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Hua-Feng Yin,1,2 Chun-Ming Yin,3 Ting Ouyang,4 Shu-Ding Sun,1 Wei-Guo Chen,1 Xiao-Lin Yang,5 Xin He,6 Chun-Feng Zhang1 1School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 210009, Jiangsu, People’s Republic of China; 2Jiangxi QingFeng Pharmaceutical Co., Ltd, Ganzhou, 341000, Jiangxi, People’s Republic of China; 3Emergency Department, The First Affiliated Hospital of Gannan Medical University, Ganzhou, 341000, Jiangxi, People’s Republic of China; 4School of Chinese Materia Medical, Beijing University of Chinese Medicine, Beijing, People’s Republic of China; 5Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China; 6School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, People’s Republic of ChinaCorrespondence: Xin HeSchool of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, People’s Republic of ChinaTel +852-54445989Email xhe06@qub.ac.ukChun-Feng ZhangSchool of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 210009, People’s Republic of ChinaTel +86-25-86185129Email zhangchunfeng67@163.comPurpose: The aim of the present study was to develop an optimized Genkwanin (GKA)-loaded self-nanoemulsifying drug delivery system (SNEDDS) formulation to enhance the solubility, intestinal permeability, oral bioavailability and anti-colitis-associated colorectal cancer (CAC) activity of GKA.Methods: We designed a SNEDDS comprised oil phase, surfactants and co-surfactants for oral administration of GKA, the best of which were selected by investigating the saturation solubility, constructing pseudo-ternary phase diagrams, followed by optimizing thermodynamic stability, emulsification efficacy, self-nanoemulsification time, droplet size, transmission electron microscopy (TEM), drug release and intestinal permeability. In addition, the physicochemical properties and pharmacokinetics of GKA-SNEDDS were characterized, and its anti-colitis-associated colorectal cancer (CAC) activity and potential mechanisms were evaluated in AOM/DSS-induced C57BL/6J mice model.Results: The optimized nanoemulsion formula (OF) consists of Maisine CC, Labrasol ALF and Transcutol HP in a weight ratio of 20:60:20 (w/w/w), in which ratio the OF shows multiple improvements, specifically small mean droplet size, excellent stability, fast release properties as well as enhanced solubility and permeability. Pharmacokinetic studies demonstrated that compared with GKA suspension, the relative bioavailability of GKA-SNEDDS was increased by 353.28%. Moreover, GKA-SNEDDS not only significantly prevents weight loss and improves disease activity index (DAI) but also reduces the histological scores of inflammatory cytokine levels as well as inhibiting the formation of colon tumors via inducing tumor cell apoptosis in the AOM/DSS-induced CAC mice model.Conclusion: Our results show that the developed GKA-SNEDDS exhibited enhanced oral bioavailability and excellent anti-CAC efficacy. In summary, GKA-SNEDDS, using lipid nanoparticles as the drug delivery carrier, can be applied as a potential drug delivery system for improving the clinical application of GKA.Keywords: insoluble herbal drug, nanoparticle-based drug delivery system, intestinal permeability, pharmacokinetics, anticancer efficacy

Objectives As a traditional Chinese medicine, lotus leaf was reported to have significant hepatoprotective effect. To explore the hepatoprotective mechanism of lotus leaf, a rapid and reliable UPLC-MS/MS method was conducted to simultaneously determine six specific endogenous substances including 5-oxoproline, phenylalanine, tryptophan, C18-phytosphingosine, lysophosphatidylcholine (16 : 0) and lysophosphatidylcholine (18 : 1). Methods With the help of HPLC–FT-ICR-MS, the chemical constituents of louts leaf extract were elucidated. By observing histopathological changes and determining hepatotoxicity-related biochemical indicators, rat model of liver injury was developed and the hepatoprotective effect of lotus leaf was verified. With the developed UPLC-MS/MS method, six endogenous metabolites related to hepatotoxicity were monitored to investigate the hepatoprotective mechanism of lotus leaf. Key findings In the qualitative analysis, a total of twenty compounds including ten flavonoids, nine alkaloids and one proanthocyanidin were identified. Based on the results of determining six endogenous metabolites related to hepatotoxicity, it was predicted that the hepatoprotective mechanism of lotus leaf might be related to glutathione metabolism, phenylalanine metabolism, tryptophan metabolism, sphingolipid metabolism and phospholipid metabolism. Conclusions This study could be a meaningful investigation to provide mechanistic insights into the hepatoprotective effect of lotus leaf and further lay a theoretical basis for the clinical application of lotus leaf.

Daphne altaica Pall. (D. altaica; Thymelaeaceae) has long been used in traditional Kazakh medicine for the treatment of cancer and respiratory diseases. Previous studies have demonstrated the in vitro anticancer effects of D. altaica extract and its constituents in certain cancer cell lines; however, the underlying molecular mechanisms are not completely understooD. The present study aimed to investigate the molecular mechanisms underlying the activity of an ethyl acetate extract of D. altaica (Da‑Ea) by assessing its effects on cell morphology, cell apoptosis, cell cycle progression and the expression levels of peroxisome proliferator‑activated receptor γ (PPARγ) in Eca‑109 cells. Cell morphology was observed under a phase contrast microscope. Cell apoptosis and cell cycle progression were assessed by flow cytometry following Annexin V/propidium iodide (PI) double staining and PI single staining, respectively. The mRNA and protein expression levels of PPARγ were determined by reverse transcription‑quantitative PCR and western blotting, respectively. Compared with the control group, the percentage of apoptotic cells, cell cycle arrest at S phase and apoptotic morphological cell characteristics were increased in Da‑Ea‑treated Eca‑109 cells. Furthermore, Da‑Ea treatment upregulated the mRNA and protein expression levels of PPARγ compared with the control cells. High‑performance liquid chromatography with diode‑array detection indicated that daphnetin‑7‑O‑β‑D‑glucoside, daphnetin, demethyldaphnoretin‑7‑O‑β‑D‑glucopyranoside and genkwanol A were the main constituents of Da‑Ea. Collectively, the results suggested that Da‑Ea displayed antiproliferative activities in Eca‑109 cells by inducing apoptosis and S phase cell cycle arrest, as well as upregulating PPARγ expression levels.

Ethnopharmacological relevance Daphnes Cortex (Daphne Giraldii Nitsche, DGN) is a popular traditional Chinese herbal medicine for traumatic injuries and rheumatoid arthritis (RA) in the Shaanxi and Gansu provinces of China. Due to skin irritation caused by raw DGN (RDGN), licorice-processed DGN products are usually used in clinical practice. However, the efficacy and mechanisms of action between DGN and its licorice-processed DGN products in treating RA have not been compared. Aims This study compared the efficacy and elucidated the mechanisms in vitro and in vivo between RDGN and its licorice-processed DGN products in treating RA. Materials and methods A collagen-induced RA rat model was established, and treated with different doses of RDGN and its licorice-processed DGN products for 4 weeks to explore the therapeutic effects. The anti-inflammatory effects were assessed in RAW 264.7 macrophages stimulated by lipopolysaccharide (LPS). Analyses of the differential quality markers (DQMs) between DGN and its licorice-processed DGN products using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, and non-targeted metabolomics analyses of rat synovial tissues were used to systematically explore correlations between DGN processing and its efficacy. Results Licorice-processed DGN products significantly ameliorated RA symptoms in CIA rats. Licorice-processed DGN products also regulated inflammatory cytokines, matrix metalloproteinases, and vascular endothelial growth factor in the serum and cell supernatants. Licorice-processed DGN products significantly inhibited Toll-like receptor 4/nuclear factor kappa B/NOD-like receptor family, pyrin domain containing 3 (TLR4/NF-κB/NLRP3) signaling in CIA rats and LPS-induced RAW264.7 cells. The DQMs between RDGN and its licorice-processed DGN products were identified, most of which were amino acids or energy-related metabolites present in licorice-processed DGN products. Correlations between DQMs with differential metabolites and differential metabolic pathways were established. Conclusions Licorice-processed DGN products displayed better anti-inflammatory effects via the TLR4/NF-κB/NLRP3 signaling pathway on CIA rats and LPS-induced RAW264.7 cells, and regulation of the metabolic profile in treating RA.

