Steroidal Sapogenins Research Articles

Anticancer Activity of Diosgenin and Its Semi-synthetic Derivatives: Role in Autophagy Mediated Cell Death and Induction of Apoptosis.

During cancer progression, the unrestricted proliferation of cells is supported by the impaired cell death response provoked by certain oncogenes. Both autophagy and apoptosis are the signaling pathways of cell death, which are targeted for cancer treatment. Defects in apoptosis result in reduced cell death and ultimately tumor progression. The tumor cells lacking apoptosis phenomena are killed by ROS- mediated autophagy. The autophagic programmed cell death requires apoptosis protein for inhibiting tumor growth; thus, the interconnection between these two pathways determines the fate of a cell. The cross-regulation of autophagy and apoptosis is an important aspect to modulate autophagy, apoptosis and to sensibilise apoptosis-resistant tumor cells under metabolic stress and might be a rational approach for drug designing strategy for the treatment of cancer. Numerous proteins involved in autophagy have been investigated as the druggable target for anticancer therapy. Several compounds of natural origin have been reported, to control autophagy activity through the PI3K/Akt/mTOR key pathway. Diosgenin, a steroidal sapogenin has emerged as a potential candidate for cancer treatment. It induces ROS-mediated autophagy, inhibits PI3K/Akt/mTOR pathway, and produces cytotoxicity selectively in cancer cells. This review aims to focus on optimal strategies using diosgenin to induce apoptosis by modulating the pathways involved in autophagy regulation and its potential implication in the treatment of various cancer. The discussion has been extended to the medicinal chemistry of semi-synthetic derivatives of diosgenin exhibiting anticancer activity.

Synthesis of Solanum Alkaloid Demissidine Stereoisomers and Analogues.

Demissidine is an indolizidine alkaloid isolated from several potato species. A simple synthesis of demissidine stereoisomers and analogues from a common steroidal sapogenin tigogenin is presented in the paper. The key intermediate in the synthesis of these compounds is readily available tigogenoic acid. Its step-by-step transformation to indolizidine yielded 20R,25R or 20R,25S products while the direct reductive amination produced the 20S,25R compound (25-epi-demissidine).

The nephroprotective potential of diosgenin against ischemia-reperfusion acute renal damage via suppression of oxidative stress and downregulating inflammatory mediators

ABSTRACT Renal ischemia/reperfusion injury (I/R) is caused by short impairment of the blood supply to the kidney. Acute renal damage (ARD) is still a major problem in today’s clinical practice. Diosgenin (DG), a steroidal sapogenin mainly present infenugreek, yams, Costus species in high amounts. DG as a leading phytoconstituents is beneficial in several disease conditions due to its multifunction including antioxidant, hypolipidemic, anti-inflammatory, hypoglycemic, anticancer and anti-proliferative effects. The purpose of this study was to investigate the nephroprotective potential of DG on ischemia-reperfusion induced kidney damage in rats. Thirty male Wistar rats were divided into five groups. DG was given orally at doses of 20, 40 and 80 mg/kg for 10 days. On day 11th ARD was induced by unilateral ischemia for 45 min followed by 24-hour reperfusion. The levels of creatinine, blood urea nitrogen, total proteins, pro-inflammatory cytokines were decreased in DG + I/R rats compared to NC rats, along with a significant amelioration of oxidative stress. Histopathological analysis exhibited less histological damage in DG + I/R rats as compared with NC rats. Pretreatment of rats with DG (40 and 80 mg/kg) was able to improve these damages significantly, though DG (20 mg/kg) was insignificantly effective. In conclusion, DG proved nephroprotective potential against kidney damage induced by I/R injury via mitigation of inflammation, oxidative stress, and histopathologic injuries.

