Solanum Torvum Swartz

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Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE Antifungal and antimycotoxigenic potency of Solanum torvum Swartz. leaf extract: isolation and identiﬁcation of compound active against mycotoxigenic strains of Aspergillus ﬂavus and Fusarium verticillioides R.U. Abhishek, S. Thippeswamy, K. Manjunath and D.C. Mohana

Department of Microbiology and Biotechnology, Bangalore University, Bengaluru, India

Keywords Abstract aflatoxin B1, antifungal, antimycotoxigenic, Aspergillus flavus, fumonisin B1, Fusarium Aims: The main objective of this study was to investigate the antifungal effect verticillioides, Solanum torvum, torvoside K. of Solanum torvum leaves against different field and storage fungi, and to identify its active compound. In addition, to evaluate in vitro and in vivo Correspondence inhibitory efficacy on toxigenic strains of Aspergillus flavus and Fusarium Devihalli C. Mohana, Department of Microbi- verticillioides. ology and Biotechnology, Bengaluru Univer- Methods and Results: Leaves of S. torvum were sequentially extracted with sity, Jnana Bharathi, Bengaluru-560 056, India. petroleum ether, toluene, chloroform, methanol and ethanol. The antifungal E-mail: [email protected] compound isolated from chloroform extract was identified as torvoside K based on spectral analysis. The antifungal activity of chloroform extract and 2015/1115: received 3 June 2015, revised 16 torvoside K was determined by broth microdilution and poisoned food July 2015 and accepted 15 September 2015 techniques. The minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and zone of inhibition (ZOI) were recorded. doi:10.1111/jam.12956 Further, inhibitory effects of chloroform extract and torvoside K on growth of A. flavus and F. verticillioides, and their toxin productions were evaluated using in vitro and in vivo assays. Torvoside K showed the significant activity against tested fungi with ZOIs and MICs ranging from 334to874% and 3125– 250 lgml 1, respectively. Further, torvoside K showed concentration- dependent antimycotoxigenic activity against aflatoxin B1 and fumonisin B1 production by A. flavus and F. verticillioides, respectively. Conclusions: It was observed that the compound torvoside K significantly inhibited the growth of all fungi tested. Growth of A. flavus and F. verticillioides, and aflatoxin B1 and fumonisin B1 productions were completely inhibited in vitro and in vivo by torvoside K with increasing concentration. Significance and Impact of the Study: Control of mycotoxigenic fungi requires compounds that able to inhibit both fungal growth and mycotoxin production. The antimycotoxigenic potential of torvoside K of S. torvum is described in this study for the first time. The results indicate the possible use of S. torvum as source of antifungal agents against postharvest fungal infestation of food commodities and mycotoxin contaminations.

contamination of various foodstuffs and agricultural Introduction commodities is a major problem in the tropics and sub- Fungi have been reported to cause damage to grains and tropics, where climatic conditions and agricultural and other foodstuffs (>12%) during pre- and post-harvest storage practices are favourable to fungal growth and processing and storage (Al-Reza et al. 2010). Mycotoxin toxin production (Kumar et al. 2008). Approximately

25–40% of foodstuffs worldwide are contaminated with Solanum torvum Swartz. is a small shrub of the Solana- mycotoxins, which are well known to have hazardous ceae family, distributed widely in India, Malaya, China, effects on human beings and animals (Lutfullah and Hus- Phillipines and tropical America. The fruits of S. torvum sain 2012). Many species of Aspergillus, Fusarium and are edible and traditionally used for the treatment of Penicillium are not only recognized plant pathogens but abscesses, jigger wounds, skin infections and athlete’s foot are also sources of the important mycotoxins of concern (Balachandran et al. 2012). Pharmacological studies on this in animal and human health (Placinta et al. 1999). plant have demonstrated antiviral (Arthan et al. 2002), Among the mycotoxins, aflatoxins and fumonisins are the immunosecretory (Israf et al. 2004), antioxidant (Sivapriya most toxic secondary metabolites mainly produced by and Srinivas 2007; Loganayaki et al. 2010; Ramamurthy species of Aspergillus and Fusarium. Aspergillus flavus and et al. 2012), analgesic, anti-inflammatory (Ndebia et al. Fusarium verticillioides are two important mycotoxigenic 2007) and anti-ulcerogenic (Nguelefack et al. 2008) activi- moulds that colonize different kinds of food grains. They ties. Solanum torvum contains a number of potential phar- spoil various types of foods viz., cereals, legumes, spices, macologically active chemicals like isoflavonoid sulphate vegetables, fruits etc. and also produce mycotoxins that and steroidal glycosides (Yahara et al. 1996; Arthan et al. can be mutagenic, teratogenic, carcinogenic causing feed 2002), chlorogenone and neochlorogenone (Cuervo et al. refusal and emesis in humans or animals (Nguefack et al. 1991), triacontane derivatives (Mahmood et al. 1983, 2009; Shukla et al. 2012a). Aflatoxins are highly toxic 1985), 22-b-O-spirostanol oligoglycosides (Iida et al. 2005) polyketide secondary metabolites produced mainly by and 26-O-b-glucosidase (Arthan et al. 2006). Antimicro- A. flavus. The human health impact of aflatoxin B1 bial activity of the leaf and fruit of this plant have been pre- (AFB1) exposure is widespread in developing countries. viously reported (Chah et al. 2000; Balachandran et al. It is known that AFB1 causes teratogenicity, immunotoxi- 2012; Lalitha et al. 2010). Balachandran et al. (2012) city, hepatotoxicity and even death in humans and farm reported the antimicrobial and antimycobacterial activities animals (Reddy et al. 2010; Rosas-Taraco et al. 2011). of methyl caffeate isolated from S. torvum fruit. Although Exposure to fumonisins, mainly produced by F. verticil- the antimicrobial activity of crude extracts of S. torvum lioides, has been associated with several diseases in ani- leaves has been reported earlier (Bari et al. 2010; Lalitha mals including leucoencephalomalacia in equines, et al. 2010), there are no reports on active compound pulmonary oedema in swine, liver cancer in rats and responsible for antifungal activity of S. torvum leaves. In immunosuppression in poultry (Ficoseco et al. 2014). this context, an attempt has been made to identify the Both aflatoxins and fumonisins are relevant in food and active compound responsible for antifungal activity as well feedstuffs due to their widespread occurrence and co- as its effect on mycotoxin biosynthesis by A. flavus and occurrence (Chulze 2010). F. verticillioides. To prevent mycotoxin contamination in grain-based foods and feeds, control of growth of mycotoxigenic Materials and methods moulds is necessary. Management of fungal contamination, biodeterioration and mycotoxin accumulation in Fungal strains foodstuffs is generally achieved using synthetic chemicals (Tripathi et al. 2004; Garcia et al. 2012). However, resi- Fusarium oxysporum (NCIM 1043) was obtained from the dues of these chemicals in agricultural produce and National Chemical Laboratory, Pune (India). Alternaria by-products cause damage to animal and human health, brassicicola (Sunflower isolate) and Penicillium expansum further, continuous and indiscriminate use of chemical (Apple isolate) were obtained from the Department of preservatives can lead to the development of resistance in Microbiology, University of Mysore, and Fusarium lateri- micro-organisms (Al-Reza et al. 2010; Shukla et al. tium (Mulberry isolate) was collected from the Central 2012b). Botanicals, being the natural derivatives, are Sericulture Research and Training Institute, Mysore, biodegradable and do not leave toxic residues or by- India. Maize isolates of Aspergillus flavus (aflatoxigenic products to contaminate the environment, hence gaining strain), Aspergillus fumigatus, Aspergillus ochraceus, Asper- attention as alternative chemical control measures (Tri- gillus tamari, Aspergillus terreus, Fusarium equiseti, Fusar- pathi et al. 2004; Marin et al. 2011). Natural antimicro- ium udum, Fusarium verticillioides (fumonisinogenic bials have also shown important antifungal properties, strain) and Penicillium citrinum, and sorghum isolates of the identification of antifungal compounds from plants is Alternaria geophila and Curvularia tetramera were used one of the promising and alternative strategy for prevent- for the study, which were reported in our previous studies ing fungal-deterioration and mycotoxin contaminations (Thippeswamy et al. 2013, 2014). These test fungal strains (Al-Reza et al. 2010; Marin et al. 2011; Shukla et al. were maintained on Sabouraud dextrose agar (SDA), and 2012a). 7 days old cultures were used for further assays.

