Over Production Of Anthraquinones Biology Essay

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Earlier studies on the Ceratotheca triloba Hook.f. plant have shown that its roots contain three anthraquinones. Two of these compounds; 9,10- anthracenedione and 2-methyl-4-hydroxy anthraquinone are structurally similar to mitoxantrone which is the current drug of choice for the treatment of breast cancer and acute leukemias. In this study, the aim was to elicit the overproduction of anthraquinones in C. triloba cell suspension cultures. Cell suspension cultures were initiated from callus leaf explants and elicited with methyl jasmonate after 21 days when the suspended biomass concentration was 34 g.L-1 (6.8 fold increase in wet weight). 2-methyl-4-hydroxy anthraquinone was detected by comparison to the root extract using TLC. HPLC analysis showed that the production yield of 2-methyl-4-hydroxy anthraquinone in the elicited culture increased 37.5 - fold after the one month culture period compared to the control culture. This is the first study that shows C. triloba can be micro-propagated by plant cell culture techniques and produce a potential bioactive compound for cancer therapy.

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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 consists of the basic structure of 9, 10-anthracenedione (Bajaj, 1999). Their derivatives currently represent one of the most effective cytostatic and front line therapy for a variety of systematic and solid tumors (ref). Examples of drugs containing the 9,10- anthracenedione moiety include: daunorubicin and mitoxantrone (McClendon and Osheroff, 2007) From previous studies we have isolated two anthraquinones from the root extracts of Ceratotheca triloba; 9,10- anthracenedione and 2-methyl-4-hydroxy anthraquinone which are structurally similar to mitoxantrone. The production of anthraquinones from root extracts is however limited as it takes approximately 1-2 years for the C. triloba plant to mature to a feasible size, prior to harvesting. This also leads to the destruction of already established plants. Furthermore 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 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 relatively easily scaled up from the shake flask stage to large-scale bioreactors (Schlatmann et al., 1996; Wen, 1995).

Studies have been conducted on the production of various derivatives of 9, 10- anthracenedione in plant cell cultures (Examples include: Rubia cordifolia, Rudgea jasminoides, Rubia tinctorum L, Morinda elliptica, Cinchona robusta) (Bulgakov et al., 2002, Oliveira et al., 2007, Orban et al., 2008 Jasril et al., 2003, Han et al., 2002), however there is no literature that reports the production of 9,10- anthracenedione and 2-methyl-4-hydroxy anthraquinone from the C. triloba cell culture. C. triloba is a South African plant that is widely distributed in the summer rainfall areas, especially grass lands, rocky places and on disturbed ground and along roadsides. This flowering plant belongs to the family Pedaliaceae and is commonly known as wild foxglove (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 (Tredgold, 1986). Previous nutritional, chemical and antioxidant studies were conducted on the C. triloba plant in order 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. 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 biosynthesis 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 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, examples include; paclitaxel and baccatin III from Taxus species (Yukimune et al., 1996) and Ajmalicine from Catharanthus roseus (ref).

MATERIALS AND METHODS

Plant material

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C. triloba was collected in Durban, Province of Kwazulu Natal, South Africa, and identified by Professor H. Baijnath, a botanist of the University of Kwazulu Natal (Westville), where a voucher specimen was deposited (Durban Botanical Herbar­ium).

Plant cell culture

The leaves of the C. triloba plant were removed and washed with distilled water three times at 1 minute intervals. Thereafter, they were sterilized with HgCI2 (0.1 %) and NaCIO (30 % and 40%). 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. Leaf disks submerged in distilled water for 20 minutes, to serve as the control for the experiment. After completion of the surface sterilization process, 0.5 cm square leaf disks were placed on MS medium (Murashige and Skoog, 1962) (six disks per plate) which was prepared using MS basal powder with sucrose and agar (Sigma-Aldrich, Inc). The medium supplemented with 1 mg.L-1 of 2.4-D and 6-BAP (Sigma-Aldrich, Inc). All treatments were tested by using two plates of medium and 12 replicates of leave disks. The prepared explant plates were incubated in the dark phase at 26ËšC for a period of one week. The explants were visually screened on day 7; the percentage of contamination and the level of tissue damage on each explant were recorded. The sterilization method to mass produced callus from sterilized explants incubated for 4-5 weeks at 26 C in the dark phase. Callus cultures induced from the explants were transferred onto the fresh medium. Maintenance of callus cultures was achieved by sub-culturing callus tissue of a 0.5 cm diameter at 3 week intervals on fresh medium.

