Hormone-sensitive lipase is involved in the action of hydroxysafflor yellow A (HYSA) inhibiting adipogenesis of 3T3-L1cells

BackgroundSafflor yellow A (SY) has been demonstrated to be beneficial to cardiovascular system. Our previous study showed that hydroxysafflor yellow A (HSYA), a main component of SY, could increase peroxisome proliferator-activated receptor γ mRNA expression. In this study, we investigate the effect of HSYA on the proliferation and adipogenesis of mouse 3T3-L1 preadipocytes.MethodsThe proliferation and adipogenesis of 3T3-L1 cells treated with HSYA was studied by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) spectrophotometry, Oil Red O staining and intracellular triglyceride assay methods. HSL mRNA expression and promoter activity were studied by real-time quantitative RT-PCR, transient transfection and dual luciferase reporter gene methods.ResultsHSYA (0.1 mg/L) significantly inhibited the proliferation of 3T3-L1 cells when compared with control cells in 8 h. This effect was further enhanced with the extension time (24 to 96 h) and an increase of concentration of HSYA (1–10 mg/L). The maximal inhibitory action was observed at 0.1 mg/L HSYA in 72 h (86 ± 11.8% vs. 100 ± 4.1%, p < 0.01). HSYA notably reduced the amount of intracellular lipid and triglyceride content in adipocytes to 85% (1 mg/L) and 75% (100 mg/L) on Day 4 following the differentiation, respectively, while increased HSL mRNA expression and promoter activities to 2.7 fold and 1.55 fold, respectively (p < 0.01), in differentiated 3T3-L1 adipocytes.ConclusionsHSYA inhibits the proliferation and adipogenesis of 3T3-L1 preadipocytes. The inhibitory action of HYSA on adipogenesis may be due to the promotion of lipolytic-specific enzyme HSL expression by increasing HSL promoter activity.     

Phytic acid enhances the oral absorption of isorhamnetin,quercetin, and kaempferol in total flavones of Hippophae rhamnoides L.

AimTotal flavones of Hippophae rhamnoides L. (TFH) have a clinical use in the treatment of cardiac disease. The pharmacological effects of TFH are attributed to its major flavonoid components, isorhamnetin, kaempferol, and quercetin. However, poor oral bioavailability of these flavonoids limits the clinical applications of TFH. This study explores phytic acid (IP₆) enhancement of the oral absorption in rats of isorhamnetin, kaempferol, and quercetin in TFH.MethodsIn vitro Caco-2 cell experiments and in vivo pharmacokinetic studies were performed to investigate the effects of IP₆. The aqueous solubility and lipophilicity of isorhamnetin, quercetin, and kaempferol were determined with and without IP₆, and mucosal epithelial damage resulting from IP₆ addition was evaluated by MTT assays and morphology observations.ResultsThe P_app of isorhamnetin, kaempferol, and quercetin was improved 2.03-, 1.69-, and 2.11-fold in the presence of 333 μg/mL of IP₆, respectively. Water solubility was increased 22.75-, 15.15-, and 12.86-fold for isorhamnetin, kaempferol, and quercetin, respectively, in the presence of 20 mg/mL IP₆. The lipophilicity of the three flavonoids was slightly decreased, but their hydrophilicity was increased after the addition of IP₆ in the water phase as the logP values of isorhamnetin, kaempferol, and quercetin decreased from 2.38 ± 0.12 to 1.64 ± 0.02, from 2.57 ± 0.20 to 2.01 ± 0.04, and from 2.39 ± 0.12 to 1.15 ± 0.01, respectively. The absorption enhancement ratios were 3.21 for isorhamnetin, 2.98 for kaempferol, and 1.64 for quercetin with co-administration of IP₆ (200 mg/kg) in rats. In addition, IP₆ (200 mg/kg, oral) caused neither significant irritation to the rat intestines nor cytotoxicity (400 μg/mL) in Caco-2 cells.ConclusionsThe oral bioavailability of isorhamnetin, kaempferol, and quercetin in TFH was enhanced by the co-administration of IP₆. The main mechanisms are related to their enhanced aqueous solubility and permeability in the presence of IP₆. In summary, IP₆ is a potential absorption enhancer for pharmaceutical formulations that is both effective and safe.     

