Secoiridoid Glycosides Research Articles

Enrichment of secoiridoid glycosides from Gentiana rigescens extracts in amino acid-based deep eutectic solvents via macroporous resin adsorption

The simultaneous enrichment of the secoiridoid glycosides gentiopicroside, loganic acid, swertiamaroside, and sweroside from amino acid-based deep eutectic solvent (AADES) extracts of Gentiana rigescens roots and rhizomes was developed. To screen suitable resins, the performance and characterization of 11 widely used macroporous resins for the adsorption and desorption of target secoiridoid glycosides were considered. Compared with the other resins, the H103 resin was found to have greater adsorption and desorption capacities for gentiopicroside, loganic acid, swertiamaroside, and sweroside. Therefore, this resin was chosen as the best resin for secoiridoid glycosides enrichment. In a column packed with H103 resin, the dynamic adsorption and desorption factors for the target secoiridoid glycosides were investigated and optimized. After dynamic testing on the H103 packed column, the optimal operating conditions were determined as follows: a sample volume of 12 bed volumes (BVs) and a loading flow rate of 2 BV/h were used, the volume of deionized water used for the aqueous washing process was 8 BV, and the aqueous washing flow rate was 3 BV/h. Ethanol with a volume fraction of 10–90 % was used as the eluent in the ethanol desorption process with a gradient of 10 %. Each ethanol volume fraction was used for 2 BV, with a flow rate of 3 BV/h. The eluent fractions containing 40–80 % ethanol were collected, combined, and concentrated, followed by vacuum drying to obtain the final product. The purities of gentiopicroside, loganic acid, swertiamaroside, and sweroside in the final product were 58.11, 13.73, 3.86, and 2.77 %, respectively, with corresponding recovery rates of 74.09, 72.19, 77.50, and 76.45 %, respectively. Additionally, the reuse of AADES proved to be effective.

Major specialized natural products from the endangered plant Heptacodium miconioides, potential medicinal uses and insights into its longstanding unresolved systematic classification

A comprehensive phytochemical investigation of the flower buds and leaves/twigs of Heptacodium miconioides, a cultivated ornamental plant native to China and categorized as ‘vulnerable’, has led to the isolation of 45 structurally diverse compounds, which comprise 18 phenylpropanoids (1–4, 7–20), 11 pentacyclic triterpenoids (5, 6, 21–29), eight secoiridoid glycosides (30–37), three quinic acid derivatives (38–40), and a few miscellaneous components (41–45). Among them, (+)-α-intermedianol (1), (+)-holophyllol A (2), and (−)-pseudolarkaemin A (3) represent previously unreported enantiomeric lignans, while (+)-7′(R)-hydroxymatairesinol (4) is an undescribed naturally occurring lignan. Heptacoacids A (5) and B (6) are undescribed 24-nor-urs-28-oic acid derivatives. Their chemical structures were determined by 2D-NMR, supplemented by evidence from specific rotations and circular dichroism spectra. Given the uncertainty surrounding the systematic position of Heptacodium, integrative taxonomy (ITA), a method utilized to define contentious species, is applied. Chemotaxonomy, a vital aspect of ITA, becomes significant. By employing hierarchical clustering analysis (HCA) and syntenic pattern analysis methods, a taxonomic examination based on the major specialized natural products from the flower buds of H. miconioides and two other Caprifoliaceae plants (i.e., Lonicera japonica and Abelia × grandiflora) could offer enhanced understanding of the systematic placement of Heptacodium. Additionally, compounds 39 and 40 displayed remarkable inhibitory activities against ATP-citrate lyase (ACL), with IC50 values of 0.11 and 1.10 μM, respectively. In summary, the discovery of medical properties and refining systematic classification can establish a sturdy groundwork for conservation efforts aimed at mitigating species diversity loss while addressing human diseases.

