Biotechnological production of ruscogenins in plant cell and organ cultures of Ruscus aculeatus.

Ruscus aculeatus is a threatened medicinal plant whose main bioactive components, the ruscogenins, have long been used in the treatment of hemorrhoids and varicose veins, but recently demonstrated activity against some types of cancer. Plant cell biofactories could constitute an alternative to the whole plant as a source of ruscogenins. In this pipeline, despite the in vitro recalcitrance of R. aculeatus, after many attempts we developed friable calli and derived plant cell suspensions, and their ruscogenin production was compared with that of organized in vitro plantlet and root-rhizome cultures. Root-rhizomes showed a higher capacity for biomass and ruscogenin production than the cell suspensions and the yields were greatly improved by elicitation with coronatine. Although ruscogenins accumulate in plants mainly in the root-rhizome, it was demonstrated that the aerial part could play an important role in their biosynthesis, as production was higher in the whole plant than in the root-rhizome cultures.

Biotransformation of Neoruscogenin by the Endophytic Fungus Alternaria eureka.

Biotransformation of neoruscogenin (NR, 1, spirosta-5,25(27)-diene-1ß,3ß-diol), the major bioactive sapogenin of Ruscus preparations, was carried out with the endophytic fungus Alternaria eureka. Fourteen new biotransformation products (2-15) were isolated, and their structures were elucidated by NMR and HRESIMS data analyses. A. eureka affected mainly oxygenation, oxidation, and epoxidation reactions on the B and C rings of the sapogenin to afford compounds 8-15. In addition to these, cleavage of the spiroketal system as in compounds 2-7 and subsequent transformations provided unusual metabolites. This is the first study reporting conversion of the spirostanol skeleton to cholestane-type metabolites 2-5. Additionally, the cleavage of the C-22/C-26 oxygen bridge yielding a furostanol-type steroidal framework and subsequent formation of the epoxy bridge between C-18 and C-22 in 7 was encountered for the first time in steroid chemistry.

Biotransformation of ruscogenins by Cunninghamella blakesleeana NRRL 1369 and neoruscogenin by endophytic fungus Neosartorya hiratsukae.

Biotransformation of steroidal ruscogenins (neoruscogenin and ruscogenin) was carried out with Cunninghamella blakesleeana NRRL 1369 and endophytic fungus Neosartorya hiratsukae yielding mainly P450 monooxygenase products together with a glycosylated compound. Fermentation of ruscogenins (75:25, neoruscogenin-ruscogenin mixture) with C. blakesleeana yielded 8 previously undescribed hydroxylated compounds. Furthermore, microbial transformation of neoruscogenin by endophytic fungus N. hiratsukae afforded three previously undescribed neoruscogenin derivatives. While hydroxylation at C-7, C-12, C-14, C-21 with further oxidation at C-1 and C-7 were observed with C. blakesleeana, N. hiratsukae biotransformation provided C-7 and C-12 hydroxylated compounds along with C-12 oxidized and C-1(O) glycosylated derivatives. The structures of the metabolites were elucidated by 1-D (1H, 13C and DEPT135) and 2-D NMR (COSY, HMBC, HMQC, NOESY, ROESY) as well as HR-MS analyses.

Investigation on the mechanisms for biotransformation of saponins to diosgenin.

In order to improve the efficiency of biotransformation of saponins in Dioscorea zingiberensis to diosgenin, a new enzymatic model was developed to investigate the mechanism of the metabolic systems. Four main saponin hydrolases (E1, E2, E3 and E4) were purified from Trichoderma reesei. Using progracillin as substrate, the enzymatic hydrolysis experiments with E1, E2, E3 and E4 were carried out respectively. Saponin concentrations during each biotransformation reaction were constructed with a kinetic model consisting of a few Michaelis-Menten equations. During biotransformation, C-26 glycoside and C-3 terminal glycoside were cleaved sequentially from saponins by E1, E2, E3 and E4. Then C-3 terminal rhamnoside and C-3 glycoside were released from the aglycone stepwisely by E2 and E3, to yield diosgenin. E2 and E3 were the key enzymes in the system, and cleavage of the C-3 glycoside from saponins was the rate-limiting step in the biotransformation process. The proposed enzymatic model might be used to analyze the mechanism for biotransformation of saponins to diosgenin.

