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.

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.

THE BLOCKING EFFECTS OF GLYCYRRHIZE URALENSIS AND CHELIDONIUM MAJUS ON MUTAGENESIS INDUCED BY AFLATOXIN B1

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Thin Layer Chromatography Screening for Glycyrrhiza uralensis

[Objectives] To establish a simple and rapid,specific and reproducible thin layer chromatography( TLC) method,and accordingly establish a thin layer identification method for quality control of Glycyrrhiza uralensis. [Methods] According to the physical and chemical properties,components of Glycyrrhiza uralensis were identified by thin layer chromatography. According to Veterinary Pharmacopoeia of the People's Republic of China 2010( Volume Ⅱ) and related literature report,the thin layer chromatography identification method was studied and improved for Glycyrrhiza uralensis.[Results] When the preparation method for test sample solution was 6g Glycyrrhiza uralensis added with 30 mL saturated n-butanol,ultrasonic processing 20 min,filtered,filtrate dried,added 3 mL methanol and obtained Glycyrrhiza uralensis test sample solution. Then,chloroform-methanol-formic acid-ethyl acetate( 10∶ 3∶ 2∶ 3) as developing solvent,clear spot with high degree of separation and reproducibility could be obtained,and can be used for thin layer chromatography identification of ammonium glycyrrhetate in Glycyrrhiza uralensis.[Conclusions] The modified method omitted the step of ether treatment of samples,and directly adopted saturated n-butanol to extract components. It does not influence the detection of chromatographic spots,and solves the issue of sample gelatinizing sticking and filtration,simplifies the test process,and reduces the harm to human body.

Improving the accumulation of 18α-and 18β-glycyrrhizins by over-expressing GuHMGR, GuSQS1, and GuBAS genes in Glycyrrhiza uralensis

Objective:To study the influence of over-expression of three functional genes involved in GC biosynthetic pathway,GuHMGR,GuSQS1,and GuBAS on GC production.Methods:Three plant expression vectors were constructed and transformed into Agrobacterium tumefaciens EHA105,which were used to infect Glycyrrhiza uralensis hypocotyls explants.After induction,selection,differentiation,culture,and transplantation,12,15,and 5 regenerated plants over-expressing GuHMGR,GuSQS1,and GuBAS,were obtained,respectively.Results:RT-PCR analysis showed these transgenic regenerated G.uralensis plants had 2-6 copies of GuHMGR,GuSQS1,or GuBAS.HPLC analysis showed the contents of 18α-and 18β-GC in all transgenic regenerated samples were both higher than that in the blank control.With the increase of copy numbers of GuHMGR,GuSQS1,and GuBAS,the contents of 18α-and 18β-GC were both increased in most samples.The highest 18α-and 18β-GC contents in transgenic regenerated plants were about 3.05 times and 2.80 times higher than that in the blank control,respectively.Conclusion:Over-expression of the GuHMGR,GuSQS1,and GuBAS genes enhance the accumulation of 18α-and 18β-GC in the roots and rhizomes of G.uralensis.We hope this work can lay a foundation for the molecular breeding research of G.uralensis and improving the quality of the roots and rhizomes of G.uralensis cultivars.

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.

Synthesis of amino-silane modified magnetic silica adsorbents and application for adsorption of flavonoids from Glycyrrhiza uralensis Fisch

Magnetic separation technology was applied in the separation of flavonoids from the licorice root in this work. Licorice flavonoids (LF) displayed a remarkable array of biological and pharmacological activities. The magnetic adsorbents with functional -NH2 groups were synthesized by immobilization of amino-silane on the surface of the magnetic silica supports, which were prepared by co-precipitation method. The adsorption and desorption characteristics of the magnetic adsorbents for the separation of LF have been evaluated. The purity of an enriched extract with this method was 16.7% while the crude extract only had about 6.8% purity. Therefore, it can be concluded that these kinds of magnetic adsorbents have selectivity to the flavonoids to some extent. The affinity selectivity of the adsorbents is based on the formation of hydrogen bonding between the -NH2 on the magnetic adsorbents and -OH,-CO on the flavonoids.