Chromatographic purification of the roots of Daphne genkwa led to 11 new guaiane-type sesquiterpenoids (1-11), named genkwanoids A-K, and six known analogues (12-17). A comprehensive set of spectroscopic methods including IR, UV, HRESIMS, and 1D/2D NMR were used to elucidate the structures and relative configurations of 1-11. The absolute configurations were determined by the optical rotation calculations and the modified Mosher's method. The possible biosynthetic pathways for 1-11 are proposed. Furthermore, the neuroprotective effects of 1-17 on H2O2-induced damage in human neuroblastoma SH-SY5Y cells were screened using an MTT assay. Compounds 9, 10, and 16 exhibited moderate neuroprotective effects, with survival rates of 79.34%, 79.94%, and 75.64% after treatment with 12.5 μM, values that were comparable to the positive control, Trolox (78.65%). Furthermore, annexin V-FITC/PI staining of cells treated with 9, 10, and 16 showed that the neuroprotective effects of these compounds may arise from inhibiting cell apoptosis.

Background Since long-term or high-dose use of COPD medication causes adverse effects in patients with COPD, more effective and safer ways to manage COPD symptoms are required. Daphne kiusiana Miquel is a medicinal plant, but its anti-COPD efficacy was little studied. Purpose We investigated the anti-COPD activity and molecular mechanism of action of active compounds isolated from D. kiusiana to find drug candidates for COPD. Methods We isolated seven compounds (1–7) in an ethyl acetate (EtOAc) fraction from D. kiusiana, and determined that seven compounds effectively control the inflammatory responsiveness in both PMA-stimulated lung epithelial cells (in vitro) and/or in COPD model mice using cigarette smoke- and lipopolysaccharides-exposed animals in vivo. Results We show that the ethyl acetate (EtOAc) fraction from D. kiusiana. suppresses inflammatory response in both PMA-stimulated human lung epithelial cells (in vitro) and COPD model mice (in vivo). The EtOAc fraction effectively suppresses various inflammatory responses, such as mucus secretion, ROS production, bronchial recruitment of inflammatory cells, and release of proinflammatory cytokines. Additionally, we isolated three compounds with anti-inflammatory efficacy from the EtOAc fraction, out of which daphnodorin C was the most effective. Finally, we demonstrated that daphnodorin C negatively regulates inflammatory gene expression by suppressing NF-κB and specific MAPK signaling pathways (JNK and p38) in vitro and in vivo. Conclusions These results suggest that daphnodorin C could be a promising therapeutic alternative for managing COPD symptoms.

Daphnegiravone D (DGD), a prenylated flavonoid from Daphne giraldii Nitsche, significantly inhibited cell growth of several cancer cell lines without cytotoxicity on human normal cells. Our previous study showed that DGD could induce apoptosis in hepatocellular carcinoma Hep3B and HepG2 cells, but the detailed mechanism was still unclear. The present study provides that DGD-induced oxidative and nitrosative stress contribute to apoptotic cell death in Hep3B and HepG2 cells. Furthermore, there is a positive loop between oxidative stress and p38 activation, similar result is observed between nitrosative stress and p38. N-Acetylcysteine (NAC), a reactive oxygen species scavenger, could relieve DGD-induced oxidative stress, but exerts little effect on nitrosative stress. In addition, carboxy-PTIO (PTIO, a well-known scavenger of reactive nitrogen species) down-regulates the induction of nitrosative stress without obvious effect on oxidative stress in DGD-treated cells. In conclusion, the induction of oxidative and nitrosative stress could enhance p38-mediated apoptosis in DGD-treated Hep3B and HepG2 cells. Moreover, we speculated that OS and NS could not ultimately affect each other in DGD-treated HCC cells. This study gives a new insight on the mechanism of DGD-induced apoptotic cell death via oxidative and nitrosative stress in HCC cells.

1. To investigate Genkwa Flos hepatotoxicity, a cell metabolomics strategy combined with serum pharmacology was performed on human HL-7702 liver cells in this study. 2. Firstly, cell viability and biochemical indicators were determined and the cell morphology was observed to confirm the cell injury and develop a cell hepatotoxicity model. Then, with the help of cell metabolomics based on UPLC-MS, the Genkwa Flos group samples were completely separated from the blank group samples in the score plots and seven upregulated as well as two down-regulated putative biomarkers in the loading plot were identified and confirmed. Besides, two signal molecules and four enzymes involved in biosynthesis pathway of lysophosphatidylcholine and the sphingosine kinase/sphingosine-1-phosphate pathway were determined to investigate the relationship between Genkwa Flos hepatotoxicity and these two classic pathways. Finally, the metabolic pathways related to specific biomarkers and two classic metabolic pathways were analyzed to explain the possible mechanism of Genkwa Flos hepatotoxicity. 3. Based on the results, lipid peroxidation and oxidative stress, phospholipase A2/lysophosphatidylcholine pathway, the disturbance of sphingosine-1-phosphate metabolic profile centered on sphingosine kinase/sphingosine-1-phosphate pathway and fatty acid metabolism might be critical participators in the progression of liver injury induced by Genkwa Flos.

Screening for new natural anti-neuroinflammatory compounds was performed with the traditional folk medicine Genkwa Flos, which potently inhibited nitric oxide (NO) production by LPS-activated microglial BV-2 cells. Two new lathyrane-type diterpenes, genkwalathins A (1) and B (2), and 14 known daphnane-type diterpenes (3–16) were isolated. The lathyrane-type diterpenes were isolated for the first time from the Thymelaeaceae family in this study. Compounds 1 and 2 moderately inhibited LPS-induced NO production in BV-2 cells without affecting cell viability, while six daphnane-type diterpenes (3, 4, 6, 7, 9 and 10) potently reduced NO production with IC50 values less than 1 μM, although they did display weak cytotoxicity. A structure–activity relationship study on the daphnane-type diterpenes indicated that the stereochemistry at C-19, the benzoate group at C-20, and the epoxide moiety could be important for their anti-neuroinflammatory effects.

