane peptides with β-glucosidase allows the enzyme to penetrate capillary endothelial cells and clear extracellular deep solid tumors to kill the cells therein. The Chinese medicine amarogentin and β-glucosidase will become an important treatment for various tumors when an appropriate monoclonal antibody is developed.

Topics: Amygdalin; Antibodies, Monoclonal; Antineoplastic Agents; beta-Glucosidase; Cell-Penetrating Peptides; Humans; Iridoids; Prodrugs

Topics: Actins; Albumins; Animals; Antioxidants; Carbon Tetrachloride; Cell Line; Dose-Response Relationship, Drug; Down-Regulation; Gentiana; Glycosides; Humans; Hydroxyproline; Iridoids; Liver Cirrhosis; Malondialdehyde; Mice; Mice, Inbred C57BL; Nucleotides, Cyclic; Oxidative Stress; Phytotherapy; Plant Extracts; Plant Roots; Swertia; Tissue Distribution

Amarogentin has been reported to have a preventive effect on liver cancer via inducing cancer cell apoptosis. We attempted to elucidate the roles of p53-associated apoptosis pathways in the chemopreventive mechanism of amarogentin. The findings of this study will facilitate the development of a novel supplementary strategy for the treatment of liver cancer.. The purity of amarogentin was assessed by high-performance liquid chromatography. The inhibitory ratios of the liver cell lines were determined using a Cell Counting Kit-8 following treatment with a gradient concentration of amarogentin. Cell apoptosis was detected by flow cytometry using annexin V-fluorescein isothiocyanate/propidium iodide kits. The gene and protein expression of p53-associated molecules, such as Akt, human telomerase reverse transcriptase, RelA, and p38, was detected by real-time quantitative polymerase chain reaction, Western blotting, and immunohistochemical staining in liver cancer cells and mouse tumor tissues after treatment with amarogentin.. The inhibitory effect of amarogentin on cell proliferation was more obvious in liver cancer cells, and amarogentin was more likely to induce the apoptosis of liver cancer cells than that of normal liver cells. The gene and protein expression levels of Akt, RelA, and human telomerase reverse transcriptase were markedly higher in the control group than in the preventive group and treatment groups. Only the expression of human telomerase reverse transcriptase was downregulated, accompanied by the upregulation of p53.. The results of our study suggest that amarogentin promotes apoptosis of liver cancer cells by the upregulation of p53 and downregulation of human telomerase reverse transcriptase and prevents the malignant transformation of these cells.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Humans; Iridoids; Liver Neoplasms; Male; Mice; Signal Transduction; Telomerase; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Amarogentin, a secoiridoid glycoside isolated from medicinal plant Swertia chirata, was found to restrict CCl4 /N-nitrosodiethyl amine (NDEA) induced mouse liver carcinogenesis by modulating G1/S cell cycle check point and inducing apoptosis. To understand its therapeutic efficacy on stem cell self renewal pathways, prevalence of CD44 positive cancer stem cell (CSC) population, expressions (mRNA/protein) of some key regulatory genes of self renewal Wnt and Hedgehog pathways along with expressions of E-cadherin and EGFR were analyzed during the liver carcinogenesis and in liver cancer cell line HepG2. It was observed that amarogentin could significantly reduce CD44 positive CSCs in both pre and post initiation stages of carcinogenesis than carcinogen control mice. In Wnt pathway, amarogentin could inhibit expressions of β-catenin, phospho β-catenin (Y-654) and activate expressions of antagonists sFRP1/2 and APC in the liver lesions. In Hedgehog pathway, decreased expressions of Gli1, sonic hedgehog ligand, and SMO along with up-regulation of PTCH1 were seen in the liver lesions due to amarogentin treatment. Moreover, amarogentin could up-regulate E-cadherin expression and down-regulate expression of EGFR in the liver lesions. Similarly, amarogentin could inhibit HepG2 cell growth along with expression and prevalence of CD44 positive CSCs. Similar to in vivo analysis, amarogentin could modulate the expressions of the key regulatory genes of the Wnt and hedgehog pathways and EGFR in HepG2 cells. Thus, our data suggests that the restriction of liver carcinogenesis by amarogentin might be due to reduction of CD44 positive CSCs and modulation of the self renewal pathways. © 2015 Wiley Periodicals, Inc.