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.

Over-expressing root-specific β-amyrin synthase gene increases glycyrrhizic acid content in hairy roots of glycyrrhiza uralensis

Objsective: Glycyrrhizia uralensis, one of the most widely-used traditional Chinese medicines, is mainly cropped in China. However, many cultivars are less in glycyrrhizic acid than Chinese Pharmacopoeia requires. In this paper, we improved glycyrrhizic acid by regulating β-amyrin synthase gene(GuBAS).Methods: Tobacco root-specific promoter TobRB7 and Gu BAS c DNA were obtained and combined with linearized pCAMBIA1305.1 to construct root-specific plant expression vector which was later transformed into Agrobacterium rhizogenes ACCC10060 by electrotransformation. The cotyledons and hypocotyls of G.uralensis were infected by the recombinant A. rhizogenes ACCC10060 to induce hairy roots. The GA content was quantified by HPLC.Results: The PCR and sequencing results both showed that three transgenic hairy root lines were obtained. The copy number of Gu BAS in these transgenic hairy roots was intended by q RT-PCR to be 3, 7,and 4. GA was detected by HPLC, and the results showed that GA was present in the three transgenic hairy roots, while absent in wild hairy roots.Conclusion: Over-expressing Gu BAS root-specifically in hairy roots of G. uralensis enhanced GA accumulation.

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.

Geographical clines in the size of the herb field mouse(Apodemus uralensis)

Patterns of body size variation along geographical gradients have long been searched for and generalized into eco-geographical rules.However,no rodent species has yet been analyzed in relation to the 3 dimensions of latitude,longitude and altitude.We analyzed geographical clines and dimorphism of body and skull size in the herb field mouse(Apodemus uralensis)across the species range,based on field data and on data from the literature.Sexual dimorphism in adult A.uralensis was not expressed at a large scale,while local patterns were inconsistent.Age-dependent size changes were most expressed in adult individuals:most characters of adults exceeded in size those of subadults,while subadult-juvenile size differences were only significant in body weight and length,zygomatic skull width,length of cranial diastema and breadth of braincase.Despite central morphological niches along the clines being separated,A.uralensis populations showed a high degree of size overlap in morphological space.We found the species to be characterized by high size variability,with the largest individuals inhabiting the eastern and southern edges of the distribution range.Tail,hind foot and ear lengths were largest in the southern part of the range,in agreement with Allen’s rule.The main measurements that we analyzed,namely body mass,zygomatic skull width and condylobasal skull length,show the presence of 3 clines in the size of adult A.uralensis:(i)a decreasing south-north cline,opposing Bergmann’s rule;(ii)an increasing west-east cline,in accordance with Murphy’s rule;and(iii)an increasing altitudinal cline.

甘草粗提物对鲫的抗应激及免疫保护功效研究

采用腹腔注射和混饲投喂两种方式研究中药甘草(Glycyrrhiza uralensis Fiseh)粗提物对鲫(Carassius auratus)的抗应激和免疫保护功效。将一部分粗提物按0.5%和2%的质量分数制成药饵投喂实验鲫,分别在第14 d、28 d、35 d、42 d、56 d采集血样;将另一部分粗提物用无菌生理盐水按0.5%和2%的浓度配成针剂腹腔注射鲫,分别在第3 d、7 d、14 d、21 d、28 d采集血样。结果显示:投喂实验中药物组和对照组血清皮质醇水平均呈先升后降趋势,除第14 d外,药物组皮质醇水平都低于同期对照组。在最后一次采样中(56 d),对照组、低剂量组和高剂量组的皮质醇水平分别为147.85 ng/mL、79.41 ng/mL和66.47 ng/mL。腹注实验中对照组皮质醇水平一直呈上升趋势,最后一次采样(28 d)高达274.23 ng/mL,显著高于药物组(P<0.01)。投喂甘草提取物56 d后,用Aeromonas hydrophila对鲫进行人工感染,低剂量组和高剂量组一周后的死亡率分别为45.0%和55.0%,显著低于对照组的70.0%(P<0.05)。腹注甘草提取物后第28 d,鲫受A.hydrophila菌人工感染,对照组、低剂量组和高剂量组的死亡率分别为60.0%、55.0%和35.0%,高剂量组显著低于对照组(P<0.05)。实验表明,投喂和腹注甘草粗提物后,可明显提高鲫的抗应激能力,并能增强鱼体对A.hydrophila的抵抗力。

