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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.

Two new biflavonoids, daphnogirins A (1) and B (2), were obtained from the roots of Daphne giraldii. Their structures were established on the basis of the spectral data and X-ray diffraction data of the co-crystal of 1 and 2. Daphnogrins A and B have the same configuration at C-1 and opposite configurations at C-16 and C-17. Oxygen radical scavenging assay has indicated that they are of significant antioxidative activity.

Genus Daphne belongs to the Thymelaeaceae family and consists of 70 species. Its various species exist in Europe, Philippine Islands, temperate and subtropical Asia, North Africa, Australia and Pacific. In Pakistan, Daphne is represented by three species. Our focused Daphne oleoides is widely found in diverse climatic conditions from northern cold to central hot regions which creates a rich diversity and novelty in biosynthetic levels of its chemical constituents and hence is a great opportunity. Daphne oeloides is a proven rich source of a variety of unique and interesting nature-made skeletons with a wide range of therapeutic properties. D. oleoides possesses effective therapeutic properties, therefore, has been used in herbal medicines and is still being used to treat various diseases. The modern research by various groups, including ourselves, has resulted in the isolation of a number of natural molecules including some novel tris- and bis- coumarins, daphnane diterpenoids and lignoids. Therefore, due to novelty and richness of the nature-made molecules, and their therapeutic potential combined with our significant work on D. oleoides, this report covers chemical constituents isolated from D. oleoides. The pharmacological activities of the isolated compounds and use of this species in folk medicine have also been reviewed.

Genkwadaphnin (GD), an extract from the flower buds of Daphne genkwa Siebold & Zucc. (Thymelaeaceae) has been reported a significant anti-leukemic activity. However, its functional mechanism has not been defined well. To study the biological mechanism of GD function, we have investigated whether GD affects CD44 expression, which has a role in the regulation of immune cell motilities, and identified the related signaling pathways. GD treatment induced the increase of CD44 expression in a time- and concentration-dependent manner, which was specific for immune cells. GD activated PKD1/NF-κB signaling to induce CD44 expression, and resulted in the increased migration of K562 cells. In invasion assay, cell migratory ability was induced by GD and the transfection with CD44-specific short hairpin RNA resulted in reduction of its cell migration. GD treated human peripheral blood mononuclear cell (PBMC) were also shown the increased CD44 expression and migration. These data suggest that the induction of CD44 expression by GD treatment promotes immune cell transmigration resulting in the enhanced innate immunity.

The allegation of "Glycyrrhiza antagonistic to Sargassum, Euphorbia Pekinensis, Kansui, and Genkwa", being one of the hypotheses of "18 antagonisms" in TCM pharmacology, is referring to the antagonistic action among the Radix et Rhizoma Glycyrrhiza and Radix Euphorbiae Kansui, Radix Euphorbiae Pekinensis, Flos Genkwa, and Sargassum when compounded together in a single recipe. By reviewing its history concerted with modern knowledge, it can be found that the theory of "seven emotions" was originated from Shennong's Classic of Materia Medica; while the Variorum of the Classic of Materia Medica firstly and definitely records that Radix et Rhizoma Glycyrrhizae is forbidden to be used with Radix Kansui, Flos Genkwa, Radix Euphorbiae Pekinensis, Sargassum together in a single formula. It was summarized into a Chinese poetic sentence as above-mentioned later. In the works of later ages, including Chinese Pharmacopoeia, A Great Dictionary of Chinese Materia Medica, and China's Herbology, etc., all enhance the understanding of the prohibited combination of Radix et Rhizoma Glycyrrhizae and its incompatible herbs. Nevertheless, there are discrepancies between the results of modern experimental and clinical studies on this problem, which, needless to say, should be resolved by further investigations.

Daphne genkwa Sieb.et Zucc. is a well-known medicinal plant. This study was designed to investigate the anticancer effects of total flavonoids in D. genkwa (TFDG) in vitro and in vivo. HT-29 and SW-480 human colorectal cancer cells were cultured to investigate the anticancer activity of TFDG. In addition, the Apc(Min/+) mouse model was applied in the in vivo experiment. Results of the cell experiment revealed that TFDG possessed significant inhibitory effects on HT-29 and SW-480 human colorectal cancer cells (both p 0.01). Furthermore, our in vivo data showed that after treatment with TFDG, there was a significant increase in life span (both p 0.01) and tumor numbers were reduced in the colon (both p 0.01), which was supported by the data of tumor distribution, body weight changes and organ index. Our results also indicated that expressions of interleukin (IL)-1α, IL-1β, IL-6, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in gut tissue were downregulated by treatments of TFDG, and immunity cytokine secretions in the serum were regulated after oral administration of TFDG. Taken together, these findings suggested that TFDG has a potential clinical utility in colorectal cancer therapeutics, and TFDG's action is likely linked to its ability to regulate immune function and inhibit the production of inflammatory cytokines.

