The screened compounds from Ligustri Lucidi Fructus using the immobilized calcium sensing receptor column exhibit osteogenic activity in vitro.

Calcium sensing receptor (CaSR) has become the novel target of treating osteoporosis with herbal medicine Ligustri Lucidi Fructus (LLF), however, the bioactive compounds responsible for anti-osteoporosis are hard to clarify due to the complexity and diversity of chemical constituents in it. Herein, the immobilized CaSR column was packed with stationary phase materials, which were derived from integrating CLIP-tagged CaSR directly out of crude cell lysates onto the surface of silica gels (5.83 mg/g) in a site-specific covalent manner. The column had a great specificity of recognizing agonists and kept a good stability for at least 3 weeks. The two compounds from LLF extract were screened and identified as olenuezhenoside and ligustroflavone using the immobilized CaSR column in conjunction with mass spectrometry. Molecular docking predicted that both compounds were bound in venus flytrap (VFT) domain of CaSR by the formation of hydrogen bonds. Cellular results showed that both compounds exhibited the distinct osteogenic activity by enhancing the proliferation, differentiation and mineralization of osteoblastic cells. Our study demonstrated that, the immobilized protein column enables to screen the bioactive compounds rapidly from herbal extract, and the newly discovered natural product ligands towards CaSR, including olenuezhenoside and ligustroflavone, will be the candidates for the treatment of osteoporosis.

Antibacterial and Anti-Inflammatory Polysaccharide from Fructus Ligustri Lucidi Incorporated in PVA/Pectin Hydrogels Accelerate Wound Healing.

In cutaneous wound healing, an overproduction of inflammatory chemokines and bacterial infections impedes the process. Hydrogels can maintain a physiologically moist microenvironment, absorb chemokines, prevent bacterial infection, inhibit bacterial reproduction, and facilitate wound healing at a wound site. The development of hydrogels provides a novel treatment strategy for the entire wound repair process. Here, a series of Fructus Ligustri Lucidi polysaccharide extracts loaded with polyvinyl alcohol (PVA) and pectin hydrogels were successfully fabricated through the freeze-thaw method. A hydrogel containing a 1% mixing weight ratio of FLL-E (named PVA-P-FLL-E1) demonstrated excellent physicochemical properties such as swellability, water retention, degradability, porosity, 00drug release, transparency, and adhesive strength. Notably, this hydrogel exhibited minimal cytotoxicity. Moreover, the crosslinked hydrogel, PVA-P-FLL-E1, displayed multifunctional attributes, including significant antibacterial properties, earlier re-epithelialization, production of few inflammatory cells, the formation of collagen fibers, deposition of collagen I, and faster remodeling of the ECM. Consequently, the PVA-P-FLL-E1 hydrogel stands out as a promising wound dressing due to its superior formulation and enhanced healing effects in wound care.

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.

Comparative Analysis of Phytochemical Profiles and Selected Biological Activities of Various Morphological Parts of Ligustrum vulgare.

Ligustrum vulgare (LV), widely cultivated in Europe and often used in hedges, has been historically recognized in folk medicine for its potential health benefits. This study focused on exploring the untargeted identification of secondary metabolites in ethanol extracts (70% v/v) from different morphological parts (young shoots, leaves, flowers and fruits) of LV at various stages of plant development, using ultra-high-performance liquid chromatography with high-resolution mass spectrometry (UHPLC-HRMS). Additionally, the selected biological activities (antioxidant activity, cyclooxygenase-2 inhibition (COX-2), α-amylase inhibition and cytotoxicity) of the tested extracts were determined. Untargeted metabolomics showed that LV extracts were a rich source of phenylethanoid compounds, flavonoids, iridoids and their derivatives. The flowers of LV had the highest content of oleuropein (33.43 ± 2.48 mg/g d.w.). The lowest antioxidant activity was obtained for ripe and post-seasonal fruits, while in the case of other samples, the activity was at a similar level. All tested extracts showed α-amylase and COX-2 inhibitory activity. In addition, LV extracts showed strong antiproliferative properties in colorectal (HT29) and liver (HepG2) cancer cell lines. The obtained results show the difference in the content of bioactive compounds in various morphological parts of Ligustrum vulgare. These differences may influence the multifaceted medicinal potential of this plant.

