Identification of genetic variants controlling diosgenin content in Dioscorea zingiberensis tuber by genome-wide association study.

BACKGROUND: Diosgenin is an important steroidal precursor renowned for its diverse medicinal uses. It is predominantly sourced from Dioscorea species, particularly Dioscorea zingiberensis. Dioscorea zingiberensis has an ability to accumulate 2-16% diosgenin in its rhizomes. In this study, a diverse population of 180 D. zingiberensis accessions was used to evaluate the genomic regions associated with diosgenin biosynthesis by the genome wide association study approach (GWAS). RESULTS: The whole population was characterized for diosgenin contents from tubers by gas chromatography mass spectrometry. The individuals were genotyped by the genotyping-by-sequencing approach and 10,000 high-quality SNP markers were extracted for the GWAS. The highest significant marker-trait-association was observed as an SNP transversion (G to T) on chromosome 10, with 64% phenotypic variance explained. The SNP was located in the promoter region of CYP94D144 which is a member of P450 gene family involved in the independent biosynthesis of diosgenin from cholesterol. The transcription factor (TF) binding site enrichment analysis of the promoter region of CYP94D144 revealed NAC TF as a potential regulator. The results were further validated through expression profiling by qRT-PCR, and the comparison of high and low diosgenin producing hybrids obtained from a bi-parental population. CONCLUSIONS: This study not only enhanced the understanding of the genetic basis of diosgenin biosynthesis but also serves as a valuable reference for future genomic investigations on CYP94D144, with the aim of augmenting diosgenin production in yam tubers.

22R- but not 22S-hydroxycholesterol is recruited for diosgenin biosynthesis.

Diosgenin is an important compound in the pharmaceutical industry and it is biosynthesized in several eudicot and monocot species, herein represented by fenugreek (a eudicot), and Dioscorea zingiberensis (a monocot). Formation of diosgenin can be achieved by the early C22,16-oxidations of cholesterol followed by a late C26-oxidation. This study reveals that, in both fenugreek and D. zingiberensis, the early C22,16-oxygenase(s) shows strict 22R-stereospecificity for hydroxylation of the substrates. Evidence against the recently proposed intermediacy of 16S,22S-dihydroxycholesterol in diosgenin biosynthesis was also found. Moreover, in contrast to the eudicot fenugreek, which utilizes a single multifunctional cytochrome P450 (TfCYP90B50) to perform the early C22,16-oxidations, the monocot D. zingiberensis has evolved two separate cytochrome P450 enzymes, with DzCYP90B71 being specific for the 22R-oxidation and DzCYP90G6 for the C16-oxidation. We suggest that the DzCYP90B71/DzCYP90G6 pair represent more broadly conserved catalysts for diosgenin biosynthesis in monocots.

The Functional Characterization of DzCYP72A12-4 Related to Diosgenin Biosynthesis and Drought Adaptability in Dioscorea zingiberensis.

Dioscorea zingiberensis is a perennial herb famous for the production of diosgenin, which is a valuable initial material for the industrial synthesis of steroid drugs. Sterol C26-hydroxylases, such as TfCYP72A616 and PpCYP72A613, play an important role in the diosgenin biosynthesis pathway. In the present study, a novel gene, DzCYP72A12-4, was identified as C26-hydroxylase and was found to be involved in diosgenin biosynthesis, for the first time in D. zingiberensis, using comprehensive methods. Then, the diosgenin heterogenous biosynthesis pathway starting from cholesterol was created in stable transgenic tobacco (Nicotiana tabacum L.) harboring DzCYP90B71(QPZ88854), DzCYP90G6(QPZ88855) and DzCYP72A12-4. Meanwhile, diosgenin was detected in the transgenic tobacco using an ultra-performance liquid chromatography system (Vanquish UPLC 689, Thermo Fisher Scientific, Bremen, Germany) tandem MS (Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer, Thermo Fisher Scientific, Bremen, Germany). Further RT-qPCR analysis showed that DzCYP72A12-4 was highly expressed in both rhizomes and leaves and was upregulated under 15% polyethylene glycol (PEG) treatment, indicating that DzCYP72A12-4 may be related to drought resistance. In addition, the germination rate of the diosgenin-producing tobacco seeds was higher than that of the negative controls under 15% PEG pressure. In addition, the concentration of malonaldehyde (MDA) was lower in the diosgenin-producing tobacco seedlings than those of the control, indicating higher drought adaptability. The results of this study provide valuable information for further research on diosgenin biosynthesis in D. zingiberensis and its functions related to drought adaptability.