Canadian sainfoin and fenugreek as forage and functional foods

AbstractSainfoin (Onobrychis viciifolia Scop.) and fenugreek (Trigonella foenum‐graecum L.) are two legumes that are being developed as forage crops in Canada with potential benefits for animal and human health. Sainfoin, a perennial crop containing condensed tannins (CTs), is gaining popularity in western Canada because of its benefits for cattle. Its CTs make the crop bloat‐free for grazing cattle while improving protein digestibility and reducing greenhouse gas emissions. The CT‐containing fenugreek is also considered as a bloat‐free annual forage legume that was developed to serve in short‐term crop rotations in western Canada. These crops are known to provide health and nutritional benefits to cattle with their high protein content and other beneficial nutraceuticals such as crude fiber, 4‐hydroxyisoleucine, steroid sapogenins, and galactomannans. Some of these nutraceuticals have the potential to benefit human health; however, such attributes have not been studied enough to harness the full potential of these legume crops in Canada. Recent research suggests that legumes are a healthy substitute for meat. However, metabolite analysis of sainfoin is mostly limited to proteins and CTs. The CTs reported in sainfoin are involved in reduction of blood pressure and detoxification and providing anticancer properties in humans. Recent studies on fenugreek have highlighted the beneficial nutraceuticals associated with human health but most of those claims are not backed by relevant clinical studies. In this article, we reviewed the nutritional quality attributes of sainfoin and fenugreek and assessed their potential as functional foods and nutraceuticals for animal and human health based on scientific evidence.

Invitro anti‑bacterial activity of diosgenin on Porphyromonasgingivalis and Prevotellaintermedia.

Diosgenin (Dios), a natural steroidal sapogenin, is a bioactive compound extracted from dietary fenugreek seeds. It has a wide range of applications, exhibiting anti-oxidant, anti-inflammatory and anti-cancer activities. However, whether the extracts have beneficial effects on periodontal pathogens has so far remained elusive. The aim of the present study was to investigate the anti-bacterial effects of Dios on Porphyromonas gingivalis (P. gingivalis) and Prevotella intermedia (P. intermedia) in vitro. The anti-microbial effect of Dios on P. gingivalis and P. intermedia was assessed by a direct contact test (DCT) and the Cell Counting Kit (CCK)-8 assay at 60, 90 and 120 min. In addition, counting of colony-forming units (CFU) and live/dead cell staining were used to evaluate the anti-bacterial effects. The results of the DCT and CCK-8 assays indicated that Dios had beneficial dose-dependent inhibitory effects on P. gingivalis and P. intermedia. The CFU counting results also indicated that Dios had dose-dependent anti-bacterial effects on P. gingivalis and P. intermedia. Of note, Dios had significant anti-bacterial effects on the biofilms of P. gingivalis and P. intermedia in vitro as visualized by the live/dead cell staining method. In conclusion, the present results demonstrated that Dios had a marked anti-bacterial activity against P. gingivalis and P. intermedia in vitro, both in suspension and on biofilms. The present study highlighted the potential applications of Dios as a novel natural agent to prevent and treat periodontitis through its anti-bacterial effects.

Anti-inflammatory steroidal sapogenins and a conjugated chalcone-stilbene from Dracaena usambarensis Engl.

