Transcriptome and metabolome analysis revealed the dynamic change of bioactive compounds of Fructus Ligustri Lucidi.

BACKGROUND: The Fructus Ligustri Lucidi, the fruit of Ligustrum lucidum, contains a variety of bioactive compounds, such as flavonoids, triterpenoids, and secoiridoids. The proportions of these compounds vary greatly during the different fruit development periods of Fructus Ligustri Lucidi. However, a clear understanding of how the proportions of the compounds and their regulatory biosynthetic mechanisms change across the different fruit development periods of Fructus Ligustri Lucidi is still lacking. RESULTS: In this study, metabolite profiling and transcriptome analysis of six fruit development periods (45 DAF, 75 DAF, 112 DAF, 135 DAF, 170 DAF, and 195 DAF) were performed. Seventy compounds were tentatively identified, of which secoiridoids were the most abundant. Eleven identified compounds were quantified by high performance liquid chromatography. A total of 103,058 unigenes were obtained from six periods of Fructus Ligustri Lucidi. Furthermore, candidate genes involved in triterpenoids, phenylethanols, and oleoside-type secoiridoid biosynthesis were identified and analyzed. The in vitro enzyme activities of nine glycosyltransferases involved in salidroside biosynthesis revealed that they can catalyze trysol and hydroxytyrosol to salidroside and hydroxylsalidroside. CONCLUSIONS: These results provide valuable information to clarify the profile and molecular regulatory mechanisms of metabolite biosynthesis, and also in optimizing the harvest time of this fruit.

Effects of the combination of Epimedii Folium and Ligustri Lucidi Fructus on apoptosis and autophagy in SOP rats and osteoblasts via PI3K/AKT/mTOR pathway.

BACKGROUND: This study aimed to investigate the effects of the combination of Epimedii Folium (EF) and Ligustri Lucidi Fructus (LLF) on regulating apoptosis and autophagy in senile osteoporosis (SOP) rats. METHODS: Firstly, we identified the components in the decoction and drug-containing serum of EL (EF&LLF) by Ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS). Secondly, SOP rats were treated with EF, LLF, EL and caltrate to evaluate the advantages of EL. Finally, H2O2-, chloroquine-, and MHY1485-induced osteoblasts were treated with different doses of EL to reveal the molecular mechanism of EL. We detected bone microstructure, oxidative stress levels, ALP activity and the expressions of Bax, Bcl-2, caspase3, P53, Beclin-1, p-PI3K, PI3K, p-Akt, Akt, p-mTOR, mTOR, and LC3 in vivo and in vitro. RESULTS: 36 compounds in EL decoction and 23 in EL-containing serum were identified, including flavonoids, iridoid terpenoids, phenylethanoid glycosides, polyols and triterpenoids. EL could inhibit apoptosis activity and increase ALP activity. In SOP rats and chloroquine-inhibited osteoblasts, EL could improve bone tissue microstructure and osteoblasts functions by upregulating Bcl-2, Beclin1, and LC3-II/LC3-I, while downregulating p53 in all treatment groups. In H2O2-induced osteoblasts, EL could upregulate the protein and mRNA expressions of Bcl-2 while downregulate LC3-II/LC3-I, p53 and Beclin1. Besides, EL was able to down-regulate PI3K/AKT/mTOR pathway which activated in SOP rats and MHY1485-induced osteoblasts. CONCLUSIONS: These findings demonstrate that EL with bone protective effects on SOP rats by regulating autophagy and apoptosis via PI3K/Akt/mTOR signaling pathway, which might be an alternative medicine for the treatment of SOP.

Salidroside, 8(E)-Nuezhenide, and Ligustroside from Ligustrum japonicum Fructus Inhibit Expressions of MMP-2 and -9 in HT 1080 Fibrosarcoma.