[Screening and identification of key enzyme genes for steroidal saponin biosynthesis in Dioscorea zingiberensis under low phosphorus stress].

To investigate the responses of key enzymes involved in steroidal saponin biosynthesis of Dioscorea zingiberensis to low phosphorus stress, we designed three treatments of severe phosphorus stress, moderate phosphorus stress, and normal phosphorus level. The D. zingiberensis plants were collected at the early, middle, and late stages of treatment. The content of total steroidal saponins in different tissues of D. zingiberensis was determined by spectrophotometry for the identification of the critical stage in response to low phosphorus stress. BGI 500 sequencing platform was employed to obtain the transcript information of D. zingiberensis samples at the critical stage of low phosphorus stress, and then a transcriptome library was constructed. The correlation between the expression of genes involved in steroidal saponin biosynthesis and the content of total steroidal saponins was analyzed for the screening of the key enzyme genes in response to low phosphorus stress. Further, the expression patterns of these genes were analyzed by real-time fluorescence PCR(qRT-PCR). The content of total steroidal saponins in D. zingiberensis had obvious tissue specificity under low phosphorus stress, and the early stage of stress was particularly important for D. zingiberensis to respond to low phosphorus stress. A total of 101 593 unigenes were obtained by transcriptome sequencing, of which 77.35% were annotated in NT, NR, SwissProt, KOG, GO, and KEGG. A total of 256 transcripts of known key enzyme genes in the biosynthetic pathway of steroidal saponins were identified. The expression levels of 69 transcripts encoding 18 catalytic enzymes were significantly correlated with the content of total steroidal saponins. The qRT-PCR results showed that several key enzyme genes presented different expression patterns in four tissues under low phosphorus stress. The results indicated that the content of total steroidal saponins and the expression of key enzyme genes regulating steroidal saponin biosynthesis in D. zingensis changed under low phosphorus stress. This study provides the biological information for elucidating the molecular mechanism of steroidal saponin biosynthesis in D. zingensis exposed to low phosphorus stress.

Genome-Wide Identification of CYP72A Gene Family and Expression Patterns Related to Jasmonic Acid Treatment and Steroidal Saponin Accumulation in Dioscorea zingiberensis.

Dioscorea zingiberensis is a medicinal herb containing a large amount of steroidal saponins, which are the major bioactive compounds and the primary storage form of diosgenin. The CYP72A gene family, belonging to cytochromes P450, exerts indispensable effects on the biosynthesis of numerous bioactive compounds. In this work, a total of 25 CYP72A genes were identified in D. zingiberensis and categorized into two groups according to the homology of protein sequences. The characteristics of their phylogenetic relationship, intron-exon organization, conserved motifs and cis-regulatory elements were performed by bioinformatics methods. The transcriptome data demonstrated that expression patterns of DzCYP72As varied by tissues. Moreover, qRT-PCR results displayed diverse expression profiles of DzCYP72As under different concentrations of jasmonic acid (JA). Likewise, eight metabolites in the biosynthesis pathway of steroidal saponins (four phytosterols, diosgenin, parvifloside, protodeltonin and dioscin) exhibited different contents under different concentrations of JA, and the content of total steroidal saponin was largest at the dose of 100 µmol/L of JA. The redundant analysis showed that 12 DzCYP72As had a strong correlation with specialized metabolites. Those genes were negatively correlated with stigmasterol and cholesterol but positively correlated with six other specialized metabolites. Among all DzCYP72As evaluated, DzCYP72A6, DzCYP72A16 and DzCYP72A17 contributed the most to the variation of specialized metabolites in the biosynthesis pathway of steroidal saponins. This study provides valuable information for further research on the biological functions related to steroidal saponin biosynthesis.

Protective effect of Dioscorea zingiberensis ethanol extract on the disruption of blood-testes barrier in high-fat diet/streptozotocin-induced diabetic mice by upregulating ZO-1 and Nrf2.