Topics: Animals; Carbon Tetrachloride; Cell Proliferation; Cell Survival; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Hep G2 Cells; Humans; Hyaluronan Receptors; Iridoids; Liver Neoplasms; Mice; Neoplastic Stem Cells; Wnt Signaling Pathway

Gentian root extract is used as a bitter food additive in Japan. We investigated the constituents of this extract to acquire the chemical data needed for standardized specifications. Fourteen known compounds were isolated in addition to a mixture of gentisin and isogentisin: anofinic acid, 2-methoxyanofinic acid, furan-2-carboxylic acid, 5-hydroxymethyl-2-furfural, 2,3-dihydroxybenzoic acid, isovitexin, gentiopicroside, loganic acid, sweroside, vanillic acid, gentisin 7-O-primeveroside, isogentisin 3-O-primeveroside, 6'-O-glucosylgentiopicroside, and swertiajaposide D. Moreover, a new compound, loganic acid 7-(2'-hydroxy-3'-O-β-D-glucopyranosyl)benzoate (1), was also isolated. HPLC was used to analyze gentiopicroside and amarogentin, defined as the main constituents of gentian root extract in the List of Existing Food Additives in Japan.

Topics: Chromatography, High Pressure Liquid; Food Additives; Gentiana; Iridoid Glucosides; Iridoids; Molecular Structure; Plant Extracts; Plant Roots

Here, we report potential transcripts involved in the biosynthesis of therapeutic metabolites in Swertia japonica , the first report of transcriptome assembly, and characterization of the medicinal plant from Swertia genus. Swertia genus, representing over 170 plant species including herbs such as S. chirata, S. hookeri, S. longifolia, S. japonica, among others, have been used as the traditional medicine in China, India, Korea, and Japan for thousands of years. Due to the lack of genomic and transcriptomic resources, little is known about the molecular basis involved in the biosynthesis of characteristic key bioactive metabolites. Here, we performed deep-transcriptome sequencing for the aerial tissues and the roots of S. japonica, generating over 2 billion raw reads with an average length of 101 bps. Using a combined approach of three popular assemblers, de novo transcriptome assembly for S. japonica was obtained, yielding 81,729 unigenes having an average length of 884 bps and N50 value of 1452 bps, of which 46,963 unigenes were annotated based on the sequence similarity against NCBI-nr protein database. Annotation of transcriptome assembly resulted in the identification of putative genes encoding all enzymes from the key therapeutic metabolite biosynthesis pathways. Transcript abundance analysis, gene ontology enrichment analysis, and KEGG pathway enrichment analysis revealed metabolic processes being up-regulated in the aerial tissues with respect to the roots of S. japonica. We also identified 37 unigenes as potential candidates involved in the glycosylation of bioactive metabolites. Being the first report of transcriptome assembly and annotation for any of the Swertia species, this study will be a valuable resource for future investigations on the biosynthetic pathways of therapeutic metabolites and their regulations.

Topics: Biosynthetic Pathways; Gene Expression Profiling; Gene Expression Regulation, Plant; Gene Ontology; Genes, Plant; Glucosyltransferases; High-Throughput Nucleotide Sequencing; Iridoid Glucosides; Iridoids; Metabolome; Microsatellite Repeats; Molecular Sequence Annotation; Phylogeny; Plant Roots; Pyrones; Swertia; Transcriptome; Up-Regulation

Amarogentin is a bitter-tasting secoiridoid glycoside isolated from an herb. Inhibition of aldose reductase by amarogentin has been documented as an antidiabetic action. However, the mechanisms of action of amarogentin in diabetic disorders remain unknown. The present study employed streptozotocin-induced type 1 diabetic (T1DM) rats to investigate the antihyperglycemic action of amarogentin. Changes in the protein expression of glucose transporter 4 (GLUT4) and phosphoenolpyruvate carboxykinase (PEPCK) in skeletal muscle and liver, respectively, were also detected by Western blotting. Additionally, a type 2 diabetes (T2DM) animal model induced using a fructose-rich diet was also applied to assess the effect of amarogentin on insulin resistance according to the homeostasis model assessment-insulin resistance (HOMA-IR). Amarogentin dose-dependently attenuated hyperglycemia in the T1DM rats lacking insulin. The action of amarogentin was further supported in rats administered the oral glucose tolerance test. Western blotting showed that amarogentin reversed the decreased GLUT4 level in skeletal muscle and reduced the elevated PEPCK expression in livers isolated from the T1DM rats. Moreover, amarogentin decreased the HOMA-IR and increased insulin sensitivity in the T2DM rats. These data show that amarogentin may ameliorate glucose homeostasis in diabetic rats, indicating its potential for future development as an antidiabetic drug.