SRL, Mumbai, India) was eluted with solvents of increas- Plant material and preparation of plant extracts ing polarity using a gradient of chloroform and methanol Fresh leaves of S. torvum were collected from the Jnanab- (10:0,9:1? 0 : 10, v/v). The eluates were collected harathi Campus, Bangalore University, Bengaluru (India) in different fractions and concentrated. Based on antifun- in the month of July, 2012. The plant sample was authen- gal activity and TLC analysis, the active fractions of simi- ticated by Prof. Sankara Rao (JCB National Herbarium, lar profile were pooled together. The active compound Indian Institute of Science, Bengaluru, India) by compar- was obtained as a white amorphous powder, then anal- ison with the voucher specimens already deposited in the ysed for purity of the active compound following TLC Herbarium. The collected plant material was washed with analysis and tested with Ehrlich reagent as previously distilled water and shade-dried at room temperature. The indicated. The isolated compound was subjected to Four- dried leaves were ground well and stored in airtight con- ier transform infrared (IR), electrospray ionization-mass tainers. spectrometry (ESI-MS), 1H and 13C NMR analyses. In Fifty grams of the powdered plant sample was succes- the negative ion mode [M + H] of ESI-MS, active com- sively extracted in 200 ml solvent with increasing polarity pound of S. torvum showed a molecular ion peak at m/z viz., petroleum ether, toluene, chloroform, methanol and 740 corresponding to the molecular formula C39H64O13 ethanol, using a Soxhlet apparatus (Labline, Mumbai, (calculated m/z 74092). Further, the obtained data were India). All the solvent extracts were concentrated separately compared with chemical database and published values in under reduced pressure using a rotary flash evaporator literature (Yahara et al. 1996; Iida et al. 2005; Challal (Superfit, Mumbai, India) and stored in airtight glass tubes et al. 2014). (Harborne 1998), then checked for antifungal activity. Screening for antifungal activity against different fungi Bioautographic method by poisoned food technique In preliminary antifungal activity assay, the chloroform Antifungal activity of the chloroform extract (CE) and its extract showed fungal inhibitory activity, which was further active constituent torvoside K (TK) were tested against subjected to bioautographic method for identification of 15 different storage and field fungi using poisoned food antifungal band following the procedure of Ficoseco et al. technique as described by Mohana et al. (2008). CE and (2014). A spot of active chloroform extract was deposited TK were dissolved in DMSO and incorporated into SDA on thin layer chromatography (TLC) plate (silica gel to achieve the media of requisite concentration G60 F254, Merck, Darmstadt, Germany) and eluted using 1000 lgml 1. The control media without test samples a solvent system of chloroform/methanol (75:25, v/v), was added by the DMSO, the solvent which was used for to find the active band of inhibition. TLC plates were pre- dissolving the samples. The prepared media were auto- pared in duplicate, one plate was used for bioautography claved, 20 ml of media poured into Petri dishes and assay and other was kept for comparison. In direct bioauto- allowed to cool. Five millimetre discs of 7 day-old culture graphy assay, the plate was overlaid by the SDA (HiMedia, of test fungi were placed at the centre of the Petri dishes. Mumbai, India) and swabbed by the suspension of spores The inoculated plates were incubated at 28 2°C for of F. verticillioides (105 spores ml 1). The plate was incu- 7 days. Triplicates were maintained for each concentra- bated at 28 2°C for 48 h and observation of the inhibition and control. The fungitoxicity of test samples in tion was based on the inhibition caused by the active band. terms of percentage of mycelial growth inhibition was Antifungal band was recorded by comparing with visual- calculated as follows: ized bands on uninoculated TLC plate sprayed with Ehrlich Growth inhibitionð%Þ¼ðC T=CÞ100 reagent (20gofp–dimethylaminobenzaldehyde in 50 ml of 95% ethanol and 50 ml of concentrated hydrochloric where, C is the diameter of mycelial growth in control acid) and/or 10% H2SO4-acetic anhydride-chloroform plates, and T is the diameter of mycelial growth in trea- (1 : 10 : 25, v/v/v) followed by further exposure to 105°C. ted plates.