Gentiopicroside and swertiamarin induce non-selective oxidative stress-mediated cytotoxic effects in human peripheral blood mononuclear cells

Gentiopicroside (Gp) and swertiamarin (Sm), secoiridoid glycosides commonly found in plants of the Gentianaceae family, differ in one functional group. They exhibit promising cytotoxic effects in cancer cell lines and overall protective outcomes, marking them as promising molecules for developing novel pharmaceuticals. To investigate potential variations in cellular sensitivity to compounds of similar molecular structures, we analyzed the mode of Gp and Sm induced cell death in human peripheral blood mononuclear cells (PBMCs) after 48 h of treatment. The lowest tested concentration that significantly reduces cell viability, 50 μM, was applied. Oxidative stress parameters were estimated by measuring the levels of prooxidative/antioxidative balance, lipid peroxidation products, and 8-oxo-7,8-dihydro-2-deoxyguanosine, while gene expression of DNA repair enzymes was evaluated by employing quantitative real-time PCR. Cellular morphology was analyzed by fluorescent microscopy, and immunoblot analysis of apoptosis and necroptosis-related proteins was used to assess the type of cell death induced by the treatments. The discriminatory impact of Gp/Sm treatments on apoptosis and necroptosis-induced cell death was evaluated by monitoring the cell survival in co-treatment with specific cell death inhibitors. Obtained results show greater cytotoxicity of Gp than Sm suggesting that variations in the molecular structures of the tested compounds can substantially affect their biological effects. Gp/Sm co-treatment with apoptosis and necroptosis inhibitors revealed a distinct, albeit non-specific mechanism of PBMCs cell death. Although the therapeutic may not directly cause a specific type of cell death, its extent can be pivotal in assessing the safety of therapeutic application and developing phytopharmaceuticals with improved features. Since phytopharmaceuticals affect all exposed cells, identification of cytotoxic mechanisms on PBMCs after Gp and Sm treatment is important for addressing the formulation and dosage of potential phytopharmaceuticals.

Seco-iridoid glycosides and flavonoid glycosides from the Gentiana olivieri Griseb and their anti-inflammatory activities

Three undescribed seco-iridoid glycosides, one undescribed flavonoid glycoside, and three known glycosides were isolated and identified from Gentiana olivieri Griseb. The structures of these compounds were determined through spectroscopic analysis and ECD calculations. Olivierisecosides NP (1–3) were identified as aromatic conjugated seco-iridoid glucosides, among them olivierisecoside N was representing a particularly rare subtype known as the morroniside seco-iridoids. The compounds 2, 3, 5, and 6 exhibited significant inhibition of COX-2 expression, particularly compound 5 which demonstrated the most pronounced inhibitory activity with IC50 value of 23.33 ± 0.51 μM. This study provides evidence for the potential development and utilization of G. olivieri as a source of anti-inflammatory components.

Gentiopicroside improves non-alcoholic steatohepatitis by activating PPARα and suppressing HIF1.

Gentiopicroside (GPS) is a highly water-soluble small-molecule drug and the main bioactive secoiridoid glycoside of Gentiana scabra that has been shown to have hepatoprotective effects against non-alcoholic steatohepatitis (NASH), a form of non-alcoholic fatty liver disease (NAFLD) that can progress to cirrhosis and hepatocellular carcinoma. However, the effects of GPS on NASH and the underlying mechanisms remain obscure. Firstly, a high-fat, high-cholesterol (HFHC) diet and a high-sugar solution containing d-fructose and d-glucose were used to establish a non-alcoholic steatohepatitis (NASH) mice model. Secondly, we confirmed GPS supplementation improve metabolic abnormalities and reduce inflammation in NASH mice induced by HFHC and high-sugar solution. Then we used metabolomics to investigate the mechanisms of GPS in NASH mice. Metabolomics analysis showed GPS may work through the Peroxisome Proliferator-Activated Receptor (PPAR) signaling pathway and glycine, serine, and threonine metabolism. Functional metabolites restored by GPS included serine, glycine, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Western blot and qRT-PCR analysis confirmed GPS improve NASH by regulating PPARα and Hypoxia-Inducible Factor-1α (HIF-1α) signaling pathways. In vitro, studies further demonstrated EPA and DHA enhance fatty acid oxidation through the PPARα pathway, while serine and glycine inhibit oxidative stress through the HIF-1α pathway in palmitic acid-stimulated HepG2 cells. Our results suggest GPS's anti-inflammatory and anti-steatosis effects in NASH progression are related to the suppression of HIF-1α through the restoration of L-serine and glycine and the activation of PPARα through increased EPA and DHA.