Topics: Animals; Biomarkers; Blood Glucose; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Dietary Carbohydrates; Dose-Response Relationship, Drug; Fructose; Glucose Tolerance Test; Glucose Transporter Type 4; Hypoglycemic Agents; Insulin; Insulin Resistance; Intracellular Signaling Peptides and Proteins; Iridoids; Liver; Male; Muscle, Skeletal; Phosphoenolpyruvate Carboxykinase (GTP); Rats, Wistar; Streptozocin

The root of Gentiana lutea L., famous for its bitter properties, is often used in alcoholic bitter beverages, food products and traditional medicine to stimulate the appetite and improve digestion. This study presents a new, fast, and accurate HPLC method using HPLC/ESI-MS and HPLC/DAD for simultaneous analysis of iridoids (loganic acid), secoiridoids (gentiopicroside, sweroside, swertiamarin, amarogentin) and xanthones (isogentisin) in different populations of G.lutea L., cultivated in the Monti Sibillini National Park, obtained wild there, or purchased commercially. Comparison of HPLC/ESI-MS and HPLC/DAD indicated that HPLC/ESI-MS is more sensitive, reliable and selective. Analysis of twenty samples showed that gentiopicroside is the most dominant compound (1.85-3.97%), followed by loganic acid (0.11-1.30%), isogentisin (0.03-0.48%), sweroside (0.05-0.35%), swertiamarin (0.08-0.30%), and amarogentin (0.01-0.07%). The results confirmed the high quality of the G.lutea cultivated in the Monti Sibillini National Park.

Topics: Alcoholic Beverages; Chromatography, High Pressure Liquid; Gentiana; Iridoid Glucosides; Iridoids; Mass Spectrometry; Plant Roots; Pyrones; Taste; Xanthones

Recent studies have shown that human bitter taste receptors (TAS2Rs) are not only expressed in mucous epithelial cells of the tongue, but also in epithelial cells of the colon, stomach and upper respiratory tract. These cell types come in close contact with external bitter compounds by ingestion or breathing. In the present work we addressed the question whether bitter taste receptors might also be expressed in cornified epithelial cells of the skin. Here, we show for the first time the expression of TAS2R1 and TAS2R38 in human skin. Double staining of HaCaT cells and primary keratinocytes demonstrated the colocalization of TAS2R1 and TAS2R38 with the adaptor protein α-gustducin that is essential for signal transduction upon ligand binding. To test if TAS2Rs in keratinocytes are functional, we stimulated HaCaT cells with diphenidol, a clinically used bitter-tasting antiemetic, or amarogentin, the bitterest plant substance, that binds TAS2Rs, including TAS2R1 and TAS2R38. Diphenidol and amarogentin induced calcium influx. Furthermore, in keratinocytes diphenidol and amarogentin stimulated the expression of the differentiation markers keratin 10, involucrin and transglutaminase. Therefore, apart from the known role in mucous membranes of the gastrointestinal tract, TAS2Rs are expressed in the epidermis and might play a role in keratinocyte differentiation.

Topics: Adult; Animals; Cell Line; Cells, Cultured; Humans; Iridoids; Keratinocytes; Mice; NIH 3T3 Cells; Piperidines; Receptors, G-Protein-Coupled; Tongue

Swertia chirayita is an endangered medicinal herb widely used as an antidiabetic. It contains two major classes of metabolites, secoiridoids and xanthones, i.e. swertiamarin, mangiferin, amarogentin and amaroswerin. The biosynthetic pathways for these chemical constituents are not completely deciphered due to gaps and redundancy of routes proposed such as for mangiferin. The missing intermediates in pathways were detected through LC-ESI-QToF-HRMS/MS, including the detection of new secoiridoids, amaronitidin and gentiopicroside. The study also reports that the biosynthesis of amaronitidin occurs through the coupling of gentiopicroside and biphenyl acid derivatives such as amarogentin and amaroswerin. This study reports for the first time complete biosynthetic pathways for gentiopicroside, mangiferin, amarogentin, amaroswerin and amaronitidin in S. chirayita with the detection of intermediate metabolites iriflophenone, maclurin, deoxyloganic acid, loganic acid and 1,3,6,7-tetrahydroxy-9H-xanthen-9-one.

Topics: Biosynthetic Pathways; Glucosides; Hypoglycemic Agents; Iridoid Glucosides; Iridoids; Plants, Medicinal; Pyrans; Secondary Metabolism; Swertia; Tandem Mass Spectrometry; Xanthones

Swertia chirayita, an endangered medicinal herb, contains three major secondary metabolites swertiamarin, amarogentin and mangiferin, exhibiting valuable therapeutic traits. No information exists as of today on the biosynthesis of these metabolites in S. chirayita. The current study reports the expression profiling of swertiamarin, amarogentin and mangiferin biosynthesis pathway genes and their correlation with the respective metabolites content in different tissues of S. chirayita. Root tissues of greenhouse grown plants contained the maximum amount of secoiridoids (swertiamarin, 2.8% of fr. wt and amarogentin, 0.1% of fr. wt), whereas maximum accumulation of mangiferin (1.0% of fr. wt) was observed in floral organs. Differential gene expression analysis and their subsequent principal component analysis unveiled ten genes (encoding HMGR, PMK, MVK, ISPD, ISPE, GES, G10H, 8HGO, IS and 7DLGT) of the secoiridoids biosynthesis pathway and five genes (encoding EPSPS, PAL, ADT, CM and CS) of mangiferin biosynthesis with elevated transcript amounts in relation to corresponding metabolite contents. Three genes of the secoiridoids biosynthesis pathway (encoding PMK, ISPD and IS) showed elevated levels (∼57-104 fold increase in roots), and EPSPS of mangiferin biosynthesis showed an about 117 fold increase in transcripts in leaf tissues of the greenhouse grown plants. The study does provide leads on potential candidate genes correlating with the metabolites biosynthesis in S. chirayita as an initiative towards its genetic improvement.

