Methyl Protodioscin Research Articles

Methyl Protodioscin from the Roots of Asparagus cochinchinensis Attenuates Airway Inflammation by Inhibiting Cytokine Production.

The present study was designed to find pharmacologically active compound against airway inflammation from the roots of Asparagus cochinchinensis. The 70% ethanol extract of the roots of A. cochinchinensis (ACE) was found to inhibit IL-6 production from IL-1β-treated lung epithelial cells (A549) and the major constituent, methyl protodioscin (MP), also strongly inhibited the production of IL-6, IL-8, and tumor necrosis factor- (TNF-) α from A549 cells at 10–100 μM. This downregulating effect of proinflammatory cytokine production was found to be mediated, at least in part, via inhibition of c-Jun N-terminal kinase (JNK) and c-Jun activation pathway. When examined on an in vivo model of airway inflammation in mice, lipopolysaccharide- (LPS-) induced acute lung injury, ACE, and MP significantly inhibited cell infiltration in the bronchoalveolar lavage fluid by the oral treatment at doses of 100–400 mg/kg and 30–60 mg/kg, respectively. MP also inhibited the production of proinflammatory cytokines such as IL-6, TNF-α, and IL-1β in lung tissue. All of these findings provide scientific evidence supporting the role of A. cochinchinensis as a herbal remedy in treating airway inflammation and also suggest a therapeutic value of MP on airway inflammatory disorders.

Methyl protodioscin induces G2/M cell cycle arrest and apoptosis in A549 human lung cancer cells.

Background:Methyl protodioscin (MPD) is a furostanol bisglycoside with antitumor properties. It has been shown to reduce proliferation, cause cell cycle arrest.Objective:The present study elucidates the mechanism underlying MPD's apoptotic effects, using the A549 human lung cancer cell line.Materials and Methods:The human pulmonary adenocarcinoma cell line A549 was obtained from the Cell Bank of the Animal Experiment Center, North School Region, Sun Yat-Sen University. All of the cells were grown in RPMI 1640 supplemented with 10% fetal calf serum (Hyclone, Logan, UT, USA), penicillin (10,000 U/l), and streptomycin (100 mg/l) at 37°C in a 5% CO2 humidified atmosphere. The induction of apoptosis was observed in flow cytometry and fluorescent staining experiments.Results:MPD showed growth inhibitory effects in A549 cells in a dose- and time-dependent manner. The significant G2/M cell cycle arrest and apoptotic effect were also seen in A549 cells treated with MPD. MPD-induced apoptosis was accompanied by a significant reduction of mitochondrial membrane potential, release of mitochondrial cytochrome c to cytosol, activation of caspase-3, downregulation of Bcl-2, p-Bad, and upregulation of Bax.Conclusion:Our results show that the induction of apoptosis by MPD involves multiple molecular pathways and strongly suggest that Bcl-2 family proteins signaling pathways. In addition, mitochondrial membrane potential, mitochondrial cytochrome c and caspase-3 were also closely associated with MPD-induced apoptotic process in human A549 cells.

Two Spirostan Steroid Glycoside Fatty Esters from Dioscorea cayenensis

Two new fatty acid-spirostan steroid glycoside esters, progenin III palmitate (1) and progenin III linoleate (2), were isolated from the MeOH extract of Dioscorea cayenensis rhizomes. The extract also yielded seven previously known spirostan and furostan steroid glycosides (3-9). The structures of the new compounds were established as (25R)-spirost-5-en-3beta-yl O-alpha-L-rhamnopyranosyl-(1 --> 2)-[6-O-palmitoyl]-O-beta-D-glucopyranoside (1) and (25R)-spirost-5-en-3beta-yl O-alpha-L-rhamnopyranosyl-(1 --> 2)-[6-O-linoleoyl]-O-beta-D-glucopyranoside (2) by chemical and spectroscopic methods, including 1D and 2D NMR. The known compounds were identified as progenin III (3), dioscin (4), deltonin (5), asperin (6), gracillin (7), protodioscin (8)], and methyl protodioscin (9).

