Identifying Anti-NSCLC Bioactive Compounds in Scutellaria via 2D NMR-Based Metabolomic Analysis of Pharmacologically Classified Crude Extracts.

We presented a strategy utilizing 2D NMR-based metabolomic analysis of crude extracts, categorized by different pharmacological activities, to rapidly identify the primary bioactive components of TCM. It was applied to identify the potential bioactive components from Scutellaria crude extracts that exhibit anti-non-small cell lung cancer (anti-NSCLC) activity. Four Scutellaria species were chosen as the study subjects because of their close phylogenetic relationship, but their crude extracts exhibit significantly different anti-NSCLC activity. Cell proliferation assay was used to assess the anti-NSCLC activity of four species of Scutellaria. 1H-13C HSQC spectra were acquired for the chemical profiling of these crude extracts. Based on the pharmacological classification (PCA, OPLS-DA and univariate hypothesis test) were performed to identify the bioactive constituents in Scutellaria associated with the anti-NSCLC activity. As a result, three compounds, baicalein, wogonin and scutellarin were identified as bioactive compounds. The anti-NSCLC activity of the three potential active compounds were further confirmed via cell proliferation assay. The mechanism of the anti-NSCLC activity by these active constituents was further explored via flow cytometry and western blot analyses. This study demonstrated 2D NMR-based metabolomic analysis of pharmacologically classified crude extracts to be an efficient approach to the identification of active components of herbal medicine.

Hypoglycemic and hypolipidemic dual activities of extracts and flavonoids from Desmodium caudatum and an efficient synthesis of the most potent 8-prenylquercetin.

Since glucolipid metabolism disorders is often the mono-target therapy fails in managing blood glucose and lipid levels and the other complications, it is urgent and necessary to seek for the new potential drugs or functional food acting on multi-targets. The hypoglycemic and hypolipidemic dual activities of the root, stems and leaves of Desmodium caudatum, which is used for traditional Chinese medicine, was evaluated. Twelve extracts with different extraction conditions were prepared and extract 9 was find to exhibit potential inhibitory activities of fructose-1, 6-bisphosphatase (FBPase), α-glucosidase, and pancrelipase, as well as promote cellular glucose consumption and reduce cellular content of lipid. Five flavonoids were isolated and identified from extract 9, among which 8-prenylquercetin exhibited potent α-glucosidase (IC50 = 4.38 µM) and FBPase (IC50 = 3.62 µM) dual inhibitory activity, which were 75-fold higher than acarbose (IC50 = 330.10 µM) and comparable with AMP (IC50 = 2.92 µM). In addition, 8-prenylquercetin was able to promote glucose consumption and reduce lipid content. Besides, an efficient synthesis of the most potent 8-prenylquercetin was developed from inexpensive and commercially available rutin in 21% overall yield by 6 steps, which lay the foundation of preparation sufficient amount for follow-up study.

Microextraction of Reseda luteola-Dyed Wool and Qualitative Analysis of Its Flavones by UHPLC-UV, NMR and MS.

Detailed knowledge on natural dyes is important for agronomy and quality control as well as the fastness, stability, and analysis of dyed textiles. Weld (Reseda luteola L.), which is a source of flavone-based yellow dye, is the focus of this study. One aim was to reduce the required amount of dyed textile to ≤50 µg for a successful chromatographic analysis. The second aim was to unambiguously confirm the identity of all weld flavones. By carrying out the extraction of 50 µg dyed wool with 25 µL of solvent and analysis by reversed-phase UHPLC at 345 nm, reproducible chromatographic fingerprints could be obtained with good signal to noise ratios. Ten baseline separated peaks with relative areas ≥1% were separated in 6 min. Through repeated polyamide column chromatography and prepHPLC, the compounds corresponding with the fingerprint peaks were purified from dried weld. Each was unequivocally identified, including the position and configuration of attached sugars, by means of 1D and 2D NMR and high-resolution MS. Apigenin-4'-O-glucoside and luteolin-4'-O-glucoside were additionally identified as two trace flavones co-eluting with other flavone glucosides, the former for the first time in weld. The microextraction might be extended to other used dye plants, thus reducing the required amount of precious historical textiles.

Evaluation of cholinesterase inhibitory and antioxidant activity of Wedelia chinensis and isolation of apigenin as an active compound.

