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ObjectiveAgrobacterium tumefaciens-mediated transformation （ATMT） of Clonostachys rosea， an endophytic fungus of Glycyrrhiza uralensis seeds， was established and optimized， and orthogonal test was designed to optimize the colonization conditions of C. rosea for G. uralensis seeds， so as to lay foundation for the development of biofertilizer and the breeding of high-quality G. uralensis. MethodThe conditions of ATMT were optimized from three aspects， including the concentration of acetosyringone， co-culture time and the concentration of conidia of recipient fungi. Then， high-quality transformants were selected. Orthogonal test was used to optimize the colonization conditions by taking co-culture temperature， co-culture time and spore concentration as factors and colonization rate as index. ResultWhen spore concentration was 1×107 cfu·mL-1， acetosyringone concentration was 150 μmol·L-1 and the co-culture time was 60 h， the transformation efficiency of C. rosea was the highest， which was 135 transformants per 1×107 recipient fungal spores. The accuracy and stability of the transformations were tested by cloning the marker gene green fluorescent protein （GFP） and β-glucuronidase （GUS） staining. When co-culture temperature was 25 ℃， co-culture time was 36 h and the spore concentration was 1×106 cfu·mL-1， the colonizing rate for C. rosea back dyeing into G. uralensis seeds by seed soaking method was the highest， which was 71.11%. ConclusionThis study successfully establishes stable and efficient technical systems not only of ATMT in C. rosea， but also of colonization of the transformants into G. uralensis seeds， which can lay a foundation for the development of biofertilizer of G. uralensis.
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The present study investigated the chemical constituents from the aerial parts of Glycyrrhiza uralensis. The ethanol extract of the aerial parts of G. uralensis was separated and purified by different column chromatographies such as macroporous resin, silica gel, and Sephadex LH-20, and through preparative HPLC and recrystallization. Thirteen compounds were isolated and identified as(2S)-6-[(Z)-3-hydroxymethyl-2-butenyl]-5,7,3'-trihydroxy-4'-methoxy-dihydroflavanone(1),(2S)-8-[(E)-3-hydroxymethyl-2-butenyl]-5,7,3',5'-tetrahydroxy-dihydroflavanone(2), α,α'-dihydro-5,4'-dihydroxy-3-acetoxy-2-isopentenylstilbene(3), 6-prenylquercetin(4), 6-prenylquercetin-3-methyl ether(5), formononetin(6), 3,3'-dimethylquercetin(7), chrysoeriol(8), diosmetin(9),(10E,12Z,14E)-9,16-dioxooctadec-10,12,14-trienoic acid(10), 5,7,3',4'-tetrahydroxy-6-prenyl-dihydroflavanone(11), naringenin(12), dibutylphthalate(13). Compounds 1-3 are new compounds, and compounds 10 and 13 are isolated from aerial parts of this plant for the first time.
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Glycyrrhiza uralensis/chemistry , Plant Components, Aerial/chemistryABSTRACT
OBJECTIVE To optimize the preparation technology of baicalin （BCN）-glycyrrhizic acid （GA） solid nanocrystals （BCN-GA-SN）， to characterize them and investigate their in vitro release characteristics. METHODS According to the compatibility ratio of classic couplet medicinals “Scutellaria baicalensis-Glycyrrhiza uralensis”， the compatibility ratio of BCN and GA was determined as 6∶1 （m/m）； BCN-GA nanosuspension was prepared by precipitation method combined with high-pressure homogenization method. The preparation technology of BCN-GA nanosuspension was optimized by using mean particle size and polydispersity index （PDI） as indexes and with types and dosage of stabilizers， stirring speed and time， high-pressure homogenization pressure and frequency as factors. The freeze-dried consolidation process of BCN-GA nanosuspension was optimized to prepare BCN-GA-SN using average particle size， PDI and redispersibility index （RDI） as indicators， with the type and dosage of freeze-dried protective agents as factors； then， the physicochemical properties and in vitro release of BCN-GA-SN were investigated. RESULTS The optimal preparation technology of BCN-GA-SN was as follows： BCN-GA nanosuspension was prepared by using 15% sodium dodecyl sulfate as a stabilizer， stirring at 1 000 r/min for 15 minutes， and homogenizing at 100 MPa for 20 times； then， BCN-GA nanosuspension was freeze-dried and solidified with 5% mannitol （corresponding to the dosage of BCN）. The average particle size of prepared BCN-GA-SN was （442.2±5.7） nm with PDI of 0.225±0.015 and RDI of 1.055± 0.013. The prepared BCN-GA-SN presented as the irregularly spherical shape with more uniform size； the drug-loading amount of BCN in the nanocrystal was （62.5±0.7）%， and that of GA was （9.4±0.2）%； the in vitro release results showed that the cumulative dissolution of BCN-GA-SN was higher than that of the physical mixture of BCN and GA. CONCLUSIONS BCN-GA-SN is prepared successfully in this study with uniform particle size and even distribution， which can effectively improve the dissolution of BCN.
