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Daphne mezereum L., an important medicinal plant in Scandinavian folk medicine, was used to treat ailments such as diarrhea, swelling and stomach pain. A range of natural compounds have been isolated, but little attention has been given to the polysaccharides in this plant. Previous work in our group have shown that a polysaccharide enriched fraction from the bark of D. mezereum exhibited pro-inflammatory effects. To pursue this further, the aim of the present work was to isolate and characterize these polysaccharides. From the ethanol-precipitate of a water extract, one neutral (DMP-NF) and one acidic (DMP-AF) fraction was isolated by anion-exchange chromatography. GC, GC-MS and 1D- and 2D-NMR were used to characterize the polysaccharide structures. DMP-NF appeared to be a mixture of arabinan, arabinogalactan and hemicelluloses such as xyloglucan, mannan and xylan. DMP-AF contained a pectic polysaccharide mainly consisting of an unusually long homogalacturonan backbone. Enzymatic treatment by pectinase of DMP-AF yielded DMP-ED, which contained a rhamnogalacturonan-I backbone with arabinan, galactan and arabinogalactan side chains. Both DMP-NF and DMP-ED induced IFN-γ and TNF-α secretion in peripheral blood mononuclear cells (PBMCs), DMP-ED being the most potent fraction. DMP-AF was less active, which might be due to a less sterically available rhamnogalacturonan-I domain., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Six undescribed guaianolide sesquiterpenes (1-6) were obtained from the aerial parts of Daphne penicillata. Their structures and absolute configuration were elucidated by HRESIMS, NMR analyses, ECD calculations and single-crystal X-ray diffraction analysis. Structurally, all compounds possess the typical 5,7-fused system of 8,12-guaianolides and this guaianolide-type was first reported to be isolated from Daphne penicillata. All compounds (1-6) were evaluated for anti-inflammatory and cytotoxic activity. Among them, compounds 1 and 5 showed moderate inhibitory effects on LPS-induced NO production in BV2 cells and 4 displayed potential inhibition against Hep3B cells with an IC 50 value of 7.33 μM., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2023. Published by Elsevier B.V.)

Phytochemical investigation into the leaves and branches of Daphne genkwa afforded 25 meroterpenoids (1-16) including nine pairs of enantiomers (1a/1b-8a/8b and 12a/12b), among which 20 compounds have been reported in the present work for the first time. The structures with absolute configurations of the new molecules (excluding 10-13) were established via comprehensive spectroscopic analyses especially electronic circular dichroism (ECD) and Mosher's methods. A preliminary in vitro cell viability assay revealed remarkable cytotoxicities of selective compounds against A549 (lung), Hela (cervical), MDA-MB231 (breast) and MCF-7 (breast) cancer cells, and compound 8a showed the best inhibitory activity with IC 50 values in the range of 3.12-4.67 μM toward the four cell lines. Subsequent in vitro antitumor evaluation of 8a disclosed that it could inhibit the proliferation and metastasis, as well as induce significant apoptosis and cycle arrest, of A549 cells. Further mechanistic investigations revealed that 8a could exert its antitumor activity via inhibiting the PI3K/Akt/mTOR signaling pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Premise: Under uncertain pollinator visit conditions, plants often exhibit long flowering periods and generalized pollination systems. Flowering of the gynodioecious shrub Daphne jezoensis occurs in early spring in cool temperate forests. Pollination by nocturnal moths is expected, given the species' tubular-shaped flowers with sweet fragrance and nectar. However, the effectiveness of nocturnal moths under cool conditions is unknown. We evaluated the relative importance of diurnal and nocturnal visitors as pollinators in early spring., Methods: We investigated flowering duration, flower visitors, and floral scents in a natural population. We experimentally exposed flowers to visitors only during daytime or nighttime using bagging treatments and evaluated the contributions of diurnal and nocturnal insects to fruit set, pollen dispersal distance, and paternity diversity using 16 microsatellite markers., Results: Female flowers lasted ~3 wk, which was ~8 d longer than the flowering period of hermaphrodites. Various insects, including Coleoptera, Diptera, Hymenoptera, and Lepidoptera, visited the flowers during both daytime and nighttime. Flowers emitted volatiles, such as lilac aldehyde isomers and β-ocimene, which are known to attract moths. Fruit-set rate in the night-open treatment was similar to or higher than that in the day-open treatment. However, pollen dispersal distance in the night-open treatment was shorter than that in the day-open treatment. Paternity diversity was similar in day-open and night-open treatments., Conclusions: Early-blooming plants ensure pollen receipt and dispersal by having a long flowering period and using both diurnal and nocturnal flower visitors, suggesting the importance of a generalized pollination system under uncertain pollinator visit conditions., (© 2023 Botanical Society of America.)