Methyl jasmonate and salicylic acid as powerful elicitors for enhancing the production of secoiridoid glycoside from cell suspension cultures of Enicostema axillare (Poir. ex Lam.) A. Raynal

Methyl jasmonate and salicylic acid as powerful elicitors for enhancing the production of secoiridoid glycoside from cell suspension cultures of Enicostema axillare (Poir. ex Lam.) A. Raynal

Gentiopicroside Ameliorates Sepsis-Induced Acute Lung Injury via Inhibiting Inflammatory Response.

Sepsis is a systemic inflammatory reaction syndrome caused by infections. Acute lung injury (ALI) occurs first and most frequently in patients with sepsis. Gentiopicroside (GPS), which originates mostly from Gentiana, is classified as a secoiridoid glycosides. Terpenoid glycosides have various biological effects, including liver protection, blood glucose and cholesterol level management, and anti-inflammatory and antitumor effects. However, presently, the biochemical foundation and mechanism of the anti-inflammatory effects of GPS in sepsis-induced ALI have not been explained. In the present study, we established a rat model of sepsis ALI induced by cecal ligation and puncture. This enables us to observe the effects of GPS therapy, which significantly reduced the inflammatory response (TNF-α, IL-1β, and IL-6), nitrogen stress, oxidative stress, and severity of ALI at both the whole animal and molecular levels. In addition, GPS ameliorates LPS-induced ALI via regulation of inflammatory response and cell proptosis in BEAS-2B. This study provides a theoretical basis for treating sepsis-induced ALI with GPS.

Exploring the mechanism of Gentiana rigescens Franch. in the treatment of pulmonary fibrosis based on network pharmacology

Pulmonary fibrosis (PF) is a severe chronic interstitial lung disease with high mortality, and there is currently a lack of definite and effective treatment methods. Studies have found that gentiopicroside, a secoiridoid glycoside compound derived from plants of the Gentiana genus, can significantly improve pulmonary inflammation and fibrosis lesions in mice with pulmonary fibrosis. However, the mechanism of its anti-fibrotic effect is unclear. Therefore, in this study, we employed network pharmacology's virtual computer technology to investigate the potential mechanism underlying the anti-pulmonary fibrosis effect of Gentianae Radix et Rhizome (Longdan in Chinese, GRR). Our findings offer valuable theoretical insights and serve as a guiding reference for future experimental investigations. A total of 10 active compounds and 920 drug-target proteins were identified from the TCMSP database. The compound-target-pathway-disease network showed that GRR could potentially treat PF by regulating the MDM2, ERBB2 and VEGFA, signaling pathways through its key targets, including AKT1, TNF, and MAPK1. The protein‒protein interaction network revealed that these targets had strong interactions with each other, indicating a potential synergistic effect of GRR in treating PF. The GO and KEGG enrichment analyses further supported the potential anti-pulmonary fibrosis mechanisms of GRR, including regulating the inflammatory response, ECM-receptor interaction, and TGF-beta signaling pathways. Our study provides a systematic analysis of the potential anti-pulmonary fibrosis mechanisms of GRR based on network pharmacology. These findings could contribute to the development of novel treatments for PF and provide a basis for further experimental studies.

Secoiridoid glycosides from the fruits of Ligustrum lucidum and their in vitro anti-inflammatory activity

Seven new secoiridoid glycosides (1–7), together with a known analogue (8), were isolated from the fruits of Ligustrum lucidum. Their structures with absolute configurations were determined by HR-ESI-MS, 1D and 2D NMR, and electronic circular dichroism (ECD) spectroscopic analysis, as well as biogenetic consideration. Compounds 1 and 2 are the first examples of secoiridoid glycoside dimers featuring a rare rearranged oleoside-type secoiridoid moiety, and compounds 3–7 represent a new class of oleoside-type secoiridoid glycosides with unusual stereochemistry at C-1 position. A plausible biosynthetic pathway for this group of unusual secoiridoid glycosides was also proposed herein. In addition, the isolates were evaluated for their in vitro anti-inflammatory activity, and all tested compounds exhibited modest inhibitory effects against nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 macrophages.