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OBJECTIVE@#To evaluate the antidiarrheal effect of ethanol extract of Glycyrrhiza uralensis Fisch root (GFR) in vivo and jejunal contraction in vitro.@*METHODS@#In vivo, 50 mice were divided into negative control, positive control (verapamil), low-, medium- and high-dose GFR (250, 500, 1,000 mg/kg) groups by a random number table, 10 mice in each group. The antidiarrheal activity was evaluated in castor oil-induced diarrhea mice model by evacuation index (EI). In vitro, the effects of GFR (0.01, 0.03, 0.1, 0.3, 1, 3, and 10 g/L) on the spontaneous contraction of isolated smooth muscle of rabbit jejunum and contraction of pretreated by Acetylcholine (ACh, 10 µmol/L) and KCl (60 mmol/L) were observed for 200 s. In addition, CaCl2 was accumulated to further study its mechanism after pretreating jejunal smooth muscle with GFR (1 and 3 g/L) or verapamil (0.03 and 0.1 µmol/L) in a Ca2+-free-high-K+ solution containing ethylene diamine tetraacetic acid (EDTA).@*RESULTS@#GFR (500 and 1,000 mg/kg) significantly reduced EI in castor oil-induced diarrhea model mice (P<0.01). Meanwhile, GFR (0.01, 0.03, 0.1, 0.3, 1, 3, and 10 g/L) inhibited the spontaneous contraction of rabbit jejunum (P<0.05 or P<0.01). Contraction of jejunums samples pretreated by ACh and KCl with 50% effective concentration (EC50) values was 1.05 (0.71-1.24), 0.34 (0.29-0.41) and 0.15 (0.11-0.20) g/L, respectively. In addition, GFR moved the concentration-effect curve of CaCl2 down to the right, showing a similar effect to verapamil.@*CONCLUSIONS@#GFR can effectively against diarrhea and inhibit intestinal contraction, and these antidiarrheal effects may be based on blocking L-type Ca2+ channels and muscarinic receptors.
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Mice , Rabbits , Animals , Antidiarrheals/adverse effects , Jejunum , Glycyrrhiza uralensis , Castor Oil/adverse effects , Calcium Chloride/adverse effects , Diarrhea/drug therapy , Plant Extracts/adverse effects , Verapamil/adverse effects , Muscle ContractionABSTRACT
(3S)-Linalool synthase (LIS) is a key enzyme involved in the monoterpene biosynthetic pathway. Based on our previous transcriptome study, the expression level of LIS gene was exceedingly related to glycyrrhizic acid (GA) biosynthesis. Therefore, we used hairy root culturing to further investigate the effect of LIS on the GA biosynthesis. A LIS gene (GenBank accession number: MZ169552) was cloned from Glycyrrhiza uralensis. The plant binary overexpression vector pCA-LIS was constructed by gene fusion. G. uralensis hairy roots overexpressing LIS were induced by the Agrobacterium rhizogenes ATCC15834. The expression levels of LIS were analyzed by real-time quantitative PCR (RT-qPCR) and the contents of GA in hairy root lines were determined by UPLC. It was found that in the hairy root lines overexpressing LIS, the expression levels of LIS were significantly higher than that in the wild type, while the contents of GA were remarkably lower than those in the wild type and negative control. These findings indicate that the expression level of LIS is negatively correlated with the accumulation of GA. In this study, LIS was cloned from G. uralensis for the first time and the negative regulatory effect of LIS on GA biosynthesis was confirmed by reverse genetics. This work provides support for further improvement of the molecular regulatory network of GA biosynthesis in G. uralensis.
