ABSTRACT
Cell cultures of Alstonia scholaris were treated with homogenates of Candida albicans, Fusarium oxysporum, Penicillium avelanium and Saccharomyces cerevisiae. The impact caused by the concentration, exposure time and the type of synergetic organisms on the accumulation of pentacyclic triterpenes was observed. When exposed to biotic elicitors for longer periods, some cell lines doubled the production of those triterpenes. S. cerevisiae homogenate was the best elicitor of triterpenes in all cell lines investigated.
Introduction
The species Alstonia scholaris, belonging to the family Apocynaceae, is widely distributed throughout tropical regions of Africa and Asia. A. scholaris, which is commonly called blackboard tree or devil tree, is a tropical evergreen tree native to South and South-east Asia. The chemical constituents of Alstonia spp. have been extensively investigated, and almost 400 compounds have been reported.[Citation1–6] Further, the leaves of A. scholaris have historically been used in ‘Dai’ ethnopharmacology to treat chronic respiratory diseases.[Citation7,Citation8] Extracts of A. scholaris not only possess a wide spectrum of pharmacological activities,[Citation7–11] but also inhibit germination and seedling growth of several weed species.[Citation12,Citation13] Recently, Javaid et al. [Citation14] showed that aqueous leaf extracts of A. scholaris contain potent herbicidal constituents for the management of parthenium weed. Therefore, A. scholaris may have strong allelopathic potential; further, allelopathic interactions may play an important role in the dominance of A. scholaris plant communities.[Citation15–17] Although allelopathic substances may comprise polyphenols, flavonoids, alkaloids and triterpenoids, only indole alkaloids and triterpenoids have been reported from A. scholaris. Acid triterpenes are of great interest due to the diversity of pharmacological activities they display: anti-inflammatory, hepatoprotective, antitumoural, among others. Fungal elicitation has been an effective tool to enhance the yield of secondary metabolites, also helping in the elucidation of mechanisms of plant responses to biotic stress agents. Most fungal elicitation strategies utilize rules undefined mixtures such as autoclaved fungal homogenates or fungal culture filtrates. Several reports have shown that the use of yeast and fungi as elicitors caused an accumulation of triterpenoid phytoalexins. In elicited cultures of Catharanthus roseus (L.), the qualitative profile of terpenoid products detected was similar to that found in control cultures, but different from the observation with Tabernaemontana divaricata and Uncaria tomentosa.[Citation5,Citation6,Citation13,Citation18–20] In this study, four different types of biotic elicitors, yeast and fungi, were tested to stimulate triterpene production. The effects of concentration and exposure time of those elicitors on A. scholaris cell lines were reported here.
Materials and methods
Callus cultures were induced from disinfested leaf explants and maintained on solid MS medium supplemented with 30 g. L−1 sucrose in two different hormone combination: AI (1.0 mg L−1 2,4-dichlorophenoxyacetic acid and 1.0 mg L−1 kinetin) and AII (1.0 mg L−1 2,4-dichlorophenoxyacetic acid and 0.1 mg L−1 kinetin). Two different cell lines – chlorophyllated cells and non-chlorophyllated cells – were selected from cultures carried out in AII medium, while only one cell line was obtained in AI medium. Cells were subcultured every 30 days and maintained at 28 ± 2 ºC under 16-hour-day photoperiod.
Ten-week-old callus cultures were inoculated into a 250 mL erlenmeyer flask containing 100 mL of MS medium with 30 g sucrose L−1. The suspension cultures were established using different callus lines: calli previously cultured on AI semi-solid MS medium were inoculated into AI liquid MS medium (Culture 2); non-chlorophyllated calli cultivated on AII semi-solid MS medium were inoculated into both AI liquid MS medium (Culture 1) and AII liquid MS medium (Culture 4) and chlorophyllated calli cultured on AII solid MS medium were inoculated into AII liquid MS medium (Culture 3). Cultures were subcultured every 30 days and maintained in a growth room, under agitation (110 rpm) on orbital shaker, at 28 ± 2 ºC exposed to a 16-hour-day photoperiod.
Candida albicans, Fusarium oxysporum and Penicillium avelanium were cultured in liquid potato/dextrose medium under agitation (110 rpm) on orbital shaker at 28 ºC. After 96 h of incubation, the cultures were autoclaved and the micelia were collected by filtration and then dried. C. albicans, F. oxysporum, P. Avelanium and Saccharomyces cerevisiae micelia (5, 10 and 50 mg mL−1) were homogenized in deionized water and autoclaved. Fungal homogenates were added separately to the suspension cultures either at the early (14 days) or late exponential phase of growth (20 days). No elicitor was added to the control cultures. All experiments were conducted with four A. scholaris suspension lines (Cultures 1–4). For a time course study, untreated and elicited suspension cultures were harvested at different time intervals (24, 48 and 72 h) by vacuum filtration. Triplicate flasks were run for each treatment and controls.
Dry and powered material (200 mg) was extracted overnight, successively with chloroform and methanol (5 mL each) at room temperature. Chloroform and methanol extracts were grouped and the solvent evaporated at room temperature. Crude extract was then re-suspended in water and partitioned three times with ethyl acetate. The combined organic extracts were evaporated under vacuum, residues were dissolved in 1 mL of methanol and analysed by HPLC. HPLC analysis conditions: Shimadzu LC10ADvp system equipped with Supelco LC18 column (250 × 4.6 mm), coupled to a diode array detector. Samples were eluted with methanol: H2O (Acetic acid, 0.1%), 85:15, at 1 mL min−1 under isocratic condition and monitored at 210 nm. Twenty microlitre of each solution from oleanolic acid, ursolic acid and samples were used for quantitative analysis. The identification of oleanolic and ursolic acid was done by comparing their retention times and spectral data with those of standard compounds. All quantifications were performed in triplicate the independent experiments: elicited and control cultures. The quantification was evaluated using external calibration curves. Calibration curves with their respective standards were developed with diluted samples of standard compounds within the range of 1.0–0.01 mg mL−1. Authentic ursolic acid and oleanolic acid were purchased from Aldrich Co. Relative standard deviation was obtained as appropriate. Analyses of variance, followed by LSD post hoc determinations, were performed. All computations were done using the statistical software STATISTICA 6.0 (StatSoft Italia srl).