This study evaluated several secondary metabolites, essential oils (EOs) compositions, and antioxidant activity in four medicinal plants that originated in Isfahan rangelands. The species were Astragalus verus, Astragalus adscendens, Daphne mucronata, and Phlomis olivieri. Thirty-two genotypes of these species were evaluated for different biochemical traits. Based on the evaluation of EOs compounds, GC/MS analysis revealed the total number of identified compounds. These compounds were 25, 22, 12, and 22 for A. adscendens, A. verus, D. mucronata, and P. olivieri, respectively. The dominant compounds were phthalate (59.88 %) in A. adscendens, phytol (38.02 %) in A. verus, hexanoic acid (32.05 %) in D. mucronata and β-cubebene (30.94 %) in P. olivieri. Phytochemical analysis showed that D. mucronata, A. adscendens, and P. olivieri had the highest total phenolics content (TPC) (18.24 mg gallic acid equivalent/g dry weight), total flavonoids content (5.57 mg QE/g DW), and total anthocyanins content (0.23 mg/g DW), respectively. The highest total chlorophyll (0.27 mg/g DW), total carotenoids (0.03 mg/g DW), and antioxidant activity (71.36 %) were observed in A. adscendens, A. adscendens and A. verus, respectively. Among all genotypes, the highest TPC (20.1 mg GAE/g DW) was observed in genotype 5 of D. mucronata. This study provided new information on the chemical compounds within the distribution range of these ecologically dominant rangeland species in Isfahan province, Iran. The data revealed that superior genotypes from these species are rich in natural antioxidants and bioactive compounds. Thus, they can be used in ethno pharmacological fields, food, and industrial applications., (© 2023 Wiley-VHCA AG, Zurich, Switzerland.)

Odd ploidy-level cytotypes in sexually reproducing species are considered a dead end due to absent or reduced fertility. If sterility is only partial, however, their contribution to the population gene pool can be augmented by longevity and clonal growth. To test this, we investigated the cytotype origin and spatial pattern, and pollen viability in three relict shrub species of the genus Daphne (Thymelaeaceae Juss.) in central Europe. Daphne cneorum subsp. cneorum is a widespread European species that has a broad ecological amplitude, whereas D . cneorum subsp. arbusculoides and D. arbuscula are narrow endemics of the western Pannonian Plain and the Western Carpathians, respectively. Our study confirmed that all three taxa are diploid. However, of more than a thousand analysed individuals of D. cneorum subsp . cneorum , five in four different populations were triploid. Our data indicate that these triploids most likely originate from recurrent autopolyploidization events caused by the fusion of reduced and unreduced gametes. High pollen viability was observed in all three taxa and in both diploid and triploid cytotypes, ranging from 65 to 100 %. Our study highlights the significant role of odd ploidy-level cytotypes in interploidy gene flow, calling for more research into their reproduction, genetic variability, and overall fitness. Interestingly, while the endemic D. arbuscula differs from D. cneorum based on genetic and genome size data, D. cneorum subsp. arbusculoides was indistinguishable from D. cneorum subsp. cneorum . However, our study reveals that the subspecies differ in the number of flowers per inflorescence. This is the first comprehensive cytogeographic study of this intriguing genus at a regional scale, and in spite of its karyological stability, it contributes to our understanding of genomic evolution in plant species with a wide ecological amplitude., Competing Interests: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Annals of Botany Company.)

