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Many herbal species in the genus Ligustrum have been shown to contain compounds with anti-cancer biological activity. This study aimed to isolate some compounds from the leaves of Ligustrum robustum (Roxb.) Blume (L. robustum) and evaluate their effects against liver cancer cells. As a result, seven previously reported compounds (1-7) were isolated, including four lignans (1-4) and three phenolic derivatives (5-7). The structures of these compounds were determined using spectroscopic methods and comparison with reported data. All isolates were assessed for their inhibitory effects on HepG2 liver cancer cells. Screening results revealed that two compounds, isocubein (3) and 4-(2-acetoxyethyl)phenol (7), exhibited strong inhibitory activity against cell proliferation, with IC 50 values of 3.1±0.9 and 4.5±14 μM, respectively. Further analyses demonstrated that both compounds could suppress the formation and development of 3D tumorspheres in terms of quantity and size. Additionally, isocubein (3) and 4-(2-acetoxyethyl)phenol (7) exhibited the ability to inhibit the migration of HepG2 cells. This study represents the first report on the inhibitory activity against HepG2 liver cancer cells of extracts and isolated compounds from L. robustum, providing valuable information for future research aiming to develop products for liver cancer treatment., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)

This study aimed to explore the pharmacodynamics and mechanisms of different processing methods of Ligustrum lucidum Ait. (LLA) in addressing kidney-yin deficiency (KYD). Forty-eight Sprague-Dawley rats were divided into eight groups based on their weight. The KYD model was established by intragastric administration of levothyroxine sodium. Each group was administered the corresponding treatment for 15 consecutive days. The general condition of the rats during the treatment period was observed. In addition, the levels of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and the ratio of cAMP to cGMP in the serum of rats from different groups were measured. Serum samples were analyzed using the ultra-performance liquid chromatography (UPLC)-Orbitrap Fusion MS technique for metabolomics analysis. Compared with the model group, the general condition of the rats in the wine-steamed L. lucidum group (WL) and salt-steamed L. lucidum group (SSL) groups showed significant improvement. The serum levels of cAMP, cGMP, and the cAMP-to-cGMP ratio tended to return to normal. Metabolic analysis identified 38 relevant biomarkers and revealed 3 major metabolic pathways: phenylalanine, tyrosine, and tryptophan biosynthesis; phenylalanine metabolism; and sphingolipid metabolism. The different processing methods of LLA demonstrated therapeutic effects on KYD in rats, likely related to the restoration of disturbed metabolism by adjusting the levels of endogenous metabolites in the kidney. The SSL demonstrated significantly superior effects compared with the other four types of LLA processed products., (© 2024 John Wiley & Sons Ltd.)

Atmospheric particulate matter (PM) pollution has become a major environmental risk, and green plants can mitigate air pollution by regulating their enzymatic activity, osmoregulatory substances, photosynthetic pigments, and other biochemical characteristics. The present investigation aims to evaluate the mitigation potential of five common evergreen tree species ( Photinia serrulata , Ligustrum lucidum , Eriobotrya japonica , Euonymus japonicus , Pittosporum tobira ) against air pollution and to assess the effect of dust retention on plant physiological functions exposed to three different pollution levels (road, campus, and park). The results found that the amount of dust retained per unit leaf area of the plants was proportional to the mass concentration of atmospheric particulate matter in the environment, and that dust accumulation was higher on the road and campus than in the park. There were significant differences in dust retention among the five tree species, with the highest leaf dust accumulation observed for E. japonica (5.45 g·m -2 ), and the lowest for P. tobira (1.53 g·m -2 ). In addition, the increase in PM adsorption by different plants was uneven with increasing pollution levels, with significant decreases in chlorophyll content, photosynthetic and transpiration rate. From a physiological perspective, P. tobira exhibited greater potential to respond to PM pollution. Biochemical indicators suggested that PM pollution caused changes in plant protective enzyme activities, with a decrease in superoxide dismutase (SOD) and peroxidase (POD) activities, as well as promoting membrane lipid peroxidation, and appropriate stress also enables plants to counteract oxidative damage. In particular, PM exposure also induced stomatal constriction. Overall, PM retention was significantly associated with physiological and photosynthetic traits. In conclusion, our study contributes to the understanding of the effects of PM on plant physiology. Furthermore, it also provides insights into the selection of plants that are tolerant to PM pollution., Competing Interests: The authors declare that they have no competing interests., (© 2024 He et al.)

Ligustri Lucidi Fructus (LLF) is a traditional Chinese medicine (TCM) to treat hepatopathy and osteopathy. Wine-processed LLF (WLLF) was much more widely used than raw LLF (RLLF) in clinical practice, however, there is no consensus on processing time. To investigate the processing status of WLLF and transformation rules during processing, a UHPLC-Q-Orbitrap-MS method combined with data-independent acquisition (DIA) mode was firstly established and 227 compounds were identified or tentatively identified. Subsequently, a novel strategy using integration weighted gene co-expression network analysis (WGCNA) with LC-MS data was proposed. A total of 73 differential metabolites were screened out between RLLF and WLLF (wine steaming for 18 h). Meanwhile, the concentration of 11 differential compounds for WLLF was quantified. Finally, correlations between compounds were analyzed by WGCNA and the top five compounds negatively correlated with salidroside were validated, revealing that G13, specnuezhenide, oleuropein, acteoside, and neonuzhenide could be transformed into salidroside and its analogues during processing, respectively. The results indicated that our proposed strategy could be effectively employed to evaluate the processing status of TCMs., 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 © 2024 Elsevier B.V. All rights reserved.)

In this study, we propose a novel strategy utilizing deep eutectic solvents (DESs) as both the extraction solvent and dispersing liquid, with nanometer zinc oxide (ZnO) serving as the adsorbent. This method incorporates ultrasound-assisted matrix solid phase dispersion (UA-MSPD) for the extraction of six active components (salidroside, echinacoside, acteoside, specnuezhenide, nuezhenoside G13, and oleanolic acid) from Ligustri Lucidi Fructus samples. The extracts were then analyzed using high-performance liquid chromatography equipped with a diode array detector. The effects of various parameters such as dispersant dosage, DESs volume, grinding time, ultrasonication duration, and eluent volume on extraction recovery were investigated and optimized using a central composite design under response surface methodology. The optimized conditions yielded detection limits ranging from 0.003 to 0.01 mg/g and relative standard deviations of 8.7% or lower. Extraction recoveries varied between 93% and 98%. The method demonstrated excellent linearity for the analytes (R 2 ≥ 0.9997). The simple, green, and efficient DESs/ZnO-UA-MSPD technique proved to be rapid, accurate, and reliable for extracting and analyzing the six active ingredients in Ligustri Lucidi Fructus samples., (© 2024 Wiley‐VCH GmbH.)

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., 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 © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s).)