Daphne gnidium L. (Thymeleacees) is a famous Moroccan plant with cancer-related ethnobotanical use. Previously, we demonstrated that ethyl acetate extract of D. gnidium had antiproliferative and pro-apoptotic potential on human breast tumor MCF-7 cells. The purpose of this study was to investigate if the antiproliferative effect of this extract was similar for different human cancer cell lines such as A549 lung cancer and SMMC-7721 hepatoma cells. Moreover, this work essentially focused on the intrinsic apoptotic signaling pathway. Antiproliferative activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide on A549 and SMMC-7721 cells. The characterization of the mechanisms involved in this effect was determined by lactate dehydrogenase test, apoptosis assays and western blot analyses. Our present study has shown that this extract strongly inhibited proliferation of A549 (IC50: 213 ± 15 μg/ml) and SMMC-7721 (IC50: 170 ± 13 μLg/ml) cells. The characterization of antiproliferative effect demonstrated that this extract was an apoptosis inducer in both cell lines tested. The results of western blot analyses have shown in SMMC-7721 cells that this extract activated caspase signaling triggered by the modulation of Bcl-2 family proteins. These findings suggest that this natural extract-induced effects may have novel therapeutic applications for the treatment of different cancer types.

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.

Genkwa Flos (GF) is a well-known traditional medicine that is used to treat tumors and to relieve inflammation-related symptoms. GF tends to be taken in repeated doses for a long period of time, and although many reports on the toxicity of raw GF have led to a processing method to remove the toxicity, little information is currently available with regards to the toxic effects of subchronic exposure to processed GF (PGF). The aim of this study was to assess the possible genotoxicity and subchronic toxicity of PGF extract in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. A 13-week repeat-dose oral toxicity study was carried out with rats, and the change in body weight observed in rats receiving PGF extract was normal. It is worth noting that the PGF extract groups exhibited an obvious increase in liver weight along with a significant increase in serum alkaline phosphatase activity at doses of 667 and 2000mg/kg, providing evidence of hepatotoxic potential. More importantly, the results of the Ames test indicated that the PGF extract presented a mutagenic potential. Altogether, these results are the first to determine the subchronic toxicity and genotoxicity of the PGF extract, indicating that when GF is used for medicinal purposes, the period of use should be considered despite the manner in which the extract is processed.

« Violence et douceur de la poésie », titre de la conférence de Michael Edwards donnée à la Sorbonne le 28 mars 2013, pourrait à lui seul résumer cet article tant les poèmes de Sylvia Plath inspirés du mythe de Daphné dans Les Métamorphoses d’Ovide évoquent ces deux extrêmes. Son ton acerbe et ironique semble condamner avec intransigeance la virginité. Pourtant, la virginité semble également protéger la femme à travers sa représentation symbolique sous la forme de l’arbre. En captant l’instant que lui offre cette muse poétique, Plath parvient à dompter Daphné, l’éternelle insoumise. Tantôt d’une extrême violence, tantôt doux-amers, ces vers apportent de nouvelles perspectives à un mythe maintes fois conté. “Sweet violence” This is how Sir Philip Sidney described tragedy in The Defence of Poesy (1583). Actually, these two words perfectly fit the content of my article. Both an ironic criticism of virginity and a metaphor of protection for Sylvia Plath, the myth of Daphne from Ovid’s Metamorphoses has been written and rewritten as if trying to capture the uncatchable Muse. Plath’s poem tells her contemporary vision of the myth, at once violent and sweet, beautiful and ugly, biased and genuine.

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.

Two new phenolic constituents, daphnenone (1) and daphneone (2), were isolated from the stem bark of Daphne odora Thunb. var. marginata. Their structures were established on the basis of spectroscopic analysis. Compounds 1 and 2 were tested for cytotoxic activity by MTT assays on five human tumour cell lines, K562, A549, MCF-7, LOVO and HepG2. Compound 1 showed obvious cytotoxic activity against all the five cell lines.

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.

