Pathways for the steroidal saponins conversion to diosgenin during acid hydrolysis of Dioscorea zingiberensis C. H. Wright

Diosgenin is an important starting material for the synthesis of steroidal hormone drugs in pharmaceutical industry. Acid hydrolysis of Dioscorea zingiberensis C. H. Wright (DZW) was the key step in the conversion of steroidal saponins to diosgenin in its manufacture factories. The pathways for the conversion of steroidal saponins to diosgenin during acid hydrolysis of DZW were studied experimentally. Three intermediate products and one byproduct were identified. The conversion pathways were from raw steroidal saponins zingiberensis newsaponin (1), deltonin (2), prosapogenin A of dioscin (3) to three intermediate products diosgenin-triglucoside (4), diosgenin-diglucoside (5) and trillin (6), and then to the product diosgenin (7) and byproduct 25-spirosta-3, 5-dienes (8). A maximum diosgenin yield of 2.5% was achieved when reacting in 0.8 M sulfuric acid for 6 h at 120 °C, while the intermediate products were just exhausted. After 6 h, the diosgenin yield decreased, due to the conversion of diosgenin to 25-spirosta-3, 5-dienes (8). The knowledge of the pathways for the conversion of steroidal saponins to diosgenin provide a scientific basis for assessing the performance of acid hydrolysis reaction in diosgenin manufacture factories by monitoring the content of three intermediate products (4-6). It also gives an alternative method for the synthesis of diosgenyl saponins (4-6) from plant material.     

Elicitation of Diosgenin Production in Trigonella foenum-graecum (Fenugreek) Seedlings by Methyl Jasmonate

The effects of methyl jasmonate (MeJA), an elicitor of plant defense mechanisms, on the biosynthesis of diosgenin, a steroidal saponin, were investigated in six fenugreek (Trigonella foenum-graecum) varieties (Gujarat Methi-2, Kasuri-1, Kasuri-2, Pusa Early Branching, Rajasthan Methi and Maharashtra Methi-5). Treatment with 0.01% MeJA increased diosgenin levels, in 12 days old seedlings, from 0.5%–0.9% to 1.1%–1.8%. In addition, MeJA upregulated the expression of two pivotal genes of the mevalonate pathway, the metabolic route leading to diosgenin: 3-hydroxy-3-methylglutaryl-CoA reductase (HMG) and sterol-3-β-glucosyl transferase (STRL). In particular, MeJA increased the expression of HMG and STRL genes by 3.2- and 22.2-fold, respectively, in the Gujarat Methi-2 variety, and by 25.4- and 28.4-fold, respectively, in the Kasuri-2 variety. Therefore, MeJA may be considered a promising elicitor for diosgenin production by fenugreek plants.     

纤维素酶催化与三液相萃取偶联制备盾叶薯蓣皂苷元

利用三液相萃取与酶催化转化耦合技术对盾叶薯蓣中薯蓣皂苷进行水解,并分离提取薯蓣皂苷元。考察了几种甾体皂苷和水解下的葡萄糖在三液相中的分配、酶的分配以及酶活力在三液相体系中的保留情况,并对比了三液相、有机相、水相中纤维素酶催化转化与薯蓣皂苷元的生成情况。实验结果表明,正己烷/1,4-二氧六环/硫酸铵三液相体系能够满足酶催化和产物分离同步进行的要求,使薯蓣皂苷元分配在正己烷相,底物和酶保留在1,4-二氧六环相,大部分葡萄糖进入硫酸铵相,反应进行96h薯蓣皂苷元的得率为69.4%,比有机相、水相酶催化分别提高一倍和27.6倍,显示出耦合技术的明显优势。     

Glycosylation of Ganoderic Acid G by Bacillus Glycosyltransferases

Ganoderma lucidum is a medicinal fungus abundant in triterpenoids, its primary bioactive components. Although numerous Ganoderma triterpenoids have already been identified, rare Ganoderma triterpenoid saponins were recently discovered. To create novel Ganoderma saponins, ganoderic acid G (GAG) was selected for biotransformation using four Bacillus glycosyltransferases (GTs) including BtGT_16345 from the Bacillus thuringiensis GA A07 strain and three GTs (BsGT110, BsUGT398, and BsUGT489) from the Bacillus subtilis ATCC 6633 strain. The results showed that BsUGT489 catalyzed the glycosylation of GAG to GAG-3-o-β-glucoside, while BsGT110 catalyzed the glycosylation of GAG to GAG-26-o-β-glucoside, which showed 54-fold and 97-fold greater aqueous solubility than that of GAG, respectively. To our knowledge, these two GAG saponins are new compounds. The glycosylation specificity of the four Bacillus GTs highlights the possibility of novel Ganoderma triterpenoid saponin production in the future.     