Four new steroidal sapogenins, dracaenogenins CF (1–4), a new conjugated chalcone-stilbene, 3′′-methoxycochinchinenene H (5) together with eight known compounds namely, (25S)-spirosta-1,4-dien-3-one (6), trans-resveratrol (7), 4,4′-dihydroxy-3′-methoxychalcone (8), N-trans-coumaroyltyramine (9), N-trans-p-coumaroyloctopamine (10), N-trans-feruloyloctopamine (11), 7-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-N2,N3-bis(4-hydroxyphenethyl)-6-methoxy-1,2-dihydronaphthalene-2,3-dicarboxamide (12) and grossamide (13) were isolated from the stems of Dracaena usambarensis Engl. from Kenya. It is important to note that compounds 12 and 13 are being reported from this genus for the first time. Structural elucidation of the isolated compounds was done using spectroscopic (NMR, UV, IR, optical rotation) and spectrometric (HRESIMS) techniques. The absolute and relative configurations of the isolated compounds were determined by employing single crystal X-ray crystallography analysis, NOESY correlations and coupling constants. The anti-inflammatory potencies of the isolated compounds were evaluated by measuring the levels of four cytokines (IL-1β, IL-2, GM-CSF and TNF-α) in the supernatant media of human peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide (LPS). At the tested concentration of 100 μM, the new conjugated chalcone-stilbene 5, the dihydrochalcone, 8 and the lignanamide, 13 were substantially more potent than the standard drug, ibuprofen, inhibiting the release of all the cytokines, IL-1β, IL-2, GM-CSF and TNF-α from 0.06–58.04% compared to LPS control. These compounds should therefore be considered for development into anti-inflammatory drug candidates. Compound 7 significantly decreased the release of GM-CSF (6.11% of LPS control) and TNF-α (18.35% of LPS control). The cytokine TNF-α was sensitive to all the tested compounds 1-13.

Method Development for Quantitative Determination of Diosgenin from the Seeds of Fenugreek, <i>Trigonella foenum-graecum</i> L.

Introduction . Modern pharmacognostic research is aimed at searching for plant biologically active individual compounds (RBAIS) isolated from plant extracts. Aim . Study of the content of the steroid sapogenin diosgenin in fenugreek seeds in plant extracts by HPLC-UV method. Materials and methods . The object of study was raw materials-fenugreek seeds produced as medicinal plant raw materials by LLC «Sage» (Irkutsk). Series of extraction and chromatographic separation by HPLC-UV method were performed. Results and discussion . In this work, the method of diosgenin extraction was successfully optimized (after acid hydrolysis of dioscin with a 5 % aqueous solution of hydrochloric acid when heated for at least 4 hours in a sand bath at a power of 150 W) from raw fenugreek seeds using a 50 % aqueous solution of isopropanol, h.h. A simplified technique for chromatographic separation of diosgenin from fenugreek seed extracts using an isocratic mode of 99.9 % acetonitrile elution (h.h.) was proposed. Conclusion . The average content of diosgenin (after acid hydrolysis of dioscin) in fenugreek seeds, taking into account the humidity of raw materials, is (M ± σ ) 5,3 ± 0,05 mg/g (95 % CI: 5,1-5,4 mg/g; n = 6).

Diosgenin as a Novel Alternative Therapy for Inhibition of Growth, Invasion, and Angiogenesis Abilities of Different Glioblastoma Cell Lines.

Fenugreek (Trigonella foenum-graecum) seeds and roots of wild yam (Dioscorea villosa) possess nutritional and medicinal properties and have been used for centuries in traditional medicine to treat different diseases and inflammatory responses. Diosgenin is a natural steroidal sapogenin extracted from fenugreek and wild yam and it is one of the major bioactive compounds used in the treatment of diabetes, hypercholesterolemia, and inflammation. Recent studies have shown a promising effect of diosgenin as ananti-tumor agent for inhibition of cell proliferation and induction of apoptosis in many cancers such as colon cancer, leukemia, breast cancer, and liver cancer. We examined the effects of different concentrations (5, 10, 15, 20, and 25µM) of diosgenin on proliferation of rat C6 and human T98G glioblastoma cell lines. We noticed that diosgenin had a high inhibitory effect on the growth of both C6 and T98G cell lines. Diosgenin induced the differentiation of glioblastoma cells, as determined by the increasein the expression of the differentiation marker glial fibrillary acidic protein (GFAP); and decreased the dedifferentiation of the cells, as shown by the decreasein the abundance of the dedifferentiation marker proteins Id2, N-Myc, telomerase reverse transcriptase (TERT), and Notch-1. It also induced apoptosis in C6 and T98G cell lines and themolecular mechanisms involved in the induction of apoptosis included increase in pro-apoptotic Bax protein and decrease in anti-apoptotic Bcl-2 protein. Further, the diosgenin-induced suppression of cell migration was correlated with the decrease in expressionof matrix metalloproteinase 2 (MMP2) and MMP9; and the inhibition of angiogenesis, as determined by the tube formation assay, was correlated with a decrease in the protein levels of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2). In conclusion, diosgenin showed anti-tumoreffects in glioblastoma cells by induction of differentiation and apoptosis and inhibition of migration,invasion, and angiogenesis.