Estrogenic and anti-estrogenic activities of hispolon from Phellinus lonicerinus (Bond.) Bond. et sing

Hispolon was the main antitumor active ingredient in Phellinus sensu lato species. In order to confirm the dual regulating estrogenic ingredient and obtain more effective natural estrogen replacement drugs, hispolon was separated from Phellinus lonicerinus (Bond.) Bond. et sing. Hispolon exhibited significant anti-proliferative effect against estrogen-sensitive ER (+) MCF-7 cells in the absence of estrogen, and exhibits antagonistic effects on 17β-estradiol (E₂)-induced MCF-7 cell proliferation when E₂ and the different concentrations of hispolon were treated simultaneously. Hispolon also inhibited the proliferation of estrogen-negative ER (−) MDA-MB-231 cells at the concentration of 5.00 × 10^− 5 M. The yeast two-hybrid experiments showed that hispolon had strong and non-selective effects on the estrogen receptor (ER) α and ERβ at a concentration of 1.00 × 10^− 6 M. The ERβ-binding ability of hispolon was larger than ERα in the concentration range of 1.00 × 10^− 9 M and 1.00 × 10^− 7 M. Hispolon could increase the body weight coefficient, serum E₂ and progesterone contents in immature female mice at dose of 9.10 × 10^− 6 mol/kg, and increase coefficient of thymus and spleen in mice. The Gscores of hispolon-ERα and hispolon-ERβ docked complexes were − 7.93 kcal/mol and − 7.79 kcal/mol in docking simulations. Hispolon presented dual regulating estrogenic activities, which showed estrogenic agonist activity at low concentration or lack of endogenous estrogen, and the estrogenic antagonistic effect was stimulated at high concentrations or too much endogenous estrogen. Hispolon could be used for treating the estrogen deficiency-related disease with the benefit of non-toxic to normal cells, good antitumor effects and estrogenic activity.     

Isoflavone content and estrogenic activity of different batches of red clover (Trifolium pratense L.) extracts: An in vitro study in MCF-7 cells

The estrogenicity of different batches of red clover (Trifolium pratense L., Fabaceae; RCL) extracts and its relationship with the isoflavone content were assessed by measuring MCF-7 cell proliferation by flow cytometry and propidium iodide staining. RCL extracts were compared to estradiol (E2) and to the main RCL isoflavones biochanin A, daidzein, genistein and formononetin. Isoflavone content in the extracts was assayed by HPLC.E2 and isoflavones increased MCF-7 proliferation in a concentration-dependent fashion, with the following potency order: E2 >>> genistein > biochanin A = daidzein > formononetin. Extracts increased MCF-7 proliferation with different potencies, which in four out of five extracts correlated with the ratios 5,7-dihydroxyisoflavones/7-hydroxyisoflavones. The efficacy of all extracts increased with decreasing genistein contents. A solution containing the main isoflavones at the average concentration of RCL extracts increased MCF-7 proliferation with higher potency and steeper concentration–response curve. The effects of E2, of RCL extracts and of the isoflavone solution were inhibited by the estrogen receptor antagonist 4-hydroxytamoxifen.Flow cytometric analysis of MCF-7 proliferation is a suitable bioassay for the estrogenicity of RCL extracts, thus expanding the characterization of individual batches beyond assessment of chemical composition and contributing to improved standardization of quality and activity.     