The increasing concerns about health have led to a growing demand for high-quality fried foods. The potential uses of Ligustrum robustum (Rxob.) Blume, a traditional tea in China, as natural additives to enhance the quality of starchy food during frying was studied. Results indicated that L. robustum polyphenols extract (LREs) could improve the quality of fried starchy food, according to the tests of color, moisture content, oil content, texture property, and volatile flavor. The in vitro digestion results demonstrated that LRE reduced the final glucose content from 11.35 ± 0.17 to 10.80 ± 0.70 mmol/L and increased the phenolic content of fried starch foods from 1.23 ± 0.04 to 3.76 ± 0.14 mg/g. The appearance and polarizing microscopy results showed that LRE promoted large starch bulges on the surface of fried starchy foods. Meanwhile, X-ray diffraction results showed that LRE increased the intensity of characteristic diffraction peak of fried starch with a range of 21.8%-28%, and Fourier transform infrared results showed that LRE reduced the damage to short-range order structure of starch caused by the frying process. In addition, LRE increased the aggregation of starch granules according to the SEM observation and decreased the enthalpy of starch gelatinization based on the differential scanning calorimetry results. The present results suggest that LREs have the potential to be utilized as a natural additive for regulating the quality of fried starchy food in food industries. PRACTICAL APPLICATION: The enhancement of L. robustum polyphenols on the quality of starchy food during frying was found, and its mechanisms were also explored. This work indicated that L. robustum might be used as a novel economic natural additive for producing high-quality fried foods., (© 2024 Institute of Food Technologists.)

Ligustrum robustum is one of the traditional teas in China with a long history of drinking and medicinal use. Through Response surface optimization, the yield of polysaccharides extracted by ultrasonic-assisted complex enzyme (UAE-EN) method was increased to 14.10 ± 0.56 %. Neutral homogeneous polysaccharide (LRNP) and acidic homogeneous polysaccharide (LRAP-1, LRAP-2, LRAP-3) from L. robustum were purified. The molecular weights of them were 5894, 4256, 4621 and 3915 Da. LRNP was composed of glucose (Glc), galactose (Gal), arabinose (Ara) with molar percentage of 24.97, 42.38 and 30.80. Structure analysis revealed that the backbone of LRNP consisted of 1,5-linked α-Araf, 1,4-linked β-Galp, 1,6-linked β-Galp, and 1,4-linked β-Glcp with the branches of 1,2-linked α-Araf, 1,3-linked α-Araf, 1,3-linked β-Glcp and 1,6-linked β-Galp residues, some terminal residues of α-Araf, β-Glcp and α-Galp were also included. In vitro experiments showed that the four polysaccharides possessed excellent antioxidant, antitumor and hypoglycemic activities. LRNP possessed the protective effect against oxidative stress. The studies provide a basis for further exploitation of L. robustum., 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 © 2024 Elsevier B.V. All rights reserved.)

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, H 2 O 2 -, 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 H 2 O 2 -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., 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 © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 + E 2 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.

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., (© 2023 Wiley-VCH GmbH.)

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 T 10 ( 1a ), T 11 ( 1b ), T 12 ( 2a ), T 13 ( 2b ), T 14 ( 3a ), T 15 ( 3b ), F 1 ( 4b ), T 16 ( 5a ), T 17 ( 5b ), and E 1 ( 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 (IC 50 : 4.38 ± 0.11 μM) was as strong as orlistat (IC 50 : 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 (IC 50 : 6.27 ± 0.23 ~ 8.59 ± 0.09 μM) were stronger than l-(+)-ascorbic acid (IC 50 : 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.

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., (© 2023 Institute of Food Technologists.)

A quantitative analysis method and a chemical pattern recognition method were developed to evaluate raw Ligustri Lucidi Fructus (LLF) from different regions and different processed products. In this study, a comprehensive strategy using ultra-high-performance liquid chromatography-mass spectrometry quantitative analysis method was established for the simultaneous determination of 16 components in 47 batches of LLF covering 19 regions belonging to 8 provinces and 24 batches of different processed products (steamed LLF without auxiliary material, wine-steamed LLF, salt-steamed LLF, and vinegar-steamed LLF). The results of this study indicated that the proposed method was reliable and accurate for the rapid analysis proved by detection limit, quantification limit, precision, and accuracy. Furthermore, principal component analysis and hierarchical cluster analysis were employed to analyze the experimental data, showing that the best-quality samples of 47 batches of raw LLF were S47 (Lantian, Shaanxi), S39 (Pingyang-2, Shandong), S38 (Pingyang-1, Shandong), and S45 (Lingbao, Henan), whereas the worst-quality samples were S7-S16 (Huzhou, Zhejiang). In 24 batches of processed products, the best-quality samples were S48 (salt steamed 2 h), S60 (wine steamed 2 h), and S61 (wine steamed 4 h). Meanwhile, the heat map showed that the contents of triterpenoid saponins, including C16 (ursolic acid), C15 (oleanic acid), and C14 (maslinic acid), were higher than those of other compounds in 71 batches of samples. These results suggested that the quality of raw LLF in the central and northern regions was better than that in the southern regions, and regarding the processed products, different auxiliary materials had little effect on the quality of LLF, but steaming time of 2 h was appropriate. Briefly, this study proposed a multiparameter quantitative analysis method for the overall quality control of raw LLF samples covering different regions in China and different processed LLF., (© 2022 John Wiley & Sons Ltd.)

Ethnopharmacological Relevance: Fructus Ligustri Lucidi (FLL), the fruit of Ligustrum lucidum Ait., is a traditional Chinese medicine that has been used for tonifying the kidney and liver for decades., Aim of the Study: This study aimed to explore and identify polysaccharides from FLL and elucidate its protective effect against renal fibrosis., Materials and Methods: Polysaccharides were extracted and isolated from FLL. The purified fraction was identified by serial phytochemical work, such as gel-permeation chromatography, ion chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance. Mice with unilateral ureteral obstruction (UUO) were applied as a renal fibrosis model. The male UUO mice were pretreated with heteropolysaccharide (Poly) 1 week prior to surgery and continuously treated for 7 days after the operation. Renal fibrosis was assessed by Periodic Acid-Schiff (PAS) staining and Masson's trichrome staining in paraffin-embedded slides. The murine mesangial cells SV40-MES13 upon angiotensin II (Ang II) treatment were developed as an in vitro fibrotic model. The cells were treated by Poly in the presence of Ang II. Molecular expression was detected by RT-PCR, immunoblotting, and immunofluorescence staining., Results: We identified a heteropolysaccharide composed of arabinose and galactose (molar ratio, 0.73:0.27) with a predicted chemical structure characterized by a backbone composed of 1,5-α-Araf, 1,3,5-α-Araf, 1,6-α-Galp, and 1,3,6-β-Galp and side chains comprised of T-α-Araf, T-α-Arap, and 1,3-α-Araf. Pretreatment of UUO mice with Poly effectively alleviated glomerulosclerosis and tubulointerstitial fibrosis. Moreover, Poly pretreatment down-regulated the expression of extracellular matrix (ECM) protein fibronectin (FN), profibrotic factor VEGF, proinflammatory cytokines MCP-1 and Rantes in the obstructed kidney. Similarly, the incubation of SV40-MES13 cells with Poly significantly inhibited Ang II-induced elevation in accumulation and expression level of FN and attenuated Ang II-evoked up-regulation in protein expression of MCP-1 and Rantes., Conclusions: Our study isolated and identified a naturally occurring heteropolysaccharide in FLL and revealed its potential in protecting the kidneys from fibrosis., 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 © 2022 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Ligustri Lucidi Fructus (LLF) is one of the usual Chinese herbs that has long been used with high therapeutic and condition value. LLF is used for the treatment of dizziness and tinnitus, soreness and weakness of the waist and knees, premature greying of the hair, the darkness of the eyes, internal heat and thirst, bone steam and hot flashes and other symptoms., Aim of the Study: This review reviews botany, traditional uses, processing, phytochemistry, quality control, pharmacology, toxicity and pharmacokinetics to better understand its therapeutic potential., Materials and Methods: The literature on LLF was obtained from Google Scholar and Baidu Scholar, PubMed, ScienceDirect, SciFinder, Web of Science, China National Knowledge Infrastructure (CNKI), WAN FANG DATA and libraries. Some local books, official websites, PhD or MS's dissertations were also included. Phytochemical constituents' structures were drawn by ChemDraw software., Results: So far, Multiple chemical components were isolated and identified from LLF, mainly including terpenoids and flavonoids. Modern studies have shown that LLF extracts and compounds have a wide range of pharmacological effects, including antitumor, liver protection, blood glucose, lipid-lowering, immune regulation, and other aspects., Conclusions: LLF occupies an important position in the traditional medical system. It is cost-effective and is a significant plant with therapeutic applications in modern medicine. However, further in-depth studies are needed to determine the medical use of this plant and its chemical composition, pharmacological activity, quality control, toxicity and pharmacokinetics., 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 © 2022 Elsevier B.V. All rights reserved.)

