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Ceratotheca triloba Hook.f. produce two important compounds 9,10- anthracenedione and 1-hydroxy-4-methylanthaquinone in their roots. These compounds are structurally similar to mitoxantrone which is the current drug of choice for the treatment of breast cancer and acute leukemias. In this study, cell suspension cultures of C. triloba were established and the production of 9,10- anthracenedione and 1-hydroxy-4-methylanthaquinone was increased by elicitation with methyl jasmonate. The compounds produced were verified by comparison to known standards using HPLC analysis. The production of 1-hydroxy-4-methylanthaquinone in the elicited culture increased by 37.5 - fold after the one month culture period, compared to the control culture. A strategy to formulate growth medium, culture age and an appropriate elicitor was established. By adding methyl jasmonate to a 21 day old culture (with a biomass concentration of 19 g.l-1) cultivated in MS medium at an incubation temperature of 26ËšC, 1-hydroxy-4-methylanthaquinone can be produced at 0.75 µg.ml-1.
Abbreviations: 6-BAP- 6-benzylaminopurine; 2,4-D- 2,4-dichlorophenoxyacetic acid; AN-
9,10- anthracenedione; MA-1-hydroxy-4-methylanthaquinone; R- root extract; IC- intracellular extract; EC- extracellular extract
Currently cancer treatment involves radiotherapy or chemotherapy. These treatment regimens are effective but have many side effects; hence new bioactive compounds such as anthraquinones are being investigated. Anthraquinones are a class of natural compounds that consist of the basic structure of 9, 10-anthracenedione (Sajc et al. 1999). Their derivatives currently represent one of the most effective cytostatic agents used as front line therapy for a variety of systematic and solid tumors (Weiss et al., 1986). Examples of drugs containing the 9,10- anthracenedione moiety include: doxorubicin and mitoxantrone (McClendon and Osheroff 2007). From our earlier studies we have isolated two anthraquinones, 9,10- anthracenedione and 1-hydroxy-4-methylanthaquinone from root extracts of Ceratotheca triloba (Figure 1 and 2). These compounds are structurally similar to mitoxantrone. The production of anthraquinones from root extracts is however limited as it require harvesting of large quantities of field grown material. Plant growth is also negatively affected by biological influences (pathogen sensitivity and insects) during the winter months. To overcome these limitations the production of the potential anticancer anthraquinones from C. triloba was investigated by plant cell culture technology. Plant cell suspension cultures are the preferred mode of producing phyto-pharmaceutical compounds because they are amendable to good manufacturing practice (GMP) procedures and the production of the compound can be relatively easily scaled up from the shake flask stage to large-scale bioreactors (Schlatmann et al. 1996; Wen 1995). Studies have been conducted (Bulgakov et al. 2002, Oliveira et al. 2007, Orban et al. 2008 Jasril et al. 2003, Han et al. 2002) on the production of various derivatives of 9, 10- anthracenedione in plant cell cultures (Example: Rubia cordifolia, Rudgea jasminoides, Rubia tinctorum L, Morinda elliptica and Cinchona robusta). However there, is no literature that reports the production of 9,10- anthracenedione and 1-hydroxy-4-methylanthraquinone from the C. triloba cell culture. C. triloba is a South African plant that belongs to the family Pedaliaceae. It is widely distributed in the summer rainfall areas, especially grass lands, rocky places and on disturbed ground and along roadsides. (Tredgold 1986). The whole plant soaked in water may be used as a substitute for soap or shampoo. C. triloba can also serve as source of traditional medicine to treat painful menstruation, stomach cramps, nausea, fever and diarrhea (Hutchings 1996)Tredgold 1986). Previous nutritional, chemical and antioxidant studies were conducted on C. triloba to preliminary assess of the nutritional value. In terms of traditional leafy vegetables, C. triloba serves as a good source of energy and magnesium (Odhav et al. 2007).
In this study an attempt was made to enhance the yield of anthraquinones produced in C. triloba cell suspension cultures using methyl jasmonate as an elicitor. Generally, when plant cells are exposed to chemical and environmental elicitors via specific plant receptors, certain biological responses are triggered which lead to the activation of biosynthetic genes and subsequently the production of plant secondary metabolites (Yukimune et al. 1996). The main advantage of using this strategy is that it reduces the time taken to obtain high yields of the secondary metabolites (Barz et al. 1988; Eilert 1987; DiCosmo and Tallevi 1985). Jasmonates play a key role in eliciting biological responses that lead to the accumulation of secondary metabolites (Gundlach et al. 1992). Methyl jasmonate was used in this study as it has been proven to increase the production of phyto-pharmaceutically valuable compounds, such as paclitaxel and baccatin III from Taxus species (Yukimune et al. 1996) and Ajmalicine from Catharanthus roseus (Parsons et al. 2004).
