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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.
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Ligustrum/genética , Sementes , Frutas , Reação em Cadeia da Polimerase , Projetos de PesquisaRESUMO
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.
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Frutas , Genoma de Cloroplastos , Ligustrum/genética , Filogenia , Melhoramento VegetalRESUMO
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.
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Objective: To investigate the relationship between the anti-osteoporotic effect of Ligustrum lucidum and the growth hormone (GH)/insulin-like growth factor 1 (IGF-1) signaling pathways. Methods: L. lucidum aqueous extract was orally administrated to ovariectomized (OVX) rats for 14 weeks. Then the femurs were removed and stained with hematoxylin & eosin (HE) and Safranin O/Fast Green staining, respectively, to evaluate the change of bone microstructure. The histomorphological parameters and bone mineral density (BMD) of the femurs were measured by micro-CT. Furthermore, rat serum GH level was determined by ELISA assay, and IGF-1 protein expression in liver and bone was determined by Western blotting and immunohistochemical staining. Results: L. lucidum prevented the disorganized femoral trabeculae and inhibited the decrease in BMD and glycosaminoglycan content in OVX rats. In addition, L. lucidum significantly inhibited the decrease of serum GH levels and improved IGF-1 protein expression of liver and bone in OVX rats. Conclusion: L. lucidum may prevent against osteoporosis through inhibition of bone loss and improvement of bone microstructure via regulating GH/IGF-1 signaling pathway.
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Objective: To study the relative molecular mass and composition of Ligustrum lucidum polysaccharide purified by dialysis, and provide the theories for the relationship between the biological activity and the internal composition. Methods: In this paper, L. lucidum as an object was studied. Polysaccharide was isolated by water extraction and ethyl alcohol precipitation and purified by Sevage method, hydrogen peroxide and dialysis. After the acidolysis of trifluoroacetic acid, the molecular weight was measured by GPC, and the composition of polysaccharide was analyzed by HPLC-RID. Results: The Mw of polysaccharide was 10 721, and the Mn of polysaccharide was 10 673. L. lucidum polysaccharide consisted of glucose, rhamnose, and arabinose, the molar ratio of these monosaccharide was 9.148.105.18. Conclusion: The purified polysaccharide composition is more homogeneous, and the monosaccharides of polysaccharides was easily analyzed by HPLC-RID without column derivatization.
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Objective To optimize the extraction and purification process of oleanolic acid in Ligustrum lucidum fruits with macroporous resin. Methods Grinding degree, temperature, liquid ratio, extraction time, extraction times, ethanol volume fraction and pH were selected as factors, which may influence the extraction of oleanolic acid from L. lucidum. Single factor experiments were carried out on these factors. The optimal extraction conditions were determined, and the best type of resin was chosen for separation and purification of oleanolic acid in the optimal conditions. Results The optimal technological conditions were as follows: the temperature was controlled at 80°C, ethanol solution was 60%, liquid material ratio was 1:11, extraction time was 120 min, and extraction times were twice. The crude extraction was processed at pH 12, and then the pH was adjusted to 2 to further purify the processed crude extraction. The content of oleanolic acid in the extract was 9.5% by UV visible spectrophotometric method. This extraction was further separated and purified by AB- 8 resin, and the content of oleanolic acid detected by ultraviolet visible spectrophotometr was 15.2%o Conclusion The optimal process of the oleanolic acid extraction is reproducible, simple and feasible. It may provide reference for using macroporous resin to extract oleanolic acid from L. lucidum in the industry production.
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OBJECTIVE: To explore the active ingredients of wine fried Ligustrum lucidum fructus by studying the relationship between the HPLC chromatogram and antioxidant activity. METHODS: The characteristic chromatogram of wine fried Ligustrum lucidum fructus processed by means of classical homothermal acceleration was established by HPLC. DPPH, ABTS, and FRAP methods were established to determine the antioxidant activity. The spectrum-effect relationship was studied by using partial least squares regression (PLSR), and the chromatographic peaks related to antioxidant activity were identified. RESULTS: Peaks 1, 3, 8, 10, 14 and 15 were found to have positive relationship with DPPH free radical and ABTS free radical scavenging activity among the 17 matching characteristic chromatograms. Peaks 3, 8, 12, and 13 were significantly positively related to Fe3+ resuction capacity. Among the chromatographic peaks, peak 3 and peak 8 were positively related to the activities of scavenging DPPH free radical and ABTS free radical and reducing Fe3+. Peaks 3, 12, 13, 14, 15, and 17 were determined as salidroside, luteolin-7-O-glucoside, specnuezhenide, oleuropein, ligustroflavone, and luteolin, respectively. CONCLUSION: The pharmcodynamic effects of wine fried Ligustrum lucidum fructus do not depend on the contents of several index components such as specnuezhenide and salidroside. The quality of traditional Chinese medicines should be represented by the compound groups associated with pharmcodynamic effect.