Steroidal saponins and sapogenins from fenugreek and their inhibitory activity against α-glucosidase

The seed of Trigonella foenum-graecum L. (fenugreek) has been reported to be rich in saponins, especially the dioscin or diosgenin, which are natural anti-diabetic agents with relatively low toxicity. Thus, the present study was to purify the saponins and sapogenins from fenugreek and to evaluate their α-glucosidase inhibitory activity in vitro. As a result, 33 steroidal saponins and sapogenins were isolated, including six undescribed ones and 27 previously known molecules. Among them, compounds 10, 12, 17, 22 and 29 were five 25R and 25S isomer mixtures of spirostanol saponins or sapogenins. The structures of compound 1–6 were established by 1D and 2D NMR spectroscopic analyses, high-resolution mass spectrometry, and chemical evidence. Compared to the positive control, sapogenins 26, 27, 14 and saponins 18 and 23 considerably inhibited α-glucosidase at IC50 values of 15.16, 8.98, 7.26, 5.49 and 14.01 μM, respectively. These results support the therapeutic potential of fenugreek in the treatment of diabetes with saponins and sapogenins as the active constituents.

Phytochemical, purification and spectral analysis of compounds from root tuber extract of Aparagus racemosus (Willd)

Asparagus racemosus the queen of herbs is an important medicinal plant regarded as a ayurvedic rasayana, a plant drug which prevent ageing, increase longevity, impart immunity, improve mental function, vigour and add vitality to the body. It recommended for female health in habitual abortions, weakness of the uterus, excessive bleeding during menstruation etc. Based on the fragmentary information an investigation of the crude methanolic extract of the roots for the presence of bioactive components was done adopting Harborne method. The phytochemical screening revealed the presence of chemical constituents such as alkaloids, flavonoids, steroidal saponins and carbohydrates. The TLC profiling of the methanolic extract showed four active spots for flavonoids, saponins and other secondary metabolites. The spectral analysis using Gas Chromatography-Mass Spectrometry identified about 13 major and 5 minor active constituents. Identification of steroidal sapogenin from the GC/MS spectra was the highlight of this present study. Diosgenin, the steroidal sapogenin is the precursor for the semisynthesis of progesterone.

Stereospecific synthesis and rearrangement of 22S-23-acetylsapogenins

The BF3·Et2O-catalysed acetolysis of steroid sapogenins diosgenin, sarsasapogenin and tigogenin in dichloromethane as the solvent instead of acetic anhydride afforded (20S)- and (20R)-22,26-epoxycholestanes (compounds 1 and 2). 22S-23-Acetylsapogenins (compounds 4) were synthesized stereospecifically from 20R-22,26-epoxycholestanes (compounds 2) in good yield. The rearrangement of 22S-23-acetylsapogenins (compounds 4) afforded novel disubstituted dihydropyran furostanol frameworks. Exhaustive NMR characterization of the obtained compounds is provided. Additionally, the structures of the critical compounds (6a and 7a) were unequivocallyconfirmed by single crystal X-ray diffraction studies.

