Atropurosides A-G, new steroidal saponins from Smilacina atropurpurea

Atropurosides A-G (1-7), seven new steroidal saponins, which possess new polyhydroxylated aglycones, were isolated from the rhizomes of Smilacina atropurpurea (Convallariaceae), together with a known saponin, dioscin (8). Their structures were elucidated on the basis of detailed spectroscopic analysis, including 1D and 2D NMR techniques and chemical methods. Antifungal testing of the eight compounds indicated that atropurosides B (2) and F (6) were fungicidal against Candida albicans, Candida glabrata, Cryptococcus neoformans, and Aspergillus fumigatus with minimum fungicidal concentrations (MFCs) < or = 20 microg/ml, while dioscin (8) was selectively active against C. albicans and C. glabrata (MFC < or = 5.0 microg/ml). Furthermore, the antifungal saponins 2, 6, and 8 were evaluated for their in vitro cytotoxicities in a panel of human cancer cell lines (SK-MEL, KB, BT-549, SK-OV-3, and HepG2) and non-cancerous Vero cells. All showed moderate cytotoxicities. It appears that the antifungal activity of these steroidal saponins correlates with their cytotoxicity against mammalian cells.     

A novel insulin-releasing substance, phanoside, from the plant Gynostemma pentaphyllum

Extracts from Gynostemma pentaphyllum Makino (Cucurbitaceae), a Southeast Asian herb, has been reported to affect numerous activities resulting in antitumor, cholesterol-lowering, immunopotentiating, antioxidant, and hypoglycemic effects. We have isolated one active compound by ethanol extraction, distribution in n-butyl alcohol/water, solid phase extraction/separation, and several rounds of reverse phase high pressure liquid chromatography. We have shown by NMR and mass spectrometry that this active compound is a novel saponin, a gypenoside, which we have named phanoside (21-,23-epoxy-,3beta-,20-,21-trihydroxydammar-24-ene-3-O-([alpha-d-rhamnopyranosyl(1-->2)]-[beta-d-glycopyranosyl(1-->3)]-beta-d-lyxopyranoside)), with a molecular mass of 914.5 Da. Phanoside is a dammarane-type saponin, and four stereoisomers differing in configurations at positions 21 and 23 were identified, each of which were found to stimulate insulin release from isolated rat pancreatic islets. We have also found that the stereoisomers are interconvertible. Dose-dependent insulin-releasing activities at 3.3 and 16.7 mM glucose levels were determined for the racemic mixture containing all four stereoisomers. Phanoside at 500 microM stimulates insulin release in vitro 10-fold at 3.3 mM glucose and potentiates the release almost 4-fold at 16.7 mM glucose. At these glucose levels, 2 microm glibenclamide stimulates insulin release only 2-fold. Interestingly, beta-cell sensitivity to phanoside is higher at 16.7 mM than at 3.3 mM glucose, although insulin responses were significantly increased by phanoside below 125 microM only at high glucose levels. Also when given orally to rats, phanoside (40 and 80 mg/ml) improved glucose tolerance and enhanced plasma insulin levels at hyperglycemia.     

Bioactive oleanolic acid saponins and other constituents from the roots of Viguiera decurrens

The bisdesmoside oleanolic acid saponin, 3-0-(methyl-beta-D-glucuronopyranosiduronoate)-28-0-beta-D-glucopyranosyl-oleanolate along with nine known compounds (two diterpenic acids, one chromene, three triterpenes, one steroidal glycoside, and two monodesmoside oleanolic acid saponins), were obtained from Viguiera decurrens roots. The chemical structure of the bisdesmoside oleanolic saponin was determined by chemical and NMR spectral evidence. A mixture of monodesmoside saponins displayed cytotoxic activity against P388 and COLON cell lines (ED50= 2.3 and 3.6 microg/ml, respectively). Two of the known compounds showed insecticidal activity against the Mexican bean beetle larvae (Epilachna varivestis).     