Chemical investigation of the Daphne genkwa has led to the isolation of four sesquiterpenes (1a/1b, 2 and 3), including one pair of sesquiterpene enantiomers (1a/1b), 1a is a new compound (+)-4-hydroxy-10-epirotundone, and twelve lignans (4-15). Their structures were elucidated by spectroscopic analysis, and the absolute configurations of 1a/1b were determined by CD analysis. All compounds were examined for their inhibitory effects on the nitric oxide (NO) production induced by lipopolysaccharide (LPS) in BV-2 microglial cells, and compounds 7-10 exhibited pronounced inhibition on NO production with IC50 values in the range of 5.8-10.2 μM, being more active than the positive control, quercetin (IC50 = 17.0 μM).

The flower bud of Daphne genkwa (Genkwa Flos) is a commonly used herbal medicine in Asian countries. Luteolin and apigenin are two recognized active flavonoids in Genkwa Flos. The aim of this study was to investigate the intestinal absorption mechanisms of Genkwa Flos flavonoids using in situ single-pass intestinal perfusion rat model. Using HPLC, we determined its major effective flavonoids luteolin, apigenin, as well as, hydroxygenkwanin and genkwanin in biological samples. The intestinal absorption mechanisms of the total flavonoids in Genkwa Flos (TFG) were investigated using in situ single-pass intestinal perfusion rat model. Comparing the TFG absorption rate in different intestinal segments, data showed that the small intestine absorption was significantly higher than that of the colon ([Formula: see text]). Compared with duodenum and ileum, the jejunum was the best small intestinal site for TFG absorption. The high TFG concentration (61.48[Formula: see text][Formula: see text]g/ml) yielded the highest permeability ([Formula: see text]). Subsequently, three membrane protein inhibitors (verapamil, pantoprazole and probenecid) were used to explore the TFG absorption pathways. Data showed probenecid, a multidrug resistance protein (or MRP) inhibitor, effectively enhanced the TFG absorption ([Formula: see text]). Furthermore, by comparing commonly used natural absorption enhancers on TFG, it was observed that camphor was the most effective. In Situ single-pass intestinal perfusion experiment shows that TFG absorption is much higher in the small intestine than in the colon, and the TFG is absorbed mainly via an active transport pathway with MRP-mediated efflux mechanism. Camphor obviously enhanced the TFG absorption, and this could be an effective TFG formulation preparation method to increase clinical effectiveness after Genkwa Flos administration. Our study elucidated the TFG absorption mechanisms, and provided new information for its formulation preparation.

Methylisothiazolone (MIT) is a common biocide that is widely used in water-desalination reverse-osmosis processes. The transformation of MIT during water treatment processes is poorly understood. The kinetics and mechanisms involved in the degradation of MIT during ozonation were investigated in this study. Ozonation was found to be a useful way of degrading MIT in water, and the degradation rate constant was 0.11 (±0.1) × 10 3 L/(mol·s). The degradation rate constant did not change when the pH was increased from 3 to 9. The pre-exponential factor A and the activation energy E a for the ozonation process were 7.564 × 10 13 L/(mol·s) and 66.74 kJ/mol, respectively. The decrease in the MIT concentration and the amount of ozone consumed were measured, and the stoichiometric factor α for the ozone consumption to MIT removal ratio was found to be 1.8. Several ozonation products were detected using time-of-flight mass spectrometry. Almost 32% of the organic sulfur in the MIT was oxidized to release sulfate ions, which caused a decrease in pH. Sulfur atoms were oxidized to sulfone species and then hydrolyzed to give sulfate during ozonation. Addition reactions involving carbon–carbon double bonds and the oxidation of α-carbon atoms also occurred. MIT was found to be lethal to Daphnia magna Straus ( D. magna ) with a median lethal concentration of 18.2 μmol/L. Even though the primary ozonation products of MIT still showed some toxicity to D. magna , ozone could minimize the toxic effect after a long reaction time.

PURPOSE: Daphne mucronata Royle grown in Iran has shown anticancer activities against different cancer cell lines. Therefore, within this study, we investigate the phytochemical pattern of this plant. METHOD: Phytochemical investigation was done using standard column chromatography system: The structures were recognized by the interpretation of one and two-dimensional nuclear magnetic resonance (NMR) spectra and the help of High-Resolution Electrospray Ionization Mass spectroscopy (HR-ESIMS) and Infrared spectroscopy (IR) data. Stereochemistry was determined using 2D and 3D NOESY, and comparison of coupling constant values with literature. The absolute configuration was determined and confirmed using specific rotation and electronic circular dichroism experiments. Cytotoxicity was done against HeLa cells by standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Luciferase assay was used to check if the compounds can inhibit the activation of cancer-related signaling pathways. Molecular docking simulation was done for biological activity evaluation and to examine the interaction of the ligand with each of the proteins. RESULTS: A new sesquiterpenoid, 4,11(12)-guiadiene-1-ol-3-one (4), together with eight specialized metabolites, betulinic acid (1), coniferyl aldehyde (2), oleanolic acid (3), daphnetoxin (5), apigenin (7), syringin (8), and genkwanol A (9) were isolated and reported for the first time from the shoots of the plant. Compound 4 as an undescribed compound was submitted for cytotoxicity assay and showed moderate activity with the IC(50) value of 51.3 ± 4.2 μM against HeLa cancer cells. It showed selective inhibition of Interleukin-6 mediated signal transducer and activator of transcription 3 pathway (STAT-3/ IL-6), and Smad protein / transforming growth factor beta (TGF-β) transcription factors when screened through an array of cancer signaling pathways. Molecular docking confirmed biological tests and showed the interaction with STAT3 and Smad proteins. CONCLUSION: An undescribed sesquiterpenoid: 4,11(12)-guiadiene-1-ol-3-one in addition to eight known compounds were isolated. The new sesquiterpene was evaluated for the luciferase assay on 14 main cancer-related signaling pathways and showed selective inhibition of STAT3/IL6, and Smad/ TGF-β transcription factors. Molecular docking simulation showed more interactions with STAT3 than Smad, which confirms better interaction of compound 4 with STAT3 than Smad proteins. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s40199-020-00336-x) contains supplementary material, which is available to authorized users.

A phytochemical study on the roots of Daphne genkwa yielded seven new guaiane-type sesquiterpenoids (1–7), daphne A-G. Their structures were elucidated through comprehensive spectroscopic analyses. The absolute configurations were determined by comparison between experimental electronic circular dichroism (ECD) and calculated ECD spectra via time-dependent density functional theory (TDDFT) and the modified Mosher’s method. Furthermore, all isolates were evaluated for their neuroprotective activities against H2O2-induced injury in human neuroblastoma SH-SY5Y cells. Among them, compounds 1 (78.42%) and 4 (79.34%) exhibited potent neuroprotective effects against H2O2-induced neurotoxicity at 25 μM. Further Annexin V-FITC/propidium iodide (PI) doubling staining exhibited that the neuroprotective effects of compounds 1 and 4 appeared to be mediated via suppressing cell apoptosis. Flow cytometry assays also proved that compounds 1 and 4 could attenuate mitochondrial dysfunction in SH-SY5Y cells.

