Study on the extraction and antifungal activity of volatile oil from stems，leaves and roots of Glehnia littoralis / 中国药房

OBJECTIVE To explore the extraction process of volatile oil from the stems， leaves and roots of Glehnia littoralis， analyze the chemical components of the volatile oil from the stems， leaves and roots of G. littoralis， and preliminarily evaluate its in vitro antifungal activity. METHODS Based on the steam distillation method， single factor test and orthogonal experiment were conducted to optimize the extraction method of volatile oil from the stems， leaves and roots of G. littoralis. The chemical components of the volatile oil from the stems， leaves and roots of G. littoralis were identified by using gas chromatography-mass spectrometry （GC-MS) technology and their relative contents were calculated. The antifungal activity of volatile oils from the stems， leaves and roots of G. littoralis against Fusarium solani， Fusarium incarnatum， Fusarium oxysporum， Aspergillus parasiticus and Aspergillus flavus was determined by paper diffusion method. RESULTS The optimal extraction process of G. littoralis was solid-liquid ratio of 1∶15， distillation time of 5 hours， and KCl concentration of 15%. Eleven components were identified from the volatile oil of the stems and leaves of G. littoralis， and a total of eight components were identified from the volatile oil of the roots. Ginsenethinol was a common component in the volatile oil from the stems， leaves and roots of G. littoralis， its contents in the stems and leaves， roots were 38.21% and 74.02%， respectively. The volatile oil from the stems， leaves and roots of G. littoralis had a certain E-mail：zwhjzs@126.com inhibitory effect on F. solani， F. incarnatum， F. oxysporum， A. parasiticus and A. flavus， especially volatile oil from the stems and leaves. CONCLUSIONS There is a significant difference in chemical components of the volatile oil between the roots， stems and leaves of G. littoralis， both of which have certain in vitro antifungal activity.

Study on quality standard for rice-fired Glehnia littoralis / 中国药房

OBJECTIVE To establish a quality standard for rice-fired Glehnia littoralis . METHODS Appearance observation ， powder microscopic identification and thin-layer chromatography （TLC）identification were performed for the samples of rice-fired G. littoralis decoction piece. According to the relevant methods stated in 2020 edition of Chinese Pharmacopoeia （part Ⅳ），the contents of moisture ，total ash ，acid-insoluble ash ，water-soluble extract and acid-soluble extract were determined. The contents of psoralen，zanthoxylin，bergapten，imperatorin and isoimperatorin were determined by high performance liquid chromatography （HPLC）. RESULTS The rice-fired G. littoralis decoction pieces were round-like small segments ，slightly rough ，yellow（peeled） or dark yellowish brown （with peel ），special gas and slightly sweet taste. The powder was yellowish white. Under the microscope ， secretions and secretory cells ，ducts，gelatinized starch granules ，ray cells ，parenchyma cells ，etc. could be seen. TLC showed the spots developed clearly. In the chromatogram of the test sample ，there was the same blue fluorescent spot at the corresponding position of the chromatogram of isoimperatorin control sample. The moisture ，total ash ，acid-insoluble ash ，water-soluble extract and ethanol-soluble extract from 9 batches of samples were 5.82％-6.27％，3.19％-3.59％，0.21％-0.27％，24.91％-30.30％ and 20.66％ -25.83％ ，respectively. The linear range of psoralen ，zanthotoxin，bergapten，imperatorin and isoimperatorin were 0.240-2.400，0.320-3.200，0.224-2.240，0.292-2.920，0.208-2.080 µg/mL（all r＞0.999 0）. Limits of quantitation were 0.032 0， 0.030 0，0325 0，0.032 0，0.045 0 µg，respectively. Limits of detection were 0.100 8，0.089 6，0.071 5，0.090 0，0.132 0 µg， respectively. RSDs of prescision ，stability（24 h）and reprodu- cibility tests were less than 3％. Average recoveries were 100.56％ （RSD＝1.36％ ，n＝6），100.73％（RSD＝2.25％ ，n＝6）， 100.36％（RSD＝0.98％，n＝6），98.24％（RSD＝0.40％，n＝6） E-mail：853063968@qq.com and 99.40％（RSD＝0.35％，n＝6），respectively. The contents of above five components were 5.85-13.31，8.63-33.38，6.23- E-mail：shixiaofeng2005@sina.com 15.25，6.12-12.98，5.52-10.77 µg/g，respectively. The total contents were 34.20-83.47 µg/g. CONCLUSIONS It is preliminarily proposed that the moisture ，total ash and acid-insoluble ash should not exceed 7.30％，4.10％，0.30％. The water-soluble extract and ethanol-soluble extract are no less than 21.00％ and 18.00％，respectively. The total content of coumarin should not be less than 52.03 µg/g（with peel ）and 26.34 μg/g（peeled）. Established quality standard can be used for the quality control of rice-fired G. littoralis .