Separation and identification of the antifungal Determination of minimal inhibitory concentration compound from the chloroform extract of Solanum (MIC) and minimal fungicidal concentration (MFC) torvum The MIC and MFC of CE and TK were determined by The TLC band with antifungal activity identified in chlo- employing the broth microdilution method following stan- roform extract was separated by column chromatography. dard procedures (NCCLS 2002; Khaledi et al. 2014). The The column containing silica gel (mesh size: 60–120; CE and TK dissolved in 10% DMSO were first diluted to

the highest concentration (1000 lgml 1) to be tested, and 05 ml of DMSO was maintained as control. The broth then serial two-fold dilutions were made in a concentra- cultures were filtered through Whatman no. 1 filter tion range from 3125 to 1000 lgml 1 in 96-well microti- paper, mycelia dried at 100°C for 12 h and mycelial dry tre plate with Sabouraud dextrose broth (SDB). The cell weight (MDW) was recorded. The MDW of CE and TK suspension of overnight incubated test fungi in broth cul- treated samples were compared with control for their ture was adjusted to 104 spores ml 1. Each well of 96-well MDW losses. The filtrate was used for the extraction of microtitre plate was containing 200 ll of two-fold diluted AFB1 by adding equal volume of chloroform (25 ml) and broth of different concentrations and inoculated with shaken well in a separating funnel. The chloroform layer

15 ll of cell suspension of test fungi. The well containing was passed through anhydrous sodium sulphate (Na2SO4) DMSO without the test samples and inoculated with test and evaporated in dark condition at room temperature. fungi was used as the negative control. Synthetic fungicides The residue was re-dissolved in 1 ml of chloroform and copper oxychloride 50% WP (Fungicop-50, Karnatak 10 ll of sample was spotted onto the TLC plate adjacent

Agrochemicals Pvt Ltd, Bengaluru, India) and zinc ethylene to AFB1 standard (Sigma, Steinheim, Germany). The plate bisthiocarbamate 75% WP (Indifil Z-75, Indofil Chemicals was developed in chloroform-acetone (96 : 4, v/v), air- Company, Mumbai, India) were used as positive controls dried and visualized under ultra-violet light (365 nm; in conditions identical to tests samples. The inoculated UV-cabinet, Labline). Qualitative identification of AFB1 microtitre plate was sealed with parafilm, then agitated with content was done by visual comparison of intensity of flu- a microtitre plate shaker (Bio-Rad, Hercules, CA, USA) and orescence of the samples with standard spots. For quanti- incubated at 28 2°C for 72 h. The inoculated plates were tative estimation, the fluorescent spots were scrapped off observed for the presence or absence of fungal growth. After the plates, dissolved in 5 ml cold CH3OH, and centrifuged macroscopic observation, a 10 ll of treated broths were at 3000 g for 5 min. The absorbance of supernatant was radially streaked onto the SDA plates and incubated at measured at 265 nm using a spectrophotometer (UV-

28 2°C for 72 h. After the incubation period, the lowest 1700, Shimadzu, Kyoto, Japan) and the amount of AFB1 concentration at which the micro-organism tested did not content was calculated following formula: demonstrate visible growth that value was recorded as Aflatoxin content ðlgkg 1Þ¼½D M=E L1000 MIC. The complete absence of growth on the agar surface in the lowest concentration of the sample tested was defined where, D is absorbance, M is molecular weight of AFB1 as MFC. For further confirmation, 50 ll of iodo-nitro-te- (312), E is molar extinction coefficient of AFB1 (21 800), trazolium chloride (INT, SRL; 200 lgml 1) was added to and L is path length (1 cm). each well and incubated at 30°C for 30 min. The pale yel- The efficacy of CE and TK on growth of F. verticil- low-coloured INT was reduced to pink colour which indi- lioides and FB1 production was determined in vitro fol- cates the presence of viable microbial cells, while yellow lowing the method of Bailly et al. (2005) with some colour remained same where the microbial growth was modifications. Briefly, 100 ll of a spore suspension (104 inhibited (Hajji et al. 2010). microconidia ml 1)ofF. verticillioides was inoculated into SDB containing the requisite amount of CE and TK (625, 125, 250, 500 and 1000 lgml 1), and incubated at In vitro efficacy of CE and TK on mycelial growth and 28 2°C for 10 days. Fungal biomass of F. verticillioides mycotoxin production by Aspergillus flavus and obtained after the filtration of the SDB medium was mea- Fusarium verticillioides sured by weighing the mycelial mat after 48 h of freeze- Inhibitory effects of CE and TK on growth of mycotoxi- drying. To estimate FB1 production, mycelial mat of each genic A. flavus and F. verticillioides, and production of culture was ground in acetonitrile : water (1 : 1, v/v) and their toxins aflatoxin B1 and fumonisin B1 were evalu- filtered through 045 lm membrane filter. The filtrate ated using an in vitro assay. Sucrose-magnesium sul- was evaporated on water bath at 60°C and the residue phate-potassium nitrate- yeast extract (SMKY) liquid was redissolved in 1 ml of methanol. From this, 10 llof medium was used to determine the efficacy of CE and sample was spotted adjacent to standard FB1 (Sigma) on TK against growth of A. flavus and AFB1 production TLC plate, then eluted in a solvent system comprising of (Shukla et al. 2012a; Thippeswamy et al. 2013). SMKY butanol : acetic acid : water (20 : 10 : 10, v/v/v) and medium (25 ml) was taken in 100 ml flasks, to which allowed to air dry. The air dried TLC plate was sprayed requisite amount of test samples were added to get 625, with 05% p-anisaldehyde in methanol : acetic 1 125, 250, 500 and 1000 lgml concentrations. The acid : H2SO4 (85 : 10 : 05, v/v/v) solution followed by flasks were aseptically inoculated with suspension of toxi- heating at 110°C for 10 min. After that, the amount of 4 1 1 genic strain of A. flavus (10 spores ml , 100 ll flask ) FB1 on TLC plate was estimated qualitatively and quanti- and incubated at 28°C for 10 days. The flask containing tatively using spectrophotodensitometer (Bio-Rad,

