Earlier surveies on the Ceratotheca triloba Hook.f. works 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 pick for the intervention of chest malignant neoplastic disease and acute leukaemia. In this survey, the purpose was to arouse the overrun of anthraquinones in C. triloba cell suspension civilizations. Cell suspension civilizations were initiated from callus leaf explants and elicited with methyl jasmonate after 21 yearss when the suspended biomass concentration was 34 g.L-1 ( 6.8 fold addition in wet weight ) . 2-methyl-4-hydroxy anthraquinone was detected by comparing to the root infusion utilizing TLC. HPLC analysis showed that the production output of 2-methyl-4-hydroxy anthraquinone in the evoked civilization increased 37.5 – crease after the one month civilization period compared to the control civilization. This is the first survey that shows C. triloba can be micro-propagated by works cell civilization techniques and bring forth a possible bioactive compound for malignant neoplastic disease therapy.
Presently malignant neoplastic disease intervention involves radiation therapy or chemotherapy. These intervention regimens are effectual but have many side effects ; therefore new bioactive compounds such as anthraquinones are being investigated. Anthraquinones are a category of natural compounds that consists of the basic construction of 9, 10-anthracenedione ( Bajaj, 1999 ) . Their derived functions presently represent one of the most effectual cytostatic and front line therapy for a assortment of systematic and solid tumours ( ref ) . Examples of drugs incorporating the 9,10- anthracenedione mediety include: daunorubicin and mitoxantrone ( McClendon and Osheroff, 2007 ) From old surveies we have isolated two anthraquinones from the root infusions of Ceratotheca triloba ; 9,10- anthracenedione and 2-methyl-4-hydroxy anthraquinone which are structurally similar to mitoxantrone. The production of anthraquinones from root infusions is nevertheless limited as it takes about 1-2 old ages for the C. triloba works to maturate to a executable size, prior to reaping. This besides leads to the devastation of already established workss. Furthermore works growing is besides negatively affected by biological influences ( pathogen sensitiveness and insects ) during the winter months. To get the better of these restrictions the production of the possible anticancer anthraquinones from C. triloba was investigated works cell civilization engineering. Plant cell suspension civilizations are the preferable manner of bring forthing phyto-pharmaceutical compounds because they are correctable to good fabrication pattern ( GMP ) processs and the production of the compound can comparatively easy scaled up from the shingle flask phase to large-scale bioreactors ( Schlatmann et al. , 1996 ; Wen, 1995 ) .
Surveies have been conducted on the production of assorted derived functions of 9, 10- anthracenedione in works cell civilizations ( 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 ) , nevertheless there is no literature that reports the production of 9,10- anthracenedione and 2-methyl-4-hydroxy anthraquinone from the C. triloba cell civilization. C. triloba is a South African works that is widely distributed in the summer rainfall countries, particularly grass lands, bouldery topographic points and on disturbed land and along waysides. This blossoming works belongs to the household Pedaliaceae and is normally known as wild digitalis ( Tredgold, 1986 ) . The whole works soaked in H2O may be used as a replacement for soap or shampoo. C. triloba can besides function as beginning of traditional medical specialty to handle painful menses, tummy spasms, sickness, febrility and diarrhoea ( Tredgold, 1986 ) . Previous nutritionary, chemical and antioxidant surveies were conducted on the C. triloba works in order to preliminary buttocks of the nutritionary value. In footings of traditional leafy veggies, C. triloba serves as a good beginning of energy and Mg ( Odhav et al. , 2007 ) .
In this survey an effort was made to heighten the output of anthraquinones produced in C. triloba cell suspension cultures utilizing methyl jasmonate. By and large, when works cells are exposed to chemical and environmental elicitors via specific works receptors, certain biological responses are triggered which lead to the activation of biogenesis cistrons and later the production of works secondary metabolites ( Yukimune et al. , 1996 ) . The chief advantage of utilizing this scheme is that it reduces the clip taken to obtain high outputs of the secondary metabolites ( Barz et al. , 1988 ; Eilert, 1987 ; DiCosmo and Tallevi, 1985 ) . Jasmonates play cardinal function in arousing biological responses that lead to the accretion of secondary metabolites ( Gundlach et al. , 1992 ) . Methyl jasmonate was used in this survey as it has been proven to increase the production of phyto-pharmaceutically valuable compounds, illustrations include ; paclitaxel and baccatin III from Taxus species ( Yukimune et al. , 1996 ) and Ajmalicine from Catharanthus roseus ( ref ) .