Background and Aims: The evolution of mating systems from outcrossing to self-fertilization is a common transition in flowering plants. This shift is often associated with the 'selfing syndrome', which is characterized by less visible flowers with functional changes to control outcrossing. In most cases, the evolutionary history and demographic dynamics underlying the evolution of the selfing syndrome remain poorly understood., Methods: Here, we characterize differences in the demographic genetic consequences and associated floral-specific traits between two distinct geographical groups of a wild shrub, Daphne kiusiana, endemic to East Asia; plants in the eastern region (southeastern Korea and Kyushu, Japan) exhibit smaller and fewer flowers compared to those of plants in the western region (southwestern Korea). Genetic analyses were conducted using nuclear microsatellites and chloroplast DNA (multiplexed phylogenetic marker sequencing) datasets., Key Results: A high selfing rate with significantly increased homozygosity characterized the eastern lineage, associated with lower levels of visibility and herkogamy in the floral traits. The two lineages harboured independent phylogeographical histories. In contrast to the western lineage, the eastern lineage showed a gradual reduction in the effective population size with no signs of a severe bottleneck despite its extreme range contraction during the last glacial period., Conclusions: Our results suggest that the selfing-associated morphological changes in D. kiusiana are of relatively old origin (at least 100 000 years ago) and were driven by directional selection for efficient self-pollination. We provide evidence that the evolution of the selfing syndrome in D. kiusiana is not strongly associated with a severe population bottleneck., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Annals of Botany Company.)

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., (© 2022 John Wiley & Sons Ltd.)

This study aimed to assess the effects of total flavonoid extracts (TFDG) and the monomers of Daphne genkwa on the CYP2C8 activity in vitro and in vivo.The 50% inhibitory concentration (IC 50 ) values were used to determine the inhibitory effect of TFDG and its four monomers for the CYP2C8 activity by recombinant human CYP2C8 (RHCYP2C8) yeast microsome system in vitro, and the volume per dose index (VDI) was predicted the potential inhibition in vivo. The effects of multiple-dose administration of TFDG on the pharmacokinetic parameters of rosiglitazone in rats were evaluated.The IC 50 values of apigenin, luteolin, hydroxy-genkwanin, genkwanin, and TFDG were 7.27 μmol/L, 11.9 μmol/L, 28.1 μmol/L, 127 μmol/L, and 13.4 μg/mL, respectively. The VDI values of apigenin and TFDG were 2.15 L and 6.60 L. In vivo study, compared with the control group, the elimination phase half-life and mean residence time in the TFDG treatment group were significantly increased by 96.9% and 106.8% ( p < .05), respectively.Apigenin showed a moderate inhibitory effect on the CYP2C8 activity in vitro, while the other three monomers were weak inhibitors. TFDG had a strong inhibitory effect on CYP2C8 in vitro and in vivo, and also inhibited the metabolism of rosiglitazone in rats.

The present study was undertaken to establish micromorphological standards of Daphne mucronata Royle and Myrtus communis (L.). Daphne mucronata Royle is a xerophytic perennial shrub while, Myrtus communis is an evergreen shrub or small tree. Micromorphological investigation is one of the cheapest methods. It will be helpful in the accurate identification of these plants from other species. Results of macroscopic evaluation showed various organoleptic features. Anatomy of the selected plant parts was carried out. It showed a typical dicot structure as it revealed the presence of epidermal cells, crystals, trichomes, cuticle, mesophyll tissues, xylem, phloem, paracytic, and dicytic stomata. Anatomical study facilitates the detection of adulteration with inferior commercial varieties, exhausted drugs, cheaper natural substances, and chemicals. Stomatal number, types, and size of stomata, and palisade ratio of Myrtus communis were observed. These observations will be helpful in the preparation of taxonomic key for the identification of selected species. Powder drug study [scanning electron microscopy (SEM)] showed characteristics histological differentiation. The methods cited here can be measured as characteristics to identify and validate these drugs., (© 2021 Wiley Periodicals LLC.)

To investigate in vitro anticancer, antimicrobial, antioxidant and in vivo hypoglycaemic effects of crude methanolic extracts (CMEs) of Berberis baluchistanica and Daphne oleoides. MTT assay for cytotoxicity on HeLa and NIH cells, disc diffusion protocols for antimicrobial and DPPH assay for antioxidant potential were applied. In vivo hypoglycaemic effect was investigated on Alloxan-induced diabetic rabbits. D. oleoides CME exhibited moderate cytotoxic behaviour against HeLa cells (IC 50 77.87μg/mL) whereas B. baluchistanica CME was found deficient (IC 50 170.02μg/mL). P. aeruginosa was susceptible to both CMEs. M. luteus and B. subtilis was prone to the bactericidal effects of D. oleoides and B. baluchistanica CMEs respectively. D. oleoides CME inhibited more than 80% S. cerevisiae and 60% C. glabrata colonies. B. baluchistanica CME showed significant antioxidant activity (IC 50 52.86μg/ml) than D. oleoides CME (IC 50 87.30μg/ml) and standard resveratrol (IC 50 109.46μg/ml). B. baluchistanica CME showed superior antidiabetic effect (135.75 mg/dl ±0.53) as compared to D. oleoides CME (191.50 mg/dl ± 0.48) but less antidiabetic effect than metformin hydrochloride (standard). All the above potentials exhibited by D. oleoides and B. baluchistanica CMEs propose further investigations to isolate and purify responsible biologically active lead molecule(s) with diverse capabilities.