Seco-iridoid glycosides from the Gentiana olivieri Griseb and their bioactivities

The ethanol extract of the Gentiana olivieri Griseb plant was subjected to an investigation to ascertain the presence of its iridoid constituents. By means of HPLC and TLC monitoring, a total of thirteen previously unreported seco-iridoid glucosides olivierisecoside A-M, as well as seven known seco-iridoid glycosides and one known iridoid glycoside were isolated. Their structures were elucidated by a comprehensive spectroscopic data analysis and ECD calculations. The absolute configuration of olivierisecoside D was further confirmed through single-crystal X-ray diffraction analysis. All the identified compounds were characterized as aromatic conjugated seco-iridoid glucosides, with olivierisecoside F–I representing a particularly rare subtype known as the morroniside type seco-iridoids. In vitro testing of the isolated compounds revealed their potential anti-inflammatory and hepatoprotective effects. The results showed olivieroside B and 6′-gentisoyl-8-epi-kingiside have good anti-inflammatory activities in LPS induced RAW264.7 cells. Additionally, olivierisecoside M exhibited some improvements in PA-induced L02 and HepG2 cells damage, known compound loganin showed slight hepatoprotective effect in PA-induced HepG2 cells damage.

Gentiopicroside modulates glucose homeostasis in high-fat-diet and streptozotocin-induced type 2 diabetic mice.

Gluconeogenesis is closely related to the occurrence and development of type 2 diabetes mellitus (T2DM). Gentiopicroside (GPS) is the main active secoiridoid glycoside in Gentiana manshurica Kitagawa, which can improve chronic complications associated with diabetes and regulate glucose metabolism. However, the effects and potential mechanisms by which GPS affects T2DM understudied and poorly understood. In this study, we systematically explored the pharmacological effects of GPS on T2DM induced by a high-fat diet (HFD) and streptozotocin (STZ) as well as explored its related mechanisms. The results showed that GPS supplementation discernibly decreased blood glucose levels, food intake and water consumption, ameliorated glucose intolerance, abnormal pyruvate tolerance, insulin resistance and dyslipidemia. Furthermore, GPS discernibly ameliorated pathological morphological abnormalities of the liver and pancreas, reduced hepatic steatosis and maintain the balance between α-cells and β-cells in pancreas. Moreover, GPS significantly inhibited gluconeogenesis, as evidenced by the suppressed protein expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) in the liver. Additionally, the results of Western blot analysis revealed that GPS increased p-PI3K, p-AKT, and p-FOXO1 expression levels, and decreased FOXO1 expression at protein level in the liver. Furthermore, the results of the immunostaining and Western blot analysis demonstrated that GPS supplementation increased the expression of zonula occludens-1 (ZO-1) and occludin in the ileum. Collectively, these results indicate that GPS may inhibit hepatic gluconeogenesis by regulating the PI3K/AKT/FOXO1 signaling pathway and maintain intestinal barrier integrity, and ultimately improve T2DM. Together, these findings indicate that GPS is a potential candidate drug for the prevention and treatment of T2DM, and the results of our study will provide experimental basis for further exploration of the possibility of GPS as a therapeutic agent for T2DM.

Amarogentin Inhibits Tumor Stem Cell-Like Features of Lung Cancer by Regulating Notch Signaling

Amarogentin, a secoiridoid glycoside isolated from the Indian medicinal plant Swertia chirata, is widely known for its antileishmanial, antioxidative, hypoglycemic, antihepatotoxic, antimalarial, anti-inflammatory, and antimicrobial activities. It has been evaluated for its possible anticancerous role in lung cancer using A549 and H1299 cells as models. The results of these studies show a dose-dependent decrease in cell proliferation, cell migration, and invasion by amarogentin. Furthermore, amarogentin repressed stemness in lung cancer, as shown by a diminution in the expression of DLL4, NICD, and Hes1 proteins. In conclusion, we report for the first time the potential regulatory impact of amarogentin on lung cancer. These findings reveal that amarogentin treatment inhibits tumor stem cell-like features in lung cancer by regulating Notch signaling. Therefore, amarogentin may be deemed as a useful therapeutic drug for lung cancer.