MATERIALS AND METHODS
C. triloba was collected in Durban, Province of Kwazulu Natal, South Africa, and identified by Professor H. Baijnath, a phytologist of the University of Kwazulu Natal ( Westville ) , where a verifier specimen was deposited ( Durban Botanical Herbarium ) .
Plant cell civilization
The foliages of the C. triloba works were removed and washed with distilled H2O three times at 1 minute intervals. Thereafter, they were sterilized with HgCI2 ( 0.1 % ) and NaCIO ( 30 % and 40 % ) . The sterilisation agents were tested separately and in combinations utilizing the exposure time/s indicated in Table 1. Excess detergent staying on the foliages was washed off with unfertile distilled H2O at each interval. Leaf discs submerged in distilled H2O for 20 proceedingss, to function as the control for the experiment. After completion of the surface sterilisation procedure, 0.5 centimeter square foliage discs were placed on MS medium ( Murashige and Skoog, 1962 ) ( six discs per home base ) which was prepared utilizing MS basal pulverization 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 interventions were tested by utilizing two home bases of medium and 12 replicates of leave discs. The prepared explant home bases were incubated in the dark stage at 26EsC for a period of one hebdomad. The explants were visually screened on twenty-four hours 7 ; the per centum of taint and the degree of tissue harm on each explant were recorded. The sterilisation method to mass produced callosity from sterilized explants incubated for 4-5 hebdomads at 26 C in the dark stage. Callus civilizations induced from the explants were transferred onto the fresh medium. Care of callosity civilizations was achieved by sub-culturing callus tissue of a 0.5 centimeter diameter at 3 hebdomad intervals on fresh medium.
Table 1 Different sterilisation interventions and exposure times for leave explants
Exposure clip ( proceedingss )
( proceedingss )
HgCl2 ( 0.1 % )
NaClO ( 30 % )
NaClO ( 40 % )
NaClO ( 30 % )
HgCl2 ( 0.1 % )
NaClO ( 40 % )
HgCl2 ( 0.1 % )
Approximately 2 g of xanthous callosity ( three hebdomads old ) from the 2nd sub-culture was transferred into 250 milliliters Erlenmeyer flasks incorporating 50 milliliter 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 milliliter ) at 100 revolutions per minute and incubated at 26EsC in the dark stage. The cell suspension civilizations were sub-cultured after one hebdomad of cultivation by reassigning an aliquot of 30 milliliter of civilization into 500 milliliters Erlenmeyer flasks incorporating 100 milliliter of MS medium. Cultures at 100 milliliters graduated table provided inoculant for carry oning shake flask experiments at a 400 milliliter graduated table. A growing curve was constructed to obtain sufficient cell mass for evocation. All flasks were sampled at 7 twenty-four hours intervals to find the measure of cell mass in the flask by wet weight analysis. Triplicate samples of the cell suspension civilization ( 2 milliliter ) were vacuity filtered through pre-weighed filters ( 0.22 µm, 47mm, white grid, Millipore ) after which each filter incorporating wet biomass was measured on an analytical balance ( Adventurer ohaus ) . The wet weight was determined by the undermentioned equation: [ ( Wet weight + filter ) – ( filter ) ] * 500 = moisture weight ( g. L-1 ) . Methyl jasmonate ( Sigma-Aldrich, Inc ) solution was prepared at a concentration of 100 µM in ethyl alcohol. An aliquot of 1.100 milliliter of the elicitor solution ( 2.5 µl of ethanol per milliliter of civilization ) was filter sterilized ( 0.22 µm filter ) into two flasks on twenty-four hours 21. An equal volume of ethyl alcohol was filter sterilized into two flasks to function as control civilizations. The evocation was conducted for a 9 twenty-four hours period and cell suspension civilizations were harvested at twenty-four hours 30 to execute extraction and chromatographic analyses.