Testicular injury is the primary pathogenesis of diabetes-induced male infertility. Dioscorea zingiberensis (DZ), a traditional Chinese medicine (TCM) including saponins, flavonoids and cellulose, is used to treat diseases in the reproductive system. But the protective effects of DZ on diabetes-induced testicular injury remain poorly understood. In this study, the therapeutic effects of chronic oral DZ treatment on testis impairment in a diabetic mouse model were explored by assessing sperm morphology, blood-testes barrier (BTB) integrity and testicular histological examination. Our results showed that DZ significantly reversed BTB disruption, testicular tissue injury and abnormal sperm morphology in diabetic mice. Interestingly, diabetes-induced disruption of the BTB was associated with a decrease in the tight junction (TJ) protein zonula occludens-1 (ZO-1). Dioscorea zingiberensis effectively increased ZO-1 expression in testis tissue to restore the integrity of the BTB. Moreover, DZ treatment significantly reduced hyperglycaemia-induced increases in malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels. Further mechanistic studies revealed that DZ substantially enhanced the expression of Nrf2, NOQ1 and HO-1, which indicated that DZ exerts potential antioxidant effects against testicular tissue damage via the activation of Nrf2. In conclusion, the protective effects of DZ rely on repairing the integrity of the BTB and on reducing oxidative stress damage by mediating ZO-1 and Nrf2. The study contributes to discovering the DZ possible mechanism of action.

Functional characterization of squalene epoxidase and NADPH-cytochrome P450 reductase in Dioscorea zingiberensis.

Dioscorea zingiberensis is a perennial medicinal herb rich in a variety of pharmaceutical steroidal saponins. Squalene epoxidase (SE) is the key enzyme in the biosynthesis pathways of triterpenoids and sterols, and catalyzes the epoxidation of squalene in coordination with NADPH-cytochrome P450 reductase (CPR). In this study, we cloned DzSE and DzCPR gene sequences from D. zingiberensis leaves, encoding proteins with 514 and 692 amino acids, respectively. Recombinant proteins were successfully expressed in vitro, and enzymatic analysis indicated that, when SE and CPR were incubated with the substrates squalene and NADPH, 2,3-oxidosqualene was formed as the product. Subcellular localization revealed that both the DzSE and DzCPR proteins are localized to the endoplasmic reticulum. The changes in transcription of DzSE and DzCPR were similar in several tissues. DzSE expression was enhanced in a time-dependent manner after methyl jasmonate (MeJA) treatments, while DzCPR expression was not inducible.

Multiplatform Metabolomics Investigation of Antiadipogenic Effects on 3T3-L1 Adipocytes by a Potent Diarylheptanoid.

Obesity is fast becoming a serious health problem worldwide. Of the many possible antiobesity strategies, one interesting approach focuses on blocking adipocyte differentiation and lipid accumulation to counteract the rise in fat storage. However, there is currently no drug available for the treatment of obesity that works by inhibiting adipocyte differentiation. Here we use a broad-based metabolomics approach to interrogate and better understand metabolic changes that occur during adipocyte differentiation. In particular, we focus on changes induced by the antiadipogenic diarylheptanoid, which was isolated from a traditional Chinese medicine Dioscorea zingiberensis and identified as (3 R,5 R)-3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-heptane (1). Targeted aqueous metabolic profiling indicated that a total of 14 metabolites involved in the TCA cycle, glycolysis, amino acid metabolism, and purine catabolism participate in regulating energy metabolism, lipogenesis, and lipolysis in adipocyte differentiation and can be modulated by diarylheptanoid 1. As indicated by lipidomics analysis, diarylheptanoid 1 restored the quantity and degree of unsaturation of long-chain free fatty acids and restored the levels of 171 lipids mainly from 10 lipid classes in adipocytes. In addition, carbohydrate metabolism in diarylheptanoid-1-treated adipocytes further demonstrated the delayed differentiation process by flux analysis. Our results provide valuable information for further understanding the metabolic adjustment in adipocytes subjected to diarylheptanoid 1 treatment. Moreover, this study offers new insight into developing antiadipogenic leading compounds based on metabolomics.