Raw aqueous extracts and acid-hydrolysed extracts from leaves and stems of Daphne gnidium L. were investigated by HPLC-PDA under two different separation conditions. This approach provides improved characterisation of natural compounds, especially those with similar structures, thanks to better resolution of critical pairs which can be difficult to separate by modification of gradient parameters only. In the leaf extracts, the presence of luteolin-3',7-di-O-glucoside was found according to the results obtained for a standard sample. This compound is reported for the first time in this species. The presence of some other, previously reported flavonoids is confirmed. The stem extract is rich in coumarins and seems not to contain any significant amounts of flavonoids. Further investigation of hydrolysed extracts is a useful additional step in the indirect identification of flavonoid and coumarin glycosides.

Colony PCR was developed for detection of pythiaceous species recovered on selective agar plates without DNA extraction. A minute amount of mycelia from a single colony was picked up with a pipette tip and added directly to the PCR mix as template for DNA amplification. Successful amplification was achieved in over 95% of the colonies recovered from plant tissues, irrigation water and soil with species-specific primers or oomycete ITS-1 primers. PCR was inhibited in the case of colonies emerging from unwashed pine bark potting mix plates. Direct colony PCR with ITS-1 primers combined with single-strand conformation polymorphism analysis (SSCP) was used to determine population levels of single and multiple species in plant and environmental samples. Application of this technique for disease diagnosis and monitoring pathogen sources was explored, and the potential for studying diversity and population dynamics of other cultivated microbial communities in the environment is discussed.

En este artículo se destaca la figura de Dafne por ser la primera protagonista de un drama musical cantado, antecedente de lo que hoy se denomina ópera. Se ponen de relieve los orígenes de esta última como producto de las discusiones sobre el futuro de la música en el seno de la Camerata Florentina, fundada por Jacopo Corsi alrededor de 1580 y se comentan los aportes teóricos de Vincenzo Galilei que dio forma escrita a las inquietudes vigentes sobre el devenir musical. Los miembros de la Camerata fundaban su concepción de la música en el modelo de los antiguos griegos y tendían hacia el ideal de una música capaz de expresar cabalmente el sentimiento de la palabra y de influir sobre el ánimo del oyente, capacidad que atribuían al arte musical de la antigüedad clásica, según los escritos de poetas y filósofos. Dicha concepción marca un punto de ruptura con el modelo polifónico vigente en el momento, que privilegiaba el sonido por sobre el significado del texto. Se analiza el 'afortunado malentendido' del cual provino la ambición de componer una pieza teatral con parlamentos cantados: la idea de que la tragedia griega era cantada en toda su extensión, noción que con el tiempo se reveló como inexacta. A continuación se presentan libreto y breve descripción de la música de la ópera primigenia La Dafne y otras posteriores del período barroco, para culminar con la creación que Richard Strauss dedica al personaje en el primer cuarto del s. XX.This article deals with the origins of Opera, which started as a musical drama based on the legend of Daphne, and the subsequent musical versions of the text written originally by Ottavio Rinuccini, composed by Peri, Caccini and other musicians of the XVIIth Century. A brief discussion follows about the ideals and aspirations of the Camerata Fiorentina, a society of artists who met at the Palazzo Bardi in Florence at the end of the XVIth century and who are considered the intellectual fathers of Opera. The above mentioned musicians were members of this group, one of whose main leaders was Vincenzo Galilei who resumed his and the musical thinking of the Camerata in his Dialogo della Musica Antica e della Moderna. This theoretical work marks the break point between polyphonic and hetherophonic music, the former more appropriate for the church, the latter more capable of expressing human feelings. The belief, shared by the members of the Camerata, that Greek tragedy was sang in all its extension was later proved as untrue but it gave born to the opera as a side product. Daphne was also chosen as opera subject by Richard Strauss who composed a beautiful piece of music for a text written by Josef Georg, in 1934.

The effects of daphnetoxin on isolated rat liver mitochondria and freshly isolated rat hepatocytes were investigated. Daphnetoxin (in the microM range) increased mitochondrial state 4 respiration and decreased both state 3 and FCCP-uncoupled respiration. The transmembrane potential was strongly depressed by daphnetoxin in a concentration-dependent manner. The protonophoric activity of daphnetoxin was evidenced by the induction of mitochondrial swelling in hyposmotic K+ acetate medium in the presence of valinomycin. In isolated hepatocytes, daphnetoxin decreases intracellular ATP and simultaneously increases ADP and AMP concentrations. The addition of uncoupling concentrations of daphnetoxin to Ca2+-loaded mitochondria treated with Ruthenium Red results in non-specific membrane permeabilization, as evidenced by mitochondrial swelling in isosmotic sucrose medium. Mitochondrial swelling in the presence of Ca2+ was prevented by cyclosporine A and was drastically inhibited by catalase and dithiothreitol, indicating the participation of mitochondrial generated reactive oxygen species in this process. From this study we can conclude that the bioenergetic lesion promoted by daphnetoxin seems to be sufficient to explain the lethal hapatocyte injury.