Topics: Iridoid Glucosides; Iridoids; Plant Roots; Plants, Medicinal; Pyrones; Swertia; Xanthones

The ability to taste bitterness evolved to safeguard most animals, including humans, against potentially toxic substances, thereby leading to food rejection. Nonetheless, bitter perception is subject to individual variations due to the presence of genetic functional polymorphisms in bitter taste receptor (TAS2R) genes, such as the long-known association between genetic polymorphisms in TAS2R38 and bitter taste perception of phenylthiocarbamide. Yet, due to overlaps in specificities across receptors, such associations with a single TAS2R locus are uncommon. Therefore, to investigate more complex associations, we examined taste responses to six structurally diverse compounds (absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine) in a sample of the Caucasian population. By sequencing all bitter receptor loci, inferring long-range haplotypes, mapping their effects on phenotype variation, and characterizing functionally causal allelic variants, we deciphered at the molecular level how a subjects' genotype for the whole-family of TAS2R genes shapes variation in bitter taste perception. Within each haplotype block implicated in phenotypic variation, we provided evidence for at least one locus harboring functional polymorphic alleles, e.g. one locus for sensitivity to amarogentin, one of the most bitter natural compounds known, and two loci for sensitivity to grosheimin, one of the bitter compounds of artichoke. Our analyses revealed also, besides simple associations, complex associations of bitterness sensitivity across TAS2R loci. Indeed, even if several putative loci harbored both high- and low-sensitivity alleles, phenotypic variation depended on linkage between these alleles. When sensitive alleles for bitter compounds were maintained in the same linkage phase, genetically driven perceptual differences were obvious, e.g. for grosheimin. On the contrary, when sensitive alleles were in opposite phase, only weak genotype-phenotype associations were seen, e.g. for absinthin, the bitter principle of the beverage absinth. These findings illustrate the extent to which genetic influences on taste are complex, yet arise from both receptor activation patterns and linkage structure among receptor genes.

Topics: Alleles; Animals; Genetic Association Studies; Genotype; Haplotypes; Humans; Iridoids; Phenylthiourea; Polymorphism, Single Nucleotide; Quassins; Quinine; Receptors, G-Protein-Coupled; Sesquiterpenes; Sesquiterpenes, Guaiane; Taste Buds; Taste Perception; White People

Keratinocytes express the bitter taste receptors TAS2R1 and TAS2R38. Amarogentin as an agonist for TAS2R1 and other TAS2Rs promotes keratinocyte differentiation. Similarly, mast cells are known to express bitter taste receptors. The aim of this study was to assess whether bitter compounds display immunomodulatory effects on these immunocompetent cells in the skin, so that they might be a target in chronic inflammatory diseases such as atopic dermatitis and psoriasis. Here, we investigated the impact of amarogentin on substance P-induced release of histamine and TNF-α from the human mast cell line LAD-2. Furthermore, the effect of amarogentin on HaCaT keratinocytes costimulated with TNF-α and histamine was investigated. Amarogentin inhibited in LAD-2 cells substance P-induced production of newly synthesized TNF-α, but the degranulation and release of stored histamine were not affected. In HaCaT keratinocytes histamine and TNF-α induced IL-8 and MMP-1 expression was reduced by amarogentin to a similar extent as with azelastine. In conclusion amarogentin displays immunomodulatory effects in the skin by interacting with mast cells and keratinocytes.

Topics: Cells, Cultured; Histamine; Humans; Immunologic Factors; Interleukin-6; Interleukin-8; Iridoids; Keratinocytes; Mast Cells; Matrix Metalloproteinase 1; Receptors, G-Protein-Coupled; Tumor Necrosis Factor-alpha