Mining genes associated with furanocoumarin biosynthesis in an endangered medicinal plant, Glehnia littoralis

The endangered medicinal plant Glehnia littoralis is one of the important natural source of furanocoumarin, which has been used as mucolytic, antitussive, antitumour and antibacterial. However, the genetic information of furanocoumarin biosynthesis in G. littoralis is scarce at present. The objective of this study was to mine the putative candidate genes involved in the biosynthesis pathway of furanocoumarin and provide references for gene identification, and functional genomics of G. littoralis. We carried out the transcriptome analysis of leaves and roots in G. littoralis, which provided a dataset for gene mining. Psoralen, imperatorin and isoimperatorin were detected in G. littoralis by high performance liquid chromatography analysis. Candidate key genes were mined based on the annotations and local BLAST with homologous sequences using BioEdit software. The relative expression of genes was analysed using quantitative real-time polymerase chain reaction. Further, the CYP450 genes were mined using phylogenetic analyses using MEGA 6.0 software. A total of 156,949 unigenes were generated, of which 9021 were differentially-expressed between leaves and roots. A total of 82 unigenes encoding eight enzymes in furanocoumarin biosynthetic pathway were first obtained. Seven genes that encoded key enzymes in the downstream furanocoumarin biosynthetic pathway and expressed more in roots than leaves were screened. Twenty-six candidate CYP450 unigenes expressed abundantly in roots and were chiefly concentrated in CYP71, CYP85 and CYP72 clans. Finally, we filtered 102 differentially expressed transcription factors (TFs) unigenes. The transcriptome of G. littoralis was characterized which would help to elucidate the furanocoumarin biosynthetic pathway in G. littoralis and provide an invaluable resource for further study of furanocoumarin.

Extract Promotes Neurogenesis in the Hippocampal Dentate Gyrus of the Adult Mouse through Increasing Expressions of Brain-Derived Neurotrophic Factor and Tropomyosin-Related Kinase B / 中华医学杂志(英文版)

<p><b>Background</b>Glehnia littoralis has been used for traditional Asian medicine, which has diverse therapeutic activities. However, studies regarding neurogenic effects of G. littoralis have not yet been considered. Therefore, in this study, we examined effects of G. littoralis extract on cell proliferation, neuroblast differentiation, and the maturation of newborn neurons in the hippocampus of adult mice.</p><p><b>Methods</b>A total of 39 male ICR mice (12 weeks old) were randomly assigned to vehicle-treated and 100 and 200 mg/kg G. littoralis extract-treated groups (n = 13 in each group). Vehicle and G. littoralis extract were orally administrated for 28 days. To examine neurogenic effects of G. littoralis extract, we performed immunohistochemistry for 5-bromo-2-deoxyuridine (BrdU, an indicator for cell proliferation) and doublecortin (DCX, an immature neuronal marker) and double immunofluorescence staining for BrdU and neuronal nuclear antigen (NeuN, a mature neuronal marker). In addition, we examined expressional changes of brain-derived neurotrophic factor (BDNF) and its major receptor tropomyosin-related kinase B (TrkB) using Western blotting analysis.</p><p><b>Results</b>Treatment with 200 mg/kg, not 100 mg/kg, significantly increased number of BrdU-immunoreactive () and DCX cells (48.0 ± 3.1 and 72.0 ± 3.8 cells/section, respectively) in the subgranular zone (SGZ) of the dentate gyrus (DG) and BrdU/NeuN cells (17.0 ± 1.5 cells/section) in the granule cell layer as well as in the SGZ. In addition, protein levels of BDNF and TrkB (about 232% and 244% of the vehicle-treated group, respectively) were significantly increased in the DG of the mice treated with 200 mg/kg of G. littoralis extract.</p><p><b>Conclusion</b>G. littoralis extract promots cell proliferation, neuroblast differentiation, and neuronal maturation in the hippocampal DG, and neurogenic effects might be closely related to increases of BDNF and TrkB proteins by G. littoralis extract treatment.</p>