Protective effect of diosgenin on LPS/D-Gal-induced acute liver failure in C57BL/6 mice

Acute liver failure (ALF) is a deadly clinical syndrome, which leads to a rapid loss of normal liver function. Diosgenin is a natural steroidal sapogenin found in various plant families. Various studies have shown that diosgenin have therapeutic or preventive effect in various diseases such as cancer, cardiovascular disorders, type 2 diabetes, and neurodegenerative disorders. In this study, we evaluated effects of diosgenin on mice model of ALF. Animal model of ALF was induced by intraperitoneal injection of lipopolysaccharide (LPS)/d-galactosamine (D-Gal). The male C57BL/6 mice were randomly divided into 3 groups: control group, LPS/D-Gal group, and LPS/D-Gal + diosgenin group (50 mg/kg). Mice in the LPS/D-Gal group received a combination of LPS (50 μg/kg) and D-Gal (400 mg/kg) intraperitoneally. LPS/D-Gal + diosgenin group received diosgenin twice orally 24 h and 1 h before receiving LPS/D-Gal. Markers of liver injury including ALT, AST and ALP were measured in blood samples in addition to determination of oxidative stress and inflammatory markers including MDA, nitrite, ROS, catalase, SOD, Nrf2, IL-1β, IL-6, TLR4, TNF-α and NF-κB in hepatic tissue. Administration of diosgenin could greatly reduce serum levels of ALT, AST, and ALP. Besides, hepatic levels of MDA, ROS, IL-1β, IL-6, TLR4, TNF-α, and NF-κB significantly decreased and SOD activity and Nrf2 level increased in comparison with the LPS/D-Gal group. In addition, myeloperoxidase activity as a marker of neutrophil infiltration decreased following diosgenin administration. In summary, diosgenin led to reduction of liver injury indices and oxidative stress and inflammatory events and diosgenin has probably hepatoprotecive effects in ALF.

New anti-neuroinflammatory steroids against LPS induced NO production in BV2 microglia cells by microbial transformation of isorhodeasapogenin.

Microbial transformation of isorhodeasapogenin (1), the major steroidal sapogenin of Tupistra chinensis, was performed with the fungus Syncephalastrum racemosum (AS 3.264). As a result, nine new biotransformation metabolites (2-10) were isolated and their structures were elucidated by spectroscopic analysis. Hydroxylation, oxidation and glycosylation reactions were observed on the B, C, D and F rings of steroidal skeleton. Substrate (1) and its biotransformed metabolites 2-6, 8-10 were evaluated for their anti-neuroinflammatory effect on the NO accumulation induced by LPS in BV-2 cells. All the tested metabolites were found to have more potential anti-neuroinflammatory activity than the substrate. Especially, metabolites 2, 5 and 6 exhibited significant inhibition on NO production after hydroxylation at C-12 or C-15. Moreover, metabolite 2 dose-dependently reduced the LPS-induced protein expression of iNOS and COX-2.

BF3·OEt2-Catalyzed Aldol Condensation of Steroid Sapogenins and 2-Oxoacids: A Single Step Conversion of Steroid Spiroketals into Branched α,β-Unsaturated Spirolactones

BF3·OEt2-catalyzed aldol reaction of steroid sapogenins with glyoxylic acid or pyruvic acid led to compounds in which the novel spirocyclic side chain bears an α,β-unsaturated lactone. The structures of the new spirolactones were established with the aid of combined 1D and 2D NMR techniques. A plausible mechanism is described.

Metabolomic profiling of five Agave leaf taxa via UHPLC/PDA/ESI-MS inrelation to their anti-inflammatory, immunomodulatory and ulceroprotective activities