The genus Swertia is reported to contain potent bitter compounds like iridoids, xanthones and c-glucoflavones that are known to heal many human disorders. In contrast to high ethnomedicinally valued Swertia chirayita, its other species have not been studied extensively, in spite of their common use in traditional medicinal system in Nepalese communities. So, the present study attempts to investigate the content of total polyphenols, flavonoids, antioxidant activity and estimate the rough content of amarogentin, swertiamarin and mangiferin from different species of Swertia from Nepalese Himalayas.. Whole plant parts of S. chirayita (SCH), S. angustifolia (SAN), S. paniculata (SPA), S. racemosa (SRA), S. nervosa (SNE), S. ciliata (SCI) and S. dilatata (SDI) were collected; total phenolic and flavonoid contents were quantified spectrophotometrically and in vitro DPPH free radical scavenging assay was measured. Thin layer chromatography was performed on TLC aluminium plates pre-coated with silica gel for identification of swertiamarin, amarogentin and mangiferin from those species and semi quantitative estimation was done using GelQuant.NET software using their standard compounds.. The phenolic content was highest in the methanol extract of SCH (67.49 ± 0.5 mg GAE/g) followed by SDI, SRA, SNE, SCI, SPA and SAN. The contents of flavonoids were found in the order of SCH, SPA, SRA, SNE, SDI, SCI and SAN. Promising concentration of phenolics and flavonoids produced promising DPPH free radical scavenging values. The IC50 values for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test was lowest in SCH (23.35 ± 0.6 μg/ml), even lower than the standard ascorbic acid among the seven studied species. A significant correlation of 0.977 was observed between the polyphenol content and antioxidant values. The TLC profile showed the presence of all three major phytochemicals; amarogentin, swertiamarin and mangiferin in all of the plant samples.. Among the seven studied species, SCH showed anticipating results in total phenol content, flavonoid content and DPPH radical scavenging test. The less considered species of Swertia can be a potential source of bioactive amarogentin, and other useful therapeutic compounds in the alarming status of Swertia chirayita as shown by the phytochemical analysis.

Topics: Antioxidants; Chromatography, Thin Layer; Flavonoids; Free Radical Scavengers; Humans; Iridoid Glucosides; Iridoids; Methanol; Nepal; Phytochemicals; Plant Extracts; Plants, Medicinal; Polyphenols; Pyrones; Species Specificity; Swertia; Xanthones

Swertia chirayita, a medicinal herb inhabiting the challenging terrains and high altitudes of the Himalayas, is a rich source of essential phytochemical isolates. Amarogentin, a bitter secoiridoid glycoside from S. chirayita, shows varied activity in several patho-physiological conditions, predominantly in leishmaniasis and carcinogenesis. Experimental analysis has revealed that amarogentin downregulates the cyclooxygenase-2 (COX-2) activity and helps to curtail skin carcinogenesis in mouse models; however, there exists no account on selective inhibition of the inducible cyclooxygenase (COX) isoform by amarogentin. Hence the computer-aided drug discovery methods were used to unravel the COX-2 inhibitory mechanism of amarogentin and to check its selectivity for the inducible isoform over the constitutive one. The generated theoretical models of both isoforms were subjected to molecular docking analysis with amarogentin and twenty-one other Food and Drug Authority (FDA) approved lead molecules. The post-docking binding energy profile of amarogentin was comparable to the binding energy profiles of the FDA approved selective COX-2 inhibitors. Subsequent molecular dynamics simulation analysis delineated the difference in the stability of both complexes, with amarogentin-COX-2 complex being more stable after 40ns simulation. The total binding free energy calculated by MMGBSA for the amarogentin-COX-2 complex was -52.35 KCal/mol against a binding free energy of -8.57 KCal/mol for amarogentin-COX-1 complex, suggesting a possible selective inhibition of the COX-2 protein by the natural inhibitor. Amarogentin achieves this potential selectivity by small, yet significant, structural differences inherent to the binding cavities of the two isoforms. Hypothetically, it might block the entry of the natural substrates in the hydrophobic binding channel of the COX-2, inhibiting the cyclooxygenation step. To sum up briefly, this work highlights the mechanism of the possible selective COX-2 inhibition by amarogentin and endorses the possibility of obtaining efficient, futuristic and targeted therapeutic agents for relieving inflammation and malignancy from this phytochemical source.

Topics: Amino Acid Motifs; Biosynthetic Pathways; Catalytic Domain; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Enzyme Stability; Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Iridoids; Molecular Docking Simulation; Molecular Dynamics Simulation; Prostaglandins; Protein Binding; Structural Homology, Protein; Swertia; Thermodynamics

Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60  μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC) γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLC γ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenine; Adenosine Triphosphate; Animals; Arachidonic Acid; Collagen; Cyclic GMP; Guanylate Cyclase; Humans; Iridoids; MAP Kinase Signaling System; Mice; Oxadiazoles; Phospholipase C gamma; Plant Extracts; Platelet Activation; Protein Kinase C; Quinoxalines; Thrombin; Thromboembolism; Thrombosis