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Objective To optimize the extraction and purification process of oleanolic acid in Ligustrum lucidum fruits with macroporous resin. Methods Grinding degree,temperature,liquid ratio,extraction time,extraction times,ethanol volume fraction and pH were selected as factors,which may influence the extraction of oleanolic acid from L. lucidum. Single factor experiments were carried out on these factors. The optimal extraction conditions were determined,and the best type of resin was chosen for separation and purification of oleanolic acid in the optimal conditions. Results The optimal technological conditions were as follows:the temper?ature was controlled at 80℃,ethanol solution was 60%,liquid material ratio was 1∶11,extraction time was 120 min,and extraction times were twice. The crude extraction was processed at pH 12,and then the pH was adjusted to 2 to further purify the processed crude extraction. The content of oleanolic acid in the extract was 9.5%by UV visible spectrophotometric method. This extraction was further separated and purified by AB-8 resin,and the content of oleanolic acid detected by ultraviolet visible spectrophotometr was 15.2%.Conclusion The optimal process of the oleanolic acid extraction is reproducible,simple and feasible. It may provide refer?ence for using macroporous resin to extract oleanolic acid from L. lucidum in the industry production.
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OBJECTIVE:To establish a method for the contents determination of 4 ingredients in Ligustrum lucidum decoration piece by reference extract method. METHODS:HPLC was performed on the column of XBridge C18 with mobile phase of acetoni-trile-0.1% formic acid(gradient elution) at flow rate of 1.0 ml/min,detection wavelength was 254 nm,column temperature was 30℃and volume injection was 10μl. RESULTS:The linear range was 0.037-0.598 mg for neonuezhenide,0.006-0.101 mg for ac-teoside,0.189-3.023 mg for nuezhenide and 0.314-5.027 mg for specnuezhenide(r≥0.999 0);RSDs of precision,reproducibility and stability tests were no more than 3.8%;recoveries were 98.46%-104.83%(RSD=2.43,n=6),95.55%-104.57%(RSD=3.63, n=6),100.09%-104.39%(RSD=1.45,n=6)and 98.84%-104.97%(RSD=2.02,n=6). CONCLUSIONS:The method is simple, good reproducibility,and can be used for the contents determination of 4 ingredients in L. lucidum decoration piece.
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Objective To study the chemical constituents from the fruits of Ligustrum lucidum. Methods The chemical constituents in ethyl acetate fraction of 70% ethanol extract from the fruits of L. lucidum were isolated and purified by column chromatography over silica gel, ODS, Sephadex LH-20, and HPLC. The structures were identified on the basis of physicochemical properties and spectral data analyses. Results A compound, secoiridoid glucoside, was isolated and identified as 6'-O-cinnamoyl-8-ep/-kingisidic acid (1). Conclusion Comoound 1 is a new one.
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Ligustrum lucidum can be used as immunomodulatory and to treat acute hepatitides and chronic hepatitis, and has many other bioactivities such as anti-aging activity, decreasing the level of the blood sugar, anti-inflammatory activity and anti-tumor activity. The chemical constituents of its fruits include pentacyclic triterpenoid, dammarane tetracyclic triterpenoids, flavones, secoiridoids, phenolic glucosides, and other components such as polysaccharide, amino acid, fatty acid, volatile components, pigment, microelement, and so on. This paper summarizes the research advances on the chemical constitutents of Ligustrum lucidum and the structure-activity relationship.
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Objective To study molecular target of total flavonoid in Ligustrum Lucidum Ait(TFL) on lipometabolism regulation of HepG2 cell.Methods Treatment doses of total flavonoid in Ligustrum Lucidum Ait were determined by MTT and LDH.The expression of lipoprotein lipase(LPL),3-hydroxy-3-methylglutary-coenzyme A redutasein(HMGCR)and peroxisome proliferator-actived receptorsα(PPARα)mRNA of HepG2 cell were determined by reversed transcription polymerase chain reaction(RT-PCR).Results Increased expression of LPL mRNA and PPARαmRNA with TFL(10-6～10-4g/ml)were found in HepG2 cells after treated with TFL(10-5g/ml),but decreased expression of HMGCR mRNA was found after treated with TFL(10-5,10-4 g/ml).Conclusion The potential mechanisms of total flavonoid in Ligustrum Lucidum Ait's hypolipidmic effects may be with its functions of regulating lipoprotein degradation through PPARα-LPL pathway,or regulating steroid biosynthesis by reducing HMGCR.