Reversal of dopamine neurons and locomotor ability degeneration in aged rats with smilagenin.

The purpose of this paper is to study the effect of smilagenin (SMI) (PYM50028), a sapogenin compound originally identified from Chinese medicinal herb, on dopamine neurons and locomotor ability in aged rats. Experiments were carried out on young and aged Sprague-Dawley rats, which were daily administered with either SMI (18mg/kg/day) or vehicle (0.5% sodium carboxymethycellulose [CMCNa]). Open-field and rotarod performance tests revealed that behavioral ability was impaired in aged rats and was improved by oral administration of smilagenin. Immunohistochemical data showed that tyrosine hydroxylase (TH)-positive neuron numbers in the substantia nigra pars compacta (unbiased stereological counting) were altered with aging and were increased by smilagenin treatment. Likewise, the dopamine receptor density and the striatal dopamine transporter (DAT) density ((125)I-2ß-carbomethoxy-3ß-(4-iodophenyl)-N-(3-fluoropropyl) nortropane [(125)I-FP-CIT] autoradiography) were significantly lowered in aged rats as compared to young rats, and treatment with smilagenin significantly elevated the dopamine receptor and DAT density in aged rats. Furthermore, smilagenin enhances glial cell-derived neurotrophic factor (GDNF) release both in the striatum and midbrain. These results indicate a possible role of smilagenin in the treatment of age-related extrapyramidal disorders.

A novel ß-glucosidase from Aspergillus fumigates releases diosgenin from spirostanosides of Dioscorea zingiberensis C. H. Wright (DZW).

A ß-glucosidase effectively releasing diosgenin from spirostanosides of Dioscorea zingiberensis C. H. Wright (DZW), named AfG, was purified from a strain of Aspergillus fumigates. The molecular weight of AfG was 113 kDa. Analysis of protein fragments by ESI-Q-TOF indicated that AfG was a ß-glucosidase. The circular dichroism spectrum suggested that the main secondary structure of AfG in Milli-Q water was α-helixes. Atomic force microscopy revealed that it was a globular protein. AfG maintained high activity from pH 3.6 to 5.0 and from 50 to 90°C. With the strong heat stability, AfG retained 55% of its original activity at 65°C for 120 h. AfG utilized muti-3-O-glycosides of various steroidal saponins from DZW as substrate, such as trillin, diosgenin diglucoside, dioscin, deltonin and gracillin, to yield diosgenin, suggesting the possibility of producing diosgenin from total saponins of DZW using a single enzyme.

One unique steroidal sapogenin obtained through the microbial transformation of ruscogenin by Phytophthora cactorum ATCC 32134 and its potential inhibitory effect on tissue factor (TF) procoagulant activity.

With the aim to obtain more effective tissue factor (TF) inhibitors, the microbial transformation of three steroidal sapogenins, ruscogenin (1), diosgenin (2) and sarsasapogenin (3), was carried out and only ruscogenin was selectivity converted to 1-hydroxy-spirost-4-en-3-one (4) by Phytophthora cactorum ATCC 32134. The in vitro anti-TF procoagulant activity of this metabolite was enhanced almost 10 times to an IC(50) value of 0.29 microM. The chemical assignments of compound 4 were made unambiguously using ESI-MS, IR and 2D NMR spectroscopy.

Inhibitory effects of steroidal timosaponins isolated from the rhizomes of Anemarrhena asphodeloides against passive cutaneous anaphylaxis reaction and pruritus.