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Licorice, one of the most commonly used medicinal materials in China, grows mainly in arid and semi-arid regions and has important economic and ecological values. Basic leucine zipper (bZIP) transcription factors in plants play an important role in regulating biological or abiotic stress responses, growth, and secondary metabolite synthesis. bZIP transcription factors in the published whole genome database of Glycyrrhiza uralensis were identified using bZIP sequences found in Arabidopsis thaliana genome as reference, and ABA-dependent bZIP genes were identified by using Illumina high-throughput sequencing. The physical and chemical properties, structure of the encoded proteins, and the gene expression patterns with exogenous ABA stress were analyzed. A total of 69 bZIP transcription factor genes were identified in G. uralensis, named Gubzip1-69, and they were divided into 10 subfamilies (A-I and S) according to their similarity to bZIPs of A. thaliana. By calculating the relative expression levels of the 69 GubZIPs genes under different concentrations of exogenous ABA stress, genes that may be involved in the regulation of ABA signaling pathways were identified, namely GubZIP1, GubZIP5, GubZIP8, GubZIP30, GubZIP33 and GubZIP56. The results of expression pattern analysis of these GubZIPs genes under exogenous ABA stress showed that the expression pattern of GubZIPs genes changed significantly with 50 mg·L-1 ABA. The relative expression levels of these genes decreased 3 h after treatment, and gradually increased 6 h after treatment. Except for GubZIP8, the relative expression levels of these genes were significantly increased after 12 h. Further research on the function of bZIP transcription factors of G. uralensis and elucidating their regulatory mechanisms should be of interest and will provide a scientific basis for cultivating high-quality cultivars of G. uralensis through molecular breeding methods.
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Objective:To investigate the immunoregulatory effects of Glycyrrhiza uralensis ethanol extract(GUEE) on the maturation of dendritic cells (DCs) and the adjuvant effect of GUEE on OVA in na?ve BALB/c mice and an ovalbumin (OVA)-induced asthma mouse model. Methods:GUEE was prepared, and the effects of different concentrations of GUEE on the maturation of DCs and the secreted cytokines as well as the effects of GUEE on bacterial lipopolysaccharide (LPS)-induced DC maturation were examined in vitro. The effect of GUEE on the morphology of mouse bone marrow derived DCs was observed using microscopy. Molecular expression levels on the surface of DCs were detected using flow cytometry. The levels of interleukin-1β (IL-1β), IL-6, IL-12, and tumor necrosis factor-α (TNF-α) in the supernatant of DCs cultures were measured by enzyme-linked immunosorbent assay (ELISA). The maturation status of DCs was detected by flow cytometry by injecting different concentrations of GUEE into the paws of mice and isolating the draining lymph nodes 24 h later. The naive BALB/c mice were co-immunized with OVA, and the changes in regulatory T cells (Treg) were detected by flow cytometry. An OVA-protein-induced mouse asthma model was established to investigate whether GUEE as a tolerogenic adjuvant has an antigen-specific therapeutic effect on asthmatic mice. Pulmonary pathological changes were analyzed by hematoxylin-eosin staining (HE) and PAS staining. OVA-specific antibodies in serum and the frequencies of Tregs, CD4 + IFN-γ + and CD4 + IL-4 + T cells in the spleen were detected by ELISA and flow cytometry, respectively. Results:GUEE suppressed DCs maturation induced by LPS both in vitro and in vivo (all P<0.05), and reduced proinflammatory cytokine production, including IL-1β, IL-6, IL-12 and TNF-α in the absence or presence of LPS (all P<0.05). Moreover, co-immunization with OVA and GUEE increased the amount of Tregs in na?ve BALB/c mice ( P<0.05). In OVA-induced asthmatic mice, OVA and GUEE co-immunization and GUEE alone treatment substantially ameliorated the inflammation of lung tissues, decreased the levels of IgG 1 and the amount of CD4 + IL-4 + T cells, and increased the amount of Tregs (all P<0.05). Conclusions:GUEE alone or as the tolerogenic adjuvant can ameliorate allergic diseases through inhibition of DC maturation and type 2 helper T cell responses and induction of Tregs.