Spirostanol saponins from the rhizomes of Tacca chantrieri and their cytotoxic activity

The rhizomes of Tacca chantrieri have been analysed for steroidal saponin constituents, resulting in the isolation of four new spirostanol saponins (1-4), along with one known saponin (5); their structures were elucidated on the basis of extensive spectroscopic analysis, including 2D NMR, and the results of hydrolytic cleavage. The isolated compounds were evaluated for their cytotoxic activity against HL-60 human promyelocytic leukemia cells.     

FDS,a novel saponin isolated from Felicium decipiens: Lectin interaction and biological complementary activities

Filicium decipiens saponin (FDS) is the first saponin purified from F. decipiens seeds using ion-exchange and gel filtration chromatographies. In the present study, FDS and the aqueous crude extract of F. decipiens seeds were examined for their antifungal properties, hemolytic/cytotoxicity activity. The dried seeds were powdered and homogenized in 10% (w/v) for the preparation of crude extract. Sample obtained from Sephadex-LH-20 demonstrated FDS with a peak at 752.35 g/mol. FDS showed the highest toxicity against Aspergillus flavus, presenting with a low fungicidal concentration at 12.8 μg/mL. Under hemolytic induction, rabbit and cow erythrocytes were more affected by both samples, and the inhibition effect of PpyLL lectin was observed. These characteristics provide fundamental understanding of F. decipiens secondary metabolites, which would benefit future research to prevent dangerous traditional uses and provide agricultural solution.     

Synthesis and cytotoxic activity of diosgenyl saponin analogues

Diosgenyl saponins are steroidal glycosides that are often found as major components in many traditional oriental medicines. Recently, a number of naturally occurring diosgenyl saponins have been shown to exert cytotoxic activity against several strains of human cancer cells. Use of these saponin compounds for cancer treatment is hampered due to the lack of understanding of their action mechanism as well as limited access to such structurally complicated molecules. In the present paper, we have prepared a group of diosgenyl saponin analogues which contain a beta-D-2-amino-2-deoxy-glucopyranose residue having different substituents at the amino group. Moderate cytotoxic activity is found for most analogues against neuroblastoma (SK-N-SH) cells, breast cancer (MCF-7) cells, and cervical cancer (HeLa) cells. The analogue 13 that contains an alpha-lipoic acid residue exhibits the highest potency against all three cancer cell lines with IC(50) ranging from 4.8 microM in SK-N-SH cells to 7.3 microM in HeLa cells. Preliminary mechanistic investigation with one saponin analogue (10) shows that the compound induces cell cycle arrest at G(1) phase in SK-N-SH cells, but the same compound induces cell cycle arrest at G(2) phase in MCF-7 cells. This result suggests that the cytotoxic activity of these saponin analogues may involve different action mechanisms in cell lines derived from different cancer sites.     

New neuritogenic steroidal saponin from Ophiopogon japonicus (Thunb.) Ker-Gawl

A new steroidal saponin was isolated from Ophiopogon japonicus. This saponin possesses a modification by 2-hydroxy-3-methylvalerylation of the hydroxyl group at C-4' of the sugar, linked to C-1 of the aglycone. It exhibited significant neuritogenic activity for PC12 cells. The structure-activity relationship revealed the aglycone, rather than the sugar moieties and acylation, to be important for the neuritogenic activity.     

玉米根V-ATPase的A亚基磷酸化位点的鉴定

以玉米 (Zea mays L.) 根的高纯度液泡膜为材料进行的磷酸化反应表明,液泡膜蛋白的磷酸化可明显提高V型H -ATPase (V-ATPase) 的ATP水解活性和H 转运活性。进一步研究表明,纯化的液泡膜蛋白能被硫代磷酸化,用V-ATPase的A亚基抗体将一条约69 kD的条带鉴定为A亚基。为了测定V-ATPase的A亚基的磷酸化位点,从硫代磷酸化的凝胶中切下A亚基条带并用胰蛋白酶彻底消化。用RP-HPLC分离纯化酶解片断,收集纯化的硫代磷酸化肽段进行质谱分析所测定的分子量为573.83 Da。A亚基胰蛋白酶彻底消化后能产生61个肽段,只有F56肽段的分子量573.66 Da与573.83 Da最接近,而且F56肽段上只有第525位的丝氨酸可以被磷酸化。因此可以确定,玉米根V-AT-Pase A亚基的潜在磷酸化位点为Ser525。就我们所知,这是首次确定植物V-ATPase A亚基的磷酸化位点。     