盐碱条件下接种丛枝菌根真菌对甘草种苗生长的影响

[目的]研究接种丛枝菌根真菌对甘草生长和矿质元素吸收的影响。[方法]测定丛枝菌根侵染率和幼苗干重及N、P、K和Na的含量变化,研究菌根真菌与对甘草生长的作用。[结果]接种丛枝菌根真菌提高了甘草的生物量,提高了对N、P和K的吸收,但降低对Na的吸收。[结论]接种菌根真菌,增强了甘草的生长和抗盐碱能力,其机制可能与对元素的选择性吸收有关。

超临界流体萃取甘草中角鲨烯的方法研究

采用超临界流体萃取技术提取甘草(Glycyrrhiza uralensis)中的角鲨烯,并用高效液相色谱法测定其含量。结果显示,在丙酮为夹带剂的萃取产物中角鲨烯含量高达0.47%,超临界流体萃取法对甘草角鲨烯的总提取率为0.018%,高于传统索氏提取法及超声辅助提取法所得提取率。

甘草和连翘对牙鲆CYP3A活性影响的研究

研究甘草(Glycyrrhiza uralensis)和连翘(Forsythia suspensa)对牙鲆(Paralichthys olivaceus)肝细胞色素P4503A(CYP3A)活性的影响。将牙鲆随机分为3组,即对照组、甘草组和连翘组,对照组每日口灌0.9%的生理盐水,试验组每日1次口灌甘草30 mg/kg和连翘100 mg/kg,连续口灌6 d,第7天时,采用直接测定法和间接测定法(探针药物)进行CYP3A酶活性的测定。结果显示:在肝微粒体水平上,甘草和连翘均可明显提高红霉素-N-脱甲基酶活性,分别提高了1.79倍、4.87倍。与对照组相比,甘草组和连翘组氨苯砜的半衰期分别降低了4.51%(P>0.05)和36.8%(P<0.01);药时曲线下面积分别减小了8.28%(P>0.05)和32.3%(P<0.01);总清除率分别增加了5.63%(P>0.05)和99.3%(P<0.01)。说明连翘对牙鲆CYP3A的活性具有极显著诱导作用,甘草诱导作用不显著。提示其他药物与连翘合用时,其有效性和安全性可能会受到影响。

AM真菌对甘草侵染及生长的影响

为了探索AM真菌对甘草(Glycyrrhiza uralensis Fisch.)的影响,试验在盆栽条件下,比较了接种AM真菌对甘草幼苗生长的影响。结果表明,接种的4种AM真菌能与甘草幼苗形成菌根,显著促进甘草幼苗的生长,形成菌根的甘草幼苗均能显著提高成活率,增加甘草幼苗的苗高、地径、鲜重、干重,提高水分利用率,提高叶绿素含量,增强抗旱性。

甘草采挖收获机的研制

针对甘草（Glycyrrhiza uralensis Fisch.）主要品种及农村动力状况,设计了一种甘草采挖收获机。结果表明,该挖掘机的挖掘深度大于40 cm,损失率小于3%,生产率达0.2~0.4 hm2/h。该机的挖掘深度、配套动力及相关性能指标均达到设计要求。

离子束注入甘草种子的当代刺激效应

将内蒙本地产甘草种子用浓硫酸处理,吹干,再经离子束注入处理,结果显示其发芽率和出苗率均表现"马鞍型"曲线特征,拐点在4×1016ion/cm2剂量。离子束注入对10d龄的幼苗的抗氧化酶活性,在30d龄幼苗的茎高、主根长、侧根数和全株干重等方面,具有明显的刺激效应。