BACKGROUND: Wedelia chinensis has been reported as a folk medicine for the treatment of different diseases including neurodegenerative disease. Although the plant has been studied well for diverse biological activities, the effect of this plant in neurological disorder is largely unknown. The present study was undertaken to evaluate the cholinesterase inhibitory and antioxidant potential of W. chinensis. METHODS: The extract and fractions of the plant were evaluated for acetylcholinesterase and butyrylcholinesterase inhibitory activity by modified Ellman method. The antioxidant activity was assessed in several in vitro models/assays such as reducing power, total antioxidant capacity, total phenolic and flavonoid content, scavenging of 2,2'-diphenyl-1-picrylhydrazyl (DPPH) free radical and hydroxyl radical, and inhibition of brain lipid peroxidation. Chromatographic and spectroscopic methods were used to isolate and identify the active compound from the extract. RESULTS: Among the fractions, aqueous fraction (AQF) and ethylacetate fraction (EAF) exhibited high inhibition against acetylcholinesterase (IC50: 40.02 ± 0.16 µg/ml and 57.76 ± 0.37 µg/ml) and butyrylcholinesterase (IC50: 31.79 ± 0.18 µg/ml and 48.41 ± 0.05 µg/ml). Similarly, the EAF and AQF had high content of phenolics and flavonoids and possess strong antioxidant activity in several antioxidant assays including DPPH and hydroxyl radical scavenging, reducing power and total antioxidant activity. They effectively inhibited the peroxidation of brain lipid in vitro with IC50 values of 45.20 ± 0.10 µg/ml and 25.53 ± 0.04 µg/ml, respectively. A significant correlation was observed between total flavonoids and antioxidant and cholinesterase inhibitory activity. Activity guided chromatographic separation led to the isolation of a major active compound from the EAF and its structure was elucidated as apigenin by spectral analysis. CONCLUSIONS: The potential ability of W. chinensis to inhibit the cholinesterase activity and peroxidation of lipids suggest that the plant might be useful for the management of AD.

The Contents of Polyphenols in Perilla frutescens (L.) Britton var. frutescens (Egoma) Leaves are Determined by Vegetative Stage, Spatial Leaf Position, and Timing of Harvesting during the Day.

The leaf of Perilla frutescens (L.) Britton var. frutescens (egoma) is a rich source of polyphenolic compounds, including rosmarinic acid. However, there is still a lack of detailed information concerning the content of phenolic compounds in these leaves. Since some flavonoids were found as a conjugated form, leaves were used untreated or hydrolyzed using ß-glucuronidase for analysis. Enzymatic hydrolysis method successfully identified some polyphenols, which have not been reported before. Scutellarin, a flavone glucuronide with a molecular mass similar to that of luteolin 7-O-glucuronide, was present in egoma leaves. Scutellarin was the second most abundant polyphenolic compound, after rosmarinic acid. Egoma leaves at the top of the plant contained a higher amount of rosmarinic acid and scutellarin compared to that in the leaves below. The difference in plant growth stage also influenced the rosmarinic acid and scutellarin contents, while the time of harvesting during the day did rosmarinic acid contents only. This is the first time that scutellarin, a traditional Chinese medicine, widely used for the treatment of cerebrovascular disease, was quantitatively determined in egoma leaves. The present study may help adding value to egoma leaves, developing dietary supplements, functional foods, and cosmetics.

Ficus deltoidea: Potential inhibitor of pro-inflammatory mediators in lipopolysaccharide-induced activation of microglial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Ficus deltoidea Jack (FD) is widely consumed in traditional medicine as a treatment for various diseases in Malaysia. Each part of the plant such as its leave, stem, fruit and root are used traditionally to treat different types of diseases. Vitexin and isovitexin are bioactive compounds abundantly found in the leaves of FD that possessed many pharmacological properties including neuroprotection. Nonetheless, its effects on key events in neuroinflammation are unknown. AIM OF THE STUDY: To determine the inhibitory properties of FD aqueous extract on pro-inflammatory mediators involved in lipopolysaccharide (LPS)-induced microglial cells. METHODS: Vitexin and isovitexin in the extract were quantified via high performance liquid chromatography (HPLC). The extract was evaluated for its cytotoxicity activity via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Pre-treatment with the extract on LPS-induced microglial cells was done to determine its antioxidant and anti-neuroinflammatory properties by measuring the level of reactive oxygen species (ROS), nitric oxide (NO), tumour necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) via 2'-7'-dichlorofluorescin diacetate (DCFDA) assay, Griess assay and Western blot respectively. RESULTS: The extract at all tested concentrations (0.1 µg/mL, 1 µg/mL, 10 µg/mL, 100 µg/mL) were not cytotoxic as the percentage viability of microglial cells were all above ~80%. At the highest concentration (100 µg/mL), the extract significantly reduced the formation of ROS, NO, TNF-α, IL-1ß and IL-6 in microglial cells induced by LPS. CONCLUSION: The extract showed neuroprotective effects by attenuating the levels of pro-inflammatory and cytotoxic factors in LPS-induced microglial cells, possibly by mediating the nuclear factor-kappa B (NF-κB) signalling pathway.