Pharmacological mechanism of indigenous herb Exacum lawii on cisplatin instigated toxicity in human embryonic kidney cells (HEK-293)

BackgroundExacum lawii C.B. Clarke (Gentianaceae) bitter medicinal herb, has been extensively applied for the treatment of kidney diseases and ailments by Indigenous medical practitioners and local communities in Maharashtra and Karnataka states of India. The pharmacognostical and phytochemical standardization has been performed, but its therapeutic mechanism is still unknown. Swertiamerin, a secoiridoid glycoside established as marker component. The aim of the current research is to investigate the cytoprotective ability and examine targets of action of Exacum lawii standardised extract against cisplatin exposed Human embryonic kidney cell line (HEK-293). MethodsSwertiamerin was isolated from standardised extract using column chromatography and characterized by various analytical techniques. In-vitro cellular assay (MTT cytotoxicity assay, iNOS expression, proinflammatory cytokine level, ROS estimation and cell cycle progression, DNA fragmentation assay and morphology of cells) were employed to understand the efficacy and mechanism of action of standardised extract and swertiamerin against cisplatin induced toxicity in HEK-293 cells. ResultsExtract and swertiamerin summed up to be nontoxic to HEK-293 cells and the IC50 for cisplatin was 2.261 µg/ml. Molecular docking study showed the binding capacity of swertiamerin for iNOS enzyme (responsible for the nitrosative stress in cells). Various Scientific techniques validates the potential of extract and swertiamerin to reduce the nitrosative stress, oxidative stress, and inflammation in HEK-293 cells significantly (P < 0.05) at the dose of 50 µl (2 mg/ml) and 20 µl (0.5 mg/ml) respectively. ConclusionThis study validates the ethnomedicinal use herb Exacum lawii in nephrotoxicity. Swertiamerin as the characteristic bioactive compound might works synergistically with other phytoconstituents to prevent the cellular damage from nitrosative stress, oxidative stress, and inflammation. Therefore, present study emphasizes on the mechanism of action involved in pharmacological activity.

Design and optimization of “greener” and sustainable ultrasound-assisted extraction of valuable bioactive compounds from common centaury (Centaurium erythraea Rafn) aerial parts: A comparative study using aqueous propylene glycol and ethanol

The design of two environmentally friendly protocols regarding ultrasound-assisted extraction (UAE) of valuable bioactive compounds from common centaury (Centaurium erythraea Rafn, Gentianaceae) aerial parts with aqueous mixtures of „green“ organic solvents (ethanol and propylene glycol) was conducted. In order to ensure the maximum total polyphenolic content (TPC), Box-Behnken experimental design (BBD) was used for both UAE/aqueous ethanol (UAE/aEtOH) and UAE/aqueous propylene glycol (UAE/aPPG) systems to optimize time, solvent concentration, liquid-to-solid ratio and temperature. The liquid-to-solid ratio had the highest effect on the TPC for tested extraction systems. For UAE/aEtOH system under the optimal conditions the predicted and experimentally obtained TPC were close (4.11 and 4.13 ± 0.04 g GAE/100 g d.w., respectively). The predicted and experimentally obtained TPC for UAE/aPPG system under the optimum conditions were 4.12 and 4.16 ± 0.04 g GAE/100 g d.w., respectively. According to UHPLC-DAD-MS/MS analysis, key bioactive components in both extracts were organic acids and derivates, flavanols, xanthones and secoiridoid glycosides. UAE/aPPG and UAE/aEtOH extracts contained 45 and 42 compounds, respectively. Since these extracts can be incorporated into different products as natural active ingredients, the extraction protocols are likely to ensure easy transfer to a large-scale production and further utilization in different industries, specifically ensuring cleaner production with reduced impact on the environment. Moreover, the usage of ethanol and its presence in the final industrial products has downsides: evaporation, flammability, irritation of different organs, discouraged use for some consumers due to preexisting medical conditions, interactions with various prescribed medicines, ethical reasons, etc. Thus, a much safer UAE/aPPG system, providing extract with more biologically functional phytochemicals and higher TPC at a lower temperature, is more recommended for eco-friendly future industrial use.

Gentiopicroside Ameliorates Cerebrovascular Angiogenesis, Neuronal Injury and Immune Disorder in Rats with Cerebral Ischemia/Reperfusion Injury via VEGF and Phosphorylated Nrf2 Elevation.