The crude extract of Swertia chirayita, an important medicinal plant of Nepal, is locally used for many diseases including type 2 diabetes. In this study, crude aqueous and 12% ethanol solution extracts of S. chirayita collected from nine districts of Nepal were analyzed for anti-diabetic-linked anti-hyperglycemia potential using in vitro biochemical assays. There was moderate-to-high positive correlation between antioxidant activity and total phenolic content of both extracts and moderate-to-high α-glucosidase inhibitory activity. Although the anti-diabetic property of S. chirayita is mainly attributed to the phytochemical swerchirin present in its hexane fraction, we propose that the crude extract of this plant used in local healing also has anti-hyperglycemia potential. The crude extracts indicated the presence of three main phytochemicals mainly mangiferin, swertiamarin, and amarogentin and their derivatives. Among the standard compounds (mangiferin, swertiamarin, and amarogentin), mangiferin showed α-glucosidase and 2,2-diphenyl-1-picrylhydrazyl radical inhibitory activity indicating anti-hyperglycemia potential.

Topics: Angiotensin-Converting Enzyme Inhibitors; Antioxidants; Biphenyl Compounds; Chromatography, High Pressure Liquid; Glycoside Hydrolase Inhibitors; Hypoglycemic Agents; Inhibitory Concentration 50; Iridoids; Nepal; Phenols; Picrates; Plant Extracts; Swertia; Xanthones

Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2) activity has been implicated in the development of various secondary complications of diabetes. Thus, ALR2 inhibition could be an effective strategy in the prevention or delay of certain diabetic complications. Gentiana lutea grows naturally in the central and southern areas of Europe. Its roots are commonly consumed as a beverage in some European countries and are also known to have medicinal properties. The water, ethanol, methanol, and ether extracts of the roots of G. lutea were subjected to in vitro bioassay to evaluate their inhibitory activity on the ALR2. While the ether and methanol extracts showed greater inhibitory activities against both rat lens and human ALR2, the water and ethanol extracts showed moderate inhibitory activities. Moreover, the ether and methanol extracts of G. lutea roots significantly and dose-dependently inhibited sorbitol accumulation in human erythrocytes under high glucose conditions. Molecular docking studies with the constituents commonly present in the roots of G. lutea indicate that a secoiridoid glycoside, amarogentin, may be a potential inhibitor of ALR2. This is the first paper that shows G. lutea extracts exhibit inhibitory activity towards ALR2 and these results suggest that Gentiana or its constituents might be useful to prevent or treat diabetic complications.

Topics: Aldehyde Reductase; Animals; Biological Assay; Erythrocytes; Gentiana; Glucose; Humans; Iridoid Glycosides; Iridoids; Male; Methanol; Plant Extracts; Rats; Sorbitol

Amarogentin, a secoiridoid glycoside, is an active component of the medicinal plant Swertia chirata. In this study, chemopreventive and chemotherapeutic actions of amarogentin were evaluated in a carbon tetrachloride (CCl(4))/N-nitrosodiethylamine (NDEA)-induced liver carcinogenesis mouse model system during continuous and posttreatment schedule. Better survival, no toxicity and increased body weight were noted in amarogentin-treated mice. Reduction in proliferation and increase in apoptosis frequency were evident in amarogentin-treated groups. In carcinogen control group moderate dysplasia, severe dysplasia and hepatocellular carcinoma were evident at 10th, 20th and 30th week, respectively. Amarogentin was found to prevent progression of liver carcinogenesis at mild dysplastic stage. Exposure to CCl(4)/NDEA resulted in upregulation of ppRb807/811, cyclinD1 and cdc25A at 10th week and additional activation of cMyc and mdm2 along with downregulation of LIMD1, p53 and p21 at 20th week. This was followed by activation of ppRb567 and downregulation of Rbsp3 at 30th week. Prevention of carcinogenesis by amarogentin in both groups might be due to cumulative upregulation of LIMD1, RBSP3, p16 and downregulation of cdc25A at 10th week along with activation of p53 and p21 and downregulation of ppRb807/811 and ppRb567 at 20th week, followed by downregulation of cyclinD1, cMyc and mdm2 at 30th week. During carcinogenesis reduction of apoptosis was evident since 20th week. However, amarogentin treatment could significantly induce apoptosis through upregulation of the Bax-Bcl2 ratio, activation of caspase-3 and poly ADP ribose polymerase cleavage. This is the first report of chemopreventive/therapeutic role of amarogentin during liver carcinogenesis through modulation of cell cycle and apoptosis.