Materials and methods
Specimens of C. triloba in flower were was collected in Durban, Province of Kwazulu Natal, South Africa, and identified at the Ward Herbarium using taxonomic keys. Voucher specimens were deposited at the Durban Botanical Herbarium.
Plant cell culture
Leaves of the C. triloba plant were removed and washed with distilled water three times and sterilized with HgCI2 and NaCIO. The sterilization agents were tested individually and in combinations using the exposure time/s indicated in Table 1. Excess detergent remaining on the leaves was washed off with sterile distilled water at each interval. Leaves submerged in distilled water for 20 minutes, served as the control. The sterilized and control leaves were cut into 0.5 cm square disks and were placed on MS medium (Murashige and Skoog 1962) (Sigma-Aldrich, Inc) which was supplemented with 1 mg.L-1 2.4-D and 6-BAP (Sigma-Aldrich, Inc). The leaf disks (12 replicates for each treatment) were incubated in the dark phase at 26ËšC for one week and were visually screened on the seventh day. The percentage of contamination and level of tissue damage on each explant were recorded. The sterilization regimen that yielded no contamination was subsequently used to mass produce callus cultures. These cultures were transferred onto fresh medium and maintained by sub-culturing at 3 week intervals.
Approximately 2 g of callus (three weeks old) from the second sub-culture was transferred into 250 ml Erlenmeyer flasks containing 50 ml of MS liquid medium supplemented with1 mg.L-1 of 2.4-D and 6-BAP. The flasks were agitated on a shaker (Infors Ecotron) at 100 rpm and incubated at 26ËšC in the dark phase for one week. These cultures were scaled up to 400 ml volumes in 1000 ml flasks by transferring 70 ml of medium to 30 ml of culture. After one week of culturing 300 ml of medium was added into the flasks. A growth curve was constructed to obtain sufficient cell mass for elicitation. All flasks were sampled at 7 day intervals to determine the quantity of cell mass by wet weight analysis. Triplicate samples of the cell suspension culture (2 ml) were vacuum filtered through pre-weighed filters (0.22 µm, 47mm,white grid, Millipore) after which each filter containing wet biomass was measured. The wet weight was determined by the following equation: [(Wet weight + filter) - (filter)] x 500 = wet weight (g. L-1). Methyl jasmonate (Sigma-Aldrich, Inc) solution was prepared at a concentration of 100 µM in ethanol. An aliquot of 1000 of the elicitor solution (2.5 µl of 100 µM of methyl jasmonate per ml of culture) was filter sterilized (0.22 µm filter) into two flasks on day 21. An equal volume of ethanol was filter sterilized into two flasks to serve as control cultures. The elicitation was conducted for a 9 day period and cell suspension cultures were harvested at day 30 to perform extraction and chromatographic analyses.
Extraction of anthraquinones from C. triloba roots
Healthy, uninfected roots of the C. triloba plant were dried room temperature for 5-8 days. Once completely dry, plant material was ground to a fine powder using a Wareing blender and stored in a closed container at room temperature until required. The ground material was extracted with the least polar solvent, hexane. The hexane extract was evaporated and stored. The macerate was reconstituted in hexane and the extract was combined with the initial extract. This procedure was repeated three times.
Extraction of anthraquinones from C.triloba cell suspension cultures
The cell mass of suspension cultures were harvested by centrifuging 50 ml volumes at 4000 rpm for 10 minutes at 20ËšC. The cell mass was disrupted by sonication (Virsonic, Virtis) at 4 psi for 10 minutes. Anthraquinones were extracted by agitating the disrupted cell mass on a shaker at 180 rpm for 24 hours at room temperature in 100 ml of hexane. The supernatant was agitated in 200 ml hexane. Hexane fractions were separated and concentrated by using a roto-evaporator (Heidolph Laborota 400 efficient) with the water bath set at a temperature of 50°C and the flask rotated at 60 rpm. The residues where dissolved in 10 ml of hexane while the excess residue that was fixed to the flask was dissolved in 5 ml of ethyl acetate. The hexane and ethyl acetate fractions were then pooled and air dried for 3 days to further concentrate the extract preparation for chromatographic analyses.