ChemInform Abstract: An Improved Synthesis of Methyl Protodioscin. Part 2. A Direct E‐Ring Opening by BF3—Et2O/Ac2O from Dioscin Ester.

AbstractThe direct E‐ring opening in diosgenin‐derived glycoside derivative (I) by the system acetic anhydride/boron trifluoride etherate is studied.

An Improved Synthesis of Methyl Protodioscin. II. A Direct E-ring Opening by BF3–Et2O/Ac2O from Dioscin Ester

Abstract A direct E-ring opening of dioscin ester by BF3–Et2O/Ac2O was studied, and methyl protodioscin was synthesized from dioscin ester in three steps with a total yield of 44%. The details of the E-ring opening reaction was discussed, and a prominent stability of C16–OAc was obversed.

Pregnane glycosides and steroid saponins from Smilax bockii Warb. and their NGF-potentiating activity

The chemical constituents of the roots of Smilax bockii Warb. were investigated and two pregnane glycosides and three steroid saponins were isolated. Their structures were established as 3β-hydroxypregna-5,16-dien-20-one-3-O-α-L-rhamnopyranosyl(1 → 4)-α-L-rhamnopyranosyl(1 → 4)-[α-L-rhamnopyranosyl(1 → 2)]-β-D-glucopyranoside (1), 3β-hydroxypregna-5,16-dien-20-one-3-O-α-L-rhamnopyranosyl(1 → 2)-[α-L-rhamnopyranosyl(1 → 4)]-β-D-glucopyranoside (2), dioscin (3), methyl protodioscin (4), 26-O-β-D-glucopyranosyl-22α-methoxyl-(25R)-furost-5-en-3β, 26-diol 3-O-α-L-rhamnopyranosyl(1 → 4)-α-L-rhamnopyranosyl(1 → 4)-[α-L-rhamnopyranosyl(1 → 2)]-β-D-glucopyranoside (5) on the basis of spectral analysis and chemical methods. Compound 1 is a new natural product and compounds 2, 5 were first isolated from the genus Smilax. Compound 5 showed enhancing activity of nerve growth factor (NGF)-induced neurite outgrowth in PC 12D cells.

An Improved Synthesis of Methyl Protodioscin: Tautomerization and Direct Access to the 3-O-Substituted Kryptogenin

Abstract The acid-catalyzed tautomerization of 3-O-substituted kryptogenin 2 was studied, and the effects of acids such as silica gel, TMSOTf, acetic acid, and CDCl3, are discussed. Additionally, a Zn/KI/HOAc reduction based on this tautomerization was adopted, which afforded 2 directly, and provided a facile procedure for the synthesis of methyl protodioscin and other furostanol saponins in a mild way.

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the quantification of methyl protodioscin in rat plasma: application to a pharmacokinetic study

A high performance liquid chromatography/tandem mass spectrometry assay was first developed and validated for the quantification of methyl protodioscin (MPD), a natural furostanol saponin with distinct antitumor activity, in rat plasma with 17alpha-ethinylestradiol as internal standard (IS). Methanol-mediated protein precipitation was employed for plasma sample pretreatment. The separation was achieved on a C(18) column (150 x 4.6 mm, i.d., 5 microm) by isocratic elution with methanol-water (72:28, v/v) as mobile phase at a flow rate of 1.0 mL/min. Ion acquisition was performed in selective reaction monitoring positive mode by monitoring the transition of m/z 1085.7 --> 1053.7 for MPD, and in selective ion monitoring negative mode by monitoring the deprotonated ion m/z 295.5 for IS. The assay was linear over the concentration range of 2.024-270.0 microg/mL with 2.024 microg/mL as the lower limit of quantification. It was specific, accurate, precise and reproducible with intra- and inter-run RSD <8.3% and RE between -11.5 and 12.8%. The assay was successfully applied to a preclinical pharmacokinetic study after an intravenous dose of 40 mg/kg MPD to rats.