A phenyl ethanoid, salidroside (SAL), and two secoiridoids, 8(E)-nuezhenide (NZD) and ligustroside (LIG), were isolated from fruits of Ligustrumjaponicum, used as traditional folk medicine, and their chemical structures were elucidated by the comparison of spectral data with published literature. Matrix metalloproteinases (MMPs) are major enzymes that play crucial roles in the metastasis and invasive behavior of tumors. In particular, MMP-2 and MMP-9, regulated by the MAPK signaling pathways, including p38, ERK and JNK, are known to play a key role in the degradation of the basement membrane. In the present study, the effects of SAL, NZD and LIG on the expression of MMP-2 and -9 were examined in phorbol 12-myristate 13-acetate (PMA)-induced HT 1080 cells. All the compounds significantly lowered the amount of MMP-2 and MMP-9 released, as determined by gelatin zymography and ELISA. In addition, the mRNA and protein expression levels of MMP-2 and MMP-9 were significantly suppressed, as measured by RT-PCR and Western blotting. According to the Western blotting assay, SAL and LIG effectively reduced the expression of MMP-2 in a dose-dependent manner. NZD lowered the expression of MMP-9 in a similar way. The phosphorylation of p38, ERK and JNK was also significantly suppressed by these compounds. These findings suggest that all the compounds regulate the release and expression of MMP-2 and MMP-9 via MAPK signaling pathways.

Integration of multicomponent characterization, untargeted metabolomics and mass spectrometry imaging to unveil the holistic chemical transformations and key markers associated with wine steaming of Ligustri Lucidi Fructus.

Processing of traditional Chinese medicine (TCM) can enhance the efficacy and/or reduce the toxicity. Currently available approaches regarding TCM processing generally focus on a few markers, rendering a one-sided strategy that fail to unveil the involved global chemical transformation. We herein present a strategy, by integrating enhanced multicomponent characterization, untargeted metabolomics, and mass spectrometry imaging (MSI), to visualize the chemical transformation and identify the markers associated with the wine steaming of Ligustri Lucidi Fructus (LLF), as a case. An ultra-high-performance liquid chromatography/quadrupole-Orbitrap mass spectrometry-based polarity-switching (between the negative and positive modes), precursor ions list-including data-dependent acquisition approach was developed, which enabled the simultaneous targeted/untargeted characterization of 158 components from LLF via one injection analysis. Holistic, continuous, and time-dependent chemical variation trajectory, among different processing time (0-12 h) for LLF, was depicted by principle component analysis. Pattern recognition chemometrics could unveil 20 markers, among which the peak area ratios of eight components to oleuropein aglycone, used as an internal standard, were diagnostic to identify the processed (both the commercial and in-house prepared) from the raw LLF. Four markers (10-hydroxyoleoside dimethylester, 8-demethyl-7-ketoliganin, elenolic acid, and salidroside) showed an increasing trend, while another four (neonuezhenide/isomer, verbascoside/isomer, luteoline, and nuzhenal A) decreased in LLF after processing. MSI visualized the spatial distribution in the fruit and indicated consistent variation trends for four major markers deduced by the untargeted metabolomics approach. This integral strategy, in contrast to the conventional approaches, gives more convincing data supporting the processing mechanism investigations of TCM from a macroscopic perspective.

Photosystem II of Ligustrum lucidum in response to different levels of manganese exposure.

The toxic effect of excessive manganese (Mn) on photosystem II (PSII) of woody species remains largely unexplored. In this study, five Mn concentrations (0, 12, 24, 36, and 48 mM) were used, and the toxicity of Mn on PSII behavior in leaves of Ligustrum lucidum was investigated using in vivo chlorophyll fluorescence transients. Results showed that excessive Mn levels induced positive L- and K- bands. Variable fluorescence at 2 ms (VJ) and 30 ms (VI), absorption flux (ABS/RC), trapped energy flux (TRo/RC), and dissipated energy flux (DIo/RC) increased in Mn-treated leaves, whereas the performance index (PIABS), electron transport flux (ETo/RC), maximum quantum yield (φPo), quantum yield of electron transport (φEo), and probability that an electron moves further than QA- (ψo) decreased. Also, excessive Mn significantly decreased the net photosynthesis rate and increased intercellular CO2 concentration. The results indicated that Mn blocked the electron transfer from the donor side to the acceptor side in PSII, which might be associated with the accumulation of QA-, hence limiting the net photosynthetic rate.