The leaves of Ligustrum robustum have been consumed as Ku-Ding-Cha for clearing heat and removing toxins, and they have been used as a folk medicine for curing hypertension, diabetes, and obesity in China. The phytochemical research on the leaves of L. robustum led to the isolation and identification of two new hexenol glycosides, two new butenol glycosides, and five new sugar esters, named ligurobustosides X ( 1a ), X 1 ( 1b ), Y ( 2a ), and Y 1 ( 2b ) and ligurobustates A ( 3a ), B ( 3b ), C ( 4b ), D ( 5a ), and E ( 5b ), along with seven known compounds ( 4a and 6 - 10 ). Compounds 1 - 10 were tested for their inhibitory effects on fatty acid synthase (FAS), α-glucosidase, and α-amylase, as well as their antioxidant activities. Compound 2 showed strong FAS inhibitory activity (IC 50 4.10 ± 0.12 μM) close to that of the positive control orlistat (IC 50 4.46 ± 0.13 μM); compounds 7 and 9 revealed moderate α-glucosidase inhibitory activities; compounds 1 - 10 showed moderate α-amylase inhibitory activities; and compounds 1 and 10 displayed stronger 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) ammonium salt (ABTS) radical scavenging effects (IC 50 3.41 ± 0.08~5.65 ± 0.19 μM) than the positive control l-(+)-ascorbic acid (IC 50 10.06 ± 0.19 μM). This study provides a theoretical foundation for the leaves of L. robustum as a functional tea to prevent diabetes and its complications.

In this study, we aimed to demonstrate the therapeutic effect of Ligustri Lucidi Fructus on chemotherapy-induced myelosuppression and elucidate its mechanism. A pharmacological study was conducted to investigate the mechanism of the inhibiting effects of Ligustri Lucidi Fructus on cyclophosphamide-induced bone marrow suppression in mice. HPLC was used to measure the chemical components. We demonstrated that medium and high doses of Ligustri Lucidi Fructus increased the amount of white blood cells and bone marrow nucleated cells (p < 0.05) in the cyclophosphamian-induced mouse model, and at the same time reduced granulocyte-macrophage-colony stimulating factor and thrombopoietin in the serum of myelosuppression mice (p < 0.01). Medium and high doses of Ligustri Lucidi Fructus can also adjust the thymus index and spleen index(p < 0.05). Ligustri Lucidi Fructus regulates the balance of bcl-2/bax, inhibits the expression of Caspase-3 and meanwhile stimulates the expression of mitogen-activated protein (MEK) and phospho extracellular regulated protein kinases (p-ERK) on the MAPK pathway. Five chemical constituents of Ligustri Lucidi Fructus, which may be related to myelosuppression, were analyzed. The content of specnuezhenide was 0.281%, that of ligustroflavone was 0.004%, that of salidroside was 0.094%, that of hydroxytyrosol was 0.060% and that of tyrosol was 0.069%. The effect of Ligustri Lucidi Fructus on myelosuppression after chemotherapy may be related to its multicomponent and multitarget nature. Ligustri Lucidi Fructus may be a promising potential drug for treatment after chemotherapy., (© 2022 John Wiley & Sons Ltd.)

Background: Ulcerative colitis (UC) is pathologically characterized by an inappropriate immune response to the gut commensal microbes accompanied by persistent epithelial barrier dysfunction, and its progression increases the susceptibility to colitis-associated cancer (CAC), as well as other complications. Fructus ligustri lucidi (FLL) has a long historical application in traditional Chinese medicine due to its various pharmacological effects, including antioxidation and anti-inflammation. The present study aimed to explore the molecular and cellular mechanisms of FLL in treating colitis., Methods: A high-performance liquid chromatography (HPLC) combined with ultraviolet (UV) was performed to validate the quality of FLL; Network pharmacology analysis and weighted gene co-expression network analysis (WGCNA) based on The Cancer Genome Atlas (TCGA) database predicted the therapeutic value of FLL against UC and CAC; 2% dextran sodium sulfate (DSS) was administered to mice to establish murine models of experimental colitis, and FLL was given for the next 14 days at different concentrations; 16S rRNA sequencing and untargeted metabolomics were performed on fecal samples to delineate the alteration in microbiome profile; Western blotting, flow cytometry, and immunocytochemistry experiments were conducted to confirm the predicted cellular mechanisms., Results: Network pharmacology analysis and WGCNA predicted that the targets of the FLL were associated with the progression of UC and the survival of patients with colorectal cancer by regulating tumor necrosis factor (TNF) and IL-17 signaling pathways, immune cell functions, responses to bacterial and reactive oxygen species (ROS), and cell proliferation. In vivo experiments corroborated that the high dose of FLL significantly attenuated the progression of experimental colitis by reversing the weight loss and bloody stool, reconstructing the integrity of colorectal epithelium, and suppressing the concentration of pro-inflammatory cytokines. Moreover, FLL treatment reduced the transition of macrophages (Mφs) to the proinflammatory phenotype and promoted Mφs-regulated wound healing, and suppressed the production of ROS in intestinal organoids (IOs) and crypts. 16S rRNA and untargeted metabolomics showed that the administration of FLL inhibited DSS-caused colonization of the potentially pathogenic gut microorganisms and reversed DSS-influenced metabolic profile., Conclusion: FLL is a potent anti-colitis drug by suppressing inflammation and rescuing dysbiosis., 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 © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The potential uses of Ligustrum robustum (Rxob.) Blume extract as a natural antioxidant to protect the quality of different oils during storage and frying process were studied. The results showed that L. robustum extract has been shown to retard the decline in the quality of both oils based on the tests of acid value, peroxide value, p-anisidine value, color, volatile flavor, and fatty acid compositions, and the protective effect of L. robustum extract on the quality of peanut oil was better than that of palm oil. By the component analysis, L. robustum extract was found to have a total phenols content of 140.75 ± 1.52 mg/g, and ligurobustoside C was identified as the main phenolic compound. The thermogravimetric and differential scanning calorimetry results showed that L. robustum extract enhanced the oxidative stability of peanut and palm oils. In addition, Fourier transform infrared results indicated that L. robustum extract had protective effects on the C=C bond and ester bond of oil molecule. Moreover, by using electron spin resonance technique, L. robustum extract showed the ability to inhibit and scavenge alkyl-free radicals in both oils. The present results suggested that L. robustum extract may protect the quality of oils during the storage and frying process by inhibiting the oxidation of unsaturated fatty acids and might be a potential natural antioxidant in the food industry. PRACTICAL APPLICATIONS: The excellent antioxidant ability of Ligustrum robustum (Rxob.) Blume extract on the oxidation of different oils and its low price indicated that it could be used as a new low-cost natural antioxidant in oil processing., (© 2022 Institute of Food Technologists®.)