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., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Daphnenoids A-C ( 1 - 3 ), three unusual sesquiterpenes with distinctive ring skeletons, together with a biogenetically related daphnenoid D ( 4 ) were obtained from the herb of Daphne penicillata by molecular networking strategies. Daphnenoid A ( 1 ) possesses a unique caged tetracyclo [5.3.2.0 1 , 6 .0 4 , 11 ] dodecane scaffold by unexpected cyclizations of C-1/C-11 and C-2/C-14. Daphnenoids B and C ( 2 and 3 ) were the first discovered natural sesquiterpenes with unique 5/5 spirocyclic systems in nature. Their structures were determined by NMR spectroscopic analysis, computer-assisted structure elucidation methods, quantum chemical calculations, and X-ray diffraction. A hypothetical biogenetic pathway begins with typical guaiane sesquiterpene ( a ), including a key intermediate ( 4 ) was proposed. Daphnenoids B and C ( 2 and 3 ) exhibited potential inhibitory activities on the production of NO against LPS-induced BV2 microglial cells.

There are abundant natural diterpenoids in the plants of the genus Daphne from the Thymelaeaceae family, featuring a 5/7/6-tricyclic ring system and usually with an orthoester group. So far, a total of 135 diterpenoids has been isolated from the species of the genus Daphne , which could be further classified into three main types according to the substitution pattern of ring A and oxygen-containing functions at ring B. A variety of studies have demonstrated that these compounds exert a wide range of bioactivities both in vitro and in vivo including anticancer, anti-inflammatory, anti-HIV, antifertility, neurotrophic, and cholesterol-lowering effects, which is reviewed herein. Meanwhile, the fascinating structure-activity relationship is also concluded in this review in the hope of providing an easy access to available information for the synthesis and optimization of efficient drugs.

