Impact of different processing methods of Ligustrum lucidum Ait. on kidney-yin deficiency: a study based on pharmacodynamics and metabolomics research.

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.

A review of botany, phytochemistry, pharmacology, and applications of the herb with the homology of medicine and food: Ligustrum lucidum W.T. Aiton.

Ligustrum lucidum W.T. Aiton is an outstanding herb with the homology of medicine and food. Its ripe fruits are traditionally used as an important tonic for kidneys and liver in China. Ligustrum lucidum W.T. Aiton is rich in nutritional components and a variety of bioactive ingredients. A total of 206 compounds have been isolated and identified, they mainly include flavonoids, phenylpropanoids, iridoid glycosides, and triterpenoids. These compounds exert anti-osteoporosis, anti-tumor, liver protective, antioxidant, anti-inflammatory, and immunomodulatory effects. Ligustrum lucidum W.T. Aiton has been traditionally used to treat many complex diseases, including osteoporotic bone pain, rheumatic bone, cancer, related aging symptoms, and so on. In the 2020 Edition of Chinese Pharmacopoeia, there are more than 100 prescriptions containing L. lucidum W.T. Aiton. Among them, some classical preparations including Er Zhi Wan and Zhenqi fuzheng formula, are used in the treatment of various cancers with good therapeutic effects. Additionally, L. lucidum W.T. Aiton has also many excellent applications for functional food, ornamental plants, bioindicator of air pollution, algicidal agents, and feed additives. Ligustrum lucidum W.T. Aiton has rich plant resources. However, the application potential of it has not been fully exploited. We hope that this paper provides a theoretical basis for the high-value and high-connotation development of L. lucidum W.T. Aiton in the future.

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.

Tree Physiological Variables as a Proxy of Heavy Metal and Platinum Group Elements Pollution in Urban Areas.

Physiological variables (the content of chlorophyll, flavonoids and nitrogen, together with Fv/Fm) and the content of ten heavy metals (As, Cd, Cu, Hg, Mn, Ni, Pb, Sb, V and Zn) and two platinum group elements (PGEs: Pd and Rh) were measured in the leaves of 50 individuals of Ligustrum lucidum trees regularly distributed in the city of Logroño (Northern Spain). Three of these variables increased with increasing physiological vitality (chlorophyll, nitrogen and Fv/Fm), whereas flavonoids increased in response to different abiotic stresses, including pollution. Our aim was to test their adequacy as proxies for the pollution due to heavy metals and PGEs. The three vitality indicators generally showed high values typical of healthy plants, and they did not seem to be consistently affected by the different pollutants. In fact, the three vitality variables were positively correlated with the first factor of a PCA that was dominated by heavy metals (mainly Pb, but also Sb, V and Ni). In addition, Fv/Fm was negatively correlated with the second factor of the PCA, which was dominated by PGEs, but the trees showing Fv/Fm values below the damage threshold did not coincide with those showing high PGE content. Regarding flavonoid content, it was negatively correlated with PCA factors dominated by heavy metals, which did not confirm its role as a protectant against metal stress. The relatively low levels of pollution usually found in the city of Logroño, together with the influence of other environmental factors and the relative tolerance of Ligustrum lucidum to modest atmospheric pollution, probably determined the only slight response of the physiological variables to heavy metals and PGEs.

[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.

Effect and Mechanism of Specnuezhenide on Chemotherapy-induced Myelosuppression.