Table 1 Different sterilization treatments and exposure times for leave explants

Sterilization

agent 1

Sterilization

Agent 2

Exposure time (minutes)

agent 1

Exposure time

(minutes)

Agent 2

HgCl2(0.1 %)

5

NaClO (30 %)

15

NaClO (40 %)

20

NaClO (30 %)

HgCl2 (0.1 %)

15

5

NaClO (40 %)

HgCl2 (0.1 %)

15

5

Control

Water

20

Approximately 2 g of yellow calli (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, Polychem supplies cc) at 100 rpm and incubated at 26ËšC in the dark phase. The cell suspension cultures were sub-cultured after one week of cultivation by transferring an aliquot of 30 ml of culture into 500 ml Erlenmeyer flasks containing 100 ml of MS medium. Cultures at 100 ml scale provided inocula for conducting shake flask experiments at a 400 ml scale. 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 in the flask 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 on an analytical balance (Adventurer ohaus). The wet weight was determined by the following equation: [(Wet weight + filter) - (filter)]* 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 1.100 ml of the elicitor solution (2.5 µl of ethanol 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 and analysis of anthraquinones

The plant portals of C. triloba were carefully examined and old, insect-damaged; fungus-infected roots were removed. Healthy roots were spread out and dried in the laboratory at room temperature for 5-8 days or until they broke easily by hand. Once completely dry, plant material was ground to a fine powder using a Wareing blender. Larger quantities were crushed using a Retsch Mühle mill at the Department of Biotechnology and Food Technology (DUT), to grind material to a fine powder of c. 1.0 mm diameter. Material was stored in a closed container at room temperature until required. The extraction protocol

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Cell suspension cultures were harvested by centrifugation (Eppendorf centrifuge 5810) at 4000 rpm for 10 minutes at 20ËšC. Thereafter cell mass was separated from the supernatant and sonicated (Virsonic, Virtis) at 4 psi for 10 minutes. Anthraquinones were extracted by agitating the cell mass on a shaker (Infors Ecotron, Polychem suppliers cc) at 180 rpm for 24 hours at room temperature in 100 ml of hexane and the supernatant 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 two 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 standards; 9,10- anthracenedione and 2-methyl-4-hyroxy anthraquinone (Sigma-Aldrich, Inc) were prepared at 1 mg.ml-1 concentration using ethyl acetate as a diluent. Approximately 10 µl of each standard solution, 20 µl of the root extract (100 mg/ml of hexane) and 50 µl of the cell extract (dissolved in ethyl acetate) was applied TLC silica gel plate (Merck TLC F254 or Silica gel 60 plates). The TLC plates developed in two mobile phases; petroleum ether: ethyl acetate: formic acid (75:25:1) and ethyl acetate: methanol: water (100:13.5:10). 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 (Wagner et al., 1984).

Anthraquinones were also detected by comparison of the root and cell and supernatant extracts. Approximately 20 µl of the root extract (100 mg/ml of hexane) and 50 µl of the cell extract were applied to the TLC silica gel plate (Merck TLC F254 or Silica gel 60 plates). The TLC plate developed in a hexane: ethyl acetate (90:10) mobile phase. Separated anthraquinones were visualized under visible and ultraviolet light (254 and 360 nm, Camac Universal UV lamp TL-600) after the TLC plates were sprayed with 5 % KOH in ethanol (according to Wagner et al., 1984, with modifications).

