Impact of Preparative Isolation of C-Glycosylflavones Derived from Dianthus superbus on In Vitro Glucose Metabolism.

Dianthus superbus L. has been extensively studied for its potential medicinal properties in traditional Chinese medicine and is often consumed as a tea by traditional folk. It has the potential to be exploited in the treatment of inflammation, immunological disorders, and diabetic nephropathy. Based on previous studies, this study continued the separation of another subfraction of Dianthus superbus and established reversed-phase/reversed-phase and reversed-phase/hydrophilic (RPLC) two-dimensional (2D) high-performance liquid chromatography (HPLC) modes, quickly separating two C-glycosylflavones, among which 2″-O-rhamnosyllutonarin was a new compound and isomer with 6‴-O-rhamnosyllutonarin. This is the first study to investigate the effects of 2″-O-rhamnosyllutonarin and 6‴-O-rhamnosyllutonarin on cellular glucose metabolism in vitro. First, molecular docking was used to examine the effects of 2″-O-rhamnosyllutonarin and 6″-O-rhamnosyllutonarin on AKT and AMPK; these two compounds exhibited relatively high activity. Following this, based on the HepG2 cell model of insulin resistance, it was proved that both of the 2″-O-rhamnosyllutonarin and 6‴-O-rhamnosyllutonarin demonstrated substantial efficacy in ameliorating insulin resistance and were found to be non-toxic. Simultaneously, it is expected that the methods developed in this study will provide a basis for future studies concerning the separation and pharmacological effects of C-glycosyl flavonoids.

Phytoecdysteroids from Dianthus superbus L.: Structures and anti-neuroinflammatory evaluation.

Six undescribed C27-phytoecdysteroid derivatives, named superecdysones A-F, and ten known analogs were extracted from the whole plant of Dianthus superbus L. Their structures were identified by extensive spectroscopy, mass spectrometric methods, chemical transformations, chiral HPLC analysis, and the single-crystal X-ray diffraction analysis. Superecdysones A and B possess a tetrahydrofuran ring in the side chain and superecdysones C-E are rare phytoecdysones containing a (R)-lactic acid moiety, whereas superecdysone F is an uncommon B-ring-modified ecdysone. Notably, based on the variable temperature (from 333 K to 253 K) NMR experiments of superecdysone C, the missing carbon signals were visible at 253 K and assigned. The neuroinflammatory bioassay of all compounds were evaluated, and 22-acetyl-2-deoxyecdysone, 2-deoxy-20-hydroxyecdysone, 20-hydroxyecdysone, ecdysterone-22-O-benzoate, 20-hydroxyecdysone-20,22-O-R-ethylidene, and acetonide derivative 20-hydroxyecdysterone-20, 22-acetonide significantly suppressed the LPS-induced nitric oxide generation in microglia cells (BV-2), with IC50 values ranging from 6.9 to 23.0 µM. Structure-activity relationships were also discussed. Molecular docking simulations of the active compounds confirmed the possible mechanism of action against neuroinflammations. Furthermore, none compounds showed cytotoxicity against HepG2 and MCF-7. It is the first report about the occurrence and anti-neuroinflammatory activity of the phytoecdysteroids in the genus Dianthus. Our findings demonstrated that ecdysteroids may be used as potential anti-inflammatory drugs.

Probing the effect of quercetin 3-glucoside from Dianthus superbus L against influenza virus infection- In vitro and in silico biochemical and toxicological screening.

Investigation of antiviral and cytotoxic effect of quercetin 3-glucoside (Q3G) from Dianthus superbus L over influenza virus infection and replication were studied. Moreover, anti-influenza mechanism was screened by time-dependent antiviral assay, virus-induced symptoms and related gene expressions. The blockade of cap-binding domain of polymerase basic protein subunit were analysed by molecular docking study. The Q3G demonstrated potent antiviral activity showing 4.93, 6.43, 9.94, 8.3, and 7.1 µg/mL of IC50 for A/PR/8/34, A/Victoria/3/75, A/WS/33, B/Maryland/1/59, and B/Lee/40, respectively. The cellular toxicity of Q3G and oseltamivir (control) were tested and >100 µg/mL of CC50 value considered as nontoxic. Influenza A virus infection induced a higher ROS production, however potentially reduced by Q3G treatment and significantly blocked virus infection induced acidic vesicular organelles (AVO). Moreover, Q3G has no inhibitory effect for neuraminidase activity but blocked virus replication through time dependent assay and showed more competitive binding affinity (-8.0 kcal/mal) than GTP (-7.0 kcal/mol) to block polymerase basic protein-2 subunit of influenza virus. Q3G from D. superbus showed potent antiviral activity against influenza A and B viruses with suppressive effect on virus-induced cellular ROS generation and AVO formation. Thus, this study provided a new line of research for Q3G to develop possible natural anti-influenza drug.

