Amarogentin Research Articles

Effects on keratinocytes of the traditional combination of herb extract (Royal Oji Complex) implicated the improvement of young children's skin moisture and barrier.

Natural products are often friendly and can be used on children's skin after systematic and careful research. Therefore, in this study, the Royal Oji Complex (ROC), a product with natural ingredients, was used to study their effectiveness on keratinocytes taken from the skin of children from 0 to 3 years old. Normal human epidermal keratinocytes and tissue-isolated keratinocytes (TIKC) from young donors were treated with three different concentrations of ROC: 0.1, 1, and 10ppm. The mRNA expression of the epidermal barrier's essential genes, such as hyaluronic acid synthase 3 (Has3), involucrin (IVL), loricrin (LOR), and claudin-1 (CLD1) was investigated using qRT-PCR. Ceramide content was measured by ELISA, with retinoic acid (R.A.) and amarogentin (AMA) serving as positive controls. ROC significantly elevated HAS3 gene expression in HEKn cells, especially at 10ppm, indicating potential advantages for skin hydration in young infants. IVL increased at first but decreased as ROC concentrations increased. LOR was upregulated at lower ROC concentrations but reduced at higher doses. CLD1 gene expression increased considerably in HEKn but reduced with increasing ROC doses. Ceramide concentration increased somewhat but not significantly at 10ppm. ROC shows potential in altering keratinocyte gene expression, with unique responses in HEKn and TIKC from young donors. While changes in ceramide content were insignificant, these results help to comprehend ROC's multiple effects on young children's skin.

Anti-inflammatory effects of amarogentin on 2,4-dinitrochlorobenzene-induced atopic dermatitis-like mice and in HaCat cells.

Amarogentin (AMA) is a secoiridoid glycoside extracted from Swertia and Gentiana roots and exhibits many biological effects such as antioxidative, anti-inflammatory, and antitumor activities. Atopic dermatitis (AD) is a chronic inflammatory skin disease caused by disorders in the regulation of multiple inflammatory cytokines. No effective cure has been found for AD now. We constructed the HaCat and splenocyte model and tested the inhibitory effect of AMA on IL-4, IL-6, and IL-13 secretions using enzyme-linked immunosorbent assay (ELISA). The AD mouse model was constructed and treated with AMA, the severity of skin lesions was observed, epidermal tissue was collected, and epidermal thickness and mast cell infiltration were observed using hematoxylin and eosin and toluidine blue staining, respectively. The expression of kallikrein-related peptidase 7 (KLK7) and filaggrin (FLG) was detected using immunostaining and Western blot analysis. The mRNA expression of KLK7 and FLG was detected using quantitative polymerase chain reaction (qPCR). Blood immunoglobulin E (IgE) secretion was detected. AMA inhibited IL-6 secreted by tumor necrosis factor (TNF)-α-induced HaCaT cells and reduced IL-4 and IL-13 secreted by phytohemagglutinin (PHA)-induced primary cells in the mice spleen. It was found that the treatment of AMA with 2,4-dinitrochlorobenzene-induced AD-like mice could promote the recovery of dermatitis, reduce the score of dermatitis severity and the scratching frequency, treat the skin lesions, reduce the epidermal thickness, decrease the infiltration of mast cells, reduce the IgE level in serum, decrease the expression levels of AD-related cytokines, increase protein and mRNA expression of FLG, and reduce the protein and mRNA expression of KLK7 in the skin tissues of AD-like mice. In conclusion, AMA inhibits inflammatory response at the cellular level, and AMA reduces the validation response of specific dermatitis mice, relieves pruritus, and repairs the damaged skin barrier.

Amarogentin has protective effects against sepsis-induced brain injury via modulating the AMPK/SIRT1/NF-κB pathway

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.

Impact of Amarogentin on Gastric Carcinoma Cell Multiplication, Apoptosis and Migration via circKIF4A/miR-152-3p

Objective 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. Methods 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. Results 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. Conclusion AG can decrease GC multiplication, clone formation and migration and induce apoptosis via modulating circKIF4A/miR-152-3p expression.

The Insulin Receptor: A Potential Target of Amarogentin Isolated from Gentiana rigescens Franch That Induces Neurogenesis in PC12 Cells.