Synergistic effects of arbuscular mycorrhizal fungi and biochar are highly beneficial to Ligustrum lucidum seedlings in Cd-contaminated soil.

Cadmium (Cd) contamination is a widespread environmental issue. There is a lack of knowledge about the impacts of applying arbuscular mycorrhizal fungi (AMF) and biochar, either alone or in their combination, on alleviating Cd phytotoxicity in Ligustrum lucidum. Therefore, a pot experiment was conducted in a greenhouse, where L. lucidum seedlings were randomly subjected to four regimes of AMF treatments (inoculation with sterilized AMF, with Rhizophagus irregularis, Diversispora versiformis, alone or a mixture of these two fungi), and two regimes of biochar treatments (with or without rice-husk biochar), as well as three regimes of Cd treatments (0, 15, and 150 mg kg-1), to examine the responses of growth, photosynthetic capabilities, soil enzymatic activities, nutritional concentrations, and Cd absorption of L. lucidum plants to the interactive effects of AMF, biochar, and Cd. The results demonstrated that under Cd contaminations, AMF alone significantly increased plant total dry weight, soil pH, and plant nitrogen (N) concentration by 84%, 3.2%, and 13.2%, respectively, and inhibited soil Cd transferring to plant shoot by 42.2%; biochar alone significantly enhanced net photosynthetic rate, soil pH, and soil catalase of non-mycorrhizal plants by 16.4%, 9%, and 11.9%, respectively, and reduced the soil Cd transferring to plant shoot by 44.7%; the additive effect between AMF and biochar greatly enhanced plant total dry weight by 101.9%, and reduced the soil Cd transferring to plant shoot by 51.6%. Furthermore, dual inoculation with D. versiformis and R. irregularis conferred more benefits on plants than the single fungal species did. Accordingly, amending Cd-contaminated soil with the combination of mixed-fungi inoculation and biochar application performed the best than either AMF or biochar alone. These responses may have been attributed to higher mycorrhizal colonization, soil pH, biomass accumulation, and biomass allocation to the roots, as well as photosynthetic capabilities. In conclusion, the combined use of mixed-fungi involving D. versiformis and R. irregularis and biochar addition had significant synergistic effects on enhancing plant performance and reducing Cd uptake of L. lucidum plants in Cd-contaminated soil.

Exploring the boost of steaming with wine on Ligustri Lucidi Fructus in treating postmenopausal osteoporosis based on superior "multi-component structure" and iron/bone metabolism coregulation.

