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Objective: To investigate the chemical components from the 80% EtOH extract of Atractylodes lancea, as well as the inhibitory activities of the isolated compounds on LPS-induced NO production of microglia BV2 cells. Methods: The n-BuOH-soluble fraction of the crude extract was successively chromatographed with Diaion HP-20, Sephadex LH-20, and preparative HPLC C18-column. At last, the planar and stereochemical structures of these obtained compounds were established on the basis of extensive spectroscopic data (HRESIMS, NMR, and ECD, etc). Results: Ten glycosides were isolated from the n-BuOH-soluble fraction of the 80% EtOH extract of A. lancea, including (2E,8R)-decene-4,6-diyne-1,8-diol-1-O-β-D- apiofuranosyl-(1→6)-β-D-glucopyranoside (1), (8S)-decane-4,6-diyne-1,8-diol-8-O-β-D-glucopyranoside (2), (2E,8R)-decene-4,6- diyne-1,8-diol-8-O-β-D-glucopyranoside (3), (2E,8S)-decene-4,6-diyne-1,8-diol-8-O-β-D-glucopyranoside (4), (2E,8E)-2,8- decadiene-4,6-diyne-1,10-diol-1-O-β-D-glucopyranoside (5), (7R,8S)-3',9,9'-trihydroxyl-3-methoxyl-1'-propanol-7,8-dihydrobenzo- funanneoligan-4-O-β-D-glucopyranoside (6), (7'R*,8S*,8'S*)-lyoniresinol 9'-O-β-D-glucopyranoside (7), (7S,8R)-4,9,9'-trihydroxy- 3'-methoxy-8-O-4'-neolignan 7-O-β-D-glucopyranoside (8), methyl salicylate 2-O-α-L-xylopyranosyl-(1→6)-β-D-glucopyranoside (9), and phenylmethanol 7-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (10). Conclusion: Compounds 1 and 2 are named atractyeneyneglycoside A and atractyeneyneglycoside B, while compounds 6, 8-10 are first isolated from the rhizomes of A. lancea. At the concentration of 10 μmol/L, compound 10 exhibited the strongest inhibitory effects on LPS-induced NO production of microglia BV2 cells with the value of 31.18%, while compounds 1 and 2 just showed weaker inhibitory effects with values of 22.01% and 14.09%, respectively.
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Objective: To explore the effect on the accumulation of medicinal compositions β-eudesmol, atractylon, atractylodin and key enzyme genes 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and farnesyl pyrophosphate synthase (FPPS) expression in biosynthesis of Atractylodes lancea under copper stress. Methods: Under copper stress, the expression of key enzyme genes HMGR and FPPS in A. lancea was determined by real-time fluorescence quantitative PCR; the content of three medicinal components in A. lancea were determined by HPLC; The correlation analysis was performed with SPSS, and DPS software for grey correlation analysis. Results: When the copper stress concentration was within 100 mg/kg, the expression of FPPS and the content of atractylon in the rhizomes of A. lancea increased slightly. However, when the copper concentration continued to increase, the expression levels of HMGR and FPPS and three medicinal components content of A. lancea showed a different degrees of downward trend. The expression levels of HMGR and FPPS were positively correlated with the content of β-eudesmol, atractylon, and atractylodin (P < 0.05) under copper stress. Grey relational analysis showed that the content of β-eudesmol and atractylon in the rhizomes was significantly correlated with the expression of HMGR and FPPS of A. lancea under copper stress. The expression of FPPS gene had the larger contribution on the composition of β-eudesmol and atractylon. However, the correlation between the content of atractylodin and the expression of these two key enzyme genes was relatively small. Conclusion: This study clarified the change regulation of two key enzyme gene expression and the content of three medicinal compositions, and revealed the relationship between β-eudesmol, atractylon and HMGR and FPPS, the key enzymes in terpene biosynthesis of A. lancea under copper stress. It contributed to the further study of the molecular regulation mechanism of the synthesis of medicinal constituents under copper stress and provided a theoretical basis for improving the quality of A. lancea.