Extraction and analysis of anthraquinones
The works 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 research lab at room temperature for 5-8 yearss or until they broke easy by manus. Once wholly dry, works stuff was land to a all right pulverization utilizing a Wareing liquidizer. Larger measures were crushed utilizing a Retsch M & A ; uuml ; hle factory at the Department of Biotechnology and Food Technology ( DUT ) , to crunch stuff to a all right pulverization of c. 1.0 millimeter diameter. Material was stored in a closed container at room temperature until required. The extraction protocol
Cell suspension civilizations were harvested by centrifugation ( Eppendorf extractor 5810 ) at 4000 revolutions per minute for 10 proceedingss at 20EsC. Thereafter cell mass was separated from the supernatant and sonicated ( Virsonic, Virtis ) at 4 pounds per square inch for 10 proceedingss. Anthraquinones were extracted by fomenting the cell mass on a shaker ( Infors Ecotron, Polychem providers cc ) at 180 revolutions per minute for 24 hours at room temperature in 100 milliliter of hexane and the supernatant in 200 milliliters hexane. Hexane fractions were separated and concentrated by utilizing a roto-evaporator ( Heidolph Laborota 400 efficient ) with the H2O bath set at a temperature of 50 & A ; deg ; C and the flask rotated at 60 revolutions per minute. The residues where dissolved in 10 milliliter of hexane while the extra residue that was fixed to the flask was dissolved in 5 milliliter of ethyl ethanoate. The hexane and ethyl ethanoate fractions were so pooled and air dried for two yearss to farther concentrate the extract readying for chromatographic analyses.
Detection of anthraquinones produced in C. triloba cells by TLC
Thin bed chromatography was performed to observe anthraquinones in cell and supernatant infusions by utilizing criterions ; 9,10- anthracenedione and 2-methyl-4-hyroxy anthraquinone ( Sigma-Aldrich, Inc ) were prepared at 1 mg.ml-1 concentration utilizing ethyl ethanoate as a dilutant. Approximately 10 µl of each standard solution, 20 µl of the root infusion ( 100 mg/ml of hexane ) and 50 µl of the cell infusion ( dissolved in ethyl ethanoate ) was applied TLC silica gel home base ( Merck TLC F254 or Silica gel 60 home bases ) . The TLC home bases developed in two nomadic stages ; crude oil quintessence: ethyl ethanoate: formic acid ( 75:25:1 ) and ethyl ethanoate: methyl alcohol: H2O ( 100:13.5:10 ) . Separated anthraquinones were visualized under seeable and ultraviolet visible radiation ( 254 and 360 nanometer, Camag Universal UV lamp TL-600 ) after the TLC home bases were sprayed with 5 % KOH in ethyl alcohol ( Wagner et al. , 1984 ) .
Anthraquinones were besides detected by comparing of the root and cell and supernatant infusions. Approximately 20 µl of the root infusion ( 100 mg/ml of hexane ) and 50 µl of the cell infusion were applied to the TLC silicon oxide gel home base ( Merck TLC F254 or Silica gel 60 home bases ) . The TLC home base developed in a hexane: ethyl ethanoate ( 90:10 ) Mobile stage. Separated anthraquinones were visualized under seeable and ultraviolet visible radiation ( 254 and 360 nanometer, Camac Universal UV lamp TL-600 ) after the TLC home bases were sprayed with 5 % KOH in ethyl alcohol ( harmonizing to Wagner et al. , 1984, with alterations ) .