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OBJECTIVE To study the effects of self-assembled nanoparticles from Shaoy ao gancao decoction （SGD-SAN）on the in vitro release and intestinal absorption of the main components of Glycyrrhiza uralensis . METHODS Gancao single decoction （GSD），Shaoyao single decoction （SSD），mixed suspension of Shaoyao and Gancao single decoction （MSSGD）and SGD （i.e. Shaoyao-Gancao decoction ）were prepared ，and SAN was characterized. HPLC method was adopted to determine the contents of 7 main components （liquiritin apioside ， liquiritin， isoliquiritin apioside ， isoliquiritin， liquiritigenin， glycyrrhizic acid ， isoliquiritigenin）in G. uralensis . The dialysis bag method was used to investigate the effects of the formation of SGD-SAN on in vitro release of 7 main components in G. uralensis with pH 1.2 HCl solution and pH 6.8 phosphate buffered solution （PBS）as release media. Single-pass intestinal perfusion study was performed to investigate the effects of the formation of SGD-SAN on the intestinal absorption of 7 main components from G. uralensis . RESULTS SAN with particle size of 200-300 nm and polydispersity index of 0.3-0.5 was found in GSD ，MSSGD and SGD. GSD-SAN and MSSGD-SAN were in rod shape while SGD-SAN was irregularly spherical under transmission electron microscope. The results of in vitro release study showed that the formation of SGD-SAN could significantly increase in vitro release of liquiritigenin ，isoliquiritigenin and glycyrrhizic acid ，and had no effect on other components of G. uralensis in pH 1.2 HCl solution. The formation of SGD-SAN also had no effect on the release of each component from G. uralensis in pH 6.8 PBS. The results of intestinal perfusion experiments showed that the formation of SGD-SAN could significantly promote the absorption of each component from G. uralensis in the ileum. CONCLUSIONS- The formation of SGD-SAN significantly improves the in vitro release of poorly soluble components from G. uralensis and promotes the intestinal absorption of main components from G. uralensis ，which is the physical structure basis for the compatibility and synergy of Paeonia lactiflora and G. uralensis .
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Pseudo-allergic reactions (PARs) widely occur upon application of drugs or functional foods. Anti-pseudo-allergic ingredients from natural products have attracted much attention. This study aimed to investigate anti-pseudo-allergic compounds in licorice. The anti-pseudo-allergic effect of licorice extract was evaluated in rat basophilic leukemia 2H3 (RBL-2H3) cells. Anti-pseudo-allergic compounds were screened by using RBL-2H3 cell extraction and the effects of target components were verified further in RBL-2H3 cells, mouse peritoneal mast cells (MPMCs) and mice. Molecular docking and human MRGPRX2-expressing HEK293T cells (MRGPRX2-HEK293T cells) extraction were performed to determine the potential ligands of MAS-related G protein-coupled receptor-X2 (MRGPRX2), a pivotal target for PARs. Glycyrrhizic acid (GA) and licorice chalcone A (LA) were screened and shown to inhibit Compound48/80-induced degranulation and calcium influx in RBL-2H3 cells. GA and LA also inhibited degranulation in MPMCs and increase of histamine and TNF-α in mice. LA could bind to MRGPRX2, as determined by molecular docking and MRGPRX2-HEK293T cell extraction. Our study provides a strong rationale for using GA and LA as novel treatment options for PARs. LA is a potential ligand of MRGPRX2.