Bioassay-Guided Identification of the Antiproliferative Compounds of Cissus trifoliata and the Transcriptomic Effect of Resveratrol in Prostate Cancer Pc3 Cells.

The bioassay-guided fractionation of a CHCl3-MeOH extract from the stems of Cissus trifoliata identified an active fraction against PC3 prostate cancer cells. The treatment for 24 h showed an 80% reduction in cell viability (p ≤ 0.05) by a WST-1 assay at a concentration of 100 µg/mL. The HPLC-QTOF-MS analysis of the fraction showed the presence of coumaric and isoferulic acids, apigenin, kaempferol, chrysoeriol, naringenin, ursolic and betulinic acids, hexadecadienoic and octadecadienoic fatty acids, and the stilbene resveratrol. The exposure of PC3 cells to resveratrol (IC25 = 23 µg/mL) for 24 h induced significant changes in 847 genes (Z-score ≥ ±2). The functional classification tool of the DAVID v6.8 platform indicates that the underlying molecular mechanisms against the proliferation of PC3 cells were associated (p ≤ 0.05) with the process of differentiation and metabolism. These findings provide experimental evidence suggesting the potential of C. trifoliata as a promising natural source of anticancer compounds.

Anti-inflammatory effect of Adelia ricinella L. aerial parts.

OBJECTIVE: To investigate the main chemical components and the anti-inflammatory activity of extracts of Adelia ricinella L. aerial parts. METHODS: Three extracts obtained by soxhlet extraction and ethanol/water mixtures were evaluated in their chemical composition by UPLC-DAD-MS/MS. The in vitro anti-inflammatory activity of the prepared extracts was assessed through three different assays: COX-1 and COX-2 enzymatic inhibition, cell-based COX assays on RAW264.7 macrophages (ATCC) measuring the COX-2 protein expression by Western blot and the measurement of the PGE2 concentration in the supernatants of the culture medium. Also was determinate the effect of the three extracts on the RAW 264.7 cell viability. KEY FINDINGS: Few differences in the phytochemical profile were found between the three prepared extracts, identifying a blend of thirteen flavonoids derived from luteolin and apigenin, with orientin as main constituent. Plant extracts (alcoholic and aqueous) did not affect the macrophage cell viability (IC50 > 256 µg/ml) and significantly reduced COX-1 and COX-2 enzyme activities. Additionally, COX-2 expression and PGE2 release were suppressed after 24 h of LPS stimulation and treatment with plant extracts (8-64 µg/ml). CONCLUSIONS: A. ricinella extracts showed the ability to reduce the inflammatory effect exerted by LPS in murine macrophages. However, further studies should confirm their anti-inflammatory activity.

Vitexin, a fenugreek glycoside, ameliorated obesity-induced diabetic nephropathy via modulation of NF-κB/IkBα and AMPK/ACC pathways in mice.

Obesity is one of the most critical risk factors for diabetes mellitus and plays a significant role in diabetic nephropathy (DN). The present investigation aimed to evaluate the possible mechanism of action of vitexin on obesity-induced DN in a high-fat diet (HFD)-fed experimental C57BL/6 mice model. Obesity was induced in male C57BL/6 mice by chronic administration of HFD, and mice were concomitantly treated with vitexin (15, 30, and 60 mg/kg, p.o.). HFD-induced increased renal oxido-nitrosative stress and proinflammatory cytokine levels were significantly inhibited by vitexin. The Western blot analysis suggested that alteration in renal NF-κB, IκBα, nephrin, AMPK, and ACC phosphorylation levels was effectively restored by vitexin treatment. Histological aberration induced in renal tissue after chronic administration of HFD was also reduced by vitexin. In conclusion, vitexin suppressed the progression of obesity-induced DN via modulation of NF-κB/IkBα and AMPK/ACC pathways in an experimental model of HFD-induced DN in C57BL/6J mice.