Characterization of steroidal saponins in crude extract from Dioscorea nipponica Makino by liquid chromatography tandem multi-stage mass spectrometry

The liquid chromatography–electrospray ionization-tandem multi-stage mass spectrometry (LC–ESI-MS n ) method was developed for the analyses and characterization of steroidal saponins in plant extract from the rhizome of Dioscorea nipponica Makino. The HPLC experiments were performed by means of a reversed-phase C 18 column and a binary mobile phase system consisting of water and acetonitrile under gradient elution conditions. Pseudoprotodioscin, methyl protodioscin and dioscin were identified by comparing the retention times, UV spectra and the fragmentation properties of [ M − H] − ions with the authentic standards. Four groups of steroidal saponin isomers possessed the [ M − H] − ions at m/ z 1063, 1045, 901 and 1047, respectively, were observed during the LC–ESI(−)-MS analysis, and three groups of them except the pair of isomers with the [ M − H] − ions at m/ z 1047 could be differentiated by LC–ESI(−)-MS 3. Furthermore, the ESI-MS n fragmentation behaviors of the [ M + Li] + ions of pseudoprotodioscin and methyl protodioscin have been investigated, and the observed information helped the structural elucidation of the more abundant isomer with the [ M − H] − ion at m/ z 1047. As the result, a special sugar sequence of the saccharide chains was observed that not glucose but rhamnose might be connected with the hydroxyl group at C-3 position of the steroidal aglycone.

Structural identification of methyl protodioscin metabolites in rats’ urine and their antiproliferative activities against human tumor cell lines

Methyl protodioscin (MPD), a furostanol saponin, is a preclinical drug shown potent antiproliferative activities against most cell lines from leukemia and solid tumors. The metabolites of MPD in rats’ urine after single oral doses of 80 mg/kg were investigated in this research. Ten metabolites were isolated and purified by liquid–liquid extraction, open-column chromatography, medium-pressure liquid chromatography, and preparative high-performance liquid chromatography. The structural identification of the metabolites was carried out by high resolution mass spectra, NMR spectroscopic methods including 1H NMR, 13C NMR and 2D NMR, as well as chemical ways. The 10 metabolites were elucidated to be dioscin (M-1), pregna-5,16-dien-3β-ol-20-one- O-α- l-rhamnopyranosyl-(1 → 2)-[α- l-rhamnopyranosyl-(1 → 4)]-β- d-glucopyranoside (M-2), diosgenin (M-3), protobioside (M-4), methyl protobioside (M-5), 26- O-β- d-glucopyrannosyl(25 R)-furan-5-ene-3β, 22α, 26-trihydroxy-3- O-α- l-rhamnopyranosyl-(1 → 4)-β- d-glucopyranoside(M-6),26- O-β- d-glucopyranosyl(25 R)-furan-5-ene-3β,26-dihydroxy-22-methoxy-3- O-α- l-rhamnopyranosyl-(1 → 4)-β- d-glucopyranoside (M-7), prosapogenin A of dioscin (M-8), prosapogenin B of dioscin (M-9), and diosgenin-3- O-β- d-glucopyranoside (M-10), respectively. M-1 was the main urinary metabolite of MPD in rats. Some metabolites showed potent antiproliferative activities against HepG2, NCI-H460, MCF-7 and HeLa cell lines in vitro.

Facile Conversion of Spirostan Saponin into Furostan Saponin: Synthesis of Methyl Protodioscin and Its 26-Thio-analogue

[reaction: see text] A facile approach for the conversion of a spirostan saponin into its furostan counterpart, as illustrated by the transformation of dioscin to methyl protodioscin (and its 26-thio-analogue), has been developed.

Microbial metabolism of methyl protodioscin by Aspergillus niger culture—A new androstenedione producing way from steroid

Methyl protodioscin ( 1), a natural furostanol biglycoside steroid, was a preclinical anticancer drug, which showed potent activity against most cell lines from leukemia and solid tumors in the National Cancer Institute's (NCI) human cancer panel. Metabolism of methyl protodioscin by Aspergillus niger was investigated. Seven metabolites were isolated and identified. Two main metabolites were pregnane glycosides and four were furostanol glycosides, together with the aglycone. It was found that steroidal saponin skeleton could be converted to pregnenolone skeleton only using microbial methods, which must have chemical procedures in the reported literatures. The proposed biosynthetic pathways of the microbial conversion products of methyl protodioscin were drawn. The found enriched the reaction types of microbial bioconversion and provided a new producing way of androstenedione from steroid. Most metabolites showed strong cytotoxic activities against HepG2, NCI-H460, HeLa, and MCF-7 cell lines.