Diameter increment patterns among 106 tree species in a logged and silviculturally treated Costa Rican rain forest

Studies of growth rates of trees in managed neotropical forests have rarely employed complete botanical identification of all species, while published information for Central American lowland rain forests largely concerns forests free of recent disturbance. We studied diameter increments of trees in a managed Costa Rican rain forest. The Pentaclethra macroloba-dominated forest was located on low hills with Ultisols in Holdridge's Tropical Wet Forest life zone. The 540 m × 540 m (29.2 ha) experimental area was lightly logged during 1989–1990. The 180 m × 180 m (3.24 ha) experimental plots comprised a 100 m × 100 m (1.0 ha) central permanent sample plot (PSP) with a 40-m wide buffer strip. Post-harvest silvicultural treatments were liberation/refinement (in 1991) and shelterwood (in 1992), applied under a complete randomized block design with three replicates, using logged but untreated plots as controls. All live trees ≥10 cm DBH in the PSPs, were identified to species; data reported are for 1993–1996. Cluster analysis was used to group species on the basis of the median and quartiles of their diameter increment distributions, separating data by silvicultural treatments; five diameter increment groups were established and subdivided on the basis of the adult height of each species (four categories), giving 17 species groups in the final classification. Adult height and silvicultural treatment made a significant contribution to growth rate variation. Median annual increments of the slowest-growing species groups, which featured many under- and middle story species, were ca. 1 mm; those for the fastest growing species, which were mainly canopy and emergents, were ca. 16 mm. All species in the groups of very fast growth were pioneers, whether short or long-lived, though many other pioneer species did not show fast growth. The proportions of species found in groups of moderate, fast or very fast growth were greater in the silviculturally treated plots than in the controls, and one complete diameter increment group, of fast growth, was only represented in the treated plots. Crown form, crown illumination and presence of lianas in the crown, showed significant correlations with diameter increments, though the importance of these latter two variables varied with silvicultural treatment. The very fast growth groups differed from the others in having higher proportions of trees with well-formed, well-illuminated crowns and an irregular diameter distribution with relatively few individuals in the smallest DBH class. Comparison with data from other neotropical forest sites shows that long-lived pioneers such as Vochysia ferruginea and Jacaranda copaia grow fast or very fast at all sites, while non-commercial canopy and emergent species of Chrysobalanaceae and Sapotaceae appear to be uniformly slow-growing. Growth data for the majority of species are, however, published for the first time.     

The inhibition of resveratrol to human skin squamous cell carcinoma A431 xenografts in nude mice

Squamous cell carcinoma (SCC) is one of the commonest dermatological malignancies. Resveratrol (Res) is one type of polyphenolic compound which was first identified from the roots of Veratrum grandinorum in 1940. The previous studies found that Res can promote apoptosis of a variety of tumor cell, especially SCC cells. However it is rare to study the inhibition mechanism of Res in the animal model. In this study, through the establishment of human cutaneous SCC A431 xenografts in nude mice, we observed Res inhibition effect and investigated the inhibition mechanism by checking the expression of apoptosis-related factors, p53, ERK and survivin. The results showed that the xenograft volume and weight of Res groups were less than those of the control groups (P < 0.05), but the net body mass of nude mice of Res groups was not significantly different from the control groups (P > 0.05). The apoptotic index of Res groups were significantly higher than the control groups (P < 0.05). The protein and mRNA expression of p53 and ERK were statistically positively correlated (P < 0.05) and significantly increased in Res high- and medium-dose groups compared with the control groups (P < 0.05). Moreover, the protein and mRNA expression of SVV were negatively correlated with p53 (P < 0.05) and lower than the control groups (P < 0.05). The results demonstrate Res inhibitory effect and indicate that the inhibition mechanism of Res is to upgrade the protein and mRNA expression of p53 and to downgrade the protein and mRNA expression of SVV, thus inducing the apoptosis of tumor cells.     

