Antifungal Saponins Research Articles

Production of antifungal saponins in an airlift bioreactor with a cell line transformed from Solanum chrysotrichum and its activity against strawberry phytopathogens

Biotechnology through plant cell cultures in bioreactors is a tool that allows increasing the production of secondary metabolites of commercial interest. The hydrodynamic characterization, in addition to the transfer (OTR) and uptake (OUR) of oxygen through the dynamic method with different aeration rate, were used to see their influence on the production of biomass and saponins. The culture poisoning technique was used to determine the antifungal activity of the SC-2 and SC-3 saponins in vitro. Likewise, the shear or hydrodynamic stress of 273.6 mN/m2 were calculated based on the Reynolds Number. The oxygen supply (OTR) was always greater than the demand (OUR) for all the aeration rate evaluated. Dry weight values of 8.6 gDW/L and a concentration of 2.7 mg/L and 187.3 mg/L of the saponins SC-2 and SC-3 respectively were obtained with an air flow of 0.1 vvm. In addition, it was possible to inhibit the growth of phytopathogenic fungi in vitro by up to 93%, while in vivo it was possible to reduce the infections of strawberry seeds inoculated with phytopathogens, obtaining up to 94% of germinated seeds. This information will facilitate the rational operation of the bioreactor culture system that produces secondary metabolites.

Analysis of Two New Arabinosyltransferases Belonging to the Carbohydrate-Active Enzyme (CAZY) Glycosyl Transferase Family1 Provides Insights into Disease Resistance and Sugar Donor Specificity.

Glycosylation of small molecules is critical for numerous biological processes in plants, including hormone homeostasis, neutralization of xenobiotics, and synthesis and storage of specialized metabolites. Glycosylation of plant natural products is usually performed by uridine diphosphate-dependent glycosyltransferases (UGTs). Triterpene glycosides (saponins) are a large family of plant natural products that determine important agronomic traits such as disease resistance and flavor and have numerous pharmaceutical applications. Most characterized plant natural product UGTs are glucosyltransferases, and little is known about enzymes that add other sugars. Here we report the discovery and characterization of AsAAT1 (UGT99D1), which is required for biosynthesis of the antifungal saponin avenacin A-1 in oat (Avena strigosa). This enzyme adds l-Ara to the triterpene scaffold at the C-3 position, a modification critical for disease resistance. The only previously reported plant natural product arabinosyltransferase is a flavonoid arabinosyltransferase from Arabidopsis (Arabidopsis thaliana). We show that AsAAT1 has high specificity for UDP-β-l-arabinopyranose, identify two amino acids required for sugar donor specificity, and through targeted mutagenesis convert AsAAT1 into a glucosyltransferase. We further identify a second arabinosyltransferase potentially implicated in the biosynthesis of saponins that determine bitterness in soybean (Glycine max). Our investigations suggest independent evolution of UDP-Ara sugar donor specificity in arabinosyltransferases in monocots and eudicots.

The bioassay-guided isolation of antifungal saponins from Hosta plantaginea leaves

Four new steroidal saponins hostaside Ⅰ (1), hostaside Ⅱ (2), hostaside Ⅲ (3), and hostaside Ⅳ (4), together with five known steroidal saponins (5–9), were isolated by the bioassay-guided fractionation from the leaves of Hosta plantaginea (Lam.) Aschers, a worldwide well-known ornamental plant. Hostasides Ⅰ and Ⅱ showed significant antifungal activities, and they could inhibit the growth of Candida albicans and Fusarium oxysporium with MIC values as low as 4 μg/ml.

