α-Glucosidase Inhibitors from Glycyrrhiza uralensis Fisch.

Aim To screen for α-glucosidase inhibitor from Glyeyrrhiza uralensis Fisch.. Methods Glycyrrhizic acid, glycyrrhetinic acid, flavonoids of glycyrrhiza, alkaloids of glycyrrhiza, and glycyrrhiza polysaccharides were isolated from the root of Glycyrrhiza uralensis Fisch. respectively. Three compounds were isolated from the flavonoids of glycyrrhiza as guided by the α-glucosidase inhibitory test in vitro. Moreover, the characteristics of inhibitory kinetics of glycyrol and glycyrrhetinic acid were investi- gated. Results The flavonoids of glycyrrhiza and glycyrrhetinic acid had the strongest α-glucosidase inhibitory activity. Glycyrol,β-sitosterol and liquifitin were isolated and identified. Glycyrol was a fast- binding, reversible, noncompetitive α-glucosidase inhibitor, showing IC50 at 0.26 μg·mL^-1 Glycyrrhetinic acid was a fast-binding, irreversible α-glucosidase inhibitor, showing IC50 at 102.4 μg·mL^-1. Conclusion Glycyrol is an effective α-glucosidase inhibitor.

Effect of Different Concentrations of Mo on Yield and Accumulation of Active Constituents of Glycyrrhiza uralensis Fisch

[Objective] The aim was to explore the effects of different concentrations of molybdenum（Mo）on the growth,yield and the accumulations of active constituents of Glycyrrhiza uralensis.[Method] The seedlings of one-year-old G.uralensis were subjected to four concentrations of Mo（0,0.52,5.20 and 10.40 mg/L Mo）culturing in vermiculite and 0.52 mg/L Mo was the normal concentration in the complete Hoagland nutrition solution.The solution was irrigated to the pots every week.The fresh and dry weights of shoots and roots of the plants were measured by electronic balance.The contents of glycyrrhizic acid and liquiritin of roots were determined by HPLC.[Result] This result showed that both fresh and dry weights of the shoots and roots increased with the increasing of Mo concentrations.During the later period of this experiment,they showed significant differences among different Mo treatments.On the 105 d,both fresh and dry weights of the shoots and roots under 0 mg/L Mo reached the lowest,and the 5.20 and 10.40 mg/L Mo reached the highest.However,the contents of glycyrrhizic acid and liquiritin increased first and then decreased with increasing of Mo concentrations.During the whole experiment,the contents of glycyrrhizic acid and liquiritin under 5.20 mg/L Mo were always higher than that under other Mo treatments.[Conclusion] In conclusion,both the concentrations of 5.20 and 10.40 mg/L Mo could promote the accumulation of the shoots and roots of G.uralensis,but the 5.20 mg/L Mo was more useful for the accumulation of glycyrrhizic acid and liquiritin in the roots.

Measurement and comparison of glycyrrhizic acid contents in root of licorice(Glycyrrhiza uralensis Fisch.)from different cultivating areas

The samples of licorice (Glycyrrhiza uralensis Fisch.) from 14 different cultivated areas were determined by the method of high Performance Capillary Electrophoresis (HPCE) for the contents of glycyrrihizic acid (GA) in root. The results showed that the licorice plants come from various cultivated areas of China has different contents of GA. The GA content of licorice from Zhaodong in Heilongjiang Province is the highest, followed by those from E抰uoke, Chifeng, and Hangjin Banner in Inner Mongolia. Some suggestions for establishing the production base of licorice were put forward based on the study.

Primary Speciation Analysis on 6 Kinds of Microelements in Glycyrrhiza uralensis Fisch.

[Objective]The aim was to analyze the primary speciation of 6 microelements in Glycyrrhiza uralensis Fisch. and provide theoretical basis for explaining pharmacodynamic principle of liquorice and discussing quality control of liquorice planting. [Method]The 6 elements Cu,Zn,Ca,Fe,Mg and Mn in roots of G.uralensis were extracted based on traditional decoction method and were separated into water-soluble state and suspension state by micro porous filtering film. The elements in water-soluble state were detected by flame atomic adsorption spectrophotometry （FAAS）. [Result]The results showed that extractive rates of the elements were in the range of 1.71%-60.06%,and immerse-residue ratio in 0.018 3-1.682 0; the results also indicated that the immerse-residue ratio of Zn was biggest （1.68）,Zn played an important medical role and might be considered as the best characteristic element in G.uralensis; the recoveries of the elements were ranged from 95.72% to 103.15% and relative standard deviations （RSD） were less than 2.38%. [Conclusion]Because of its high accuracy,FAAS method is feasible for analyzing primary speciation of microelements in G.uralensis.