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Objective: To clone the full-length cDNA encoding farnesyl pyrophosphate synthase (FPPS) from Atractylodes lancea and analyze its expression. Methods: The full-length cDNA of FPPS in A. lancea was cloned via homology-based cloning and rapid amplification of cDNA ends approach. Also, the characterization of gene was revealed by bioinformatic analysis. The expression of FPPS was determined by qRT-PCR while the content of sesquiterpenes of rhizome in different growth stages in A. lancea was measured by GC-MS. Meanwhile the correlation between them was analyzed. Results: The full-length cDNA (1320 bp) of FPPS gene was obtained (AlFPPS, GenBank accession number KX443242), with an open reading frame of 1029 bp, encoding 342 amino acids. The deduced AlFPPS protein sequence contained five conserved motifs, two of which is full of Asp (DDXXD). qRT-PCR and GC-MS results showed a significant positive correlation between the content of sesquiterpenes and AlFPPS expression level. Conclusion: It can be primarily deduced that AlFPPS gene should be an important control point in the biosynthetic pathway of sesquiterpenes in A. lancea. This work provides scientific basis for clarifying the biosynthetic pathway of sesquiterpenes and application of biological engineering.
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Objective: To study the mechanism of the content of 5-hydroxymethylfurfural (5-HMF) increasing after Atractylodes lancea in bran-processed. Methods: The fructose and glucose were processed with A. lancea bran-processed conditions and their contents were determined by HPLC. Besides, the content of fructose was measured and the changes were compared before and after processing. Results: Glucose samples did not detect 5-HMF in the processing conditions, but fructose samples detected 5-HMF in the same conditions. In addition, the content of fructose was significantly decreased in the best conditions while the content of 5-HMF increased the most. Conclusion: The content of 5-HMF remarkably increases mainly due to the conversion of fructose.
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Objective: The aim of present study was to identify Atractylodes lancea and its closely related species (Atractylodes chinesis and Atractylodes macrocephala) using the ITS2 barcode. Methods: The total genomic DNAs were extracted from twenty-nine samples of A. lancea and its closely related species from different habitats. The ITS2 sequences of these samples were amplified and bidirectional sequenced by PCR. The obtained sequences were submitted to the GenBank and the ITS2 sequences of 45 samples belonging to ten species were downloaded from the GenBank. Total 73 ITS2 sequences were aligned and the genetic distances were analyzed using the MEGA 5.1. Identification analyses were performed using the BLAST1 and the nearest distance methods, and were presented intuitively by constructing Neighbor-joining (NJ) tree. Results: The lengths of all ITS2 sequences of A. lancea were 229 bp presented as one haplotype pattern. There was significant divergence between the interspecific and intraspecific genetic distances of the ITS2 sequences. The NJ tree showed that A. lancea could differed obviously from its closely related species, which showed high monophyly. The secondary structures of ITS2 in the helical regions displayed clear differences in stem loop number, size, position, and screw angle among the medicinal plants of Compositae. Conclusion: As a DNA barcode, ITS2 sequences can stably and accurately distinguish A. lancea from its closely related species and also provide a new technique to ensure the clinical safety in utilization of Chinese materia medica.
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Objective: To investigate the signal molecules and signal transduction involved in endophytic fungal elicitor-induced atractylodin biosynthesis and the effect of an endophytic fungal elicitor on the key enzyme activity in Atractylodes lancea. Methods: Content changes of nitric oxide (NO), salicylic acid (SA), and atractylodin were detected under the endophytic fungal elicitor treatment by plant cell suspension culture technology. Results: The endophytic fungal elicitor remarkably promoted NO burst and the biosynthesis of SA and atractyodin by activating nitric oxide synthase (NOS), phenylalanine ammonia lyase (PAL), and acetyl coenzyme A carboxylase (ACC), respectively. NOS inhibitor PBITU could inhibit the NO and SA accumulation and the atractyodin biosynthesis induced by the elicitor. And atractyodin biosynthesis could also be triggered by exogenous NO or SA. The results indicated that NO and SA were the necessary signal molecules and NO burst was mediated by NOS induced by endophytic fungal elicitor. NO quencher cPITO could effectively remove NO burst in A. lancea cell induced by endophytic fungal elicitor and notably inhibit the biosynthesis promotion of SA and atractyodin in A. lancea cell induced by endophytic fungal elicitor. Exogenous SNP could reverse the cPITO inhibition on the activity of PAL and ACC and the synthesis of SA and atractylodin. This suggested that NO was an upstream signal molecule mediated endophytic fungal elicitor to accelerate the biosynthesis of SA and atractyodin. Conclusion: Endophytic fungal elicitor mediated through NO followed by SA could promote atractyodin biosynthesis by activating ACC in A. lancea.