Agave plants are popular for their myriad applications in traditional medicine attributed to their reported anti-inflammatory, immunomodulatory, cytotoxic and antifungal activities. The aim of this study was to examine the anti-inflammatory, immunomodulatory and ulceroprotective activity of Agave species in relation to their metabolite fingerprint via a metabolome based ultra-performance liquid chromatography-mass spectrometry (UPLC–MS) approach coupled to chemometrics. The metabolomic differences among five examined Agave leaves viz. Agave americana L., A. americana var. marginata Trel, A. angustifolia Haw. cv. marginata, A. desmettiana Jacobi, A. pygmaea Gentry were determined via a total of 56 annotated metabolites. Identification based on MSn and UV spectra revealed 25 steroidal saponins and sapogenins, 6 flavonoids, 2 homoisoflavonoids, 7 phenolic acids, 6 fatty acids and 3 fatty acid amides, some of which are reported for the first time in Agave. Metabolites heterogeneity was assessed among leaf taxa via multivariate data analyses for samples classification, showing that saponins is the major metabolite contributing to their classification. The carrageenan induced acute inflammatory rat model was used to assess the anti-inflammatory activity of Agave extracts via monitoring of blood cytokine levels. Additionally, their effects on ethanol-induced gastric ulcer in rats were evaluated. A. pygmaea showed the most significant anti-inflammatory and immunomodulatory activity, while A. angustifolia var. marginata possessed the highest ulceroprotective activity, which could be attributable to the high abundance of various saponins and homoisoflavonoids in those taxa.

Synthesis of 23E-ethylidene spirostanes via the BF3·Et2O-catalyzed aldol condensation of steroid sapogenins and acetaldehyde

The treatment of acetylated steroid sapogenins with acetaldehyde and BF3·Et2O in CH2Cl2 led to moderate yields of the corresponding 23E-ethylidene spirostanes. The obtained compounds were characterized using a combination of 1D and 2D NMR techniques. Single crystal X-ray diffraction corroborated the proposed structures.

Characterization of the interaction of diosgenin with human serum albumin and α1-acid glycoprotein using biophysical and bioinformatic tools

Diosgenin (DIO), a steroidal sapogenin with high therapeutic potential, has been shown to exhibit a myriad of pharmacologically significant properties and is commonly used as a template for synthesizing steroidal drugs. The efficacy of bioactive compounds as a potential drug depends mainly upon their interaction with proteins in the circulatory system. Therefore, the interaction of DIO with the major carrier proteins in humans, i.e. human serum albumin (HSA) and α 1 -acid glycoprotein (AAG), was characterized using multispectroscopic and molecular docking techniques. Analysis of fluorescence enhancement data showed intermediate binding affinity for both DIO–HSA and DIO–AAG interactions. Circular dichroism spectral results revealed minimal conformational alterations in HSA upon DIO binding, but the effect on AAG was more significant. Competitive ligand displacement results suggested that DIO does not bind strongly to the main drug binding sites of HSA. However, DIO was found to favor binding to subdomain IB of HSA based on molecular docking data. For AAG, the binding location of DIO was determined for the two variants of AAG (A and F1*S variants) using molecular docking. For variant A, the binding site was situated in lobes I and II of the central binding pocket of the protein, while in variant F1*S, DIO was docked to a cleft on the protein surface. Hydrophobic interactions and van der Waals forces were mainly responsible for complexation between DIO and both proteins. Additionally, there was involvement of hydrogen bonding between DIO and both variants of AAG, but not between DIO and HSA. • AAG binds to DIO with slightly stronger affinity than HSA. • Hydrophobic forces are mainly responsible for DIO binding to both proteins. • DIO binds to HSA at subdomain IB. • DIO binds to variant F1*S of AAG at a cleft at the surface. • DIO binds to variant A of AAG at the central cavity.

Therapeutic Potential of Diosgenin and Its Major Derivatives against Neurological Diseases: Recent Advances.