Identification and quantification of anthraquinones by HPLC analysis

Cell extracts were dried at room temperature for 2-3 days and dissolved in 1 ml of ethanol; the filtrates were used for HPLC analysis (according to Fernand et al., 2008 with modifications). Separation and quantitative analyses of anthraquinones were performed on a Merck- Hitachi LaChrom system (Darmstadt, Germany) consisting of an 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 -C 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 flow rate of 0.4mL/min using an acetonitrile/methanol/buffer (25:55:20,v/v), where the buffer is 10mM ammonium acetate (NH4Ac) 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 the developing 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 bacterial contamination in respective explants was 8 % as the bacterial contamination remained localized to the affected explants (Table 2). Thus the type of contamination can affect the percentage of sterile explants obtained after the treatment. Explant plates contaminated with fungi were discarded and the sterile explants that remained unaffected by bacterial contamination were transferred to fresh MS medium. Therefore in cases where the plant tends to have a high degree of fungal contamination, it should be recommended that one leaf explant be placed one plate of medium. An anti-fungal agent can also be incorporated into the medium at an appropriate concentration.

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 meristamatic tissue in an explant the more liable it could be to tissue damage caused by the surface sterilization agents. Therefore leaves (green leaves) with a lower level of meristematic tissue were selected and surface sterilized with NaCIO (30%) and HgCI2 (0.1%) to induce callus cultures from the C. triloba plant.

Table 2 Percentage of contamination and level of tissue damage after sterilization with different treatments

Treatments

Type of contamination

Percentage contamination

Std deviation

Degree of tissue damage

1

Fungal

100

0

++++a

2

Fungal

100

0

+c

3

Bacterial

16.67

11.78

+++b

4

-

0

0

+++b

5

Bacterial

16.67

11.78

+c

6 (control)

Fungal

100

0

++++a

n=12, ±: standard deviation, 1) 0.1% HgCI2, 2) 30% NaCIO, 3) 40 % NaCIO , 4) 30 % NaCIO and HgCI2 , 5) 40 % NaCIO and HgCI2 , 6) Water (control), a) highest degree of tissue damage, b) high degree of tissue damage, c) Low degree of tissue damage.

Callus initiation was observed on the surface or cut ends of the explants after 2-3 weeks of inoculation. After five weeks the entire leaf explant was transformed into callus tissue (Figure 1). Callus cultures induced MS medium were orange-yellow. Sub-cultured callus tissue produced root hairs and root- like structures after three weeks. The tips of the root-like structures contained a red-orange pigment (Figure 1). The callus morphological properties were evaluated as previous studies have shown this parameter can be used to predict whether anthraquinones are being produced in culture. The orange-yellow color of the C. triloba callus could be due to the medium used and the production of anthraquinones (Figure 1). M. elliptica leaf explants also produced yellow callus cultures on MS medium and the liquid medium turn yellow when anthraquinones were released in the from the cells (Abdullah et al., 1998). The red- orange pigmented observed at the tips of the root like structures of the C. triloba callus could be the presence of anthraquinones. Studies conducted by Bais et al., (2002) have shown that pigmented regions of the Hyperium perforatum (St John's worts) callus contained the polyketide (anthraquinone belongs to this group of compounds) Hypericin. In addition since anthraquinones are natural pigments (Hattori et al., 1993), the pigments present in the callus can be used as a marker for selecting high yielding cell lines. According to a study conducted by Mischenko et al., (1999), the orange calli accumulated higher anthraquinone content than yellow calli.

Root hairs

Root-like structures

Figure 1 Orange- yellow callus was induced from C. triloba leave explant and sub-cultured callus developed root hairs and root- like structures.

Elicitation of cell suspension cultures

Figure 2 Production of biomass in C. triloba cell suspension cultures

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 (ref). 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 2). 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 (Figure 2). After 20 days dense, orange- yellow cell suspension cultures with large aggregates formed. Therefore cell suspension cultures were treated methyl jasmonate on day 21 to elicit the production of anthraquinones. The production of cell suspension cultures with 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 2, a significant increase in biomass occurred after day 21 (Figure 2). This could be due the high level of aggregation that occurred in the control 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 3B and 3D 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).

Cell suspension cultures turned dark brown 2 days after the addition of methyl jasmonate while control cultures remained orange-yellow (Figure 3A and 3C). As a result brown aggregates formed in elicited cultures compared to the control culture which produced orange-yellow (Figure 3B and 3D). M. elliptica cells turn brown when anthraquinones are 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.

