Iridoid constituents from the branches of Viburnum chinshanense and their inhibitory effects on α-amylase and α-glucosidase.

Ten previously undescribed iridoid constituents, viburnshosins A-E (1-5) and viburnshosides A-E (6-10), together with one known analogue (11), were isolated from the branches of Viburnum chinshanense. Their structures were unambiguously elucidated by a comprehensive analysis of 1D and 2D NMR data, together with HRESIMS spectroscopic data. The absolute configurations of compounds 1-10 were assigned by means of the calculated ECD spectra. Interestingly, compounds 2 and 3 are the first iridoids with an unusual C-3-C-7 oxo bridge. Compounds 4, 5, and 10 displayed remarkable inhibitory effects against α-amylase (IC50: 38.42, 37.65, and 21.64 µM, respectively) and α-glucosidase (IC50: 12.97, 19.34, and 25.71 µM, respectively), comparable to those of the positive control acarbose (IC50: 39.75 and 23.66 µM, respectively). The interaction modes of compounds 4 and 10 with two enzymes were analyzed by molecular modeling.

Lignan constituents with α-amylase and α-glucosidase inhibitory activities from the fruits of Viburnum urceolatum.

Eleven previously undescribed lignan constituents, including five 8-O-4' type neolignans, viburnurcosides A-E (1-5), three benzofuran type neolignans, viburnurcosides F-H (6-8), and three tetrahydrofuran type lignans, viburnurcosides I-K (9-11), were isolated from the fruits of Viburnum urceolatum. The structures of all isolates were elucidated by an extensive analysis of the NMR and HRESIMS data. The absolute configurations of these compounds were determined by quantum-chemical electronic circular dichroism calculation and comparison. The sugar units of viburnurcosides A-K were identified by acid hydrolysis and HPLC analysis of the chiral derivatives of monosaccharides. The in vitro enzyme inhibition assay exhibited that viburnurcoside J (10) had the most potent inhibitory activity against α-amylase and α-glucosidase with the IC50 values of 19.75 and 9.14 µM, respectively, which were stronger than those of the positive control acarbose (37.31 and 26.75 µM, respectively). The potential binding modes of viburnurcoside J (10) with α-amylase and α-glucosidase were also analyzed by molecular modeling.

23-Hydroxyursolic acid from Viburnum lutescens inhibits osteoclast differentiation in vitro and lipopolysaccharide-induced bone loss in vivo by suppressing c-Fos and NF-κB signalling.

Bone homeostasis is maintained by a combination of osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Excessive osteoclast activity is linked to several bone-related disorders, including osteoporosis and rheumatoid arthritis. Pharmacological therapy might have a number of adverse effects. Therefore, the development of natural anti-osteoclastogenic drugs with greater efficacy and fewer adverse effects is desirable. In this study, the anti-osteoclastogenic effects of 23-hydroxyursolic acid (HUA), a triterpene isolated from Viburnum lutescens, were investigated in vitro and in vivo. HUA significantly inhibited receptor activator of nuclear factor kappa-B ligand (RANKL)-induced mature osteoclast differentiation by reducing the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and F-actin ring formation. It also inhibited the expression of osteoclast-specific marker genes such OSCAR, MMP-9, TRAP, DC-STAMP, and CtsK, as well as transcription factors, c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) in response to RANKL. Mice orally administered with HUA (25 and 50 mg/kg) exhibited significant protection against bone loss and osteoclast formation induced by lipopolysaccharide (LPS). HUA suppressed RANKL-induced nuclear factor kappa B (NF-κB) activation and phosphorylation of JNK and ERK mitogen-activated protein kinases (MAPKs). These results suggest that HUA attenuates osteoclast formation in vitro and in vivo by suppressing the RANKL-mediated AP1, NF-κB, and NFATc1 pathways. Therefore, HUA may be a lead compound for the prevention or treatment of osteolytic bone disorders.

Recent Advance on Chemistry and Bioactivities of Secondary Metabolites from Viburnum Plants: An Update.