OBJECTIVE: This study aimed to investigate the therapeutic effect of Specnuezhenide on myelosuppression induced by chemotherapy and clarify its mechanism. METHODS: In this study, we measured peripheral blood cells, thymus index, spleen index, bone marrow nucleated cells (BMNCs), and the number of cell colonies counted in vitro by hematopoietic progenitor cells (HPCs) to determine the effect of SPN on cyclophosphamide (CTX)-induced myelosuppression. The alterations in the expression of relevant proteins, the cell cycle, and cytokines associated with hematopoietic cells were examined to better understand how it works. RESULTS: In the cyclophosphamide-induced mouse model, our study discovered that SPN can increase the number of peripheral blood cells and BMNCs after treatment, increase the thymus index and decrease the spleen index, and promote the proliferation and differentiation of HPCs. SPN can improve the production of cultured colonies in vitro, reduce the level of hematopoietic factors in vivo, regulate the proportion of G0/G1 phase cells, and promote the normal growth and development of cells. SPN can increase the expression levels of key proteins MEK and p-ERK in the MAPK signaling pathway, which may be one of the important mechanisms for improving myelosuppression. CONCLUSION: SPN can enhance the hematological and immunological functions of myelosuppressionmice, and it is hypothesized that SPN is extremely helpful to the hematopoietic and immune functions of tumor patients following chemotherapy. SPN might be used to treat myelosuppression. Additionally, high doses of SPN have a stronger therapeutic effect than low levels of SPN.

Species identification of Ligustrum lucidum / 中国中药杂志

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.

Therapeutic Potentials of Secoiridoids from the Fruits of Ligustrum lucidum Aiton against Inflammation-Related Skin Diseases.

Ligustrum lucidum Aiton is a flowering plant of the Oleaceae family, and its fruits have been traditionally used for skin nourishment and the treatment of skin diseases. However, the anti-inflammatory constituents for skin disease are not well-characterized. Phytochemical investigation of L. lucidum fruits resulted in the isolation of a new secoiridoid, secoligulene (1), together with (E)-3-(1-oxobut-2-en-2-yl)pentanedioic acid (2) and trans-(E)-3-(1-oxobut-2-en-2-yl)glutaric acid (3). Secoligulene (1) displayed the potent inhibitory effect on NO production with an IC50 value of 12.0 µg/mL. Secoligulene (1) also downregulated mRNA transcriptional levels of pro-inflammatory cytokines such as IL-1 α, IL-1ß, IL-6 and COX-2 in LPS-stimulated RAW264.7 cells. Further investigation showed that secoligulene (1) inhibited the phosphorylation of IκB and JNK activated by LPS. In addition, secoligulene (1) downregulated the expression of chemokines such as CXCL8 and CCL20 in the TNF-α/IL-17/IFN-γ induced HaCaT psoriasis model. Taken together, these findings support the beneficial effects of L. lucidum and its constituents on inflammation-related skin diseases and can be further developed as therapeutic treatments for related diseases.

[Complete chloroplast genome of Ligustrum lucidum and highly variable marker identification for Ligustrum].

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.

Ligustrum lucidum Leaf Extract-Assisted Green Synthesis of Silver Nanoparticles and Nano-Adsorbents Having Potential in Ultrasound-Assisted Adsorptive Removal of Methylene Blue Dye from Wastewater and Antimicrobial Activity.

Present study was conducted to investigate the adsorption and ultrasound-assisted adsorption potential of silver nanoparticles (AgNPs) and silver nanoparticles loaded on chitosan (AgCS composite) as nano-adsorbents for methylene blue (MB) removal. AgNPs were synthesized using leaf extract of Ligustrum lucidum, which were incorporated on the chitosan's surface for modification. UV−Vis Spectroscopy, FTIR, XRD, SEM, and EDX techniques were used to confirm the synthesis and characterization of nanomaterials. Batch adsorption and sono-adsorption experiments for the removal of MB were executed under optimal conditions; for fitting the experimental equilibrium data, Langmuir and Freundlich's isotherm models were adopted. In addition, the antimicrobial potential of the AgNPs and AgCS were examined against selected bacterial and fungal strains. UV−Vis spectroscopy confirmed AgNPs synthesis from the leaf extract of L. lucidum used as a reducer, which was spherical as exposed in the SEM analysis. The FTIR spectrum illustrated phytochemicals in the leaf extract of L. lucidum functioning as stabilizing agents around AgNPs and AgCS. Whereas, corresponding crystalline peaks of nanomaterial, including a signal peak at 3 keV indicating the presence of silver, were confirmed by XRD and EDX. The Langmuir model was chosen as an efficient model for adsorption and sono-adsorption, which exposed that under optimum conditions (pH = 6, dye initial concentration = 5 mg L−1, adsorbents dosage = 0.005 g, time = 120 min, US power 80 W), MB removal efficiency of AgNPs was >70%, using ultrasound-assisted adsorption compared to the non-sonicated adsorption. Furthermore, AgNPs exhibited promising antibacterial potential against Staphylococcus aureus with the maximum zone of inhibition (14.67 ± 0.47 mm). It was concluded that the green synthesis approach for the large-scale production of metallic nanoparticles is quite effective and can be recommended for efficient and cost-effective way to eradicate dyes, particularly from textile wastewater.