Structural analogues of diosgenyl saponins: Synthesis and anticancer activity

Saponins display various biological activities including anti-tumor activity. Recently intensive research has been focused on developing saponins for tumor therapies. The diosgenyl saponin dioscin is one of the most common steroidal saponins and exhibits potent anticancer activity in several human cancer cells through apoptosis-inducing pathways. In this paper, we describe the synthesis of several diosgenyl saponin analogues containing either a 2-amino-2-deoxy-β-d-glucopyranosyl residue or an α-l-rhamnopyranosyl-(1→4)-2-amino-2-deoxy-β-d-glucopyranosyl residue with different acyl substituents on the amino group. The cytotoxic activity of these compounds was evaluated in MCF-7 breast cancer cells and HeLa cervical cancer cells. Structure–activity relationship studies show that the disaccharide saponin analogues are in general less active than their corresponding monosaccharide analogues. The incorporation of an aromatic nitro functionality into these saponin analogues does not exhibit significant effect on their cytotoxic activity.     

Growth and steroidal saponin production in hairy root cultures of Solanum aculeatissimum

Summary Hairy root cultures of Solanum aculeatissimum were established by trans-formation using Agrobacterium rhizogenes strain 15834. Root growth and production of steroidal saponin were investigated under various culture conditions. Transformed roots grew better in Gamborg's B5 medium containing 3 % sucrose under continuous light than in the dark. Also, the roots turned light green when cultured under continuous light. Green hairy roots produced aculeatiside A (6.71mg ·) L^–1 and aculeatiside B (6.39mg · L^–1) after 8 weeks of culture, while no steroidal saponin was detected in hairy roots cultured in the dark. Of the three culture media tested, Gamborg's B5 medium was superior for growth and steroidal saponin production. Growth and steroidal saponin production were enhanced when 100g · L^–1 auxin except for 2,4-D was added to the medium. The addition of 2,4-D inhibited growth. Production of steroidal saponin was highest with NAA. Transformed roots used in this experiment were confirmed that hairy roots examined contain both TL-DNA and TR-DNA region of Ri plasmid by PCR amplification analysis of DNA.Abbreviations MS medium Murashige and Skoog's medium (1962) - B5 medium Gamborg's B5 medium (1968) - LS medium Linsmaier and Skoog's medium(1965) - HPLC High performance liquid chromatography - NAA -Naphthaleneacetic acid - IAA Indole-3-acetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - PCR polymerase chain reaction     

Steroidal alkaloid saponins and steroidal saponins from Solanum surattense

A rare 16β-H steroidal alkaloid saponin (1), an avenacoside-type saponin (2), two steroidal saponins (4, 5), one revised-structure steroidal saponin (3) and six known compounds (6-11) were isolated from aerial parts of Solanum surattense Burm. f. Their structures were established on the basis of physical data, as well as by using spectroscopic (HRESIMS, 1D and 2D NMR), and chemical analysis methods. Compounds 1 and 11 showed cytotoxicity against A549 cell line with IC₅₀ values of 20.3 and 15.7 μM, respectively.     

New insights into the structure-cytotoxicity relationship of spirostan saponins and related glycosides

A variety of spirostan saponins and related glycosides were synthesized and evaluated for their cytotoxicity against the human myeloid leukemia cell line (HL-60). A linear glycosylation strategy allowed for accessing a variety of functionalization patterns at both the spirostanic and the saccharide moieties, which provides new information regarding the structure-cytotoxicity relationship of this family of steroidal glycosides. Intriguing results were achieved with respect to hecogenyl and 5α-hydroxy-laxogenyl β-chacotriosides, turning out to be the former very cytotoxic and the latter no cytotoxic at all. Importantly, the partially pivaloylated β-d-glucosides of 5α-hydroxy-laxogenin were the most potent cytotoxic compounds among all tested glycosides. This comprises the first report on acylated spirostanyl glucosides displaying significant cytotoxicity, and therefore, it opens up new opportunities toward the development of saponin analogues as anticancer agents.     