Purification,structural elucidation and antitumor activity of a novel mannogalactoglucan from the fruiting bodies of Lentinus edodes

A mannogalactoglucan, named LE-MGG, was isolated from the basidiocarps of Lentinus edodes by hot water-extraction, ethanol precipitation anion exchange chromatography, and further purified by gel-permeation chromatography (GPC). Its structural features were investigated by high performance liquid chromatography (HPLC), high performance gel-permeation chromatography (HPGPC), methylation analysis, periodate oxidation-Smith degradation, and by IR and NMR spectroscopy, including two-dimensional (2D) NMR. HPLC analysis revealed that LE-MGG contained mannose–galactose–glucose in the molar ratio of 10:18:72. GPC and HPGPC showed that LE-MGG was a homogeneous fraction (d = 1.34) and its molecular weight was estimated to be 18 kDa. Chemical and spectroscopic studies indicated that LE-MGG consists of (1 → 6)-, (1 → 4)- and (1 → 3)-linked β-d-glucopyranosyl residues, (1 → 6)-linked α-d-galactopyranosyl residues, (1 → 3,6)- and (1 → 2,4)-linked α-d-mannopyranosyl residues and terminal residues of β-d-glucopyranosyl. Cytotoxicity assay showed that LE-MGG presented higher antitumor activities against S-180 cell with a dose-dependent manner, and exhibited lower cytotoxicity to carcinoma HCT-116 and HT-29 cells. Our studies showed also that LE-MGG presented antitumor bioactivities on Sarcoma 180 solid tumor cell implanted in Kunming mice. This finding suggests that mannogalactoglucan should be explored as potential antitumor agents and could be potentially applied as a natural antitumor drug.     

Identification of chicoric acid as a hypoglycemic agent from Ocimum gratissimum leaf extract in a biomonitoring in vivo study

Ocimum gratissimum L. is popularly used to treat diabetes mellitus. The hypoglycemic activity of this medicinal species has been confirmed by in vivo studies. The present study conducted a chemical investigation of a leaf decoction (10% p/v) of O. gratissimum monitored by in vivo hypoglycemic activity assays. Four phenolic substances were identified: l-caftaric acid (1), l-chicoric acid (2), eugenyl-β-d-glucopyranoside (3) and vicenin-2 (4). The acute hypoglycemic activity of the O. gratissimum decoction fractions Og1-S (300 mg/kg), Og1-A (240 mg/kg) and Og1-B (80 mg/kg) was evaluated intraperitoneally in normal and streptozotocin-induced diabetic mice. They reduced glycemia by 63%, 76% and 60% (in 120 min), respectively, in the diabetic mice. Subfractions of Og1-A were also evaluated under the same conditions: Og1-AS (200 mg/kg) and Og1-AP (40 mg/kg) produced a decrease of only 37% and 39%, respectively. Among the major phenolic substances, only chicoric acid (2; 3 mg/kg) reduced significantly the glycemic levels of diabetic mice by 53%, 120 min after treatment. This is the first study describing the hypoglycemic activity of chicoric acid in an animal model of diabetes mellitus. In addition, we suggest that there may be other substances contributing to this activity. Thus, for the first time, a correlation is established between the hypoglycemic activity of O. gratissimum and its chemical composition.     

Comparative pharmacokinetics and tissue distribution study of mono-, and di-caffeoylquinic acids isomers of Ainsliaea fragrans Champ by a fast UHPLC–MS/MS method

Ainsliaea fragrans Champ, as a well-known herb in Traditional Chinese Medicine, was often used in the treatment of gynecological diseases. Caffeoylquinic acids (CQAs) were the bioactive constituents of this plant medicine which primarily contains mono-CQAs (MCQA) and di-CQAs (DCQA). The biosynthesis showed that MCQAs were the precursor of DCQAs. Recent literatures manifested some particular features of DCQAs, different from MCQAs. Therefore it is apparent that a complete and scientific assessment of DCQAs and MCQAs should include not only the DCQAs' pharmacokinetics and distribution but also its degradation products. So an efficient, sensitive rapid resolution liquid chromatography/tandem mass spectrometry (UHPLC–MS/MS) method for the simultaneous determination of the active ingredients in rat plasma and different tissues had been developed and validated. Mass spectrometric detection was performed by selected reaction monitoring mode (MRM) via an electrospray ionization source operating in negative ionization mode. The method was validated in plasma and tissue samples, which showed good linearity over a wide concentration range (r² > 0.99), and obtained lower limit of quantification (LLOQ) was 2.34 ng·mL^− 1 for the analytes in biological samples. The intra- and inter-day assay variability was less than 15%, and the accuracy was between − 8.8% and 5.7%. This study provided the pharmacokinetic profiles and the tissue regional distribution of MCQAs, DCQAs and caffeic acid. The results indicated that the DCQAs isomers were absorbed quickly after oral administration and degradation products MCQAs were mostly found in tissues, not in plasma. Besides, 1,5-DCQA was the prior configuration for the isomerization phenomenon. The small intestine was the main absorption site for DCQAs. Interestingly, the content of the DCQA and MCQA isomers was all high in the ovary and uterus, and some could pass through the barrier between the blood and brain obviously.     