Utilization of Dianthus superbus L and its bioactive compounds for antioxidant, anti-influenza and toxicological effects.

Dianthus superbus (DS) is a traditional medicinal herb well known for its medicinal and therapeutic potential and widely distributed in various Asian countries. The ethyl acetate (EA), butanol (Bu) and distilled water (DW) extracts of DS assessed for extraction of bioactive compounds and their biological activities. The chemical analysis was done using LC-MS/MS and antioxidant, anticancer and antiviral activities were determined. EA extracts showed strong anticancer activity with IC50 of 9.5, 13.8 and 69.9 µg/mL on SKOV, NCL-H1299 and Caski cancer cell lines, respectively. The Bu extracts exhibited strongest antiviral activity with respect to both influenza A and B viruses with IC50 values of 4.97 and 3.9 µg/mL, respectively. Also the metabolic profile for EA, Bu and DW extracts shows high variations and influence precisely the antioxidant, anticancer and antiviral properties. The quercetin 3- rutinoside and isorhamnetin 3- glucoside showed higher neuraminidase inhibition activity in dose dependent manner. Molecular docking study revealed that flavonol glycosides have higher binding activities towards influenza polymerase membrane glycoprotein. Correlation study showed that flavonol glycosides were linked to anti-influenza activity and cyclic peptides with anticancer activities. This study provides vital information for effective utilization of DS for medicinal, food and therapeutic purposes.

Dianthus superbus Improves Glomerular Fibrosis and Renal Dysfunction in Diabetic Nephropathy Model.

Glomerular fibrosis is caused by an accumulation of intercellular spaces containing mesangial matrix proteins through either diffused or nodular changes. Dianthus superbus has been used in traditional medicine as a diuretic, a contraceptive, and an anti-inflammatory agent. The aim of this study was to investigate the effects of Dianthus superbus-EtOAc soluble fraction (DS-EA) on glomerular fibrosis and renal dysfunction, which has been implicated in diabetic nephropathy in human renal mesangial cells and db/db mice. DS-EA was administered to db/db mice at 10 or 50 mg/kg/day for 8 weeks. DS-EA treatment significantly ameliorated blood glucose, insulin, the homeostasis model assessment of insulin resistance (HOMA-IR) index, and HbA1c in diabetic mice. DS-EA decreased albumin excretion, creatinine clearance (Ccr), and plasma creatinine levels. DS-EA also ameliorated the levels of kidney injury molecules-1 (KIM-1) and C-reactive protein. DS-EA reduced the periodic acid-Schiff (PAS) staining intensity and basement membrane thickening in glomeruli of the diabetic nephropathy model. In addition, DS-EA suppressed transforming growth factor-ß (TGF-ß)/Smad signaling. Collagen type IV, a glomerular fibrosis biomarker, was significantly decreased upon DS-EA administration. DS-EA pretreatment attenuated levels of inflammation factors such as intracellular cell adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1). DS-EA inhibited the translocation of nuclear factor kappa B (NF-κB) in Angiotensin II (Ang II)-stimulated mesangial cells. These findings suggest that DS-EA has a protective effect against renal inflammation and fibrosis. Therefore, DS-EA may serve as a potential therapeutic agent targeting glomerulonephritis and glomerulosclerosis, which lead to diabetic nephropathy.

Isolation, characterization, and in rats plasma pharmacokinetic study of a new triterpenoid saponin from Dianthus superbus.