Identification and Biological Activity of Antifungal Saponins from Shallot (Allium cepa L. Aggregatum Group)

The n-butanol extract of shallot basal plates and roots showed antifungal activity against plant pathogenic fungi. The purified compounds from the extract were examined for antifungal activity to determine the predominant antifungal compounds in the extract. Two major antifungal compounds purified were determined to be alliospiroside A (ALA) and alliospiroside B. ALA had prominent antifungal activity against a wide range of fungi. The products of acid hydrolysis of ALA showed a reduced antifungal activity, suggesting that the compound's sugar chain is essential for its antifungal activity. Fungal cells treated with ALA showed rapid production of reactive oxygen species. The fungicidal action of ALA was partially inhibited by a superoxide scavenger, Tiron, suggesting that superoxide anion generation in the fungal cells may be related to the compound's action. Inoculation experiments showed that ALA protected strawberry plants against Colletotrichum gloeosporioides , indicating that ALA has the potential to control anthracnose of the plant.

Antifungal saponins from bulbs of white onion, Allium cepa L.

Three saponins, named ceposide A, ceposide B, and ceposide C were isolated from the bulbs of white onion, Allium cepa L. Elucidation of their structure was carried out by comprehensive spectroscopic analyses, including 2D NMR spectroscopy and mass spectrometry, and chemical evidences. The structures of the compounds were identified as (25R)-furost-5(6)-en-1β,3β,22α,26-tetraol 1-O-β-d-xylopyranosyl 26-O-α-d-rhamnoyranosyl-(1→2)-O-β-d-galactopyranoside (ceposide A), (25R)-furost-5(6)-en-1β,3β,22α,26-tetraol 1-O-β-d-xylopyranosyl 26-O-α-d-rhamnoyranosyl-(1→2)-O-β-d-glucopyranoside (ceposide B), and (25R)-furost-5(6)-en-1β,3β,22α,26-tetraol 1-O-β-d-galactopyranosyl 26-O-α-d-rhamnoyranosyl-(1→2)-O-β-d-galactopyranoside (ceposide C). The isolated compounds, alone and in combinations, were evaluated for their antimicrobial activity on ten fungal species. Antifungal activity of all three saponins increased with their concentration and varied with the following rank: ceposide B>ceposide A–ceposide C. We found a significant synergism in the antifungal activity of the three ceposides against Botrytis cinerea and Trichoderma atroviride, because growth of these fungi was strongly inhibited when the three saponins were applied in combination. In contrast, Fusarium oxysporum f. sp. lycopersici, Sclerotium cepivorum and Rhizoctonia solani were very little affected by saponins.

Genetic transformation of Solanum chrysotrichum by Agrobacterium tumefaciens and the production of antifungal saponins

Solanum chrysotrichum (Solanaceae) synthesizes a family of six antifungal spirostanol saponins designated as SC-1 to SC-6. The production of saponins by wild-type plants is variable depending on the environmental conditions. In order to develop an in vitro system for the sustained production of these saponins, transformed cell suspension cultures of S. chrysotrichum were established from nodal explants of 3-mo-old plantlets by infecting with the Agrobacterium tumefaciens strain C58/pBI12. From these cultures, kanamycin-resistant and phytohormone-independent cell suspension line C58 5.1.1 was obtained. PCR and Southern blot analyses were used to confirm the integration of the wild-type T-DNA into the plant genome. Batch cultures of the C58 5.1.1 cell line were grown in phytohormone-free MS liquid medium for 25 d. First-order growth kinetics and the production of the antifungal saponins (SC-2, SC-3, and SC-4) were determined by dry weight and quantified by HPLC, respectively, from the cells as well as the culture medium. Based on the cell biomass, the specific growth rate was 0.09 d−1 and the yield of SC-2 reached 5.5% of dry weight, representing 40 times higher amount than that produced in plant leaves. SC-3 was recovered with a maximum yield of 0.9% of dry weight, whereas SC-4 was accumulated at 1.1% of dry weight. Saponins SC-2 and SC-3 were also excreted into the culture medium in low concentrations.