The therapeutic effects of EGCG on vitiligo

Epigallocatechin-3-gallate (EGCG) is one of the main chemical constituents of green tea, which has been used as an important traditional Chinese medicine. Green tea has anti-inflammatory, anti-oxidant, and immunomodulatory properties. However, the effects of EGCG on vitiligo are not known. We assessed the role of EGCG in vitiligo induced by monobenzone in mice. We demonstrated that EGCG: delayed the time of depigmentation; reduced the prevalence of depigmentation; and decreased the area of depigmentation. Examination of depigmented skin treated with EGCG by reflectance confocal microscopy suggested increased numbers of epidermal melanocytes and histologic examination showed decreased perilesional accumulation of CD8⁺ T cells. To further investigate the mechanism of the anti-inflammatory effects of EGCG, levels of inflammatory mediator tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-6 were tested by enzyme-linked immunosorbent assay. Serum cytokine levels were significantly decreased after administration of EGCG compared with the model group. These results suggested that EGCG may have protective effects against vitiligo, and that it could contribute to suppression of activation of CD8⁺ T cells and inflammatory mediators. Based on these results, 5% EGCG was considered to be the most suitable concentration for treating vitiligo, and was used for further study. In addition, we investigated the gene-expression profile of this model in relation to EGCG. Using a 4 × 44 K whole genome oligo microarray assay, 1264 down-regulated genes and 1332 up-regulated genes were recorded in the 5% EGCG group compared with the model group, and selected genes were validated by real-time polymerase chain reaction. Our study demonstrated that EGCG administration was significantly associated with a decreased risk of vitiligo. EGCG could be a new preventive agent against vitiligo in the clinical setting.     

Structural characterization and antioxidant activity of a low-molecular polysaccharide from Dendrobium huoshanense

The present study aimed at investigating the structural features and antioxidant activities of a polysaccharide fraction (DHP1A) obtained from Dendrobium huoshanense, a precious herb medicine in China. DHP1A mainly consisted of mannose (Man), glucose (Glc) and a trace of galactose (Gal), with a molecular weight of 6700 Da. Its backbone contained (1 → 4)-linked α-D-Glcp, (1 → 6)-linked α-D-Glcp and (1 → 4)-linked β-D-Manp, with a branch of terminal β-D-Galp. The in vitro antioxidant evaluation revealed that DHP1A had a remarkable inhibition effect on the FeCl₂-induced lipid peroxidation. Furthermore, DHP1A pretreatment decreased the production of malondialdehyde (MDA), and restored the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), as well as the level of glutathione (GSH) in the livers of CCl₄-treated mice. These results suggested that DHP1A was a potential antioxidant component in D. huoshanense.     