Cytotoxicity evaluation of gnidilatimonoein, the most active isolated diterpene ester from Daphne mucronata [Sadeghi H, Mianabadi M, Yazdanparast R, (2002) Journal of Tropical. Medicinal Plant1 3: 169-173], revealed the strong antiproliferative activity among several different human cancer cell lines (K562, CCRF-CEM, HL-60 and MOLT-4 leukemia cell lines, LNCaP-FGC-10 a prostate cancer cell line) and a mouse BALB/C fibrosarcoma cell line (WEHI-164). Using flow cytometry technique, it was found that treatment of the most responsive cells (K562) with gnidilatimonoein inhibited the progression of cells through G1 phase by almost 15% compared to the untreated cells. The population of the treated cells in the S and G2 phases also reduced by 8.3% and 5.4%, respectively. Based on the extent of [3H]-thymidine and [3H]-uridine incorporation into DNA and RNA, respectively, the major metabolic effects of gnidilatimonoein were found to be mainly on DNA and to a less extent on RNA synthesis. Additionally, the activity of inosine-5'-monophosphate dehydrogenase (IMPDH), under the effects of genidilatimonoein, was reduced in the treated cells by 44%. These data strongly suggest that the purine biosynthetic pathway is significantly affected by gnidilatimonoein.

Alteration of the cell surface glycoproteins of cancerous cells correlate with malignancy potential. To evaluate the mediation of membrane glycoproteins in wehi-164 cancerous cells, under the effect of D. mucronata crude extract and one of its purified active components, gnidilatimonoein, their attachment to fibronectin-coated wells were investigated. The plant extract, 27 μl/ml (equivalent to 0.54 mg/of plant leaves powder per ml of culture medium), as well as gnidilatimonoein (0.94 μM), were capable of quenching by 58% and 64%, respectively, the attachment of wehi-164 cells to fibronectin-coated wells (4 μg/ml). In addition to alteration of cell adhesive properties, the morphology of the treated cells were significantly changed upon treatment with the non-toxic dose of the plant extract or gnidilatimonoein. While the untreated cells have polygonal shapes, the treated cells had spherical shapes.

This study determined the 3'-terminal nucleotide sequences of two Korean isolates of Daphne virus S (DVS), a tentative member of the genus Carlavirus, causing leaf distortion and chlorotic spot disease symptoms in daphne plants. The 3'-terminal 1,465 nucleotide sequences of the two isolates contained two open reading frames coding for proteins of 36 kDa viral coat protein (CP) and 12 kDa from the 5'-3' end, which is a typical genome structure of the 3'-terminal region of carlaviruses. Both DVS isolates were 98.1% and 93.6% amino acid identical in the CP and 12 kDa, respectively. The CP gene of DVS shares 25.2-55.2% and 42.9-56.1% similarities with that of 19 other carlaviruses at the amino acid and nucleotide levels, respectively. The 3'-proximal 12 kDa gene of DVS shares 20.2-57.8% amino acid identities with that of 18 other members of the genus. The 3' noncoding region of DVS consists of 73 nucleotides with long excluding poly A tract, and shares 69.1-77.1% identities to the known carlaviruses. In the phylogenetic analyses of the two proteins, DVS was closely related to Helenium virus S and Chrysanthemum virus B. This is the first sequence information for the DVS, and further confirms the classification of DVS as a distinct member of the genus Carlavirus.

A simple sensitive LC-MS/MS method has been developed for the simultaneous determination of giraldoid A and giraldoid B in rat plasma. The method was applied to pharmacokinetics studies of the two compounds from Daphne giraldii Nitsche. Chromatographic separation was accomplished on an Acquity UPLC™ BEH C18 column (100 × 2.1 mm, 1.7 mm) by gradient elution with a flow rate of 0.2 mL min-1. The method was linear over the concentration range of 1.0-1000 ng mL-1 , and the lower limits of quantification were 1.04 ± 0.10 and 1.04 ± 0.09 ng mL-1 , respectively. The intra- and inter-day precisions (RSD) were

The phytochemical analysis of the ethanolic extract obtained from D. oleoides collected from Sardinia Island allowed the isolation of several new constituents for the species (3, 8 and 9) together with two new coumarinyl ethers (1 and 2) besides the chemotaxonomic markers of the Daphne genus (4 - 7 and 10) which are also known to possess interesting biological activities. The structure of the new compounds were elucidated by extensive spectroscopic and spectrometric analyses. The identification of these compounds gives an experimental evidence of the variability in the secondary metabolites pattern owned by populations growing in restricted area in respect to populations not confined by geographical barrier. This article is protected by copyright. All rights reserved.