In this paper, the first phytochemical analysis of the ethanolic extract ofDaphne sericeaVahl flowering aerial parts collected in Italy and its biological activities were reported. Eleven compounds were identified i.e., α-linolenic acid (1), tri-linoleoyl-sn-glycerol (2), pheophorbideaethyl ester (3), pilloin (4), sinensetin (5), yuanhuanin (6), rutamontine (7), syringin (8),p-coumaric acid (9),p-anisic acid (10) and caffeic acid (11). To the best of our knowledge, compounds (1-4,7-8and10) were isolated fromD. sericeafor the first time during this work, whereas sinensetin (5) represents a newly identified component of the entire Thymelaeaceae family. The extract was found to possess radical scavenging against both DPPH•and 2,2′-azino-bis(3-thylbenzothiazoline-6-sulfonic acid (ABTS•+) radicals, with at least a 40-fold higher potency against the latter. Moreover, chelating abilities against both ferrous and ferric ions have been highlighted, thus suggesting a possible indirect antioxidant power of the extract. Although the precise bioactive compounds remain to be discovered, the polyphenolic constituents, including phenolic acids, tannins and flavonoids, seem to contribute to the antioxidant power of the phytocomplex. In addition, the extract produced cytotoxic effects in MDA-MB-231 and U87-MG cancer cell lines, especially at the concentration of 625 μg/mL and after 48–72 h. Further studies are required to clarify the contribution of the identified compounds in the bioactivities of the extract and to support possible future applications.

Ethnopharmacological relevance Daphne pseudomezereum var. koreana Hamaya is distributed in the Gangwon-do of South Korea and is traditionally used to treat chronic inflammatory diseases, including rheumatoid arthritis. Aim of the study: We investigated the anti-inflammatory effect of biflavonoid-rich fraction (BF) obtained from an extract of D. pseudomezereum leaves on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and mouse model of ovalbumin (OVA)-induced allergic asthma. Materials and methods Neochamaejasmin B (NB) and chamaejasmin D (CD) were spectroscopically characterized as major components of BF obtained from the leaves of D. pseudomezereum. RAW264.7 cells pretreated with NB, CD and BF and activated by LPS (500 ng/ml) were used to assess the anti-inflammatory effects of these materials in vitro. To evaluate the protective effect of BF on allergic asthma, female BALB/c mice were sensitized to OVA by intraperitoneal (i.p.) injection and treated with BF by oral administration (15 or 30 mg/kg). Results Pretreatment with BF inhibited LPS-stimulated nitric oxide (NO), TNF-α and IL-6, and led to upregulation of heme oxygenase-1 (HO-1) in RAW264.7 macrophages. Orally administered BF significantly inhibited the recruitment of eosinophils and the production of IL-5, IL-6, IL-13 and MCP-1 as judged by the analysis of BALF from OVA-induced asthma animal model. BF also decreased the levels of IgE in the serum of asthmatic mice. BF suppressed the influx of inflammatory cells into nearby airways and the hypersecretion of mucus by the airway epithelium of asthmatic mice. In addition, the increase in Penh in asthmatic mice was reduced by BF administration. Furthermore, BF led to Nrf2 activation and HO-1 induction in the lungs of mice. Conclusions These data have shown the anti-asthmatic effects of BF, and therefore we expect that BF may be a potential candidate as a natural drug/nutraceutical for the prevention and treatment of allergic asthma.

Twelve new flavan derivatives including four pairs of enantiomers, daphnegiralins A1-A4 (1) and daphnegiralins B1-B4 (2), and two pairs of epimers, daphnegiralins C1/C2 (3) and daphnegiralins D1/D2 (4), were isolated from the stem bark and roots of Daphne giraldii. Their structures were elucidated using spectroscopic analyses, computational approaches, and chemical methods. Separation of the enantiomeric mixtures (1a, 1b, 2a, and 2b) was achieved using chiral HPLC. The compounds were evaluated against a small panel of human cancer cell lines, and 1b-2, 2a, and 2b were cytotoxic against Hep3B human hepatoma cells.

Objective This study aims to select the most effective anti-Rheumatoid Arthritis (RA) component of flavonoids from Daphne genkwa Sieb. et Zucc. by anti-inflammatory and immunomodulatory effects in vitro, and to elucidate the mechanism. Methods The anti-inflammatory and immunomodulatory effects of total flavonoids (TF) and four flavonoid components (genkwanin, hydroxygenkwanin, luteolin and apigenin) were determined by pharmacological approach in LPS-induced RAW 264.7 macrophages and ConA-induced T lymphocytes. Principal component analysis (PCA) was used to obtain the optimal anti-RA component in vitro. Western blot and real-time quantitative PCR (q-PCR) were used to explore the mechanisms. Finally, the in vitro anti-RA effect was verified by human rheumatoid arthritis fibroblast-like synoviocytes (FLSs). Results TF and four flavonoids significantly reduced the expressions of NO, iNOS, TNF-α, IL-6, IFN-γ and IL-2. PCA showed that genkwanin was the most effective anti-RA component in vitro. Genkwanin inhibited nuclear factor-κB (NF-κB) pathway by decreasing the phosphorylation levels of IKK, IκB and NF-κB, and down-regulated the expressions of iNOS, COX-2 and IL-6 mRNA. Genkwanin also inhibited the abnormal proliferation of FLSs and down-regulated the secretions of NO and IL-6. Conclusion The most effective anti-RA component was genkwanin. Genkwanin exerts anti-RA effect through down-regulating the activation of NF-κB pathway and mRNA expressions of inflammatory mediators, and also by inhibiting the abnormal proliferation of FLSs and its NO and IL-6 secretion levels.

Daphne genkwa Sieb. et Zucc., as a traditional oriental herb, has been widely distributed and employed in China. The major bioactive components in D. genkwa are flavonoid compounds, which showed pharmacological activities such as anti-inflammatory, analgesic, anti-tumor and immunomodulatory activities. In this study, we analyzed total flavonoids in D. genkwa and their metabolites in normal and adjuvant arthritis (AA) rat plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). A total of 4 metabolites in plasma, 9 metabolites in urine and 15 metabolites in feces were characterized respectively by LC-Q-TOF-MS technology in normal rat. And 9 of the metabolites were observed in the AA rat urine, while there was no prototype drug or its metabolites detected in plasma and fecal samples. The metabolic pathway mainly involves hydroxylation, methylation, glucuronide, sulfate conjugation, oxidation and reduction, during the phase I and phase II biotransformation pathway. All the information gained here will be greatly helpful in elucidating the potential biological and pharmacological mechanism of flavonoid in D. genkwa, thus providing new ideas for drug development.

Daphnetin, one of the major bioactive components isolated from Daphne odora, has been reported to have anti-inflammatory and anti-oxidative effects. Inflammation and oxidative stress have been known to play critical roles in cisplatin-induced nephrotoxicity. The purpose of this study was to investigate the protective effects of daphnetin on cisplatin-induced nephrotoxicity. The levels of blood urea nitrogen (BUN) and creatinine, as well as the kidney reactive oxygen species (ROS), and malondialdehyde (MDA) activity were measured in this study. The expression of inflammatory cytokines TNF-α and IL-1β were measured by ELISA. The results showed that daphnetin protected against cisplatin-induced nephrotoxicity by attenuating kidney histological changes, serum BUN and creatinine. Furthermore, the expression of TNF-α and IL-1β, as well as ROS and MDA in kidney tissues were decreased by daphnetin. In addition, daphnetin dose-dependently inhibited cisplatin-induced NF-κB activation and up-regulated the expression of Nrf2 and HO-1. In conclusion, the results of this study suggested that daphnetin inhibited cisplatin-induced nephrotoxicity by inhibiting NF-κB and activating Nrf2 signaling pathways. Daphnetin might be a promising agent in the treatment of kidney injury.