Apigenin attenuates TGF-ß1-stimulated cardiac fibroblast differentiation and extracellular matrix production by targeting miR-155-5p/c-Ski/Smad pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Apigenin is a natural flavonoid compound present in chamomile (Matricaia chamomilla L.) from the Asteraceae family, which is used in the treatment of cardiovascular diseases by traditional healers, but its effects on differentiation and extracellular matrix (ECM) production of cardiac fibroblasts (CFs) induced by transforming growth factor beta 1 (TGF-ß1) are poorly understood. AIM OF THE STUDY: This study aimed to examine these effects and potential molecular mechanisms and to provide a new application of apigenin in the prevention and treatment of cardiac fibrosis. MATERIALS AND METHODS: The TGF-ß1-stimulated CFs or the combination of TGF-ß1-stimulated and microRNA-155-5p (miR-155-5p) inhibitor- or mimic-transfected CFs were treated with or without apigenin. The expression levels of intracellular related mRNA and proteins were detected by real-time polymerase chain reaction and Western blot methods, respectively. The luciferase reporter gene containing cellular Sloan-Kettering Institute (c-Ski) wild or mutant type 3'-UTR was used and the luciferase activity was examined to verify the direct link of miR-155-5p and c-Ski. RESULTS: After treatment of TGF-ß1-stimulated CFs with 6-24 µM apigenin, the expression of c-Ski was increased, while levels of miR-155-5p, α-smooth muscle actin, collagen Ⅰ/Ⅲ, Smad2/3, and p-Smad2/3 were decreased. After transfection of CFs with the miR-155-5p inhibitor or mimic, the similar or inverse results were respectively observed as well. The combination of TGF-ß1 and miR-155-5p inhibitor or mimic might cause an antagonistical or synergistic effect, respectively, and apigenin addition could enhance the effects of the inhibitor and antagonize the effects of the mimic. Luciferase reporter gene assay demonstrated that c-Ski was a direct target of miR-155-5p. CONCLUSION: These findings suggested that apigenin could inhibit the differentiation and ECM production in TGF-ß1-stimulated CFs, and its mechanisms might partly be attributable to the reduction of miR-155-5p expression and subsequent increment of c-Ski expression, which might result in the inhibition of Smad2/3 and p-Smad2/3 expressions.

Inhibitory Effect of Lygodium Root on the Cytochrome P450 3A Enzyme in vitro and in vivo.

PURPOSE: The aim of the present study was to investigate the interactions of the main components of Lygodium root (ie, p-coumaric acid, acacetin, apigenin, buddleoside and Diosmetin-7-O-ß-D-glucopyranoside) with cytochrome P450 3A enzyme activity both in vitro and in vivo. METHODS: In vitro inhibition of drugs was assessed by incubating rat liver microsomes (RLMs) with a typical P450 3A enzyme substrate, midazolam, to determine their 50% inhibitory concentration (IC50) values. For the in vivo study, healthy male Sprague Dawley rats were consecutively administered acacetin or apigenin for 7 days at the dosage of 5 mg/kg after being randomly divided into 3 groups: Group A (control group), Group B (acacetin group) and Group C (apigenin group). RESULTS: Among the five main components of Lygodium root, only acacetin and apigenin showed inhibitory effects on the cytochrome P450 3A enzyme in vitro. The IC50 values of acacetin and apigenin were 58.46 µM and 8.20 µM, respectively. Additionally, the in vivo analysis results revealed that acacetin and apigenin could systemically inhibit midazolam metabolism in rats. The Tmax, AUC(0-t) and Cmax of midazolam in group B and group C were significantly increased (P<0.05), accompanied by a significant decrease in Vz/F and CLz/F (P<0.05). CONCLUSION: Acacetin and apigenin could inhibit the activity of the cytochrome P450 3A enzyme in vitro and in vivo, indicating that herbal drug interactions might occur when taking Lygodium root and midazolam synchronously.