The Effect of Glehnia Littoralis on Alpha-amanitin Induced Hepatotoxicity in a Murine Model

PURPOSE: Glehnia littoralis has been reported to have several pharmacological properties but no in vivo reports describing the protective effects of this plant on α-amanitin-induced hepatotoxicity have been published. α-Amanitin is a peptide found in several mushroom species that accounts for the majority of severe mushroom poisonings leading to severe hepatonecrosis. In our previous in vitro study, we found that α-amanitin induced oxidative stress, which may contribute to its severe hepatotoxicity. The aim of this study was to investigate whether Glehnia littoralis acetate extract (GLEA) has protective antioxidant effects on α-amanitin-induced hepatotoxicity in a murine model. METHODS: Swiss mice (n=40 in all groups) were divided into four groups (n=10/group). Three hours after giving α-amanitin (0.6 mg/kg, i.p.) to the mice, they were administered silibinin (50 mg/kg/d, i.p.) or Glehnia littoralis ethyl acetate extract (100 mg/kg/d, oral) therapies once a day for 3 days. After 72 hours of treatment, each subject was killed, cardiac blood was aspirated for hepatic aminotransferase measurement, and liver specimens were harvested to evaluate the extent of hepatonecrosis. The degree of hepatonecrosis was assessed by a pathologist blinded to the treatment group and divided into 4 categories according to the grade of hepatonecrosis. RESULTS: GLEA significantly improved the beneficial functional parameters in α-amanitin-induced hepatotoxicity. In the histopathological evaluation, the toxicity that was generated with α-amanitin was significantly reduced by GLEA, showing a possible hepatoprotective effect. CONCLUSION: In this murine model, Glehnia littoralis was effective in limiting hepatic injury after α-amanitin poisoning. Increases of aminotransferases and degrees of hepatonecrosis were attenuated by this antidotal therapy.

Cloning and expression analysis of GlPS1, GlPS2 gene in Glehnia littoralis / 中草药

Objective: To clone the full-length cDNA sequences of psoralen synthase (PS) genes from Glehnia littoralis so as to perform the bioinformatic and expression pattern analysis. Methods: Based on our previous transcriptome sequencing data of G. littoralis, the gene sequences GlPS1 and GlPS2 with high expression level were screened. The 3’cDNA ends of GlPS1 and GlPS2 genes were cloned by the RACE (rapid amplification of cDNA ends) method and the full-length cDNA of genes were assembled by using DNAMAN software. And then encoded GlPS proteins were analyzed by the bioinformatics tools. The issue specific expression of GlPS1 and GlPS2 genes were detected using qPCR. Results: The full-length cDNA of GlPS1 gene was 1 885 bp, which encoding a protein of 495 amino acids with a predicted molecular weight of 55 740.7 and isoelectric point of 8.28; The full-length cDNA of GlPS2 gene was 1 971 bp, which encoding a protein of 502 amino acids with a predicted molecular weight of 56 363.9 and isoelectric point of 6.62. GlPS1 and GlPS2 proteins belong to the cytochrome P450 superfamily, which share one transmembrane zone acting as hydrophilic protein. Phylogenetic analysis showed GlPS1 and GlPS2 were genetically closely related to the PS of Pastinaca sativa, Apium graveolens, Ammi majus. Higher expression of GlPS1 gene was observed in roots than leaves. However, GlPS2 gene was expressed at a relatively higher level in flowers than in roots. Conclusion: The full-length cDNA of GlPS1 and GlPS2 genes were obtained and the expression patterns were explored in G. littoralis for the first time, which provided a foundation for further studies on gene function and genetic regulatory mechanism of GlPS.

