Screening of active components and preliminary mechanism exploration of Lycii Fructus for improving osteoporosis based on the zebrafish model / 药学学报

Juvenile zebrafish were used to screen the active components of Lycii Fructus for improving osteoporosis. The screening results were further verified by zebrafish adult osteoporosis model and the action mechanism was explored. Prednisolone was used as the inducer to build osteoporosis models of juvenile and adult zebrafish, and 9 groups of samples of different extracts and chemical parts of Lycii Fructus were given. Alizarin red staining was applied for observing the scale matrix mineralization and bone resorption. The activities of osteoblasts and osteoclasts were detected using alkaline phosphatase (ALP) and tartrate resistant acid phosphatase (TRAP/TRACP) staining. The expressions of bone metabolism-related genes alp, osteoprotectin (opn), osteoblast specific transcription factor (sp7), cathepsin K (ctsk), tracp, and Runt family transcription factor 2b (runx2b) in each group were determined using quantitative polymerase chain reaction. The results showed that all components of Lycii Fructus improved the formation area of the first vertebrae, the staining light density value, and the number of vertebrae joints in juvenile zebrafish and the Lycium barbarum polysaccharide (LBP) treatment group exerted the best effect. In addition, LBP prevented the formation of bone resorption lacunae in zebrafish scales, increased ALP activity, decreased TRAP activity, up-regulated the alp, sp7, and opn genes, and lowered the expressions of ctsk and tracp genes. In conclusion, LBP regulated the activity of osteoblasts and osteoclasts, reduced bone resorption, promoted bone formation and enhanced bone density, which might be the main anti-osteoporosis active fraction of Lycii Fructus. This study provided modern scientific evidence for the scientific connotation of the traditional effect of "strengthening bones and muscles" of Lycii Fructus, provided the reference for the evaluation of the anti-osteoporosis activity of traditional Chinese medicine based on zebrafish adult model, and provided beneficial enlightenment for the bone health needs of the aging society population.

Comparative analysis of differentially expressed genes for biosynthesis of active ingredients in fruits of different cultivars of Lycium barbarum L. based on transcriptome sequencing / 生物工程学报

To explore the differentially expressed genes (DEGs) related to biosynthesis of active ingredients in wolfberry fruits of different varieties of Lycium barbarum L. and reveal the molecular mechanism of the differences of active ingredients, we utilized Illumina NovaSeq 6000 high-throughput sequencing technology to conduct transcriptome sequencing on the fruits of 'Ningqi No.1' and 'Ningqi No.7' during the green fruit stage, color turning stage and maturity stage. Subsequently, we compared the profiles of related gene expression in the fruits of the two varieties at different development stages. The results showed that a total of 811 818 178 clean reads were obtained, resulting in 121.76 Gb of valid data. There were 2 827, 2 552 and 2 311 DEGs obtained during the green fruit stage, color turning stage and maturity stage of 'Ningqi No. 1' and 'Ningqi No. 7', respectively, among which 2 153, 2 050 and 1 825 genes were annotated in six databases, including gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) and clusters of orthologous groups of proteins (KOG). In GO database, 1 307, 865 and 624 DEGs of green fruit stage, color turning stage and maturity stage were found to be enriched in biological processes, cell components and molecular functions, respectively. In the KEGG database, the DEGs at three developmental stages were mainly concentrated in metabolic pathways, biosynthesis of secondary metabolites and plant-pathogen interaction. In KOG database, 1 775, 1 751 and 1 541 DEGs were annotated at three developmental stages, respectively. Searching the annotated genes against the PubMed database revealed 18, 26 and 24 DEGs related to the synthesis of active ingredients were mined at the green fruit stage, color turning stage and maturity stage, respectively. These genes are involved in carotenoid, flavonoid, terpenoid, alkaloid, vitamin metabolic pathways, etc. Seven DEGs were verified by RT-qPCR, which showed consistent results with transcriptome sequencing. This study provides preliminary evidences for the differences in the content of active ingredients in different Lycium barbarum L. varieties from the transcriptional level. These evidences may facilitate further exploring the key genes for active ingredients biosynthesis in Lycium barbarum L. and analyzing their expression regulation mechanism.