Universal Hood II 720BR/02170) at 600 nm by compar- described previously by Tian et al. (2012) and Prakash ing with different concentrations of standard FB1. et al. (2014) with slight modifications. Fifty ll spore suspension of toxigenic strains of A. flavus and F. verticillioides containing 106 spores ml 1 was inoculated into In vivo efficacy of CE and TK on growth of respective medium (SMKY for A. flavus and SDB for mycotoxigenic fungi and, aflatoxin and fumonisin F. verticillioides) containing different concentrations of production in viable maize TK viz.,625, 125, 250, 500 and 1000 lgml 1 and incu- Inhibitory effects of CE and TK on growth of mycotoxi- bated at 28 2°C for 4 days. A control set was kept genic A. flavus and F. verticillioides, and production of parallel to the treatment sets without treatment. After their toxins aflatoxin B1 and fumonisin B1 were evaluated incubation, mycelia were harvested and washed twice in vivo using viable maize (Zea mays L.) as model. The effi- with distilled water. The net wet weights of the cell pel- cacy of CE and TK on aflatoxin production in viable maize lets were recorded. Five millilitre of 25% alcoholic potas- was determined by following the procedure of Garcia et al. sium hydroxide solution (25 g KOH in 35 ml distilled (2012), and Probst and Cotty (2012) with slight modifica- water and made up to 100 ml with absolute ethanol) tions. The maize seeds were treated with different concen- was added to each sample and vortex mixed for 2 min, trations of CE and TK ranging from 625 to 1000 lgg 1 of followed by incubation at 85 2°C for 4 h in water maize seeds. The maize seeds without sample treatment bath. Sterols were extracted from each sample by adding were maintained as control. The sample-treated and a mixture of 2 ml distilled water and 5 ml n-heptane. untreated maize were inoculated with 100 ll spore suspen- Then, the mixture was sufficiently mixed by vortex for sion of A. flavus toxigenic strain containing 104 2 min allowing the layers to separate for 1 h at room 1 spores ml . The water activity (aw 095) of maize was temperature and n-heptane layer was analysed by scan- adjusted by aseptically adding sterile distilled water to ning spectrophotometry (UV-1700, Shimadzu) between maize kernels in sterile container as described by Garcia 230 and 300 nm. Base correction of the absorbance was et al. (2012). The inoculated maize were kept for incuba- done with control containing only respective concentration at room temperature for 15 days. After incubation, 5 g tion of test compound without inoculation of test fungi. of maize seeds were milled and extracted with 15 ml of ace- The presence of ergosterol (at 282 nm) and the late tonitrile-water (60 : 40, v/v) and shaken for 10 min. The sterol intermediate 24(28) dehydroergosterol (at 230 and extract was filtered through Whatman No. 1 filter paper 282 nm) in the n-heptane layer led to a characteristic and the filtrate was extracted with equal volume of chloro- curve. The ergosterol amount was calculated as a per- form. Further, the extracted AFB1 was estimated qualita- centage of the wet weight of the fungal mycelia, was tively and quantitatively as described above in vitro assay. based on the absorbance and wet weight of the initial The efficacy of CE and TK on FB1 production in vivo mycelial pellet. The calculated formula of the ergosterol was determined in viable maize seeds following the proce- amount is as follows: dures of Bailly et al. (2005) and Thippeswamy et al. (2014) with minor modifications. Briefly, freshly harvested maize %ergosterol þ %24ðÞ 28 dehydroergosterol samples were collected and the water activity (a 095) was w ¼ ðÞA282=290 =pellet weight; adjusted. The maize samples were treated with different concentrations of CE and TK separately (625, 125, 250, %24ðÞ 28 dehydroergosterol ¼ ðÞA230=518 =pellet weight; 500 and 1000 lgml 1) and inoculated with 100 llofa spore suspension (104 microconidia ml 1)ofF. verticillioides and incubated at 25°C up to 15 days. After incuba- %ergosterol ¼ ðÞ%ergosterol þ %24ðÞ 28 dehydroergosterol tion, the maize samples were used for FB1 extraction and %24ðÞ 28 dehydroergosterol quantification following the procedure of Bailly et al. (2005). The amount of FB1 was estimated qualitatively and where, 290 and 518 are the E values (in percentages per quantitatively using spectrophoto-densitometric method cm) determined for crystalline ergosterol and 24(28) as described above in vitro assay. dehydroergosterol, respectively, and pellet weight is the net wet weight (g). Effect of TK on ergosterol content in the plasma membrane of toxigenic Aspergillus flavus and Fusarium Statistical analysis verticillioides The experiments were performed in triplicate and values The ergosterol content in the plasma membrane of were expressed as means standard error. Analysis of A. flavus and F. verticillioides was detected by a method variance was conducted, and the differences between

values were tested for signiﬁcance by ANOVA with the SPSS (a) (b) 19 (IBM, Armonk, NY, USA) programme. Differences at P ≤ 005 were considered statistically signiﬁcant.