One new oleanolic acid triterpenoid saponin, 3-O-ß-D-glucopyranosyl olean-11, 13(18)-diene-23,28-dioic acid, (hereafter referred to as DS-1) was isolated from the traditional Chinese medicinal plant Dianthus superbus (D. superbus). DS-1 plays an important role in the bioactivity of D. superbus. Thus, a sensitive, reliable and accurate reversed-phased liquid chromatography with tandem mass spectrometry (LC-MS/MS) in negative ion mode was developed and validated for the quantification and pharmacokinetic study of DS-1 in rats plasma. The pharmacokinetic profile showed that DS-1 was rapidly absorbed and eliminated in plasma, indicating that significant accumulation of the compound in biological specimen is unlikely. In addition, poor absorption into systemic circulation was observed after oral administration of DS-1, resulting in low absolute bioavailability (0.92 %).

Effects of different soil remediation methods on inhibition of lead absorption and growth and quality of Dianthus superbus L.

Heavy metal pollution in soil poses a serious threat to the growth of plants used in traditional Chinese medicine. Therefore, a pot experiment was conducted to study the effects of various soil remediation methods on the performance of Herba Dianthi (Dianthus superbus L.) grown on Pb-contaminated soil. The results show that inoculation of Herba Dianthi with arbuscular mycorrhizal fungi (AMF) led to a significant reduction in Pb uptake (P< 0.05), and increased root development and root-to-shoot ratio compared to untreated control plants, along with the highest content of active components. When planting with Trifolium repens, the reduction effect of Pb absorption was insignificant. Herba Dianthi showed improved growth and active ingredients, and the lowest Pb content, with AMF inoculation. The addition of EDTA decreased the growth of Herba Dianthi, but promoted the absorption of Pb. The inhibition of tumor cells was highest in E2. In conclusion, inoculation with AMF can ensure that plant lead content meets testing standards, helping to improve the quality of medicinal herbs.

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A pot experiment was conducted to study the effects of inoculation with arbuscular mycorrhizal (AM) fungi, neighboring plants (Trifolium repens), addition of EDTA and phosphate fertilizer on the performance of Dianthus superbus grown on Pb contaminated soil. The growth and quality of D. superbus were investigated to provide a theoretical basis for the scientific cultivation of Chinese herbal medicine. The results showed that the D. superbus with AM resulted in a significant inhibition of Pb uptake (P＜0.05), increase of root development and root/shoot ratio compared to untreated control plants, and with the highest content of active component. The content of emodin reached 6.5 mg·g-1. When planting with T. repens the reducing effect of Pb absorption induced by AM fungi was insignificant. The quality of D. superbus decreased, and the emodin content decreased to 3.2 mg·g-1, which was lower than that in the control group. However, D. superbus showed improved growth and active ingredients, and the lowest Pb content (1.3 mg·g-1) due to AM fungal inoculation. The addition of EDTA decreased the growth of D. superbus, but promoted the absorption of Pb, with the Pb content to 340.0 mg·g-1. Calcium phosphate showed the ability to immobilize other heavy metals in the soil, therefore might be more suitable to be applied in the conditions of compound pollution. Considering all of the results, AM fungi play a positive role in protecting the safety and quality of Chinese herbal medicine.

Simultaneous Determination of Eight Bioactive Compounds in Dianthus superbus by High-performance Liquid Chromatography.

BACKGROUND: Dianthus superbus, one of traditional herbal medicine, is widely used to treat urethritis, carbuncles and carcinoma. OBJECTIVE: A simultaneous determination method was established for controlling the quality of D. superbus using the eight compounds, (E)-methyl-4-hydroxy-4-(8a-methyl-3-oxodecahydronaphthalen-4a-yl) (1), diosmetin-7-O(2'',6''-di-O-α-L-rhamnopyranosyl)-ß-D-glucopyranoside (2), vanillic acid (3), 4-hydroxyphenyl acetic acid (4), 4-methoxyphenyl acetic acid (5), (E)-4-methoxycinnamic acid (6), 3-methoxy-4-hydroxyphenylethanol (7), and methyl hydroferulate (8) isolated from D. superbus. MATERIALS AND METHODS: This analysis method was developed using high performance liquid chromatography coupled with diode array detector with a Shishedo C18 column at a column temperature of 3°C. The mobile phase was composed of 0.1% trifluoroacetic acid in water and acetonitrile. The flow rate was 1 ml/min and detection wavelength was set at 205 nm and 280 nm. Validation was performed in order to demonstrate selectivity, accuracy and precision of the method. RESULTS: The calibration curves showed good linearity (R (2) > 0.99). The limits of detection and limits of quantification were within the ranges 0.0159-0.6205 µg/ml and 0.3210-1.8802 µg/ml, respectively. Moreover, the relative standard deviations of intra- and inter-day precision were both <2.98%. The overall recoveries were in the range of 96.23-109.87%. Quantitative analysis of eight compounds in 12 D. superbus samples (D-1-D-12) from various regions were analyzed and compared by developed method. CONCLUSION: As a result, this established method was accurate and sensitive for the quality evaluation of eight compounds isolated from D. superbus and may provide a new basis for quality control of D. superbus. SUMMARY: A simultaneous determination method of eight compounds in Dianthus superbus was established by high performance liquid chromatography-diode array detectorDeveloped analysis method is validated with linearity, precious and accuracyThe newly established method was successfully evaluated contents of eight compounds in 12 D. superbus samples (D.1.D.12) from various regions and compared. Abbreviations used: HPLC: High performance liquid chromatography, LOD: Limits of detection, LOQ: Limits of quantification, RSD: Relative standard deviation.