Preliminary results on the separation of the different parts of Ligustrum lucidum Ait fruit and the main bioactive compounds analysis.

Oleanolic acid (OA) and ursolic acid (UA) possess anti-inflammatory, antioxidative, antiprotozoal, antimutagenic and anticancer properties and are the main bioactive compounds in the fruit of Ligustrum lucidum Ait. The aim of this paper was to study the method of separating the whole fruit into the main ingredients containing OA and UA for the later effective extraction with reduced organic solvents and easy separation and purification. In the present study, the sarcocarps and exocarps were separated from the whole fruits (designated exo-sarcocarps, the mass percentages (w/w, dry weight basis), 33.1%), testae (13.5%) and cores (48.7%) by using methods separated. The contents of OA and UA in whole fruits, exo-sarcocarps, testae and cores were analyzed. The OA and UA extraction yields were highest in exo-sarcocarps vs. yields from whole fruits, testae or cores. The spective yields of OA and UA from exo-sarcocarps were 24.34 ± 2.09 and 7.82 ± 0.09 mg/g; these yields were about 4 times higher than OA yields and about 4 times higher than UA yields from fruit.

Complete chloroplast genome of Ligustrum lucidum and highly variable marker identification for Ligustrum / 中国中药杂志

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.

Effects of air pollution on physiological traits of Ligustrum lucidum Ait. leaves in Luoyang, China.

Leaves of trees experience the maximum brunt of exposure and undergo certain changes in physiological traits responding to air pollution, and then, the specific leaf traits can be the indicators of air pollution in an area. However, due to the diversity of sources, the composition of air pollutants is very complex. This makes it difficult to predict air pollution using physiological differentiation of leaves. The purpose of this investigation was to examine potential of Ligustrum lucidum Ait. leaf measurement as a method to predict the air pollutants in Luoyang, China. Leaves of roadside L. lucidum were studied from the city center with serious air pollution to relatively unpolluted areas. Leaf size, stomatal traits, and non-structural carbohydrate were measured. The particulate and gaseous pollutants (including sulfur dioxide, nitrogen dioxide, carbon monoxide, and ozone) were investigated too. The results showed that the leaf area and soluble sugar content decreased, while the aspect ratio of leaves increased in heavily polluted areas. As pollution increased, the stomatal traits in different crown positions were changed differently. No significant correlation was found between ozone content and the measured traits of leaves. The responses found in the physiological differentiation of the leaves reflect acclimation to air pollution. The soluble sugar content of the leaves could be used to indicate the short-term stress of air pollution, the area, and aspect ratio of leaves are indicative of the long-term stress due to air pollution. Therefore, physiological traits of L. lucidum leaves appeared to be significant predictive factors for the air pollutants in urban areas.