Results

Antifungal activity visualized by bioautographic assay The chloroform extract of S. torvum leaves showed the highest antifungal activity than other extracts viz., petroleum ether, toluene, methanol and ethanol extracts. A TLC approach was followed to identify the active compound responsible for the observed antifungal activity in chloroform extract of S. torvum. Bioautographic and TLC analysis indicated that the band with Rf = 056 showed the observed antifungal activity. The active band was visualized in TLC plate after spraying with Ehrlich reagent and/or 10% H2SO4-acetic anhydride-chloroform, separately. The brick-red colour conﬁrmed that the compound belongs to glycoside group.

Identification of the active compound isolated from the chloroform extract of Solanum torvum The chloroform extract was subjected to column chro- Figure 1 Silica gel chromatograms of 9th and 10th fractions obtained from column chromatography of chloroform extract of Sola- matography with a gradient elution of chloroform and num torvum leaves. The plates were developed in chloro- ? methanol (10 : 0, 9 : 1 0 : 10, v/v) to afford 26 frac- form : methanol (75:25, v/v). Chromatograms were (a) observed tions. The 9th and 10th fractions of column chromatog- after spraying with 10% H2SO4 : acetic anhydride : chloroform raphy showed similar chromatographic profile and (1 : 10 : 25, v/v/v) and further exposure to 105°C (b) bioautographed bioautographic results (Fig. 1), which were pooled against Fusarium verticillioides. together. The fraction was further purified by preparative TLC, then the compound obtained as amorphous powder and subjected to spectral analysis. The compound was characterized as torvoside K based on the comparison of its spectral data with the reported values in literature, OH which was previously reported as torvoside C by Yahara et al. (1996). In the ESI-MS spectrum, the isolated com- O pound showed a molecular ion peak ([M + H] )atm/z

740 (C39H64O13, requires, 74092) corresponding to the 13 O molecular formula C39H64O13. The C-NMR spectrum showed 39 carbon signals and 1H-HMR spectrum showed 64 proton signals similar to those of reported values (Yahara et al. 1996; Iida et al. 2005; Challal et al. 2014). In an earlier report, it was reported as glycoside of neoso- HO OH OH laspigenin having 22-a-O-spirostane skeleton because its 13C-NMR signals due to the aglycone moiety were identi- O O OH cal with those of neosolaspigenin (Yahara et al. 1996). 13 However, the C-NMR signals of torvoside K were coin- O O cident with those of the sapogenol moiety of this tor- OH OH voside K (Iida et al. 2005; Challal et al. 2014). Therefore, torvoside K was determined to be 6-O-a-L-rhamnopyra- nosyl-(1?3)-b-D-quinovopyranosyl (22R,23S,25R)-3b,6 Figure 2 Structure of torvoside K isolated from Solanum torvum a, 23-trihydroxy-5a-spirostane (Fig. 2). leaves.

In broth microdilution assay, the TK showed signifi- Antifungal efficacy of CE and TK assessed using cant inhibitory activity against most of the pathogenic poisoned food technique and microdilution method fungi tested with lower MIC and MFC than that of CE The growth inhibitory effects of CE and TK were and copper oxychloride 50%. The results revealed that screened against a panel of 15 fungal pathogens contain- the antifungal activity of TK was comparable to zinc ing storage and field fungi are given in Table 1. The ethylene bisthiocarbamate 75% which showed the signifi- percentage of growth inhibition by the CE and TK was cant inhibitory activity against a wide range of fungi estimated by measuring the growth diameter of colony tested. The test samples showed significant inhibitory grown in the medium with treatment and control. Most activity against field fungi tested viz., Alternaria spp., of the treated field fungi were susceptible at Curvularia sp. and Fusarium spp., with lower MIC/MFC 1000 lgml 1 concentration of CE and TK with inhibi- values than those recorded for storage fungi viz., Aspergil- tion of spore germination. Among the fungi tested, lus spp. and Penicillium spp. On comparative evaluation P. expansum, A. flavus and A. tamari were found to be with synthetic fungicides copper oxychloride 50% and most resistant, with the mycelial growth inhibition 265, zinc ethylene bisthiocarbamate 75%, CE and TK showed 280 and 300%, respectively. The field fungi such as varying degrees of MIC and MFC values against different A. brassicicola (874%), F. verticillioides (842%), fungi tested. Among the samples tested, copper oxychlo- F. udum (841%) and C. tetramera (824%), were found ride 50% was found least effective against any of the be most susceptible organisms at 1000 lgml 1. At the fungi tested, most of the fungi were not completely concentration of 1000 lgml 1, TK caused more than inhibited even at concentration of 1000 lgml 1. 50% mycelial inhibition of most of the fungi except P. expansum, A. flavus and A. tamari. However, remark- Inhibitory effect CE and TK on fungal growth and able antifungal index (502–874%) was recorded by TK mycotoxin production in culture medium against rest of the fungi at the same concentration. Among the two mycotoxigenic fungi tested, aflatoxigenic Inhibitory effects of CE and TK on mycelial growth, and strain of A. flavus was found to be resistant, whereas production of aflatoxin B1 by A. flavus and fumonisin fumonisinogenic strain of F. verticillioides was susceptible B1 by F. verticillioides were evaluated using suitable to TK and CE. growth media. The biomass (MDW) of A. flavus and

Table 1 Antifungal activity of chloroform extract of Solanum torvum, torvoside K and synthetic fungicides against different ﬁeld and storage fungi

Chloroform extract Torvoside K CO ZEB

% Mycelial % Mycelial Test fungi inhibition* MIC MFC inhibition* MIC MFC MIC MFC MIC MFC