Viburnum species are a group of small trees or shrubs that are of great ornamental and medicinal values. Some of them have been used for a long time both as conventional and ethnic medicine. Viburnum fruits, eaten in fresh and processed forms, have been revealed to contain various health-promoting nutrients. With the increasing research on Viburnum plants, they are considered to be an abundant resource of bioactive natural products possessing diverse pharmacological properties and unique chemical structures, that is powerfully proved by the existence of structurally novel vibsane-type diterpenoids which only occur in Viburnum species, newly discovered lignan constituents with unusual side chains and other noteworthy natural components. This review describes 185 new and 228 known secondary metabolites from Viburnum genus between 2008 and 2020, including their chemical structures, sources and bioactivities, and highlights the corresponding structure-activity relationships.

Protocol for Extraction and Electron Microscopy Visualization of Lipids in Viburnum tinus Fruit Using Cryo-Ultramicrotomy.

We recently reported that Viburnum tinus fruit generates its metallic blue color using globular lipid inclusions embedded in its epicarpal cell walls. This protocol describes steps to visualize the lipidic nature of the nanostructure using cryo-ultramicrotomy, chloroform extraction, and transmission electron microscopy (TEM) imaging. This method is useful to localize and characterize novel lipidic nanostructures embedded in both plant and animal tissues at the TEM resolution. For complete details on the use and execution of this protocol, please refer to Middleton et al. (2020).

Viburnum tinus Fruits Use Lipids to Produce Metallic Blue Structural Color.

Viburnum tinus is an evergreen shrub that is native to the Mediterranean region but cultivated widely in Europe and around the world. It produces ripe metallic blue fruits throughout winter [1]. Despite its limited fleshy pulp [2], its high lipid content [3] makes it a valuable resource to the small birds [4] that act as its seed-dispersers [5]. Here, we find that the metallic blue appearance of the fruits is produced by globular lipid inclusions arranged in a disordered multilayer structure. This structure is embedded in the cell walls of the epicarp and underlaid with a dark layer of anthocyanin pigments. The presence of such large, organized lipid aggregates in plant cell walls represents a new mechanism for structural coloration and may serve as an honest signal of nutritional content.

Induction of Apoptosis in Lung Cancer Cells by Viburnum grandiflorum via Mitochondrial Pathway.

BACKGROUND Lung cancer is one of the leading causes of mortality and morbidity. Viburnum grandiflorum is a medicinal herb known for its wide spectrum of pharmacological activities, but its anti-cancer properties against lung cancer cells have not been previously investigated. The present study elucidated the antitumor effect and associated mechanism of methanol extract of Viburnum grandiflorum extract (VGE) against lung cancer cells. MATERIAL AND METHODS The viability of H1650, HCC827, and H1299 cells was measured using MTT assay. Apoptosis and cell cycle progression were determined by flow cytometry using annexin-V/PI and JC-1 stains, respectively. The Lipofectamine Plus reagent (Invitrogen) was used for transfection of caspase-9 plasmid to H1650 and H1299 cells. RESULTS The results showed decreased H1650, HCC827, and H1299 cell viability by VGE, which occurred in a concentration- and time-dependent manner. The VGE treatment significantly increased the rate of apoptosis in H1650 (P<0.05) and H1299 (P<0.02) cells at 48 and 72 h. Treatment of H1650 and H1299 cells with 10 µM of VGE significantly enhanced the number of cells in sub-G1 phase. The VGE treatment cleaved pro-caspase-8/-9 and-3 in H1650 and HCC827 cells at 72 h. The VGE treatment of H1650 and HCC827 cells reduced Mcl-1 protein expression. Treatment of H1650 and HCC827 cells with VGE markedly decreased the level of p-Akt. However, dominant-negative caspase-9 (caspase-9 dN) plasmid transfection prevented the viability-inhibitory effect of VGE on H1650 and HCC827 cells. Treatment of H1650 and HCC827 cells with VGE increased levels of cytochrome c in the cytosol. CONCLUSIONS VGE inhibited lung carcinoma cell viability by apoptosis activation through a caspase-dependent pathway. Therefore, VGE is a potent anti-cancer agent against lung cancer cells.