Bioactivity-guided isolation of immunomodulatory compounds from the fruits of Ligustrum lucidum.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruits of Ligustrum lucidum (FLL) W.T. Aiton (Oleaceae) is included in the 2020 "Chinese Pharmacopoeia" and is widely used in traditional Chinese medicine as a tonic. In recent years, FLL has been reported to improve immune function, but the bioactive compounds and mechanisms of FLL remain poorly characterized. AIM OF THE STUDY: To identify FFL compounds with strong immune activity and explore their molecular mechanisms. MATERIALS AND METHODS: The phagocytic activity of RAW264.7 macrophages and proliferation activity of spleen lymphocytes were used to guide the isolation of bioactive compounds from FLL extracts. Lymphocyte subpopulations, Ca2+ concentrations, and surface molecule expression were analyzed using flow cytometry. Cytokine secretion was examined using ELISA. FITC-OVA uptake was observed using fluorescence microscopy. NF-κB activation was analyzed using western blotting. RESULTS: The extraction and isolation produced ten compounds, namely oleuropeinic acid, nuezhenide, isonuezhenide, salidroside, isoligustrosidic acid, ligulucidumosides A, 8(E)-nuezhenide, hydroxytyrosol, oleuropein, and p-hydroxyphenethyl 7-ß-D-glucosideelenolic acid ester were isolated and identified from FLL-Bu-30%. Immunoactivity experiments showed that hydroxytyrosol had the strongest macrophage phagocytotic and lymphocyte proliferation-promoting activities. Further studies showed that hydroxytyrosol could significantly enhance lymphocyte subsets CD3+, CD4+/CD8+, and CD3+CD4-CD8-, promote IL-4, IFN-γ, and TNF-α secretion, and increase intracellular Ca2+ concentrations. In addition, the results from RAW264.7 macrophages showed that hydroxytyrosol increased FITC-OVA uptake, induced TNF-α and IL-1ß production, upregulated MHC-II, CD80, and CD86 expression, promoted cytoplasmic IκB-α degradation, and increased nuclear NF-κB p65 levels. CONCLUSION: Our study provides substantial evidence regarding the mechanism of the immunomodulatory effects of compounds from FLL.

Photosynthesis Performance and Antioxidative Enzymes Response of Melia azedarach and Ligustrum lucidum Plants Under Pb-Zn Mine Tailing Conditions.

Lead-zinc (Pb-Zn) mine tailings pose a great risk to the natural environment and human health because of their high toxicity. In this study, the responses of photosynthesis, chlorophyll fluorescence, and antioxidative enzyme of Melia azedarach and Ligustrum lucidum in the soil contaminated by Pb-Zn mine tailings were investigated. Results showed that Pb-Zn mine tailings significantly reduced net photosynthetic rates and leaf photosynthetic pigment content of both trees, and the reduction of net photosynthetic rates was mainly caused by their biochemical limitation (BL). The chlorophyll fluorescence parameters from Pb-Zn tailing stressed leaves indicated that Pb-Zn tailings affected PSII activity which was evident from the change values of energy fluxes per reaction center (RC): probability that an electron moves further than QA - (ETO/TRO), maximum quantum yield for primary photochemistry (TRO/ABS), the density of PSII RC per excited cross-section (RC/CSO), the absorption of antenna chlorophylls per PSII RC (ABS/RC), and the turnover number of QA reduction events (N). Pb-Zn mine tailings also affected the oxidation and reduction of PSI, which resulted in a great increase of reactive oxygen species (ROS) contents and then stimulated the rate of lipid peroxidation. Both trees exhibited certain antioxidative defense mechanisms as elevated superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, then declined under high level of Pb-Zn tailing treatment. Comparatively, L. lucidum showed less extent effect on photosynthesis and higher antioxidative enzyme activities than M. azedarach; thus L. lucidum was more tolerant than M. azedarach at least under the described Pb-Zn tailing treatment. These results indicate that the effect of Pb-Zn mine tailings on photosynthesis performance mainly related to imbalance of the PSII activity and PSI redox state in both trees. We propose that M. azedarach and L. lucidum could relieve the oxidative stress for phytoremediation under the appropriate Pb-Zn mine tailing content.