Nickel determination is important because of its use in many industrial areas and its negative effects on human health. In this study, an ultraviolet-based photochemical vapor generation (UV-PVG) setup was combined with a T-shaped zirconium-coated slotted quartz tube-atom trapping (T-SQT-AT) apparatus to boost the sensitivity of a flame atomic absorption spectrophotometer for nickel determination. Nickel was separated from the sample matrix by converting it into its volatile species prior to online preconcentration by trapping on the zirconium-coated T-SQT inner surface. Analytical performance was maximized by optimizing all variable conditions. The limit of detection (LOD) and limit of quantification (LOQ) were found as 10 and 33 µg/L, respectively. Daphne tea and lake water samples were analyzed under optimum conditions, and there was no detectable nickel in the samples. For this purpose, spiking experiments were carried out for the samples in order to evaluate the applicability and accuracy of the method. The percent recovery values calculated for the two samples spiked at three different concentrations ranged between 90 and 112%. To our best knowledge, this is the first study in literature where UV-PVG was combined with T-SQT-AT for the determination of nickel in daphne tea and lake water samples prior to FAAS determination., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Zagros Mountains are like an island in the Middle East and they are subjected to changes in climate. Daphne mucronata Royle is an important medicinal plant species preserved in the high elevation regions in these mountains. Maxent species distribution model was used to integrate presence data (2413 points) and environmental variables to model the current and future potential distribution of D. mucronata in Iran. The most important variables were Bio19 with 50.5% contribution, followed by Bio8 and Bio2 with 30% and 11.4% contributions, respectively. The best Maxent model included seven variables, 4 feature types (linear, quadratic, product, and hinge), and had a test AUC value of 0.894. The current potential distribution indicated that 8% of Iran's drylands are suitable for growing D. mucronata and this area could decrease to 5.2% under representative concentration pathway (RCP) 4.5, and 3.1% under RCP 8.5 due to climate change. Our results suggest that D. mucronata may lose overall about 2.8% and 4.9% of its current distribution under RCP 4.5 and RCP 8.5, respectively, by 2050. There would be only 0.7 and 0.2% gains under RCP 4.5 and 8.5, respectively. The species would locally disappear between 1500- and 2000-m elevation under RCP 4.5 and 8.5, respectively. The establishment of some stations for monitoring the changes in transition zone or lost areas especially on the southeastern parts of Zagros Mountain can help in detecting changes in the future. Additionally, stable habitats may be good target areas for future conservation planning., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Ethnopharmacological Relevance: Genkwa flos, as a traditional herb, is the dried flower buds of Daphne genkwa Sieb.et Zucc. It is used in traditional medicine for the treatment of cough, sore throats, edema., Aim of the Study: The study aimed to explore a new mathematical method for multivariate evaluation, investigate the anti-inflammatory and anti-angiogenic activities of flavonoids in Daphne Genkwa under ex vivo conditions., Materials and Methods: The flavonoids monomers in Daphne Genkwa were separated by preparative liquid chromatography and identified by HPLC-ESI-ITMS. An in vitro inflammatory model of macrophage RAW264.7 induced by LPS and an angiogenesis model of human umbilical vein endothelial cells induced by TNF-α were established. Flavonoids were extracted and prepared for intervention to detect the amount of secretion after drug intervention to reflect the anti-inflammatory and anti-angiogenic activities of each component. In addition, a new mathematical method, which combined principal component analysis and efficacy coefficient method, was adopted in pharmacodynamic evaluation., Results: Fourteen flavonoids monomers were separated by preparative liquid chromatography and identified by HPLC-ESI-ITMS including H1 (hydroxygenkwanin-5-O-β-D-glucoside), H2 (apigenin-7-O-β-D-glucoside), H3 (kaempferol-3-O-β-D-glucoside), H4 (hydroxygenkwanin-5-O-β-D-primeveroside), H5 (apigenin-5-O-β-D-primeveroside), H6 (apigenin-7-O-β-D-glucuronide), H7 (luteolin-5-O-β-D-glucopyranoside), H8 (genkwain-5-O-β-D- glucoside), H9 (luteolin), H10 (Daphnodorin G), H11 (tiliroside), H12 (apigenin), H13 (3'- hydroxygenkwain) and H14 (genkwanin). We found that most of flavonoids down-regulated VCAM and MMP-3, while H1, H8, H9, H14 reduced VEGF and ICAM was only decreased by H14., Conclusion: Genkwanin may be the most active anti-rheumatoid arthritis flavonoids in Daphne genkwa. Meanwhile, the new mathematical method used in the study provided a new direction for solving the problem of multi-index pharmacodynamic evaluation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The aim of the study was to determine the stable diagnostic traits of a Caucasian endemic plant Daphne axilliflora (Keissl.) Pobed. and study its phytochemical composition. The following stable diagnostic characteristic were identified during the microstructural analysis: leaf surface glabrous, hypostomatic, dorsoventral; epidermal cells chaotic; curved with curved walls on both the upper and lower epidermis; stomata anomocytic; well visible chloroplasts, rhomboid and needle shaped crystals in lower leaf epidermis; leaf vascular bundles reverse-collateral; vascular system monocyclic, bilateral; wood diffuse-porous; xylem parenchyma is apotracheal, scanty metatracheal; vessel walls are predominantly characterized by spiral and pitted thickening; collenchyma lamellar; radial rays in single rows, heterogeneous. Phytochemical analysis showed the presence of terpene and phenolic substances, including coumarins. Two well-known coumarins (daphnin and daphnetin) were isolated and identified.