Undifferentiated F9 embryonal carcinoma cells produce a short-chain collagen molecule.

The undifferentiated F9 embryonal carcinoma cells produce a unique collagen that decreases in amount during retinoic acid-induced differentiation of F9 cells into basement-membrane parietal endoderm. A bacterial-collagenase-sensitive protein of approx. 60,000 Da was resolved on polyacrylamide-gel electrophoresis. After pepsin digestion, two pepsin-resistant fragments containing hydroxyproline were demonstrated, suggesting that a portion of the molecule has a stable triple helix. The mRNA from the undifferentiated F9 cells translates a collagenase-sensitive protein with a molecular mass consistent with the 60,000 Da collagenous protein produced by undifferentiated F8 cells.     

Mechanism of inhibition of the class A beta -lactamases PC1 and TEM-1 by tazobactam. Observation of reaction products by electrospray ionization mass spectrometry

The reactions of class A beta-lactamases PC1 and TEM-1 with tazobactam (TZB), a potent penicillanic sulfone inhibitor for class A beta-lactamases, were studied using electrospray ionization mass spectrometry (ESI/MS). Following inactivation of the beta-lactamases by TZB, new abundant high mass components were observed including three with molecular masses of 52, 70, and 88 Da greater than PC1 and TEM-1, respectively, and a component with a molecular mass of 300 Da greater than PC1. In addition, three TZB reaction products with molecular masses of 248, 264, and 280 Da were observed. High performance liquid chromatography (HPLC)/ESI/MS analysis of the TZB-PC1 adduct digested with Glu-C revealed three new components with masses 52, 70, and 88 Da greater than that of the peptide composed of amino acid residues 58-82 and one new component with a mass 70 Da greater than that of the peptide composed of amino acid residues 125-141. HPLC/ESI/MS/MS analysis of the two digested peptides whose masses increased by 70 Da indicated that Ser-70 and Ser-130 were the most likely TZB-modified amino acid residues. Based on these data, a mechanism for the inactivation of the class A beta-lactamases by TZB is proposed. In this scheme, initial acylation of Ser-70 by TZB and opening of the lactam ring are followed by one of several different events: (1) the rapid decomposition of TZB with loss of the enamine moiety to form the propiolylated enzyme, (2) an intramolecular nucleophilic displacement of the imine or enamine moiety by Ser-130 to form a cross-linked vinyl ether, and (3) hydrolysis of the imine or enamines to form a Ser-70-linked aldehyde.     

A new steroidal saponin from AGAVE brittoniana and its biphasic effect on the Na+-ATPase activity

A new steroidal saponin, 3-{(O-6-deoxy-a-L-mannopyranosyl-(1 --> 4)-O-beta-D-glucopyranosyl-(1 --> 3)-O-[O-beta-D-glucopyranosyl-(1 --> 3)-beta-D-glucopyranosyl-(1 --> 2)]-O-beta-D-glucopyranosyl-(1 --> 4)-beta-D-galactopyranosyl)oxy}-6-hydroxy-(3beta,5alpha,6alpha,25R)-spirostan-12-one, was isolated from Agave brittoniana Trel. The structure was determined by extensive NMR spectroscopy studies and chemical conversions. Its effects on the Na+-ATPase and (Na+ + K+)-ATPase activities of the proximal tubule from pig kidney were evaluated. It was observed that this steroidal saponin exerts a biphasic effect on the Na+-ATPase activity. It is concluded that the effect of the aqueous extract as a diuretic is due, at least in part, to the action of saponin on the ouabain-insensitive Na+-ATPase.     