The genus Daphne L. comprises 100 plant species. This name is derived from the name of a nymph, one of the daughters of the rivergod Pineios, who was transformed into a laurel tree to escape the amorous Apollo. Two species, D. mezereum L. and D. cneorum L., grow in the wild in Poland. D. mezereum is more common, but it is rarely found in the whole country. D. cneorum grows in the Małopolska Upland and in the Lublin region. These taxa are fully protected in Poland. Various plant species of the genus Daphne are considered to be ornamental, medicinal, poisonous and bee plants. In the bark of D. mezereum and in leaf buds of D. odora Thunb., there is a high content of daphnin and more than 20% of coumarins. Plants of the genus Daphne are poisonous and contain harmful substances, among others a glycoside daphnin and a resinous substance mezerein. The nectaries in flowers of the family Thymelaeaceae are classified as annular or intrastaminal. The aim of this study was to analyse the location and structure of the floral nectaries as well as nectar production in flowers of D. mezereum. D. mezereum belongs to the earliest flowering (I-V) melliferous plants. Densely packed flowers are borne in clusters of 2-3 in the axils of already fallen leaves. A pink corolla with fused petals has a diameter of 1-1.5 cm. Eight stamens are attached to the corolla tube. In Daphne flowers, the nectary surrounds a superior ovary borne on a gynophore. In D. merezeum, this gland forms a ring around the base of the ovary. In Daphne flowers, the stomata secrete nectar onto the nectary surface. The parenchyma cells of the nectary (longitudinal section) consist of 4-7 layers. The vascular tissue supplying the nectary reaches the subnectariferous parenchyma, while the branches of phloem elements reach the base of the nectariferous parenchyma. Flowers of D. mezereum produce nectar in abundance. The colourful corolla filled with nectar attracts bees and butterflies.

Rhizoma Atractylodes macrocephala, Radix Isatidis, Coptis chinensis and Flos Genkwa are common herbal remedies used by pregnant woman in China. In this study, their potential embryotoxicity was assessed using the embryonic stem cell test (EST) and a prediction model. The potential embryotoxicity of the herbs was based on three endpoints: the concentrations of the compounds that inhibited the proliferation of 50% of embryonic stem cells (ESCs) (IC50ES), the concentrations that inhibited 50% of 3T3 cells (IC503T3), and the concentrations that inhibited the differentiation of 50% of ESCs (ID50ES). The results revealed that Rhizoma Atractylodes macrocephala and Radix Isatidis are non-embryotoxic compounds. Coptis chinensis extracts appeared to demonstrated weak embryotoxicity, and Flos Genkwa exhibited strong embryotoxicity. These results may be useful in guiding the clinical use of these herbs and in expanding the application of the EST to the field of traditional Chinese medicine.

Yuanhuacine (YHL-14), the major component of daphnane diterpene ester isolated from the flower buds of Daphne genkwa, has been reported to have activity against cell proliferation in various cancer cell lines. Nevertheless, the potential mechanism has not been explored yet. Here we demonstrate that YHL-14 inhibits bladder and colon cancer cell growth through up-regulation of p21 expression in an Sp1-dependent manner. We found that YHL-14 treatment resulted in up-regulation of p21 expression and a significant G2/M phase arrest in T24T and HCT116 cells without affecting p53 protein expression and activation. Further studies indicate that p21 induction by YHL-14 occurs at the transcriptional level via up-regulation of Sp1 protein expression. Moreover, our results show that p38 is essential for YHL-14-mediated Sp1 protein stabilization, G2/M growth arrest induction, and anchorage-independent growth inhibition of cancer cells. Taken together, our studies demonstrate a novel mechanism of YHL-14 against cancer cell growth in bladder and colon cancer cell lines, which provides valuable information for the design and synthesis of other new conformation-constrained derivatives on the basis of the structure of YHL-14 for cancer therapy.