Amarogentin (AMA) is a secoiridoid glycoside isolated from the traditional Chinese medicine, Gentiana rigescens Franch. AMA exhibits nerve growth factor (NGF)-mimicking and NGF-enhancing activities in PC12 cells and in primary cortical neuron cells. In this study, a possible mechanism was found showing the remarkable induction of phosphorylation of the insulin receptor (INSR) and protein kinase B (AKT). The potential target of AMA was predicted by using a small-interfering RNA (siRNA) and the cellular thermal shift assay (CETSA). The AMA-induced neurite outgrowth was reduced by the siRNA against the INSR and the results of the CETSA suggested that the INSR showed a significant thermal stability-shifted effect upon AMA treatment. Other neurotrophic signaling pathways in PC12 cells were investigated using specific inhibitors, Western blotting and PC12(rasN17) and PC12(mtGAP) mutants. The inhibitors of the glucocorticoid receptor (GR), phospholipase C (PLC) and protein kinase C (PKC), Ras, Raf and mitogen-activated protein kinase (MEK) significantly reduced the neurite outgrowth induced by AMA in PC12 cells. Furthermore, the phosphorylation reactions of GR, PLC, PKC and an extracellular signal-regulated kinase (ERK) were significantly increased after inducing AMA and markedly decreased after treatment with the corresponding inhibitors. Collectively, these results suggested that AMA-induced neuritogenic activity in PC12 cells potentially depended on targeting the INSR and activating the downstream Ras/Raf/ERK and PI3K/AKT signaling pathways. In addition, the GR/PLC/PKC signaling pathway was found to be involved in the neurogenesis effect of AMA.

Simultaneous Detection and Validation of Analytical Markers of Swertia chirata by HPLC-DAD to Evaluate the Potency of Extracts and Fractions against Antioxidant Potential

The present study is based on the selection of extract and fraction of Swertia chirata plant for the antioxidant potential with HPLC fingerprinting, which includes the simultaneous detection and quantification of four analytical markers protocatechuic acid (PCA), swertiamarin (SM), mangiferin (MF) and amarogentin (AG) by HPLC-DAD. The yield of water extract (SWA), hydroalcoholic extract (SHA) and fractions of hydroalcoholic extracts were evaluated for their antioxidant potential against 2,2-diphenyl-1-picrylhydrazyl-hydrate free radical assay (DPPH assay), 2,2′-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid radical scavenging assay (ABTS assay), total reducing assay (TRA), ferric reducing antioxidant potential assay (FRAP assay), total antioxidant capacity assay (TAC assay). The hydroalcoholic extracts (SHA) can be a better choice as compared to water extract (SWA) due to higher yield of extract (13.680 ± 0.548%) and higher antioxidant activity against DPPH assay, ABTS assay, TRA assay, FRAP assay and TAC assay. In hydroalcoholic extract (SHA), ethyl acetate fraction (SEA) showed most potent activity against DPPH (IC50 = 0.008 ± 0.002 mg/mL) and ABTS (0.025 ± 0.001 mg/mL). n-Hexane fraction of SHA showed higher FRAP (28.664 ± 3.153 μmol/mL) and TAC (3.263 ± 0.325 μmol/mL) value (equivalent to ascorbic acid in μmol/mL) but showed very low yield (0.468 ± 0.018%), SBU showed higher TRA value (0.413 ± 0.309 mg/mL). The ethyl acetate fraction (SEA) can be a choice for antioxidant as it showed second highest FRAP (19.547 ± 2.119 μmol/mL) and TAC (2.750 ± 0.466 μmol/mL) with better yield (2.473 ± 0.594%) as compared to n-hexane (SH) fractions (0.468 ± 0.018%).

Effect of peeling on amygdalin content in Pruni semen

Objective The present study aimed to explore the rationality of peeling process of Pruni semen through the determination of amygdalin. Methods The content of amarogentin in seed coat, seed kernel and total seed of Pruni semen, respectively, were determined by HPLC according to the methods of content determination under Pruni semen in Chinese Pharmacopeia (2015). The chromatographic column was Inertsil ODS-SP C18 (4.6 mm×150 mm, 5 μm). The gradient elution with acetonitrile-water as mobile phase was performed. The flow velocity was 0.6 ml/min, the column temperature was 40 ℃, and the wavelength was 210 nm. Results The average recovery rate (n=6) was 98.26%. The sample was stable within 24 h, and the RSD (n=6) was 1.12%. The quality of control products was good in the range of 0.02-0.40 μg. The analysis indicated that there is no significant differences (P>0.05) in the contents of amygdalin in seed coat, seed kernel and total seed of Pruni semen. Conclusions With the content of amygdalin as the index of evaluation, the Pruni semen had better not be peeled. Key words: Pruni semen; Processing (TCD); Amygdalin; Assay (TCD)

