Abstract: Cell suspension culture from friable callus tissue of Cereus peruvianus was established in a liquid medium, similar to that used for long-term callus culture. Its aim was to increase, in an alternative manner, alkaloid and enzymes production and to induce alkaloid release in the culture medium. Growth of callus-cell in suspension culture slightly increased, though maximum cell mass was detected after 42 days. Whereas total alkaloid production in cell suspension appeared in the early phase of the growth cycle, the second alkaloid flush appeared at the end of growth stage. In the growth medium, maximum accumulation of alkaloid also occurred after the period of maximum growth. Alkaloid production from cell suspension culture of C. peruvianus may be considered effective since the intracellular production showed that the level of alkaloids was three times higher in cell suspension than in callus tissues and extra-cellular accumulation of the alkaloid may be directly recovered from the culture medium. Suspension cell culture from callus can also be a source for enzyme extraction of the Est and ADH isozymes and may provide further possibilities in biotransformation reactions.
Introduction
Yield increase of compounds and reduced cost of time and capital are the great goal of biotechnology. When compared to the main molecules found in plants, the plant’s secondary compounds are usually low in abundance. Different strategies, including in vitro systems, have been extensively studied to improve the production of secondary plant compounds (Kutney, 1996; Boungard et al., 2001; Sato et al., 2001). In vitro cell culture is beneficial because useful metabolites are obtained under a controlled environment, regardless of climatic changes and soil conditions (Collin, 2001). Living plants are inconvenient because generally present different concentrations of the target compounds, which may depend on the specific time of the plant harvest (Salmore and Hunter, 2001; Puricelli et al., 2002; Ralphs and Gardner, 2001). Moreover, their exploitation may cause gradual genetic erosion.
Callus tissues culture of Cereus peruvianus have been used as source of alkaloids (Oliveira and Machado, 2003), polysaccharides (Machado et al., 2004) and fatty acids (Machado et al., 2006). Comparison with alkaloid production by C. peruvianus plants and by callus tissue has indicated that alkaloid levels were almost twice as high in callus tissues than in plant shoots (Oliveira and Machado, 2003). Tyramine and hordenine (N,N-dimethyltyramine) are the main alkaloids in the C. peruvianus species (Vries et al., 1971). Tyramine has shown important pharmacological applications. It has been reported that tyramine mimics insulin action in diabetic rats and in cell cultures (Bairras et al., 2003; Visentin et al., 2003; Subra et al., 2003). Whereas tyramine is also a neurotransmitter/neuromodulator that acts through G protein-coupled receptors in invertebrates (Roeder, 2004; Donini and Lange, 2004), hordenine has been reported to be an inhibitor of noradrenaline uptake (Barwell et al., 1989).
Tyramine and hordenine were obtained from callus tissues of C. peruvianus (Oliveira and Machado, 2003). However, callus culture develops special problems for downstream processing since cells should be collected and the product extracted. It would thus be beneficial to develop methods by which product excretion would be directly introduced into the growth medium (San Miguel-Chaves et al., 2003). When compared to callus culture, cell suspension recovers larger quantities of relatively homogeneous, undifferentiated cells from which compounds may be more easily isolated and makes possible the excretion of secondary products into the medium. Extra-cellular production avoids destruction of the biomass used for compound extraction since these may be directly recovered from the medium.
Most suspension culture is obtained by the transfer of friable callus lumps for agitated liquid medium of the same composition as that used for callus growth. In current study, suspension cell culture from C. peruvianus callus tissues provides an alternative method to increase alkaloid and enzymes production.
Materials and Methods
Callus tissues of C. peruvianus were induced from hypocotyls in MS medium (Murashige and Skoog, 1962) containing B5 vitamins (Gamborg et al., 1968), 0.8% agar, 3% sucrose, 4.0 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.0 mg L-1 N-(2-furanylmethyl)-1H-purine-6 amine (kinetin) and maintained at 32°C, during a 16 h photoperiod (15 m-2 sec-1 light intensity) (Oliveira et al., 1995). Long-term and non-regenerant callus tissues have been subcultured in fresh medium at 25 to 30 day intervals during 13 years in the Plant Tissue Culture and Electrophoresis Laboratory (State University of Maringa; Maringa, PR., Brazil).
The suspension culture of a cell line was initiated from the long-term cultures on a liquid medium similar to that for callus culture, excluding the agar. Medium was adjusted to pH 5.8 and then sterilized by autoclaving at 121°C, for 20 min. The suspension culture was maintained in 250 mL Erlenmeyer flasks containing 100 mL of liquid medium and 1g (fresh weight) of friable callus tissue. The mixture was stirred on a rotation shaker (120 rpm and 28±2°C).