Effects of ultrasonic pretreatment on far infrared vacuum drying properties and quality of Lycium barbarum / 中草药

Objective: To investigate the effect of ultrasonic technology on the far infrared vacuum drying process by using Lycium barbarum as the test material. Methods: The far-infrared vacuum drying test of L. barbarum was performed by ultrasonic technology. Using the independently developed far-infrared vacuum radiation drying equipment to study the far-infrared vacuum drying characteristics and quality change law of L. barbarum under different ultrasonic frequency, ultrasonic power and ultrasonic treatment time; And the Weibull function was used to simulate the drying process dynamically. Results: With the increase of ultrasonic frequency, ultrasonic power and ultrasonic treatment time, the drying time was significantly shortened, and the drying rate was significantly increased. The Weibull distribution function could better simulate the far-infrared vacuum drying process of L. barbarum under different ultrasonic treatment conditions (r2 = 0.993 9-0.999 2, χ2 = 0.000 1-0.000 6), the scale parameter α generally increased with the increase of ultrasonic frequency, ultrasonic power and ultrasonic processing time, and the shape parameter β was greater than 1; Compared with far-infrared vacuum dried products, ultrasonic treatment can not only reduce the loss of active ingredients of L. barbarum, but also shorten its drying time. Among them, the polysaccharides (812.846 mg/g) of the dried products obtained by the single factor test under the condition of 40 kHz ultrasonic frequency was the highest. When the ultrasonic power was 80 W, the total flavone content of L. barbarum after drying increased up to 28.69% in the single factor test. When the ultrasonic frequency was 60 kHz, the total phenol content of the dried L. barbarum products obtained by the single factor test increased by 10.11%. When the ultrasonic treatment time was 25 min, the corresponding DPPH value of dried L. barbarum producta obtained by the single factor test increased by 14.33%; By comparing the microstructure diagrams under different processing conditions, it was found that ultrasonic waves can not only increase the number of micropores in the surface of materials, but also reduce the damage to L. barbarum epidermal cells. Conclusion: The application of ultrasonic technology to the far-infrared vacuum drying of L. barbarum strengthens the heat and mass transfer process inside and improves the quality of L. barbarum dried fruit, which can provide technical guidance for the development of L. barbarum drying technology.

Preliminary study of medication rule of syndrome differentiation treatment for optic atrophy by TCM master Pin-zheng Liao based on data mining / 中草药

Objective: To dig out and analyze the medication rule of optic atrophy treatment by TCM master Pin-zheng Liao using the Traditional Chinese Medicine Inheritance Support System, and summarize and explore its potential new prescription. Methods: A total of 90 TCM prescriptions for the treatment of optic atrophy by Pin-zheng Liao were collected. Based on frequency statistics, association rule, entropy clustering method rule analysis and other data mining methods, the law and characteristics of drugs were excavated. Results: A total of 113 herbs were included in 90 prescriptions, the most frequently used Chinese herbs were Lycium barbarum, Ganoderma lucidum, etc. Tonic drugs were used the most, the medicated herbs were usually sweet and peace, the Chinese herbs which belong to the liver channel were the most in channel tropism drugs. Seventeen combinations of commonly used drugs were obtained by association rule analysis. Based on entropy clustering method rule analysis, 10 potential new prescriptions were obtained. Conclusion: TCM master Pin-zheng Liao believes that optic atrophy is closely related to liver, spleen and idney. Blood stasis and vein obstruction is the main pathogenesis of the disease. The drugs with effects of activating blood circulation and dredging the meridians, tonifying liver and kidney were recommended for the treating.

Composition differences of polyphenolic compounds in fruits of Lycium ruthenicum Murr. and Lycium barbarum L / 上海交通大学学报（医学版）

Objective: Both Lycium barbarum L. (LB) and Lycium ruthenicum Murr. (LR) belong to the Lycium genus of Solanaceae family but their fruits have significant phenotypic differences in terms of color and shapes. This study is aimed to investigate the inter-species difference of their fruit polyphenol composition. Methods: The polyphenol composition of fresh and dried fruits of two Lycium species were comprehensively analyzed using ultra-high performance liquid chromatography-electrospray ionization-quadrupole-time of flight mass spectrometry (UHPLC-ESIQ- TOF-MS). Results: 35 polyphenolic compounds were detected and quantified in the fresh and dried Lycium fruits including phenolic acids, anthocyanins and phenolamides (i.e., the amide-adducts of polyphenolic acids and polyamines). For both species, the dried fruits had more polyphenolic compounds than their fresh fruits with significant inter-species difference; for both fresh and dried fruits, LR had more polyphenolic compounds than LB. Conclusion: There are significant inter-species differences between these two Lycium in both fresh and dried fruits. These results offer essential information for further understanding of the biological functions of these two Lycium fruits as phytomedicines and functional food.