Epimedium has a wide range of clinical applications; however, there have been numerous reports of adverse reactions in recent years, which has resulted in it being changed from a widely recognized "nontoxic" to a "potentially toxic" traditional Chinese medicine. The combination of Epimedium and Ligustri lucidi fructus is commonly used in the clinic. The purpose of this study was to investigate the pharmacokinetic characteristics of Epimedium and Ligustri lucidi fructus to explore the possible synergism and reduction in toxicity. Based on liquid chromatography tandem mass spectrometry, a method was established for the determination of icariin, epimedin A, epimedin B, epimedin C, baohuoside Ⅰ, and specnuezhenide in biological samples and was successfully applied to study the pharmacokinetics of the drug pair. The results showed that the five flavonoids (specnuezhenide could not be detected) could be rapidly absorbed into the blood, and the second peak time in vivo was earlier after the combination, indicating that the metabolic pathway may be changed. In addition, combination with Ligustri lucidi fructus could significantly reduce the concentration of 5 flavonoids in vivo and increase their elimination rate, which may attenuate their virulence, thus providing a reference for the rational clinical use of Epimedium., (© 2022 Wiley-VCH GmbH.)

Background: Benign prostatic hyperplasia (BPH) is a urinary system disease with high prevalence among the middle and elder men. In BPH, proliferation of prostate cells and the imbanlance between androgen and estrogen are both important inducers. Previous studies have demonstrated that compounds from Ligustri Lucidi Fructus (LLF) and Ecliptae Herba (EH) are of phytoestrogenic or phytoandrogenic activities. The combination of LLF with EH at the ratio of 1:1 on crude drugs quantity is called Erzhi formula (EZF), which is used for in vivo research of our study., Purpose: This study aimed to investigate potential mechanisms of EZF and its active pharmaceutical ingredients on BPH in vitro and in vivo., Methods: Therapeutic effects of EZF was evaluated in E 2 /testosterone (1:100) induced BPH rats model. The pathological changes of prostate, concentrations of testosterone, DHT, E 2 , PSA in rats' plasma and prostate were detected. The expressions of PCNA, AR, ERα, ERβ, SRD5A1, SRD5A2 were measured in BPH rat prostates and E 2 -stimulated human benign prostatic epithelial cells (BPH-1)., Results: EZF treatment significantly attenuated rat prostate enlargement, alleviated BPH pathological features, and decreased the expression of PCNA. The up-regulation of AR, ERα, SRD5A1/2 expressions, and down-regulation of ERβ expression at prostate of rat BPH model were significantly blocked by EZF administration. The expression levels of testosterone, DHT, E 2 , PSA were strongly inhibited by EZF treatment. At the cellular level, ligustrosidic acid and echinocystic acid inhibited E 2 -induced BPH-1 cell proliferation and PCNA expressions, which were consistent with the results in vivo. And these two ingredients also down-regulated the expressions of AR, ERα, SRD5A1/2 and up-regulated the expression of ERβ in BPH-1 cells., Conclusion: EZF, ligustrosidic acid from LLF and echinocystic acid from EH showed inhibitive effects on BPH via down-regulating prostatic AR, ERα, SRD5A1/2 expressions and up-regulating ERβ expression., (Copyright © 2022. Published by Elsevier GmbH.)

The leaves of Ligustrum robustum have been applied as Ku-Ding-Cha, a functional tea to clear heat, remove toxins, and treat obesity and diabetes, in Southwest China. The phytochemical research on the leaves of L. robustum led to the isolation and identification of eight new monoterpenoid glycosides ( 1 - 8 ) and three known monoterpenoid glycosides ( 9 - 11 ). Compounds 1 - 11 were tested for the inhibitory activities on fatty acid synthase (FAS), α -glucosidase, α -amylase, and the antioxidant effects. Compound 2 showed stronger FAS inhibitory activity (IC 50 : 2.36 ± 0.10 μM) than the positive control orlistat (IC 50 : 4.46 ± 0.13 μM), while compounds 1 , 2 , 5 and 11 displayed more potent ABTS radical scavenging activity (IC 50 : 6.91 ± 0.10~9.41 ± 0.22 μM) than the positive control L-(+)-ascorbic acid (IC 50 : 10.06 ± 0.19 μM). This study provided a theoretical basis for the leaves of L. robustum as a functional tea to treat obesity.

Ligustri Lucidi Fructus, the sun-dried mature fruit of Ligustrum lucidum, is cool, plain, sweet, and bitter, which can be used as both food and medicine, with the effects of improving vision, blacking hair, and tonifying liver and kidney. It takes effect slowly. However, little is known about the genetic information of the medicinal plant and it is still a challenge to distinguish Ligustrum species. In this study, the complete chloroplast genome of L. lucidum was obtained by genome skimming and then compared with that of five other Ligustrum species, which had been reported. This study aims to evaluate the interspecific variation of chloroplast genome within the genus and develop molecular markers for species identification of the genus. The result showed that the chloroplast genome of L. lucidum was 162 162 bp with a circular quadripartite structure of two single-copy regions separated by a pair of inverted repeats. The Ligustrum chloroplast genomes were conserved with small interspecific difference. Comparative analysis of six Ligustrum chloroplast genomes revealed three variable regions(rbcL-accD, ycf1a, and ycf1b), and ycf1a and ycf1b can be used as the species-specific DNA barcode for Ligustrum. Phylogeny analysis provided the best resolution of Ligustrum and supported that L. lucidum was sister to L. gracile. This study clarified the genetic diversity of L. lucidum from provenance, which can serve as a reference for further analysis of pharmacological differences and breeding of excellent varieties with stable drug effects.

Ligustri Lucidi Fructus is a dried and mature fruit of Ligustrum lucidum Ait., which has the effects of nourishing liver and kidney. Herein, an accurate and sensitive method was established for the separation and identification of the absorbed constituents and metabolites of Ligustri Lucidi Fructus in rat plasma based on ultra-high-performance liquid chromatography-Q-Exactive Orbitrap tandem mass spectrometry. A total of 73 prototype constituents and 148 metabolites were identified or characterized in administered plasma, and the possible metabolic pathways of constituents mainly involved hydroxylation, sulfation, demethylation, and glucuronidation. Besides, the network pharmacology was further investigated to illuminate its potential mechanism of treatment for liver injury by the biological targets regulating related pathways. Network pharmacological analysis showed that target components through 399 targets regulate 220 pathways. The docking results showed that 36 key target components were closely related to liver injury. Overall, the study clearly presented the metabolic processes of Ligustri Lucidi Fructus and gave a comprehensive metabolic profile of Ligustri Lucidi Fructus in vivo first. Combining with network pharmacology and molecular docking discovered potential drug targets and disclose the biological processes of Ligustri Lucidi Fructus, which will be a viable step toward uncovering the secret mask of study for traditional Chinese medicine., (© 2021 Wiley-VCH GmbH.)