Cell growth was determined by measuring the increase of the culture’s dry cell weight. The biomass in the culture was thus separated from the liquid by filtration under vacuum and then lyophilized to obtain the dry cell weight.
Extraction and Isolation of Alkaloids
The freeze-dried material (Table 1) was ground and refluxed
for 1h with a solution 0.33% acetic acid prepared in 70% ethanol (50 mL, v/v).
After filtration, ethanol was evaporated under reduced pressure to produce an
acid aqueous solution.
| Table 1: | Growth (dry wt., mg mg-1) of Cereus peruvianus cell over a period of 56 days in suspension and intra-(mg mg-1 of dry wt.) and extra-cellular (mg mL-1) alkaloid production |
The solution was alkalinized with NaOH 0.5 M to reach pH 8.0-9.0 and extracted three times with the same volume of CH2Cl2. Extract was subsequently evaporated under reduced pressure and the residue was dissolved in 10 mL HCl 0.1 N.
Identification of Alkaloids
Alkaloids tyramine and hordenine were isolated and identified by their physical
and spectral properties (Oliveira and Machado, 2003). Alkaloids in the dichlorometane
extracts were identified by thin-layer chromatography, silica gel G60 saturated
with 0.1 N KOH, using CHCl3:Methanol (95:5, v/v) as eluent, whereas
ninhydrin spray was used as chromogenic reagent, by comparison with authentic
standards of tyramine (Rf = 0.12) and hordenine (Rf =
0.66).
Alkaloids Analysis
Alkaloid concentration was determined by ultraviolet-visible (UV-VIS) spectrophotometer
(Varian Cary-1E). UV detection wavelength was 274 nm; tyramine hydrochloride
(1 mg mL-1, Sigma-Aldrich Co., St. Louis, MO) was used as standard,
since tyramine and hordenine, the alkaloids previously identified from C.
peruvianus (Oliveira and Machado, 2003), presented the same chromophore
group in UV spectrum.
Electrophoresis for Enzyme Analysis
Electrophoresis evaluations were carried out on samples consisting of cells
in suspension. Cells (200 mg) were homogenized with a glass rod in an Eppendorf
microcentrifuge tube using 20 μL of 1.0 M phosphate buffer, pH 7.0, containing
5% PVP-40, 1.0 mM EDTA, 0.5% β-mercaptoethanol, 0.5% triton X-100 and 10%
glycerol solution. After homogenization, the samples were centrifuged at 25,000
rpm for 30 min at 4°C in a Sorval 3K-30 centrifuge and the supernatant (35
μL) was used for each sample.
For esterase isozymes (Est; EC 3.1.1.) analysis was used polyacrylamide gels (12%) prepared with 0.375 M Tris-HCl, pH 8.8, as buffer (Ceron et al., 1992). Electrophoresis was performed for 5 h at 4°C, at a constant voltage of 200V. Running buffer was 0.1 M Tris-glycine, pH 8.3. Staining techniques of Johnson et al. (1966) and Steiner and Johnson (1973), modified by Ceron et al. (1992), identified the esterases. The gels were soaked for 30 min in 50 mL 0.1 M sodium phosphate, pH 6.2, at room temperature and esterase activity was visualized by placing the gels for 1 h in a staining solution prepared with 50 mL of sodium phosphate solution, 30 mg of β-naphthyl acetate, 40 mg of α-naphthyl acetate, 60 mg of Fast Blue RR salt and 5 mL of N-propanol.
Electrophoresis condition and the reaction mixture for the detection of alcohol dehydrogenase isozymes (ADH; EC 1.1.1.1) of C. peruvianus tissues in starch gels were reported by Mangolin et al. (1994).
Results and Discussion
Table 1 show the weight of the dry cell mass of C. peruvianus callus-cell in suspension culture over a period of 56 days and the corresponding growth. Growth increased slightly, though the maximal cell mass was detected on the 42nd day.
Production of alkaloid was not directly growth related (Fig. 1 and Table 1). There is evidence that total alkaloid production in C. peruvianus cell suspension appeared in the early phase of the growth cycle, but the higher alkaloid production appeared at the end of growth stage (after 42 days). Maximum accumulation of the alkaloid in the growth medium occurred too after the period of maximum growth.
Bougaud et al. (2001) explained why the alkaloid production is not directly related to cellular growth. According to these authors, the lack of secondary metabolite production during the early stages of growth may be explained by carbon allocation mainly distributed for primary metabolism (building of cell structures and respiration) during which growth is active. On the other hand, when growth ceases, carbon is no longer needed in large quantities for primary metabolism and secondary compounds are more actively synthesized.