Topics: Animals; Apoptosis; Body Weight; Cell Cycle Checkpoints; Cell Proliferation; Chemoprevention; Female; G1 Phase; Iridoids; Liver; Liver Neoplasms, Experimental; Mice; Phosphorylation; Retinoblastoma Protein; S Phase

The plant Swertia chirata (Gentianaceae) is known for its multifarious medicinal value in the Indian system of medicine (Ayurveda). Its methanol extracts having antidiabetic activity contains mangiferin, amarogentin, amaroswerin, sweroside and swertiamarin as active constituents. The pharmacokinetics of mangiferin and amarogentin have been carried out after intravenous administration of pure standards and extract from S. chirata (CT) in rabbits to assess systemic interaction. The remaining three components were also monitored in plasma for pharmacokinetic estimation based on the ratio analysis method. Mangiferin was characterized by a relative low clearance ( approximately 0.14 L/h/kg) and a lesser volume of distribution ( approximately 0.15 L/kg), while amarogentin exhibited a rapid clearance ( approximately 2.62 L/h/kg) and wide distribution ( approximately 1.08 L/kg) from the systemic circulation. No significant difference was observed in pharmacokinetic parameters of mangiferin and amarogentin either administered alone or as CT formulation in rabbits.

Topics: Animals; Drug Interactions; Glucosides; Iridoids; Male; Plant Extracts; Rabbits; Swertia; Tandem Mass Spectrometry; Xanthones

Bitterness perception in mammals is mediated through activation of dedicated bitter taste receptors located in the oral cavity. Genomic analyses revealed the existence of orthologous mammalian bitter taste receptor genes, which presumably recognize the same compounds in different species, as well as species-specific receptor gene expansions believed to fulfill a critical role during evolution. In man, 8 of the 25 bitter taste receptors (hTAS2Rs) are closely related members of such an expanded subfamily of receptor genes. This study identified two natural bitter terpenoids, andrographolide and amarogentin, that are agonists for the orphan receptor hTAS2R50, the most distant member of the subfamily. This paper presents the pharmacological characterization of this receptor and analyzes its functional relationship with the previously deorphaned hTAS2R43, hTAS2R44, hTAS2R46, and hTAS2R47. Insights into the general breadth of tuning, functional redundancies, and relationships between pharmacological activation patterns and amino acid homologies for this receptor subfamily are presented.

Topics: Cell Line; Diterpenes; Gene Expression; Glucosides; Humans; Iridoids; Molecular Conformation; Receptors, G-Protein-Coupled

A novel, fast, and simple capillary electrophoresis method has been developed for the analysis of gentisin, isogentisin, and amarogentin in roots of Gentiana lutea (yellow gentian), an herb traditionally used as gastric stimulant. Gentisin and isogentisin are xanthones showing potent inhibition of monoamine oxidase type A and B, amarogentin represents one of the bitter principles of Gentiana, responsible for its gastric-roborant effects. Optimal CE-separation conditions comprise a 100 mM sodium tetraborate buffer of pH 9.3, containing 10 mM beta-cyclodextrin as additive; optimum temperature and applied voltage were found to be 30 degrees C and 25 kV, respectively. Direct diode array detection at 260 nm (gentisin, isogentisin) and 242 nm (amarogentin) was performed, and the required analysis time was only 11 min. The developed method was validated for linearity, sensitivity, precision, and accuracy, and utilized to assay several commercially available G. lutea samples. Quantitative data obtained with the developed CE method are compared with HPLC results, and the advantages of each approach are discussed.

Topics: Calibration; Chromatography, High Pressure Liquid; Electrophoresis, Capillary; Gentiana; Glucosides; Iridoids; Molecular Structure; Xanthones

Extracts from Swertia chirata (family Gentianaceae) have antidiabetics and antioxidant activity, largely attributed to the flavonoids and secoiridoids, which are a major class of functional components in methanolic extracts from aerial part of plants. In order to facilitate analysis of systemic exposure to S. chirata derived products in animals, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) based method that is capable of routinely monitoring plasma levels of flavonoids and secoiridoids. An LC-MS/MS-based method has been developed for the simultaneous estimation of two bioactive markers, mangiferin and amarogentin along with three other components, amaroswerin, sweroside and swertiamarin in rat plasma. All the analytes including the internal standard (kutkoside) were chromatographed on RP-18 column (250 mm x 4 mm i.d., 5 microm.) coupled with guard column using acetonitrile: 0.5 mM ammonium acetate buffer, pH approximately 3.0 as mobile phase at a flow rate of 1 ml/min in gradient mode. The final flow to source was splitted in 1:1 ratio. The detection of the analytes was performed on API 4000 LC-MS/MS system in the multiple reaction-monitoring (MRM) mode. The quantitation for analytes other than the pure markers was based on relative concentration. The method was validated in terms of establishing linearity, specificity, sensitivity, recovery, accuracy and precision (Intra- and Inter-day), freeze-thaw stability, peltier stability, dry residue stability and long-term stability. The recoveries from spiked control samples were >90% for all analytes and internal standard except mangiferin where recovery was >60%. Intra- and inter-day accuracy and precision of the validated method were within the acceptable limits of <15% at low and <10% at other concentrations. The quantitation method was successfully applied to generate pharmacokinetic (PK) profile of markers as well as to detect other components in plasma after intravenous dose administration of herbal preparation in male Sprague-Dawley (SD) rats.