Genome survey sequencing of Dioscorea zingiberensis.

Dioscorea zingiberensis (Dioscoreceae) is the main plant source of diosgenin (steroidal sapogenins), the precursor for the production of steroid hormones in the pharmaceutical industry. Despite its large economic value, genomic information of the genus Dioscorea is currently unavailable. Here, we present an initial survey of the D. zingiberensis genome performed by next-generation sequencing technology together with a genome size investigation inferred by flow cytometry. The whole genome survey of D. zingiberensis generated 31.48 Gb of sequence data with approximately 78.70× coverage. The estimated genome size is 800 Mb, with a high level of heterozygosity based on K-mer analysis. These reads were assembled into 334 288 contigs with a N50 length of 1079 bp, which were further assembled into 92 163 scaffolds with a total length of 173.46 Mb. A total of 4935 genes, 81 tRNAs, 69 rRNAs, and 661 miRNAs were predicted by the genome analysis, and 263 484 repeated sequences were obtained with 419 372 simple sequence repeats (SSRs). Among these SSRs, the mononucleotide repeat type was the most abundant (up to 54.60% of the total SSRs), followed by the dinucleotide (29.60%), trinucleotide (11.37%), tetranucleotide (3.53%), pentanucleotide (0.65%), and hexanucleotide (0.25%) repeat types. The 1C-value of D. zingiberensis was calibrated against Salvia miltiorrhiza and calculated as 0.87 pg (851 Mb) by flow cytometry, which was very close to the result of the genome survey. This is the first report of genome-wide characterization within this taxon.

Isolation of endophytic fungi from Dioscorea zingiberensis C. H. Wright and application for diosgenin production by solid-state fermentation.

In this study, endophytic fungi were isolated from Dioscorea zingiberensis C.H. Wright (DZW), and a novel clean process to prepare diosgenin from DZW was developed. A total of 123 strains of endophytic fungi were isolated from different plant tissues of DZW. Among them, the strain Fusarium sp. (CPCC 400709) showed the best activity of hydrolyzing steroidal saponins in DZW into diosgenin. Thus, this strain was used to prepare diosgenin from DZW by solid-state fermentation. The fermentation parameters were optimized using response surface methodology, and a high yield of diosgenin (2.16%) was obtained at 14.5% ammonium sulfate, an inoculum size of 12.3%, and 22 days of fermentation. Furthermore, the highest diosgenin yield (2.79%) was obtained by co-fermentation with Fusarium sp. (CPCC 400709) and Curvularia lunata (CPCC 400737), which was 98.9% of that obtained by ß-glucosidase pretreated acid hydrolysis (2.82%). This process is acid-free and wastewater-free, and shows promise as an effective and clean way to prepare diosgenin for use in industrial applications from DZW.

Comparative Transcriptome Analysis Identifies Putative Genes Involved in Dioscin Biosynthesis in Dioscorea zingiberensis.

Dioscorea zingiberensis is a perennial herb native to China. The rhizome of D. zingiberensis has long been used as a traditional Chinese medicine to treat rheumatic arthritis. Dioscin is the major bioactive ingredient conferring the medicinal property described in Chinese pharmacopoeia. Several previous studies have suggested cholesterol as the intermediate to the biosynthesis of dioscin, however, the biosynthetic steps to dioscin after cholesterol remain unknown. In this study, a comprehensive D. zingiberensis transcriptome derived from its leaf and rhizome was constructed. Based on the annotation using various public databases, all possible enzymes in the biosynthetic steps to cholesterol were identified. In the late steps beyond cholesterol, cholesterol undergoes site-specific oxidation by cytochrome P450s (CYPs) and glycosylation by UDP-glycosyltransferases (UGTs) to yield dioscin. From the D. zingiberensis transcriptome, a total of 485 unigenes were annotated as CYPs and 195 unigenes with a sequence length above 1000 bp were annotated as UGTs. Transcriptomic comparison revealed 165 CYP annotated unigenes correlating to dioscin biosynthesis in the plant. Further phylogenetic analysis suggested that among those CYP candidates four of them would be the most likely candidates involved in the biosynthetic steps from cholesterol to dioscin. Additionally, from the UGT annotated unigenes, six of them were annotated as 3-O-UGTs and two of them were annotated as rhamnosyltransferases, which consisted of potential UGT candidates involved in dioscin biosynthesis. To further explore the function of the UGT candidates, two 3-O-UGT candidates, named Dz3GT1 and Dz3GT2, were cloned and functionally characterized. Both Dz3GT1 and Dz3GT2 were able to catalyze a C3-glucosylation activity on diosgenin. In conclusion, this study will facilitate our understanding of dioscin biosynthesis pathway and provides a basis for further mining the genes involved in dioscin biosynthesis.