Inactivation of jack bean urease by scutellarin: Elucidation of inhibitory efficacy,kinetics and mechanism

In the present study, the inactivation effect of scutellarin (SL) on jack bean urease was investigated to elucidate the inhibitory potency, kinetics and mechanism of inhibition. It was revealed that SL acted as a concentration- and time-dependent inactivator of urease characteristic of slow-binding inhibition with an IC₅₀ of 1.35 ± 0.15 mM. The rapid formation of the initial SL–urease complex with an inhibition constant of K_i = 5.37 × 10^− 2 mM was followed by a slow isomerization into the final complex with the overall inhibition constant of K_i* = 3.49 × 10^− 3 mM. High effectiveness of thiol protectors, such as L-cysteine (L-cys), 2-mercaptoethanol (2-ME) and dithiothreitol (DTT) significantly slowed down the rate of inactivation, indicating the strategic role of the active site sulfhydryl group in the blocking process. While the insignificant protection by boric acid and fluoride from the inactivation further confirmed that the active site cysteine should be obligatory for urease inhibition, which was also rationalized by the molecular docking study. The inhibition of SL on urease proved to be reversible since SL-blocked urease could be reactivated by DTT application and multidilution. The results obtained indicated that urease inactivation resulted from the reaction between SL and the sulfhydryl group.     

Wood density of trees in open savannas of the Brazilian Amazon

Studies on basic density of woody species in Amazonian savannas are needed to convert data on woody volume to biomass. These ecosystems, which have large carbon stocks, emit greenhouse gases annually due to frequent burnings. Basic density (g cm⁻³: oven-dry weight/wet volume), measured from complete sample disks (bark, sapwood and heartwood), was calculated for the most abundant woody species in three types of open savannas (Sg: grassy-woody savanna; Sp: savanna parkland; Tp: steppe-like parkland) in Roraima, a state in the northern part of Brazil’s Amazon region. The species selected represent 90–95% of the woody biomass estimated in these ecosystem types. Seven additional species were lumped in an “others” group. In total, we sampled 107 trees: 40 in Sg, 37 in Sp and 30 in Tp. Bowdichia virgilioides (0.516 ± 0.021 (S.E.) g cm⁻³) was the species with the highest basic density, followed by the “others” group (0.485 ± 0.057 g cm⁻³), Curatella americana (0.413 ± 0.028 g cm⁻³), Byrsonima crassifolia + B. coccolobifolia (0.394 ± 0.019 g cm⁻³), Himatanthus articulatus (0.375 ± 0.020 g cm⁻³) and B. verbascifolia (0.332 ± 0.020 g cm⁻³). Basic density of the species with the greatest woody biomass in Roraima’s open savannas (C. americana and B. crassifolia + B. coccolobifolia) did not differ significantly at the 5% level (ANOVA) among the three ecosystem types studied. Wood basic density in these savannas (weighted mean = 0.404 ± 0.025 g cm⁻³) is lower than that in Amazonian forests (weighted mean = 0.680 g cm⁻³). These results reduce uncertainty in calculations of carbon stocks and of greenhouse gas emissions from clearing and burning tropical savanna.     

Effects of the extracts and an active compound curcumenone isolated from Curcuma zedoaria rhizomes on alcohol-induced drunkenness in mice

The Curcuma zedoaria rhizome has been used traditionally to treat gastrointestinal diseases as an aromatic stomachic drug, and this is currently used to treat alcohol-induced loss of appetite and nausea in Japan. We examined the effects of various fractions and isolated compounds on alcohol-induced drunkenness and blood alcohol concentrations in mice. The 30% ethanol-extract (1000 mg/kg) of C. zedoaria rhizome prevented drunkenness 60 and 120 min after 40% alcohol administration. The n-hexane-soluble fraction (300 mg/kg) and an isolated compound (3, 10 or 30 mg/kg) prevented drunkenness at 30, 60 or 120 min. The extract, n-hexane-soluble fraction and isolated compound reduced the elevation in blood alcohol concentrations 30 and 60 min after 40% alcohol administration. The isolated compound (10 and 30 mg/kg) enhanced liver ADH activity 30 and 60 min after 40% alcohol administration. The compound was identified as curcumenone by a direct comparison of ¹H- and ¹³C-NMR spectral data. In conclusion, the protective effect of the C. zedoaria extract on drunkenness might be due to an active substance, curcumenone, and decreases in the elevation of blood alcohol concentrations through increased liver alcohol dehydrogenase activity.     