Whole-plant allocation to storage and defense in juveniles of related evergreen and deciduous shrub species.

In evergreen plants, old leaves may contribute photosynthate to initiation of shoot growth in the spring. They might also function as storage sites for carbohydrates and nitrogen (N). We hence hypothesized that whole-plant allocation of carbohydrates and N to storage in stems and roots may be lower in evergreen than in deciduous species. We selected three species pairs consisting of an evergreen and a related deciduous species: Mahonia aquifolium (Pursh) Nutt. and Berberis vulgaris L. (Berberidaceae), Prunus laurocerasus L. and Prunus serotina Ehrh. (Rosaceae), and Viburnum rhytidophyllum Hemsl. and Viburnum lantana L. (Adoxaceae). Seedlings were grown outdoors in pots and harvested on two dates during the growing season for the determination of biomass, carbohydrate and N allocation ratios. Plant size-adjusted pools of nonstructural carbohydrates in stems and roots were lower in the evergreen species of Berberidaceae and Adoxaceae, and the slope of the carbohydrate pool vs plant biomass relationship was lower in the evergreen species of Rosaceae compared with the respective deciduous species, consistent with the leading hypothesis. Pools of N in stems and roots, however, did not vary with leaf habit. In all species, foliage contained more than half of the plant's nonstructural carbohydrate pool and, in late summer, also more than half of the plant's N pool, suggesting that in juvenile individuals of evergreen species, leaves may be a major storage site. Additionally, we hypothesized that concentration of defensive phenolic compounds in leaves should be higher in evergreen than in deciduous species, because the lower carbohydrate pool in stems and roots of the former restricts their capacity for regrowth following herbivory and also because of the need to protect their longer-living foliage. Our results did not support this hypothesis, suggesting that evergreen plants may rely predominantly on structural defenses. In summary, our study indicates that leaf habit has consequences for storage economics at the whole-plant level, with evergreen shrub species storing less carbohydrates (but not N) per unit plant biomass than deciduous species.

Influence of arbuscular mycorrhizal fungi and treated wastewater on water relations and leaf structure alterations of Viburnum tinus L. plants during both saline and recovery periods.

Nowadays, irrigation with low quality water is becoming an alternative to satisfy the needs of crops. However, some plant species have to deal with high salinity of reclaimed water, by adapting their physiological behaviour during both saline and recovery periods and developing morphological changes in their leaves. The application of arbuscular mycorrhizal fungi (AMF) could also be a suitable option to mitigate the negative effects of this kind of water, although the effectiveness of plant-AMF association is influenced by many factors. In this work, during forty weeks, the combined effect of Glomus iranicum var. tenuihypharum and two types of water: control, C, EC<0.9 dS m(-1) and reclaimed water, RW (with EC: 4 dS m(-1) during a first saline period and EC: 6 dS m(-1) during a second saline period) was evaluated for laurustinus plants (Viburnum tinus L.) transplanted in soil. This was followed by a recovery period of eight weeks, when all the plants were irrigated in the control irrigation conditions. Seasonal and daily changes in stem water potential (Ψstem), stomatal conductance (gs), photosynthesis (Pn) and leaf internal CO2 concentration (Ci) of laurustinus plants were evaluated. Leaf structure alterations, nutrient imbalance, height and leaf hydraulic conductivity (Kleaf) were also determined. Due to the high difficulty of absorbing water from the soil, RW plants showed a high volumetric water content (θv) in soil. The stem water potential and the stomatal conductance (gs) values were reduced in RW plants throughout the second saline period. These decreases were also found during the day. Leaf Ca(2+)/Na(+) and K(+)/Na(+) ratios diminished in RW plants respect to the C plants due to the Na(+) accumulation, although height and chlorophyll content values did not show statistical differences. Leaves from RW plants showed a significantly thicker mesophyll than Control leaves as a consequence of high EC. The area of palisade parenchyma (PP) increased while the area of spongy parenchyma (SP) decreased in RW leaves with respect to the C leaves. These structural changes could be considered as a strategy to maximize photosynthesis potential in saline conditions. Mycorrhizal inoculation improved the water status of both C and RW plants by increasing their Ψstem and gs values. As regards leaf structure, AMF showed an opposite effect to salinity for PP and SP. At the end of the recovery period, hardly any statistical differences of physiological parameters were found between treatments, although a tendency to improve them was observed in inoculated plants. In any case, the leaf structural changes and the great reduction in Kleaf observed at Ψleaf below -1.5 MPa would constitute an important mechanism for laurustinus plants to reduce the water loses produced by salinity.