Liqustri lucidi Fructus inhibits hepatic injury and functions as an antioxidant by activation of AMP-activated protein kinase in vivo and in vitro.

Medicinal herbs are used to treat or prevent various diseases, and function to regulate protective mechanisms as nutraceuticals. Fructus Ligustri lucidi is the fruit of Ligustrum lucidum and has been used for its tonic effects on the liver. This study was designed to examine the effects of Fructus Ligustri lucidi water extract (FLL) against severe oxidative stress and mitochondrial impairment in vivo and in vitro and to elucidate its cellular mechanisms of action. Treatment of HepG2 cells with arachidonic acid (AA) + iron successfully induced oxidative stress and apoptosis, as indicated by depletion of glutathione, formation of ROS, decreses in mitochondrial membrane potential (Δψm), and altered expression of apoptosis-related proteins, such as procaspase-3 and Bcl-xL. FLL treatment significantly blocked these pathological changes and the mitochondrial dysfunction caused by AA + iron, which were similar with the effect of aminoimidazole-carboxamide-ß-d-ribofuranoside (AICAR). Moreover, FLL induced the activation of AMP-activated protein kinase (AMPK), which was mediated by its upstream kinase LKB1. Inhibition or activation of AMPK revealed the role of AMPK in cellular protection conferred by FLL in LKB1-deficient cells. In mice, oral administration of 100 mg/kg FLL activated AMPK in the liver, and protected against oxidative stress and liver injury induced by CCl4 injection. Among the components of FLL, chlorogenic acid was found to be responsible for the protection of hepatocytes against AA + iron-induced cellular damage. Overall, our results confirmed that FLL has the ability to protect hepatocytes against oxidative injury through regulation of the AMPK signaling pathway.

[Leaf micro-morphology and features in adsorbing air suspended particulate matter and accumulating heavy metals in seven trees species].

The purpose of this study was to assess the relationship between tree leaf micro-morphology and features in adsorbing air suspended particulate matter and accumulating heavy metals. Seven tree species, including Ginkgo biloba, at heavy traffic density site in Huainan were selected to analyze the frequency of air particulate matter retained by leaves, the particle amount of different sizes per unit leaf area retained by leaves and its related micro-morphology structure, and the relationship between particle amount of different sizes per unit leaf area retained by leaves and its related accumulation of heavy metals. We found that the species characterized by small leaf area, special epidemis with abundant fax, and highly uneven cell wall, as well as big and dense stomata and without trichomes mainly absorbed fine particulate matter; while those species with many trichomes mainly retained coarse particulate matter. Accumulation of heavy metals in leaves of the seven species was significantly different except for Ph. Tree species with high capacities in heavy metal accumulation were Ginkgo biloba, Ligustrum lucidum, and Cinnamomum camphora. Accumulation of Cd, Cr, Ni, Zn, Cu and total heavy metal concentration for seven tree species was positively related to the amount of particulate matter absorbed. Correlation coefficients of d10 vs d2.5, d10 vs d1.0, d2.5 vs d1.0 were 0.987, 0.971, 0.996, respective, and the correlate level was significant. The ratios of d2.5/d10, d1.0/d10, d1.0/d2.5 were 0.844, 0.763, 0.822, indicating that the particulate matter from traffic was mainly fine particulates.

Seasonal variations in metabolite profiling of the fruits of Ligustrum lucidum Ait.