Detection of anthraquinones produced in C. triloba cells by TLC
Thin layer chromatography was performed to detect anthraquinones in cell and supernatant extracts by using of 9,10- anthracenedione and 1-hydroxy-4-methylanthaquinone standards (1 mg.ml-1 in ethyl acetate) (Sigma-Aldrich, Inc) . Approximately 10 µl of each standard solution, 20 µl of the root extract (100 mg.ml-1 in hexane) and 50 µl of the cell extract (dissolved in ethyl acetate) were applied to the TLC silica gel plate (Merck TLC F254). Petroleum ether: ethyl acetate: formic acid (75:25:1), ethyl acetate: methanol: water (100:13.5:10) and hexane: ethyl acetate (90:10) were used as mobile phases. Separated anthraquinones were visualized under visible and ultraviolet light (254 and 360 nm, Camag Universal UV lamp TL-600) after the TLC plates were sprayed with 5 % KOH in ethanol or p-anisaldehyde solution (13.31 ml of anisaldehyde in 250 ml of ethanol and 2.5 ml of H2SO4) (Wagner et al., 1984). Plates sprayed with p-anisaldehyde were developed by heating at 120ËšC in a oven for 20 minutes.
Identification and quantification of anthraquinones by HPLC analysis
HPLC analysis was carried out according to the method of Fernand et al. (2008). Cell extracts were dried at room temperature for 3 days and dissolved in 1 ml of ethanol, the filtrates were used for HPLC analysis. Separation and quantitative analyses of anthraquinones were performed on a Merck- Hitachi LaChrom system (Darmstadt, Germany) consisting of a D 7000 system controller, four pumps (D7400), a Merck- Hitachi LaChrom (L-7200) auto injector and an Merck- Hitachi LaChrom (L-7200) UV-VIS detector (λ = 260 nm). Separation of the analytes was performed at 40 ËšC on a Licrospher C18 (2) column, 100 ËšA pore size, 5µm particle size, 250-4.6mm i.d.column containing a guard column (Merck, Darmstadt, Germany). The analytes were eluted isocratically at a ¬‚ow rate of 0.4mL/min using an acetonitrile/methanol/buffer (25:55:20,v/v). The buffer used as 10mM ammonium acetate at pH 6.8. The injection volume was 10 µL.
Results and discussion
Sterilization of C. triloba explant material
A major challenge at the initial stage of developing the C. triloba cell culture system was to overcome the contamination in field grown plants, as this was the source of explant material. It was therefore important to study effect of two surface sterilization agents on contamination and the leaf tissue. The most prevalent type of contamination in C. triloba explants was fungal while bacterial contamination occurred randomly. The percentage explants contaminated with fungi were 100 % with respective treatments due to the spread of the contamination to all explants in the plate. The percentage of explants contaminated with bacteria was 16.67 % as the bacterial contamination remained localized to the affected explants (Table 2).
Explants treated with NaCIO (30%) and HgCI2 (0.1%) in combination eradicated all contaminants present in the leaf explants, therefore this treatment was used for initiation of callus. The effectiveness of this treatment could be due to synergistic effect of the two surface sterilization agents as contaminated explants resulted when they were used separately. However a high level of tissue damage was observed in the explants when this treatment regime was applied (Table 2). A possible reason could be that leaf (light green leaf) explants were exposed to this treatment contained a high level of meristematic tissue. The higher degree of meristematic tissue in an explant the more liable it could be to tissue damage caused by the surface sterilization agents. Therefore, leaves with a lower level of meristematic tissue (green leaves) were selected and surface sterilized with NaCIO (30%) and HgCI2 (0.1%) to induce callus cultures from the C. triloba plant.
Callus initiation was observed on the surface and cut ends of the explants after 2-3 weeks of inoculation. After five weeks the entire leaf explant was transformed into callus tissue. Callus cultures induced on MS medium were orange- yellow in color. These cultures turned brown as they aged and the medium subsequently became yellow, this could be due to the release anthraquinones. Abdullah et al. (1998) also observed the same phenomenon. Sub-cultured callus tissue produced root hairs and root- like structures after three weeks and these contained yellow pigment on tips of the root-like structures. (Figure 3). These pigments can be used as a marker for selecting high yielding cell lines.