Methyl protodioscin induces G2/M cell cycle arrest and apoptosis in HepG2 liver cancer cells

Methyl protodioscin (NSC-698790) is one of the main bioactive components in the traditional Chinese medicine Dioscorea collettii var. hypoglauca (Dioscoreaceae). In this study, we investigated the anti-proliferative effect of methyl protodioscin on the HepG2 cells and the mechanism of the induced cytotoxicity. Treatment of methyl protodioscin resulted in G2/M arrest and apoptosis in HepG2 cells. These effects were attributed to down-regulation of Cyclin B1 and the signaling pathways leading to up-regulation of Bax and down-regulation of BCL2, suggesting that methyl protodioscin may be a novel anti-mitotic agent.

Bioconversion of methyl protodioscin by Penicillium melinii cells

Bioconversion of methyl protodioscin (1) by Penicillium melinii was investigated. Seven bioconversion products were isolated and identified. Three of them were glycosidation products and were new compounds. Their structures were identified to be 3- O-[α- l-rhamnopyranosyl-(1 → 2)-{α- l-rhamnopyranosyl-(1 → 4)}-β- d-glucopyranosyl]-26- O-[β- d-glucopyranosyl-(1 → 6)-β- d-glucopyranosyl]-25( R)-furost-5,20(22)-diene-3β,26-triol (2), 3- O-[α- l-rhamnopyranosyl-(1 → 2)-{α- l-rhamnopyranosyl-(1 → 4)}-β- d-glucopyranosyl]-26- O-[β- d-glucopyranosyl-(1 → 6)-β- d-glucopyranosyl]-25( R)-furost-5-ene-3β,26-triol (3), 16β-[4′-methyl-5′- O-(β- d-glucopyranosyl-(1 → 6)-β- d-glucopyranosyl)-pentanoxyl]-pregn-5-en-3β-ol-20-one- O-α- l-rhamnopyranosyl-(1 → 2)-[α- l-rhamnopyranosyl-(1 → 4)]-β- d-glucopyranoside (4), respectively. To the best knowledge we known, this was the first time to find the type of furostanol saponins that had disugar at C-26. The proposed biosynthetic pathways of methyl protodioscin were drawn. Most bioconversion products showed considerable cytotoxic activities against HepG2, NCI-H460, MCF-7 and HeLa cell lines compared to methyl protodioscin.

Microbial transformation of methyl protodioscin by Cunninghamella elegans

Biotransformation of methyl protodioscin ( 1) by Cunninghamella elegans (AS 3.1207) was investigated. Nine bioconversion products were isolated and identified. Eight of the bioconversion products were pregnane glycoside or steroidal saponins. It was found that steroidal saponin skeleton could be converted to pregnenolone skeleton only using microbial methods, which must have chemical procedures in the reported literatures. The found enriched the types of bioconversion reaction and provided a new way for the production of androstenedione. Most bioconversion products showed considerable cytotoxic activities against HepG2, NCI-H460, MCF-7 and HeLa cell lines compared to methyl protodioscin.

Methyl protodioscin induces G 2/M arrest and apoptosis in K562 cells with the hyperpolarization of mitochondria

Methyl protodioscin is a furostanol bisglycoside with antitumor properties. The present study investigated its effects on human chronic myelogenous leukemia K562 cells. Cell cycle analysis showed that methyl protodioscin caused distinct G 2/M arrest, with the appearance of polyploidy population. The levels of cyclin B1 decreased, whereas Cdc2 kept at a steady level. Subsequent apoptosis after G 2/M blockage was demonstrated through DNA fragmentation and the annexin V staining assay. Methyl protodioscin induced a biphasic alteration (i.e. an early hyperpolarization, followed by depolarization) in mitochondrial membrane potential of K562 cells. The transient decline of intracellular Ca 2+ concentration was observed at early stage. The generation of reactive oxygen species was also detected. The anti-apoptotic Bcl-x L transiently increased and then decreased. And the pro-apoptotic Bax was markedly up-regulated. Taken together, these data demonstrated that methyl protodioscin inhibits K562 cell proliferation via G 2/M arrest and apoptosis, with mitochondrial hyperpolarization and the disruption of Ca 2+ homeostasis playing important roles.