Optimization of RAPD-PCR Reaction System for Glycyrrhiza uralensis Based on Orthogonal Design

[Objective] The aim was to obtain the optimum RAPD-PCR reaction system for Glycyrrhiza uralensis.[Method] Orthogonal design was adopted to screen the suitable concentration of four major factors（dNTPs,primers,Taq polymerase and DNA template） in PCR reaction system.[Result] The optimal reaction system obtained by orthogonal design was 25 μl in total volume,containing 2.5 μl of 10×PCR buffer solution（include MgCl2）,2.5 μl of 10 mmol/L dNTPs,2 μl（100 ng） of DNA template,2 μl of 10 μmol/L primers,0.4 μl（5 U） of Taq polymerase;the optimum annealing temperature was 34 ℃.[Conclusion] Orthogonal design was an effective method for the optimization of RAPD-PCR reaction system for G.uralensis.

Effect of Glycyrrhiza uralensis Fisch polysaccharide on growth performance and immunologic function in mice in Ural City,Xinjiang

Objective:To discuss the effect of Glycyrrhiza uralensis(G.uralensis) Fisch polysaccharide on growth performance and immunologic function in mice in Ural City,Xinjiang and to provide important data supporting the application of Glycyrrhiza polysaccharide.Methods:A total of100 Kunming mice aged 3 weeks old were randomly divided into 5 groups with 20 mice in each group(10 were females and 10 were males).About 0.5 mL normal saline was given to the mice of control group every day and 0.5 mL G.uralensis Fisch polysaccharide was given to the mice of other groups at the concentration of 1,20,50 and 100 mg/mL respectively.The growth performance(average body weight,average daily feed intake and feed efficiency),immune organ indexes(spleen index and thymus index) and immunologic function(serum IL-2,CD4^+/CD8^+ and the activity of NK cells) of mice in each group were detected continuously.Results:The average body weight,feed efficiency,serum IL-2,CD4^+/CD8^+ and the activity of NK cells of mice were increased with the increase of administrated time after administrating G.uralensis Fisch polysaccharide and were reached up the largest level on Day 28.At the same time,each index was proportional to the given dose and was significantly higher than those of control group and reached up the largest level at the administrated dose of 100 mg/mL.After administrating G.uralensis Fisch polysaccharide,the spleen index and thymus index of mice were increased with the increase of administrated dose and the spleen index and thymus index of mice administrated with the dose of 100 mg/mL were maximum which was more than 1.51 times and 1.43 times of that in control group respectively and the comparative differences showed statistical significance(P<0.05).The average daily feed intake of mice in each group was increased with the passage of lime and at the same time,the comparison of average daily feed intake of mice in each group was not significantly different(P>0.05).Conclusions:G.uralensis Fisch polysaccharide can significantly improve the growth performance and immunologic function of mice and laid a research basis for the clinical application of G.uralensis Fisch polysaccharide.

A New Dihydroflavone Glycoside from Glycyrrhiza uralensis

A new dihydroflavone glycoside was isolated from the underground parts of Glycyrrhiza uralensis. Its structure was elucidated as 7-hydroxyl-4'-O-β-D-(6'-O-α-hydroxylpropionyl) glucopyranosyl dihydroflavone by spectral methods.

Molecular Cloning and Expression Analysis of an Actin Gene from Chinese Licorice(Glycyrrhiza uralensis Fisch)

A PCR-based homologous cloning strategy was used to identify an actin gene from the roots of Chinese licorice（Glycyrrhiza uralensis Fisch）. Results of sequence analysis indicate that a 1137 bp cDNA with an open reading frame encoding 377 amino acids, actin ortholog, GuActin, was successfully cloned and characterized（GenBank accession No. EU190972）. Thus far, GuActin is the first actin of Chinese licorice that has been identified at a molecular level. Analysis by Northern blot shows that GuActin was expressed strongly in the roots, particularly in radicles than in stems and leaves. These results suggest that GuActin may be a member of the vegetative subfamily of the actin family.