Alternaria brassicicola 7934 032 125 250 874 032 3125 125 3125 500 3125 625 Alternaria geophila 6040 026 125 250 760 026 625 125 125 >1000 3125 500 Aspergillus ﬂavus† 2800 014 250 >1000 334 014 125 500 250 >1000 125 1000 Aspergillus fumigatus 4278 076 250 1000 502 076 125 250 250 >1000 250 1000 Aspergillus ochraceus 4950 008 125 500 568 008 125 250 250 500 625 500 Aspergillus tamari 3000 012 500 >1000 343 012 250 1000 500 >1000 125 250 Aspergillus terreus 4105 045 125 500 520 045 625 250 250 >1000 125 250 Curvularia tetramera 6932 076 625 250 824 076 3125 125 250 >1000 3125 500 Fusarium equiseti 5290 033 125 250 635 033 625 125 250 >1000 3125 125 Fusarium lateritium 6778 014 125 250 798 014 625 250 500 >1000 3125 125 Fusarium oxysporum 6800 043 125 250 795 043 625 125 500 >1000 3125 125 Fusarium udum 7356 022 625 125 841 022 3125 625 250 >1000 3125 125 Fusarium verticillioides‡ 7642 027 125 250 842 027 625 250 1000 >1000 625 125 Penicillium expansum 2625 011 500 >1000 295 011 250 >1000 500 >1000 500 >1000 Penicillium citrinum 3044 000 500 >1000 336 000 250 >1000 1000 >1000 500 >1000

CO: Copper oxychloride 50%; ZEB: Zinc ethylene bisthiocarbamate 75%. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values are expressed in lgml1. Data given are the mean values of three replicates standard error (P ≤ 005). *Per cent mycelial inhibition at sample concentration of 1000 lgml1. †Aﬂatoxin B1 producing strain. ‡Fumonisin B1 producing strain.

Table 2 In vitro efﬁcacy of chloroform extract of Solanum torvum and torvoside K on mycelial dry weight (MDW), FB1 production from Aspergil- lus ﬂavus and FB1 production from Fusarium verticillioides

Effect on growth of A. ﬂavus and AFB1 production in SMKY Effect on growth of F. verticillioides and FB1 production in medium SDB medium

Chloroform extract Torvoside K Chloroform extract Torvoside K

Concentration* AFB1 content AFB1 content FB1 content FB1 content (lgml1) MDW (mg) (lgl1) MDW (mg) (lgl1) MDW (mg) (lgmg1) MDW (mg) (lgmg1)

Control 5480 93 14784 287 5480 93 14784 287 1408 87450 004 1408 87450 004 625 4730 96 13310 255 4230 102 11080 289 1120 88430 002 940 81280 003 125 3943 89 9446 286 1746 53 1026 215984 73224 003 730 65085 002 250 2923 65 5178 254789 38ND 543 52098 001 323 64ND 500 1356 329571 214ND ND 370 45ND ND ND 1000 370 27NDNDNDNDNDNDND

Data given are the mean of three replicates standard error (P ≤ 005). ND, not detected; SMKY, Sucrose-magnesium sulphate-potassium nitrate- yeast extract; SDB, Sabouraud dextrose broth. *In vitro treatment with different concentrations of samples in lgml1 of growth medium.

F. verticillioides and mycotoxin production were signifi- (Table 3). The amount of AFB1 and FB1 production in cantly inhibited by CE and TK in a dose-dependent man- control were found to be 12940 201 lgkg 1 and 1 ner (Table 2). The amount of AFB1 production in 265 003 lgg , respectively. AFB1 production in 1 1 control was found to be 14784 287 lgl and FB1 maize was inhibited completely by TK at 1000 lgkg , 1 was 450 004 lgmg of fungal biomass. AFB1 and but CE did not inhibit AFB1 production completely. FB1 productions were completely inhibited by TK at con- Similarly, FB1 production was inhibited by TK at concen- centrations higher than 250 lgml 1, while fungal bio- trations higher than 500 lgkg 1, whereas CE inhibited mass were completely inhibited at concentrations higher at 1000 lgkg 1. 1 than 500 lgml . Similarly, in the case of CE, AFB1 production was inhibited at 1000 lgml 1 with no complete Effectiveness of active compound TK on ergosterol inhibition of fungal biomass, whereas FB1 production was content in the plasma membrane of toxigenic fungi inhibited at concentrations higher than 500 lgml 1 with complete inhibition of fungal biomass at 1000 lgml 1. The inhibitory effects of TK on ergosterol content in the plasma membrane of A. flavus and F. verticillioides are shown in Fig. 3. When compared to control, the reduc- Inhibitory effect of CE and TK on aflatoxin and tion percentage of ergosterol content in the plasma mem- fumonisin production in viable maize brane of A. flavus by TK was recorded to be 4867%, In vivo inhibitory efficacy of CE and TK on production 5730%, 7043% at 625, 125, and 250 lgml 1, respec- of aflatoxin B1 by A. flavus and fumonisin B1 by F. verti- tively (data not presented). Similarly, in F. verticillioides, cillioides were evaluated using viable maize as model a reduction percentage of the ergosterol content as com-

Table 3 In vivo efﬁcacy of chloroform l 1 l 1 extract of Solanum torvum and torvoside K AFB1 production ( gkg )in FB1 production ( gg )in on AFB1 production from Aspergillus ﬂavus maize maize and FB1 production from Fusarium verticil- Concentration* Chloroform Chloroform lioides in maize 1 (lgg ) extract Torvoside K extract Torvoside K

Control 12940 201 12940 201265 003 265 003 125 11910 185 10085 194220 004 156 003 250 10146 191 7168 175124 002 019 002 500 6778 154 2413 121039 001 ND 1000 1634 133ND ND ND

Data given are the mean of three replicates standard error (P ≤ 005). ND, not detected. *In vivo treatment with different concentrations of samples in lgg1 of viable maize.