BACKGROUND: Clinical studies indicated that postmenopausal osteoporosis (PMOP) often accompanied by iron overload risk factor, which exacerbated bone metabolism disorders and accelerated PMOP. Previous research found that multicomponent in Ligustri Lucidi Fructus (FLL) or wine-steamed FLL (WFLL) acted on the common targets of iron overload and PMOP simultaneously, which indicated that FLL and WFLL probably regulated iron/bone metabolism dually. Additionally, WFLL had more superior effect according to the theory of Chinese medicine for thousands of years. PURPOSE: To reveal the "superior multi-component structure (SMCS)" and its molecular mechanisms in parallelly down-regulating iron overload and rescuing bone metabolism by WFLL. DESIGNS AND METHODS: HPLC fingerprinting was established to compare the chemical profiles of FLL and WFLL; Then, the chemical compositions and quality markers of FLL and WFLL were analyzed by UPLC-Orbitrap-MS/MS coupled with OPLS-DA; the dynamic contents of quality markers and the multi-component structure at different wine steaming times (WST) were simultaneously determined by HPLC-DAD. Meanwhile, the dynamic efficacy of FLL at different WST were hunt by systematic zebrafish model. Subsequently, potential mechanism of WFLL in treating PMOP accompanied with iron overload was obtained from network pharmacology (NP) and molecular docking (MD). Finally, zebrafish and ovariectomy rat model were carried out to validate this potential mechanism. RESULTS: HPLC fingerprints similarity of 15 batches in FLL and WFLL were among 0.9-1.0. 126 compositions were identified, including 58 iridoids, 25 terpenes, 30 phenylethanoids, 7 flavonoids and 6 others. 20 quality markers associated with WFLL was revealed, and the ratio of phenylethanols: Iridoids: Triterpenes (P/I/T) was converted from 1: 15: 4.5 to 1: 0.8: 0.9 during steaming (0 - 24 h) calculated by the quantification of 11 quality markers; the bone mineralization and motor performance of zebrafish larvae indicated that the optimum efficacy of WFLL at 12 h (p < 0.05) in which the SMCS of P/I/T was converted to 1: 4: 1.8. NP discovered that BMP-Smad pathway is one of the potential mechanisms of FLL in anti PMOP and then regulated bone formation and iron overload simultaneously. MD revealed that 17 active ingredients and 10 core targets genes could spontaneously bind with appropriate affinity. Rats model verified that FLL and WFLL significantly reversed PMOP, based on the improvement in bone formation indexes (ALP, OPG, OGN), iron metabolism indicators (hepcidin, ferritin), bone microstructure (BMD, BV/TV, Tb. Th, Tb. N); Moreover, WFLL significant enhanced reversal effect in anti-PMOP compared to FLL (p < 0.05). FLL and WFLL increased genes and proteins expression (Hep, BMP-6, p-Smad1/5, Smad4) related to BMP-Smad pathway compared with model group, and WFLL was more superior than FLL (p< 0.05). CONCLUSION: The SMCS of FLL was optimized by wine-steam, WFLL represented a dual effect in downregulating iron overload and promoting bone formation, and the BMP-Smad pathway is one of the potential molecular mechanisms.

Post-Chromatographic Derivatization Coupled with Mass Spectrometry as a Method of Profiling and Identification of Antioxidants; Ligustrum vulgare Phytocomplex as an Example.

High-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) coupled with radical scavenging assays, such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) can be both used for the detection of the antioxidants in plant extracts. In this study, the ethanolic (70% v/v) extracts from different morphological parts of Ligustrum vulgare collected at different stages of maturity were used as the source of antioxidants. The final identification of antioxidants was performed using high-resolution mass spectroscopy (HRMS). As a result, 19 compounds with antioxidant properties detected with HPLC-ABTS assay and 10 compounds detected with HPTLC-DPPH/ABTS assay were identified, mostly from the group of iridoids, phenylethanoids, and flavonoids. When comparing different L. vulgare samples, it was found that the extracts obtained from leaves contained the greatest number of antioxidants. The results of this study suggest that HPTLC-DPPH/ABTS as well as HPLC-ABTS derivatization coupled with the HRMS can be successfully used for profiling and identification of antioxidants from natural sources. Planar chromatography is more suitable for screening multiple samples because of its simplicity, whereas more challenging liquid chromatography provides more detailed information and is therefore better for a selected set of samples.

Monoterpenoid Glycosides from the Leaves of Ligustrum robustum and Their Bioactivities (II).

Ligustrum robustum has been not only used as a heat-clearing and detoxicating functional tea (Ku-Ding-Cha) but also consumed as a hypotensive, anti-diabetic, and weight-reducing folk medicine. From the leaves of L. robustum, ten new monoterpenoid glycosides named ligurobustosides T10 (1a), T11 (1b), T12 (2a), T13 (2b), T14 (3a), T15 (3b), F1 (4b), T16 (5a), T17 (5b), and E1 (6b), together with five known ones (4a, 6a, 7, 8a, 8b), were separated and identified using the spectroscopic method and chemical method in this research. The results of biological tests exhibited that the fatty acid synthase (FAS) inhibitory action of compound 5 (IC50: 4.38 ± 0.11 µM) was as strong as orlistat (IC50: 4.46 ± 0.13 µM), a positive control; the α-glucosidase inhibitory actions of compounds 1-4 and 7-8, and the α-amylase inhibitory actions of compounds 1-8 were medium; the ABTS radical scavenging capacities of compounds 1-3 and 5-8 (IC50: 6.27 ± 0.23 ~ 8.59 ± 0.09 µM) were stronger than l-(+)-ascorbic acid (IC50: 10.06 ± 0.19 µM) served as a positive control. This research offered a theoretical foundation for the leaves of L. robustum to prevent diabetes and its complications.

