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Direct acid hydrolysis of Dioscorea zingiberensis rhizomes for preparation of diosgenin is wildly used in the traditional industry， which uses a large amount of inorganic acid catalysts， with high wastewater discharge and serious environmental pollution. Therefore， exploring clean and efficient preparation methods and processes has become an inevitable choice to realize the sustainable development of industrial production of diosgenin. Herein， the author reviewed and analyzed the research progress and problems of enzymatic hydrolysis， microbial transformation and modified acid hydrolysis in the preparation of diosgenin from D. zingiberensis rhizomes during the last ten years， and their application prospects are analyzed. Enzymatic hydrolysis has mild reaction conditions， but the yield of diosgenin is low， the economic cost is high， and the purification process of active enzyme is complicated. Microorganism shows specific activity to the substrate and high efficiency for diosgenin production， and microbial transformation is clean and environmentally friendly， but microbial transformation is time-consuming and the metabolic intermediates are complicated. For the modified acid hydrolysis， two-phase acid hydrolysis can reduce the amount of acid catalyst， and sulfonic acid-functionalized ionic liquid displays good recyclable performance by replacing the traditional inorganic acid， however， the wastewater discharge should still be considered. Solid acid catalysts are non-corrosive and easy to be recycled， but the need to use ethanol as the reaction solvent has certain safety hazards， and the catalyst preparation process is cumbersome. In conclusion， exploring clean and efficient conversion methods is an important research trend for preparation of diosgenin from D. zingiberensis rhizomes. For the enzymatic hydrolysis， the key glycoside hydrolases in the bioconversion process should be explored in depth， the conversion pathway of enzymatic saponins and enzyme specificity should be fully elucidated， and efforts should be made to improve the efficiency of enzymatic hydrolysis. For the microbial transformation， we should accelerate its industrial application process based on selecting and breeding efficient transformation strains， and optimizing stable transformation systems and processes， and in-depth investigation of the mechanism of microbial transformation， fully elucidating the specific key hydrolases and its catalytic properties， and striving to improve the efficiency of microbial transformation. For the modified acid hydrolysis， novel acid catalytic system with simple structure， stable performance and good biodegradability should be explored and applied， which can effectively solve the problems of environmental pollution and production safety.
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ObjectiveTo investigate the metabolites and gene expression characteristics in fibrous roots of Dioscorea zingiberensis in response to low phosphorus stress. MethodThe severe stress group， the moderate stress group， and the normal group were set up to stimulate the low phosphorus stress experiment. The fibrous roots of D. zingiberensis were collected during initial stress. The metabolites and transcriptomic characteristics were analyzed by gas chromatography-mass spectrometry （GC-MS） derivatization and RNA-seq techniques. Through multivariate statistical analysis of metabolites treated by different methods，functional analysis of differentially expressed genes， and data mining， the metabolism markers produced in fibrous roots of D. zingiberensis under low phosphorus stress were screened out， and the metabolic pathway characteristics of different genes were analyzed. ResultA total of 116 GC-MS metabolites were detected from the fibrous roots of D. zingiberensis. The metabolic characteristics of fibrous roots of D. zingiberensis under different low phosphorus treatments were obviously different. Orthogonal partial least squares discriminant analysis（OPLS-DA） model was used to screen six differential metabolites represented by sugars and alcohols from metabolites of fibrous roots treated with different methods，and these components were presumedly metabolism markers of fibrous roots of D. zingiberensis in response to low phosphorus stress. The differential genes screened out from the severe stress group and the normal group were mainly enriched in peroxidase pathway，phosphate and hypophosphate metabolism pathway，while the differential genes screened out from the severe stress group and the moderate stress group were mainly enriched in glutathione metabolism pathway and phosphopentose pathway. A total of 177 differential genes in response to low phosphorus stress were screened out from fibrous roots， involving many pathways such as terpenoid skeleton and inositol biosynthesis，which was consistent with the fact that the metabolic differential components in fibrous roots in response to low phosphorus stress were mainly saccharides and inositol. ConclusionThe metabolites and gene expression in fibrous roots of D. zingiberensis responded to low phosphorus stress，and the differential metabolites were closely related to differentially expressed genes. This study is expected to provide a theoretical basis for the research on the molecular mechanism of D. zingiberensis in response to low phosphorus stress.