Comparison of in vitro antiviral activity of tea polyphenols against influenza A and B viruses and structure–activity relationship analysis

Influenza poses a particular risk of severe outcomes in the elderly, the very young and those with underlying diseases. Tea polyphenols are the natural phenolic compounds in teas, and principally consist of catechins, proanthocyanidins, flavonols, and theaflavins, which antiviral activities have been reported recently. This study is to gain a further insight into potential of various tea polyphenols for inhibiting influenza virus infection. Five tea polyphenols exhibited inhibitory activity against influenza A virus in the trend of theaflavin > procyanidin B-2 > procyanidin B-2 digallate > (−)-epigallocatechin(EGC) > (−)-epigallocatechingallate(EGCG) with IC₅₀ values in the range of 16.2–56.5 μg/ml. Six of the tested compounds showed anti-influenza B virus activity in the order of kaempferol > EGCG > procyanidin B-2 > (−)-EGC ~ methylated EGC > theaflavin with IC₅₀ values in the range of 9.0–49.7 μg/ml. Based on these results, the structure–activity relationship (SAR) was explained as follows. First, the dimeric molecules, such as theaflavin and procyanidin B-2, generally displayed more potent antiviral activity against both influenza A and B viruses than the catechin monomers. Second, the kaempferol for inhibition of influenza B virus indicated that the more planar flavonol structure with only one C-4′ phenolic hydroxyl group in the B ring is necessary for the anti-influenza B virus activity. A similar SAR can be drawn from the assays of another enveloped RNA virus, such as respiratory syncytial virus. These results are expected to provide guides for rational design of antiviral drugs based on polyphenols.     

A potent acetylcholinesterase inhibitor from Pancratium illyricum L.

Plants belonging to the Amaryllidaceae contain an exclusive group of alkaloids, known as sources of important biological activities. In the present work, Pancratium illyricum L., a species belonging to this family and endemic of Sardinia (Italy), was investigated for its alkaloid content. Fresh bulbs and leaves were processed separately. Standard extraction and purification procedures were applied to obtain fractions and compounds for GC–MS and NMR analysis. In addition to eight already known alkaloids (1–8), 11α-hydroxy-O-methylleucotamine (9) was isolated for the first time and its structure completely determined by one and two-dimensional ¹H and ¹³C NMR spectroscopy. This new galanthamine-type compound exhibited a pronounced in vitro acetylcholinesterase (AChE) inhibitory activity (IC₅₀ = 3.5 ± 1.1 μM) in comparison to the reference standard galanthamine hydrobromide (IC₅₀ = 1.5 ± 0.2 μM).     

In vitro permeability analysis,pharmacokinetic and brain distribution study in mice of imperatorin,isoimperatorin and cnidilin in Radix Angelicae Dahuricae

Coumarins are important constituents of Radix Angelicae Dahuricae, a well-known traditional Chinese medicine possess several known bioactivities with potentials in the treatment of central nervous system diseases. By using an HPLC–MS/MS method, we analyzed the in vivo plasma and brain pharmacokinetics of three ingredients of coumarins, including imperatorin, isoimperatorin and cnidilin in mice after oral administration of Dahuricae extract at doses of 800 mg/kg. The biosamples were prepared using acetonitrile precipitation and the separation was achieved on an XDB-C18 column by gradient elution. The BBB permeability and P-gp-mediated efflux were further examined in Madin Canine kidney cells transfected with full length cDNA for human multidrug resistance gene1 (MDCKII-MDR1). Our results demonstrate that the method has excellent and satisfactory selectivity, sensitivity, linearity, precision, and accuracy for simultaneous determination of imperatorin, isoimperatorin and cnidilin. The pharmacokinetics parameters were determined by using noncompartmental analyses, including the AUC_(0 − t) in plasma (1695.22, 1326.45 and 636.98 mg*h/L), the AUC_(0 − t) in brain (1812.35, 2125.17 and 1145.83 ng*h/g) as well as the T_1/2 in plasma (0.66, 0.82, 0.97 h) and brain (0.96, 1.1, 0.99 h) for imperatorin, isoimperatorin and cnidilin, respectively, suggesting that the three coumarins could easily pass through the BBB in vivo. In the in vitro model we observed high permeability of imperatorin and isoimperatorin with the P-gp-mediated efflux ratios of 0.53 and 0.06, as well as medium permeability of cnidilin with 0.82. All data suggest that these three coumarins have high BBB permeability and have pharmacokinetic potentials for the treatment of central nervous system diseases.     