Anti-Inflammatory Mechanism of Ligustrum Lucidum Ait Based on Network Pharmacology and Molecular Docking.

OBJECTIVE: To investigate possible targets of Ligustrum lucidum and its anti-inflammatory mechanism. Core targets were screened by the established networks of component target and potential protein interaction, accompanied by GO and KEGG analyses. The findings were confirmed using molecular docking. RESULTS: eight active components including Quercetin, luteolin, kaempferol and corresponding 163 potential targets, including CCL2, IL6, CXCL8, TNF, IL1B, were identified with a threshold of OB ≥ 30% and DL ≥ 0.18%. 1018 anti-inflammatory targets were acquired, with 101 common targets identified by mutual mapping. 172 nodes and 287 edges are readable in the "component target" visualization network graph. The KEGG analysis identified 20 significantly differentiated signaling pathways, including IL-17, toll-like receptor, TNF, HIF-1, lipid and atherosclerosis. CONCLUSION: Molecular docking has verified the binding sites and energies of the chemical components in Ligustrum lucidum with key anti-inflammatory proteins, providing a theoretical basis for its clinical use and development.

Secoiridoid glycosides from the fruits of Ligustrum lucidum and their in vitro anti-inflammatory activity.

Seven new secoiridoid glycosides (1-7), together with a known analogue (8), were isolated from the fruits of Ligustrum lucidum. Their structures with absolute configurations were determined by HR-ESI-MS, 1D and 2D NMR, and electronic circular dichroism (ECD) spectroscopic analysis, as well as biogenetic consideration. Compounds 1 and 2 are the first examples of secoiridoid glycoside dimers featuring a rare rearranged oleoside-type secoiridoid moiety, and compounds 3-7 represent a new class of oleoside-type secoiridoid glycosides with unusual stereochemistry at C-1 position. A plausible biosynthetic pathway for this group of unusual secoiridoid glycosides was also proposed herein. In addition, the isolates were evaluated for their in vitro anti-inflammatory activity, and all tested compounds exhibited modest inhibitory effects against nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 macrophages.

Metabolomics analysis of Ligustri Lucidi Fructus at different harvest times during the whole growing period based on ultra-high-performance liquid chromatography with mass spectrometry.

After medicinal market research, it was found that the harvest time of Ligustri Lucidi Fructus (LLF) was chaotic in practice. In order to determine the optimal harvest period of LLF to ensure its pharmacological activity, metabolomics analysis of LLF at different harvest times based on ultra-high-performance liquid chromatography-triple quadrupole-(linear ion trap)-tandem mass spectrometry was established. In this study, 166 differential metabolites (DMs) in 448 metabolites at different harvest times were screened out based on variable importance in projection value, and among them, 94 DMs with regular trends of change in relative content (59 increased and 35 decreased with the growth period) were chosen to further research. The result of the multivariate statistical analysis showed that November was the optimal harvest period of LLF. Additionally, 10-hydroxyligustroside, oleoside 11-methyl ester, and salidroside were screened out to be used as the evaluation indicators of immature LLF, while specnuezhenide, nuezhenoside G13, and neonuezhenide were the evaluation indicators of mature LLF. This study provides fundamental insight for metabolite identification and proposes the best harvest period of LLF to avoid confusion in the medicinal market.

A prospective study on the endometrium properties and the pregnancy rate of infertile women with thin endometrium receiving Fructus ligustri lucidi used in traditional Chinese medicine.