Main conclusion The results provide a significant verification of the activity of exogenously applied phytohormones: gibberellic acid, jasmonic acid, and brassinolide in the modulation of the plant’s response to nickel treatment. Abstract The study investigated nickel accumulation and its toxicity to Daphne jasminea shoots cultured in vitro with or without exogenous supplementation with phytohormones: gibberellic acid (GA3), jasmonic acid (JA), and brassinolide (BL). The aim was to verify the modulatory effect of exogenous plant growth regulators (PGRs) on plant reaction to Ni excess. The combined action of Ni and PGRs was evaluated at the anatomical, ultrastructural, and biochemical levels. Nickel toxicity was manifested in decreased biomass accretion and growth tolerance index (83–53.6%), attributed to enhanced synthesis of growth inhibitors, mainly abscisic acid. As a defence reaction, endogenous gibberellins accumulated. Exogenous GA3 ameliorated the plant reaction to Ni stress, inducing proliferation and growth rate. Ni tolerance in the presence of GA3 was attributed to peroxisomal reactions that stimulated the synthesis of endogenous JA. In contrast, the application of BL caused enhanced Ni accumulation. Plants suffered from pronounced stress due to massive oxidation. The defence strategy of plants subjected to Ni and BL involved cell wall rearrangements. Exogenous JA stimulated the synthesis of active auxins and salicylic acid, contributing to enhanced mitotic activity within explants. However, JA disturbed the integrity of chloroplasts and lamellar compartments. Our study revealed that an action of exogenous PGRs may either enhance tolerance to Ni or increase metal toxicity in D. jasminea. Particularly in in vitro culture, where explants are subjected to external phytohormonal stimuli, the combined effects of supplemental PGRs may enhance stress and substantially affect plant development. Our results provide a significant verification of exogenous PGRs activity in the modulation of plant response to nickel. Electronic supplementary material The online version of this article (10.1007/s00425-018-2979-6) contains supplementary material, which is available to authorized users.

Hepatocellular carcinoma (HCC) is a primary malignant tumor with very poor prognosis. To search for more effective compounds for HCC treatment, two new pairs of flavan enantiomers, daphnegiranol C1/C2 (1a/1b) and daphnegiranol D1/D2 (2a/2b), were isolated from the stem bark and roots of Daphne giraldii by using chiral chromatography. MTT assay was applied to evaluate their cytotoxicity against three hepatocarcinoma cell lines (Hep3B, MHCC97H, HepG2) as well as a normal liver cell line L02. The results showed that these compounds preferred to inhibit the growth of Hep3B cells (p53 null). Among them, 2a/2b (the IC50 value was 4.87 and 3.35 μM, respectively) exhibited a stronger cytotoxic effect than sorafenib (IC50 = 6.59 μM) in Hep3B cells. A further study demonstrated that 2a/2b could induce apoptotic cell death with an increase in reactive oxygen species (ROS) in Hep3B cells. In addition, the IC50 values of 2a/2b in HepG2 and MHCC97H cells (both harbored p53 gene) were more than 10-folds greater when compared with Hep3B cells, indicating that 2a/2b selectively exhibited cytotoxicity in p53-null hepatocarcinoma cells. Moreover, inhibition of p53 increased the inhibitory effect in p53-wild hepatocarcinoma cells, as well as apoptotic cells and ROS generation. Taken together, flavan enantiomers 2a/2b selectively induced apoptosis in Hep3B cells because of p53 deficiency.

Methanol extract of Daphne gnidium leaves was assessed for its antigenotoxic and neuroprotective effects through antioxidant and antibutyrylcholinesterase activities. Antigenotoxic activity was evaluated against methyl methanesulfonate injected intraperitoneally to mice, using the comet assay. The protective effect of D. gnidium reached 99.12%, at the lowest tested dose (44 mg/kg b.w.) in kidney cells, and 92.16% at the dose of 88 mg/kg b.w. in blood cells. The extract was dissolved in water and administrated to mice by intraperitoneal injection. Antioxidant activity was tested against DPPH radicals. It reached a maximum of 74.52% with an IC50 value of 45 µg/ml. Anticholinesterase activity was determined against butyrylcholinesterase, an enzyme linked to Alzheimer disease. The extract exhibited antibutyrylcholinestrase effect with an inhibition percentage of 35.82% at the lowest tested dose (44 mg/kg b.w.).

A selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for investigating the pharmacokinetics of umbelliferone, apigenin, genkwanin and hydroxygenkwanin after oral administration of Daphne genkwa extract. Plasma samples were treated by protein precipitation with acetonitrile. Analytes were detected by triple-quadrupole MS/MS with an ESI source in negative selection reaction monitoring mode. The transitions of m/z 161 → 133 for umbelliferone, m/z 269 → 117 for apigenin, m/z 283 → 268 for genkwanin and m/z 299 → 284 for hydroxygenkwanin were confirmed for quantification. Chromatographic separation was conducted using an Eclipse XDB-C18 column, and the applied isocratic elution program allowed for simultaneous determination of the four analytes for a total run time of 2.5 min. The linearity was validated over the plasma concentration ranges of 1.421-1421 ng/mL for umbelliferone, 0.845-845 ng/mL for apigenin, 1.025-1025 ng/mL for genkwanin and 0.845-845 ng/mL for hydroxygenkwanin. The extraction recovery rate was >82.7% for each analyte. No apparent matrix effect was observed during the bioanalysis. After full validation, the proposed method was successfully applied to compare the pharmacokinetics of these analytes between normal and arthritic rats.

Daphne genkwa Sieb.et Zucc. is a well-known medicinal plant. This study was designed to apply the ultra-high performance liquid chromatography (UPLC) system to establish a quality control method of Daphne genkwa. Data revealed that there were fifteen common peaks in ten batches of D. genkwa Sieb. Et Zucc.(Thymelaeaceae) from different provinces of China. On this basis, the fingerprint chromatogram was established to provide references for quality control. Afterwards, the chemical constitutions of these common peaks were analyzed using the UPLC-Q-TOF-MS system and nine of them were identified primarily. In addition, LPS-stimulated RAW264.7 murine macrophages and DPPH assay were adopted to study the anti-inflammatory and anti-oxidation effects of Daphne Genkwa. Then the fingerprint-efficacy relationship between UPLC fingerprints and pharmacodynamic data were studied with canonical correlation analysis (CCA). Analysis results indicated that the anti-inflammatory and anti-oxidation effects were discrepant among the ten Daphne genkwa samples due to their inherent differences of chemical compositions. Taken together, this research established a fingerprint-efficacy relationship model of Daphne Genkwa plants by combining the UPLC analytic technique and pharmacological researches, which provided references for the detection of the principle components of traditional Chinese medicine on bioactivity.