Safety investigation on total steroid saponins extracts from Dioscorea zingiberensis C.H. Wright: Sub-acute and chronic toxicity studies on dogs.

Sub-acute and chronic toxic effects of total steroidal saponins (TSSN) extracts from Dioscorea zingiberensis C.H. Wright on various internal organs and biochemical indicators have never been studied before and this study is the first of its kind to demonstrate sub-acute and chronic toxicities of TSSN on dogs. Administration of TSSN extracts at doses up to 3000 mg/Kg daily for 14 days, no biochemical and organ changes were observed on the experimental groups of dogs. Further, chronic toxicity study through oral administration of TSSN extracts at the gradual doses of 50, 250 and 500 mg/Kg for 90 days followed by a 2-week recovery assay revealed absence of significant architectural and morphological changes in internal organs which were confirmed through histopathological examination and merely no significant alteration in the biochemical indicators including hematologic and urine analysis and electrocardiogram compared to the control dogs. This toxicological evaluation came across with the finding that the herbal preparation can be considered as nontoxic and animals could tolerate the extracts at doses up to 500 mg/Kg with LD50 greater than 3000 mg/Kg. It may serve as a preliminary scientific evidence for further therapeutic investigations.

Enhancement of Palmarumycin C12 and C13 Production by the Endophytic Fungus Berkleasmium sp. Dzf12 in an Aqueous-Organic Solvent System.

The endophytic fungus Berkleasmium sp. Dzf12, isolated from Dioscorea zingiberensis, was found to produce palmarumycins C12 and C13 which possess a great variety of biological activities. Seven biocompatible water-immiscible organic solvents including n-dodecane, n-hexadecane, 1-hexadecene, liquid paraffin, dibutyl phthalate, butyl oleate and oleic acid were evaluated to improve palmarumycins C12 and C13 production in suspension culture of Berkleasmium sp. Dzf12. Among the chosen solvents both butyl oleate and liquid paraffin were the most effective to improve palmarumycins C12 and C13 production. The addition of dibutyl phthalate, butyl oleate and oleic acid to the cultures of Berkleasmium sp. Dzf12 significantly enhanced palmarumycin C12 production by adsorbing palmarumycin C12 into the organic phase. When butyl oleate was fed at 5% (v/v) in medium at the beginning of fermentation (day 0), the highest palmarumycin C12 yield (191.6 mg/L) was achieved, about a 34.87-fold increase in comparison with the control (5.3 mg/L). n-Dodecane, 1-hexadecene and liquid paraffin had a great influence on the production of palmarumycin C13. When liquid paraffin was added at 10% (v/v) in medium on day 3 of fermentation, the palmarumycin C13 yield reached a maximum value (134.1 mg/L), which was 4.35-fold that of the control (30.8 mg/L). Application of the aqueous-organic solvent system should be a simple and efficient process strategy for enhancing palmarumycin C12 and C13 production in liquid cultures of the endophytic fungus Berkleasmium sp. Dzf12.

Direct Biotransformation of Dioscin into Diosgenin in Rhizome of Dioscorea zingiberensis by Penicillium dioscin.