OBJECTIVE: To evaluate whether Zi Gui Nv Zhen capsules (ZGNZC) can increase the fertility rate of Chinese women with infertility due to thin endometrium. METHODS: Prospective, randomized, open-labeled 3-monthly study; 104 patients (aged 20-40 years) receiving either ZGNZC (experimental group, n = 55) or not (control group, n = 49). Main outcomes: thickness/type of the endometrium during ovulation and pregnancy rate. Between-group analysis (A) compares the experimental vs. control group, and within-group analysis (B) compares data at baseline and after study in the experimental group. RESULTS: (A) Between-group-analysis: Patients with ZGNZC had a higher endometrium thickness (0.8 ± 0.27 vs. 0.68 ± 0.10; p < .05) and higher type A endometrium rates (34.2% vs. 13.2%; p < .05) than the control group. Pregnancy rates were higher in the experimental than in the control group (43.6% vs. 22.4%; p < .05). (B) Within-group-analysis: ZGNZC increased endometrium thickness (0.58 ± 0.13 vs. 0.87 ± 0.24 vs. 0.83 ± 0.26 vs. 0.80 ± 0.27), and type A endometrium rates (10.9% vs. 60.0% vs. 49.0% vs. 34.2%) (all p < .05). Univariate analysis of pregnancy with other study parameters showed positive and significant correlations between pregnancy and administration of ZGNZC (p < .05). All hepato-renal biomarkers remained within the norm. There were no adverse events. CONCLUSIONS: In infertile women with thin endometrium who wish to conceive, two months' application of ZGNZC can improve endometrial properties and proliferation, which is necessary for a healthy pregnancy, and increase the clinical pregnancy rate in our prospective randomized observational study.

A New HPLC-UV Method Using Hydrolyzation with Sodium Hydroxide for Quantitation of Trans-p-Hydroxycinnamic Acid and Total Trans-p-Hydroxycinnamic Acid Esters in the Leaves of Ligustrum robustum.

Trans-p-hydroxycinnamic acid and its esters in the leaves of Ligustrum robustum might be a new resource to prevent diabetes and its complications. In the present study, a new HPLC-UV method using hydrolyzation with sodium hydroxide for quantitation of trans-p-hydroxycinnamic acid and total trans-p-hydroxycinnamic acid esters in the leaves of L. robustum was developed, since it was difficult and troublesome to analyze no less than 34 trans-p-hydroxycinnamic acid esters by usual HPLC. The extract of L. robustum was hydrolyzed with sodium hydroxide at 80 °C for 2 h, and then, hydrochloride was added. HPLC analysis was performed in reverse phase mode using a C-18 column, eluting with methanol-0.1% acetic acid aqueous solution (40:60, v/v) in isocratic mode at a flow rate of 1.0 mL·min-1 and detecting at 310 nm. The linear range for trans-p-hydroxycinnamic acid was 11.0-352.0 µg·mL-1 (r2 = 1.000). The limit of detection and limit of quantification were 2.00 and 6.07 µg·mL-1, respectively. The relative standard deviations of intra-day and inter-day variabilities for trans-p-hydroxycinnamic acid were less than 2%. The percentage recovery of trans-p-hydroxycinnamic acid was 103.3% ± 1.1%. It is the first HPLC method using hydrolyzation for quantification of many carboxylic esters. Finally, the method was used successfully to determine trans-p-hydroxycinnamic acid and total trans-p-hydroxycinnamic acid esters in various extracts of the leaves of L. robustum. The 60-70% ethanol extracts of L. robustum showed the highest contents of free trans-p-hydroxycinnamic acid (3.96-3.99 mg·g-1), and the 50-80% ethanol extracts of L. robustum displayed the highest contents of total trans-p-hydroxycinnamic acid esters (202.6-210.6 mg·g-1). The method can be applied also to the quality control of the products of L. robustum.

Eco-friendly and efficient extraction of polyphenols from Ligustrum robustum by deep eutectic solvent assisted ultrasound.