Neuroprotective Properties of Compounds Extracted from Dianthus superbus L. against Glutamate-induced Cell Death in HT22 Cells.

BACKGROUND: Dianthus superbus L. has been used in Chinese herbal medicine as a diuretic and anti-inflammatory agent. OBJECTIVE: In this study, we isolated ten bioactive compounds from D. superbus and evaluated their neuroprotective activity against glutamate-induced cell death in the hippocampal neuronal HT22 cells. MATERIALS AND METHODS: New compound, (E)-methyl-4-hydroxy-4-(8a-methyl-3-oxodecahydronaphthalen-4a-yl) (1) and, nine known compounds, diosmetin-7-O (2'',6''-di-O-α-L-rhamnopyranosyl)-ß-D-glucopyranoside (2), 4-hydroxy-3-methoxy-pentyl ester benzenepropanoic acid (3), vanillic acid (4), 4-hydroxy-benzeneacetic acid (5), 4-methoxybenzeneacetic acid (6), (E)-4-methoxycinnamic acid (7), 3-methoxy-4-hydroxyphenylethanol (8), hydroferulic acid (9), and methyl hydroferulate (10), were isolated by bioactivity-guided separation. Structures of the isolated compounds were identified on the basis of (1)H nuclear magnetic resonance (NMR), (13)C NMR, and two-dimensional NMR spectra, while their neuroprotective properties were evaluated by performing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS: D. superbus extract had a neuroprotective effect and isolated 10 compounds. Among the compounds, compounds 5 and 6 effectively protected HT22 cells against glutamate toxicity. CONCLUSION: In conclusion, the extract of D. superbus and compounds isolated from it exhibited neuroprotective properties, suggesting therapeutic potential for applications in neurotoxic diseases. SUMMARY: D. superbus extract significantly protected on glutamate-induced cell death in HT22 cellsNew compound, (E)-methyl-4-hydroxy-4-(8a-methyl-3-oxodecahydronaphthalen-4a-yl) (1) and, nine known compounds, diosmetin-7-O(2'',6''-di-O-α-L-rhamnopyranosyl)-ß-D-glucopyranoside (2), 4-hydroxy-3-methoxy-pentyl ester benzenepropanoic acid (3), vanillic acid (4), 4-hydroxy-benzeneacetic acid (5), 4-methoxybenzeneacetic acid (6), (E)-4-methoxycinnamic acid (7), 3-methoxy-4-hydroxyphenylethanol (8), hydroferulic acid (9), and methyl hydroferulate (10) were isolated from D. superbus extract4-hydroxy-benzeneacetic acid and 4-methoxybenzeneacetic acid showed significant protective activity against glutamate-induced toxicity in HT22 cells. Abbreviations used: CNS: Central nervous system, ROS: Reactive oxygen species, CHCl3: Chloroform, EtOAc: Ethyl acetate, BuOH: Butanol, HPLC: High performance liquid chromatography, TLC: Thin layer chromatography, MPLC: Middle performance liquid chromatography, MeOH: Methanol, OD: Optical density, COSY: Correlation spectroscopy, HMQC: Heteronuclear multiple-quantum correlation, HMBC: Heteronuclear multiple-bond correlation, HR-MS: High-resolution molecular spectroscopy, MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

The complete chloroplast genome sequence of Dianthus superbus var. longicalycinus.