Apigenin, flavonoid component isolated from Gentiana veitchiorum flower suppresses the oxidative stress through LDLR-LCAT signaling pathway.

Flower of Gentiana veitchiorum has traditionally been used as an herbal medicine in Tibet for treatment of variola, respiratory infection, and pneumonia. However, the effective components contained in flower are not identified yet, and the underlying mechanisms for anti-inflammatory, antibacterial, and antioxidative activities remain to be elucidated. Here, we first extracted the flavonoid mixture from G. veitchiorum flower. The mixture was then further isolated and the within compounds was identified through the high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The results showed that apigenin (4',5,7-trihydroxyflavone) was the most abundant flavonoid in G. veitchiorum flower. We next examined the antioxidative activity of the extracted apigenin using the ferric reducing/antioxidant power (FRAP), the 1,1-diphenyl-2-picrylhydrazyl (DPPH), and the 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) assays and found that a positive correlation between apigenin concentration and reactive oxygen species (ROS) scavenging rate. The biochemical assays further revealed that the levels of total cholesterol (TC), triglyceride (TG), and malondialdehyde (MDA) were reduced, while the activity of superoxide dismutase (SOD) was increased after apigenin treatment in hyperlipidemic rats. Moreover, we performed histopathological investigations and found that the lipidic deposition patterns were recovered and the amount of lipid vacuoles was significantly reduced in apigenin-treated hyperlipidemic rat liver. Western blotting assay showed that the expression of low-density lipoprotein receptor (LDLR) and lecithin-cholesterol acyltransferase (LCAT) were up-regulated in the apigenin-treated samples. Overall, our results demonstrated that the apigenin isolated from G. veitchiorum flower exhibited radical scavenging activities, and reversed the high fat diet-induced oxidative damage in rats. Its antioxidative activities are probably achieved via LDLR-LCAT signaling pathway.

Improved preparation of vitexin from hot water extract of Basella alba, the commercially available vegetable Malabar spinach ("Tsurumurasaki" in Japanese) and the application to semisynthesis of chafuroside B.

Hot water extraction of D-arabinofuranosylvitexin from the raw leaves of commercially available Basella alba "Tsurumurasaki" and subsequent acidic hydrolysis was improved to be a procedure using a high-pressure steam sterilizer to afford vitexin. The amount was estimated to be 14.1 mg from 1 g of dry weight of the raw leaves, whose recovery was calculated to be 95% based on the estimated content of D-arabinofuranosylvitexin in B. alba raw leaves. The product was dehydratively cyclized between hydroxy groups on the carbohydrate and flavone skeletons under modified Mitsunobu reaction conditions in N,N-dimethylformamide to give chafuroside B, which is known to be a bioactive Oolong tea polyphenol. Through these transformations, 10.2 mg of chafuroside B could be semisynthesized from 1 g of dry weight of the raw leaves, and the efficiency was improved compared to that from the extraction from Oolong tea (3.4 µg from 1 g of dry weight).

Targeted Isolation of Antioxidant Constituents from Plantago asiatica L. and In Vitro Activity Assay.

Plantago asiatica L. is widely distributed in Eastern Asia and a commonly used drug in China, Korea, and Japan for diuretic and antiphlogistic purposes. In this experiment, the present study was performed to isolate antioxidant molecules based on the DPPH scavenging activity assay and discover the bioactive compounds which contributed to performing the function of Plantago asiatica L. Each faction was chosen for further isolation guided by DPPH scavenging activity assay. Afterwards, two potential bioactive molecules, aesculetin and apigenin, were isolated for in vitro antioxidant activity in cells. Hydrogen-peroxide-induced oxidative stress led to decreased cell viability, impaired intercellular junction, and damage to the cell membrane and DNA. Furthermore, aesculetin ameliorated decreased cell viability induced by hydrogen peroxide via upregulation of antioxidant related genes, and apigenin also protected against H2O2 mainly by improving the glutathione (GSH) antioxidant system, such as increasing the activity of glutathione peroxidase (GPX), glutathione reductase (GR), and the ration of GSH/glutathione disulfide (GSSG). Above all, these findings suggest that aesculetin and apigenin may be bioactive compounds for antioxidant function in Plantago asiatica L.