Diosgenin is an important precursor for synthesis of more than 200 steroidal hormone medicines. Rhizome of Dioscorea zingiberensis C. H. Wright (RDZ) contained the highest content of diosgenin in Dioscorea plant species. Diosgenin is traditionally extracted by acid hydrolysis from RDZ. However, the acid hydrolysis process produces massive wastewater which caused serious environment pollution. In this study, diosgenin extraction by direct biotransformation with Penicillium dioscin was investigated. The spawn cultivation conditions were optimized as: Czapeks liquid culture medium without sugar and agar (1,000 ml) + 6.0 g dioscin/6.0 g DL, 30 °C, 36 h; solid fermentation of RDZ: mycelia/RDZ of 0.05 g/kg, 30 °C, 50 h; the yield of diosgenin was over 90 %. Spawn cultivation was crucial for the direct biotransformation. In the spawn cultivation, amount and ratio of dioscin/DL were the key factors to promote biotransformation activity of P. dioscin. This biotransformation method was environment-friendly, simple and energy saving, and might be a potential substitute for acid hydrolysis in diosgenin extraction industry.

Conversion of Dioscorea zingiberensis saponins to prosapogenin A by enzymatic hydrolysis.

Prosapogenin A is a secondary saponin in Dioscorea zingiberensis, and it showed remarkable pharmacological effects. Due to very low content and lack of well-developed biotransformation, its preparation was not efficient and clean. This study aims to establish an eco-friendly strategy for preparation of Prosapogenin A from plant material. Physical separation was employed to recycle starch and cellulose, and then D101 resin and polyamide packed-bed column was incorporated for purification of total steroidal saponins (TSS). After these pretreatments, purity of TSS was largely increased to 83.2% with recovery at 87.6%, which was subjected to enzymatic hydrolysis. Optimized reaction system was constructed in 0.20 M HAc-NaAc buffer (pH4.2) containing cellulase/TSS (3:1, w/w), and the hydrolysis was performed at 53 °C for 6 h. Consequently, TSS was almost completely hydrolyzed to Prosapogenin A, while the highest yield reached 5.62%. The newly proposed approach is promising for efficient preparation of Prosapogenin A in industrial applications.

A pH-sensitive intelligent packaging film harnessing Dioscorea zingiberensis starch and anthocyanin for meat freshness monitoring.

Abundant starch was isolated from Dioscorea zingiberensis C.H. Wright, a novel and underutilized industrial crop resource. In this study, an intelligent packaging film able to indicate food freshness was developed and characterized. D. zingiberensis starch (DZS) was bleached first, and its particle size, total starch content, amylose content, and gelatinization temperature were then measured. Butterfly pea (Clitoria ternatea Linn.) flowers were selected as the source of polyphenols, which rendered the prepared film intelligent and progressively blue-violet. SEM and FT-IR analyses showed the homogeneous dispersion of butterfly pea flower extract (BPE) in the film. The BPE-loaded film showed improved flexibility and resistance to UV and oxidation while maintaining sufficient mechanical strength and physical properties. Moreover, the film underwent a distinguishable color change from red to blue-violet and finally to green-yellow with increasing pH from 2 to 13. Similar color alteration also occurred when the film was exposed to ammonia. When the film was used to monitor the freshness of chicken stored at room temperature, it exhibited an obvious color change, implying its deterioration. Therefore, the newly developed BPE-DZS film, which was produced from readily accessible natural substances, can serve as an intelligent packaging material, indicating food freshness and prolonging shelf life.

Research progress of diosgenin extraction from Dioscorea zingiberensis C. H. Wright: Inspiration of novel method with environmental protection and efficient characteristics.

Diosgenin was the starting materials to synthesize various hormone drugs and mainly generated from Dioscorea zingiberensis C. H. Wright by acidolysis, enzymolysis, microbiological fermentation, and integrated manner. Only acidic hydrolysis with strong acid such as hydrochloric acid or sulfuric acid was used in practice in diosgenin enterprises due to their feasibility and simplicity, nevertheless finally resulting in a great deal of unmanageable wastewater and severely polluted the surrounding environment. Aiming to provide a comprehensive and up-to date information of researches on diosgenin production from this plant, 151 cases were collected from scientific databases including Web of Science, Pubmed, Science Direct, Wiley, Springer, and China Knowledge Resource Integrated (CNKI). Their advantages and disadvantages with different production methods were analyzed based on these available data in this review paper. Considering the fact that nearly all of diosgenin enterprises were closed for the environmental protection and the life health of the people, this review paper was beneficial for providing useful guidelines to develop novel technologies with environmentally-friendly and cleaner features for diosgenin production or facilitate the transformation of other methods like enzymolysis, microbiological fermentation, or integrated methods from laboratory scale to industry scale.