The complete chloroplast genome (cpDNA) sequence of Dianthus superbus var. longicalycinus is an economically important traditional Chinese medicine was reported and characterized. The cpDNA of Dianthus superbus var. longicalycinus is 149,539 bp, with 36.3% GC content. A pair of inverted repeats (IRs) of 24,803 bp is separated by a large single-copy region (LSC, 82,805 bp) and a small single-copy region (SSC, 17,128 bp). It encodes 85 protein-coding genes, 36 tRNA genes and 8 rRNA genes. Of 129 individual genes, 13 genes encoded one intron and three genes have two introns.

Probing the impact of quercetin-7-O-glucoside on influenza virus replication influence.

BACKGROUND: Influenza virus is still at large and seriously affects social welfare and health. Dianthus superbus is a well-known medicinal plant widely used in Mongolian and Chinese traditional medicine for anti-inflammatory purposes. PURPOSE: To investigate the influence of this novel herbal medicinal product over virus infection and virus-induced symptoms METHOD: Quercetin-7-O-glucoside was isolated by bioassay (anti-influenza)-guided fractionation. The structural elucidation was made with 1H-NMR and 13C-NMR. Influenza A/Vic/3/75 (H3N2), A/PR/8/34 (H1N1), B/Maryland/1/59 and B/Lee/40 viruses were used for the evaluation of the antiviral activity. Virus-induced reactive oxygen species and autophagy formation levels were studied. The antiviral mechanism was elucidated via time-dependent, pre-, post-incubation assay methods. The viral RNA replication inhibition of Q7G was analyzed using quantitative RT-PCR method. The blocking of polymerase basic protein subunits of influenza viral RNA polymerase by Q7G was detected by in silico molecular docking assays using AutoDock Vina program with m(7)GTP. Additionally, Q7G was tested against M-MuLV RNA polymerase. RESULTS: Q7G was not cytotoxic (CC50>100µg/ml) in MDCK cells and it showed 3.1µg/ml, 6.61µg/ml, 8.19µg/ml and 5.17µg/ml IC50 values against influenza A/PR/8/34, A/Vic/3/75, B/Lee/40 and B/Maryland/1/59 virus strains, respectively. Treatment of Q7G highly reduced ROS and autophagy formation induced by influenza virus infection. Q7G did not reduce NA activity and did not directly interact with the virus particles. Since viral RNA synthesis was blocked by treatment of Q7G. We targeted viral RNA polymerase for further probing. Interestingly, the binding energy of Q7G on viral PB2 protein was -9.1kcal/mol and was higher than m(7)GTP recorded as -7.5kcal/mol. It also was observe to block M-MuLV RNA polymerase. CONCLUSION: Isolated compound Q7G showed strong inhibition activity against influenza A and B viruses. It also reduced virus-induced ROS and autophagy formation. Q7G does not directly bind to the virus particles and did not affect NA activity. These results indicated that Q7G inhibits viral RNA polymerase, and that it occupies the binding site of m(7)GTP on viral PB2 protein.

Cognitive-Enhancing Effect of Dianthus superbus var. Longicalycinus on Scopolamine-Induced Memory Impairment in Mice.

Dianthus superbus (D. superbus) is a traditional crude drug used for the treatment of urethritis, carbuncles and carcinomas. The objective of this study was to confirm the cognitive enhancing effect of D. superbus in memory impairment induced mice and to elucidate the possible potential mechanism. Effect of D. superbus on scopolamine induced memory impairment on mice was evaluated using the Morris water maze and passive avoidance tests. We also investigated acetylcholinesterase (AChE) activity and brain-derived neurotropic factor (BDNF) expression in scopolamine-induced mice. HPLC-DAD analysis was performed to identify active compounds in D. superbus. The results revealed that D. superbus attenuated the learning and memory impairment induced by scopolamine. D. superbus also inhibited AChE levels in the hippocampi of the scopolamine-injected mice. Moreover, D. superbus increased BDNF expression in the hippocampus. Eight compounds were identified using HPLC-DAD analysis. The content of 4-hydroxyphenyl acetic acid was higher than contents of other compounds. These results indicated that D. superbus improved memory functioning accompanied by inhibition of AChE and upregulation of BDNF, suggesting that D. superbus may be a useful therapeutic agent for the prevention or treatment of Alzheimer's disease.