International audience; The antiproliferative potential of extracts of Daphne gnidium L. (Thymelaeaceae) on K562 cells was assessed, and the capacity of these extracts to disturb the cell cycle of K562 cells and to inhibit human P-glycoprotein was evaluated. The antiproliferative activity was evaluated using the MTT assay. The cell cycle analysis and the inhibition of P-glycoprotein were tested by flow cytometry. All the tested extracts exhibited significant anti-proliferative effects. Ethyl acetate extract has the strongest cytotoxic effect with an IC50 of 18.5 μg/ml. Furthermore, cell cycle analysis revealed that cells treated with chloroform, butanol and aqueous extracts were arrested predominantly in G2-M phase. Butanol extract was the most active extract. Percentage of cells arrested in G2-M was 34 %, 36.67 % and 42.63 % respectively, after treatment with 25, 75 and 100 μg/ml of the extract, versus 19 % in the cells treated with the vehicle solvent. In addition, chloroform extract had the ability to inhibit human P-glycoprotein-mediated daunorubicin in K562/R7 leukaemic cells in a dose-dependent manner compared to the positive control, cyclosporin A. These findings demonstrate that extracts from D. gnidium leaves have antileukaemic activity by perturbing the cell cycle of K562 and inhibiting human P-glycoprotein in K562/R7 cell line.

Genkwa Flos (GF), the dried flower bud from Daphne genkwa Sieb. et Zucc. (Thymelaeaceae), is a well-known and widely used traditional Chinese medicine. However, we know little about the in vivo mechanism of GF toxicity. Nematode Caenorhabditis elegans has been considered as a useful toxicity assay system by offering a system best suited for asking the in vivo questions. In the present study, we employed the prolonged exposure assay system of C. elegans to perform the full in vivo toxicity assessment of raw-processed GF. Our data show that GF exposure could induce the toxicity on lifespan, development, reproduction, and locomotion behavior. GF exposure not only decreased body length but also induced the formation of abnormal vulva. The decrease in brood size in GF exposed nematodes appeared mainly at day-1 during the development of adult nematodes. The decrease of locomotion behavior in GF exposed nematodes might be due to the damage on development of D-type GABAergic motor neurons. Moreover, we observed the induction of intestinal reactive oxygen species (ROS) production and alteration of expression patterns of genes required for development of apical domain, microvilli, and apical junction of intestine in GF exposed nematodes, implying the possible dysfunction of the primary targeted organ. In addition, GF exposure induced increase in defecation cycle length and deficits in development of AVL and DVB neurons controlling the defecation behavior. Therefore, our study implies the usefulness of C. elegans assay system for toxicity assessment from a certain Chinese medicine or plant extract. The observed toxicity of GF might be the combinational effects of oxidative stress, dysfunction of intestine, and altered defecation behavior in nematodes.

Ethnobotanical surveys revealed that Daphne oleoides Schreber has been used against rheumatic pain and for wound healing in Turkish folk medicine. The aim of the present study is to verify the folkloric assertion of D. oleoides ssp. oleoides (DOO) by bioassay-guided fractionation procedures leading to determination of the active component(s) and to elucidate the activity mechanisms of the isolated compounds. The wound healing activity of the methanol extract, its subextracts, fractions and isolates was evaluated by using two different in vivo wound healing experimental techniques. Anti-inflammatory and antioxidant activities of the test materials were also evaluated. For the determination of the activity mechanisms, the isolated compounds were screened for hyaluronidase, collagenase and elastase enzyme inhibitory activities. The methanolic extract of DOO was found to possess potent wound healing activity. This extract was then subjected to successive solvent extractions with n-hexane, dichloromethane, ethyl acetate (EtOAc) and n-butanol. EtOAc subextract yielded three compounds, quercetin 3-O-glucoside, triumbellin and rutarensin by using chromatographic separation techniques. The experimental study revealed that D. oleoides subsp. oleoides methanolic extract possesses significant wound healing effect and quercetin 3-O-glucoside was determined as the active component responsible from the activity. © 2014 ACG Publications. All rights reserved.

Daphnetin, a natural coumarin derivative, is known to display anti-inflammatory properties and has been used to treat inflammatory diseases. A novel finding suggested that daphnetin might have a neuroprotective effect in stressed mice, leading us to explore its role in the microglial inflammatory response, as well as its underlying mechanism of action. We found that the production of pro-inflammatory mediators, including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), induced by lipopolysaccharide (LPS) or β-amyloid (Aβ) was significantly suppressed by daphnetin in a dose-dependent manner in BV2 microglia. Also, daphnetin inhibited LPS-induced nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and NO formation by microglia. Mechanistically, daphnetin blunted the transcriptional activity of nuclear factor-kappa B (NF-κB), which was associated with the down-regulation of the phosphorylation and nuclear translocation of RelA/p65. Inhibitors of kappa B (IκB) phosphorylation and degradation were also affected by daphnetin, which was likely due to the reduced activation of IκB kinase (IKK). Additionally, LPS-induced activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK, were, to a varying extent, altered by daphnetin. Finally, daphnetin blocked phosphatidylinositol-3 kinase (PI-3K)/protein kinase B (Akt) signaling in LPS-activated microglia, which appeared to at least partially account for the reduction in NF-κB transcriptional activity. Thus, daphnetin inhibited microglial activation and proinflammatory responses by modulating a series of intracellular signaling pathways, including IKK/IκB, MAPKs and PI-3K/Akt.