Conditions and Stimulation for Germination in Glycyrrhiza uralensis Fisch Seeds

Evaluation of seed quality is the key to seed distributing and seeding of Glycyrrhiza uralensis Fisch, as an important species for pharmacy and soil conservation. Here, we study the effects of light and temperature on seed germination and mechanical and chemical scarification on breaking the seed coat. Seeds were collected in 2004, 2005, and 2006, placed in Petri dishes, and incubated at constant temperature 20, 25, and 30℃, and alternating temperature 15-25, 20-30, 15-30, and 20-35℃ under either an 8h photoperiod or total darkness for 28 consecutive days. Different methods were used to break the dormancy owing to hard seededness in this species such as chemical scarification by immersing in concentrated sulphuric acid for 5, 10, 15, 20, 25, 30, 45, and 60 min, in 0.2% KNO3 solution to saturate the seedbed, and prechilling for 7 d at 7℃ and mechanical scarification by cutting. The results showed that alternating temperature at 20-30℃ with 8 h photoperiod and 16 h darkness was optimum for G. uralensis seed germinating in the laboratory. Hard seeds were broken by concentrated sulphuric acid soaking or mechanical scarification by cutting. Germination of seeds harvested in two different years was both promoted by immersing for 30-45 min in concentrated sulphuric acid. KNO3 solution was ineffective for reducing hard seeds. During seed germinating, the first count was on the 7th day and the last count was on the 14th day.

TWO NEM IS0PRENYL PLAVONOIDS FROM THE LEAVES OP GLYCYRRHIZA URALENSIS FISOH

Three flavonoids have been isolated from the leaves of Glycyrrhiza uralensis Fisch(licorice,Leguminosae).The structures have been identified as 5,7,3',4'-tetrahydroxy-3-methoxy-5'-isoprenyl flavone(I,uralenol-3-methylether),5,6,3',4'-tetrahydroxy-3-methoxy-6'-isoprenyl flavone (Ⅱ, unalene) and quercetin(Ⅲ).Ⅰ and Ⅱ are new compound.

Effects of Different Arbuscular Mycorrhizal Fungi on Growth and Protective Enzyme Activity of Glycyrrhiza uralensis

[Objective] This study aimed to investigate the effects of 5 kinds of arbuscular mycorrhizal fungi( Acaulospora mellea,Glomus mosseae,Glomus versiforme,Glomus aggregatum,Glomus etunicatum) on the growth and protective enzyme activity of Glycyrrhiza uralensis Fisch. [Method] The growth indicators and protective enzymes activity of glycyrrhiza plants inoculated and uninoculated with fungi were compared. [Result] The plant height,basal diameter,main root length,aboveground fresh weight,underground fresh weight,aboveground dry weight and underground dry weight of the inoculated glycyrrhiza plants were increased significantly compared with those in the control( non-inoculation) group. In the inoculated glycyrrhiza plants,the growth index was significantly increased compared with that in the control group( P <0. 05); the activities of superoxide dismutase( SOD) and peroxidase( POD) increased first and then decreased; and the activity of catalase( CAT) showed a continuous rising trend. The effects of different inoculants on the growth of G. uralensis were significantly different.[Conclusion]G. etunicatum,G. mosseae and G. aggregatum had a significant effect on the growth of G. uralensis,and were superior to other fungi in resisting the adverse environment.

Effect of Ar Ion Beam Implantation on Morphological and Physiological Characteristics of Liquorice(Glycyrrhiza uralensis Fisch)Under Short-Term Artificial Drought Conditions

Ar^＋ ion beam with low energy of 30 keV was implanted into liquorice （Glycyrrhiza uralensis Fisch） seeds at the doses of 0, 600, 900 and 1200 × （2.6 × 10^13） ions/cm^2, respectively. The seeds were sowed in pots and after one month the plants were subjected to different drought conditions for two months. Then the plants＇ morphological and physiological characteristics, antioxidation enzymes and levels of endogenous hormones were investigated. The results showed that ion implantation at a proper dose can greatly enhance the liquorice seedlings＇ resistance against drought stress.