Compatibility effects of ginseng and Ligustrum lucidum Ait herb pair on hematopoietic recovery in mice with cyclophosphamide-induced myelosuppression and its material basis.

BACKGROUND: Ginseng (G) and Ligustrum lucidum Ait (LLA) are core traditional Chinese medicines in treating myelosuppression formula. The present study was designed to profile effect of G and LLA herb pair (G-LLA) on myelosuppressed mice. METHODS: The mice myelosuppression model was established by intraperitoneal (i.p.) injection of cyclophosphamide (Cy). Hematopoietic function of bone marrow was measured by hemopoietic progenitor cell culture and peripheral blood count, and serum hemopoietic factors were tested by enzyme-linked immunosorbent assay. Bone marrow cell cycle was performed by flow cytometry. HPLC was used to measure 20 potential chemical components related to myelosuppression, including ginsenoside Rg1, Re, Rb1, Rc, Rb2, Rb3, Rd, Rk3, Rh4, 20 (S)-Rg3, 20 (R)-Rg3, Rk1, Rg5, salidroside, and so on. RESULTS: G, LLA, and G-LLA improved the amount of peripheral blood cells and bone marrow cells of myelosuppressed mice (P < 0.01). They significantly increased the colony quantity of colony-forming unit-granulocyte macrophage, burst-forming unit-erythroid, colony-forming unit-erythroid, and colony-forming unit-megakaryocyte and amount of G2/M and S phase cells (P < 0.01). They also significantly decreased the amount of hematopoiesis-related cytokines (P < 0.01). The content of chemical components in G-LLA changed, and the change of rare saponin was the most obvious. CONCLUSION: These results show that G-LLA herb pair might produce synergistic or complementary compatibility effects on bone marrow suppression after chemotherapy. It suggests that the substance basis of G-LLA for treating bone marrow suppression may be effective chemical components.

Optimization of the Purification Technology of Total Triterpenoids of Ligustrum lucidum by Box-Behnken Re- sponse Surface Methodology / 中国药房

OBJECTIVE：To i mprove the transfer rate and purity of oleanolic acid and ursolic acid in total triterpenoids from Ligustrum lucidum ，so as to optimize the purification technology. METHODS ：Oleanolic acid and ursolic acid were used as representative components of total triterpenoids ，and their contents were determined by HPLC. The determination was performed on Thermo BDS Hypersil C 18 column with mobile phase consisted of methanol- 0.02％ ammonium acetate solution （80∶20，V/V）at the flow rate of 1.0 mL/min. The detection wavelength was set at 210 nm，and column temperature was 30 ℃. The sample size was 20 μ L. In single factor tests，using transfer rate of oleanolic acid and ursolic acid as index ，the effects of water precipitation temperature and time ，the amount of redissolved ethanol on the purification technology was investigated ；using transfer rate and purity of two components as indexes ，the effects of the amount of activated carbon and volume fraction of crystallization ethanol were investigated. Based on it ，using the amount of redissolved ethanol and activated carbon ，volume fraction of crystallization ethanol as factors ，Box-Behnken response surface methodology was used to optimize the purification technology ，and validation tests were performed. RESULTS ：The optimal purification technology was adding 4-fold（mL/g，the same below ）water in L. lucidum concentrated solution ，placing for 2 hours at 0 ℃（water precipitation ）；adding 1-fold ethanol to dissolve （redissolution）； adding 4％ activated carbon （edulcoration）；finally adding water to adjust the volume fraction of ethanol to 80％，placing at 4 ℃ for 12 hours（crystallization），centrifuging and drying. The results of 3 times of validation tests showed that the transfer rates of oleanolic acid and ursolic acid in total triterpenoids prepared by optimized technology were 61.11％ and 65.78％，the purities of them were 53.44％ and 19.79％，and RSDs were both lower than 3％. CONCLUSIONS ：The optimized purification technology has high extraction efficiency and simple operation ，which can be used for industrial production of purification of total triterpenoids from L. lucidum and the development of corresponding preparations.