A New Steroidal Saponin from Dioscorea cayenensis

The new 26-O-beta-D-glucopyranosyl-3beta,26-dihydroxy-20,22-seco-25(R)-furost-5-en-20,22-dione-3-O-alpha-L-rhamnopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->4)-[alpha-L-rhamnopyranosyl-(1-->2)]-beta-D-glucopyranoside (1), along with the known methyl protodioscin (2), asperoside (3) and prosapogenin A of dioscin (4) were isolated from the rhizomes of Dioscorea cayenensis LAM.-HOLL (Dioscoreaceae). Their structures were established mainly on the basis of 600 MHz 2D-NMR spectral data. 4 exhibited antifungal activity against the human pathogenic yeasts Candida albicans, C. glabrata and C. tropicalis (MICs of 20.8, 6.25, 25 microg/ml, respectively), whereas saponins 1-3 were inactive.

Isolation and identification of steroidal saponins in Taiwanese yam cultivar (Dioscorea pseudojaponica Yamamoto).

A new furostanol pentaoligoside and spirostanol tetraoligoside were isolated for the first time from yam tubers (Dioscorea pseudojaponica Yamamoto) from Taiwan, together with four known yam saponins, methyl protodioscin, methyl protogracillin, dioscin, and gracillin. Their structures were characterized as 26-O-beta-D-glucopyranosyl-22alpha-methoxyl-(25R)-furost-5-en-3beta,26-diol, 3-O-alpha-L-rhamnopyranosyl-(1-->2)-O-([alpha-L-rhamnopyranosyl-(1-->4)]-O-[alpha-L-rhamnopyranosyl-(1-->4)])-beta-D-glucopyranoside, and (25R)-spirost-5-en-3beta-ol 3-O-alpha-L-rhamnopyranosyl-(1-->2)-O-([alpha-L-rhamnopyranosyl-(1-->4)]-O-[alpha-L-rhamnopyranosyl-(1-->4)])-beta-D-glucopyranoside. The structural identification was performed using LC-MS and 1H and 13C NMR. The methanol extract of yam tubers was fractionated by XAD-2 column chromatography using a methanol/water gradient elution system to yield furostanol and spirostanol glycoside fractions. Preparative high-performance liquid chromatography, employing a C18 column and a mobile phase of methanol/water (69:31, v/v), was used to separate each furostanol glycoside, whereas a mobile phase of methanol/water (79:21, v/v) was used to resolve the individual spirostanol glycosides. The conversions from steroid saponins to diosgenin after acid hydrolysis were around 68 and 90% for furostanol and spirostanol glycosides, respectively.

Total Synthesis of Methyl Protodioscin: A Potent Agent with Antitumor Activity.

Methyl protodioscin (1), otherwise known as 3-O-[α-l-rhamnopyranosyl-(1→2)-{α-l-rhamnopyranosyl-(1→4)}-β-d-glucopyranosyl]-26-O-[β-d-glucopyranosyl]-22-methoxy-25(R)-furost-5-ene-3β,26-diol, has been synthesized for the first time from diosgenin through nine steps in an overall yield of 7.8%.

Total synthesis of methyl protodioscin: a potent agent with antitumor activity.

Methyl protodioscin (1), otherwise known as 3-O-[alpha-L-rhamnopyranosyl-(1-->2)-[alpha-L-rhamnopyranosyl-(1-->4)]-beta-d-glucopyranosyl]-26-O-[beta-D-glucopyranosyl]-22-methoxy-25(R)-furost-5-ene-3 beta,26-diol, has been synthesized for the first time from diosgenin through nine steps in an overall yield of 7.8%.