Chemical constituents and allelopathic activity of Machaerium eriocarpum Benth.

The analysis by HPLC-PDA of the hydroalcoholic extract of the leaves of M. eriocarpum together with the injection of the fractions containing the already identified metabolites allowed the detection of at least 5 flavonoids, of which two are derived from apigenin and three from luteolin. After isolating larger amounts of isovitexin (I), assays were performed to evaluate the allelopathic activity together with the crude extract. The results show that the initial inhibition indexes were very similar to those observed in the treatments with F17 (Fraction enriched in isovitexin) and F18 (isovitexin), mainly in the concentrations of 500 and 1000 mg L-1. The index of the number of lateral roots, an increase of the inhibitory effect is observed with the increase of the concentration of M. eriocarpum extract.

Liquid chromatography fingerprint analysis and antioxidant activity of selected lavender species with chemometric calculations.

The extracts of seven Lavandulae species (Lavandula stoechas, Lavandula lanata, Lavandula viridis, Lavandula angustifolia "Rosea", Lavandula angustifolia "Afropurpurea", Lavandula angustifolia and one unknown) were analyzed using the reversed-phase-high performance liquid chromatography-diode array detection (RP-HPLC-DAD) with gradient elution technique to obtain the chromatographic fingerprint profiles. The HPLC analysis was performed using the Kinetex RP18 chromatographic column and eluent consisting of methanol-water-0.1% formic acid (5-100% (v/v)) at 30 °C with the run time of 60 min. and the detection wavelength 280 nm. The chromatograms were preliminary processed with the smoothing, noise reduction, background subtraction and alignment using the SpecAlign program (version 2.4.1). The presence of selected standards (apigenin, myricetin, luteolin, luteolin 7-glucoside, chlorogenic acid, caffeic acid, ferulic acid) in the extracts was confirmed. The chemical similarity between studied plants was evaluated using the Cluster Analysis (Pearson correlation coefficient, r, and Euclidean) and PCA. The preliminary antioxidant activity of studied extracts was evaluated based on the total phenolic content (Folin-Ciocalteu method), ferric ion reducing antioxidant parameter (FRAP) and α,α-diphenyl-ß-picrylhydrazyl (DPPH) free radical scavenging method using the spectrophotometric technique.

Preparation of flavone di-C-glycoside isomers from Jian-Gu injection (Premna fulva Craib.) using recycling counter-current chromatography.

Jian-Gu Injection (JGI, Premna fulva Craib) has been demonstrated to be effective in the clinical treatment of bone hyperplasia. However, an effective purification method of the JGI flavone C-glycosides as reference materials is not available. The present work developed a recycling counter-current chromatography approach to prepare these materials in high quality. An optimized biphasic solvent system composed of ethyl acetate/n-butanol/water (1:9:10, v/v/v) was employed to purify the five congeneric flavone C-glycosides, identified as apigenin 6,8-di-C-ß-d-glucopyranoside (vicenin 2), apigenin 6-C-ß-d-xylopyranosyl-8-C-ß-d-glucopyranoside (vicenin 1), apigenin 6-C-ß-d-xylopyranosyl-8-C-ß-d-galactopyranoside, apigenin 6-C-ß-d-glucopyranosyl-8-C-ß-d-xylopyranoside (vicenin 3), and apigenin 6-C-ß-d-galactopyranosyl-8-C-ß-d-xylopyranoside, by means of UHPLC-PDA-ESI-IT-ToF-MSn and NMR spectroscopy.

Protective Effects of Ligustroflavone, an Active Compound from Ligustrum lucidum, on Diabetes-Induced Osteoporosis in Mice: A Potential Candidate as Calcium-Sensing Receptor Antagonist.

Ligustroflavone is one major compound contained in active fraction from Fructus Ligustri Lucidi (the fruit of Ligustrum lucidum), which could regulate parathyroid hormone (PTH) levels and improve calcium balance by acting on calcium-sensing receptors (CaSR). This study aimed to explore the potency of ligustroflavone as a CaSR antagonist and its protective effects against diabetic osteoporosis in mice. LF interacted well with the allosteric site of CaSR shown by molecular docking analysis, increased PTH release of primary parathyroid gland cells and suppressed extracellular calcium influx in HEK-293 cells. The serum level of PTH attained peak value at 2 h and maintained high during the period of 1 h and 3 h than that before treatment in mice after a single dose of LF. Treatment of diabetic mice with LF inhibited the decrease in calcium level of serum and bone and the enhancement in urinary calcium excretion as well as elevated circulating PTH levels. Trabecular bone mineral density and micro-architecture were markedly improved in diabetic mice upon to LF treatment for 8 weeks. LF reduced CaSR mRNA and protein expression in the kidneys of diabetic mice. Taken together, ligustroflavone could transiently increase PTH level and regulate calcium metabolism as well as prevent osteoporosis in diabetic mice, suggesting that ligustroflavone might be an effective antagonist on CaSR.