Amarogentin, a secoiridoid glycoside, activates AMP- activated protein kinase (AMPK) to exert beneficial vasculo-metabolic effects

IntroductionAMP-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. MethodsMolecular docking of Gentiana lutea constituents was performed with crystal structure of human α2β1γ1 trimeric AMPK (PDB ID: 4CFE). Binding of Amarogentin (AG) to α2 subunit was confirmed through isothermal titration calorimetry (ITC) and in vitro kinase assays were performed. L6 myotube, HUH7 and endothelial cell cultures were employed to validate in silico and in vitro observations. Lipid lowering and anti-atherosclerotic effects were confirmed in streptozotocin induced diabetic mice via biochemical measurements and through heamatoxylin and eosin, Masson's trichrome and Oil Red O staining. ResultsAG interacts with the α2 subunit of AMPK and activates the trimeric kinase with an EC50 value of 277 pM. In cell culture experiments, AG induced phosphorylation of AMPK as well as its downstream targets, acetyl-coA-carboxylase (ACC) and endothelial nitric oxide synthase (eNOS). Additionally, it enhanced glucose uptake in myotubes and blocked TNF-α induced endothelial inflammation. Oral supplementation of AG significantly attenuated diabetes-mediated neointimal thickening, and collagen and lipid deposition in the aorta. It also improved circulating levels of lipids and liver function in diabetic mice. ConclusionIn conclusion, AG exerts beneficial vasculo-metabolic effects by activating AMPK. General significanceAmarogentin, 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.

Magnetic particles-based enzyme immunoassay for rapid determination of secoiridoid glycoside, amarogentin

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−1, with a limit of detection of 8.58 ng mL−1. Validation analysis revealed that MPs-EIA is a sufficiently sensitive and rapid for the quantitative analysis of AG in plant samples. To the best of our knowledge, this is the first MPs-EIA that has been applied to plant samples.

Sensitive quantitative analysis of the bitter glycoside amarogentin by specific monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay.

Amarogentin (AG) is a naturally occurring secoiridoid glycoside produced mainly in the plant genera Swertia and Gentiana. Extracts of these plants have a long history of use in Japan as bitter stomachics because of their strong bitterness. Because the AG content directly reflects the potential activity of the extract, the quality control of these plant extracts through the quantitative analysis of AG is important. In the present study, we aimed to develop a quantitative analysis of AG using a monoclonal antibody (MAb) against AG (MAb 1E9) in the plant family Gentianaceae. Surprisingly, production of MAb 1E9 was successfully achieved by immunizing AG–bovine serum albumin (BSA) conjugates into mice although the number of AG bound to BSA was only one. The characterization of MAb 1E9 revealed that it shows high specificity to AG, enabling the development of an icELISA for the specific determination of AG. In addition, the detectable concentration of AG in the developed icELISA ranged from 1.95 to 62.5 ng mL−1 with a limit of detection of 1.28 ng mL−1, which is approximately 30–625 times higher in sensitivity compared with the conventional HPLC method. Validation analysis revealed that the icELISA using MAb 1E9 is precise (intra-assay variation <3.9%, inter-assay variation <8.8%) and accurate (recovery rates of spiked samples were between 91.0% and 106.4%). This method can be used for the quality control of plant extracts using AG as an index.

Serum metabonomics study of the hepatoprotective effect of amarogentin on CCl4-induced liver fibrosis in mice by GC-TOF-MS analysis

Amarogentin (AG) is a secoiridoid glycoside that is mainly extracted from the traditional Chinese medicine Swertia and Gentiana, which have been widely used in clinical practice to treat liver disease. However, the exact hepatoprotective mechanism of AG was still looking forward to further elucidation by far. In this study, C57BL/6 mice were divided into the following three groups: control, model and AG. Fibrosis was induced by CCl4. Mice were orally treated with 100mg/kg AG or with normal saline as a control. At the end of the experiment, the validity of the model and the hepatoprotective effects of AG were examined by histopathology and biochemical indicators. Metabonomics technology was further performed to systematically evaluate the endogenous metabolite profiles. Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) technology with pattern recognition analysis, including principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA), showed a clear separation of the model group and the control group, with the AG treatment group located much closer to the control group than the model group, which was consistent with the results of biochemical and histopathological assays. Moreover, nine potential biomarkers were identified to elucidate the drug mechanism of AG, which may be related to pathways of amino acid and fatty acid metabolism.