Japonicasins A and B,two new isoprenylated flavanones from Sophora japonica

Two new flavanones with a C₁₅ isoprenoid group, japonicasins A and B (1 and 2), were isolated from the leaves of Sophora japonica. This is the first report on the presence of the (2E,7E)-6-isopropyl-3,9-dimethyldeca-2,7,9-trien-1-yl group (C₁₅ isoprenoid group) in isoprenylated flavonoids. Their structures were determined by spectroscopic methods, including UV, IR, 1D and 2D NMR, HRESIMS, and CD experiments. In addition, the antioxidant activities of compounds 1 and 2 were determined through DPPH radical scavenging assays. They exhibited potential antioxidant activities, with IC₅₀ values of 35.1 ± 0.8 μM and 88.7 ± 1.1 μM for compounds 1 and 2, respectively.     

New cytotoxic triterpenoid saponins from the whole plant of Clematis lasiandra Maxim

Four new oleanane type triterpenoid saponins (1–4) and three known saponins (5–7) were isolated from the whole plant of Clematis lasiandra Maxim. The structures of the four new compounds were elucidated as 3-O-β-d-ribopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)-[β-d-glucopyranosyl-(1 → 4)]-β-d-xylopyranosyl hederagenin (1), 3-O-β-d-ribopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)-β-d-xylopyranosyl oleanolic acid 28-O-β-d-glucopyranosyl ester (2), 3-O-β-d-ribopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)-β-d-xylopyranosyl hederagenin (3) and 3-O-β-d-ribopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)-[β-d-glucopyranosyl-(1 → 4)]-α-l-arabinopyranosyl hederagenin (4) on the basis of extensive spectroscopic analysis and chemical evidence. Compounds 1–7 were evaluated for their cytotoxicity against human tumor cell lines HL-60, Hep-G2 and SGC-7901, and all of the evaluated saponins showed significant cytotoxicity to those three tumor cell lines with IC₅₀ in the range from 1.40 to 19.50 μmol/L except for compounds 2 and 6.     

Pharmacokinetic and metabolism studies of rohitukine in rats by high performance liquid-chromatography with tandem mass spectrometry

A sensitive, selective, and rapid high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the quantification of rohitukine in rat plasma. HPLC was performed using a Symmetry-Shield C₁₈ (5 μ, 4.6 × 150 mm) column, and isocratic elution with ammonium acetate buffer (pH 4; 10 mM):methanol (08:92, v/v) at a flow rate of 0.6 mL/min. Sample clean-up involved solid phase extraction (SPE) of analyte and internal standard (phenacetin) from 100 μL plasma. The parent → product ion transitions (MRM) for analyte and IS were 306.1 → 245.1 m/z and 180.1 → 138.1 m/z respectively, and were monitored on a triple quadrupole mass spectrometer, operating in positive ion mode. The method was validated across the dynamic concentration range of 5–500 ng/mL for rohitukine, with a fast run time of 4.5 min. The analytical method measured concentrations of rohitukine with accuracy (% bias) of <± 10% and precision (% RSD) of <± 12%. Rohitukine was stable during the battery of stability studies viz., bench-top, auto-sampler, freeze/thaw cycles and 30 days of storage in a freezer at − 70 ± 10 °C. Finally, the applicability of this assay has been successfully demonstrated in vivo pharmacokinetic and in vitro metabolism studies in Sprague–Dawley rat. This method will therefore be highly useful for future preclinical and clinical pharmacokinetic studies of rohitukine.