Activity, polypeptide and gene identification of thylakoid Ndh complex in trees: potential physiological relevance of fluorescence assays.

Three evergreen (Laurus nobilis, Viburnum tinus and Thuja plicata) and two autumnal abscission deciduous trees (Cydonia oblonga and Prunus domestica) have been investigated for the presence (zymogram and immunodetection) and functionality (post-illumination chlorophyll fluorescence) of the thylakoid Ndh complex. The presence of encoding ndh genes has also been investigated in T. plicata. Western assays allowed tentative identification of zymogram NADH dehydrogenase bands corresponding to the Ndh complex after native electrophoresis of solubilized fractions from L. nobilis, V. tinus, C. oblonga and P. domestica leaves, but not in those of T. plicata. However, Ndh subunits were detected after SDS-PAGE of thylakoid solubilized proteins of T. plicata. The leaves of the five plants showed the post-illumination chlorophyll fluorescence increase dependent on the presence of active Ndh complex. The fluorescence increase was higher in autumn in deciduous, but not in evergreen trees, which suggests that the thylakoid Ndh complex could be involved in autumnal leaf senescence. Two ndhB genes were sequenced from T. plicata that differ at the 350 bp 3' end sequence. Comparison with the mRNA revealed that ndhB genes have a 707-bp type II intron between exons 1 (723 bp) and 2 (729 bp) and that the UCA 259th codon is edited to UUA in mRNA. Phylogenetically, the ndhB genes of T. plicata group close to those of Metasequoia, Cryptomeria, Taxodium, Juniperus and Widdringtonia in the cupresaceae branch and are 5' end shortened by 18 codons with respect to that of angiosperms.

[Diurnal rhythm of Viburnum awabuki and Betula luminifera volatiles and electroantennogram response of Batocera horsfieldi].

By the method of Tenax-TA absorbent adsorption combined with GC-MS, this paper analyzed the changes of the diurnal rhythm of the volatiles in the healthy branches and Batocera horsfieldi-damaged branches of Viburnum awabuki and Betula luminifera, and electroantennogram technique was used to perform a comparative analysis on the electroantennogram (EAG) responses of unmated male and female B. horsfieldi to the volatiles. After the feeding by B. horsfieldi, there was a decrease in the contents of limonene, nonanal, hexadecane, butyl acrylate, and 3-methyl-butanoic acid in damaged branches of V. awabuki and in the neohexane and hexadecane contents in damaged branches of B. luminifera. Simultaneously, new materials such as permethyl 99A, octyl alcohol, iodo, decanal, hexanal, and bioallethrin etc. were newly synthesized in the damaged branches. The EAG response values of unmated male and female B. horsfieldi adults to the volatiles in the damaged branches of B. luminifera were the highest, being 1.23 mV and 1.38 mV, while to the healthy branches of V. awabuki were the lowest, being 0.95 mV and 1.01 mV, respectively. As for the time period, the EAG response values of the adults to the volatiles were the lowest from 12:00 to 14:00, which accorded with the feeding behaviors of the adults, i. e., taking food in the field in early morning or at dusk.

[Characteristics of foliar delta13C values of common shrub species in various microhabitats with different karst rocky desertification degrees].