Toxicity of the goji berry fruit associated with artificial excipients and dried without additives / Toxicidade do fruto goji berry associado a excipientes artificiais e desidratado sem aditivos

The aim of this study was to evaluate the cytotoxic and genotoxic potential of goji berry fruit-based pharmaceutical powders obtained from three pharmaceutical laboratories. The product A was tested at concentrations of 0.012; 0.025 and 0.05 g mL-1, and B and C at concentrations 0.02; 0.04 and 0.08 g mL-1. It was also evaluated the tea of the dried goji berry fruit (non-additives) in the concentrations 0.035; 0.07 and 0.14 g mL-1 for comparison to the results obtained with powdered goji berry. Tea concentrations in the two exposure times did not cause inhibition of cell division nor cellular alterations to meristem tissues. For the industrialized goji products, all concentrations analyzed caused significant antiproliferative effect to the tissues evaluated at the shortest time of analysis. There were no significant cellular changes in tissues exposed to industrialized goji. Therefore, under the conditions of analysis, goji berry powder, at the three concentrations evaluated, was cytotoxic to root meristems.

Objetivou-se na presente pesquisa avaliar, em células meristemáticas de raízes de A. cepa, nos tempos de exposição 24 e 48 horas, o potencial citotóxico e genotóxico de produtos farmacêuticos do fruto goji berry em pó, provenientes de três laboratórios farmacêuticos. O produto A foi avaliado nas concentrações 0,012; 0,025 e 0,05 g mL-1, e B e C nas concentrações 0,02; 0,04 e 0,08 g mL-1. Avaliou-se também o chá do fruto seco de goji (não aditivado), nas concentrações 0,035; 0,07 e 0,14 g mL-1, para comparação com os resultados obtidos do fruto em pó. Verificou-se que o chá nas concentrações avaliadas, nos dois tempos de exposição estabelecidos, não ocasionaram inibição da divisão celular e nem alterações celulares aos meristemas de raízes. Para os goji industrializados, todas as concentrações analisadas causaram efeito antiproliferativo significativo aos tecidos avaliados logo no menor tempo de análise considerado. Nenhum dos produtos industrializados causou número significativo de alterações aos meristemas analisados. Assim, os goji em pó foram citotóxicos ao bioensaio utilizado por terem acarretado relevante instabilidade genética aos meristemas de raízes.

Research and application of Lycii Fructus in medicinal field / 中草药·英文版

Lycii Fructus (Lycium barbarum, Gouqizi in Chinese name) is one of the most popular Chinese material medica and a common ingredient in tonic food. This fruit has been paid rapidly growing attention for its nutrient value and noticeable pharmacological properties. The present paper focuses on the legal resource of Gouqizi and reviews the main research in medicinal field including botanical identification, ethnopharmacological functions, phytochemistry, pharmacological effects, clinic usages, and safety issues. In addition, some issues needed address will be also discussed. We strongly believe that further investigation will deepen our knowledge of Gouqizi and promote the industrial development in the world.

Research progress on extraction, purification, and molecular structural elucidation of active polysaccharide from Lycii Fructus and analysis on its industrial development status and prospect / 中草药

Lycii Fructus is a precious traditional medicinal and edible herb with multifunction, in which polysaccharide is believed to be a predominant active substance with multifarious biological functions and broad utilization prospect. In this review, the new technology application in extraction, purification, and structural elucidation of Lycii Fructus polysaccharide was systematically untangled, the current research situation and development trend were outlined, and the potential product groups and industry prospect in Lycii Fructus polysaccharide industrialization were analyzed to provide scientific reference for the enhancement of effective utilization and industrial chain of Lycii Fructus resources.

Determination of polysaccharide in Lycium barbarum L. extract powder containing malt dextrin / 国际药学研究杂志

Objective To establish a method for determination of polysaccharide in Lycium barbarum L . extract powder containing with malt dextrin. Method Maltodextrin was treated with glucoamylase hydrolysis and ethanol precipitation successively;then the phenol-sulfuric acid method was used to determine the content of polysaccharide. The glucoamylase hydrolysis technolgy of malt dextrin was optimized, and the variation of polysaccharide content before and after hydrolysis, the difference of polysaccharide contents between direct determination and truth were compared. Morever, the hydrolysis effect of glucoamylase on Lycium barbarum L. was investigated. Results The optimum hydrolysis condition was: temperature 60℃, the ratio of enzyme to substrate 1∶5, hydrolysis time 30 min. The polysaccharide content determined directly was significantly higher than the real value (P0.05). Glucoamylase showed no hydrolysis effects on Lycium barbarum L. polysaccharide. Conclusion Glucoamylase hydrolysis can deplete the influence of malt dextrin on determination of polysaccharide in Lycium barbarum L. extract powder, the procedure is simple and effective.