Diosgenin (DG), a well-known steroidal sapogenin, is present abundantly in medicinal herbs such as Dioscorea rhizome, Dioscorea villosa, Trigonella foenum-graecum, Smilax China, and Rhizoma polgonati. DG is utilized as a major starting material for the production of steroidal drugs in the pharmaceutical industry. Due to its wide range of pharmacological activities and medicinal properties, it has been used in the treatment of cancers, hyperlipidemia, inflammation, and infections. Numerous studies have reported that DG is useful in the prevention and treatment of neurological diseases. Its therapeutic mechanisms are based on the mediation of different signaling pathways, and targeting these pathways might lead to the development of effective therapeutic agents for neurological diseases. The present review mainly summarizes recent progress using DG and its derivatives as therapeutic agents for multiple neurological disorders along with their various mechanisms in the central nervous system. In particular, those related to therapeutic efficacy for Parkinson's disease, Alzheimer's disease, brain injury, neuroinflammation, and ischemia are discussed. This review article also critically evaluates existing limitations associated with the solubility and bioavailability of DG and discusses imperatives for translational clinical research. It briefly recapitulates recent advances in structural modification and novel formulations to increase the therapeutic efficacy and brain levels of DG. In the present review, databases of PubMed, Web of Science, and Scopus were used for studies of DG and its derivatives in the treatment of central nervous system diseases published in English until December 10, 2019. Three independent researchers examined articles for eligibility. A total of 150 articles were screened from the above scientific literature databases. Finally, a total of 46 articles were extracted and included in this review. Keywords related to glioma, ischemia, memory, aging, cognitive impairment, Alzheimer, Parkinson, and neurodegenerative disorders were searched in the databases based on DG and its derivatives.

Study on steroidal saponins in crude and stir-fried Fructus Tribuli by ultra-high-performance liquid chromatography-mass spectrometry coupled with multivariate statistical analysis.

Fructus Tribuli is a traditional Chinese medicine used clinically for many years. Crude Fructus Tribuli and stir-fried Fructus Tribuli are recorded in the Pharmacopoeia of the People's Republic of China. However, the differences between steroidal saponins in crude Fructus Tribuli and stir-fried Fructus Tribuli have not been compared. In this study, ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry along with multivariate statistical analysis was developed to discriminate the chemical profiles and identify the steroidal saponins of crude Fructus Tribuli and stir-fried Fructus Tribuli. Additionally, an ultra-high-performance liquid chromatography triple-quadrupole mass spectrometer was used for the simultaneous quantification of nine major steroidal saponins to analyze the variations between crude Fructus Tribuli and stir-fried Fructus Tribuli. Finally, a total of 30 steroidal saponins whose structures or contents changed significantly after processing were found and identified. The mechanism of structural transformations deduced indicated that during the stir-frying of Fructus Tribuli, C-22 hydroxy furostanol saponins were converted to the corresponding furostanol saponins containing C-20-C-22 double bonds by dehydroxylation and deglycosylation reactions that occurred in the spirostanol saponins causing the generation of steroidal sapogenins. This study was successfully applied to the global analysis of crude Fructus Tribuli and stir-fried Fructus Tribuli. The results of this research will be beneficial to explore the processing mechanism of Fructus Tribuli.

Antioxidative enzymes activities and accumulation of steroids in hairy roots of Trigonella.

Steroidal sapogenins and phytosterols are a group of secondary metabolites which are very considerable in the pharmaceutical industry. Fenugreek (Trigonella foenum-graecum L.) is the good source of these compounds. In recent decades, there is a great interest to production of these compounds by cultivation of transformed roots. In present study, hairy roots induction in two Trigonella species (T. foenum-graeceum, T. monantha) with three strains of Agrobacterium rhizogenes (15,834, A4 and wt) was investigated. Transgenic status of roots was confirmed by PCR using rolB specific primers. Virulence of these strains was examined on explants of leaf, leaf cotyledone and hypocotyle in both species. The best strain was wt for hairy root induction in hypocotyle and leaf explants of T. foenum-graeceum and T. monantha. Significant quantitative differences were showed between shoot, root and hairy roots in both species. Protein content in root and hairy root of both species was significantly lower in comparison with shoot. Superoxide dismutase (SOD) and peroxidase (POX) activities in hairy roots of both species were higher as compared to other organs. The hairy roots of both species showed an ability to synthesize steroidal sapogenins. These results presented that hairy roots could be a suitable procedure for producing sapogenins compounds that have medicinal value in Trigonella.