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OBJECTIVE：To establish a method for the content determination of diosgenin in Dioscorea zingiberensis，and to compare the contents of diosgenin in wild D. zingiberensis from different habitats. METHODS：HPLC method was adopted. The determination was performed on Inertsil ODS-3 column with mobile phase consisted of acetonitrile-water （50 ∶ 50，V/V） at the flow rate of 1.0 mL/min. The column temperature was set at 30 ℃. The detection wavelength was set at 203 nm，and sample size was 10 μL. Established method was used to determine the contents of diosgenin in wild D. zingiberensis from different habitats （Shaanxi Shangluo，Shaanxi Ankang，Henan Neixiang，Yunnan Xuanwei，Sichuan Deyang，Hubei Shiyan，Hunan Zhangjiajie，Hunan Changde）. The differences of diosgenin content were compared. RESULTS：The linear range of diosgenin were 4.16-165.6 μg/mL （r＝ 0.999 9）. RSDs of precision，reproducible and stability tests were all lower than 2％ （n＝5 or n＝6）. The average recovery of diosgenin was 101.18％ （RSD＝1.27％，n＝6）. The content of diosgenin in wild D. zingiberensis from different habitats were in descending order，i.e. Sichuan Deyang （1 405.36 μg/g）＞Shaanxi Shangluo （1 201.79 μg/g）＞Hunan Zhangjiajie （1 035.18 μg/g）＞Shaanxi Ankang （632.64 μg/g）＞Hunan Changde （598.64 μg/g）＞Yunnan Xuanwei （425.34 μg/g）＞Henan Neixiang （350.13 μg/g）＞Hubei Shiyan （338.39 μg/g）. CONCLUSIONS：Established method is simple，accurate and suitable for the content determination of diosgenin in D. zingiberensis. The contents of diosgenin in wild D. zingiberensis from different habitats are different significantly，and the content of diosgenin in the samples from Sichuan Deyang is the highest.
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Objective To compare and analyze the transcriptome of rhizome and leaves of Dioscorea zingiberensis, and excavate the key enzyme genes related to the saponin biosynthetic pathway in D. zingiberensis. Methods The transcriptome of rhizome and leaves of D. zingiberensis were sequenced by Illumina HiSeq2000 high-throughput sequencing technique. According to sequence annotate results to find the differentially expressed genes. Then the key enzyme genes related to the biosynthesis of diosgenin were identified according to the content of saponins in rhizomes and leaves of D. zingiberensis. The expression levels of some candidate genes were analyzed by real-time fluorescence quantitative PCR (qRT-PCR). Results A total of 81 660 Unigenes were gained and 64.33% of them were annotated in NT, NR, Swiss-Prot, KOG, GO, and KEGG databases. Based on their expression and KEGG annotation, totally 227 catalytic enzyme genes of 29 kinds that may participate in D. zingiberensis saponin biosynthetic pathway were screened. The expression pattern of some catalytic enzymes was correlated with the content of saponin. Also were found five D. zingiberensis endophyte genes. Conclusion This experiment obtained candidate key enzyme genes tentatively that involved in the biosynthesis of saponin. Some candidate enzyme genes may participate in the post-modification process of steroidal saponins in D. zingiberensis. In additon, it was found that D. zingiberensis endotrophic bacteria may be involved in the saponin biosynthesis. The results laid a foundation to further elucidate the molecular mechanism of sapogenin synthesis pathway.
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Objective: To identify WRKY genes from the rhizomes of Dioscorea zingiberensis and analyze the protein characteristics and expression level of these genes. Methods: The transcriptional EST database of the rhizomes of D. zingiberensis was used to search the analogs of AtWRKY genes by BLASTn, the full-length open reading frames (ORF) of DzWRKY and its protein characteristics were studied using bioinformatic method, and the expression levels of DzWRKY genes in rhizomes and leaves were detected from transcriptional data of the rhizomes of D. zingiberensis. Results: Twenty-seven DzWRKY transcription factors family genes with full length ORF were isolated from the rhizomes of D. zingiberensis, and two WRKY domains were confirmed in six WRKY genes. All the DzWRKY proteins were predicted as hydrophilic proteins and nucleoproteins with highly conserved WRKY domains. The 27 DzWRKY and 19 AtWRKY proteins were divided into three groups by phylogenetic analysis. All DzWRKY genes showed higher expression level in the leaves compared to the rhizomes, and highly expressed genes were mainly in groups I and IId. DzWRKY proteins exhibited lower sequence identity. Conclusion: The DzWRKY genes are successfully isolated for the first time, which would provide a reference for the study on the roles of WRKY in development and active components biosynthesis of the rhizomes of D. zingiberensis.