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Animals , Humans , Mice , Rats , Anti-Allergic Agents/therapeutic use , Calcium/metabolism , Cell Degranulation , Glycyrrhiza , HEK293 Cells , Hypersensitivity/drug therapy , Mast Cells/metabolism , Mice, Inbred C57BL , Molecular Docking Simulation , Nerve Tissue Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Neuropeptide/therapeutic useABSTRACT
Objective:To clone uridine diphosphate （UDP）-glucose dehydrogenase （<italic>UGDH</italic>） gene of <italic>Glycyrrhiza uralensis</italic> and analyze its bioinformatics and expression. Method:Total RNA was extracted from roots， stems， and leaves of 6-week-old seedlings of <italic>G. uralensis</italic>， the complementary deoxyribonucleic acid （cDNA） sequence of <italic>GuUGDH</italic>1 gene （Gu was short for <italic>G. uralensis</italic>） was cloned by reverse transcription polymerase chain reaction （RT-PCR）， then sequencing and bioinformatic analysis were performed， and the specificity of the tissue was analyzed by real-time fluorescence quantitative PCR （Real-time PCR）. Result:The open reading frame（ORF）of <italic>GuUGDH</italic>1 gene was 1 443 bp in length and encoded 480 amino acid residues （GenBank accession number of MT968993）. Bioinformatics analysis showed that GuUGDH1 was a stable acidic hydrophilic protein with a relative molecular weight of 53.056 kDa， an isoelectric point of 5.89， no signal peptide and no transmembrane helix， and all of them were outside the membrane. There were three typical conserved domains， which belonged to the UDP-glucose/guanosine diphosphate （GDP）-mannose dehydrogenase family. Phylogenetic analysis showed that the <italic>GuUGDH</italic>1 gene was closely related to <italic>Glycine max</italic> and <italic>Spatholobus suberectus</italic>. The results of Real-time PCR showed that the expression of <italic>GuUGDH</italic>1 gene could be detected in the roots， stems， and leaves of 6-week-old seedlings of <italic>G. uralensis</italic>， and the expression level in the roots was significantly higher than that in the stems and leaves. Conclusion:In this study， the <italic>UGDH</italic>1 gene of <italic>G. uralensis</italic> was cloned and its protein sequence characteristics were systematically analyzed， which can provide theoretical basis for further research on the catalytic function of UGDH1 protein.
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Chemical constituents from aerial parts of Glycyrrhiza uralensis were analyzed and identified using ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry(UPLC-Q-Exactive Orbitrap-MS). The chromatographic column of Waters Acquity UPLC BEH-C_(18)(2.1 mm×100 mm, 1.7 μm) was adopted, with acetonitrile-water(0.5% formic acid) as mobile phase at a flow rate of 0.2 mL·min~(-1). Data was collected in positive and negative modes of electrospray ionization(ESI). A total of 55 compounds, including 42 flavonoids, 9 stilbenes, 2 coumarins, 1 lignin and 1 phenolic acid, which were characterized in the aerial parts of G. uralensis based on accurate molecular mass information of molecular and product ions provided by UPLC-Q-Exactive Orbitrap-MS based on comparison with standard substances and references. It is an effective and accurate method to provide chemical information of constituents in aerial parts of G. uralensis, and can provide a reference for further study on pharmacodynamic material basis and resources development and utilization.