Ethnopharmacological Relevance: Fructus Ligustri Lucidi (FLL) is an edible herb with anti-osteoporotic activity, yet whether and how the aqueous extract of this herb affect calcium metabolism in preservation of bone quality remain unclear., Aim of the Study: To investigate the effects of FLL aqueous extract on calcium balance and short-chain fatty acids (SCFAs) production in ovariectomized (OVX) rats., Materials and Methods: OVX rats were daily and orally administrated with FLL aqueous extract (3.5 g/kg) for 14 weeks. The levels of N-terminal propeptide of type I collagen (PINP) and C-terminal telopeptide of type I collagen (CTx-I) in rat serum were evaluated by ELISA assays. The concentration of calcium in serum, urine, and feces were determined by biochemical assays. Bone quality was determined by Micro-CT, a three-point bending assay, and Fourier Transform Infrared (FTIR) Spectrometry. The expressions of Calbindin D28K and Calcium-sensing receptor (CaSR) in kidney as well as the Vitamin D receptor (VDR), the transient receptor potential vanilloid receptor 6 (TRPV6), Calbindin D9k in the duodenum were measured by immunohistochemistry, western blotting, or real-time PCR. The short-chain fatty acids (SCFAs) levels in the feces of the cecum were tested by gas chromatograghy., Results: The administration of FLL to OVX rats resulted in a significant improvement in bone mineral density and biomechanical strength as well as in maintaining bone microstructures and material quality. Meanwhile, the decreased levels of PINP and increased levels of CTx-I in OVX rats were restored by FLL treatment. Additionally, FLL treatment increased calcium absorption, upregulated VDR, TRPV6, Calbindin D9k expressions in the duodenum, Calbindin D28K in kidney, and down-regulated CaSR expression in the kidney, as well as enhanced SCFAs levels in the feces of OVX rats., Conclusions: FLL aqueous extract may preserve bone quality through regulation of the calcium balance and intestinal SCFAs production in OVX rats. This offers translational value of FLL into osteoporosis clinical trial., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Scope: Atherosclerosis (AS) is closely related to gut microbiota. Previous studies demonstrates that Ligustrum robustum (LR), a flavonoid-rich tea like plant, can mitigate several AS-related risk factors and modulate gut microbiota in animal models and human subjects. But its anti-AS effect and mechanisms remain unclear. Therefore, in this study, impacts of LR on AS development are investigated and the potential underlying mechanisms in C57BL/6J and Apoe -/- mice are explored., Methods and Results: Female C57BL/6J and Apoe -/ - mice are fed a chow diet or high-choline diet, supplemented with vehicle (water) or LR water extract (700 mg kg -1 ) by gavage for 17 weeks. It is found that LR attenuates diet-induced AS by reducing serum trimethylamine and trimethylamine-N-oxide (TMAO) levels likely by modulating gut microbiota. Moreover, LR increases the abundance of the genus Bifidobacterium, which generates bile salt hydrolase, and thus presumably enhances bile acid (BA) deconjugation and increases fecal BA excretion. Meanwhile, LR increases fecal cholesterol excretion, decreases the levels of serum and hepatic cholesterol, but did not affect short-chain fatty acids in feces., Conclusion: LR attenuates AS development presumably by decreasing serum TMAO levels and increasing fecal BA excretion likely via gut microbial modulation. These effects are accompanied by increases in fecal cholesterol excretion and decreases in serum and hepatic cholesterol., (© 2021 Wiley-VCH GmbH.)

Ethnopharmacological Relevance: Erzhi formula (EZF) consists of Ecliptae herba (EH) and Fructus Ligustri Lucidi (FLL) at a ratio 1:1, and constitutes a well-known formula in China that is commonly used for treating menopausal diseases., Aim of the Study: In this study, we explored the pharmacologic actions and potential molecular mechanisms underlying EZF's action in preventing and treating osteoporosis., Materials and Methods: The active components and related targets of EZF's anti-osteoporotic effects were predicted by network pharmacology, and functional enrichment analysis was also performed. We then used an osteoporosis model of ovariectomized (OVX) mice to detect the effects of EZF on osteoporosis., Results: The results from network pharmacology identified a total of 10 active ingredients from EH and 13 active ingredients from FLL that might affect 65 potential therapeutic targets. GO enrichment analysis revealed that EZF affected bone tissue primarily via hormone (particularly estradiol)-related pathways and bone resorption by osteoclast differentiation. KEGG analysis demonstrated that bone-related factors such as Runt-related transcription factor 2 (Runx2), Ca2, estrogen receptor1 (ESR1), androgen receptors (AR), and TNFα served as the primary targets during osteoclastic differentiation. In vivo experiments showed that the formula significantly improved the diminution in estrogen and the subsequent uterine atrophy induced by ovariectomy (P < 0.01 or 0.05), implying that the EZF exerted its actions via regulation of estradiol and the nourishing effects of the uterus in OVX mice. Dual-energy X-ray absorptiometry and micro-CT showed that EZF significantly inhibited bone loss and improved bone micro-architecture by statistically increasing the number of bone trabeculae and decreasing the separation of bone trabeculae in OVX mice (P < 0.01 or 0.05); EZF also inhibited bone loss and enhanced bone-fracture load. Furthermore, we confirmed that EZF reduced the calcium concentrations, augmented protein and mRNA levels for Runx2 in the bone marrow, and reduced PPARγ levels. RANKL-a key downstream regulatory protein of many targets that was referred to in our results of network pharmacology as being involved in the regulation of osteoclastogenesis-was significantly diminished by EZF; it also elevated OPG content. In addition, we used monocytes of bone-marrow origin to detect the effects of the potential components of EZF on osteoclast differentiation and found that wedelolactone, oleanolic acid, echinocystic acid, luteolin, and luteolin-7-o-glucoside significantly inhibited osteoclast differentiation from monocytes induced by 25 ng/mL MCSF and 50 ng/mL RANKL (P < 0.01 or 0.05)., Conclusions: Our present study indicated that EZF significantly inhibited the bone loss induced by OVX in mice by its regulation of estradiol combined with the nourishing effect of the uterus, and that it also attenuated bone resorption by decreasing the RANKL/OPG ratio so as to inhibit osteoclast maturation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ligustri Lucidi Fructus (LLF) is the dried and mature fruit of Ligubtrum lucidum Ait., which has the effect of nourishing the liver and kidney, brightening the eyes and promoting the growth of black hair. Wine-processed LLF is commonly used in traditional Chinese medicine; however, the processing mechanisms are still unclear. Herein, a system data acquisition and mining strategy was designed to investigate the chemical profile differences between the raw and wine-processed LLF, based on high-performance liquid chromatography-Orbitrap high resolution mass spectrometry coupled with multivariate statistical analysis including principal component analysis and partial least square analysis. Afterwars, a total of 55 components were found to be the main contributors to the significant difference between raw and wine-processed LLF by comparison with chromatographic behaviors, intact precursor ions, and characteristic MS fragmentation patterns. In addition, 10 main constituents of raw and wine-processed LLF were simultaneously determined by UHPLC-MS/MS for analyzing the content variations. Some structural transformation mechanisms during wine processing were deduced from the results. The results may provide a scientific foundation for deeply elucidating the wine-processing mechanism of LLF., (© 2020 John Wiley & Sons Ltd.)