(a) 0·9

0·8

0·7

0·6

0·5

0·4 Absorbance 0·3

0·2

0·1

0 230 240 250 260 270 280 290 300 Wavelength (nm) (b) 0·9

0·8

0·7

0·6

0·5

0·4 Absorbance 0·3

0·2 Figure 3 Effect of different concentrations of 0·1 torvoside K on ergosterol content in the plasma membrane of toxigenic strains of (a) 0 Aspergillus flavus and (b) Fusarium 230 240 250 260 270 280 290 300 verticillioides.(○) Control; (□)625 lgml1; Wavelength (nm) (●) 125 lgml1 and (■) 250 lgml1. pared with the control was observed at 6370% for S. torvum Swartz. leaf extract on growth of A. flavus and 625 lgml 1,7485% for 125 lgml 1,8403% for F. verticillioides, and mycotoxin production in viable 1 250 lgml . A dose-dependent decrease in ergosterol maize grain at adjusted aw and inoculum level. Condi- production in both A. flavus and F. verticillioides was tions studied in vivo were close to conditions that may observed when isolates were grown in the presence of occur during pre- and post-harvest of cereals. Further, we TK. At 500 and 1000 lgml 1 concentrations of TK, also investigated the antifungal potency against a panel of 100% reduction in ergosterol content was observed in 15 different field and storage fungi using in vitro assays. both A. flavus and F. verticillioides. The results demon- The antifungal and antimycotoxigenic properties of tor- strated that the ergosterol content (at 282 nm) in the voside K isolated from S. torvum leaves have been plasma membrane of both A. flavus and F. verticillioides reported here for the first time. was completely inhibited at concentrations higher than The chloroform extract of S. torvum yielded two active 500 lgml 1. fractions- 9th and 10th of column chromatography, which were pooled together due to similar chromatographic profile and bioautographic results. The bioauto- Discussion graphic assay showed that only one band inhibited fungal In this study, we evaluated the antifungal and antimyco- growth in each of these fractions. TLC chromatogram toxigenic capability of torvoside K isolated from sprayed with 10% H2SO4: acetic anhydride: chloroform

(1 : 10 : 25, v/v/v) showed two brick-red coloured bands, as it has been earlier reported by Prakash et al. (2014). among which the first band (Rf 056) showed antifungal MIC values of TK were comparable to synthetic fungicide activity. The active band was separated as white amor- zinc ethylene bisthiocarbamate; however, copper oxychlo- phous powder, which was identified as torvoside K based ride was found lease effective than all the samples tested. on spectral analysis. In an earlier report by Yahara et al. The antifungal effect of both CE and TE was stronger (1996), torvoside C has been reported from aerial parts than that observed for copper oxychloride 50%, a syn- of S. torvum, a glycoside of neosolaspigenin having 22- thetic contact fungicide used against a wide range of a-O-spirostane skeleton because its 13C-NMR signals due plant diseases. to the aglycone moiety were identical with those of Antimycotoxigenic efficacy of CE and TK on inhibition neosolaspigenin. Later, Iida et al. (2005) have reported of mycelial growth and production of aflatoxin B1 by the same compound from the fruits of S. torvum, A. flavus and fumonisin B1 by F. verticillioides were eval- the compound was determined to be torvoside K as the uated using in vitro and in vivo assays. During antimyco- 13C-NMR signals were coincident with those of the sapo- toxigenic assay in culture medium, a gradual decrease in genol moiety of this torvoside K. Further, Challal et al. MDW and mycotoxin production by A. flavus and F. ver- (2014) reported torvoside K from aerial parts of S. torvum, ticillioides, was observed with increasing concentration of all these data available in literature were coincident with CE and TK. Results showed a positive correlation our spectral values. Solanum torvum has been reported for between the subsequent decrease in mycelial growth and a number of potential pharmacologically active compounds mycotoxin production in vitro with increasing concentra- like isoflavonoid sulphate and steroidal glycosides (Yahara tions of TK and CE. The TK was more efficacious as et al. 1996; Arthan et al. 2002), chlorogenone and neo- mycotoxin suppressor in culture medium than viable chlorogenone (Carabot et al. 1991), triacontane derivatives maize as it caused complete inhibition of aflatoxin B1 (Mahmood et al. 1983, 1985), 22-b-O-spirostanol oligogly- secretion by A. flavus and fumonisin B1 by F. verticil- cosides (Iida et al. 2005) and 26-O-b-glucosidase (Arthan lioides at 250 lgml 1. Both aflatoxin and fumonisin pro- et al. 2006). Torvoside K was reported as novel compound ductions were completely inhibited by TK in maize at in S. torvum by Yahara et al. (1996) and Iida et al. (2005), higher concentrations, at 1000 and 500 lgml 1, respec- and it is reported for anticonvulsuion activity (Challal tively. The maize, which is susceptible to common fungal et al. 2014). There are no reports on antifungal and infestation, was selected as model for this study. Condi- antimycotoxigenic effects of torvoside K. tions studied in vivo were close to conditions that may In antifungal assays viz., poisoned food technique and occur during pre- and post-harvest of maize. In most of broth microdilution method, the differences in the degree the cases, seed treatment was most effective in suppres- of fungal inhibition were evident between both storage sion of seed borne fungi with no effect on seedling vigour and field fungal species tested, which showed that storage (data not shown). Maize is one of the most important fungi viz., species of Aspergillus and Penicillium, were crops worldwide; however, a good substrate for growth, more resistant when compared to field fungi viz., species development and activity of filamentous fungi. Maize is of Alternaria, Curvularia and Fusarium. Antifungal activ- associated with a large number of fungal species belong- ity of test samples was recorded in term of MIC/MFC. ing to the species of Aspergillus, Fusarium and Penicil- The MFC of TK for complete inhibition of growth of the lium, that can cause spoilage and mycotoxin aflatoxigenic strain of A. flavus was recorded at contaminations (Soares et al. 2013). Results of this study 500 lgml 1 in broth microdilution method, similarly in revealed that the TK and CE were found effective in antiaflatoxigenic assay, mycelia was completely inhibited inhibiting the growths of different test fungi belong to at 500 lgml 1. The MFC of TK for complete inhibition species of Aspergillus, Fusarium and Penicillium. However, of fumonisinogenic strain of F. verticillioides was Penicillium spp. was found more resistant among the 250 lgml 1, whereas mycelia was completely inhibited fungi tested, but toxigenic strains of A. flavus and F. ver- at 500 lgml 1 in antiaflatoxigenic assay. It was interest- ticillioides were effectively inhibited in vitro and in vivo. ing to note that the minimum inhibitory concentrations Both aflatoxins and fumonisins are relevant in maize and for toxigenic strains of A. flavus and F. verticillioides were maize-based foods and feeds due to their widespread 125 and 625 lgml 1, respectively, the values were occurrence and co-occurrence (Chulze 2010). almost similar to standard fungicide zinc ethylene The results from the measurement of ergosterol con- bisthiocarbamate. The MIC values were determined tent demonstrated that the TK caused adverse effect on against different test fungal species using broth microdi- plasma membrane of the toxigenic strains of A. flavus lution method. This method offers better opportunity to and F. verticillioides. The results revealed that the ergos- test samples to come in close contact with fungi as both terol content (at 282 nm) in the plasma membrane of of them are homogenously distributed inside the medium both A. flavus and F. verticillioides was completely inhib-