In vitro Protective Effects of Glehnia Littoralis on Alpha-amanitin Induced Hepatotoxicity

PURPOSE: Glehnia littoralis has been used to treat ischemic stroke, phlegm, cough, systemic paralysis, antipyretics and neuralgia. The pharmacological mechanisms of Glehnia littoralis include calcium channel block, coumarin derivatives, anticoagulation, anti-convulsive effect, as well as anti-oxidant and anti-inflammatory effects. Alpha-amanitin (α-amanitin) is a major toxin from extremely poisonous Amanita fungi. Oxidative stress, which may contribute to severe hepatotoxicity was induced by α-amanitin. The aim of this study was to investigate whether Glehnia littoralis ethyl acetate extract (GLEA) has the protective antioxidant effects on α-amanitin -induced hepatotoxicity. METHODS: Human hepatoma cell line HepG2 cells were pretreated in the presence or absence of GLEA (50, 100 and 200µg/ml) for 4 hours, then exposed to 60µmol/L of α-amanitin for an additional 4 hours. Cell viability was evaluated using the MTT method. AST, ALT, and LDH production in a culture medium and intracellular MDA, GSH, and SOD levels were determined. RESULTS: GLEA (50, 100 and 200µg/ml) significantly increased the relative cell viability by 7.11, 9.87, and 14.39%, respectively, and reduced the level of ALT by 10.39%, 34.27%, and 52.14%, AST by 9.89%, 15.16%, and 32.84%, as well as LDH by 15.86%, 22.98%, and 24.32% in culture medium, respectively. GLEA could also remarkably decrease the level of MDA and increase the content of GSH and SOD in the HepG2 cells. CONCLUSION: In the in vitro model, Glehnia littoralis was effective in limiting hepatic injury after α-amanitin poisoning. Its antioxidant effect is attenuated by antidotal therapy.

Cloning and Bioinformatics Analysis of GI4CL Gene in Glehnia littoralis / 世界科学技术-中医药现代化

This study was aimed to clone and analyze the open reading frame (ORF) of 4-coumarate:coenzyme A ligase (Gl4CL) gene in Glehnia littoralis.Based on the high-throughput sequencing of G.littoralis,the full-length cDNA of Gl4CL gene was cloned by the rapid amplification of cDNA ends (RACE) method.Physical and chemical properties,secondary structure and three-dimensional structure of Gl4CL protein were predicted.Real-time PCR was used to detect the expression of Gl4CL gene in roots and leaves of G.littoralis.A total of 1951 bp full-length cDNA of Gl4CL gene was obtained,which encoded a protein of 544 amino acids with a predicted molecular weight of 59.481 kDa and the isoelectric point of 8.20.The cDNA of Gl4CL gene included 1 635 bp of ORF,153 bp of 5'untranslated regions (5'UTR) and 163 bp of 3'UTR.The result of real-time PCR showed that Gl4CL gene was both expressed in roots and leaves of G.littoralis,while the expression of gene in roots was significantly higher than that in leaves.It was concluded that the study will lay the foundation for further study of Gl4CL gene in function and gene regulation.Through in-depth study of the relationship between the expression of Gl4CL gene and lignin,as well as the plant growth phenotypes,it is expected to obtain high yield and quality lines of Glehniae Radix with strong resistance to diseases and insect pests.