A functional yogurt was prepared with Ligustrum robustum (Rxob.) Blume extract (LRE), while its antioxidant and hypoglycemic activities were evaluated and its action mechanism was further explored. With the cofermentation of LRE, the yogurt showed the fine quality characteristics, including pH, titratable acidity, texture, syneresis susceptibility, color, microbiological content, and chemical composition. Meanwhile, the yogurt exhibited the remarkable antioxidant capability to enhance the activities of antioxidant enzymes and reduce the malondialdehyde level in animal serums. In addition, the yogurt showed the obvious hypoglycemic activity to inhibit the decrease of glucose tolerance and the increase of postprandial hyperglycemia of diabetes mice. Furthermore, using the analysis of molecular docking, the main compounds of LRE could combine tightly with α-amylase and α-glucosidase by hydrogen bond and hydrophobic interaction, so as to change their spatial structure and inhibit their biocatalytic activity in glucose metabolism. All present results suggested that LRE showed the potential value to be used as supplement to enhance the quality and functions of yogurt in food industry. PRACTICAL APPLICATION: The hypoglycemic and antioxidant activities of a functional yogurt cofermented with LRE were found and its relative action mechanism was also explored. This work provide the experimental and theoretical basis for the application of this yogurt as nutraceuticals to protect human health in food industry., (© 2021 Institute of Food Technologists®.)

Fructus Ligustri Lucidi (FLL) has been preclinically and clinically used to treat musculoskeletal diseases. However, whether and how FLL affect the canonical Wnt/β-catenin signaling in the management of osteoporosis remains largely unknown. To this end, ovariectomized (OVX) rats and primary osteoblasts were administrated with FLL aqueous extract and medicated serum, respectively. Supplement of FLL to OVX rats maintains bone quality by attenuating the reduction in bone mineral density, strength and microstructure. The maintenance may be associated with upregulating the expression of insulin-like growth factor-1, osteoprotegerin, phospho (p)-low-density lipoprotein receptor-related protein 6, p-glycogen synthase kinase 3 beta (GSK3β), β-catenin, Runx2 and c-Myc, and downregulating the expressions of sclerostin (SOST), dickkopf-related protein 1 (DKK1), GSK3β and p-β-catenin in rat femurs and tibias. In addition, the medicated serum promotes osteoblastic bone formation through activation of Wnt/β-catenin signaling via inhibition of DKK1 and SOST overexpression. Salidroside may be one of the active ingredients in FLL that are beneficial for bone homeostasis. In summary, our results suggest that FLL may preserve bone quality through induction of canonical Wnt/β-catenin signaling via inhibition of DKK1 and SOST overexpression. And FLL may offer a new source of the DKK1 or SOST inhibitors in protection against osteoporosis., (© 2020 John Wiley & Sons Ltd.)

Depression is partially caused by inflammation in central nervous system. This study investigated the ameliorative effects of phenol glycosides (PG) from Ligustrum lucidum Ait. (Oleaceae) on neuroinflammation and depressive-like behavior in mice hypothalamus as well as the molecular mechanism. Mice were administered with PG extract for 2 weeks prior to treatment with LPS. The mice treated with PG extract showed resistance to LPS-induced reduction in body weight and LPS-induced depressive-like behaviors shown by sucrose preference, tail suspension test, forced swimming test and open field test. LPS-induced activation of microglial cells and elevation in protein expression of inflammatory cytokines including IL-1β, RANTES and MCP-1 in hypothalamus of mice were abrogated by pre-treatment with PG extract. This extract down-regulated expression of TLR4, MyD88, NLRP3, renin and angiotensin II and decreased proportional area of Iba-1 + microglias in hypothalamus. Pre-treatment with PG extract inhibited LPS-triggered activation of CaSR/G α11 signaling, stimulated 1-OHase expression in hypothalamus, and enhanced circulating 1,25(OH) 2 D 3 level. Overall, pre-treatment with PG extract ameliorated LPS-induced depressive-like behaviors by repressing neuroinflammation in mice hypothalamus which was attributed to its suppression on activation of microglia and production of inflammatory cytokines via acting on TLR4 pathway, CaSR and RAS cascade associated with improving vitamin D metabolism., (© 2020 John Wiley & Sons, Ltd.)

An accurate and sensitive ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry method was established and validated for the determination of nine bioactive compounds of Ligustri Lucidi Fructus in rat plasma. Separation was performed on Halo ® C 18 column with a mobile phase of acetonitrile and 0.1% formic acid in water. The eluate was detected by multiple reaction monitoring scanning operating in the negative ionization mode. This assay method was validated for selectivity, linearity, intra- and interday precision, accuracy, recovery, matrix effect, and stability, and all methodological parameters fulfilled the Food and Drug Administration criteria for bioanalytical validation. The established method was successfully applied to a comparative pharmacokinetic study of raw and wine-processed Ligustri Lucidi Fructus in rats for the first time. It was found that the AUC 0-24 and C max value of salidroside, hydroxytyrosol, and nuezhenidic acid were increased significantly after processing, while the AUC 0-24 and C max value of oleoside 11-methyl ester, 1'''-O-β-d-glucosylformoside, specnuezhenide, G13, oleonuezhenide, and oleanolic acid were decreased, which suggested that processing affects the absorption and bioavailability of Ligustri Lucidi Fructus. The results might be valuable for the clinical reasonable application and understanding the processing mechanism of Ligustri Lucidi Fructus., (© 2020 Wiley-VCH GmbH.)

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., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020. Published by Elsevier B.V.)

The identification and quantization of traditional Chinese medicine (TCM) are a challenge for researchers and industry. Using untargeted analytical methods, the in vivo detection and identification of TCM compounds are difficult because of the significant interference of endogenous substances. Fortunately, the ongoing development of new analytical technologies, especially Q-Orbitrap-MS, offers some solutions. Our team developed a holistic MS method, combining untargeted data-dependent MS 2 (dd-MS 2 ) modes to extensively identify TCM prototypes in vivo. The method was successfully applied to the analysis of Ligustri Lucidi Fructus (LLF). LLF is a widely used TCM with a remarkable nourishing effect on the liver and kidney. In the study, we aimed to identify the prototypes in rat plasma after oral administration of LLF extract. Following separation on an HSS T 3 column, LLF extract and rat plasma were performed in untargeted dd-MS 2 mode. Forty-seven compounds were characterized in rats plasma as prototypes of LLF extract. Furthermore, seven major prototypes were chosen as pharmacokinetic markers to investigate LLF's pharmacokinetic properties. The results provides comprehensive determination of compounds in LLF both in vitro and in vivo, which is important for quality control, pharmacology studies and clinical use of LLF., (© 2020 John Wiley & Sons, Ltd.)