ited at concentrations higher than 250 lgml 1. Ergos- the compound TK has considerable antifungal and terol, a sterol, is specific to fungi and is the major sterol antimycotoxigenic activity, deserving further toxicological component of the fungal cell membrane. It is also investigations for future application. responsible for maintaining the cell function and integrity (Tian et al. 2012). Hence, ergosterol is a potential target Acknowledgements for antifungal drug. Different classes of antifungal agents viz., polyene antibiotics, azole derivatives and allylamines/ Authors are grateful to the Department of Science and thiocarbamates, they target ergosterol, the major sterol in Technology, Government of India, New Delhi (India) for the fungal plasma membrane (Georgopapadakou 1998). the financial support. The authors thank the Indian Insti- The primary mechanism of action by which azole anti- tute of Science, Bengaluru (India) for providing spectral fungal drugs inhibit fungal growth is disruption of nor- analyses and interpretation. mal sterol biosynthetic pathways, leading to a reduction in ergosterol biosynthesis (Kelly et al. 1995). The depletion of ergosterol disrupts the structure of the plasma Conflict of Interest membrane, making it more vulnerable to further damage, No conflict of interest declared. and alters the activity of several membrane-bound enzymes, such as those associated with nutrient transport and chitin synthesis. Severe ergosterol depletion may References additionally interfere with the hormone-like functions of Al-Reza, S.M., Rahman, A., Ahmed, Y. and Kang, S.C. (2010) ergosterol, affecting cell growth and proliferation (Geor- Inhibition of plant pathogens in vitro and in vivo with gopapadakou 1998). Keeping this point in view, the anti- essential oil and organic extracts of Cestrum nocturnum L. fungal mode of action of TK was assessed by measuring Pestic Biochem Physiol 96,86–92. the total intracellular ergosterol contents in fungal cells Arthan, D., Svasti, J., Kittakoop, P., Pittayakhachonwut, D., with increasing concentrations of TK. Our observations Tanticharoen, M. and Thebtaranonth, Y. (2002) Antiviral revealed that there was an adverse effect on ergosterol isoflavonoid sulfate and steroidal glycosides from the content, therefore, it confirms the considerable impair- fruits of Solanum torvum. Phytochemistry 59, 459–463. ment of the biosynthesis of ergosterol in plasma mem- Arthan, D., Kittakoop, P., Esen, A. and Svasti, J. (2006) brane of A. flavus and F. verticillioides. However, further Furastanol-26-O-b-glucosidase from the leaves of Solanum experiments are required to understand the exact mode torvum. Phytochemistry 67,27–33. of action of the TK on plasma membrane and its correla- Bailly, J.D., Querin, A., Tardieu, D. and Guerre, P. (2005) tion with the inhibition of aflatoxin biosynthesis. Production and purification of fumonisins from a highly Plant products are expected to be more advantageous toxigenic Fusarium verticillioides strain. Rev Med Vet 156 – over synthetic fungicides because of their biodegradable (Toulouse) , 547 554. nature, and the abundance of raw materials because of Balachandran, C., Duraipandiyan, V., Al-Dhabi, N.A., luxuriant growth of the plants and their renewable nature Balakrishna, K., Kalia, N.P., Rajput, V.S., Khan, I.A. and Ignacimuthu, S. (2012) Antimicrobial and makes use of plant products economical for practical antimycobacterial activities of methyl caffeate isolated application (Marin et al. 2011). The identification of from Solanum torvum Swartz. fruit. Indian J Microbiol 52, antifungal compounds from plants is one of the promis- 676–681. ing and alternative strategies for preventing fungal-deteri- Bari, M.A., Islam, W., Khan, A.R. and Mandal, A. (2010) oration and mycotoxin contaminations. Results presented Antibacterial and antifungal activity of Solanum torvum in this study confirmed that the TK isolated from (Solanaceae). Int J Agric Biol 12, 386–390. S. torvum leaves was effective for the inhibition of growth Carabot, C.A., Blunden, G. and Patel, V.A. (1991) of important mycotoxigenic fungi commonly associated Chlorogenone and neochlorogenone from the unripe with deterioration of food and feedstuffs. TK inhibited fruits of Solanum torvum. Phytochemistry 30, 1339–1341. the growth of toxigenic strains of A. flavus and F. verticil- Chah, K.F., Muko, K.N. Oboegbulem, S.I. (2000) lioides, and their toxin productions at even high water Antimicrobial activity of methanolic extract of Solanum activity levels, and was also found effective against a wide torvum fruit. Fitoterapia 71, 187–189. range of field and storage fungi. Solanum torvum, being Challal, S., Buenafe, O.E.M., Queiroz, E.F., Maljevic, S., the plant used in traditional medicine, possesses broad Marcourt, L., Bock, M., Kloeti, W., Dayrit, F.M. et al. spectrum antifungal activity against important field and (2014) Zebrafish bioassay-guided microfractionation storage moulds that would probably be a good source of identifies anticonvulsant steroid glycosides from the antifungal agents for prevention of fungal-deterioration philippine medicinal plant Solanum torvum. ACS Chem and mycotoxin contaminations. This study indicates that Neurosci 5, 993–1004.

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