To investigate the antiallergic effect of the rhizome of Anemarrhena asphodeloides (AA, family Liliaceae), which was found to inhibit the mouse passive cutaneous anaphylaxis (PCA) reaction induced by the antigen-immunoglobulin E (IgE) complex in preliminary experiments, main steroidal saponins, timosaponins AIII, BIII, and D, were isolated and their inhibitory effects against PCA reaction and scratching behaviors investigated in mice. Oral administration of three main steroidal sapogenins blocked the PCA reaction and scratching behaviors, timosaponin AIII was the most potent. However, intraperitoneal administration of timosaponin AIII showed weak inhibition. To understand its metabolism and antiallergic mechanism, timosaponin AIII was anaerobically incubated with human intestinal microflora to afford a main metabolite, sarsasapogenin. Intraperitoneal administration of sarsasapogenin inhibited allergic reaction more potently than timosaponin AIII. In addition, sarsasapogenin more potently inhibited degranulation and IL-4 protein expression of RBL-2H3 cells induced by IgE-antigen complex than timosaponin AIII. On the basis of these findings, antiallergic effect of AA may be due to those of its steroidal constituents, and that of timosaponin AIII may be activated by using intestinal microflora.

Microbial conversion of ruscogenin by Gliocladium deliquescens NRRL1086: glycosylation at C-1.

The glycosylation of ruscogenin (1) by Gliocladium deliquescens NRRL 1086 was observed and gave a regioselectively glycosylated product identified as ruscogenin 1-O-beta-D-glucopyranoside (2) by infrared, mass spectrometry, and nuclear magnetic resonance spectra. Time-course studies indicated that it appeared to be favorable to accumulate 2 when ruscogenin was added to the 24-h-old stage II culture, and the yield of 2 was about 20.1% during 120 approximately 168 h. It was noted that additional carbohydrates could significantly increase glycoside formation and the yield of 2 even reached as high as 68% compared with the control 20.1%. The primary investigation about the characteristics of the enzyme resulted that the reaction was blocked by beta-glycosidase inhibitor imidazole, however, was enhanced remarkably by glycosyltransferase inhibitor sodium dodecyl sulfate. To our knowledge, this is the first reported case of producing steroidal saponin by microbial transformation, and G. deliquescens NRRL1086 would be a practical and highly efficient tool in producing natural ruscogenin monoside.

[Studies on accumulation of active ingredients, N, P and K in Anemarrhena asphodeloides].

OBJECTIVE: To study the accumulation of active ingredients, the absorption and transformation of N, P and K in Anemarrhena asphodeloides and provide basis for determination of the harvest time and fertilizing. METHOD: Samples were collected in different phrases and the weight of dry matter, the content of N, P and K of different organs and the content of sarsasapogenin were determined. RESULT: Absorption of N, P and K started by the root and rhizoma after July. At the end of August, the N and K of the aerial part transfered largely into rhizome. The content of sarsasapogenin in rhizome was the highest in early spring. CONCLUSION: Additional fertilizer is helpful to increase the yield in July of the second year after the transplantation. The quality is the best when harvest in early spring.

Evidence for essential histidine and dicarboxylic amino-acid residues in the active site of UDP-glucose : solasodine glucosyltransferase from eggplant leaves.

Effects of several chemical probes selectively modifying various amino-acid residues on the activity of UDP-glucose : solasodine glucosyltransferase from eggplant leaves was studied. It was shown that diethylpyrocarbonate (DEPC), a specific modifier of histidine residues, was strongly inhibitory. However, in the presence of excessive amounts of the enzyme substrates, i.e. either UDP-glucose or solasodine, the inhibitory effect of DEPC was much weaker indicating that histidine (or histidines) are present in the active site of the enzyme. Our results suggest also that unmodified residues of glutamic (or aspartic) acid, lysine, cysteine, tyrosine and tryptophan are necessary for full activity of the enzyme. Reagents modifying serine and arginine residues have no effect on the enzyme activity.