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AIM@#To investigate the chemical constituents of Dioscorea zingiberensis C. H. Wright.@*METHODS@#The compounds were isolated by various chromatographic techniques, and the structures of the new steroidal saponins were elucidated by extensive 1D- and 2D-NMR, MS, and IR spectral analysis.@*RESULTS@#The 70% EtOH extract of the rhizomes of Dioscorea zingiberensis afforded two new steroidal saponins, zingiberenosides A (1) and B (2), along with eight known analogues, 3β, 26-dihydroxy-25(R)-furosta-Δ(5, 20(22))-diene-3-O-α-L- rhamnopyranosyl-(1→2)-O-β-D-glucopyranoside (3), methyl parvifloside (4), deltoside (5), methyl deltoside (6), zingiberensis new saponin (7), deltonin (8), progenin III (9) and diosgenin-diglucoside (10).@*CONCLUSION@#Two new steroidal saponins were isolated from Dioscorea zingiberensis and their structures determined.
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Dioscorea , Chemistry , Diosgenin , Chemistry , Molecular Structure , Plant Extracts , Chemistry , Rhizome , Chemistry , Saponins , Chemistry , Spirostans , Chemistry , Steroids , ChemistryABSTRACT
OBJECTIVE: To preliminarily establish the technology of fermentation pretreatment followed by two-phase combination acid hydrolysis for the extraction of diosgenin from the dry tubers of Dioscorea zingiberensis C. H. Wright. METHODS: Orthogonal experiment design was used with the yield of diosgenin as the monitoring index to optimize the conditions for fermentation pretreatment and two-phase combination acid hydrolysis. Two types of conventional methods were compared with the newly developed method. RESULTS: Fine powder of the tubers of Dioscorea zingiberensis C. H. Wright was mixed with water and fermented at 30°C for 72 h under stirring at 500 r · min-1, After that, sulfuric acid and methanol were added into the suspension to reach their final concentration of 10%, and petroleum ether was added to form a two-phase immiscible reaction system which was extracted in oil bath at 120°C for 4 h. As a consequence, the average yield of diosgenin achieved 4.28%, significantly higher than the 3.01% and 3.28% of the two conventional methods. CONCLUSION: This newly developed method of fermentation pretreatment followed by two-phase combination acid hydrolysis is proved to be more convenient than the traditional methods with a higher yield.
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Background: The perennial medicinal herb Dioscorea zingiberensis is a very important plant used for steroid drug manufacturing for its high level of diosgenin in rhizome. Although the stimulation of diosgenin accumulation by ethylene has been reported in a few of plant species, its regulation is not yet characterized at the molecular level, the underlying molecular mechanism remains elusive. Results: In this study, the effects of ethylene on diosgenin biosynthesis in in vitro cultures of D. zingiberensis were described. The results showed that, in samples treated with ethylene at concentration E3 (10(4) dilution of 40% ethephon), the diosgenin biosynthesis was significantly promoted in comparison with the control samples. Treatment with high concentrations of ethylene had inhibitory effect, whereas with low concentration of the gas elicitor brought about no detectable deleterious effect on the growth rate and diosgenin content of the cultures. The considerable increase of diosgenin level in in vitro cultured Dioscorea zingiberensis by ethylene application is accompanied by the concomitant increase of soluble proteins and chlorophyll content. The gene expressions of cycloartenol synthase and 3-hydroxy-3-methylglutaryl-CoA reductase but not of squalene synthase or farnesyl pyrophosphate synthase were up-regulated by applied ethylene. Conclusions: Our results suggest that ethylene treatment enhanced diosgenin accumulation via up-regulation of the gene expressions of cycloartenol synthase and 3-hydroxy-3-methylglutaryl-CoA reductase.