By measuring the foliar delta13C values of 5 common shrub species (Rhamnus davurica, Pyracantha fortuneana, Rubus biflorus, Zanthoxylum planispinum, and Viburnum utile) growing in various microhabitats in Wangjiazhai catchment, a typical karst desertification area in Guizhou Province, this paper studied the spatial heterogeneity of plant water use at niche scale and the response of the heterogeneity to different karst rocky desertification degrees. The foliar delta13C values of the shrub species in the microhabitats followed the order of stony surface > stony gully > stony crevice > soil surface, and those of the majority of the species were more negative in the microhabitat soil surface than in the others. The foliar delta13C values decreased in the sequence of V. utile > R. biflorus > Z. planispinum > P. fortuneana > R. davurica, and the mean foliar delta13C value of the shrubs and that of typical species in various microhabitats all increased with increasing karst rocky desertification degree, differed significantly among different microhabitats. It was suggested that with the increasing degree of karst rocky desertification, the structure and functions of karst habitats were impaired, microhabitats differentiated gradually, and drought degree increased.

Intraspecific differences in drought tolerance and acclimation in hydraulics of Ligustrum vulgare and Viburnum lantana.

An adequate general drought tolerance and the ability to acclimate to changing hydraulic conditions are important features for long-lived woody plants. In this study, we compared hydraulic safety (water potential at 50% loss of conductivity, Psi(50)), hydraulic efficiency (specific conductivity, k(s)), xylem anatomy (mean tracheid diameter, d(mean), mean hydraulic diameter, d(h), conduit wall thickness, t, conduit wall reinforcement, (t/b)(h)(2)) and stomatal conductance, g(s), of forest plants as well as irrigated and drought-treated garden plants of Ligustrum vulgare L. and Viburnum lantana L. Forest plants of L. vulgare and V. lantana were significantly less resistant to drought-induced cavitation (Psi(50) at -2.82 +/- 0.13 MPa and -2.79 +/- 0.17 MPa) than drought-treated garden plants (- 4.58 +/- 0.26 MPa and -3.57 +/- 0.15 MPa). When previously irrigated garden plants were subjected to drought, a significant decrease in d(mean) and d(h) and an increase in t and (t/b)(h)(2) were observed in L. vulgare. In contrast, in V. lantana conduit diameters increased significantly but no change in t and (t/b)(h)(2) was found. Stomatal closure occurred at similar water potentials (Psi(sc)) in forest plants and drought-treated garden plants, leading to higher safety margins (Psi(sc) - Psi(50)) of the latter (L. vulgare 1.63 MPa and V. lantana 0.43 MPa). These plants also showed higher g(s) at moderate Psi, more abrupt stomatal closure and lower cuticular conductivity. Data indicate that the development of drought-tolerant xylem as well as stomatal regulation play an important role in drought acclimation, whereby structural and physiological responses to drought are species-specific and depend on the plant's hydraulic strategy.

Resorption protection. Anthocyanins facilitate nutrient recovery in autumn by shielding leaves from potentially damaging light levels.

The resorption protection hypothesis, which states that anthocyanins protect foliar nutrient resorption during senescence by shielding photosynthetic tissues from excess light, was tested using wild-type (WT) and anthocyanin-deficient mutants of three deciduous woody species, Cornus sericea, Vaccinium elliottii (Chapmn.), and Viburnum sargentii (Koehne). WT Betula papyrifera (Marsh) was included to compare the senescence performance of a species that does not produce anthocyanins in autumn. Plants were subjected to three environmental regimes during senescence: an outdoor treatment; a 5-d high-stress (high light and low temperature) treatment followed by transfer to a low-stress environment and a low-stress treatment that served as control. In the outdoor treatment, the appearance of anthocyanins in senescing leaves of WT plants was concomitant with the development of photo-inhibition in mutant plants of all three anthocyanin-producing species. In the high-stress environment, WT plants maintained higher photochemical efficiencies than mutants and were able to recover when transferred to the low-stress environment, whereas mutant leaves dropped while still green and displayed signs of irreversible photooxidative damage. Nitrogen resorption efficiencies and proficiencies of all mutants in both stressful treatments were significantly lower than the WT counterparts. B. papyrifera displayed photochemical efficiencies and nitrogen resorption performance comparable with the highest of the anthocyanin-producing species in all three senescing environments, indicating a photoprotective strategy divergent from the other species studied. These results strongly support the resorption protection hypothesis of anthocyanins in senescing leaves.