Ovine metabolism of lithogenic sapogenins. Synthesis of [2,2,4,4-(2)h(4)]sarsasapogenone, [2,2,4,4-(2)h(4)]sarsasapogenin, and [2,2,4,4-(2)h(4)]episarsasapogenin and evaluation of deuterium retention in a sheep-dosing trial.

The suitability of [2,2,4,4-(2)H(4)]sarsasapogenone (1b), [2,2,4,4-(2)H(4)]sarsasapogenin (2b), and [2,2,4,4-(2)H(4)]episarsasapogenin (3b) as isotopically labeled dosing substrates to determine the levels of free and conjugated sapogenins present in feces from sheep grazing saponin-containing plants implicated in the development of ovine heptagenous photosentization diseases was investigated. A 1:4 mixture of [2,2,4,4-(2)H(4)]sarsasapogenin (2b) and [2,2,4,4-(2)H(4)]episarsasapogenin (3b), obtained by reduction of [2,2,4,4-(2)H(4)]sarsasapogenone (1b), was found to retain 94% of incorporated deuterium, when dosed to one sheep. The recovery of the dosed mixture of genins 2b and 3b was calculated to be 85%. Considerable loss of deuterium and a lower recovery of genin material were observed when [2,2,4,4-(2)H(4)]sarsasapogenone (1b) was dosed.

Phospholipids modulate the substrate specificity of soluble UDP-glucose:steroid glucosyltransferase from eggplant leaves.

UDP-glucose-dependent glucosylation of solasodine and diosgenin by a soluble, partially purified enzyme fraction from eggplant leaves is affected in a markedly different way by some phospholipids. While glucosylation of diosgenin and some closely related spirostanols, e.g. tigogenin or yamogenin, is strongly inhibited by relatively low concentrations of several phospholipids, the glucosylation of solasodine is unaffected or even slightly stimulated. These effects depend both on the structure of the polar head group and the nature of the acyl chains present in the phospholipid. The most potent inhibitors of diosgenin glucosylation are choline-containing lipids: phosphatidylcholine (PC) and sphingomyelin (SM) but the removal of phosphocholine moiety from these phospholipids by treatment with phospholipase C results in an almost complete recovery of the diosgenin glucoside formation by the enzyme. Significant inhibition of diosgenin glucoside synthesis and stimulation of solasodine glucosylation was found only with PC molecular species containing fatty acids with chain length of 12-18 carbon atoms. PC with shorter or longer acyl chains had little effect on glucosylation of either diosgenin or solasodine. Our results indicate that interaction between the investigated glucosyltransferase and lipids are quite specific and suggest that modulation of the enzyme activity by the nature of the lipid environment may be of importance for regulation of in vivo synthesis of steroidal saponins and glycoalkaloids in eggplant.

Ovine metabolism of saponins: evaluation of a method for estimating the ovine uptake of steroidal saponins from Narthecium ossifragum.

A sheep was dosed three times per day over six consecutive days with 70 g Narthecium ossifragum, and once on the seventh day with 70 g N. ossifragum. Additionally, it was dosed once on days 1-7 with 20 mg of [20,23,23-2H3]sarsasapogenin. After 7 days, the sheep was killed and GC-MS analysis of the free and conjugated sapogenin content in bile, urine, rumen, duodenum, jejunum, colon and rectum samples collected from the sheep, faecal samples collected on days 4-7, and dosed plant material was performed. The N. ossifragum contained mainly sarsasapogenin and smilagenin. Only neglible levels of deuterium-labelled sarsasapogenins were detected in the samples from the animal. Ingested saponins were quickly hydrolysed in the rumen to free sapogenins and, in part, epimerized at C-3 to afford episapogenins. The absorption of free sapogenins appeared to occur in the jejunum. The concentration of sapogenins in faeces reached a plateau 108 h after dosing started.

Constituents and the antitumor principle of Allium victorialis var. platyphyllum.