Contents Determination of 5 Coumarins in Sulphur-fumigated and No Sulphur-fumigated Glehnia littoralis by HPLC / 中国药房

OBJECTIVE:To establish a method for the contents determination of 5 coumarins in sulphur-fumigated and no sul-phur-fumigated Glehnia littoralis,and investigate the effect of sulphur fumigation on the coumarins in G. littoralis. METHODS:HPLC was performed on the column of Hypersil ODS C18 with mobile phase of acetonitrile-0.1% phosphoric acid (gradient elu-tion)at a flow rate of 1.0 ml/min,detection wavelength was 295 nm,column temperature was 30 ℃,and the volume was 20 μl. RESULTS:The linear range was 0.121 5-12.15 μg/L for psoralen,0.389-38.9 μg/L for xanthotoxin,0.055-5.5 μg/L for bergapten, 0.070 5-7.05 μg/L for imperatorin and 0.092 5-9.25 μg/L for isoimperatorin(r≥0.999 7);RSD of precision test was lower than 1.0,RSDs of reproducibility and stability tests were lower than 3.0;recovery was 98.01%-101.97%(RSD<3.0%,n=6). CON-CLUSIONS:The content of coumarins in G. littoralis was obviously decreased after sulphur fumigation. the method is simple and accurate,and canbe used for the quality control of sulphur-fumigated and no sulphur-fumigated G. littoralis.

Study on antimicrobial activity and classification of endophytic fungi from Glehnia littoralis / 中草药

Objective: To screen the endophytic fungi with antimicrobial activity from Glehnia littoralis and identify the taxonomy of these fungi. Methods: The endophytic fungi were isolated from G. littoralis by method of tissue isolation. The oxford-cup test was used in screening the endophytic fungi with antimicrobial activity for Escherichia coli, Staphylococcus aureus, and Candida albicans respectively. And the rDNA ITS1-5.8S-ITS2 sequences were applied for the taxonomy of endophytic fungi. Results: Nineteen strains of fungi with antimicrobial activity were attributed to four genera, five families, seven orders, and eight species. In these fungi, there were four strains with antimicrobial activity to E. coli (the max d/D value was 1.07), Fifteen strains with antimicrobial activity to S. aureus (the max d/D value was 0.65), and three strains with antimicrobial activity to C. albicans (the max d/D value was 1.27). Conclusion: Many kinds of fungi with antimicrobial activity inhabit in G. littoralis. This study is helpful to develop the recourses of endophytic fungi and to evaluate the genuineness of G. littoralis.

Identification of chemical constituents of volatile oil in coastal glehnia root / 第二军医大学学报

Objective To identify the chemical constituents of volatile oil from the Chinese traditional medicine coastal glehnia root and to compare the differences between coastal glehnia root of different locations. Methods A total of 16 batches of the coastal glehnia root were collected from several major production areas from September 2012 to March 2013, and then they were ground into powder. The volatile oil was extracted from the powder samples using the methods described in Chinese Pharmacopoeia Appendix. Gas chromatography-mass spectometry (GC-MS) was used to get the spectra of volatile oil of each sample and NIST 11. 0 database was used to identify the chemical constituents of coastal glehnia root. Results and Conclusion From 16 batches of 48 coastal glehnia root volatile oil samples, we identified 12 common components. The 12 common chemical constituents can serve as the characteristic composition of volatile oil of the coastal glehnia root, and falcarinol is the major main chemical constituent. The three batches collected from Hebei province had fewer chemical components and lower contents. We also found that the coastal glehnia root samples with root bark had more volatile oil components and higher contents than the samples without bark; moreover, eicosapentaenoic acid was only found in the samples with root bark. Peeling the bark may reduce the contents of some volatile oils such as eicosapentaenoic acid, which may affect the medicinal activity of the coastal glehnia root.

Chemical constituents from root and rhizome of Glehnia littoralis / 中草药

Object To study the chemical constituents of the underground part of Glehnia littoralis Fr. Schimidt ex Miq. Methods Eight compounds were isolated from EtOAc-soluble fraction of ethanol extract of G. littoralis by repeated chromatography over silica gel. Their structures were determined on the basis of their spectral data. Results They were identified as falcalindiol (Ⅰ), (8E)-1, 8-heptadecadiene-4, 6-diyne-3, 10-diol (Ⅱ), bergapten (Ⅲ), cnidilin (Ⅳ), xanthotoxin (Ⅴ), salicylic acid (Ⅵ), vanillic acid (Ⅶ), ferulic acid (Ⅷ). Conclusion Compounds Ⅱ and Ⅵ-Ⅷ were isolated from this plant for the first time.