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Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Glycyrrhiza uralensis , Mass Spectrometry , Plant Components, AerialABSTRACT
A metabolomics method was used to search for chemical markers in prepared slices of Glycyrrhiza uralensis with different degrees of honey processing. Coupled with these metabolomics analytical methods, ultra-performance liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS) was used to generate global chemical profiles of the raw material of Glycyrrhiza uralensis and the prepared slices. The samples were collected in Shanxi, Hebei Zhangjiakou and Inner Mongolia. A total of 57 chemical components were identified in Glycyrrhiza uralensis by using the UNIFI theoretical database combined with the library of reference samples. Among them, 37 compounds were identified in positive ion mode and 56 compounds were identified in negative ion mode. Unsupervised principal component analysis (PCA) showed that the chemical ingredients differed considerably depending on the extent of processing. Supervised orthogonal partial least squares discriminant analysis (OPLS-DA) was used to differentiate the moderate processing group and the raw group, and partial least squares discriminant analysis (PLS-DA) was used to differentiate the less, the moderate, and the excessive processing groups. The results showed that the contents of glycyrrhizic acid, licoricesaponin G2, and licoricesaponin E2 varied with the extent of processing. The content of these components increased after processing, and reached the highest level when the extent of processing was moderate (P < 0.05). Glycyrrhizic acid, licoricesaponin G2 and licoricesaponin E2 can be regarded as the chemical markers to differentiate the samples with different degrees of processing. These three compounds can be used to monitor the processing of Glycyrrhiza uralensis.
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The purpose of this study was to explore the interaction mechanism between glycyrrhiza protein and berberine in the decocting process of Rhizoma Coptidis and Liquorice and its effect on the pharmacodynamic effect. In this experiment, licorice crude protein was obtained from licorice decoction pieces, and it was found that licorice crude protein and berberine could form spherical supramolecular particles after decocting together. Morphological characterization was carried out by using Malvin particle size analyzer and emission scanning electron microscopy, and the supramolecular particles were observed to be nanoscale, which was significantly different from the morphology of licorice protein and berberine. The results of ultraviolet, infrared and fluorescence spectroscopy showed that the mechanism of molecular interaction was induced by weak bonds such as electrostatic attraction and hydrophobic interaction. Furthermore, the antimicrobial activity of berberine was significantly affected by the supramolecular particles of licorice protein-berberine, which were significantly different from the mechanical mixture. This study reveals the pharmacological value of macromolecular substances such as proteins in the decoction of licorice and Coptis chinensis from a new perspective, which is helpful to promote the secondary development of clinical effective prescriptions, especially the research on the pharmacological substance basis of classic famous prescriptions.
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OBJECTIVE：To establish a method for online detection of antioxidant active components in Glycyrrhiza uroalensis decoction pieces ，and to identify it. METHODS ：The free radical scavenging rate of 1，1-diphenyl-2-trinitrobenzene hydrazine （DPPH）was determined to evaluate the antioxidant activity of G. uralensis decoction pieces. HPLC-UV-DPPH method was used to screen the anti oxidant active components of G. uralensis decoction pieces. HPLC-TOF/MS was used to obtain mass spectrum data and Qualitive Analyst B 06.00 Build 6.0.633.0 software was used to analyze data. Through contrast analysis of UV absorption spectrum，online chromatogram ，mass spectrum information of G. uralensis and the retention time of each compound ，accurate molecular weight ，antioxidant active components were identified by referring to relevant literature. Validation test was also conducted. RESULTS ：DPPH free radical scavenging rate in 8 batches of G. uralensis decoction pieces ranged 55.71％-60.17％. Seven antioxidative active compounds ，including avolomotor ，8-isopentenyl naringin ，yellow lupulin weitone ，isoflavone B ，3′， 4′-dimethoxy3-hydroxy-6-methyl flavone ，glycyrrhizin E and glycyrrhizin H ，could be screened from G. uralensis decoction pieces. After validation ，the peak area of inverted peak generated by online reaction was positively correlated with DPPH free radical scavenging rate. CONCLUSIONS ：Established method is simple and accurate ，and can be used to quickly screen and identify the main antioxidant components of G. uralensis decoction pieces ；the peak area of inverted peak can be used to evaluate the antioxidant active components of G. uralensis decoction pieces.