Enantiomers account for quite a large percentage of compounds in natural products. Our team is interested in the separation and biological activity of racemic compounds. In this report, four pairs of prenylated flavan enantiomers [(±)-1-(±)-4], including five new compounds, were isolated from the stem and root bark of Daphne giraldii, and separated successfully by using chiral chromatographic column. Their planar structures and absolute configurations were established by comprehensive spectroscopic analyses as well as circular dichroism (CD) spectroscopy. The isolates had a selective cytotoxicity towards hepatic carcinoma cell lines. Among them, new compound (+)-4 showed a more potent inhibitory effect on Hep3B cells with an IC50 value of 30.3 μM, compared with its racemic mixture 4. Therefore, the action mechanism of (+)-4in vitro was subsequently investigated. The morphological observation and Western blot analysis demonstrated that (+)-4 could markedly induce apoptosis through intrinsic and extrinsic pathways, and also cause autophagy by increasing the phosphorylation of AMP-activated protein kinase (AMPK) in Hep3B cells. After treatment with the autophagic inhibitor bafilomycin A1 (Baf A1), (+)-4-induced apoptosis increased significantly, suggesting that the autophagy induced by (+)-4 performed a protective effect on apoptotic cell death.

The aim of the study was to investigate the anti-rheumatoid arthritic activity of four flavonoids from Daphne genkwa (FFD) in vivo and in vitro. Flavonoids of D. genkwa were extracted by refluxing with ethanol and purified by polyamide resin. An in vivo carrageenan-induced paw edema model, tampon-granuloma model and Freund's complete adjuvant (FCA)-induced arthritis mouse model were used to evaluate the anti-rheumatoid arthritic activities of FFD. Moreover, nitric oxide (NO) release and neutral red uptake (NRU) in lipopolysaccharide (LPS)-induced murine macrophage RAW264.7 cells were used to evaluate the anti-inflammatory effect in vitro. In addition, antioxidant effect of FFD was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. A high dose of FFD significantly reduced the degree of acute inflammatory paw edema in mice as a response to carrageenan administration (p

Ethnopharmacological relevance Genkwa flos ( Daphne genkwa Sieb. et Zucc.), a Chinese herbal medicine, has been traditionally used for over two thousand years in China for inflammation related symptoms, including joint pain. To evaluate the antioxidative effects of flavonoid aglycones (FA) isolated from Genkwa flos on adjuvant arthritis in rats and to identify the relationship between antioxidant potential and whole blood viscosity (WBV). Materials and methods FA compounds were identified using LC–MS and the content was assayed by HPLC. Arthritis was induced by an intradermal injection of Freund׳s complete adjuvant in the footpad. The effects of FA on paw volumes, secondary arthritis scores, histopathology of joints, and body and organ weights were measured. The antioxidant effects of FA and WBV were determined. Results LC–MS analysis showed that the FA contained four major compounds: luteolin, apigenin, hydroxygenkwanin and genkwanin. FA significantly decreased paw edema, arthritis scores, and weight loss. These observations were consistent with the reduction of oxidative stress and the improvement of the WBV. Conclusion FA significantly decreased arthritis in a rat model through antioxidant and hemorheological modulatory mechanisms. The Genkwa flos flavonoids may have clinical potential for the treatment of rheumatoid arthritis.

The bioassay-guided fractionation of Daphne retusa Hemsl. has led to the isolation of a new aryl tetrahydronaphthalene lignan derivative named as daphnretusic acid (1), along with six new source compounds such as 5,7-dihydroxyflavone (2), 7-hydroxyflavone (3), 6-methoxyflavone (4), (+) pinoresinol (5), (+) sesamin (6), and β-sitosterol-3-O-β-D-glucopyranoside (7). Their structures were elucidated by (1)H NMR, (13)C NMR, 1D, 2D NMR, UV, IR, and EIMS analyses. All the fractions (n-hexane, CHCl3, AcOEt, CH3OH, and water) and pure compounds (1-7) were subjected to the assay of urease and α-chymotrypsin inhibitory activities. Chloroform and methanol soluble fractions showed moderate urease inhibition. Compound 2 exhibited significant urease inhibition with IC50 value 60.4 ± 0.72 μM, whereas compounds 1 and 3-7 remained inactive during urease inhibition and α-chymotrypsin bioassays.

New dimeric and a trimeric coumarin glucosides namely Daphneretusin A (1) Daphneretusin B (2) along with three known oligomers (3-5) were obtained as a result of bioassay guided fractionation of Daphne retusa Hemsl. Fractions (n-hexane, CHCl3, AcOEt, CH3OH and water) exhibited potent radical scavenging activity in relevant non-physiological bioassays. The structures of isolated compounds were elucidated by UV, IR, EIMS, FAB-MS, 1D, and 2D NMR spectroscopic analysis.

Two new daphnane-type diterpene esters, daphneresiniferins A (1) and B (2), along with seven known diterpenes, yuanhuacine (3), yuanhuadine (4), yuanhuahine (5), genkwadaphnin (6), genkwanine A (7), genkwanine F (8), and genkwanine H (9), were isolated from the methanol extract of the flower buds of Daphne genkwa. Their structures were elucidated on the basis of spectroscopic methods, especially 2D NMR spectra such as HMQC, HMBC, and NOESY. All the isolates were evaluated for their inhibitory effects of the melanogenesis against α-melanocyte stimulating hormone (α-MSH)-activated B16 melanoma cells.

Four new daphnane-type diterpenes, genkwadanes A-D (1-4), together with 19 known ones, were isolated from ethanol extract of the flower buds of Daphne genkwa. Their structures were determined on the basis of extensive spectroscopic data. Among them, daphnane-type diterpene with a 1,10-double bond (1) was isolated from this plant for the first time. The cytotoxicity of all compounds 1-23 against the 10 selected human cancer cell lines was assayed. A number of compounds exhibited significant activities against the 10 cancer cell lines (IC50

To predict the mechanism of liver injury induced by Genkwa Flos, we investigated the effect of chloroform extract on UGTs and UGT1A1 activities of the liver microsomes in rat and human. In the present study, 4-nitrophenol(4-NP) and β-estradiol were elected as substrates to determine activities of UGTs and UGT1A1 by UV and HPLC. The results showed that there were 1.00% of apigenin, 6.40% of hydroxygenkwanin and 18.38% of genkwanin in chloroform extract; and total diterpene mass fraction was 31.40%. Compared with the control group, chloroform extract could significantly inhibit the activity of UGTs in rat liver microsomes(RLM) system, while the inhibitory effect was not obvious in human liver microsomes(HLM) system. UGT1A1 activity was inhibited by chloroform extract in rat liver microsomes and human liver microsomes (based on genkwanin, IC₅₀=8.76, 10.36 μmol•L⁻¹). The inhibition types were non-competitive inhibition(RLM) and uncompetitive inhibition(HLM). In conclusion, the results indicated that chloroform extract showed different inhibitory effects on UGTs and UGT1A1 activity, which may be one of the mechanisms of liver injury induced by Genkwa Flos.