Results and discussion
Callus culture was initiated from axenic leaf explants of A. scholaris inoculated on MS solid medium supplemented with two combinations of 2,4-D and kinetin (1:1 and 1:0.1). Calli cultured on AI medium developed homogenous friable of yellow pigmented biomass and calli maintained on AII medium developed heterogeneous friable biomass with chlorophyllated and non-chlorophyllated cells. Chlorophyllated and non-chlorophyllated cells were subcultured in AII liquid medium. Thereafter, all callus and suspension cultures were maintained and subcultured in AII or AI media.
As a response to the biotic stress caused by microorganisms, the A. scholaris cells were stimulated, increasing the biosynthesis of triterpenoids (–). The experiments were carried out with A. scholaris cells either in the early or late exponential phase of growth (14- or 20-day-old). Cultures were homogenous in appearance, but when stressed with fungal homogenates, there was a rapid change in their colour. The effects of various elicitors on triterpene accumulation in A. scholaris suspension cultures are shown in –. Addition of cell wall homogenates, no matter the concentration or type of microorganism source tested, resulted in increased triterpene levels. Cultures 2 and 3 were more susceptible to fungal homogenate treatments showing higher yields of oleanolic and ursolic acids. Among the elicitors tested, S. cerevisiae showed better results in terms of triterpene accumulation, producing 5 and 7 mg g−1 dw, of ursolic and oleanolic acids, respectively, followed by C. albicans, which produced 2 and 3 mg g−1 dw and P. avelanium 1–2 mg g−1. Triterpene accumulation in cultured cells treated with F. oxysporum homogenates was slightly stimulated. In most experiments, the maximum production of triterpenes was achieved around 72 h of culture in an elicitor dose related accumulation. When fungal homogenates were added to the suspension cultures at the early exponential phase, 14 days of culture, the production of triterpenes was not significant compared to control cells. This situation was verified twice in the third and sixth subcultures (Experiments 1 and 2).
Table 1. Acquiring of triterpenes in A. scholaris cultures after fungal elicitation (Culture 1).
Table 2. Acquiring of triterpenes in A. scholaris cultures after fungal treatment (Culture 2).
Table 3. Acquiring of triterpenes in suspension cultures of A. scholaris after fungal treatment (Culture 3).
Table 4. Acquiring of triterpenes in suspension cultures of A. scholaris after fungal treatment (Culture 4).
Several reports have shown that secondary metabolism in plants’ cell culture are stimulated or inhibited with fungal homogenates.[Citation1,Citation2,Citation21] In our study, elicitors induced a rapid stimulation of the secondary metabolism pathway of A. scholaris cells increasing the biosynthesis of triterpenoids. The rapid change in suspension cultures with fungal homogenates was also reported in elicitation experiments with other plant cells. Obtained results corroborate the theory that components of the cell wall homogenates act as signalling molecules of triterpenoid biosynthetic pathway, triggering the production of oleanolic acid and ursolic acid by treated cells.[Citation3,Citation22–24] When the extracts of A. scholaris unelicited cells were quantified, triterpenes were almost not detected. The regulation and enzymology of pentacyclic triterpenoid phytoalexin biosynthesis in cell suspension cultures of T. divaricata have been investigated.[Citation4,Citation25,Citation26] In T. divaricata, C. albicans elicitor preparations resulted in the production of considerable amounts of ursolic acid type pentacyclic triterpenoids, while alkaloid production was blocked under such conditions.[Citation27–29] A similar regulatory mechanism would be expected in A. scholaris cells. Neither TLC nor HPLC analysis were performed in this work for alkaloid production. A. scholaris extracts and ursolic acid exhibited inhibitory activities against B. pilosa, indicating that ursolic acid is the major allelopathic substance of A. Scholar.[Citation7,Citation9,Citation10,Citation14] Limited data regarding the allelopathic activity of triterpenes or triterpenoids are available.[Citation11,Citation30,Citation31] Macias et al. [Citation32] reported that lupane triterpenes stimulated germination of L. sativa seeds at both high and low concentrations. Triterpenoid acids are known to be mainly active against monocotyledon species and to show higher activity levels than other triterpenes.[Citation32–34] Kong et al. [Citation35] reported that fallen leaves of L. camara reduced the growth of the aquatic weed Eichhornia crassipes and the alga Microcystis aeruginosa.[Citation36–38] Two fractions with high inhibitory activity were isolated and identified as the pentacyclic triterpenoids lantadene A and lantadene B. Both compounds significantly inhibited growth of E. crassipes and M. aeruginosa at low concentrations.[Citation39–41]
Conclusions
In the present study, the effects of C. albicans, F. oxysporum, P. avelanium and S. cerevisiae homogenates, with varying concentrations, exposure time and the type of synergetic organisms, on triterpene production by A. scholaris cultures were examined. The observations support the hypothesis that triterpenoids present in A. scholaris may play a major role in allelopathic interactions with neighbouring plants. The procedures described in this work may be employed in strategies for enhancement in productivity of secondary metabolites and for investigating the complex secondary metabolite pathways in plant tissue cultures.
Disclosure statement
The authors declare no potential conflict of interest.
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