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Chalcone isomerase (CHI) is the second rate-limiting enzyme involved in the biosynthetic pathway of flavonoids in Glycyrrhiza uralensis. Based on our previous studies, we selected the specific CHI haplotype (GenBank Accession No. KY115232) to maximize flavonoid accumulation. We constructed a plant binary expression vector for overexpression of this CHI gene by the gene fusion method and transfected the plasmid into Agrobacterium tumefaciens ACCC10060 by electroporation. The recombinant A. tumefaciens ACCC10060 subsequently was used to infect cotyledons and hypocotyls of G. uralensis to obtain transgenic hairy roots. A qRT-PCR method was used to determine the copy number of CHI and a UPLC method was used to assay the content of four flavonoids in different hairy root lines. The qRT-PCR results showed that the copy number of CHI in hairy roots was 1 or 5. UPLC results showed that the content of total flavonoids, liquiritin, liquiritigenin, and isoliquiritigenin in transgenic hairy root samples was significantly higher than that in wild-type samples. This study demonstrates that overexpression of CHI significantly increases the content of flavonoids in hairy roots of G. uralensis. This work provides a theoretical basis for clarifying the function of CHI. Three transgenic hairy root lines of G. uralensis were isolated which can be used to increase the accumulation of licorice flavonoids in vitro.
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OBJECTIVE: To establish the HPLC specific chromatogram of licorice flavonoids based on chemometric analysis method, and search for differentiated components of Glycyrrhiza uralensis Fisch. cultivated for different years. METHODS: The analysis was performed on a Shiseido Capcell Pak C18 column(4.6 mm×250 mm,5 μm) by gradient elution with acetonitrile-0.1% formic acid solution as mobile phase at a flow rate of 1.0 mL•min-1. The column temperature was maintained at 40℃ and the detection wavelength was set at 280 nm. The HPLC specific chromatograms of Glycyrrhiza uralensis Fisch. cultivated for different years were further evaluated by chemometric methods including similarity analysis (SA),hierarchical clustering analysis (HCA) and principal component analysis (PCA). RESULTS: Four-years-old licorice was confirmed as the best one. Liquiritin, liquiritin apioside, liquiritigenin and isoliquiritin had significant differences, which can be used as key indicators to distinguish Glycyrrhiza uralensis Fisch. cultivated for different years. CONCLUSION: This method can provide reference for standardized cultivation and quality standard improvement of licorice.
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OBJECTIVEP: To evaluate comprehensively the quality of Glycyrrhiza uralensis Fisch. in different harvest periods by chemometric analysis based on the HPLC specific chromatogram and multi-component assay. METHODS: The similarity was analyzed with "Similarity Evaluation System for Chromatographic Fingerprint of Chinese Materia Medica 2012". t-test, correlation analysis, clustering analysis(HCA), principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used for data analysis. RESULTS: Twenty-one peaks were selected as common peaks of the fingerprint. The similarity of the samples were above 0.9. The contents of liquiritin, glycyrrhizic acid, liquiritin apioside, isoliquiritin apioside, isoliquiritin, neoisoliquiritin, echinatin, and liquiritigenin were determined.There were some differences in the quality of Glycyrrhiza uralensis Fisch. in different harvest periods, with liquiritin apioside, neoisoliquiritin and echinatin as the main compounds of difference. CONCLUSION: Autumn is confirmed as the best harvesting period of Glycyrrhiza uralensis Fisch. by chemometric analysis. It is suggested that liquiritin apioside should be used as the key quality control indicator for evaluating Glycyrrhiza uralensis Fisch.in different harvest periods.