Nurr1 is an orphan nuclear receptor that is essential for the differentiation and maintenance of dopaminergic neurons in the brain, and it is a therapeutic target for Parkinson’s disease (PD). During the screening for Nurr1 activators from natural sources using cell-based assay systems, a methanol extract of the combined stems and roots of Daphne genkwa was found to activate the transcriptional function of Nurr1 at a concentration of 3 μg/mL. The active components were isolated and identified as genkwanine N (1) and yuanhuacin (2). Both compounds 1 and 2 significantly enhanced the function of Nurr1 at 0.3 μM. Nurr1-specific siRNA abolished the activity of 1 and 2, strongly suggesting that transcriptional activation by 1 and 2 occurred through the modulation of Nurr1 function. Additionally, treatment with 1 and 2 inhibited 6-hydroxydopamine (6-OHDA)-induced neuronal cell death and lipopolysaccharide (LPS)-induced neuroinflammation. Moreover, in a 6-OHDA-lesioned rat model of PD, intraperitoneal administration of 2 (0.5 mg/kg/day) for 2 weeks significantly improved behavioral deficits and reduced tyrosine hydroxylase (TH)-positive dopaminergic neuron death induced by 6-OHDA injection and had a beneficial effect on the inflammatory response in the brain. Accordingly, compounds 1 and 2, the first reported Nurr1 activators of natural origin, are potential lead compounds for the treatment of PD.

Two new compounds, one flavanol dimer dapholosericol A (1) and one tigliane diterpene dapholosericin A (2), together with ten known ones, were isolated from the AcOEt extract of Daphne holosericea (Diels) Hamaya. Their structures were elucidated by their spectroscopic data analysis. Compounds 1 and 2 showed moderate activities against acetylcholinesterase with inhibition ratio of 36% and 29% at a concentration of 100 μmol/l, respectively.

In this study, we have investigated inhibitory capacity of ethyl acetate, total oligomer flavonoid (TOF), aqueous extracts and beta amyrin acetate, a triterpene isolated from ethyl acetate extract obtained from leaves of Daphne gnidium, on mouse melanoma (B16-F0 and B16-F10 cells) proliferation. Influence of these products on percentage cell distribution in cycle phases and melanogenesis was also studied. Cell viability was determined using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and flow cytometry was used to analyse effects of tested compounds on progression through the cell cycle. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475 nm. Ethyl acetate, TOF and aqueous extracts exhibited significant anti-proliferative activity after incubation with the two types of tumour skin cells B16-F0 and B16-F10. Furthermore, cell cycle analysis revealed that cells treated with ethyl acetate and TOF extracts were arrested predominantly in G2-M phase. Ethyl acetate extract has also the ability to enhance melanogenesis and tyrosinase activity of B16-F0 melanoma cells. Copyright © 2012 John Wiley & Sons, Ltd.

Seven previously unreported daphnane-type diterpene esters named acutilobins A-G, together with 12 known ones, were isolated from EtOAc extract of Daphne acutiloba Rehd. Their structures were elucidated based on the spectroscopic data. The cytotoxic and anti-HIV-1 activities of these daphnane-type diterpene esters were evaluated through bioassays. Fourteen of these isolates exhibited definite cytotoxic activities against the five human tumor cell lines HL-60, SMMC-7721, A-549, MCF-7, and SW480. Additionally, anti-HIV-1 activities were observed in.13 daphnane-type diterpene esters, among which acutilobins A-G exhibited significant anti-HIV-1 activities with EC50 below 1.5 nM and SI over 10,000. Particularly, genkwanineVIII showed the strongest activity with EC50 0.17 nM and SI 187,010. (C) 2011 Elsevier Ltd. All rights reserved.

Chemical examination of the methanolic extract from the stem bark of Daphne feddei led to the isolation of three new dicoumarin glucosides (1-3), and eight known coumarins, dicoumarins and dicoumarin glucosides. Their structures were elucidated by extensive analysis of spectral data and comparison with the literature values. All compounds were tested for inhibitory activity against lipopolysaccharide-induced NO production in RAW 264.7 macrophages, and compounds 4 and 5 showed potent inhibitory activity with IC₅₀ values of 0.161 and 0.127 μM, respectively.

In this paper, macroporous resin column chromatography and counter-current chromatography (CCC) were applied for large-scale preparative separation of three flavonoids from the flower of Daphne genkwa, a famous Chinese medicinal herb. Nine kinds of resins were investigated by adsorption and desorption tests and D101 macroporous resin was selected for the first cleaning-up, in which 40% aqueous ethanol was used to remove the undesired constituents and 90% aqueous ethanol was used to elute the targets. The crude extract after the first step was directly subjected to the preparative CCC purification using the solvent system composed of n-hexane-ethyl acetate-methanol-water (4:5:4:5, v/v). The compounds apigemin (823 mg), 3-hydroxyl-genkwanin (842 mg) and genkwanin (998 mg) with the purities of 98.79, 97.71 and 93.53%, respectively, determined by HPLC were produced from 3-g crude extract only in one CCC run. Their chemical structures were identified by MS, UV and the standards.

Daphnecin (1), a new coumarinolignan, has been isolated from the ethyl acetate-soluble fraction of Daphne mucronata along with three known compounds, aquillochin (2), umbelliferone (3), and coumarin (4). Their structures were determined by spectroscopic studies.

Two new daphnane-type diterpene esters, yuanhuahine (1) and yuanhualine (2), were isolated from the flowers of Daphne genkwa (Thymelaeaceae) along with three known diterpene esters, yuanhuacine (3), yuanhuadine (4), and yuanhuagine (5). Their structures were determined by a combination of 1D and 2D NMR experiments, including correlation spectroscopy (COSY), heteronuclear multiple quantum correlation (HMQC), heteronuclear multiple bond correlation (HMBC), and rotating frame Overhauser enhancement spectroscopy (ROESY) sequences, and mass spectrometry. All the isolated compounds were tested against A549 human lung cancer cells and MRC-5 human normal lung epithelial cells. Compounds 1-5 exhibited potent anti-proliferative effects against A549 lung cancer cells with IC(50) values of 12-53 nM, whereas these compounds were relatively non-cytotoxic against MRC-5 normal lung epithelial cells.

Chemical examination of the methanolic extract from the stem bark of Daphne feddei led to the isolation of five new phenylpropanoids, 4,4'-dihydroxy-3,3'-dimethoxy-9-butoxy-9,9'-epoxylignan (1), 4,4'-dihydroxy-3,3'-dimethoxy-9-ethoxy-9,9'-epoxylignan (2), daphneresinol (3), armaosigenin (4), and isocubebin (5), together with 33 known phenylpropanoids. All 38 compounds were isolated for the first time from D. feddei. All compounds were tested for inhibitory activity against LPS-induced NO production in RAW 264.7 macrophages. Compounds 2, 8, 9, 12, 13, and 15 showed potent inhibitory activities against the production of NO with IC(50) values of 0.091, 0.047, 0.005, 0.088, 0.004, and 0.074 μM/mL, respectively.