Alkaloid production from cell suspension culture of C. peruvianus may be considered efficient since it is higher (about 2% yield; Table 1) than alkaloid production from callus tissue (about 0.6% yield; Oliveira and Machado, 2003). Thus, suspension cell culture from C. peruvianus callus tissues provides an alternative to increase alkaloid production.
| Fig. 1: | Intracellular alkaloid production by Cereus peruvianus cell in suspension and accumulation of alkaloid in growth medium |
| Fig. 2: | α-and β-Esterase isozymes from callus (lanes 1-5) and suspension cell (lanes 6-8) of Cereus peruvianus in polyacrylamide gel showing specific esterase (Est-s) induced in suspension cells (arrow) and a increased expression of the isoesterases produced by Est-2, Est-3, Est-5 and Est-8 loci |
In fact, intra-cellular production showed that alkaloid level was three times higher in cell suspension than in callus tissues. Additionally, extra-cellular accumulation of the alkaloid may be directly recovered from the culture medium.
Extra-cellular production of alkaloid is an important positive aspect in the present study; extra-cellular accumulation of products is relevant in industrial processes involving bioreactor systems. It seems that there is an increase in intra-cellular concentration of alkaloid preceding further release into the medium. However, alkaloid concentration in the medium (μg mL-1) was not in all cases lower than the concentration in the cells (μg mL-1) (Table 1). It may be possible that, through long-term adaptation of C. peruvianus cell suspension into an optimal medium, higher alkaloid releases could be produced into the medium. The long-term adaptation of cell suspension culture to the optimal medium may produce changes in the alkaloid’s quantitative and qualitative rates (Rhodes et al., 1986; Robins et al. 1986). Subsequent long-term adaptation to lower growth regulators medium has led to an increase in accumulation and in the relative proportion of alkaloids. Other strategies that can be used to induce the alkaloid releaser is the cell immobilized system.
| Fig. 3: | Alcohol dehydrogenase isozymes (ADH) from callus (samples 1-3) and suspension cell (samples 4-5) of Cereus peruvianus detected in starch gel electrophoresis showing the increased expression of the ADH-3 isozyme (product of the Adh-2 locus; Mangolin et al., 1994) |
Higher levels of alkaloid from C. peruvianus cell-suspension culture may also be obtained by the addition of tyrosine in culture medium. In fact, tyramine content increased by 11% in callus tissues under original conditions when compared with control callus tissues to which tiramine was added in solid culture medium (Oliveira and Machado, 2003). The feeding of the culture with amino acids and its synthetic analogous (precursors of phenylpthylomins) can go to induction of different alkaloids from tyramine group.
The time course for intra-and extra-cellular alkaloid accumulation may be obtained by transference of higher callus tissues quantities for liquid medium. Preliminary researches have shown that transference of 5 g to 100 mL of medium reduced the maximal period for cellular growth to 28 days (Oliveira et al., 2005).
Besides alkaloid production other positive aspects for C. peruvianus cell-suspension culture is that specific Est-s isozyme was detected in cell suspension (Fig. 2, arrow) and carboxylesterases (EC 3.1.1.1) produced by Est-2, Est-3 and Est-8 loci and arylesterase (EC 3.1.1.2) p reduced by Est-5 locus, were detected as most intensely stained band, from 5 days after transfer of callus cells to suspension culture (Fig. 2). Cell suspension cultured for 5, 10, 15 and 21 days also showed ADH-3 being more stronger stained (Fig. 3). The more intensely stained Est and ADH isozymes are an indication of differential gene expression and quantitative differences of isozymes production in callus-cell in suspension culture. Thus, suspension cell culture from callus tissues of C. peruvianus may be a source for esterase and alcohol dehydrogenase enzymes extraction and/or that can will be used in biotransformation reactions using induced enzymes. Suspension cell of C. peruvianus have been used for biocatalysis in the selective interconversion of functional groups such as carbonyl reduction and ester hydrolysis (unpublished results).
Callus-cell in suspension culture of C. peruvianus is beneficial because useful metabolites and enzymes are obtained under a controlled environment, free from microbe and insect contamination, regardless of climatic changes and soil conditions and the cell-suspension may be subcultured and indefinitely propagated on the optimized medium, substituting the use of adult plants. Cloning of C. peruvianus cell lines may provide further optimization for end product yield and is a suitable source for industrial procedures of extraction because the same protocol can be quickly and easily standardized by using genetically uniform material.
Acknowledgments
The authors would like to thank Lucílio Gobbi Filho and Sérgio Luiz Calvi for their excellent technical assistance.