INHIBITORY EFFECT OF GLYCYRRHIZA URALENSIS FISH AND CHELIDONIUM MAJUS L ON GASTROCARCINOGENESIS . INDUCED BY N-METHYL-N' -NITRO-N-NITROSOGUANIDINE

In order to investigate the antagonistic effect of Glycyrrhlza Uralensis Fish (GUF) and Chelidonium maJus L (CML) on gastrccarcinogenesls Induced by MNNG in Wastar rats, we treated the rats with MNNG alone (group 1) and with MNNG plus GUF and CML (group 2 and 3) respectively. The Incidence of Infiltrating adenocarcinoma of the glandular stomach and duodenum in group 2 was significantly lower than that in group 1 (26.7% vs. 67.8%). The differentiation and aggresslvenees of carcinomas occured in group 2 were much better and mild than those in group 1. Present study also demonstrated that the Inhibitory effect of CML on proliferation of human stomach carcinoma cell line MGC-803 was very remarkable; in addition, GUF and CML were able to antagonise the mutagenlc activation of MNNG. These results suggest that GUF and CML may be empoyed In prevention of gastric carcinoma.

Impact of Ion Implantation on Licorice(Glycyrrhiza uralensis Fisch) Growth and Antioxidant Activity Under Drought Stress

Low energy ion beams are known to have stimulation effects on plant generation and to improve plants＇ intrinsic quality. In the present study, the growth and physiological index of licorice implanted with 0, 8, 10; 12 and 14× （2.6× 10^15） ions/cm^2 were investigated under well-watered and drought-stress conditions. The results showed that a proper dose of ion implan- tation was particularly efficient in stimulating the licorice growth and improving the plant biomass significantly in both the well-watered and drought-stress conditions. The physiological results of licorice measured by leaf water potential, lipid oxidation, soluble protein and antioxidant system showed a significant correlation between ion implantation and water regime except for leaf water potential. Therefore, the study indicated that ion implantation can enhance licorice＇s drought tolerance by increasing the activity of superoxide dismutase （SOD）, eatalase （CAT） and DPPH （1,1-diphenyl-2-picrylhydrazyl） radical scavenging ability to lower oxidative damage to lipids in plants. Ion beam implantation, therefore, provides an alternative method to enhance licorice drought tolerance.

Ethanol Extract of Glycyrrhiza uralensis Fisch:Antidiarrheal Activity in Mice and Contraction Effect in Isolated Rabbit Jejunum

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(El).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,10μmol/L)and KCl(60 mmol/L)were observed for 200 s.In addition,CaCl_(2)was accumulated to further study its mechanism after pretreating jejunal smooth muscle with GFR(1 and 3 g/L)or verapamil(0.03 and 0.1μmol/L)in a Ca^(2+)-free-high-K+solution containing ethylene diamine tetraacetic acid(EDTA).Results:GFR(500 and 1,000 mg/kg)significantly reduced El 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 KCI 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 CaCl_(2)down to the right,showing a similar effect to verapamil.Conclusions:GFR can effectively reduce diarrhea and inhibit intestinal contraction,and these antidiarrheal effects may be based on blocking L-type Ca^(2+) channels and muscarinic receptors.

Enhancing production of ergosterol in Pichia pastoris GS115 by over-expression of 3-hydroxy-3-methylglutaryl CoA reductase from Glycyrrhiza uralensis

The rate-limiting enzyme in the mevalonic acid(MVA)pathway which can lead to triterpenoid saponin glycyrrhizic acid(GA)is 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR).In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway,the HMGR gene from Glycyrrhiza uralensis Fisch.(GuHMGR)was cloned and over-expressed in Pichia pastoris GS115.Six recombinant P.pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC.The results showed that all the recombinant P.pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains.However,as the copy number increased,the content of ergosterol showed an increasing–decreasing–increasing pattern.This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G.uralensis.

Cloning and characterization of the gene encoding β-amyrin synthase in the glycyrrhizic acid biosynthetic pathway in Glycyrrhiza uralensis

Glycyrrhiza uralensis is considered to be one of the most important herbs in traditional Chinese medicine due to its numerous pharmacological effects particularly its ability to relieve cough and act as a mucolytic.Based on previous research,these effects are mediated by a number of active ingredients,especially glycyrrhizic acid(GA).In the present study,a gene encodingβ-amyrin synthase(β-AS)involved in GA biosynthesis in G.uralensis has been cloned and expressed in Saccharomyces cerevisiae.The cloned enzyme showed similar activity to native enzymes isolated from other Glycyrrhiza species to catalyze the conversion of 2,3-oxidosqualene intoβ-amyrin.In fact theβ-AS gene is particularly important in the GA biosynthetic pathway in G.uralensis.The complete sequence of the enzyme was determined and a phylogenetic tree based on theβ-AS gene of G.uralensis and 20 other species was created.This showed that Glycyrrhiza glabra had the closest kinship with G.uralensis.The results of this work will be useful in determining how to improve the efficacy of G.uralensis by improving its GA content and in exploring the biosynthesis of GA in vitro.