Cerebral ischemia-reperfusion (CI/R) injury is induction of blood flow restoration after an ischemic stroke. Gentiopicroside (GPC) is the principal active secoiridoid glycoside of Gentiana Manshurica Kitagawa. This research aimed to illuminate the function of GPC and its mechanism in CI/R injury. After CI/R injury models were constructed, GPC (25, 50 or 100 mg/kg) was then administered by gavage to rats. Rats were grouped into Sham, CI/R, CI/R+25 mg/kg GPC, CI/R+50 mg/kg GPC, and CI/R+100 mg/kg GPC. Neuronal cells were exposed to oxygen-glucose deprivation and reperfusion (OGD/R) injury to establish ischemic-like conditions in vitro, and cells were further treated with 25, 50, or 100 μM GPC. Cells were grouped into control, OGD/R, OGD/R+25 μM GPC, OGD/R+50 μM GPC, and OGD/R+100 μM GPC. GPC's function on rat cerebral injury, angiogenesis, oxidative stress, neuronal injury and immune dysfunction in vivo was estimated using hematoxylin-eosin staining, Western blot, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, commercial kits and enzyme linked-immunosorbent assay. Meanwhile, GPC's mechanism in CI/R injury was examined via Western blot. GPC's function in vitro was estimated via Cell Counting Kit-8 assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, flow cytometry. GPC alleviated cerebral injury through decreasing cerebral infarction volume, cerebral indexes, brain water contents (p < 0.05). GPC reduced oxidative stress and boosted cerebral angiogenesis in CI/R rats (p < 0.05). Meanwhile, GPC weakened neuronal cell apoptosis, and decreased neuron-specific enolase and S100beta protein levels in CI/R rats. GPC reduced inflammatory cytokines contents in serum and brain tissues of CI/R rats (p < 0.05). Moreover, GPC increased the viability and proliferation in OGD/R-treated neuronal cells, but decreased cell apoptosis (p < 0.05). Mechanistically, GPC upregulated vascular endothelial growth factor (VEGF) and phosphorylated nuclear factor E2-related factor 2 (p-Nrf2) levels in CI/R rat brain tissues (p < 0.05). GPC reduced cerebrovascular angiogenesis, neuronal injury and immune disorder in CI/R injury through elevating VEGF and p-Nrf2.

Neuroprotective Effect of Swertiamarin in a Rotenone Model of Parkinson's Disease: Role of Neuroinflammation and Alpha-Synuclein Accumulation.

Parkinson's disease (PD) is a progressive neurodegenerative disease with no permanent cure affecting around 1% of the population over 65. There is an urgency to search for a disease-modifying agent with fewer untoward effects. PD pathology involves the accumulation of toxic alpha-synuclein (α-syn) and neuronal inflammation leading to the degeneration of dopaminergic (DAergic) neurons. Swertiamarin (SWE), a well-studied natural product, possesses a strong anti-inflammatory effect. It is a secoiridoid glycoside isolated from Enicostemma littorale Blume. SWE showed a reversal effect on the α-syn accumulation in the 6-hydroxydopamine (6-OHDA)-induced Caenorhabditis elegans model of PD. However, there are no reports in the literature citing the effect of SWE as a neuroprotective agent in rodents. The present study aimed to evaluate the anti-inflammatory activity of SWE against lipopolysaccharide (LPS)-induced C6 glial cell activation and its neuroprotective effect in the intrastriatal rotenone mouse PD model. SWE treatment showed a significant reduction in interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) levels in LPS-induced C6 glial cell activation. Further, our studies demonstrated the suppression of microglial and astroglial activation in substantia nigra (SN) after administration of SWE (100 mg/kg, intraperitoneally) in a rotenone mouse model. Moreover, SWE alleviated the rotenone-induced α-syn overexpression in the striatum and SN. SWE ameliorated the motor impairment against rotenone-induced neurotoxicity and mitigated the loss of DAergic neurons in the nigrostriatal pathway. Therefore, SWE has the potential to develop as an adjunct therapy for PD, but it warrants further mechanistic studies.

