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To explore diversity in cold hardiness mechanisms, high resolution magnetic resonance imaging (MRI) was used to visualise freezing behaviours in wintering Daphne kamtschatica var. jezoensis flower buds, which have naked florets and no bud scales. MRI images showed that anthers remained stably supercooled to the range from -14 to -21°C or lower while most other tissues froze by -7°C. Freezing of some anthers detected in MRI images between -14 and -21°C corresponded with numerous low temperature exotherms and also with the 'all-or-nothing' type of anther injuries. In ovules/pistils, only embryo sacs remained supercooled at -7°C or lower, but slowly dehydrated during further cooling. Cryomicroscopic observation revealed ice formation in the cavities of calyx tubes and pistils but detected no ice in embryo sacs or in anthers. The distribution of ice nucleation activity in floral tissues corroborated the tissue freezing behaviours. Filaments likely work as the ice blocking barrier that prevents ice intrusion from extracellularly frozen calyx tubes to connecting unfrozen anthers. Unique freezing behaviours were demonstrated in Daphne flower buds: preferential freezing avoidance in male and female gametophytes and their surrounding tissues (by stable supercooling in anthers and by supercooling with slow dehydration in embryo sacs) while the remaining tissues tolerate extracellular freezing.

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.

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.

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.

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.

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.

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.

Twelve highly oxygenated novel daphnane-type diterpenoids genkwanines A-L (1-12), together with four known diterpenes (13-16), were isolated from the bud of Daphne genkwa, a well-known traditional Chinese medicine (TCM). The new structures were elucidated on the basis of spectral methods, especially 2D NMR spectra (HMQC, HMBC, NOESY). Inhibitory activity against endothelium cell HMEC proliferation and cytotoxic activities against two tumor cell lines were assessed for all the compounds 1-16, and found to be significant and structure related. A number of compounds showed very potent cytotoxic activities against two tumor cell lines at the IC50 levels of 0.15-8.40 microM, and most interestingly, five of the compounds 4, 8, 10, 13, and 14 exhibited strong activity to inhibit the endothelium cell HMEC at the IC50 levels of 2.90-15.0 microM.