Cultivation of cell suspension cultures for elicitation of anthraquinones
The establishment of cell suspension cultures from callus tissue was a key step in developing an efficient cell culture system for producing anthraquinones as liquid cultures have a faster growth rate compared to callus. Callus cultures were transitioned into liquid medium, the friable callus tissue dispersed into small aggregates when flasks were placed on the shaker. A growth curve of C. triloba cell suspension cultures was generated to obtain sufficient biomass to elicit the production anthraquinones (Figure 4). The biomass concentration increased from 5.50 g.l-1 to 19 g.l-1 after 20 days of cultivation; however this increase accounts only for the cell mass that remained in suspension during sampling as very large aggregates tend to sink to the bottom of the flask . After 20 days dense, orange- yellow cell suspension cultures with large aggregates formed. Therefore, cell suspension cultures were elicited on day 21 with methyl jasmonate. The production of cell suspension cultures with a highly dense cell mass is crucial for obtaining high yields of the plant-derived compound as secondary metabolites are based in intra-cellular parts of the cell (Luckner 1990). According to Figure 4, a significant increase in biomass occurred after day 21. This could be due to the high level of aggregation that occurred in the control (unelicited) and elicited cultures. A sharp decrease in cell mass occurred in the control on day 30 due to the formation of large aggregates in suspension (larger cell aggregates tend to sink to the bottom of the flask during sampling). Figure 5 shows the aggregates in the elicited culture were smaller than the control culture. A cultivated plant cell suspension culture with a high concentration of cell aggregates is an ideal target for the elicitor as cell aggregation is associated with secondary metabolite production (Bais et al. 2002).
Elicited cell suspension cultures turned dark brown 2 days after the addition of methyl jasmonate while control cultures remained orange-yellow (Figure 5). A similar trend was observed for M. elliptica cells which turned brown when anthraquinones were produced (Abdullah et al. 1998). The dark brown color of the cell aggregates can be used as an indication that anthraquinones were elicited. Since the accumulation of anthraquinones in C. triloba cell suspension cultures is coupled with cell aggregation and cell browning, the cell aggregates should be obtained and assessed for the production of anthraquinones in order to select high yielding cells for future studies.
Analysis of cell suspension culture extracts
The anthraquinones of interest, 9,10- anthracenedione and 1-hydroxy-4-methylanthaquinone could not be detected in control (unelicited) and elicited culture extracts when the TLC plate sprayed with p-anisaldehyde (Figure 6). The TLC plate sprayed with 5% KOH plate was viewed under UV light. No anthraquinones were detected under 254 nm (Figure 6) in the culture extracts. However when the TLC plate was viewed under UV light at 360 nm, 9.10-anthraquinone fluoresced orange and 1-hydroxy-4-methylanthaquinone fluoresced yellow in the root extract (Figure 6). In comparison to the root extract, only 1-hydroxy-4-methylanthaquinone (yellow fluoresces) was detected in the intra-cellular extracts of the elicited and control cultures. The anthraquinone standards confirmed the presence of anthraquinones in the intra-celluar extracts of the control and elicited cultures but individual anthraquinones could not be detected as both standards co-eluted when the two mobile phases: petroleum ether: ethyl acetate: formic acid (75:25:1) and ethyl acetate: methanol: water (100:13.5:10), were employed
TLC and HPLC analysis showed that anthraquinone accumulation was principally intracellularly based as the concentrations of the intracellular extracts were higher than that of the supernatant extracts (Figure 6 and Table 3). HPLC analysis showed the 9,10- anthracenedione and 1-hydroxy-4-methylanthaquinone standards eluted at retention times of 5.90-6.20 minutes and 6.90-7.40 minutes respectively (Figure 7). The elicited and control culture extract (intra-cellular) profiles showed a peak at 6.91 and 7.05 minutes, respectively. 1-hydroxy-4-methylanthaquinone was identified in both the extracts (Figure 8 and 9). 9,10- anthracenedione was not identified in the control and elicited cultures profiles. This was due to co-elution of 9,10- anthracenedione with other anthraquinones in the sample (Figure 7 and 8). Co-elution occurs when compounds in a sample do not separate due to the similarity of the structure between the compounds which in turn influences the elution time of the similar compounds.
A higher concentration of 1-hydroxy-4-methylanthaquinone was present in the elicited culture extract (0.75 µg.ml-1) than the control culture extract (0.02 µg.ml-1) (Table 3). Earlier studies have shown that the production of secondary compounds in cell culture systems were dramatically increased through the elicitation strategy (Wang and Zhong 2002; Yu et al. 2002). This strategy was proven to be successful in the C. trioba cell culture system as production yield of 1-hydroxy-4-methylanthaquinone in the elicited culture increased 37.5 - fold in comparison to the control culture.
This is the first study that shows C. triloba cell suspension cultures can micro-propagated by plant cell culture techniques and produce potential bioactive compounds for cancer therapy. A strategy to formulate growth medium, culture age and an appropriate elicitor was established. By adding methyl jasmonate to a 21 day old culture (with a biomass concentration of 19 g.l-1) cultivated in MS medium at an incubation temperature of 26ËšC, 1-hydroxy-4-methylanthaquinone can be produced at 0.75 µg.ml-1.