Daphne retusa Hemsl. belongs to the genus Daphne, a member of Thymelaeaceae family. The barks and stems of Daphne retusa are used as a folkloric medicine 'Zhu Shi Ma' in Western China because of its effects of detumescence and acesodyne. In this paper, we investigate the anti-inflammatory and analgesic effects of the 75% ethanol extract of the stems and barks of Daphne retusa and different fractions partitioned with petroleum ether, methylene chloride, ethyl acetate and n-butanol, respectively. The anti-inflammatory effects were evaluated using xylene-induced ear oedema in mice and carrageenan-induced paw oedema in rats, while the acetic acid-induced writhing test and hot-plate test as models for evaluating the centrally and peripherally analgesic activity. The results showed the plant has significant anti-inflammatory and analgesic effects (P

Gnidilatimonoein is a new diterpene ester, recently isolated from the leaves of Daphne macronata with potent anti-tumoral and anti-metastastic activities (Yazdanparast et al., 2004). Promyeloblastic (KG1), promyelocytic (NB4) and promonocytic (U937) cells were cultured in the presence of various concentrations of the drug (0.5-3.0 microM) for 3 days. Herein, we report that gnidilatimonoein induces differentiation and apoptosis in KG1, NB4 and U937 cells. The drug inhibited growth and proliferation of KG1, NB4 and U937 cells with IC50 values of 1.5, 1.5 and 1.0 microM, respectively, after 72 h of treatment. Cell viability was also decreased by 18%, 20% and 23%, respectively, after 72 h treatment with the drug. NBT reducing assay revealed that the inhibition of proliferation is associated with differentiation especially toward monocytes-like morphology. Indeed, the drug at 0.5-1.5 microM induced differentiation by 5-50% in the cells. Acridine orange/ethidium bromide (AO/EtBr) double staining and DNA fragmentation assays revealed that apoptosis occurred after differentiation of the cells. Based on the present data, it seems that the new compound is a good candidate for further evaluation as an effective chemotherapeutic agent acting through induction of differentiation and apoptosis.

Daphne genkwa contains a novel class of anticancer diterpene esters that inhibit DNA topoisomerase I. Fingerprint and quantitative analysis by HPLC were performed in order to characterise and evaluate D. genkwa. A standard fingerprint of Daphne diterpene esters from the root extract was first established by HPLC-UV, and the major peaks in the fingerprint profile were preliminarily determined using HPLC-MS. The principal Daphne diterpene esters, yuanhuacine (1), yuanhuadine (2), yuanhuajine (3) and yuanhuagine (4), were isolated and identified using a combination of UV, IR, MS, 1H-NMR and 13C-NMR spectral data. Quantitative analysis indicated that 1 was the principal component in the root, and that 2 was the major component in the buds. The average extraction rates of 1 and 2 were 0.0151 and 0.0033% (n = 10) from the root, respectively, and 0.0020 and 0.0078% (n = 3) from the buds, respectively. Copyright © 2006 John Wiley & Sons, Ltd.

The phytochemical investigation of the flower buds of Daphne genkwa yielded eight highly oxygenated daphnane-type diterpene esters, including a new one, genkwanine N 20-palmitate (1) and seven known ones (2-8). The molecular structures of the isolated compounds were elucidated on the basis of extensive spectroscopic analysis, including UV, IR, NMR, and MS, and comparison with literature data. Furthermore, compound 1 showed potential cytotoxic activity in vitro, against A549 and Hep G2 tumour cell lines.

Daphne genkwa Sieb. et Zucc. (Thymelaeaceae) is mainly used for the treatment of edema, asthma, and cancer in China and Korea for centuries. The major bioactive components in D. genkwa are daphnane-type diterpenoids, which showed pharmacological activities such as antileukemic, antifertility and skin irritants. In this study, the pharmacokinetics and metabolism profile of yuanhuapine, an effective and toxic diterpenoid, was investigated in rats. The plasma exposure of yuanhuapine was determined by ultra-performance liquid chromatography tandem triple-quadrupole mass spectrometry (UPLC–TQ–MS), and the pharmacokinetic parameters were calculated using the DAS 2.0 pharmacokinetic program. The metabolites were identified through ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC–Q–TOF/MS) and a Metabolynx™ (v4.1) program. After oral administration (5 mg/kg), yuanhuapine was slowly absorbed and reached a maximum concentration of 579.20 ± 212.85 ng/mL at 7.33 ± 1.03 h, it also eliminated slowly. As the cumulative excretion of yuanhuapine in urine and feces were only 0.7% and 3.3%, we supposed that biotransformation in vivo was of significant importance to this component. Not only the prototype but also twelve metabolites were found and tentatively identified in rat urine after oral administration of yuanhuapine. The metabolic pathway mainly involves hydroxylation, methylation, glucuronidation and cysteine conjugation during the phase I and phase II biotransformation pathway. All the information gained here was useful in understanding the pharmacological actions and toxic properties of yuanhuapine, and providing a meaningful basis for clinical application of such a bioactive compound of herbal medicines.

WOS: 000242242800008, Laurus nobilis L., commonly known as daphne tree, is an evergreen that belongs to the Lauraceae family. Daphne trees produce grape-sized shiny purplish berries having three parts: flesh, skin, and an inner kernel (single seed). This study examines supercritical CO2 (SC-CO2) extraction of oil from daphne seeds. The oil yield of ground seeds varied from 14 to 28% depending on the method and particle size used for oil recovery. Yields were similar for both petroleum ether and SC-CO2 extraction. The extraction yield decreased significantly with increasing particle size. The amount of extract collected increased exponentially with increasing SC-CO2 pressure. The highest extraction yield was obtained at the highest temperature studied, 75 degrees C. More than 45% of the oil was lauric acid. SC-CO2 is a viable technique to obtain high-purity L. nobilis L. seed oil, which is a potential ingredient for the cosmetic industry.

Two new daphne diterpene esters Yuanhuajine (2) and Yuanhuagine (4), together with three known daphne diterpene esters yuanhuacine (1), yuanhuadine (3), and yuanhuapine (5), were isolated and identified from Daphne genkwa, a traditional Chinese medicine. Their structures were elucidated by a combination of UV, IR, MS and NMR ((1)H NMR, (13)C NMR, HSQC, and HMBC) spectra. In order to explore the structure-activity relationship, three compounds 6, 7, and 8 were prepared as three derivatives of 1. Inhibitory activities against DNA topoisomerase I (topo I) were assessed for the compounds 1-8. These compounds, except for 8, exhibited potent inhibitory activities against DNA topo I at IC(50) levels of 11.1-53.4 microM and they are new type of topo I inhibitors bearing different structures compared with the known topo I inhibitors. The agarose-gel electrophoresis experiments showed that the orthoester group of daphne diterpene esters was necessary for the inhibitory activity against DNA topo I, and the inhibition against DNA topo I is probably one of the anti-tumor mechanisms of daphne diterpene esters.

As part of an ongoing study aimed at identifying the anti-complement active compound from the flower buds of Daphne genkwa, ten flavonoids, one biscoumarin and one amide were isolated. Their structures were identified from spectroscopic and physicochemical data as genkwanin 5-O-beta-d-primveroside (1), apigenin 7-O-beta-d-glucuronide (2), genkwanin 5-O-beta-d-glucopyranoside (3), apigenin 5-O-beta-d-glucopyranoside (4), genkwanin (5), apigenin (6), luteolin 7-methyl ether (7), luteolin (8), daphnoretin (9), velutin (10), 7-methoxyacacetin (11) and aurantiamide acetate (12). Among them, compounds 9, 10 and 12 were isolated from this plant for the first time. The anti-complement activity of these compounds was tested against the classical pathway of the complement system. Among the compounds, daphnoretin (9) exhibited significant anti-complement activity with an IC(50) value of 11.4 microm, whereas the other compounds were not active in the assay.