Bioassay-directed fractionation led to the isolation of two new biflavonoids and 13 known biflavonoids from a sample of the dried stem barks of Daphne feddei. The structures of compounds 1 and 2 were elucidated as 2''-methoxy-daphnodorin C (1) and 2''-methoxy-2-epi-daphnodorin C (2) on the basis of detailed spectroscopic analysis and X-ray crystallography. All 15 biflavonoids were tested for inhibitory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages. Compounds 1, 2, 8, 10, 14 and 15 showed varying degrees of inhibitory activities against the production of NO in tested concentration of 25, 50, 75 and 100 microg/ml.

Two new bis-coumarin glycosides were isolated from the stem barks of Daphne giraldii NITSCHE. Their structures were elucidated as 6-O-alpha-L-rhamnnopyranosyl daphnogirin (1), and 6-O-beta-D-apiofuranosyl daphnogirin (2), on the basis of detailed spectroscopic analysis. Compounds 1 and 2 were tested inhibitory effects against production of nitric oxide (NO) in RAW264.7 cells, and cytotoxicity of human tumor cell lines A549, LOVO, QGY-7703, 6T-CEM. The results showed that compounds 1 and 2 neither reduced production of NO, nor inhibited human tumor cell lines.

Phytochemical investigations on the chloroform and ethyl acetate soluble fractions of the roots of the Daphne oleoids led to the isolation of the coumarin glycosides 1-6. Compound 5 with IC50 values 22.05 and 26.30 microM repectively, was found to be the most active of these compounds when screened against Bacillus pasteurii and jack bean urease enzymes in a concentration-dependent fashion.

Aims Obesity develops when energy intake chronically exceeds total energy expenditure. We sought to assess whether the flavonoid-rich fraction of crude extracts from Daphne genkwa Siebold et Zuccarini (GFF) might inhibit adipogenesis of 3T3-L1 cells. Main methods Cell viability of 3T3-L1 preadipocytes was assessed by MTT assays, and lipid accumulation was measured by Oil Red O. Adipogenesis related factors were checked by Western blot analysis. Flow cytometry was used to analyze the mitotic cell cycle during the mitotic clonal expansion phase. Key findings Among five flavonoids isolated from GFF, only apigenin potently inhibited the differentiation of 3T3-L1 cells. Apigenin reduced CCAAT/enhancer binding protein (C/EBP) α and peroxisome proliferator-activated receptor γ levels. Apigenin-treated 3T3-L1 cells failed to undergo clonal expansion during the early phase of adipocyte differentiation. Apigenin arrested cell cycle progression at the G 0 /G 1 phase. This effect was associated with a marked decrease in cyclin D1 and cyclin-dependent kinase 4 expression, with the concomitant and sustained expression of p27 Kip1 . In addition, apigenin inhibited the DNA-binding activity of C/EBPβ in differentiating 3T3-L1 cells by down-regulating the 35 kDa isoform of C/EBPβ (liver-enriched activating protein) and up-regulating the expression of two different sets of C/EBP inhibitors: C/EBP homologous protein and the phospho-liver-enriched inhibitory protein isoform of C/EBPβ. Significance These findings suggest that apigenin can prevent 3T3-L1 preadipocyte differentiation by the inhibition of the mitotic clonal expansion and the adipogenesis related factors and upregulation of the expression of multiple C/EBPβ inhibitors.

Ethnopharmacological relevance Glycyrrhizae uralenis (GU) is often prescribed together with Cortex daphnes (CD) in traditional Chinese medicinal practice to increase the efficacy of CD on the treatment of rheumatoid arthritis (RA), but the reasons were still unknown. In order to clarify the rationality of herbaceous compatibility between CD and GU, the comparative evaluations on pharmacokinetic behaviors of daphnetin (a predominantly active ingredient in CD) after intragastric administration of CD and CD–GU (combination of CD and GU) extract were studied. In addition, the effects of glycyrrhizin and liquiritin, active ingredients of Glycyrrhiza triterpenes and Glycyrrhiza flavones respectively, on the pharmacokinetics of daphnetin were also investigated. Materials and methods Five groups of rats were orally administered with CD extract, CD–GU extract, pure daphnetin, co-administration of daphnetin and glycyrrhizin as well as co-administration of daphnetin and liquiritin at the same single dose of daphnetin (20 mg/kg). The rat plasma concentrations of daphnetin were determined by our developed UPLC–MS/MS method. The pharmacokinetics of daphnetin in above groups were investigated and compared. Results Comparing with oral administration of CD extract, AUC and Tmax of daphnetin significantly increased after giving CD–GU (p