Designation and quantification of anthraquinones by HPLC analysis
Cell infusions were dried at room temperature for 2-3 yearss and dissolved in 1 milliliter of ethyl alcohol ; the filtrates were used for HPLC analysis ( harmonizing to Fernand et al. , 2008 with alterations ) . Separation and quantitative analyses of anthraquinones were performed on a Merck- Hitachi LaChrom system ( Darmstadt, Germany ) consisting of an D 7000 system accountant, four pumps ( D7400 ) , a Merck- Hitachi LaChrom ( L-7200 ) car injector and an Merck- Hitachi LaChrom ( L-7200 ) UV-VIS sensor ( ? = 260 nanometer ) . Separation of the analytes was performed at 40 -C on a Licrospher C18 ( 2 ) column, 100 EsA pore size, 5µm atom size, 250-4.6mm i.d.column incorporating a guard column ( Merck, Darmstadt, Germany ) . The analytes were eluted isocratically at a i¬‚ow rate of 0.4mL/min utilizing an acetonitrile/methanol/buffer ( 25:55:20, v/v ) , where the buffer is 10mM ammonium ethanoate ( NH4Ac ) at pH 6.8. The injection volume was 10 µL.
RESULTS AND DISCUSSION
Sterilization of C. triloba explant stuff
A major challenge at the initial phase of the developing C. triloba cell civilization system was to get the better of the taint in field grown workss, as this was the beginning of explant stuff. It was hence of import to analyze consequence of two surface sterilisation agents on taint and the leaf tissue. The most prevailing type of taint in C. triloba explants was fungous while bacterial taint occurred indiscriminately. The per centum explants contaminated with Fungis were 100 % with several interventions due to the spread of the taint to all explants in the home base. The per centum of bacterial taint in several explants was 8 % as the bacterial taint remained localized to the affected explants ( Table 2 ) . Thus the type of taint can impact the per centum of unfertile explants obtained after the intervention. Explant home bases contaminated with Fungis were discarded and the unfertile explants that remained unaffected by bacterial taint were transferred to fresh MS medium. Therefore in instances where the works tends to hold a high grade of fungous taint, it should be recommended that one foliage explant be placed one home base of medium. An anti-fungal agent can besides be incorporated into the medium at an appropriate concentration.
Explants treated with NaCIO ( 30 % ) and HgCI2 ( 0.1 % ) in combination eradicated all contaminations present in the foliage explants, hence this intervention was used for induction of callosity. The effectivity of this intervention could be due to interactive consequence of the two surface sterilisation agents as contaminated explants resulted when they were used individually. However a high degree of tissue harm was observed in the explants when this intervention government was applied ( Table 2 ) . A possible ground could be that foliage ( light green foliage ) explants were exposed to this intervention contained a high degree of meristematic tissue. The higher grade of meristamatic tissue in an explant the more apt it could be to weave harm caused by the surface sterilisation agents. Therefore foliages ( green foliages ) with a lower degree of meristematic tissue were selected and come up sterilized with NaCIO ( 30 % ) and HgCI2 ( 0.1 % ) to bring on callus civilizations from the C. triloba works.
Table 2 Percentage of taint and degree of tissue harm after sterilisation with different interventions
Type of taint
Degree of tissue harm
6 ( control )
n=12, ± : standard divergence, 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 grade of tissue harm, B ) high grade of tissue harm, degree Celsius ) Low grade of tissue harm.
Callus induction was observed on the surface or cut terminals of the explants after 2-3 hebdomads of vaccination. After five hebdomads the full foliage explant was transformed into callus tissue ( Figure 1 ) . Callus civilizations induced MS medium were orange-yellow. Sub-cultured callus tissue produced root hairs and root- like constructions after three hebdomads. The tips of the root-like constructions contained a reddish-orange pigment ( Figure 1 ) . The callus morphological belongingss were evaluated as old surveies have shown this parametric quantity can be used to foretell whether anthraquinones are being produced in civilization. The orange-yellow colour of the C. triloba callosity could be due to the medium used and the production of anthraquinones ( Figure 1 ) . M. elliptica foliage explants besides produced xanthous callosity civilizations on MS medium and the liquid medium turn xanthous 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 constructions of the C. triloba callosity could be the presence of anthraquinones. Surveies conducted by Bais et al. , ( 2002 ) have shown that pigmented parts of the Hyperium perforatum ( St John ‘s worts ) callosity contained the polyketide ( anthraquinone belongs to this group of compounds ) Hypericin. In add-on since anthraquinones are natural pigments ( Hattori et al. , 1993 ) , the pigments present in the callosity can be used as a marker for choosing high giving up cell lines. Harmonizing to a survey conducted by Mischenko et al. , ( 1999 ) , the orange callosity accumulated higher anthraquinone content than xanthous callosity.