An eco-friendly and efficient extraction method using deep eutectic solvents assisted ultrasound extraction (DESs-UAE) for the polyphenols from Ligustrum robustum was developed. Among the 34 kinds of DESs prepared, tetraethyl ammonium bromide: 1,2,4-butanol (Teab: 1,2,4-But) was proved to be a suitable extraction solvent based on the extraction efficiency. The extraction parameters including temperature, water content, liquid-solid ratio were optimized with response surface methodology (RSM). Under the optimal conditions, the total phenolic content (TPC) and total flavonoid content (TFC) were 101.46 ± 2.96 mg GAE/g DW and 264.17 ± 5.39 mg RE/g DW, respectively. Furthermore, the extraction mechanism of DESs-UAE was investigated by extraction kinetics, molecular dynamic simulation and theory calculations of interaction. In particular, 9 kinds of polyphenols compounds from Ligustrum robustum were firstly identified by UPLC-Q-TOF-MS. Moreover, the recovered polyphenols exhibited significant antioxidant, α-glucosidase inhibition, acetylcholinesterase inhibition and anticancer activity.

Wedelolactone induces natural killer cell activity and the improvement to bioavailability using polysaccharides from Ligustri Lucidi Fructus.

Wedelolactone (WDL) is the major bioactive component in Ecliptae Herba. This present study investigated the effects of WDL on natural killer cell functions and possible underlying mechanisms. It was proved that wedelolactone enhanced the killing ability of NK92-MI by upregulating the expression of perforin and granzyme B through the JAK/STAT signaling pathway. Additionally, wedelolactone could induce the migration of NK-92MI cells by promoting CCR7 and CXCR4 expressions. However, the application of WDL is limited due to poor solubility and bioavailability. Accordingly, this study investigated the impact of polysaccharides from Ligustri Lucidi Fructus (LLFPs) on WDL. The biopharmaceutical properties and pharmacokinetic characteristics were determined to compare WDL individually and in combination with LLFPs. The results showed that LLFPs could benefit the biopharmaceutical properties of WDL. Specifically, stability, solubility, and permeability were increased by 1.19-1.82-fold, 3.22-fold, and 1.08-fold higher than those of WDL alone, respectively. Furthermore, the pharmacokinetic study revealed that LLFPs could remarkably improve AUC(0-t) (150.34 vs. 50.47 ng/mL ∗ h), t1/2 (40.78 vs. 2.81 h), and MRT(0-∞) (46.64 vs. 5.05 h) for WDL. In conclusion, WDL would be considered a potential immunopotentiator, and LLFPs could overcome the instability and insolubility, ultimately improving the bioavailability of this plant-derived phenolic coumestan.

[Species identification of Ligustrum lucidum].

Ligustrum lucidum is a woody perennial plant of genus Ligustrum in family Oleaceae. Its dried fruit has high medicinal value. In this study, the authors evaluated the variability and species identification efficiency of three specific DAN barcodes(rbcL-accD, ycf1a, ycf1b) and four general DAN barcodes(matK, rbcL, trnH-psbA, ITS2) for a rapid and accurate molecular identification of Ligustrum species. The results revealed that matK, rbcL, trnH-psbA, ITS2 and ycf1a were inefficient for identifying the Ligustrum species, and a large number of insertions and deletions were observed in rbcL-accD sequence, which was thus unsuitable for development as specific barcode. The ycf1b-2 barcode had DNA barcoding gap and high success rate of PCR amplification and DNA sequencing, which was the most suitable DNA barcode for L. lucidum identification and achieved an accurate result. In addition, to optimize the DNA extraction experiment, the authors extracted and analyzed the DNA of the exocarp, mesocarp, endocarp and seed of L. lucidum fruit. It was found that seed was the most effective part for DNA extraction, where DNAs of high concentration and quality were obtained, meeting the needs of species identification. In this study, the experimental method for DNA extraction of L. lucidum was optimized, and the seed was determined as the optimal part for DNA extraction and ycf1b-2 was the specific DNA barcode for L. lucidum identification. This study laid a foundation for the market regulation of L. lucidum.