Enhancing ergosterol production in Pichia pastoris GS115 by overexpressing squalene synthase gene from Glycyrrhiza uralensis

The present study was designed to determine the effects of copy number variations(CNVs) of squalene synthase 1(SQS1) gene on the mevalonate(MVA) pathway.SQS1 gene from G.uralensis(Gu SQS1) was cloned and over-expressed in Pichia pastoris GS115.Six recombinant P.pastoris strains containing different copy number of Gu SQS1 were constructed.HPLC was used to assay the level of ergosterol in all transgenic P.pastoris strains containing Gu SQS1.HPLC analysis showed that the contents of ergosterol in all of the transgenic P.pastoris containing Gu SQS1 were higher than that in the negative control.And with the increase of copy number of Gu SQS1,the content of ergosterol showed an increasing-decreasing-increasing pattern.The contents of ergosterol in 10-copy-Gu SQS1 P.pastoris and 47-copy-Gu SQS1 P.pastoris were significantly higher than that in the rest recombinant P.pastoris strains.In conclusion,the CNVs of Gu SQS1 influence the content of secondary metabolites in the MVA pathway.The present study provides a basis for over-expressing Gu SQS1 and increasing the content of glycyrrhizin in G.uralensis cultivars.

Genome-wide identification of abscisic acid(ABA) receptor pyrabactin resistance 1-like protein(PYL) family members and expression analysis of PYL genes in response to different concentrations of ABA stress in Glycyrrhiza uralensis

As abscisic acid(ABA)receptor,the pyrabactin resistance 1-like(PYR/PYL)protein(named PYL for simplicity)plays an important part to unveil the signal transduction of ABA and its regulatory mechanisms.Glycyrrhiza uralensis,a drought-tolerant medicinal plant,is a good model for the mechanism analysis of ABA response and active compound biosynthesis.However,knowledge about PYL family in G.uralensis remains largely unknown.Here,10 PYLs were identified in G.uralensis genome.Characterization analysis indicated that PYLs in G.uralensis(Gu PYLs)are relatively conserved.Phylogenetic analysis showed that Gu PYL1-3 belongs to subfamily I,Gu PYL4-6 and Gu PYL10 belong to subfamily II and Gu PYL7-9 belongs to subfamily III.In addition,transcriptome data presented various expression levels of Gu PYLs under different exogenous ABA stresses.The expression pattern of Gu PYLs was verified by Quantitative real-time polymerase chain reaction(q RT-PCR).The study proved that Gu PYL4,Gu PYL5,Gu PYL8 and Gu PYL9 genes are significantly up-regulated by ABA stress and the response process is dynamic.This study paves the way for elucidating the regulation mechanism of ABA signal to secondary metabolites and improving the cultivation and quality of G.uralensis using agricultural strategies.

Chemical constituents of the aerial parts of Glycyrrhiza uralensis and their inhibitory activities against PTP1B andα-glucosidase

In the present study,a total of 11 compounds were isolated from the aerial parts of Glycyrrhiza uralensis,including two new compounds,glycyuralin Q(1)and glycyuralin R(2),and nine known compounds,including licoriphenone(3),orobol(4),trifoliol(5),7,2′,4′-trihydroxy-5-methoxy-3-arylcoumarin(6),11-hydroxy-9(Z),12(Z)-octadecadienoic acid(7),11-hydroxy-9(E),12(E)-octadecadienoic acid(8),licoricone(9),glycyrin(10),and 2′-hydroxyformononetin(11).Structures of the new compounds were identified by 1 D,2 D NMR and HR-MS data analyses.Compounds 1,2 and 10 showed potent inhibitory activities against PTP1 B,with IC50 values of 1.43,4.71 and 3.79μM,respectively.Compounds 2,4 and 10 inhibitedα-glucosidase with IC50 values of 13.61,11.13 and 17.48μM,respectively.