The metabolite profiling of fruits of the herb Ligustrum lucidum Ait collected during different months has been performed using ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC/QTOFMS) and multivariate statistical analysis techniques. The markers such as oleuropein acid, neonuezhenide, specnuezhenide, oleuropein and ligustrosidic acid accountable for such variations were identified through the loadings plot of principal component analysis (PCA), and the tentative identification of the markers is completed by comparing the mass spectra and retention times with those of reference compounds and/or tentatively assigned by matching empirical molecular formulae and MS/MS data with those of the known compounds published. Furthermore, one of the chemical markers, such as specnuezhenide, which is water-soluble, biologically active and also the predominant compound in this crude drug, was quantified by ultra-performance liquid chromatography coupled with a tunable UV detector (UPLC-TUV). The developed UPLC method provides good linearity (r(2)=0.9991), repeatability (RSD=2.96%), intra- and inter-day precisions (RSD=0.21%, 0.96%), with accuracies of 99.18-100.26% and a recovery of specnuezhenide of 97.57%. The fruits of L. lucidum Ait collected from August to December were tested. The results clearly show that the fruits of L. lucidum Ait harvested in October have the highest yields of specnuezhenide. It is also noted that the variations of content of specnuezhenide obtained by both methods have a strong correlation. This suggests that the newly proposed strategy is a reliable and simple method for the rapid discrimination of subtle variations, within the same plant species or strains, due to different seasonal collection times.

Light regulation to chlorophyll synthesis and plastid development of the chlorophyll-less golden-leaf privet.

Ligustrum vicaryi L. is a hybrid of Ligustrum ovalifolium Hassk. var. aureo-marginatum and Ligustrum vulgale L., and displays a chlorophyll-less phenotype. Therefore it is widely used as a horticultural shrub because of its golden-color leaves. Its putative mechanism, light responses, chlorophyll synthesis and plastid development were studied. L. vicaryi has a higher level of 5-aminolevulinic acid (ALA), but lower levels of chlorophylls compared with L. quihoui. The yellowish phenotype of L. vicaryi upper leaves could be attributed to their hampered conversion from chlorophyllide into chlorophyll a. Despite the enhanced ALA level and the decreased thylakoid stacking in plastids, L. vicaryi golden leaves contain normal levels of Lhcb transcripts and photosystem apoproteins. Furthermore, reactive oxygen species (ROS) accumulation is almost the same in L. vicaryi and L. quihoui. The golden leaves often turn green and the contents of chlorophylls increase with decreasing light intensity. Dynamic changes of chlorophyll-synthesis-system under the light transition were also analyzed.

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.

An assessment of the tolerance of Ligustrum ovalifolium Hassk. to traffic-generated Pb using physiological and biochemical markers.

This study examined the leaf Pb content, chlorophyll fluorescence parameters (Fo, Fm, Fv, Fv/Fm, Fm/Fo and t(1/2)), photopigments (Chl a, Chl b, Chl a+b, the Chl a/b ratio, and total carotenoids), as well as total phenolics (free and bound) in privet (Ligustrum ovalifolium Hassk.) in 'Stara Zvezdara' Park, located along the avenue with heavy traffic flow (polluted site), and the Arboretum of Belgrade's Faculty of Forestry (control site). Site-dependent variations were observed for all the parameters examined. It was found that Pb accumulated in leaf tissues (3.5-4.2microg/g) originates from traffic. Results obtained showed that privet is tolerant to the accumulation of Pb generated by traffic, because it maintains optimal photosynthesis and is characterized by active protection mechanisms due to increase in leaf phenolics, which enable it to survive in habitats exposed to chronic Pb pollution stress. Therefore, L. ovalifolium has great potential for urban landscaping.

Mobilisation of nutrients and transport via the xylem sap in a shrub (Ligustrum ovalifolium) during spring growth: N and C compounds and interactions.

In open-field soilless culture there can be great deal of leaching, particularly in rainy springs. Ligneous plants have the capacity to store large quantities of nutrients in perennial organs. Knowledge of the plant's internal nutrient mobilisation during spring to supply growing organs could lead to reduction of fertiliser application. To quantify the fraction of storage mobilisation available for growth of new organs during spring, Ligustrum ovalifolium shrubs were grown for 2 years with or without fertilisation in the second spring. Nitrogen (N) absorption and N and carbon (C) mobilisation from storage were followed during spring growth via the sap quality. A mathematical combination of the sap composition with flow velocity provided the transported quantities of N and C. Nitrogen and C mobilisation towards new shoots took place during all the spring growth from bud break onwards. In unfertilised plants, C was mobilised primarily as sugars (stachyose, mannose and sucrose) and starch. In fertilised plants, the same sugars were transported in the xylem sap, but at lower concentrations. Stachyose concentration was lower in fertilised than in unfertilised plants and decreased during spring growth. Nitrogen was transported in the xylem sap mainly as amino acids in both fertilisation treatments. Glutamine was the predominant form at bud break and during shoot elongation. In fertilised plants, arginine became predominant after shoot elongation, and was related to low C availability. The interactions of N with C are discussed; specifically, insufficient availability of N limits the use of C, more of which is directed to aerial organs by sap flow.