Feasibility of measuring fusional vergence amplitudes objectively.

Two tests to measure fusional vergence amplitudes objectively were developed and validated against the two conventional clinical tests. Forty-nine adults participated in the study. Participants' negative (BI, base in) and positive (BO, base out) fusional vergence amplitudes at near were measured objectively in an haploscopic set-up by recording eye movements with an EyeLink 1000 Plus (SR Research). Stimulus disparity changed in steps or smoothly mimicking a prim bar and a Risley prism, respectively. Break and recovery points were determined offline using a custom Matlab algorithm for the analysis of eye movements. Fusional vergence amplitudes were also measured with two clinical tests using a Risley prism and a prism bar. A better agreement between tests was found for the measurement of BI than for BO fusional vergence amplitudes. The means ± SD of the differences between the BI break and recovery points measured with the two objective tests were -1.74 ± 3.35 PD and -1.97 ± 2.60 PD, respectively, which were comparable to those obtained for the subjective tests. For the BO break and recovery points, although the means of the differences between the two objective tests were small, high variability between subjects was found (0.31 ± 6.44 PD and -2.84 ± 7.01 PD, respectively). This study showed the feasibility to measure fusional vergence amplitudes objectively and overcome limitations of the conventional subjective tests. However, these tests cannot be used interchangeably due to their poor agreement.

Combined Epimedii Folium and Ligustri Lucidi Fructus with dexamethasone alleviate the proliferation of airway smooth muscle cells by regulating apoptosis/autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) theory believes kidney deficiency is the root cause of chronic refractory asthma with pathological changes of airway remodeling. Our previous experiments confirmed that the combination of Epimedii Folium and Ligustri Lucidi Fructus (ELL) with the effect of nourishing Yin and Yang of the kidney could improve the pathological changes of airway remodeling in asthmatic rats, but the specific mechanism remains unclear. AIM OF THE STUDY: This research was designed to reveal the synergy of ELL and dexamethasone (Dex) in the proliferation, apoptosis, and autophagy of airway smooth muscle cells (ASMCs). MATERIALS AND METHODS: Primary cultures of ASMCs from rats were prepared and induced with histamine (Hist), Z-DEVD-FMK (ZDF), rapamycin (Rap), or 3-Methyladenine (3-MA) at generation 3-7 for 24 or 48 h. Subsequently, the cells were treated with Dex, ELL, and ELL&Dex for 24 or 48 h. The effect of various concentrations of inducers and drugs on cell viability was detected by Methyl Thiazolyl Tetrazolium (MTT) assay, cell proliferation was tested using immunocytochemistry (ICC) by detecting Ki67 protein, cell apoptosis was measured by Annexin V-FITC/PI assay and Hoechst nuclear staining, cell ultrastructure was observed by transmission electron microscopy (TEM), and immunofluorescence (IF), Western blot (WB) combined with quantitative real-time PCR (qPCR) were used for measuring autophagy and apoptosis-related genes including protein 53 (P53), cysteinyl aspartate-specific proteinase (Caspase)-3, microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, mammalian target of rapamycin (mTOR) and p-mTOR. RESULTS: In ASMCs, Hist and ZDF promoted cell proliferation, significantly decreased Caspase-3 protein expression, and up-regulated Beclin-1 levels; Dex alone and in combination with ELL promoted Beclin-1, Caspase-3, and P53 expression, enhancing autophagy activity and apoptosis in Hist and ZDF-induced AMSCs. In contrast, Rap inhibited cell viability, increased Caspase-3, P53, Beclin-1, and LC3-II/I and decreased the levels of mTOR and p-mTOR with promoting apoptosis and autophagy; ELL or ELL&Dex reduced P53, Beclin-1, and LC3-II/I to down-regulate apoptosis and the excessive autophagic state of ASMCs induced by Rap. In the 3-MA model, cell viability and autophagy were reduced; ELL&Dex significantly upgraded the expression of Beclin-1, P53, and Caspase-3 and promoted apoptosis and autophagy of ASMCs. CONCLUSIONS: These results suggest that ELL combined with Dex may regulate the proliferation of ASMCs by promoting apoptosis and autophagy and be a potential medicine for the treatment of asthma.