Characterization of Plant-Derived Saponin Natural Products against Candida albicans

Candida albicans is an opportunistic fungal pathogen capable of life-threatening disseminated infections particularly in immunocompromised patients. Resistance to many clinically used antifungal agents has created a need to identify and develop a new generation of compounds for therapeutic use. A compound screen to identify potential antifungal natural products was undertaken, identifying 12 saponins, some of which have not been previously described. In the Caenorhabditis elegans model, some saponins conferred nematode survival comparable to that of amphotericin B. Of the 12 antifungal saponins identified, two were selected for further analysis. C. albicans isolates were inhibited by these compounds at relatively low concentrations (16 and 32 microg mL(-1)) including isolates resistant to clinically used antifungal agents. C. albicans hyphae and biofilm formation were also disrupted in the presence of these natural products, and studies demonstrate that fungal cells in the presence of saponins are more susceptible to salt-induced osmotic stress. Although saponins are known for their hemolytic activity, no hemolysis of erythrocytes was observed at three times the minimal inhibitory concentration for C. albicans, suggesting the saponins may have a preference for binding to fungal ergosterol when compared to cholesterol. Importantly, when used in combination with photosensitizer compounds, the fungus displayed increased susceptibility to photodynamic inactivation due to the ability of the saponins to increase cell permeability, thereby facilitating penetration of the photosensitizers. The large proportion of compounds identified as antifungal agents containing saponin structural features suggests it may be a suitable chemical scaffold for a new generation of antifungal compounds.

High –yield production of antifungal saponins using transformed cell suspension cultures of the Mexican species Solanum chrysotrichum

Solanum chrysotrichum (Solanaceae) is used in Mexico to treat skin mycosis. Six novel antifungal spirostanol saponins (SC-1– SC-6), were identified as the active principles [1,2]. Production of the saponins is low and varies according to geographical and ontogenic conditions. Transformed calluses and cell suspension cultures were initiated. The cell line C58 5.1.1 was selected, and batch cultures of C58 5.1.1 were established. The production of the saponins (SC-2, SC-3, and SC-4) was quantified by HPLC, The production of SC-2, which represents the most active saponin, was forty times greater than that observed in leaves of wild plants.

Molecular cloning and expression analysis of two distinct β-glucosidase genes, bg1 and aven1, with very different biological roles from the thermophilic, saprophytic fungus Talaromyces emersonii

Recent sequencing of a number of fungal genomes has revealed the presence of multiple putative β-glucosidases. Here, we report the cloning of two β-glucosidase genes ( bg1 and aven1), which have very different biological functions and represent two of a number of β-glucosidases from Talaromyces emersonii. The bg1 gene, encoding a putative intracellular β-glucosidase, shows significant similarity to other fungal glucosidases from glycosyl hydrolase family 1, known to be involved in cellulose degradation. Solka floc, methyl-xylose, gentiobiose, beech wood xylan, and lactose induced expression of bg1, whereas glucose repressed expression. A second β-glucosidase gene isolated from T. emersonii, aven1, encodes a putative avenacinase, an enzyme that deglucosylates the anti-fungal saponin, avenacin, rendering it less toxic to the fungus. This gene displays high homology with other fungal saponin-hydrolysing enzymes and β-glucosidases within GH3. A putative secretory signal peptide of 21 amino acids was identified at the N-terminus of the predicted aven1 protein sequence suggesting that this enzyme is extracellular. Furthermore, T. emersonii cultivated on oat plant biomass was shown to deglucosylate avenacin. The presence of the avenacinase transcript was confirmed by RT-PCR on RNA extracted from mycelia grown in the presence of avenacin. The expression pattern of aven1 on various carbon sources was distinctly different from that of bg1. Only methyl-xylose and gentiobiose induced transcription of aven1. Gentiobiose induces synthesis of a number of cellulase genes by T. emersonii and it may be a possible candidate for the natural cellulase inducer observed in Penicillium purpurogenum. This work represents the first report of an avenacinase gene from a thermophilic, saprophytic fungal source, and suggests that this gene is not exclusive to plant pathogens.

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) ≤20 μg/ml, while dioscin ( 8) was selectively active against C. albicans and C. glabrata (MFC ≤ 5.0 μg/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.