Figure 1 Orange- xanthous callosity was induced from C. triloba leave explant and sub-cultured callosity developed root hairs and root- like constructions.
Evocation of cell suspension civilizations
Figure 2 Production of biomass in C. triloba cell suspension civilizations
The constitution of cell suspension civilizations from callus tissue was a cardinal measure in developing an efficient cell civilization system for bring forthing anthraquinones as liquid civilizations have a faster growing rate compared to callus ( ref ) . Callus civilizations were transitioned into liquid medium, the crumbly callus tissue dispersed into little sums when flasks were placed on the shaker. A growing curve of C. triloba cell suspension civilizations was generated to obtain sufficient biomass to arouse the production anthraquinones ( Figure 2 ) . The biomass concentration increased from 5.50 g.l-1 to 19 g.l-1 after 20 yearss of cultivation ; nevertheless this addition accounts merely for the cell mass that remained in suspension during trying as really big sums tend to drop to the underside of the flask ( Figure 2 ) . After 20 yearss dense, orange- yellow cell suspension cultures with big sums formed. Therefore cell suspension civilizations were treated methyl jasmonate on twenty-four hours 21 to arouse the production of anthraquinones. The production of cell suspension cultures with extremely heavy cell mass is important for obtaining high outputs of the plant-derived compound as secondary metabolites are based in intra-cellular parts of the cell ( Luckner, 1990 ) . Harmonizing to Figure 2, a important addition in biomass occurred after twenty-four hours 21 ( Figure 2 ) . This could be due the high degree of collection that occurred in the control and evoked civilizations. A crisp lessening in cell mass occurred in the control on twenty-four hours 30 due to the formation of big sums in suspension ( larger cell sums tend to drop to the underside of the flask during trying ) . Figure 3B and 3D shows the sums in the evoked civilization were smaller than the control civilization. A cultivated works cell suspension civilization with a high concentration of cell sums is an ideal mark for the elicitor as cell collection is associated with secondary metabolite production ( Bais et al. , 2002 ) .
Cell suspension civilizations turned dark brown 2 yearss after the add-on of methyl jasmonate while control civilizations remained orange-yellow ( Figure 3A and 3C ) . As a consequence brown sums formed in evoked civilizations compared to the control civilization which produced orange-yellow ( Figure 3B and 3D ) . M. elliptica cells turn brown when anthraquinones are produced ( Abdullah et al. , 1998 ) . The dark brown colour of the cell sums can be used as an indicant that anthraquinones were elicited. Since the accretion of anthraquinones in C. triloba cell suspension civilizations is coupled with cell collection and cell Browning, the cell sums should be obtained and assessed for the production of anthraquinones in order to choose high giving up cells for future surveies.
Figure 3 Consequence of methyl jasmonate on cell suspension civilizations. The control civilization produced orange cell sums ( A and B ) and the evoked civilization produced dark brown cell sums ( C and D ) .
Analysis of elicited cell suspension civilization infusions
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 civilizations after evocation. Developed TLC home bases were sprayed with and p-anisaldehyde ( A ) and 5 % KOH ( B and C ) . The home bases were viewed under UV visible radiation at 254 nanometer ( B ) and 360 nanometer ( C )
The anthraquinones of involvement, 9,10- anthracenedione, and 2-methyl anthraquinone could non be detected in control and evoked civilization extracts when TLC plate sprayed with p-anisaldehyde ( Figure 4A ) . Therefore an alternate methodological analysis was employed which entailed of sing a 5 % KOH pre-sprayed home base under UV visible radiation. No anthraquinones were detected under 254 nanometers ( Figure 4B ) . However when the TLC home base was viewed under UV visible radiation at 360 nanometers, 9.10-anthraquinone fluoresces xanthous and 2-methyl anthraquinone fluoresces orange in the root infusion ( Figure 4C ) . In comparing to the root infusion, merely 2- methyl anthraquinone ( xanthous fluoresces ) was detected in the intra-cellular infusions of the evoked and control civilizations. The anthraquinone criterions confirmed the presence of anthraquinones in the intra-celluar infusions of the control and evoked civilizations but single anthraquinones could non be detected as both criterions had the same Rf values when the two nomadic stages: crude oil quintessence: ethyl ethanoate: formic acid ( 75:25:1 ) and ethyl ethanoate: methyl alcohol: H2O ( 100:13.5:10 ) , were employed.