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Objective: To investigate the effect of endophytic fungal elicitor on key enzyme activity, inducing pathway and mechanism involved in the secondary metabolites of Atractylodes lancea. Methods: NADPH oxidase, HMGR activities, the concentration of hydrogen peroxide (H2O2) and β-eudesmol were determined by the co-culture of endophytic fungal elicitor and A. lancea suspension cell. Results: NADPH oxidase activity was notably enhanced by Fusarium sp5 elicitor which could induce oxidative burst, significantly promote H2O2 accumulation, and activate HMGR in the sesquiterpenoids metabolic pathway. Compared with the control, the yield of β-eudesmol increased 257.6% and reached 66.59 μg/g. CAT and DPI could inhibit the HMGR activity and β-eudesmol biosynthesis in A. lancea cell induced by Fusarium sp5 elicitor. Exogenous H2O2 also induced HMGR and promoted the β-eudesmol biosynthesis. Conclusion: H2O2 is necessary to induce β-eudesmol synthetic signal molecule by activating the HMGR.
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To characterize the ultra-fine particles of Atractylodes lancea (Thunb.) DC., Phellodendron chinense Schneid and the ERMIAO PILL*, a compounded preparation of the above two drugs, for the purpose to observe a deeper view on their physico-chemical properties. Methods By comparing their particle size, porosity and specific surface area with light and scanning electron microscope. Results The size of the ultra-fine particles were more uniform in size, 90% of which were under 20 μm, their specific area were increased by 60% ～ 190% and bulk density were around 0.42 g/cm3, and the great majority of the plant cells were broken as compared with the conventional coarse powder. Conclusion Both A. lancea and P. chinense together with their combined preparation became smaller in particle size, more uniformly distributed with increased specific surface area and broken cell wall.
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Objective To establish the suspension cell line of Atractylodes lancea and to study the effect of two endophytic fungal elicitors on its essential oil production.Methods The essential oil was extracted by using ultrasonic wave after suspension cell was treated with endophytic fungal elicitors.Then,the determination of four compounds(atractylone,hinesol,?-eudesmol,and atractylodin) was carried out by gas chromatography.Results By testing in various conditions,the suspension cell line with a rapid growth rate was established.Its highest biomass(6.95 g/L) was obtained on day 21.?-Eudesmol was the only detection in the control suspension cell,and its highest content(17.469 ?g/g) was also reached on day 21.The effect of crude elicitors of two endophytic fungi(belong to Cunninghamella sp.and Gilmaniella sp.respectively,named AL4 and AL12) on the cell growth and the production of essentia1 oil were investigated.Overall AL4 elicitor got better effect.When suspension cell of 14-day-old cultures was exposed to AL4 elicitor(carbohydrate 20 mg/L medium) for 7 d,the biomass increased 3.31% over the control,and the four compounds(atractylone: 14.715 ?g/g,hinesol: 28.395 ?g/g,?-eudesmol: 38.794 ?g/g,and atractylodin: 8.310 ?g/g) were all detected.Among them,the content of ?-eudesmol reached 2.22 times as much as the control.Conclusion The cell growth and the accumulation of essential oil of A.lancea could also be promoted by adding crude elicitors of the endophytic fungi AL4 and AL12.
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Objective To clone and sequence cDNA encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMGR) from Atractylodes lancea.Methods The cDNA,encoding HMGR in A.lancea,was amplified by RACE strategy with the cDNA of the total RNA of young leaves as the template.The partial fragments of HMGR were cloned and sequenced.Results The analysis results revealed that the conserved fragments were 458 bp.At the same time,the two fragments had been obtained 84.28% identification in nucleotide acid and 92.11% identification in corresponding amino acid,named as HMGRcr1 and HMGRcr2,respectively.It was deduced that they may be members of the HMGR gene family in A.lancea.Sequencing analysis showed that HMGRcr1 and HMGRcr2 had high identity with HMGR from other plants.Conclusion The cDNA encoding HMGR from A.lancea is cloned and reported for the first time.The work will provided a foundation for exploring the mechanism of terpenes biosynthesis and application to the other medicinal plants.