Seasonal variations in water relations in current-year leaves of evergreen trees with delayed greening.

We investigated seasonal patterns of water relations in current-year leaves of three evergreen broad-leaved trees (Ilex pedunculosa Miq., Ligustrum japonicum Thunb., and Eurya japonica Thunb.) with delayed greening in a warm-temperate forest in Japan. We used the pressure-volume method to: (1) assess the extent to which seasonal variation in leaf water relations is attributable to leaf development processes in delayed greening leaves versus seasonal variation in environmental variables; and (2) investigate variation in leaf water relations during the transition from the sapling to the adult tree stage. Leaf mass per unit leaf area was generally lowest just after completion of leaf expansion in May (late spring), and increased gradually throughout the year. Osmotic potential at full turgor (Psi(o) (ft)) and leaf water potential at the turgor loss point (Psi(w) (tlp)) were highest in May, and lowest in midwinter in all species. In response to decreasing air temperature, Psi(o) (ft) dropped at the rate of 0.037 MPa degrees C(-1). Dry-mass-based water content of leaves and the symplastic water fraction of total leaf water content gradually decreased throughout the year in all species. These results indicate that reductions in the symplastic water fraction during leaf development contributed to the passive concentration of solutes in cells and the resulting drop in winter Psi(o) (ft). The ratio of solutes to water volume increased in winter in current-year leaves of L. japonicum and E. japonica, indicating that osmotic adjustment (active accumulation of solutes) also contributed to the drop in winter in Psi(o) (ft). Bulk modulus of elasticity in cell walls fluctuated seasonally, but no general trend was found across species. Over the growing season, Psi(o) (ft) and Psi(w) (tlp) were lower in adult trees than in saplings especially in the case of I. pedunculosa, suggesting that adult-tree leaves are more drought and cold tolerant than sapling leaves. The ontogenetic increase in the stress resistance of I. pedunculosa may be related to its characteristic life form because I. pedunculosa grows taller than the other species studied.

On the role of flavonoids in the integrated mechanisms of response of Ligustrum vulgare and Phillyrea latifolia to high solar radiation.

The role of flavonoids in mechanisms of acclimation to high solar radiation was analysed in Ligustrum vulgare and Phillyrea latifolia, two Mediterranean shrubs that have the same flavonoid composition but differ strikingly in their leaf morpho-anatomical traits. In plants exposed to 12 or 100% solar radiation, measurements were made for surface morphology and leaf anatomy; optical properties, photosynthetic pigments, and photosystem II efficiency; antioxidant enzymes, lipid peroxidation and phenylalanine ammonia lyase; synthesis of hydroxycinnamates and flavonoids; and the tissue-specific distribution of flavonoid aglycones and ortho-dihydroxylated B-ring flavonoid glycosides. A denser indumentum of glandular trichomes, coupled with both a thicker cuticle and a larger amount of cuticular flavonoids, allowed P. latifolia to prevent highly damaging solar wavelengths from reaching sensitive targets to a greater degree than L. vulgare. Antioxidant enzymes in P. latifolia were also more effective in countering light-induced oxidative load than those in L. vulgare. Consistently, light-induced accumulation of flavonoids in L. vulgare, particularly ortho-dihydroxylated flavonoids in the leaf mesophyll, greatly exceeded that in P. latifolia. We conclude that the accumulation of flavonoid glycosides associated with high solar radiation-induced oxidative stress and, hence, biosynthesis of flavonoids appear to be unrelated to 'tolerance' to high solar radiation in the species examined.