The genus Daphne belongs to the Thymeleaceae family and contains over 90 species that are distributed in Asia, Europe and parts of North Africa. The species of the genus Daphne are used in the traditional medicine of China, Tibet, Korea, and the Middle East for the treatment of various conditions. A broad range of studies has shown the significant biological potential of these species as sources of biologically and pharmacologically active compounds. Daphne species are a source of several classes of valuable phytochemicals such as coumarins, flavonoids, lignans, steroids and different classes of terpenes. The phytochemical diversity of this genus is demonstrated by over 350 secondary metabolites isolated from various species. The genus possesses a broad spectrum of biological activities including antibacterial, antifungal, antioxidant, analgesic, anti-inflammatory, cytotoxic, antiviral, abortive and haemostatic effects. A variety of bioactive secondary metabolites found in this genus may have potential use in pharmaceutical, cosmetic and food industries. Thus, species belonging to the genus Daphne can be considered an important source both for the treatment of various disorders, due to the presence of a plethora of bioactive constituents with potent bioactivities, and as possible leads in the discovery and synthesis of new medications., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Six undescribed sesquiterpenoids, including three acorane-type sesquiterpenoids (daphneaines A-C), three guaiane-type sesquiterpenoids (daphneaines E-G) and three known analogues were isolated from the roots of Daphne genkwa Siebold et Zucc. Their gross structures were elucidated by comprehensive spectroscopic analyses. The relative configurations of daphneaines A-C were determined by NOESY experiments. In addition, the relative configuration of daphneaine G was elucidated by performing a quantum chemical calculation of the NMR chemical shifts coupled with an advanced statistical procedure DP4+. The comparison of experimental and calculated electronic circular dichroism (ECD) data led to the establishment of the absolute configurations of daphneaines A-G. All isolates were tested for their inhibitory activity towards the LPS-induced NO production in RAW 264.7 macrophages, and daphneaine F showed inhibitory effect on NO production with an IC 50 value of 35.68 ± 3.18 μM., Competing Interests: Declaration of competing interest The authors declare that no conflict of interest exists in the submission of this manuscript., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The phytochemical investigation of the roots of Daphne genkwa yielded six secondary metabolites, including a new flavanol derivative, (2R, 3S)-5,7,4'-trihydroxy-8-methoxycarbonylflavanol ( 1 ), and five known compounds ( 2 - 6 ). The molecular structures of the isolated constituents were elucidated on the basis of extensive spectroscopic analysis, including UV, IR, NMR, and MS, and comparison with literature data. Furthermore, the cytotoxic activity of 1 and 2 against A549, HL-60, SMMC-7721, MCF-7, and SW480 cell lines was also described.

The complete plastome sequence for the type species of Daphne , Daphne laureola , was assembled and annotated in this study. The total length of the D. laureola plastome is 171,613 bp and comprises the large single copy (LSC) spanning 85,316 bp, the small single copy (SSC) spanning 2855 bp, and two inverted repeat (IR) regions each of 41,721 bp length. The SSC contains only two genes: ndhF and rpl32 . This sequence extends the list of Thymelaeaceae plastomes to be used in future systematic studies of the family and is the first European species to be sampled., Competing Interests: The authors report no conflict of interest and are alone responsible for the content and writing of the paper., (© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Ethnopharmacological Relevance: As recorded in traditional Chinese medicine (TCM) theory, Genkwa Flos (YH) and Glycyrrhizae Radix et Rhizoma (GC) compose one herbal pair of the so-called "eighteen incompatible medicaments", which indicate pairs of herbs that are mutually incompatible and that theoretically should not be applied simultaneously. However, the theory has been called into question due to a lack of evidence., Aims of Study: In this study, the incompatibility of YH and GC was investigated based on an assessment of the toxic effects of their combination by traditional safety methods and a modern metabonomic approach., Materials and Methods: Sprague-Dawley rats were used to evaluate the subacute toxicity of YH and YH-GC. The serum, urine, and several tissues were collected for biochemical analysis, histopathological examination, and metabonomic analysis., Results: Rats exposed to a dose of 1.0 g/kg YH (3 times of the Chinese Pharmacopoeia maximum dose) exhibited toxicity of the heart, liver, kidney and testes, and rats exposed to a YH-GC combination (1.0 g/kg YH + 1.0 g/kg GC) exhibited similar hepatotoxicity, which aggravated renal and reproductive toxicity. Following this, a metabonomic study tentatively identified 14 potential biomarkers in the YH group and 10 potential biomarkers in the YH-GC group, and metabolic pathways were then constructed. YH disturbed the pathways of glycerophospholipid metabolism, primary bile acid biosynthesis, and sphingolipid metabolism, while YH-GC combination induced disruptions in phenylalanine, tyrosine and tryptophan biosynthesis, tyrosine metabolism, and glycerophospholipid metabolism., Conclusion: The toxicities of YH and YH-GC combination above the Chinese Pharmacopoeia dose were obvious but different. Metabonomics combined with biochemical and histopathological methods can be applied to elucidate the toxicity mechanism of the YH-GC combination that caused liver, kidney and reproductive injuries in rats., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