Evidence for the Role of CYP51A and Xenobiotic Detoxification in Differential Sensitivity to Azole Fungicides in Boxwood Blight Pathogens

Boxwood blight, a fungal disease of ornamental plants (Buxus spp.), is caused by two sister species, Calonectria pseudonaviculata (Cps) and C. henricotiae (Che). Compared to Cps, Che is documented to display reduced sensitivity to fungicides, including the azole class of antifungals, which block synthesis of a key fungal membrane component, ergosterol. A previous study reported an ergosterol biosynthesis gene in Cps, CYP51A, to be a pseudogene, and RNA-Seq data confirm that a functional CYP51A is expressed only in Che. The lack of additional ergosterol biosynthesis genes showing significant differential expression suggests that the functional CYP51A in Che could contribute to reduced azole sensitivity when compared to Cps. RNA-Seq and bioinformatic analyses found that following azole treatment, 55 genes in Cps, belonging to diverse pathways, displayed a significant decrease in expression. Putative xenobiotic detoxification genes overexpressed in tetraconazole-treated Che encoded predicted monooxygenase and oxidoreductase enzymes. In summary, expression of a functional CYP51A gene and overexpression of predicted xenobiotic detoxification genes appear likely to contribute to differential fungicide sensitivity in these two sister taxa.     

Triterpenoid Saponins from Stauntonia chinensis Ameliorate Insulin Resistance via the AMP-Activated Protein Kinase and IR/IRS-1/PI3K/Akt Pathways in Insulin-Resistant HepG2 Cells

Inflammation and oxidative stress play crucial roles in the etiology of type 2 diabetes mellitus. In this study, we examined the anti-diabetic effects of triterpenoid saponins extracted from Stauntonia chinensis on stimulating glucose uptake by insulin-resistant human HepG2 cells. The results showed that saponin 6 significantly increased glucose uptake and glucose catabolism. Saponin 6 also enhanced the phosphorylation of AMP-activated protein kinase (AMPK) and activated the insulin receptor (IR)/insulin receptor substrate-1 (IRS-1)/phosphoinositide 3-kinase (PI3K)/Akt pathway. Therefore, our results suggest that saponins from S. chinensis improve glucose uptake and catabolism in hepatic cells by stimulating the AMPK and the IR/IRS-1/PI3K/Akt signaling pathways. The results also imply that saponins from S. chinensis can enhance glucose uptake and insulin sensitivity, representing a promising treatment for type 2 diabetes mellitus.     

Ultrasonic and fermented pretreatment technology for diosgenin production from Diosorea zingiberensis C.H. Wright

The present study evaluated the efficiency of ultrasonic and fermented pretreatment technology for diosgenin production from Diosorea zingiberensis C.H. Wright (DZW). The optimum extraction conditions were as follows: ultrasonic pretreatment condition, 20 min, 50 kHz, 100 W; fermented pretreatment condition, 30 °C, 3 d and 0.8 g yeast dosages per 100 g raw materials; acidic hydrolysis condition, 1.5 mol/L sulfuric acid, 4 h; petrol extraction condition, 5 h. Under these conditions, diosgenin yield could reach 2.30%. Scanning electron micrographs indicated morphology changes of materials during diosgenin extraction process. UV-vis and GC-MS analysis revealed wastewater from the new approach contained 20 kinds of organic compounds which were mainly acids, esters, ketones and hydrocarbons. The research indicated the new approach could not only increase 42.5% diosgenin yield, recover 81.6% starch and 50.8% cellulose, but also efficiently reduce 69.7% COD, comparing with the conventional approach.     

Silencing of the Ortholog of DEFECTIVE IN ANTHER DEHISCENCE 1 Gene in the Woody Perennial Jatropha curcas Alters Flower and Fruit Development

DEFECTIVE IN ANTHER DEHISCENCE 1 (DAD1), a phospholipase A1, utilizes galactolipids (18:3) to generate α-linolenic acid (ALA) in the initial step of jasmonic acid (JA) biosynthesis in Arabidopsis thaliana. In this study, we isolated the JcDAD1 gene, an ortholog of Arabidopsis DAD1 in Jatropha curcas, and found that it is mainly expressed in the stems, roots, and male flowers of Jatropha. JcDAD1-RNAi transgenic plants with low endogenous jasmonate levels in inflorescences exhibited more and larger flowers, as well as a few abortive female flowers, although anther and pollen development were normal. In addition, fruit number was increased and the seed size, weight, and oil contents were reduced in the transgenic Jatropha plants. These results indicate that JcDAD1 regulates the development of flowers and fruits through the JA biosynthesis pathway, but does not alter androecium development in Jatropha. These findings strengthen our understanding of the roles of JA and DAD1 in the regulation of floral development in woody perennial plants.     