Preparation and characterization of oregano essential oil-loaded Dioscorea zingiberensis starch film with antioxidant and antibacterial activity and its application in chicken preservation.

In this study, abundant starch was separated from the industrial crop Dioscorea zingiberensis C.H. Wright (DZW), and a novel bioactive packaging film loaded with oregano essential oil (OEO) was prepared and characterized. NaClO solution worked as a bleacher to prepare uniform starch powder from DZW tubers. OEO was selected from among three essential oils of Labiatae family plants for its strongest antibacterial activity. After the addition of OEO into the starch-based film, the UV-vis shielding property and antioxidant activity were enhanced. Meanwhile, the films still have a considerable performance in transparency, mechanical strength and water vapor permeability after incorporated with OEO. Furthermore, the 3% OEO-loaded starch film exhibited the strongest antibacterial activity against Bacillus subtilis, Escherichia coli and Staphylococcus aureus. It effectively lowered the total viable count of fresh chicken under 4 °C preservation conditions. These results revealed that the OEO-loaded DZW starch film can exert a positive effect on maintaining the quality and extending the shelf life of fresh meat. Therefore, readily accessible DZW tubers and oregano are very promising resources for application in degradable bioactive packaging film.

Effect of exogenous jasmonic acid on physiology and steroidal saponin accumulation in Dioscorea zingiberensis.

Dioscorea zingiberensis is a valuable medicinal herb rich in steroidal saponins. To reveal the role of jasmonic acid (JA) on physiology and steroidal saponins accumulation, D. zingiberensis were treated with different concentrations of JA. The antioxidant capacity, photosynthetic parameters, fatty acids and metabolites related to steroidal saponins biosynthesis (phytosterols, diosgenin and steroidal saponins) were examined under JA treatment. The results demonstrated that JA treatment caused a great reduction in MDA, stomatal width, photosynthetic rate and photosynthetic pigment, induced a considerable increase in proline, soluble sugar, soluble protein and antioxidant enzymes (CAT, POD and SOD), and leaded to a significant up-regulation in the expression of genes related to antioxidant system and chlorophyll degradation. Specialized metabolites displayed various changes under different concentrations of JA. The majority of fatty acids exhibited negative responses to JA treatment in leaf and rhizome. In leaf, JA treatment enhanced the accumulation of phytosterols and diosgenin, but decreased the accumulation of steroidal saponins. However, steroidal saponins were mainly accumulated in rhizome and were highly increased by JA treatment. Redundancy analysis illustrated that fatty acids were strongly associated with metabolites related to steroidal saponins. Among all fatty acids, C16:0, C18:1, C18:3, C22:0 and C24:0 contributed most to the variation in metabolites related to steroidal saponin biosynthesis. Overall, JA treatment leaded to an increase in steroidal saponins, but an inhibition of plant growth. Thus, the negative effects of JA application on plant physiology should be carefully assessed before being utilized to increase the production of steroidal saponins in D. zingiberensis.

High-value utilization of the waste hydrolysate of Dioscorea zingiberensis for docosahexaenoic acid production in Schizochytrium sp.

The starch saccharification liquid of Dioscorea zingiberensis tubers (SSLD) is a glucose-rich agro-industrial waste. Herein, SSLD was used as a novel potential carbon source for the biosynthesis of docosahexaenoic acid (DHA) in Schizochytrium sp. to achieve waste recycling and high-value utilization. Component analysis showed that SSLD contains abundant nutrients, such as glucose, amino acids, phenolics and flavonoids. When the total sugar concentration in SSLD was optimized to 90 g/L, the biomass and DHA yield reached 44.85 and 6.60 g/L, respectively, which were 32.1% and 36.92% higher than that at pure glucose culture condition. Fermentation characteristics and gene expression analysis showed that SSLD could remarkably improve cell antioxidant capacity, which is beneficial to scavenge intracellular reactive oxygen species and increase the gene expression of antioxidant enzymes in Schizochytrium sp. Hence, SSLD is an effective and economic carbon source for DHA production in Schizochytrium sp.