T-cell exhaustion has become an important issue in chronic infection because exhausted antigen-specific T cells show impaired abilities to eradicate persistently infected pathogens and produce effector cytokines, such as IFN-γ and TNF-α. Thus, strategies to either restore endogenous exhausted T cell responses or provide functional T cells are needed for therapeutics of chronic infection. Despite promising developments using antibodies and cell immunotherapy, there have been no reported attempts to restore exhausted T cells using treatment with materials derived from natural resources. Here, using a mouse model of chronic infection with lymphocytic choriomeningitis virus (LCMV), we found that vinegar-processed flowers (flos) of Daphne genkwa (vp-genkwa), which was composed mainly of four index components, restored exhausted CD4(+) and CD8(+) T cells significantly, as corroborated by evidence that vp-genkwa treatment enhanced functional LCMV-specific CD4(+) and CD8(+) T cells, both quantitatively and qualitatively. Furthermore, pretreatment with vp-genkwa prevented the generation of exhausted LCMV-specific CD8(+) T cells. Such restorations of exhausted LCMV-specific CD4(+) and CD8(+) T cells by vp-genkwa were closely associated with reduced viral burden in sera and tissues. More interestingly, vp-genkwa treatment induced down-regulation of negative molecules, such as PD-1 and Tim-3, in exhausted CD4(+) and CD8(+) T cells with more apparent down-regulation of Tim-3, suggesting that Tim-3 molecule may be a major target in restoring exhausted T cell responses. Collectively, these results provide valuable new insights into the use of vp-genkwa to develop a therapeutic strategy for chronic human diseases, such as hepatitis B and C virus, human immunodeficiency virus, and cancers., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objectives: Daphne odora var. marginata (D. marginata), an aiophyllus arbuscular plant, is one of the traditional Chinese medicines used to treat rheumatoid arthritis. This study investigated the therapeutic effects and mechanisms of daphnetin, an active monomer ingredient derived from D. marginata, on collagen-induced arthritis (CIA) in rats., Methods: The effects of daphnetin on joint diseases were assessed by hematoxylin and eosin staining and radiographic and transmission electron microscopy. The protein and mRNA expression levels of T helper (Th)1/Th2/Th17-type cytokines in the spleen were determined by flow cytometry and quantitative real-time PCR., Results: Our results showed that daphnetin significantly reduced paw swelling and was nontoxic in vivo at the tested doses. Synovial hyperplasia, joint destruction and chondrocyte degeneration in CIA rats were suppressed by daphnetin. Daphnetin treatment also reduced the levels of Th1/Th2/Th17 type cytokines in spleen lymphocytes in CIA rats. Moreover, the expression of Foxp3, which can down-regulate the activity of Th17 cells, was significantly increased in the daphnetin-treated groups., Conclusions: These results suggest that daphnetin may have therapeutic effects in down-regulating Th17-type responses in CIA rats. The beneficial effects of daphnetin on CIA may be related to its inhibition of Th17 cell priming and activation., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: In Turkish traditional medicine, the aerial parts of Daphne oleoides Schreber subsp. kurdica (DOK) have been used to treat malaria, rheumatism and for wound healing. The aim was to evaluate the ethnopharmacological usage of the plant using in vivo and in vitro pharmacological experimental models, and to perform bioassay-guided fractionation of the 85% methanolic extract of DOK for the isolation and identification of active wound-healing component(s) and to elucidate possible mechanism of the wound-healing activity., Materials and Methods: In vivo wound-healing activity was evaluated by the linear incision and the circular excision wound models. Anti-inflammatory and antioxidant activities, which are known to support the wound healing process, were also assessed by the Whittle method and the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging assays, respectively. The total phenolic content of the extract and subextracts was estimated to establish any correlation between the phenolic content and the antioxidant activity. The methanolic extract of DOK was subjected to various chromatographic separation techniques leading to the isolation and identification of the active component(s). Furthermore, in vitro hyaluronidase, collagenase and elastase enzymes inhibitory activity assays were conducted on the active components to explore the activity pathways of the remedy., Results: After confirmation of the wound-healing activity, the methanolic extract was subjected to successive solvent partitioning using solvents of increasing polarity creating five subextracts. Each subextract was tested on the same biological activity model and the ethyl acetate (EtOAc) subextract had the highest activity. The EtOAc subextract was subjected to further chromatographic separation for the isolation of components 1, 2 and 3. The structures of these compounds were elucidated as daphnetin (1), demethyldaphnoretin 7-O-glucoside (2) and luteolin-7-O-glucoside (3). Further in vivo testing revealed that luteolin-7-O-glucoside was responsible for the wound-healing activity of the aerial parts. It was also found to exert significant anti-inflammatory, antioxidant, anti-hyaluronidase and anti-collagenase activities., Conclusion: The present study explored the wound-healing potential of Daphne oleoides subsp. kurdica. Through bioassay-guided fractionation and isolation techniques, luteolin-7-O-glucoside was determined as the main active component of the aerial parts. This compound exerts its activity through inhibition of hyaluronidase and collagenase enzymes activity as well as interfering with the inflammatory stage., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Context: The present study describes the spasmogenic and spasmolytic activities of Daphne oleoides Schreb. (Thymelaeaceae), exploring the possible underlying pharmacological mechanisms., Aim: Pharmacological investigation of Daphne oleoides to provide evidence for its therapeutic application in gastrointestinal motility disorders., Materials and Methods: Methanol crude extract of Daphne oleoides (Do.Cr) was studied in gastrointestinal isolated tissues., Results: In spontaneously contracting rabbit jejunum preparations, Do.Cr at 0.3-3.0 mg/mL caused moderate stimulation, followed by relaxant effect at the next higher concentrations (5.0-10 mg/mL). In presence of atropine, spasmogenic effect was blocked and the relaxation was emerged, suggesting that the spasmogenic effect of Daphne oleoides is mediated through activation of muscarinic receptors. When tested against the high K+ (80 mM)-induced contractions, Do.Cr (0.3-5.0 mg/mL), like verapamil, inhibited the induced contractions, suggesting Ca++ channel blockade (CCB) effect. The CCB effect was further confirmed when pre-treatment of the tissue with Do.Cr shifted the Ca++ concentration-response curves to the right, similar to that caused by verapamil., Discussion and Conclusion: These results indicate that Daphne oleoides exhibits gut excitatory and inhibitory effects, occurred via cholinergic and Ca++ antagonistic pathways, respectively.