玉米根V-ATPase的A亚基磷酸化位点的鉴定

以玉米(Zea mays L)根的高纯度液泡膜为材料进行的磷酸化反应表明,液泡膜蛋白的磷酸化可明显提高v型H -ATPase(V-ATPase)的ATP水解活性和H 转运活性.进一步研究表明,纯化的液泡膜蛋白能被硫代磷酸化,用V-ATPase的A亚基抗体将一条约69 kD的条带鉴定为A亚基.为了测定V-ATPase的A亚基的磷酸化位点,从硫代磷酸化的凝胶中切下A亚基条带并用胰蛋白酶彻底消化.用RP-HPLC分离纯化酶解片断,收集纯化的硫代磷酸化肽段进行质谱分析所测定的分子量为573.83 Da.A亚基胰蛋白酶彻底消化后能产生61个肽段,只有F56肽段的分子量573.66 Da与573.83 Da最接近,而且F56肽段上只有第525位的丝氨酸可以被磷酸化.因此可以确定,玉米根V-AT-Pase A亚基的潜在磷酸化位点为Ser525.就我们所知,这是首次确定植物V-ATPase A亚基的磷酸化位点.     

Biochemical characterisation and genetic analysis of aureocin A53, a new,atypical bacteriocin from Staphylococcus aureus

Aureocin A53 is produced by Staphylococcus aureus A53. It is encoded on a 10.4 kb plasmid, pRJ9, and is active against Listeria monocytogenes. Aureocin A53 is a highly cationic 51-residue peptide containing ten lysine and five tryptophan residues. Aureocin A53 was purified to homogeneity by hydrophobic-interaction, cation-exchange, and reverse-phase chromatography. MALDI-TOF mass spectrometry yielded a molecular mass of 6012.5 Da, which was 28 Da higher than predicted from the structural gene sequence of the bacteriocin. The mass increment resulted from an N-formylmethionine residue, indicating that the aureocin A53 is synthesised and secreted without a typical bacteriocin leader sequence or sec-dependent signal peptide. The structural identity of aureocin A53 was verified by Edman sequencing after de-blocking with cyanogen bromide and extensive mass spectrometry analysis of enzymatically and laser-generated fragments. The complete sequence of pRJ9 was determined and none of the open reading frames identified in the vicinity of the structural gene aucA showed similarity to genes that are typically found in bacteriocin gene clusters. Thus, neither a dedicated protease or transporter, nor modifying enzymes and regulatory elements seemed to be involved in the production of aureocin A53. Further unique features that distinguish aureocin A53 from other peptide bacteriocins include remarkable protease stability and a defined, rigid structure in aqueous solution.     

Enterocin F4-9, a Novel O-Linked Glycosylated Bacteriocin

Enterococcus faecalis F4-9 isolated from Egyptian salted-fermented fish produces a novel bacteriocin, termed enterocin F4-9. Enterocin F4-9 was purified from the culture supernatant by three steps, and its molecular mass was determined to be 5,516.6 Da by mass spectrometry. Amino acid and DNA sequencing showed that the propeptide consists of 67 amino acid residues, with a leader peptide containing a double glycine cleavage site to produce a 47-amino-acid mature peptide. Enterocin F4-9 is modified by two molecules of N-acetylglucosamine β-O-linked to Ser37 and Thr46. The O-linked N-acetylglucosamine moieties are essential for the antimicrobial activity of enterocin F4-9. Further analysis of the enterocin F4-9 gene cluster identified enfC, which has high sequence similarity to a glycosyltransferase. The antimicrobial activity of enterocin F4-9 covered a limited range of bacteria, including, interestingly, a Gram-negative strain, Escherichia coli JM109. Enterocin F4-9 is sensitive to protease, active at a wide pH range, and moderately resistant to heat.     

A study on in vitro glycation processes by matrix-assisted laser desorption ionization mass spectrometry

The number of glucose molecules condensed on glycated bovine serum albumin have been easily determined by means of matrix-assisted laser desorption/ionization mass spectrometry. Measurements were carried out on samples from incubation of the proteins with glucose at different concentrations (0.02 M, 0.2 M, 2 M and 5 M). A clear increase in molecular mass of BSA with respect to incubation time is detected. In contrast to what is observed with fluorescence, the plots of molecular mass increase vs. incubation time show tha occurrence of a steady state, corresponding to the complete saturation of all the protein sites against glucose. Comparison of fluorescence and molecular mass data reveals that some further reactions, different from condensation, must take place, which could be in principle either intramolecular or originated by reactivity of modified condensed gluocse moieties vs. free glucose.