Figure 3 Effect of methyl jasmonate on cell suspension cultures. The control culture produced orange cell aggregates (A and B) and the elicited culture produced dark brown cell aggregates (C and D).

Analysis of elicited cell suspension culture extracts

R IC EC IC EC R IC EC IC EC R IC EC IC EC

Control Elicited Control Elicited Control Elicited

Figure 4 Detection of anthraquinones in C. triloba cell suspension cultures after elicitation. Developed TLC plates were sprayed with and p-anisaldehyde (A) and 5 % KOH (B and C). The plates were viewed under UV light at 254 nm (B) and 360 nm (C)

The anthraquinones of interest, 9,10- anthracenedione, and 2-methyl anthraquinone could not be detected in control and elicited culture extracts when TLC plate sprayed with p-anisaldehyde (Figure 4A). Therefore an alternative methodology was employed which entailed of viewing a 5% KOH pre-sprayed plate under UV light. No anthraquinones were detected under 254 nm (Figure 4B). However when the TLC plate was viewed under UV light at 360 nm, 9.10-anthraquinone fluoresces yellow and 2-methyl anthraquinone fluoresces orange in the root extract (Figure 4C). In comparison to the root extract, only 2- methyl anthraquinone (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 had the same Rf values 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.

HPLC analysis of anthraquinones in cell cultures

Figure 5 HPLC chromatogram showing 9,10- anthracenedione eluted at 5.91 minutes and 2-methyl anthraquinone eluted at 7.25 minutes

Figure 6 HPLC profile of the control culture showing the 2-methyl anthraquinone peak at 6.91 minutes

Figure 7 HPLC profile of the elicited culture showing the 2-methyl anthraquinone peak at 7.02 minutes

Table 3 Concentration of the identified anthraquinones in elicited and control cultures

Anthraquinone

Control (supernatant extract)

Control (intracellular extract)

Elicited (supernatant extract)

Elicited (intracellular extract)

9,10- anthracenedione

Co-elution

Co-elution

Co-elution

Co-elution

2-methyl anthraquinone

-

0.02 µg.ml-1

0.053 µg.ml-1

0.75 µg.ml-1

TLC and HPLC analysis showed that anthraquinone accumulation was principally intracellular based as the concentrations of the intracellular extracts were higher than that of the supernatant extracts (Figure 4C and Table 3). HPLC analysis showed the 9,10- anthracenedione and 2-methyl anthraquinone standards eluted at retention times of 5.90-6.20 minutes and 6.90-7.40 minutes respectively (Figure 5). The elicited and control culture extract (intra-cellular) profiles showed a peak at 6.91 and 7.05 minutes, respectively. 2-methyl anthraquinone was identified in both the extracts (Figure 6 and 7). In some instances anthraquinones were detected prior to elicitation as in the case of Cinchona pubescens, as observed in C. triloba (Wijnsma et al., 1984). In contrast cells of C. robusta only accumulate anthraquinones after treatment with an elicitor (Schripsema et al., 1999). The 2-methyl anthraquinone peak was larger in the elicited culture extract profile compared to the control culture extract. A higher concentration of 2-methyl anthraquinone 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 2-methyl anthraquinone in the elicited culture increased 37.5 - fold after the one month culture period compared to the control culture. The production of anthraquinones in plant cell culture has been enhanced through several other elicitors. Fungal polysaccharides increased the production anthraquinones in Rubia tinctorum L. Jasmonic acid and salicylic acid was also employed and these elicitors increased the production of pseudopurpurin and alizarin respectively (Orban et al., 2008). A yeast elicitor prepared from yeast extract increased the production of naphthoquinones in R. jasminoides and also elicited the production of 1.4- naphthohydroquinone (Oliveira et al., 2007).

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 6 and 7). 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.

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. Since elicitation with methyl jasmonate does lead to the overproduction of 2-methyl anthraquinone in C. triloba cell suspension cultures, elicitation parameters (elicitation concentration and duration of elicitation exposure) should be evaluated using methyl jasmonate as well as other elicitors to further enhance the production anthraquinones.