To search for cytotoxic components from Allium victorialis, MTT assays on each extract and an isolated component, gitogenin 3-O-lycotetroside, were performed against cancer cell lines. Cytotoxicities of most extract were shown to be comparatively weak, though IC50 values of CHCl3 fraction was found to be <31.3-368.4 microg/ml. From the incubated methanol extract at 36 degrees C, eleven kinds of organosulfuric flavours were predictable by GC-MS performance. The most abundant peak was revealed to be 2-vinyl-4H-1,3-dithiin (1) by its mass spectrum. Further, this extract showed significant cytotoxicities toward cancer cell lies. Silica gel column chromatography of the n-butanol fraction led to the isolation of gitogenin 3-O-lycotetroside (3) along with astragalin (4) and kaempferol 3, 4'-di-O-beta-D-glucoside (5). This steroidal saponin exhibited significant cytotoxic activities (IC50, 6.51-36.5 microg/ml) over several cancer cell lines. When compound 3 was incubated for 24 h with human intestinal bacteria, a major metabolite was produced and then isolated by silica gel column chromatography. By examining parent- and prominent ion peak in FAB-MS spectrum of the metabolite, the structure was speculated not to be any of prosapogenins of 3, suggesting that spiroketal ring were labile to the bacterial reaction. These suggest that disulfides produced secondarily are the antitumor principles.

Microbial transformation of dihydrosarsasapogenin with Mycobacterium sp.

Microbial transformation of sarsasapogenin (1) with Mycobacterium sp. (NRRL B-3805) gave 25(S)-neospirost-4-en-3-one (2) as the sole product in 62% yield. Incubation of dihydrosarsasapogenin (3) led to the isolation of seven products in 0.5 (4), 6.6 (5), 5 (6), 16 (7), 1 (8), 1 (9), and 4.5% (10) yields, respectively, while 15% of 3 was recovered. Among these products, 8 and 9 were C22 steroids, and 10 was a C19 steroid. Isolation of these C19 and C22 steroids indicated that this microorganism is capable of cleaving the ether linkage between C-16 and C-22 in 3. In addition, 12 alpha-hydroxylation was also observed in all these three metabolites.

Molecular cloning and bacterial expression of a cDNA encoding furostanol glycoside 26-O-beta-glucosidase of Costus speciosus.

Furostanol glycoside 26-O-beta-glucosidase (F26G) purified from Costus speciosus rhizomes was digested with endoproteinase, and several internal peptide fragments were obtained. Degenerate oligonucleotide primers based on amino acid sequences of the peptides were used for amplification of F26G cDNA fragments by applying nested polymerase chain reactions to cDNAs from in vitro cultured plantlets of C. speciosus. Using primers based on sequences of the cDNA fragments, the 5'- and 3'-end clones were isolated by rapid amplification of cDNA ends (RACE) methods. Finally, the entire coding portion of F26G cDNA was cloned by using primers designed from sequences of the RACE products. The deduced amino acid sequence of CSF26G1, the protein encoded by the cloned cDNA, consists of 562 amino acids and shows high homology to a widely distributed family of beta-glucosidases (BGA family). Cell-free homogenate of Escherichia coli expressing CSF26G1 cDNA showed beta-glucosidase activity specific for cleavage of the C-26 glucosidic bond of furostanol glycosides.

Epi-sarsasapogenin and epi-smilagenin: two sapogenins isolated from the rumen content of sheep intoxicated by Brachiaria decumbens.

Spectroscopic examination of purified extracts of the rumen content of sheep intoxicated by Brachiaria decumbens revealed the presence of two spirostanes, identified as epi-sarsasapogenin and epi-smilagenin. Sarsasapogenone was obtained by the oxidation of sarsasapogenin. The reduction of sarsasapogenone using lithium aluminum hydride yielded isomeric products, sarsasapogenin (20%) and epi-sarsasapogenin (80%).