Trifloroside Induces Bioactive Effects on Differentiation, Adhesion, Migration, and Mineralization in Pre-Osteoblast MC3T3E-1 Cells

Gentianae Scabrae Radix is used in traditional medicine and is known to possess bioactive compounds, including secoiridoid glycosides, flavonoids, lignans, and triterpenes. Trifloroside (TriFs) is a secoiridoid glycoside known for its antioxidant activity; however, its other effects have not been studied. In the present study, we investigated the biological effects of TriFs isolated from the roots of Gentianae Scabrae Radix using pre-osteoblast MC3T3E-1 cells. No cellular toxicity was observed with 1 μM TriFs, whereas 5-100 μM TriFs showed a gradual increase in cell viability. Alkaline phosphatase staining and microscopic observations revealed that 1-10 μM TriFs stimulated osteogenic activity during early osteoblast differentiation. Trifloroside also increased mineral apposition during osteoblast maturation. Biochemical analyses revealed that TriFs promoted nuclear RUNX2 expression and localization by stimulating the major osteogenic BMP2-Smad1/5/8-RUNX2 pathway. Trifloroside also increased p-GSK3β, β-catenin, p-JNK, and p-p38, but not Wnt3a, p-AKT, and p-ERK. Moreover, TriFs increased the MMP13 levels and promoted cell migration and adhesion. In contrast, TriFs-induced osteoblast differentiation and maturation had negligible effects on autophagy and necrosis. Our findings suggest that TriFs induces osteogenic effects through differentiation, adhesion, migration, and mineral apposition. Therefore, TriFs is suggested as a potential drug target in osteoblast-mediated bone diseases.

Secondary metabolites from Gentiana cruciata L. and their anti-inflammatory and analgesic activities

A previously unreported secoiridoid glycoside, cruciatoside (1) was isolated from the aerial parts of Gentiana cruciata L. along with ten known compounds eustomoside (2), eustomorusside (3), gentiopicroside (4), 6'-O-β-D-glucopyranosyl gentiopicroside (5), loganic acid (6), isoorientin (7), isovitexin (8), isovitexin 2''-(E)-ferulate (9), mangiferin (10), and 2-methyl-inositol (11). The chemical structures of the isolates were elucidated based on extensive 1 D and 2 D NMR experiments as well as HRMS analysis. All isolates were evaluated for their in vitro anti-inflammatory and analgesic activities. Compounds 9, 4, and 7 (200 µM) showed moderate anti-inflammatory activity by inhibiting nitrite production from LPS-induced RAW 264.7 macrophage cells, with the inhibition rates of 39.5%, 25.8% and 22.9% respectively without exhibiting substantial cytotoxicity. Besides, 1, 2, 4, and 7 exerted the highest decrease in IL-6 levels. Moreover, compound 4 showed in vitro analgesic activity by decreasing the PGE2 level comparable to the reference drugs.

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.

Sweroside Ameliorated Memory Deficits in Scopolamine-Induced Zebrafish (Danio rerio) Model: Involvement of Cholinergic System and Brain Oxidative Stress.

Sweroside is a secoiridoid glycoside and belongs to a large group of naturally occurring monoterpenes with glucose sugar attached to C-1 in the pyran ring. Sweroside can promote different biological activities such as antifungal, antibacterial, hepatoprotective, gastroprotective, sedative and antitumor, antioxidant, and neuroprotective activities. Zebrafish were given sweroside (12.79, 8.35, and 13.95 nM) by immersion once daily for 8 days, along with scopolamine (Sco, 100 μM) 30 min before the initiation of the behavioral testing to cause anxiety and memory loss. Employing the novel tank diving test (NTT), the Y-maze, and the novel object recognition test (NOR), anxiety-like reactions and memory-related behaviors were assessed. The following seven groups (n = 10 animals per group) were used: control, Sco (100 μM), sweroside treatment (2.79, 8.35, and 13.95 nM), galantamine (GAL, 2.71 μM as the positive control in Y-maze and NOR tests), and imipramine (IMP, 63.11 μM as the positive control in NTT test). Acetylcholinesterase activity (AChE) and the antioxidant condition of the brains were also evaluated. The structure of sweroside isolated from Schenkia spicata was identified. Treatment with sweroside significantly improved the Sco-induced decrease of the cholinergic system activity and brain oxidative stress. These results suggest that sweroside exerts a significant effect on anxiety and cognitive impairment, driven in part by the modulation of the cholinergic system activity and brain antioxidant action.