Cordycepin has recently received increased attention owing to its extensive pharmacological activity. Adenosine deaminase (ADA) is widely distributed in mammalian blood and tissues; as a result, cordycepin is quickly metabolized upon entering into the body and converted into the inactive metabolite 3'-deoxyinosine, thus limiting its activity when administered alone. We herein present a novel ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for screening ADA inhibitors against the metabolism of cordycepin. Cordycepin and 3'-deoxyinosine were chosen as substrate and product, respectively. A proper separation was achieved for all analytes within 3 min. 3'-Deoxyinosine was quantified in the presence or absence of potential ADA inhibitors to evaluate ADA activity. The assay can simultaneously determine substrate and product, with the endogenous substance and ADA inhibitors added not interfering in its activity. After optimizing the enzymatic incubation and UHPLC-MS/MS conditions, K m and V max values for ADA deamination of cordycepin were 95.18 ± 7.85 μm and 363.90 ± 12.16 μmol/min/unit, respectively. Oleanolic acid and ursolic acid from Ligustri Lucidi Fructus were chosen as ADA inhibitors with half maximal inhibitory concentration values of 21.82 ± 0.39 and 18.41 ± 0.14 μm, respectively. A non-competitive inhibition model was constructed and this assay can be used to screen other potential ADA inhibitors quickly and accurately., (© 2019 John Wiley & Sons, Ltd.)

A simple and efficient sample preparation method to extract four bioactive compounds (echinacoside, specnuezhenide, oleuropein and nuezhenoside G13) from Ligustri Lucidi Fructus was established by vortex-forced matrix solid phase dispersion (VFMSPD) method. Chitosan solution was applied as green eluent in this procedure and Celite AZO was employed as dispersant. High performance liquid chromatography (HPLC) equipped with ultraviolet (UV) detector was used to analyze the target analytes. The best result of the investigation was obtained with Celite AZO as dispersant, sample/ dispersant ratio as 1:1, grinding for 2 min, 1 mL high-viscosity chitosan solution (0.5 mg mL -1 ) used as the elution reagent and vortex mixing for 1.5 min. The method exhibit a good linearity for the analytes (r 2  > 0.999). The absolute recoveries of the four target compounds in Ligustri Lucidi Fructus ranged from 90.7% to 98.8% and the relative recoveries of the target compounds ranged from 99.2% to 102% (RSD ≤ 3.4%), which were obtained by the final optimization method. Consequently, the newly developed chitosan solution-based vortex-forced matrix solid phase dispersion (MSPD) combined with HPLC could be efficiently applied to extract and analyze the target compounds in Ligustri Lucidi Fructus samples., (Copyright © 2019. Published by Elsevier B.V.)

To evaluate the xanthine oxidase inhibitory activity of the chemical constituents of Ligustrum lucidum in vitro, the spectrum-effect relationship was investigated. The high-performance liquid chromatography fingerprint was established by ultraviolet spectrophotometry, and the xanthine oxidase inhibitory activity was tested in vitro by a high-throughput screening method. Cluster analysis, principal component analysis, gray correlation analysis, and partial least squares regression were used to explore the spectrum-effect relationships. Sixty batches of Ligustrum lucidum were collected from 16 provinces for testing. The results revealed differences among the batches of medicinal materials, and the similarity score was between 0.635 and 0.968. Thirty-three characteristic peaks (1-33) were calibrated by fingerprint evaluation software for traditional Chinese medicine. The spectrum-effect relationship study further revealed that the contents of peaks 1, 2, 4, 5, 6, 7, 14, 17, 25, 28, 31, and 33, which are potentially critical ingredients for quality control of Ligustrum lucidum fruit, were highly correlated with the inhibition of xanthine oxidase activity., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Osteoarthritis (OA) is a common degenerative disease of synovial joints caused by inflammation. Acteoside (ACT), a major component and lipase inhibitor from the Chinese tea Ligustrum purpurascens kudingcha, has been reported to regulate the inflammation and immune response. The study aims to investigate the effects of ACT on inflammatory responses and joint protection in OA rats., Methods: Cell proliferation was examined by MTT and colony formation assay. Apoptosis was analyzed using flow cytometry with Annexin V/PI staining. ELISA was employed to examine the concentration of inflammatory cytokines. OA rat model was established by surgery stimulation., Results: ACT treatment significantly inhibited the upregulation of inflammatory cytokines induced by IL-1β in primary chondrocytes, including IL-6, IL-12, TNF-α and IFN-γ. ACT stimulation also enhanced the cell proliferation, while inhibited cell apoptosis in IL-1β-treated chondrocytes. Consistently, ACT treatment led to downregulation of cleaved-caspase-3 and apoptosis regulator Bax, and upregulation of Bcl-2. Furthermore, ACT treatment inhibited IL-1β-induced activation of JAK/STAT pathway. The results were confirmed in surgery-induced OA rat model. Moreover, ACT treatment significantly inhibited synovial inflammation and articular chondrocyte apoptosis in OA rats., Conclusion: Our findings indicate that ACT has the potential therapeutic effect on OA through inhibiting the inflammatory responses via inactivating JAK/STAT signaling pathway.

Objective: To examine the effect of the aqueous extract of Ligustrum robustum on tumor growth in vitro and in vivo and explore the possible molecular mechanisms., Methods: In in vitro study, cell viabilities of human cervical carcinoma cells (HeLa), human breast cancer cells (MCF-7), human prostate cancer cells (PC-3), human hepatoma cells (7721) and human colon carcinoma cells (SW480) were evaluated with cell counting kit-8. For L. robustum-treated Hela cells, early or late apoptosis were evaluated by annexin V/PI staining. Mitochondrial membrane potential was measured by staining cells with JC-1. Apoptosis was monitored by nuclear morphology based on chromatin condensation and fragmentation by 4',6-diamidino-2-phenylinole (DAPI) staining. Caspase-3 and -8 activity levels were measured by a colorimetric assay. In vivo, to evaluate the possible mechanism of L. robustum-mediated antitumor effect, nude mouse xenograft study was also conducted., Results: In in vitro study, L. robustum was found to be toxic to HeLa, MCF-7, PC-3, 7721, SW480, with an half maximal inhibitory concentration value of 2-5 mg/mL (P<0.05). Moreover, externalization of phosphatidylserine, loss of mitochondrial membrane potential, DNA fragmentation and activation of caspase-3 and -8 were detected in L. robustum-treated Hela cells. Using a nude mouse model bearing Hela xenografts, we found that L. robustum reduced tumor volume and tumor weight (P<0.05), but had no effect on body weight and histological damage of important organs. Intraperitoneal injection of L. robustum caused a significant reduction in serum aspartate transaminase and alanine transaminase levels (P<0.05). Furthermore, cleaved caspase-3-positive and terminal nucleotidyl transferase-mediated nick end labeling (TUNEL)-positive cells were observed in L. robustum-treated tumor tissues., Conclusions: L. robustum inhibits tumor cell growth both in vitro and in vivo by inducing apoptosis in a caspase-dependent way without apparent hepatic toxicity and histological damage, which may offer partial scientific support for the ethnopharmacological claims of L. robustum as a herbal tea for its antitumor activity.