Subject(s)
Intramolecular Transferases/genetics , Intramolecular Transferases/metabolism , Dioscorea/metabolism , Hydroxymethylglutaryl CoA Reductases/genetics , Hydroxymethylglutaryl CoA Reductases/metabolism , In Vitro Techniques , RNA/isolation & purification , Gene Expression , Up-Regulation , Reverse Transcriptase Polymerase Chain Reaction , Dioscorea/growth & development , Dioscorea/genetics , Diosgenin/analysis , EthylenesABSTRACT
Objective The main economic qualities of natural triploid were evaluated and the result laid a foundation to culture new triploid variety of Dioscorea zingiberensis. Methods The ploidy level of series of D. zingiberensis from some areas of Yunnan Province were identified and the qualities of outputs per plantlet,propagation ratio,and diosgenin content etc. were measured. Results Five natural triploids were found and the qualities of the triploids all displayed diversity,output per plantlet 1 090.00—628.57 g,propagation ratio (ploidy) 72.43—29.43,dry substance ratio 36.95%—24.06%,and diosgenin content 4.40%—1.50%. The outputs of series 1 and series 3 exceed 1 000 g,the diosgenin content of series 2 reached to 4.40%,and series 3 displayed excellent quality of outputs per plantlet,propagation ratio,and diosgenin content. The synthetical index is maximum. Conclusion The series comprising good quality are obtained by the discovery of triploid and evaluation of qualities. The triploid variety is to be created with judgment and selection of progeny.
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Objective To study the chemical constituents of Dioscorea zingiberensis.Methods The fresh rhizome of D.zingiberensis was extracted three times,3 h once with EtOH-H2O at 80 ℃.The EtOH was evaporated under reduced pressure to give a residue which was suspended in water and then was exacted with petroleum ether,ethyl acetate,and n-butanol fraction.The water fraction was isolated by the reversed-phase ODS column chromatography.The chemical structures were elucidated by means of 1H-NMR,13C-NMR,135DEPT,HMQC,and HMBC spectroscopic analyses.Results Three steroidal saponins were isolated from the fresh rhizome of D.zingiberensis.The compounds were identified as:diosgenin-3-O-?-D-glucopyranosyl(1→3)-?-D-glucopyranosyl(1→4)-[?-L-rhamnopyranosyl(1→2)]-?-D-glucopyranoside(Ⅰ),(25R)-26-O-?-D-glucopyranosyl-furost-5-en-3?,22?-diol-3-O-?-D-glucopyranosyl(1→3)-?-D-glucopyranosyl(1→4)-[?-L-rhamnopyranosyl(1→2)]-?-D-glucopyranoside(Ⅱ),and(25R)-26-O-?-D-glucopyranosyl-furost-5-en-3?,22?-diol-7-carbonyl-3-O-?-D-glucopyranosyl(1→3)-?-D-glucopyranosyl(1→4)-[?-L-rhamnopyranosyl(1→2)]-?-D-glucopyranoside(Ⅲ).Conclusion Compound Ⅲ is a novel compound named as zingiberenin H.
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Objective To study water-soluble constituents of Dioscorea zingiberensis in order to seek active components. Methods Various chromatographic techniques were used to isolate the constituents. Structures of compounds were elucidated by spectroscopic analyses of 1H-NMR, 13C-NMR, 135DEPT, HMQC, HMBC, and TOCSY. Results One new steroidal saponin was isolated and identified as 26-O-(?-D-glucopyranosyl)-(25R)-furost-5-en-3?, 26-diol-22-OMe-3-O-｛?-L-rhamnopyranosyl-(1→4)-[?-D-glucopyranosl-(1→3)-?-D-glucopyranosl-(1→2)]-?-D-glucopyranoside｝. Conclusion The compound is a novel compound named as zingierenin E. The other two compounds are reported for the first time from D. zingiberensis.
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Objective To enhance the yield and product quality of diosgenin extract,the method of stepwise biocatalysis was used.Methods The optimum conditions of slepwise biocatalysis were obtained through orthogonal tests.In qualitative and quantitative analysis of diosgenin extracted through stepwise biocatalysis,IR spectrum and HPLC chromatogram were used.The yield and melting point of diosgenin were taken as investigated standard index to compare this method with others,such as acid hydrolysis,spontaneous fermentation,and enzymatic hydrolysis.Results When cellulase and pectinase compound,amlyse and diastatic enzyme were added in order in the method of stepwise biocatalysis,98% diosgenin from plants was extracted.IR spectrum of the product was in accordance with that of reference substance and the purity of it is more than 95% according to its HPLC chromatogram.Conclusion Compared with other three methods,it can improve the yield and quality with less energy consumption by stepwise biocatalysis.