In the present study, we investigated that (-)-aptosimon, isolated from flower buds of Daphne genkwa, inhibited cyclooxygenase-2 (COX-2) and inducible nitric oxide (NO) synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Similarly, (-)-aptosimon suppressed tumor necrosis factor (TNF)-alpha production. Our results clearly indicated that (-)-aptosimon inhibited LPS-induced nuclear factor-kappaB (NF-kappaB) activation, by preventing degradation of the inhibitor kappa B-alpha (IkappaB-alpha). (-)-Aptosimon also inhibited interleukin-4 (IL-4) and interleukin-13 (IL-13) production in ConA-induced splenocytes. In conclusion, the anti-inflammatory effects of (-)-aptosimon are attributed to the suppression of pro-inflammatory cytokines and mediators by blocking NF-kappaB activation. These data suggest that (-)-aptosimon as a potential therapeutic agent for inflammation-associated disorders.

A new flavanol, 5, 7, 4'-trihydroxy-8-ethoxycarbonylflavanol (1), was isolated from the ethanol extract of Daphne genkwa Sieb. et Zucc. Its structure was defined by spectroscopic methods.

The isolation of a new flavan, 5,7,4'-trihydroxy-8-ethoxycarbonylflavan (1), from the root of Daphne odora var. atrocaulis is reported.

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.

Daphne genkwa (Thymelaeaceae) has been used as a folk medicine in China. We investigated the effects of D. genkwa and Jyu-So-To on various pharmacologic models in mice including the azoxymethane (AOM)-induced colonic aberrant crypt focus formation assay, ornithine decarboxylase (ODC) activity assay, and two types of mouse ear swelling model. Administration of 236.3 ppm of Jyu-So-To in drinking water significantly suppressed AOM-induced colonic aberrant crypt focus formation (p < 0.05), with an inhibitory ratio of 46.7%. The effects of several extracts with organic solvents of D. genkwa on murine epidermal ODC activity were examined. In particular, the inhibitory ratio of the n-hexane extract was 30.8%. In the 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced ear edema model in mice, the methanol extract resulted in 56.3% inhibition compared with the control. On the other hand, there are two peaks of responses at 1 h (immediate-phase reaction; IPR) and 24 h (late-phase reaction) in biphasic cutaneous reactions, which are enhanced in the dinitrofluorobenzene model (DNFB). The water extract of D. genkwa clearly inhibited the IPR ear swelling. These results suggest that D. genkwa and Jyu-So-To should be a promising source of antitumor, antiinflammatory, and antiallergy agents.

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 microl/ml (equivalent to 0.54 mg/of plant leaves powder per ml of culture medium), as well as gnidilatimonoein (0.94 microM), were capable of quenching by 58% and 64%, respectively, the attachment of wehi-164 cells to fibronectin-coated wells (4 microg/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.