Solanum chrysotrichumHairy Root Cultures: Characterization, Scale-Up and Production of Five Antifungal Saponins for Human Use

The production of five antifungal saponins (SC-2 - SC-6) recently extracted from Solanum chrysotrichum leaves was performed using hairy root cultures grown in 250-mL flasks and in 2-L modified draught-tube internal-loop airlift reactors (2-L MR). In both scales, first order growth kinetics were observed, reaching specific growth rates of 0.08 and 0.12 d(-1), respectively. Root density at the end of the culture period was the same for the flasks and for the 2-L MR. In flasks the production of the most active saponin SC-2 was growth-associated, with a maximum yield of 0.04 % dry weight, while in the 2-L MR an SC-2 yield of 0.7 % dry weight was reached, representing a value six times greater than that observed for plant leaves. SC-3 was obtained from root biomasses grown in flasks, while SC-4 was recovered from biomasses at both levels under investigation. SC-5 and SC-6 were only detected in the culture medium of roots grown in 2-L MR. Solanum chrysotrichum hairy root cultures and their scale-up in reactors are feasible strategies for the production of these antifungal compounds for human use.

Isolation of antifungal saponins from Phytolacca tetramera, an Argentinean species in critic risk

The methanolic extract of the berries of Phytolacca tetramera, an Argentinean species submitted to a great anthropic impact, and therefore in critic risk of extinction, not previously studied, showed antifungal activity against opportunistic pathogenic fungi. Through fractionation of the extract followed by agar dilution assays, three monodesmosidic triterpenoid saponins have been isolated from the butanolic extract of P. tetramera. The structures were established as phytolaccosides: B [3-O-β- d-xylopiranosyl-phytolaccagenin], E [3-O-β- d-glucopyranosyl-(1→4)-β- d-xylopiranosyl-phytolaccagenin]. and F [3-O-α- l-rhamnopyranosyl-(1→2)-β- d-glucopyranosyl-(1→2)-β- d-xylopyranosyl-phytolaccagenic acid]. The three saponins belong to the olean-type triterpenoid saponins, with 28,30 dicarboxylic groups and an olefinic double bond on C-12. Phytolaccosides B and E but not phytolaccoside F, showed antifungal activities against a panel of human pathogenic opportunistic fungi. Phytolaccoside B was the most active compound and showed the broadest spectrum of action. The most sensitive fungus was Trichophyton mentagrophytes.

Regulation of saponin biosynthesis in primary roots of oat

The antifungal saponins, avenacins, found in the roots of oats (Avena sativa) are thought to be responsible for the resistance of oat seedlings to the ‘take-all’ fungus Gaeumannomyces graminis var. tritici. Here, we confirm the belief that avenacin biosynthesis is restricted to the growing tips of oat roots.

Cell Suspension Culture of Solanum chrysotrichum (Schldl.) - A Plant producing an Antifungal Spirostanol Saponin

The production of an antifungal spirostanol saponin designated SC-1 has been detected in cell suspension cultures of the Mexican species Solanum chrysotrichum. Batch cultures of a cell suspension obtained from hypocotyl derived calluses of this species were grown for 25 days in shake flasks containing Murashige & Skoog (MS) medium. Throughout the growth cycle, fresh and dry weight, SC-1 yield, and uptake of sucrose, glucose and fructose were determined. The effects of inoculum size and sucrose concentration on the biomass accumulation and synthesis of the active metabolite, were studied. The maximum SC-1 production, above 14 mg.g−1 (which was fifty times that of field grown plants), was reached after 20 days using a 2% inoculum and complete MS medium supplemented with 2 mgl−1 2,4-D, 2 mg l−1kinetin, and sucrose between 30 and 45 gl−1.