HPLC analysis of anthraquinones in cell civilizations
Figure 5 HPLC chromatogram demoing 9,10- anthracenedione eluted at 5.91 proceedingss and 2-methyl anthraquinone eluted at 7.25 proceedingss
Figure 6 HPLC profile of the control civilization demoing the 2-methyl anthraquinone extremum at 6.91 proceedingss
Figure 7 HPLC profile of the evoked civilization demoing the 2-methyl anthraquinone extremum at 7.02 proceedingss
Table 3 Concentration of the identified anthraquinones in evoked and control civilizations
Control ( supernatant infusion )
Control ( intracellular infusion )
Elicited ( supernatant infusion )
Elicited ( intracellular infusion )
TLC and HPLC analysis showed that anthraquinone accretion was chiefly intracellular based as the concentrations of the intracellular infusions were higher than that of the supernatant infusions ( Figure 4C and Table 3 ) . HPLC analysis showed the 9,10- anthracenedione and 2-methyl anthraquinone criterions eluted at keeping times of 5.90-6.20 proceedingss and 6.90-7.40 proceedingss severally ( Figure 5 ) . The evoked and control civilization infusion ( intra-cellular ) profiles showed a extremum at 6.91 and 7.05 proceedingss, severally. 2-methyl anthraquinone was identified in both the infusions ( Figure 6 and 7 ) . In some cases anthraquinones were detected prior to evocation as in the instance of Cinchona pubescens, as observed in C. triloba ( Wijnsma et al. , 1984 ) . In contrast cells of C. robusta merely accumulate anthraquinones after intervention with an elicitor ( Schripsema et al. , 1999 ) . The 2-methyl anthraquinone extremum was larger in the evoked civilization infusion profile compared to the control civilization infusion. A higher concentration of 2-methyl anthraquinone was present in the evoked civilization infusion ( 0.75 µg.ml-1 ) than the control civilization infusion ( 0.02 µg.ml-1 ) ( Table 3 ) . Earlier surveies have shown that the production of secondary compounds in cell civilization systems were dramatically increased through the evocation scheme ( Wang and Zhong, 2002 ; Yu et al. , 2002 ) . This scheme was proven to be successful in the C. trioba cell civilization system as production output of 2-methyl anthraquinone in the evoked civilization increased 37.5 – crease after the one month civilization period compared to the control civilization. The production of anthraquinones in works cell civilization has been enhanced through several other elicitors. Fungal polyoses increased the production anthraquinones in Rubia tinctorum L. Jasmonic acid and salicylic acid was besides employed and these elicitors increased the production of pseudopurpurin and alizarin severally ( Orban et al. , 2008 ) . A yeast elicitor prepared from yeast infusion increased the production of vitamin Ks in R. jasminoides and besides elicited the production of 1.4- naphthohydroquinone ( Oliveira et al. , 2007 ) .
9,10- anthracenedione was non identified in the control and elicited civilizations 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 non divide due to the similarity of the construction between the compounds which in bend influences the elution clip of the similar compounds.
This is the first survey that shows C. triloba cell suspension civilizations can micro-propagated by works cell civilization techniques and produce possible bioactive compounds for malignant neoplastic disease therapy. Since evocation with methyl jasmonate does take to the overrun of 2-methyl anthraquinone in C. triloba cell suspension civilizations, evocation parametric quantities ( evocation concentration and continuance of evocation exposure ) should be evaluated utilizing methyl jasmonate every bit good as other elicitors to farther heighten the production anthraquinones.