The Molecular Bases of the Interaction between a Saponin from the Roots of Gypsophila paniculata L. and Model Lipid Membranes

In view of the possible medical applications of saponins, the molecular structure of a GOTCAB saponin from the roots of Gypsophila paniculata L. was determined by NMR. The biological activity of saponins may depend on the interaction with cell membranes. To obtain more insight in the mechanism of membrane-related saponin function, an experimental and theoretical study was conducted. Ternary lipid systems composed of sphingomyelin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and cholesterol were used as models of mammalian cell membranes. The membrane–saponin interaction was studied experimentally by monitoring surface pressure in the monomolecular films formed at the air–aqueous subphase interface. The behavior of GOTCAB saponin in a water box and model monolayer systems was characterized by molecular dynamics simulations. The results obtained showed that, in the systems used, cholesterol had a decisive effect on the interaction between GOTCAB and phosphocholine or sphingomyelin as well as on its location within the lipid film.     

Larval Beetles Form a Defense from Recycled Host-Plant Chemicals Discharged as Fecal Wastes

Larvae of the leaf-feeding beetles Neolema sexpunctata and Lema trilinea carry feces on their backs that form shields. We used the generalist predatory ant, Formica subsericea, in a bioassay to determine whether shields were a physical barrier or functioned as a chemical defense. Fecal shields protected both species against ant attack. Larvae of both species reared on lettuce produced fecal shields that failed to deter ants. Commelina communis, N. sexpunctata's host, lacks noxious secondary compounds but is rich in phytol and fatty acids, metabolites of which become incorporated into the fecal defense. In contrast, the host plant of L. trilinea, Solanum dulcamara, contains steroidal glycoalkaloids and saponins, whose partially deglycosylated metabolites, together with fatty acids, appear in Lema feces. Both beetle species make modifications to host-derived precursors before incorporating the metabolites into shields. Synthetic chemicals identified as shield metabolites were deterrent when applied to baits. This study provides experimental evidence that herbivorous beetles form a chemical defense by the elimination of both primary and secondary host-derived compounds. The use of host-derived compounds in waste-based defenses may be a more widely employed strategy than was hitherto recognized, especially in instances where host plants lack elaborate secondary compounds.     

米曲霉直接转化盾叶薯蓣生产薯蓣皂苷元

利用米曲霉直接转化盾叶薯蓣制备了薯蓣皂苷元,转化中最重要的影响因素是pH、接种量和培养温度. 在pH 6.0的66.7 mmol/L Na2HPO4-KH2PO4缓冲液中,8%接种量、37℃培养84 h后提温至50℃继续转化8 h的条件下,薯蓣皂苷元得率可达17.06 mg/g,是粗酶液转化的6.77倍. 转化过程中甾体的总摩尔数分别是微波辅助提取和酸水解的1.73和1.47倍. 转化产物经质谱和核磁共振鉴定与薯蓣皂苷元同质. 直接微生物转化法优于酶解法.     

Neomycin Inhibition of (+)-7-Iso-Jasmonoyl-L-Isoleucine Accumulation and Signaling

The majority of plant defenses against insect herbivores are coordinated by jasmonate (jasmonic acid, JA; (+)-7-iso-jasmonoyl-L-isoleucine, JA-Ile)-dependent signaling cascades. Insect feeding and mimicking herbivory by application of oral secretions (OS) from the insect induced both cytosolic Ca²⁺ and jasmonate-phytohormone elevation in plants. Here it is shown that in Arabidopsis thaliana upon treatment with OS from lepidopteran Spodoptera littoralis larvae, the antibiotic neomycin selectively blocked the accumulation of OS-induced Ca²⁺ elevation and level of the bioactive JA-Ile, in contrast to JA level. Furthermore, neomycin treatment affected the downstream expression of JA-Ile-responsive genes, VSP2 and LOX2, in Arabidopsis. The neomycin-dependent reduced JA-Ile level is partially due to increased CYP94B3 expression and subsequent JA-Ile turn-over to12-hydroxy-JA-Ile. It is neither due to the inhibition of the enzymatic conjugation process nor to substrate availability. Thus, blocking Ca²⁺ elevation specifically controls JA-Ile accumulation and signaling, offering an insight into role of calcium in defense against insect herbivory.