Ethanol extract of Ligustrum lucidum Ait. leaves suppressed hepatocellular carcinoma in vitro and in vivo.

BACKGROUND: The present study investigated the pharmacological activity and mechanism of ethanol extract of Ligustrum lucidum Ait. leaves (EEL) on HCC. METHODS: Cell viability was determined using cell counting kit-8 (CCK-8) assay. The effects of EEL on cellular biological activities were analyzed by flow cytometry (FCM), cell wound scratch assay and transwell assay. The expression levels of related mRNA and protein were determined by performing quantitative real-time PCR (qRT-PCR), Western blotting assay and immunocytochemistry. Methylation-specific PCR (MSP) was carried out to investigate the possible mechanism underlying the DNA methylation of PTEN. RESULTS: EEL showed cytotoxicity to both Bel-7402 and Huh-7 cell lines. We also found that EEL enhanced the apoptosis of Bel-7402 and Huh-7 cells by regulating the expressions of Bcl-2 associated X (Bax), B cell lymphoma 2 (Bcl-2) and Cytochrome-C and the activity of caspase-3 and therefore promoted cell cycle arrest. Moreover, EEL also suppressed cell migration and invasion. EEL increased the expression of tissue inhibitor of metalloproteinases 2 (TIMP2) but decreased the expressions of matrix metalloproteinase2 (MMP2) and MMP9. Furthermore, EEL inhibited the phosphorylation of PI3K/Akt pathway. MSP results showed that EEL promoted the demethylation of PTEN, suggesting that the inactivation of PI3K/Akt may be related to DNA de-methylation of PTEN. In addition, EEL inhibited the tumor growth of HCC in vivo. CONCLUSIONS: EEL exerted anti-tumor effect on HCC in vitro and in vivo. EEL mediated by the inhibition of PI3K/Akt may be closely related to DNA de-methylation of PTEN. Thus, EEL could be used as a potential anti-cancer therapeutic agent of HCC.

Four spices prevent mice from contracting Salmonella enterica serovar Typhimurium.

Ligustrum lucidum Ait (LL), Lysimachia christinae Hance (LC), Mentha piperita Linn (MP), and Cinnamomum cassia Presl (CC) are common spices used in Asia. The present study investigated the anti-Salmonella effects of the four spices using aqueous extracts. The amount of phenolic acids and flavonoids in each spice aqueous extract was determined as indicators of purity. Mice were pretreated with LL, LC, MP or CC aqueous extract for 7 days. Following infection with Salmonella enterica serovar Typhimurium strain ST21 (ST21), the aqueous extract of each spice was subsequently administered for 4 days. ST21 infected mice had lower body weight compared with the control group. The administration of spice aqueous extracts significantly increased body weight following infection. ST21 infection increased the fecal ST21 counts compared with the control group; however, following spice aqueous extract treatments, ST21 counts significantly decreased. The spice treatments also significantly reduced ST21 count in blood and the organs. Notably, ST21 infection increased interferon (IFN)-γ and interleukin (IL)-6 levels in serum whilst spice treatments reduced these cytokines. In the spleen, spice treatment significantly lowered IFN-γ, IL-6, IL-1ß, and tumor necrosis factor (TNF)-α levels, but increased IL-12 levels. ST21 infection stimulated the production of immunoglobulin (Ig)A and IgM in serum whilst spice aqueous extract treatment significantly decreased these levels. In summary, LL and MP aqueous extract treatments had the most significant effect in protecting against ST21 infection. Results of the RAW 264.7 cell infection model suggested that the mechanisms involved in the anti-ST21 effect of each spice were individually different. All four aqueous extracts demonstrated different mechanisms in attenuating ST21 invasion with the protective effect of LC aqueous extract potentially involving TNF-α expression. The present findings suggested that the four spices may be considered as potent functional foods due to their anti-Salmonella effects.