Alterations in the phytochemical composition and antioxidant activity of Ligustrum robustum according to continuous wet- and dry-heat treatment.

Small-leaved Kuding tea (SLKDT) obtained from Ligustrum robustum is a traditional tea substitute in southern China and has a range of physiological effects. However, the changes in its phytochemical composition after various heat treatments are not reported yet. Thus, the phytochemical composition and antioxidant activities of fresh leaves of SLKDT (LrF1) and SLKDT after high-temperature wet-heat treatment (LrF2) and wet- and dry-heat treatments (LrF3) were assessed using liquid chromatography-mass spectrometry, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities and lipid peroxidation inhibition activity of LrF1 and LrF3 were investigated. The results indicated that the phytochemical composition of LrF1, LrF2, and LrF3 was significantly different. Overall, 258 and 83 differential constituents, respectively, were obtained in LrF1 versus LrF2 and LrF2 versus LrF3. The differential constituents mainly included amino acids and their derivatives, nucleosides, flavonoids, terpenoids, simple phenylpropanoids, and coumarins. After heat treatment, SLKDT exhibited obvious changes in sensory characteristics and physiological properties, which may be related to the changes in the levels of amino acids, linalool, beta-geraniol, myricetin, naringin, fraxetin, and isoacteoside. Moreover, the antioxidant activities significantly changed after heat treatment of SLKDT. Overall, our study demonstrated that heat treatment can alter the phytochemical composition of SLKDT, thus affecting its sensory properties and physiological properties. PRACTICAL APPLICATION: This study preliminarily assessed the changes in the composition of small-leaved Kuding tea (SLKDT) after various heat treatments and revealed that the composition of SLKDT tea can be adjusted by various heat and temperature treatments.

Regulation of bone and fat balance by Fructus Ligustri Lucidi in ovariectomized mice.

CONTEXT: Fructus Ligustri Lucidi (FLL), a commonly used herb of traditional Chinese medicine (TCM), is the fruit of Ligustrum lucidum Ait. (Oleaceae). The ethanol extract of FLL is a potential candidate for preventing and treating postmenopausal osteoporosis (PMOP) by nourishing the liver and kidneys. OBJECTIVE: This study determines whether an ethanol extract of FLL has anti-osteoporotic effects in ovariectomized (OVX) mice and explores the underlying mechanism. MATERIALS AND METHODS: The OVX model of eight-week-old C57BL/6J female mice was taken, and ovariectomy was used as PMOP. Mice were divided into five groups: sham-operated group (n = 10), OVX group (n = 10), OVX + E2 group (n = 10; 0.039 mg/kg), OVX + FLL group (n = 10; 2 g/kg) and OVX + FLL group (n = 10; 4 g/kg). Mice were treated by gavage with FLL or CMCNa once daily for 8 weeks. We harvested uteri, femur, and tibias from mice; bone mineral density (BMD) and bone microstructure were obtained by X-ray absorptiometry and micro-CT. Furthermore, the effect of FLL on the balance of osteoblast and adipocyte differentiation was investigated using bone marrow mesenchymal stem cells (BMMSCs). RESULTS: The results indicated that FLL did not affect OVX-induced estradiol reduction. Compared with OVX mice, FLL significantly increased BMD (63.54 vs. 61.96), Conn. D (86.46 vs. 57.00), and left tibial strength (13.91 vs. 11.27), decreased Tb. Sp (0.38 vs. 0.44) and body fat content (4.19% vs. 11.24%). FLL decreased osteoclast activity and enhanced RUNX2 expression; inhibited perilipin peroxisome proliferator-activated receptor gamma (PPARγ) expression and adipocyte differentiation from BMMSCs. CONCLUSIONS: FLL prevented additional bone loss and improved bone microstructure in OVX mice by modulating bone and fat balance, suggesting that FLL might be a therapeutic agent for PMOP.