Determination of polysaccharide in Lycium barbarum L. extract powder containing malt dextrin / 国际药学研究杂志

Objective To establish a method for determination of polysaccharide in Lycium barbarum L. extract powder containing with malt dextrin. Method Maltodextrin was treated with glucoamylase hydrolysis and ethanol precipitation successively; then the phenol-sulfuric acid method was used to determine the content of polysaccharide. The glucoamylase hydrolysis technolgy of malt dextrin was optimized, and the variation of polysaccharide content before and after hydrolysis, the difference of polysaccharide contents between direct determination and truth were compared. Morever, the hydrolysis effect of glucoamylase on Lycium barbarum L. was investigated. Results The optimum hydrolysis condition was: temperature 60" , the ratio of enzyme to substrate 1 #5, hydrolysis time 30 min. The polysaccharide content determined directly was significantly higher than the real value (P0.05). Glucoamylase showed no hydrolysis effects on Lycium barbarum L. polysaccharide. Conclusion Glucoamylase hydrolysis can deplete the influence of malt dextrin on determination of polysaccharide in Lycium barbarum L. extract powder, the procedure is simple and effective.

Cloning and bioinformation analysis of chalcone synthase gene from Lycium barbarum in Ningxia / 中草药

Objective: To obtain cDNA of chalcone synthase (CHS, EC 2.3.1.74) involved in the flavonoid/isoflavonoid biosynthesis pathway. Methods: The partial sequence of CHS in Lycium barbarum (LyCHS) was successfully cloned by RT-PCR and using a sequence homology strategy, and the bioinformation analysis was carried out. Results: The cDNA fragment of CHS gene was 1148 bp in length, which encoded a protein of 382 amino acids with the predicted relative molecular weight of 4.168 × 104. The estimated isoelectric point (pI) of the putative protein is 6.22. According to the amino acid sequence and structural analysis, it showed that this protein contained one conserved active site, namely chalcone and stilbene synthases active site. Subcellular localizations of LyCHS proteins were likely in the cytoplasm. The secondary and tertiary structures of LyCHS were abundant in α-helixs (166) and random coils (126), while were less in β-turns (25) and extended strains (65). Phylogenetic analysis showed that the genes of LyCHS were closely related to CHS in Solanum pinnatisectum, S. tuberosum, and Lycopersicon esculentum. The sequences had been registered in GenBank with the accession numbers JQ964237. Conclusion: The present results provide the foundation for the next study of CHS gene about its expression and function in L. barbarum.

Rhizosecretion of HIV-1 Recombinant Capsid Protein from Transgenic Lycium barbarum L.Hairy Roots / 中国生物工程杂志

HIV p24 core protein can induce both cellular and neutralizing antibody responses.HIV-1 CA-virus-like particles(VLPs)vaccines provide a promising approach for the development of an effective vaccination strategy against HIV infection.Rhizosecreion of the recombinant proteins provides a new manufacturing platform that can simplify the extraction and purification procedure.Lycium barbarum L.was transformed by Agrobacterium tumefaciens EHA105 harboring the plant expression vector pCAMBIA1305.2-MA4-CA with a GRP signal peptide and MA4-CA fusion gene.Transgenic hairy roots were induced and cultivated in hydroponic culture.Western blotting indicated that the recombinant CA proteins were present in two forms,a glycosylated monomer(37 kDa)and a dimer(50 kDa)in the roots and hydroponic medium.It appeared from the present immunohistochemical data that the recombinant CA proteins fused with GRP signal peptide were confined to the cytoplasm,cell wall and intercellular space,indicating targeting into the secretory pathway.It demonstrated for the first time the rhizosecretion of HIV-1 recombinant capsid protein in Lycium barbarum L.hairy roots,and may offer a novel method for expressing HIV-1 CA-VLPs vaccines in plants.

Quality in fruits of Lycium barbarum and its relationship with soil fertility factors / 中草药

Objective The contents of total sugar,polysaccharide,amino acid,betaine,carotenoid,flavone,hundred-seed weight of Lycium barbarum fruits from eight different habitats in first stubble,prosperous time,and autumn were determined and the ratios of total sugar to betaine were analyzed,in order to classify these indexes,and investigate the relationship with the soil fertility factors.MethodsHPLC and UV-spectrophotometry methods were used to determine the contents and the results were analyzed adopting the clustering,correlation,and variance analysis.Results All the indexes were divided into three parts which were total sugar,betaine,and carotenoid.Various indexes were affected in different degrees by the soil fertility factors.Conclusion The contents of total sugar,betaine,carotenoid,and the ratios of total sugar to betaine could be used to assay the quality of L.barbarum fruits reasonably;The accumulation of each ingredient in L.barbarum fruits is affected by the synergistic effects of all the soil fertility factors,not a single role.It would be beneficial to the fertilization and improvement for the quality of L.barbarum.

Extraction and Content Determination of Polysaccharide of Xinjiang Lycium Barbarum L / 中成药

We determined the polysaccharide content of different grade Xinjiang Lycium Barbarum L. in the way of colo]rimetry of phenolsulfuric acid at 490nm wavelength. All of them contained polysaccharide, But the higher content was found to be in big, red and thick fruits.