An Antifungal Saponin from White Asparagus (Asparagus officinalis L) Bottoms

An antifungal saponin was isolated from the bottom cut of white asparagus (Asparagus officinalis L), which is unusable in food processing. The structure of the saponin was identified as 3-O-[{α-L-rhamnopyranosyl (1 → 2)} {α-L-rhamnopyranosyl (1→4)}-β-D-glucopyranosyl] (25S) spirost-5-ene-3β-ol from chemical and spectral data. This saponin was shown to be identical with collettinside III from Dioscorea collettii, and to inhibit the growth of some kinds of fungi at μg ml -1 levels.

An Antifungal Saponin from White Asparagus ( Asparagus officinalis L) Bottoms

An antifungal saponin was isolated from the bottom cut of white asparagus (Asparagus officinalis L), which is unusable in food processing. The structure of the saponin was identified as 3-O-[{α-L-rhamnopyranosyl (1→2)} {α-L-rhamnopyranosyl (1→4)}-β-D-glucopyranosyl] (25S) spirost-5-ene-3β-ol from chemical and spectral data. This saponin was shown to be identical with collettinside III from Dioscorea collettii, and to inhibit the growth of some kinds of fungi at μg ml−1 levels.

Host range of a plant pathogenic fungus determined by a saponin detoxifying enzyme.

Antifungal saponins occur in many plant species and may provide a preformed chemical barrier to attack by phytopathogenic fungi. Some fungal pathogens can enzymatically detoxify host plant saponins, which suggests that saponin detoxification may determine the host range of these fungi. A gene encoding a saponin detoxifying enzyme was cloned from the cereal-infecting fungus Gaeumannomyces graminis. Fungal mutants generated by targeted gene disruption were no longer able to infect the saponin-containing host oats but retained full pathogenicity to wheat (which does not contain saponins). Thus, the ability of a phytopathogenic fungus to detoxify a plant saponin can determine its host range.

Spermicidal activity of an antifungal saponin obtained from the tropical herb Mollugo pentaphylla

The ethyl acetate fraction of Mollugo pentaphylla, a tropical herb, contains an antifungal saponin (mollugogenol-A). We report here the spermicidal effects of this saponin. Washed sperm (> 100 × 10 6with > 50% motility) from normal volunteers were incubated with varying concentrations (0–300μg/ml) of mollugogenol-A at 30 °C. Sperm motility, velocity and viability were assessed at 0, 30, 60 minutes both manually and by using computer assisted semen analysis (CASA). Samples collected at 0 and 60 minutes were evaluated for membrane lipid peroxidation, superoxide dismutase (SOD) activity and transmission electron microscopy. A dose- and time-dependent effect of this saponin on sperm motion and viability was observed. The maximal spermicidal effect (4–5 fold decrease in motility and viability) was observed with 300 μg/ml dose of saponin. A three-fold increase in sperm membrane lipid peroxidation with corresponding inhibition of SOD activity were observed after 60 minutes incubation with this spermicidal agent. Transmission electron microscopy of saponin-treated samples revealed significant damage to the sperm membrane in both head and tail regions, and the acrosomal membranes were notably swollen and disrupted. These results indicate that this natural saponin has a potential spermicidal effect besides its known antifungal activity. The likely mechanism of its action involves sperm membrane damage by increased lipid peroxidation.

An antifeedant for the yellow butterfly larvae in Camellia japonica: A revised structure of camellidin II.

Camellidin II was isolated as an antifeedant for the larvae of the yellow butterfly, Eurema hecabe mandarina de l'Orza, from the leaves of Camellia japonica L. besides a non-tested camellidin III, identified as its methyl ester, and three inactive known compounds, quinic acid, 3β, 20-dihydroxylupane, and 3, 3', 4-tri-O-methylellagic acid. Spectroscopic and chemical evidence indicates that the structure of camellidin II, reported as an antifungal saponin, is better represented as 3 β, 18 β-dihydroxy-28-norolean-12-en-16-one 3-O-β-D-glucopyranosyl- (1→2) -β-D-galactopyranosyl- (1→3) - [β-D-galactopyranosyl- (1→2)] -β-D-glucuronopyranoside.