In sexually polymorphic plant species the extent of gender divergence in floral morphology and phenology may be influenced by gender-specific selection patterns imposed by pollinators, which may change geographically. Distribution margins are areas where changes in the pollinator fauna, and thus variation in gender divergence of floral traits, are expected. We tested for pollination-driven geographic variation in the gender divergence in floral and phenological traits in the gynodioecious shrub Daphne laureola, in core and marginal areas differing in the identity of the main pollinator. Pollinators selected for longer corolla tubes in hermaphrodite individuals only in core populations, which in turn recorded higher fruit set. Consistent with these phenotypic selection patterns, gender divergence in flower corolla length was higher in core populations. Moreover, pollinators selected towards delayed flowering on hermaphrodite individuals only in marginal populations, where the two sexes differed more in flowering time. Our results support that a shift in main pollinators is able to contribute to geographic variation in the gender divergence of sexually polymorphic plant species.

The bark of Daphne mezereum L. is known as toxic drug due to the presence of diterpene esters. The phytochemical analysis of the bark used for preparation of homeopathic mother tinctures showed that gniditrin was the main diterpene constituent, only in the fruits of D. mezereum mezerein could be detected. The complete NMR data of gniditrin are published for the first time.

The phytochemical investigation of the flower buds of Daphne genkwa yielded four highly oxygenated tigliane diterpene esters (1–4), including two new phorbol derivatives, 12-O-(2′E,4′E-decadienoyl)-7-oxo-5-ene-phorbol-13-acetate (1) and 12-O-neodecanoyl-7-oxo-5-ene-phorbol-13-acetate (2). The molecular structures of the isolated compounds were elucidated on the basis of extensive spectroscopic analysis, including 1D and 2D NMR.

To study the chemical constituents from Daphne acutiloba.The constituents were separated by column chromatography, and their structures were elucidated by spectroscopic data analyses.Fifteen compounds were isolated from the EtOAc extract and identified as wikstroelide M (1), vesiculosin (2), prostratin (3), 7-hydroxy-coumarin (4), 7,8-di-hydroxy-coumarin (5), isodaphnoside (6), daphnorine (7), rutamontine (8), daphnolin (9), daphneticin (10), (+)-pinoresinol-beta-D-glucoside (11), oleodapnone (12), oleodaphnal (13), ergosterol peroxide (14) and cholest-5-en-3beta-ol (15).All the compounds except for 4, 5 and 14 were obtained from the stems of this plant for the first time.

Aim To evaluate the anti-HIV activity and mechanism of action of wikstroelide M, a daphnane diterpene from Daphne acutiloba Rehder (Thymelaeaceae). Methods The anti-HIV activities of wikstroelide M against different HIV strains were evaluated by cytopathic effect assay and p24 quantification assay with ELISA. The inhibitory effect of wikstroelide M on HIV reverse transcription was analyzed by real-time PCR and ELISA. The effect of wikstroelide M on HIV-1 integrase nuclear translocation was observed with a cell-based imaging assay. The effect of wikstroelide M on LEDGF/p75-IN interaction was assayed by molecular docking. Results Wikstroelide M potently inhibited different HIV-1 strains, including HIV-1IIIB, HIV-1A17, and HIV-19495, induced a cytopathic effect, with EC50 values ranging from 3.81 to 15.65 ng·mL−1. Wikstroelide M also had high inhibitory activities against HIV-2ROD and HIV-2CBL-20-induced cytopathic effects with EC50 values of 18.88 and 31.90 ng·mL−1. The inhibitory activities of wikstroelide M on the three HIV-1 strains were further confirmed by p24 quantification assay, with EC50 values ranging from 15.16 to 35.57 ng·mL−1. Wikstroelide M also potently inhibited HIV-1IIIB induced cytolysis in MT-4 cells, with an EC50 value of 9.60 ng·mL−1. The mechanistic assay showed that wikstroelide M targeted HIV-1 reverse transcriptase and nuclear translocation of integrase through disrupting the interaction between integrase and LEDGF/p75. Conclusion Wikstroelide M may be a potent HIV-1 and HIV-2 inhibitor, the mechanisms of action may include inhibition of reverse trascriptase activity and inhibition of integrase nuclear translocation through disrupting the interaction between integrase and LEDGF/p75.