Topics: Animals; Chromatography, Liquid; Cinnamates; Glucosides; Iridoid Glucosides; Iridoids; Male; Plant Preparations; Pyrones; Rats; Rats, Sprague-Dawley; Reference Standards; Reproducibility of Results; Swertia; Tandem Mass Spectrometry; Xanthones

Swertia chirata, is a bitter plant, used in the Indian system of medicine (Ayurveda) for various human ailments. Our laboratory was the first to report the chemopreventive effect of this plant. The antiproliferative and pro-apoptotic action of amarogentin rich fraction of S. chirata is now demonstrated on a mouse skin carcinogenesis model. Immunohistochemical localization revealed a reduction in proliferating and increase in apoptotic cells in skin lesion following treatment, also reflected in the expression of molecular markers--Cox-II and caspase-3 proteins. It may be possible to calculate relative risk, relative protection and attributable risk from the action of test agents on proliferation and apoptosis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Apoptosis; Caspase 3; Cell Proliferation; Cyclooxygenase 2; Disease Models, Animal; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Glucosides; In Situ Nick-End Labeling; Iridoids; Male; Medicine, Traditional; Membrane Proteins; Mice; Phytotherapy; Plant Extracts; Skin Neoplasms; Swertia; Up-Regulation

3,3',5-Trihydroxybiphenyl-2-carboxylic acid (6), an ester component of the bitter-tasting natural products amarogentin and amaroswerin, was synthesized in six steps in 13.6% overall yield. Its N-acetyl cysteamine thiol ester (9) and its coenzyme A thiol ester (8), a likely biosynthetic precursor of the amarums, were also prepared.

Topics: Coenzyme A; Cysteamine; Esters; Glucosides; Iridoids; Molecular Structure; Pyrans; Spectrum Analysis

Conventional and Agrobacterium rhizogenes-transformed root cultures were studied with respect to growth and amarogentin content following cultivation in various growth media. The fastest growth rate was observed using Nitsch medium. The best amarogentin content was obtained after cultivation in root culture (RC) medium for which the slowest growth rate was noticed. Addition of sucrose at 6% and 9% (w/v), respectively, also resulted in better growth rates and increased total but unaltered relative amarogentin content compared to 3% (w/v) sucrose. No change in amarogentin content was observed upon addition of elicitors, putative precursors of amarogentin biosynthesis, and plant growth hormones with the exception of salicylic acid and chitosan: at 100 mM salicylic acid a reduction and at 25 mg/L chitosan an increase of amarogentin were observed at significant levels. The cultivation of S. chirata roots in a 2-L stirred-tank bioreactor was successful only with a stainless-steel mesh fitted inside the culture vessel for immobilization of the roots. A 15-fold enhancement of amarogentin content in the medium was achieved by a root permeabilisation treatment using Tween 20 at 1.3% (v/v) final concentration in the bioreactor.

Topics: Bioreactors; Chitin; Chitosan; Culture Media; Culture Techniques; Glucosides; Iridoids; Plant Roots; Plants, Medicinal; Pyrans; Rhizobium

The antileishmanial property of amarogentin, a secoiridoid glycoside isolated from the Indian medicinal plant Swertia chirata, was examined in a hamster model of experimental leishmaniasis. The therapeutic efficacy of amarogentin was evaluated in free and two different vesicular forms, liposomes and niosomes. The amarogentin in both liposomal and niosomal forms was found to be a more active leishmanicidal agent than the free amarogentin; and the niosomal form was found to be more efficacious than the liposomal form at the same membrane microviscosity level. Toxicity studies involving blood pathology, histological staining of tissues and specific enzyme levels related to normal liver function showed no toxicity. Hence, amarogentin incorporated in liposomes or niosomes may have clinical application in the treatment of leishmaniasis.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Antiprotozoal Agents; Cricetinae; Drug Carriers; Glucosides; Iridoids; Leishmania donovani; Leishmaniasis, Visceral; Liposomes; Mesocricetus; Pyrans; Spleen; Surface-Active Agents; Treatment Outcome

A MeOH extract of Swertia chirata found to inhibit the catalytic activity of topoisomerase I of Leishmania donovani was subjected to fractionation to yield three secoiridoid glycosides: amarogentin (1), amaroswerin (2), and sweroside (3). Amarogentin is a potent inhibitor of type I DNA topoisomerase from Leishmania and exerts its effect by interaction with the enzyme, preventing binary complex formation.

Topics: Animals; DNA, Protozoan; Enzyme Inhibitors; Glucosides; India; Iridoids; Leishmania donovani; Magnetic Resonance Spectroscopy; Plants, Medicinal; Pyrans; Topoisomerase I Inhibitors