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Objective By comparing the physiological index and diosgenin content of rhizome between the artificial tetraploid and the wild type diploid of Dioscorea zingiberensis, it is aimed at revealing the potential utility of polyploid breeding in medicinal D. zingiberensis. Methods Three clones of the natural diploid and the artificially induced tetraploid of D. zingiberensis were used as materials in this study. The content of SOD, PPO, and soluble sugar of leaves was determined by spectrophotometry, CAT content was measured by using the method of KMnO_4 titration. And the diosgenin content in rhizome was analyzed by HPLC. Results The tetraploid plants showed higher level of SOD, PPO, CAT, soluble sugar content in leaves, and diosgenin content in rhizome than those of the diploid origins. The diosgenin content in the three clones of tetraploid plants increased to 27% as compared to wild type. Conclusion Artificially induced tetraploid presents high content of diosgenin and great potential in stress resistance, which would be available in good seed breeding for high yield of diosgenin.
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Objective To study the genetic relationship among Dioscorea zingiberensis with different diosgenin content at DNA level.Methods DNA of all types was amplified by 12 random primers from 40 random primers,the polymorphic bands of RAPD were counted.And the results of types were analysed by POPGENE 1.31(Tool for Population Genetic Analysis 1.31) software.Results A total of 73 bands was obtained,64 of total bands(87.67%) was polymorphic.The results indicated that there were apparent and abundant genetic variations in D.zingiberensis. Conclusion The formation and accumulation of diosgenin could be affected significantly by the genetic basis of D.zingiberensis.
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Objective To create new tetraploid resource of Dioscorea zingiberensis in the hope of potential breeding materials with high yield and high level of diosgenin. Methods Callus with tiny green buds were directly soaked in colchicine solution or cultured in medium plus with colchicine to prohibit the isolation of chromosomes and induce tetraploid mutants. Results Tetraploids were achieved successfully. The most efficient treatment for inducing tetraploid was soaking the tiny buds in 1?103 mg/L colchicine solution for 24 h, the inducing rate could be up to 35. 2%. The induced tetraploids exhibited notable difference with common wild type (diploids) in morphology, physiology, and microscopic structure. ConclusionThe tetraploid plants show the advantages of gigantic size and vigorous growth. Thus, the established technique system to induce tetraploid from tissue cultured callus would provide an efficient alternative pathway for medicinal plants of Dioscorea L. breeding.
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ObjectTo study the accumulation and distribution of dioscin in the different part of rhizome of Dioscorea zingiberensis C. H. Wright and the differences of its diosgenin. Methods Histochemistry has been used to study the accumulation and distribution of dioscin in the different part of rhizome and diosgenin was determined by HPLC. Results Dioscin distributed mainly in ground tissue. The amount of dioscin accumulated in the plant part from the region distributed with small vascular bundle (SVB) is the most abundant, and the diosgenin is also the highest in the same region; both of them in the region with no vascular bundle (NVB) are next to that of SVB; the amount of dioscin accumulated in the region distributed with big vascular bundle (BVB) is the lowest, and the diosgenin is the lowest in the same region too. Conclusion Diosgenin in every region of the three of male rhizome is higher than that of female rhizome and diosgenin in every region of the three of monoecism is between those of male and female rhizome.
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AIM:To study the application of ultrasonic extraction in process of extracting diosgenin from Dioscorea zingiberensis C.H.Wright,and to evaluate its merits.METHODS:A new process of ethanol extracting and hydrochloric acid hydrolysis was provided to extraction diosgenin,and five different extraction methods were studied and compared,and the reaction mechanism was discussed.RESULTS:The ultrasonic extraction method was more efficient.Compared with the traditional water-hydrolylis,extraction rate was 15 percent higher,dosage of hydrochloric acid was registered 90 percent decrease,and the production was purer.CONCLUSION:The grinding pretreatment of Dioscorea zingiberensis is help to enhance the effects of ultrasonic extraction,the method has some advantages in efficiency,energy-saving and low pollution.