Identification of Six Phytochemical Compounds from Asparagus officinalis L. Root Cultivars from New Zealand and China Using UAE-SPE-UPLC-MS/MS: Effects of Extracts on H2O2-Induced Oxidative Stress.

A simple, rapid, specific, and sensitive method was developed for the simultaneous identification and quantification of six major bioactive compounds, namely, caffeic acid, quercetin, apigenin, ferulic acid, baicalein, and kaempferol, from Asparagus officinalis roots (ARs) native to New Zealand (green and purple cultivars) and China (yellow, green, purple, and white cultivars) using ultrasound-assisted, solid-phase extraction (UASE-SPE) coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The method was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy (expressed as recovery %), and precision (expressed as relative standard deviation (%RSD)). The retention times, ultraviolet visible (UV-vis) data, and mass spectral patterns of the detected peaks matched those of commercial standards, allowing characterization of the target compounds. The LODs and LOQs were 23 ng/mL and 70 ng/mL, 50 ng/mL and 150 ng/mL, 10 ng/mL and 30 ng/mL, 18 ng/mL and 54 ng/mL, 14.4 ng/mL and 43.6 ng/mL, and 7.5 ng/mL and 22.5 ng/mL for caffeic acid, quercetin, apigenin, ferulic acid, baicalein, and kaempferol, respectively, and the mean recovery rates were 85.8%, 73.0%, 90.2%, 80.6%, 76.7%, and 74.5% for the six compounds, respectively. The levels of the target compounds were significantly different (p < 0.05) among the six cultivars. The Chinese yellow AR had the highest levels of bioactive compounds: 6.0, 3.9, 0.4, 1.0, 0.86, and 0.8 mg/g for caffeic acid, quercetin, apigenin, ferulic acid, baicalein, and kaempferol, respectively. The AR extracts showed protective effects against oxidative stress in the HepG2 and L929 cell lines. The results indicate that AR extracts contain high flavonoid levels that provide protective functions against oxidative stress and support the potential commercial application of AR extracts.

Structure Identification of ViceninII Extracted from Dendrobium officinale and the Reversal of TGF-ß1-Induced Epithelial⁻Mesenchymal Transition in Lung Adenocarcinoma Cells through TGF-ß/Smad and PI3K/Akt/mTOR Signaling Pathways.

ViceninII is a naturally flavonoid glycoside extracted from Dendrobium officinale, a precious Chinese traditional herb, has been proven to be valuable for cancer treatment. Transforming growth factor-ß1 (TGF-ß1), promotes the induction of epithelial⁻mesenchymal transition (EMT), a process involved in the metastasis of cells that leads to enhanced migration and invasion. However, there is no previously evidence that ViceninII has an inhibitory effect on cancer metastasis, specifically on the TGF-ß1-induced EMT process in lung adenocarcinoma cells. In this experiment, we used UV, ESIMS, and NMR to identify the structure of ViceninII.A549 and H1299 cells were treated with TGF-ß1 in the absence and presence of ViceninII, and subsequent migration and invasion were measured by wound-healing and transwell assays. The protein localization and expressions were detected by immunofluorescence and Western blotting. The results indicated that TGF-ß1 induced spindle-shaped changes, increased migration and invasion, and upregulated or downregulated the relative expression of EMT biomarkers. Meanwhile, these alterations were significantly inhibited when co-treated with ViceninII and inhibitors LY294002 and SB431542. In conclusion, ViceninII inhibited TGF-ß1-induced EMT via the deactivation of TGF-ß/Smad and PI3K/Akt/mTOR signaling pathways.This is the first time that the anti-metastatic effects of ViceninII have been demonstrated, and their molecular mechanisms provided.