Ligustrum lucidum Aiton and its berries have been used in Chinese traditional medicine for around two thousand years. In the present study, L. lucidium berries harvested in two regions of Portugal were studied. Haemolytic activity and inhibition of oxidative haemolysis as well as the enzyme inhibitory activities (α-amylase enzyme and acetylcholinesterase) were assessed. Results suggest that the different biological activities varied according to the region where samples were collected. Results demonstrated that the sample obtained from region R1 was the most efficient extract for all parameters evaluated, presenting the lowest values of IC 50 , 10.67 ± 0.46 μg/mL for the inhibition of erythrocyte oxidative haemolysis, 58.28 ± 3.77 μg/mL for the α-amylase enzyme and 67.67 ± 2.10 μg/mL for the acetylcholinesterase inhibition. L. Lucidum berries may be an interesting source of compounds for use in the development of the therapeutic armamentarium for diseases where enzymatic disruption is believed to play a role.

In order to improve the quality control level of Ligustri Lucidi Fructus(LLF) and to explore the changes of chemical components after processing,the HPLC method for fingerprint and simultaneous determination of the major polar components in LLF were established. The octadecylsilane bonded silica gel was used as the stationary phase,with acetonitrile as the mobile phase A and0. 2% formic acid as the mobile phase B in a gradient elution procedure at a flow rate of 1. 0 m L·min-1. The detection wavelength was set at 280 nm and the column temperature was 25 ℃. There were 22 common peaks,20 of which were selected from the fingerprint of LLF and its wine-steamed product,respectively,and 14 chromatographic peaks were identified with reference substances. With the same chromatographic conditions,seven components were quantitatively analyzed and the results of system adaptability and methodology investigation all met the requirements of content determination. Compared with the crude LLF,the content of 5-hydroxymethyl furfural and salidroside significantly increased in wine-steamed LLF,while the contents of iridoid glycosides generally decreased. The method provided a basis for quality control of LLF and its processed products as well as the related preparations.

The current study investigated the ability of two secoiridoids, GL-3 ( 1 ) and oleonuezhenide ( 2 ), isolated from the fruits of Ligustrum japonicum to inhibit MMP-2 and -9 activity in phorbol 12-myristate 13-acetate (PMA)-induced HT-1080 human fibrosarcoma cells. Both compound s 1 and 2 were able to exert lowered gelatin digestion activity for MMP-2 and -9 tested by gelatin zymography via suppressing the release of MMPs to culture medium according to ELISA results. Treatment with compounds was also able to suppress the expression of both mRNA and protein levels of MMP-2 and -9. Action mechanism behind the MMP inhibitory effect of the compounds was suggested to be via MAPK pathway indicated by decreased levels of phosphorylated p38, ERK and JNK proteins evaluated employing immunoblotting. Compound 1 was shown to be slightly more active to inhibit MMP-2 and -9, however, compound 2 showed more regular dose-dependency during inhibition. In conclusion, this study suggested that GL-3 and oleonuezhenide were notable natural origin potent MMP inhibitors and could serve as lead compounds for development of anti-invasive MMP inhibitors against tumor metastasis.

The content of tyrosol,salidroside,echinacoside,rutin,acteoside,ligustroflavone,specnuezhenide,and quercetin were determined by HPLC,and the color of Ligustri Lucidi Fructus was determined by comparison with color card.Hundred-seed weight was analyzed by using gravimetric method.The correlation analysis and One-way ANOVA were used to analyze the relationship between the characters,the chemical composition,the harvest time and the geographical location of Ligustri Lucidi Fructus,for giving a comprehensive evaluation of the quality of Ligustri Lucidi Fructus The results showed that 92% of Ligustri Lucidi Fructus were all up to quality standard of Chinese Pharmacopoeia,and the contents of 7 components in Ligustri Lucidi Fructus(except quercetin) were higher than those in samples with black colors.The content of salidroside in Ligustri Lucidi Fructus harvested in June was the highest and the other7 components of Ligustri Lucidi Fructus were relatively high in 8-10 months.According to the quality parameters of Ligustri Lucidi Fructus,the Ligustri Lucidi Fructus from six habitats can not be distinguished effectively.The results showed that there was a certain relationship between the color,harvest season and component content of Ligustri Lucidi Fructus,and the habitats were not related to the quality parameters of Ligustri Lucidi Fructus.The study aimsat providing data support for the resource status of native Ligustri Lucidi Fructus,and a theoretical basis for the revision of standards of Ligustri Lucidi Fructusin the future.

Ligustroflavone is one major compound contained in active fraction from Fructus Ligustri Lucidi (the fruit of Ligustrum lucidum), which could regulate parathyroid hormone (PTH) levels and improve calcium balance by acting on calcium-sensing receptors (CaSR). This study aimed to explore the potency of ligustroflavone as a CaSR antagonist and its protective effects against diabetic osteoporosis in mice. LF interacted well with the allosteric site of CaSR shown by molecular docking analysis, increased PTH release of primary parathyroid gland cells and suppressed extracellular calcium influx in HEK-293 cells. The serum level of PTH attained peak value at 2 h and maintained high during the period of 1 h and 3 h than that before treatment in mice after a single dose of LF. Treatment of diabetic mice with LF inhibited the decrease in calcium level of serum and bone and the enhancement in urinary calcium excretion as well as elevated circulating PTH levels. Trabecular bone mineral density and micro-architecture were markedly improved in diabetic mice upon to LF treatment for 8 weeks. LF reduced CaSR mRNA and protein expression in the kidneys of diabetic mice. Taken together, ligustroflavone could transiently increase PTH level and regulate calcium metabolism as well as prevent osteoporosis in diabetic mice, suggesting that ligustroflavone might be an effective antagonist on CaSR.

Objective: The present study explored the effects of the combined herbal therapy consisting of curcumin (CUR) and Fructus Ligustri Lucidi (FLL) on aspects of bone regeneration., Methods: Prior to analyzing the ability of this novel combined herbal therapy to promote aspects of bone regeneration, its cytotoxicity was determined using MC3T3-E1 cells (pre-osteoblast model). Cell proliferation was evaluated using phase-contrast microscopy and cell differentiation was estimated using alkaline phosphatase activity. The effect of the combined herbal therapy (CUR + FLL) was also assessed in terms of mineralization in the extracellular matrix (ECM) of cultured cells. Further, to explore the molecular mechanisms of bone formation, time-dependent expression of bone-regulating protein biomarkers was also evaluated., Results: Combined herbal therapy (CUR + FLL) significantly upregulated the viability, proliferation and differentiation of MC3T3-E1 cells compared to the monotherapy of CUR or FLL. The magnitude of ECM mineralization (calcium deposition) was also higher in MC3T3-E1 cells treated with combined therapy. The time-dependent expression of bone-forming protein biomarkers revealed that the tendency of expression of these bone-regulating proteins was remarkably higher in cells treated with combined therapy., Conclusion: The co-administration of CUR and FLL had superior promotion of elements of bone regeneration in cultured cells, thus could be a promising alternative herbal therapy for the management of bone erosive disorders such as osteoporosis., (Copyright © 2018 Shanghai Changhai Hospital. Published by Elsevier B.V. All rights reserved.)