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Objective:To identify the quality differential markers of different processed products of Glycyrrhiza uralensis dry roots and rhizomes. Method:Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MSE) was used to collect high-precision mass-charge ratio and ion response strength information of the components in G. uralensis dry roots and rhizomes before and after processing by negative ion mode. The data set collected after pretreatment was analyzed with principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) to quickly search the differential components in different processed products of G. uralensis dry roots and rhizomes. Differential components were identified according to the relative molecular weight, fragment ion, mass spectrum database and related literature information, then the migration of components before and after processing was studied. Result:A total of 10 quality differential markers were searched from raw products, roasted products and honey-roasted products of G. uralensis dry roots and rhizomes, mainly derivatives of liquiritin and glycyrrhizic acid. Among them, the contents of 6''-O-acetylliquiritin apioside, 6''-O-acetylliquiritin apioside isomer, 6''-O-acetylliquiritin, formononetin and 11-deoxo-18β-glycyrrhetic acid were the highest in the raw products, the contents of 6''-O-acetylisoliquiritin apioside, 6''-O-acetylisoliquiritin, isoliquiritin and glycyrrhetic acid 3-O-glucuronide were the highest in the roasted products, the content of liquiritin was the lowest in the honey-roasted products. Conclusion:There are some chemical differences among the three products. This study can provide material basis for the quality control and pharmacodynamic research of processed products of G. uralensis dry roots and rhizomes.
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Objective: To isolate and purify endophytic fungus from Glycyrrhiza uralensis, and screen the specific strain with better antibacterial and antioxidant activity (DPPH• radical scavenging, total reducing power, determination of hydroxyl radical scavenging) and anti-alpha-glucosidase activity. Methods: The endophytic fungi were isolated and purified from G. uralensis by tissue cutting. N-butanol, ethyl acetate and ethanol were used to extract the fermentation liquid and mycelium. Antibacterial activity was detected by filter paper. Three methods were used to characterize antioxidant activity. A total of 36 endophytic fungi were isolated from G. uralensis by PNPG method. Results: A total of seven genera were isolated from 108 samples by concentrated fermentation liquid extraction. Trichoderma. sp was the dominant species. The experimental results showed that 43.52% of the samples had different degrees of antibacterial effect, of which 8.33% performed well; The extracts showed different levels of antioxidant activity, of which 4.63% to 10.12% showed better performance in the three methods. 99.07% of the samples had different levels of anti-α-glucosidase activity, of which 5.56% of the samples performed well. Conclusion: The strains isolated from G. uralensis have good antibacterial activity, antioxidant activity, and anti-alpha-glucosidase activity, which further study is needed.
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OBJECTIVE：To compare the che mical composition differences of raw and fried processed Glycyrrhiza uralensis ， and provide reference for clarifying the regularity of chemical composition change before and after processing and material basis of differential use of raw and fried G. uralensis . METHODS ：UPLC-Q-TOF-MS technology was used to detect the composition of raw and fried G. uralensis . By comparing the retention time ，relative molecular weight and other information of the compounds with the databases such as METLIN ，Agilent MassHunter PCDL Manager off-line component identification workstation and references ，the chemical compositions of the compounds were preliminarily identified. Principal component analysis （PCA）method was used to observe the overall distribution trend of samples. Orthogonal partial least square （OPLS-DA）method was used to screen differential compounds [VIP ＞1.0 and | P（corr）|≥0.5 as criteria] and analyze the content changes of differential compounds. RESULTS ：A total of 31 common compounds were preliminarily identified from the raw and fried G. uralensis . PCA analysis showed that raw and fried G. uralensis could be separated well. OPLS-DA analysis result showed that 15 characteristic differential compounds were screened out from raw and fried G. uralensis ，including 13 flavonoids and 2 coumarins；the contents of 8 flavonoids compounds such as licoflavone A ，glycyrrhizin and glabridin and so on in G. uralensis were significantly increased （P＜0.05 or P＜0.01）， while the contents of 5 flavonoids components such as glycyrrhizinol ，glycyrin A and 2 coumarins components such as 2′-4′-trihydroxy-5-methoxy-3-coumarim and hedysarimcoumestan B were significantly decreased （P＜0.05） after honey-fried processing. CONCLUSIONS ：Before and after processing ，there are obvious differences between some flavonoids and coumarins ， which may be the main material basis for differential use of raw and honey-fried G. uralensis .